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Sample records for non-nmda glutamatergic pscs

  1. Glutamatergic synaptic inputs activate neurons in the subfornical organ through non-NMDA receptors.

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    Xu, S H; Inenaga, K; Honda, E; Yamashita, H

    2000-01-14

    The subfornical organ (SFO) plays an important role in central regulation of the autonomic nervous system. The synaptic transmission properties of neurons in the SFO were studied with intracellular and whole-cell patch clamp recordings in the rat slice preparations. Both the spontaneous and evoked excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were almost completely suppressed by the glutamate receptor antagonist kynurenic acid and the non-NMDA (N-methyl-D-aspartic acid) antagonist CNQX. The non-NMDA agonist kainic acid depolarized the membrane most potently, compared with NMDA and quisqualic acid. These suggest that glutamate is a main excitatory neurotransmitter in the SFO and that its action is at least partly mediated through non-NMDA receptors.

  2. Cholinergic modulation of non-N-methyl-D-aspartic acid glutamatergic transmission in the chick ventral lateral geniculate nucleus.

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    Guo, J-Z; Sorenson, E M; Chiappinelli, V A

    2010-03-17

    Neurotransmission between glutamatergic terminals of retinal ganglion cells and principal neurons of the ventral lateral geniculate nucleus (LGNv) was examined with patch clamp recordings in chick brain slices during electrical stimulation of the optic tract. Since muscarinic and nicotinic receptors are present in high densities in LGNv, the present study examined possible roles of both receptors in modulating retinogeniculate transmission. During whole-cell recordings from LGNv neurons, acetylcholine (ACh, 100 microM) caused an initial increase in amplitudes of optic tract-evoked non-N-methyl-D-aspartic acid (NMDA) glutamatergic postsynaptic currents (PSCs). This increase was unchanged when 1 microM atropine was present, indicating that this initial enhancement of PSCs was due entirely to activation of nicotinic receptors. However, during washout of ACh the amplitudes of evoked PSCs became significantly decreased by 40.4+/-5.0% for several minutes before recovering to their original amplitudes, an effect blocked by 1 microM atropine. Exogenously applied muscarine (10 microM) markedly depressed optic tract-evoked PSCs, and this decrease in amplitude was blocked by atropine. In a second set of experiments, we examined effects of releasing endogenous ACh prior to optic tract stimulation. This was accomplished by stimulation of the lateral portion of LGNv via a separate conditioning electrode. Following a brief train of low intensity conditioning stimuli, non-NMDA glutamatergic PSCs evoked by optic tract stimulation were potentiated. However, at higher conditioning stimulus intensities the PSCs were markedly decreased compared with control, and this decrease was partially blocked by atropine (1 microM). Neither ACh nor muscarine altered amplitudes of PSCs elicited by exogenously applied glutamate. Muscarine significantly reduced the frequency but not the amplitudes of miniature PSCs, consistent with a presynaptic location for muscarinic receptors mediating these

  3. Toll-like receptor 4 enhancement of non-NMDA synaptic currents increases dentate excitability after brain injury.

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    Li, Ying; Korgaonkar, Akshata A; Swietek, Bogumila; Wang, Jianfeng; Elgammal, Fatima S; Elkabes, Stella; Santhakumar, Vijayalakshmi

    2015-02-01

    Concussive brain injury results in neuronal degeneration, microglial activation and enhanced excitability in the hippocampal dentate gyrus, increasing the risk for epilepsy and memory dysfunction. Endogenous molecules released during injury can activate innate immune responses including toll-like receptor 4 (TLR4). Recent studies indicate that immune mediators can modulate neuronal excitability. Since non-specific agents that reduce TLR4 signaling can limit post-traumatic neuropathology, we examined whether TLR4 signaling contributes to early changes in dentate excitability after brain injury. Concussive brain injury caused a transient increase in hippocampal TLR4 expression within 4h, which peaked at 24h. Post-injury increase in TLR4 expression in the dentate gyrus was primarily neuronal and persisted for one week. Acute, in vitro treatment with TLR4 ligands caused bidirectional modulation of dentate excitability in control and brain-injured rats, with a reversal in the direction of modulation after brain injury. TLR4 antagonists decreased, and agonist increased, afferent-evoked dentate excitability one week after brain injury. NMDA receptor antagonist did not occlude the ability of LPS-RS, a TLR4 antagonist, to decrease post-traumatic dentate excitability. LPS-RS failed to modulate granule cell NMDA EPSCs but decreased perforant path-evoked non-NMDA EPSC peak amplitude and charge transfer in both granule cells and mossy cells. Our findings indicate an active role for TLR4 signaling in early post-traumatic dentate hyperexcitability. The novel TLR4 modulation of non-NMDA glutamatergic currents, identified herein, could represent a general mechanism by which immune activation influences neuronal excitability in neurological disorders that recruit sterile inflammatory responses. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Recovery of network-driven glutamatergic activity in rat hippocampal neurons during chronic glutamate receptor blockade.

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    Leininger, Eric; Belousov, Andrei B

    2009-01-28

    Previous studies indicated that a long-term decrease in the activity of ionotropic glutamate receptors induces cholinergic activity in rat and mouse hypothalamic neuronal cultures. Here we studied whether a prolonged inactivation of ionotropic glutamate receptors also induces cholinergic activity in hippocampal neurons. Receptor activity was chronically suppressed in rat hippocampal primary neuronal cultures with two proportionally increasing sets of concentrations of NMDA plus non-NMDA receptor antagonists: 100 microM/10 microM AP5/CNQX (1X cultures) and 200 microM/20 microM AP5/CNQX (2X cultures). Using calcium imaging we demonstrate that cholinergic activity does not develop in these cultures. Instead, network-driven glutamate-dependent activity, that normally is detected in hyper-excitable conditions, reappears in each culture group in the presence of these antagonists and can be reversibly suppressed by higher concentrations of AP5/CNQX. This activity is mediated by non-NMDA receptors and is modulated by NMDA receptors. Further, non-NMDA receptors, the general level of glutamate receptor activity and CaMK-dependent signaling are critical for development of this network-driven glutamatergic activity in the presence of receptor antagonists. Using electrophysiology, western blotting and calcium imaging we show that some neuronal parameters are either reduced or not affected by chronic glutamate receptor blockade. However, other parameters (including neuronal excitability, mEPSC frequency, and expression of GluR1, NR1 and betaCaMKII) become up-regulated and, in some cases, proportionally between the non-treated, 1X and 2X cultures. Our data suggest recovery of the network-driven glutamatergic activity after chronic glutamate receptor blockade. This recovery may represent a form of neuronal plasticity that compensates for the prolonged suppression of the activity of glutamate receptors.

  5. The role of NMDA and non-NMDA receptors in the NTS in mediating three distinct sympathoinhibitory reflexes.

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    Sartor, Daniela M; Verberne, Anthony J M

    2007-12-01

    Cholecystokinin (CCK) elicits a sympathetic vasomotor reflex that is implicated in gastrointestinal circulatory control. We sought to determine (1) the site in the solitary tract nucleus (NTS) responsible for mediating this reflex and (2) the possible involvement of excitatory amino acid (EAA) receptors. In addition, we sought to determine whether the NTS site responsible for mediating the baroreflex (phenylephrine, PE, 10 microg/kg i.v.) and the von Bezold-Jarisch reflex (phenylbiguanide, PBG, 10 microg/kg i.v) overlap with that involved in the CCK-induced reflex (CCK, 4 microg/kg, i.v.), and to compare the relative importance of NMDA and non-NDMA receptors in these reflexes. In separate experiments, the effects of PE, PBG, and CCK on mean arterial blood pressure, heart rate, and splanchnic sympathetic nerve discharge were tested before and after bilateral microinjection into the NTS of the gamma-aminobutyric acid(A) (GABA(A)) agonist muscimol, the EAA antagonist kynurenate, the NMDA receptor antagonist D: (-)-2-amino-5-phosphopentanoic acid (AP-5), the non-NMDA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), AP-5 + NBQX, or vehicle. While all treatments (except vehicle) significantly attenuated/abolished/reversed the splanchnic sympathoinhibitory responses to PE, PBG, and CCK, the extent of blockade varied between the different treatment groups. Both NMDA and non-NMDA receptors were essential to the baroreflex and the von Bezold-Jarisch reflex, whereas the CCK reflex was more dependent on non-NMDA receptors. Muscimol, kynurenate, and AP-5 + NBQX significantly attenuated the bradycardic responses to PE and PBG (P NTS responsible for eliciting all three reflexes, NMDA and non-NMDA receptors are recruited differentially for the full expression of these reflexes. The CCK-induced sympathoinhibitory reflex is unique in that it relies predominantly on non-NMDA receptors in the NTS and elicits bradycardic effects that

  6. Glutamatergic System and Schizophrenia

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    Osman Ozdemir

    2016-12-01

    Full Text Available Glutamate is the major excitatory neurotransmitter in the brain. It has a role several cognitive functions including learning, memory and perception. Glutamatergic neurotransmission is also involved in regulating neuronal migration, synaptogenesis, and the pruning neurons. Glutamatergic exci-totoxicity has been implicated in various neuropsychiatric disorders. Accumulating evidence suggests that glutamatergic dysfunction may contribute to the pathogenesis of schizophrenia. The N-methyl-D-aspartic acid (NMDA receptor antagonists such as phencyclidine and ketamine can cause both the positive and negative symptoms psychotic symptoms in normal humans, and worsen these symptoms in persons with schizophrenia. Hence, it has been hypotesized that schizophrenia may be associated with decreased NMDA-receptor activity. According to the hypothesis, NMDA reseptor hypofunction can lead to decreased inhibition of glutamatergic neurons and excessive glutamate release. Finally, the reduction of gray matter in several brain regions seen in patients with schizophrenia has been suggested to be the result of neurotoxicity mediated by NMDA receptors. [Psikiyatride Guncel Yaklasimlar - Current Approaches in Psychiatry 2016; 8(4.000: 394-405

  7. Timeline: iPSCs--The First Decade.

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    Chari, Sheila; Mao, Steve

    2016-02-04

    Research into induced pluripotent stem cells (iPSCs) has expanded at a remarkable pace in the decade since Shinya Yamanaka and Kazutoshi Takahashi first reported their groundbreaking discovery in 2006. This Timeline highlights the key events in the development of this field, including basic insights into the production of iPSCs and how they have been applied to improve our understanding and treatment of human disease. To view this Timeline, open or download the PDF. You can also listen to the associated interview with Debbie Sweet, Editor of Cell Stem Cell, and Elena Porro, Editor of Cell. PAPERCLIP.

  8. Efficient derivation of cortical glutamatergic neurons from human pluripotent stem cells: a model system to study neurotoxicity in Alzheimer's disease.

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    Vazin, Tandis; Ball, K Aurelia; Lu, Hui; Park, Hyungju; Ataeijannati, Yasaman; Head-Gordon, Teresa; Poo, Mu-ming; Schaffer, David V

    2014-02-01

    Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population, and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular, disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors, aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits, we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as "globulomers", which have shown strong correlation with the level of cognitive deficits in AD. Administration of such Aβ oligomers yielded signs of the disease, including cell culture age-dependent binding of Aβ and cell death in the glutamatergic populations. Furthermore, consistent with previous findings in postmortem human AD brain, Aβ-induced toxicity was selective for glutamatergic rather than GABAeric neurons present in our cultures. This in vitro model of cortical glutamatergic neurons thus offers a system for future mechanistic investigation and therapeutic development for AD pathology using human cell types specifically affected by this disease. © 2013.

  9. Detailed modeling of mountain wave PSCs

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    S. Fueglistaler

    2003-01-01

    Full Text Available Polar stratospheric clouds (PSCs play a key role in polar ozone depletion. In the Arctic, PSCs can occur on the mesoscale due to orographically induced gravity waves. Here we present a detailed study of a mountain wave PSC event on 25-27 January 2000 over Scandinavia. The mountain wave PSCs were intensively observed by in-situ and remote-sensing techniques during the second phase of the SOLVE/THESEO-2000 Arctic campaign. We use these excellent data of PSC observations on 3 successive days to analyze the PSCs and to perform a detailed comparison with modeled clouds. We simulated the 3-dimensional PSC structure on all 3 days with a mesoscale numerical weather prediction (NWP model and a microphysical box model (using best available nucleation rates for ice and nitric acid trihydrate particles. We show that the combined mesoscale/microphysical model is capable of reproducing the PSC measurements within the uncertainty of data interpretation with respect to spatial dimensions, temporal development and microphysical properties, without manipulating temperatures or using other tuning parameters. In contrast, microphysical modeling based upon coarser scale global NWP data, e.g. current ECMWF analysis data, cannot reproduce observations, in particular the occurrence of ice and nitric acid trihydrate clouds. Combined mesoscale/microphysical modeling may be used for detailed a posteriori PSC analysis and for future Arctic campaign flight and mission planning. The fact that remote sensing alone cannot further constrain model results due to uncertainities in the interpretation of measurements, underlines the need for synchronous in-situ PSC observations in campaigns.

  10. The BDNF Val66Met polymorphism enhances glutamatergic transmission but diminishes activity-dependent synaptic plasticity in the dorsolateral striatum.

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    Jing, Deqiang; Lee, Francis S; Ninan, Ipe

    2017-01-01

    The Val66Met polymorphism in the brain-derived neurotrophic factor (BDNF) gene disrupts the activity-dependent release of BDNF, which might underlie its involvement in several neuropsychiatric disorders. Consistent with the potential role of regulated release of BDNF in synaptic functions, earlier studies have demonstrated that the BDNF Val66Met polymorphism impairs NMDA receptor-mediated synaptic transmission and plasticity in the hippocampus, the medial prefrontal cortex and the central amygdala. However, it is unknown whether the BDNF Val66Met polymorphism affects synapses in the dorsal striatum, which depends on cortical afferents for BDNF. Electrophysiological experiments revealed an enhanced glutamatergic transmission in the dorsolateral striatum (DLS) of knock-in mice containing the variant polymorphism (BDNF(Met/Met)) compared to the wild-type (BDNF(Val/Val)) mice. This increase in glutamatergic transmission is mediated by a potentiation in glutamate release and NMDA receptor transmission in the medium spiny neurons without any alterations in non-NMDA receptor-mediated transmission. We also observed an impairment of synaptic plasticity, both long-term potentiation and depression in the DLS neurons, in BDNF(Met/Met) mice. Thus, the BDNF Val66Met polymorphism exerts an increase in glutamatergic transmission but impairs synaptic plasticity in the dorsal striatum, which might play a role in its effect on neuropsychiatric symptoms. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

  11. A new pyrrolyl-quinoxalinedione series of non-NMDA glutamate receptor antagonists: pharmacological characterization and comparison with NBQX and valproate in the kindling model of epilepsy.

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    Löscher, W; Lehmann, H; Behl, B; Seemann, D; Teschendorf, H J; Hofmann, H P; Lubisch, W; Höger, T; Lemaire, H G; Gross, G

    1999-01-01

    Antagonists at the ionotropic non-NMDA [AMPA (amino-methyl proprionic acid)/kainate] type of glutamate receptors have been suggested to possess several advantages compared to NMDA (N-methyl-D-aspartate) receptor antagonists, particularly in terms of risk/benefit ratio, but the non-NMDA receptor antagonists available so far have not fulfilled this promise. From a large series of pyrrolyl-quinoxalinedione derivatives, we selected six new competitive non-NMDA receptor antagonists. The basis of selection was high potency and selectivity for AMPA and/or kainate receptors, high in vivo potency after systemic administration, and an acceptable ratio between neuroprotective or anticonvulsant effects and adverse effects. Pharmacological characteristics of these novel compounds are described in this study with special emphasis on their effects in the kindling model of temporal lobe epilepsy, the most common type of epilepsy in humans. In most experiments, NBQX and the major antiepileptic drug valproate were used for comparison with the novel compounds. The novel non-NMDA receptor antagonists markedly differed in their AMPA and kainate receptor affinities from NBQX. Thus, while NBQX essentially did not bind to kainate receptors at relevant concentrations, several of the novel compounds exhibited affinity to rat brain kainate receptors or recombinant kainate receptor subtypes in addition to AMPA receptors. One compound, LU 97175, bound to native high affinity kainate receptors and rat GluR5-GluR7 subunits, i.e. low affinity kainate binding sites, with much higher affinities than to AMPA receptors. All compounds potently blocked AMPA-induced cell death in vitro and, except LU 97175, AMPA-induced convulsions in vivo. In the kindling model, compounds with a high affinity for GluR7 (LU 97175) or compounds (LU 115455, LU 136541) which potently bind to AMPA receptors and low affinity kainate receptor subunits were potent anticonvulsants in the kindling model, whereas the AMPA

  12. Private Security Companies (PSCs) as a Piracy Countermeasure

    DEFF Research Database (Denmark)

    Struwe, Lars Bangert

    2012-01-01

    Private Security Companies (PSC) are a part of the Best Management Practice in the shipping industry. The ship owners are using PSCs to protect their vessels against pirates, but protection of the vessels is just a part of the services that PSCs can provide. The services can be divided in four ma...... is that the use of PSCs works for the individual ship owners, but it is not a long term solution of the piracy problem unless they are used in a coordinated way by ship owners and the international society. In this way they can become an added value in the fight against piracy....

  13. Arctigenin reduces neuronal responses in the somatosensory cortex via the inhibition of non-NMDA glutamate receptors.

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    Borbély, Sándor; Jócsák, Gergely; Moldován, Kinga; Sedlák, Éva; Preininger, Éva; Boldizsár, Imre; Tóth, Attila; Atlason, Palmi T; Molnár, Elek; Világi, Ildikó

    2016-07-01

    Lignans are biologically active phenolic compounds related to lignin, produced in different plants. Arctigenin, a dibenzylbutyrolactone-type lignan, has been used as a neuroprotective agent for the treatment of encephalitis. Previous studies of cultured rat cerebral cortical neurones raised the possibility that arctigenin inhibits kainate-induced excitotoxicity. The aims of the present study were: 1) to analyse the effect of arctigenin on normal synaptic activity in ex vivo brain slices, 2) to determine its receptor binding properties and test the effect of arctigenin on AMPA/kainate receptor activation and 3) to establish its effects on neuronal activity in vivo. Arctigenin inhibited glutamatergic transmission and reduced the evoked field responses. The inhibitory effect of arctigenin on the evoked field responses proved to be substantially dose dependent. Our results indicate that arctigenin exerts its effects under physiological conditions and not only on hyper-excited neurons. Furthermore, arctigenin can cross the blood-brain barrier and in the brain it interacts with kainate sensitive ionotropic glutamate receptors. These results indicate that arctigenin is a potentially useful new pharmacological tool for the inhibition of glutamate-evoked responses in the central nervous system in vivo.

  14. The establishment of GABAergic and glutamatergic synapses on CA1 pyramidal neurons is sequential and correlates with the development of the apical dendrite.

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    Tyzio, R; Represa, A; Jorquera, I; Ben-Ari, Y; Gozlan, H; Aniksztejn, L

    1999-12-01

    We have performed a morphofunctional analysis of CA1 pyramidal neurons at birth to examine the sequence of formation of GABAergic and glutamatergic postsynaptic currents (PSCs) and to determine their relation to the dendritic arborization of pyramidal neurons. We report that at birth pyramidal neurons are heterogeneous. Three stages of development can be identified: (1) the majority of the neurons (80%) have small somata, an anlage of apical dendrite, and neither spontaneous nor evoked PSCs; (2) 10% of the neurons have a small apical dendrite restricted to the stratum radiatum and PSCs mediated only by GABA(A) receptors; and (3) 10% of the neurons have an apical dendrite that reaches the stratum lacunosum moleculare and PSCs mediated both by GABA(A) and glutamate receptors. These three groups of pyramidal neurons can be differentiated by their capacitance (C(m) = 17.9 +/- 0.8; 30.2 +/- 1.6; 43.2 +/- 3.0 pF, respectively). At birth, the synaptic markers synapsin-1 and synaptophysin labeling are present in dendritic layers but not in the stratum pyramidale, suggesting that GABAergic peridendritic synapses are established before perisomatic ones. The present observations demonstrate that GABAergic and glutamatergic synapses are established sequentially with GABAergic synapses being established first most likely on the apical dendrites of the principal neurons. We propose that different sets of conditions are required for the establishment of functional GABA and glutamate synapses, the latter necessitating more developed neurons that have apical dendrites that reach the lacunosum moleculare region.

  15. Cannabinoids: Glutamatergic Transmission and Kynurenines.

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    Colín-González, Ana Laura; Aguilera, Gabriela; Santamaría, Abel

    2016-01-01

    The endocannabinoid system (ECS) comprises a complex of receptors, enzymes, and endogenous agonists that are widely distributed in the central nervous system of mammals and participates in a considerable number of neuromodulatory functions, including neurotransmission, immunological control, and cell signaling. In turn, the kynurenine pathway (KP) is the most relevant metabolic route for tryptophan degradation to form the metabolic precursor NAD(+). Recent studies demonstrate that the control exerted by the pharmacological manipulation of the ECS on the glutamatergic system in the brain may offer key information not only on the development of psychiatric disorders like psychosis and schizophrenia-like symptoms, but it also may constitute a solid basis for the development of therapeutic strategies to combat excitotoxic events occurring in neurological disorders like Huntington's disease (HD). Part of the evidence pointing to the last approach is based on experimental protocols demonstrating the efficacy of cannabinoids to prevent the deleterious actions of the endogenous neurotoxin and KP metabolite quinolinic acid (QUIN). These findings intuitively raise the question about what is the precise role of the ECS in tryptophan metabolism through KP and vice versa. In this chapter, we will review basic concepts on the physiology of both the ECS and the KP to finally describe those recent findings combining the components of these two systems and hypothesize the future course that the research in this emerging field will take in the next years.

  16. Increased training prevents the impairing effect of intra-amygdala infusion of the non-NMDA receptor antagonist CNQX on inhibitory avoidance expression

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    Roesler R.

    1999-01-01

    Full Text Available Intra-amygdala infusion of the non-N-methyl-D-aspartate (NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX prior to testing impairs inhibitory avoidance retention test performance. Increased training attenuates the impairing effects of amygdala lesions and intra-amygdala infusions of CNQX. The objective of the present study was to determine the effects of additional training on the impairing effects of intra-amygdala CNQX on expression of the inhibitory avoidance task. Adult female Wistar rats bilaterally implanted with cannulae into the border between the central and the basolateral nuclei of the amygdala were submitted to a single session or to three training sessions (0.2 mA, 24-h interval between sessions in a step-down inhibitory avoidance task. A retention test session was held 48 h after the last training. Ten minutes prior to the retention test session, the animals received a 0.5-µl infusion of CNQX (0.5 µg or its vehicle (25% dimethylsulfoxide in saline. The CNQX infusion impaired, but did not block, retention test performance in animals submitted to a single training session. Additional training prevented the impairing effect of CNQX. The results suggest that amygdaloid non-NMDA receptors may not be critical for memory expression in animals given increased training.

  17. Mechanism of Induction: Induced Pluripotent Stem Cells (iPSCs).

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    Singh, Vimal Kishor; Kumar, Neeraj; Kalsan, Manisha; Saini, Abhishek; Chandra, Ramesh

    2015-01-01

    Induced Pluripotent Stem Cells (iPSCs) are self renewable and can differentiate to different types of adult cells, which has shown great promises in the field of regenerative medicine. iPSCs are reprogrammed from human somatic cells through ectopic expression of various transcription factors viz. Oct4, Sox2, Klf4, and c-Myc (OSKM). This novel technology enables derivation of patient specific cells, which possess a potential cure for many diseases. During the last decade, significant progresses have been achieved in enhancing the reprogramming efficiency, safety of iPSCs derivation, development of different delivery techniques by various research groups. Nevertheless, it is important to resolve and define the mechanism underlying the pluripotent stem cells. Major bottleneck which arises during iPSCs generation is the availability of source material (cells/tissues), difficulty to deliver transcription factors with no aberrant genetic modifications and limited reprogramming efficiency. Reprogramming may be achieved by employing different cocktails with number of different transcription factors, application of miRNA and some small molecules such as (Valproic acid, CHiR99021, Sodium butyrate, Vitamin C, Parnate etc). Similarly, various starting source materials have been demonstrated for iPSC based therapies including fibroblasts, cord blood, peripheral blood, keritinocytes, urine, etc., with their specific uses and limitations. Moreover, with the advent of many new reprogramming techniques, various direct delivery methods have been introduced such as using synthetic mRNA expressing pluripotent gene network has been shown to be an appropriate technique to deliver transcription factors and a dozen of small molecules which can replace transcription factors or enhance reprogramming efficiency. This article addresses the iPSCs technology mechanisms, progresses and current perspectives in the field.

  18. Central cholinesterase inhibition enhances glutamatergic synaptic transmission.

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    Kozhemyakin, Maxim; Rajasekaran, Karthik; Kapur, Jaideep

    2010-04-01

    Central cholinergic overstimulation results in prolonged seizures of status epilepticus in humans and experimental animals. Cellular mechanisms of underlying seizures caused by cholinergic stimulation remain uncertain, but enhanced glutamatergic transmission is a potential mechanism. Paraoxon, an organophosphate cholinesterase inhibitor, enhanced glutamatergic transmission on hippocampal granule cells synapses by increasing the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in a concentration-dependent fashion. The amplitude of mEPSCs was not increased, which suggested the possibility of enhanced action potential-dependent release. Analysis of EPSCs evoked by minimal stimulation revealed reduced failures and increased amplitude of evoked responses. The ratio of amplitudes of EPSCs evoked by paired stimuli was also altered. The effect of paraoxon on glutamatergic transmission was blocked by the muscarinic antagonist atropine and partially mimicked by carbachol. The nicotinic receptor antagonist α -bungarotoxin did not block the effects of paraoxon; however, nicotine enhanced glutamatergic transmission. These studies suggested that cholinergic overstimulation enhances glutamatergic transmission by enhancing neurotransmitter release from presynaptic terminals.

  19. Glutamatergic signaling in the brain's white matter.

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    Bakiri, Y; Burzomato, V; Frugier, G; Hamilton, N B; Káradóttir, R; Attwell, D

    2009-01-12

    Glutamatergic signaling has been exceptionally well characterized in the brain's gray matter, where it underlies fast information processing, learning and memory, and also generates the neuronal damage that occurs in pathological conditions such as stroke. The role of glutamatergic signaling in the white matter, an area until recently thought to be devoid of synapses, is less well understood. Here we review what is known, and highlight what is not known, of glutamatergic signaling in the white matter. We focus on how glutamate is released, the location and properties of the receptors it acts on, the interacting molecules that may regulate trafficking or signaling of the receptors, the possible functional roles of glutamate in the white matter, and its pathological effects including the possibility of treating white matter disorders with glutamate receptor blockers.

  20. Regulation of brain-derived neurotrophic factor (BDNF) expression and release from hippocampal neurons is mediated by non-NMDA type glutamate receptors.

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    Wetmore, C; Olson, L; Bean, A J

    1994-03-01

    We have examined the influence of glutamate on cortical brain-derived neurotrophic factor (BDNF) expression using in situ hybridization and immunohistochemistry. Kainic acid (KA) produced an upregulation of hippocampal and neocortical BDNF mRNA as well as BDNF protein that was blocked by a non-NMDA antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but was not affected by the NMDA antagonist 2-amino-7-phosphonoheptanoic acid (AP7). Basal levels of BDNF mRNA were not affected by NMDA, DNQX, or AP7 treatment. BDNF protein was also increased after kainate exposure with a spatial and temporal course distinct from that seen for the expression of BDNF mRNA. A dramatic shift in BDNF immunoreactivity (-IR) was observed from intracellular compartments to the neuropil surrounding CA3 pyramidal cells 2-3 hr after KA exposure. This shift in localization of BDNF-IR suggests a constitutive release of BDNF at the level of the cell body and dendrites. Moreover, we have localized mRNAs for full-length and truncated trkB, to a co-incident population of neurons and glia. These data suggest the neurons that produce BDNF also express components necessary for a biological response to the same neurotrophic factor. The present study also demonstrates increased BDNF-IR in the mossy fiber terminal zone of hippocampus after exposure to KA, as well as an increase in trkB mRNA, and provides evidence of local release of this neurotrophin into the surrounding neuropil where it would be available for local utilization. The synthesis and putative release of BDNF from somatic and/or dendritic sites within the hippocampus provide evidence of a potential autocrine or paracrine role for BDNF, and establish a local source of trophic support for the maintenance of synaptic plasticity and anatomic reorganization in the mature nervous system.

  1. Astroglia, Glutamatergic Transmission and Psychiatric Diseases.

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    Verkhratsky, Alexei; Steardo, Luca; Peng, Liang; Parpura, Vladimir

    2016-01-01

    Astrocytes are primary homeostatic cells of the central nervous system. They regulate glutamatergic transmission through the removal of glutamate from the extracellular space and by supplying neurons with glutamine. Glutamatergic transmission is generally believed to be significantly impaired in the contexts of all major neuropsychiatric diseases. In most of these neuropsychiatric diseases, astrocytes show signs of degeneration and atrophy, which is likely to be translated into reduced homeostatic capabilities. Astroglial glutamate uptake/release and glutamate homeostasis are affected in all forms of major psychiatric disorders and represent a common mechanism underlying neurotransmission disbalance, aberrant connectome and overall failure on information processing by neuronal networks, which underlie pathogenesis of neuropsychiatric diseases.

  2. Neuron-glia interactions in glutamatergic neurotransmission

    DEFF Research Database (Denmark)

    Schousboe, A; Sickmann, H M; Bak, Lasse Kristoffer;

    2011-01-01

    Glutamatergic neurotransmission accounts for a considerable part of energy consumption related to signaling in the brain. Chemical energy is provided by adenosine triphosphate (ATP) formed in glycolysis and tricarboxylic acid (TCA) cycle combined with oxidative phosphorylation. It is not clear wh...

  3. Precocious Sister Chromatid Separation (PSCS) in Cornelia de Lange Syndrome

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    Kaur, Maninder; DeScipio, Cheryl; McCallum, Jennifer; Yaeger, Dinah; Devoto, Marcella; Jackson, Laird G.; Spinner, Nancy B.; Krantz, Ian D.

    2009-01-01

    The Cornelia de Lange syndrome (CdLS) (OMIM# 122470) is a dominantly inherited multisystem developmental disorder. The phenotype consists of characteristic facial features, hirsutism, abnormalities of the upper extremities ranging from subtle changes in the phalanges and metacarpal bones to oligodactyly and phocomelia, gastroesophageal dysfunction, growth retardation, and neurodevelopmental delay. Prevalence is estimated to be as high as 1 in 10,000. Recently, mutations in NIPBL were identified in sporadic and familial CdLS cases. To date, mutations in this gene have been identified in over 45% of individuals with CdLS. NIPBL is the human homolog of the Drosophila Nipped-B gene. Although its function in mammalian systems has not yet been elucidated, sequence homologs of Nipped-B in yeast (Scc2 and Mis4) are required for sister chromatid cohesion during mitosis, and a similar role was recently demonstrated for Nipped-B in Drosophila. In order to evaluate NIPBL role in sister chromatid cohesion in humans, metaphase spreads on 90 probands (40 NIPBL mutation positive and 50 NIPBL mutation negative) with CdLS were evaluated for evidence of precocious sister chromatid separation (PSCS). We screened 50 metaphases from each proband and found evidence of PSCS in 41% (compared to 9% in control samples). These studies indicate that NIPBL may play a role in sister chromatid cohesion in humans as has been reported for its homologs in Drosophila and yeast. PMID:16100726

  4. Glutamatergic Transmission: A Matter of Three.

    Science.gov (United States)

    Martínez-Lozada, Zila; Ortega, Arturo

    2015-01-01

    Glutamatergic transmission in the vertebrate brain requires the involvement of glia cells, in a continuous molecular dialogue. Glial glutamate receptors and transporters are key molecules that sense synaptic activity and by these means modify their physiology in the short and long term. Posttranslational modifications that regulate protein-protein interactions and modulate transmitter removal are triggered in glial cells by neuronal released glutamate. Moreover, glutamate signaling cascades in these cells are linked to transcriptional and translational control and are critically involved in the control of the so-called glutamate/glutamine shuttle and by these means in glutamatergic neurotransmission. In this contribution, we summarize our current understanding of the biochemical consequences of glutamate synaptic activity in their surrounding partners and dissect the molecular mechanisms that allow neurons to take control of glia physiology to ensure proper glutamate-mediated neuronal communication.

  5. Involvement of ClC-3 chloride/proton exchangers in controlling glutamatergic synaptic strength in cultured hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Raul Enrique Guzman

    2014-05-01

    Full Text Available ClC-3 is a member of the CLC family of anion channels and transporters that localizes to early and late endosomes as well as to synaptic vesicles. Its genetic disruption in mouse models results in pronounced hippocampal and retinal neurodegeneration, suggesting that ClC-3 might be important for normal excitatory and/or inhibitory neurotransmission in central neurons. To characterize the role of ClC-3 in glutamate accumulation in synaptic vesicles we compared glutamatergic synaptic transmission in cultured hippocampal neurons from WT and Clcn3-/- mice. In Clcn3-/- neurons the amplitude and frequency of miniature as well as the amplitudes of action-potential evoked EPSCs were significantly increased as compared to WT neurons. The low-affinity competitive AMPA receptor antagonist -DGG reduced the quantal size of synaptic events more effectively in WT than in Clcn3-/- neurons, whereas no difference was observed for the high-affinity competitive non-NMDA antagonist NBQX. Paired pulse ratios of evoked EPSCs were significantly reduced, whereas the size of the readily releasable pool was not affected by the genetic ablation of ClC-3. Electron microscopy revealed increased volumes of synaptic vesicles in hippocampi of Clcn3-/- mice. Our findings demonstrate that ClC-3 controls fast excitatory synaptic transmission by regulating the amount of neurotransmitter as well as the release probability of synaptic vesicles. These results provide novel insights into the role of ClC-3 in synaptic transmission and identify excessive glutamate release as a likely basis of neurodegeneration in Clcn3-/-.

  6. Effects of NMDA and non-NMDA ionotropic glutamate receptors in the medial preoptic area on body temperature in awake rats.

    Science.gov (United States)

    Sengupta, Trina; Jaryal, Ashok Kumar; Mallick, Hruda Nanda

    2016-10-01

    Glutamate when microinjected at the medial preoptic area (mPOA) influences brain temperature (Tbr) and body temperature (Tb) in rats. Glutamate and its various receptors are present at the mPOA. The aim of this study was to identify the contribution of each of the ionotropic glutamatergic receptors at the mPOA on changes in Tbr and Tb in freely moving rats. Adult male Wistar rats (n=40) were implanted with bilateral guide cannula with indwelling styli above the mPOA. A telemetric transmitter was implanted at the peritoneum to record Tb and locomotor activity (LMA). A precalibrated thermocouple wire implanted near the hypothalamus was used to assess Tbr. Specific agonist for each ionotropic glutamate receptor was microinjected into the mPOA and its effects on temperature and LMA were measured in the rats. The rats were also microinjected with the respective ionotropic receptor antagonists, 15min prior to the microinjection of each agonist. Amongst amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-d-aspartate (NMDA) and kainic acid, AMPA increased Tb and LMA when injected at the mPOA. Specific antagonists for AMPA receptors was able to attenuate this increase (ptemperature.

  7. The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection

    NARCIS (Netherlands)

    Buganim, Yosef; Markoulaki, Styliani; van Wietmarschen, Niek; Hoke, Heather; Wu, Tao; Ganz, Kibibi; Akhtar-Zaidi, Batool; He, Yupeng; Abraham, Brian J.; Porubsky, David; Kulenkampff, Elisabeth; Faddah, Dina A.; Shi, Linyu; Gao, Qing; Sarkar, Sovan; Cohen, Malkiel; Goldmann, Johanna; Nery, Joseph R.; Schultz, Matthew D.; Ecker, Joseph R.; Xiao, Andrew; Young, Richard A.; Lansdorp, Peter M.; Jaenisch, Rudolf

    2014-01-01

    Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation.

  8. The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection

    NARCIS (Netherlands)

    Buganim, Yosef; Markoulaki, Styliani; van Wietmarschen, Niek; Hoke, Heather; Wu, Tao; Ganz, Kibibi; Akhtar-Zaidi, Batool; He, Yupeng; Abraham, Brian J.; Porubsky, David; Kulenkampff, Elisabeth; Faddah, Dina A.; Shi, Linyu; Gao, Qing; Sarkar, Sovan; Cohen, Malkiel; Goldmann, Johanna; Nery, Joseph R.; Schultz, Matthew D.; Ecker, Joseph R.; Xiao, Andrew; Young, Richard A.; Lansdorp, Peter M.; Jaenisch, Rudolf

    2014-01-01

    Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliab

  9. LU 73068, a new non-NMDA and glycine/NMDA receptor antagonist: pharmacological characterization and comparison with NBQX and L-701,324 in the kindling model of epilepsy.

    Science.gov (United States)

    Potschka, H; Löscher, W; Wlaź, P; Behl, B; Hofmann, H P; Treiber, H J; Szabo, L

    1998-11-01

    The aim of this study was to assess whether a drug which combines an antagonistic action at both NMDA and non-NMDA receptors offers advantages for treatment of epileptic seizures compared to drugs which antagonize only one of these ionotropic glutamate receptors. The novel glutamate receptor antagonist LU 73068 (4,5-dihydro-1-methyl-4-oxo-7-trifluoromethylimidazo[1,2a]quinoxal ine-2-carbonic acid) binds with high affinity to both the glycine site of the NMDA receptor (Ki 185 nM) and to the AMPA receptor (Ki 158 nM). Furthermore, binding experiments with recombinant kainate receptor subunits showed that LU 73068 binds to several of these subunits, particularly to rGluR7 (Ki 104 nM) and rGluR5 (Ki 271 nM). In comparison, the prototype non-NMDA receptor antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo[f]quinoxaline) binds with high affinity to AMPA receptors only. Both NBQX and LU 73068 were about equieffective after i.p. injection in mice to block lethal convulsions induced by AMPA or NMDA. In the rat amygdala kindling model of temporal lobe epilepsy, LU 73068 dose-dependently increased the focal seizure threshold (afterdischarge threshold, ADT). When rats were stimulated with a current 20% above the individual control ADT, LU 73068 completely blocked seizures with an ED50 of 4.9 mg kg(-1). Up to 20 mg kg(-1), only moderate adverse effects, e.g. slight ataxia, were observed. NBQX, 10 mg kg(-1), and the glycine/NMDA site antagonist L-701,324 (7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-quinoline-2(1H)one), 2.5 or 5 mg kg(-1), exerted no anticonvulsant effects in kindled rats when administered alone, but combined treatment with both drugs resulted in a significant ADT increase. The data indicate that combination of glycine/NMDA and non-NMDA receptor antagonism in a single drug is an effective means of developing a potent and effective anticonvulsant agent.

  10. Kisspeptin increases gamma-aminobutyric acidergic and glutamatergic transmission directly to gonadotropin-releasing hormone neurons in an estradiol-dependent manner.

    Science.gov (United States)

    Pielecka-Fortuna, Justyna; Moenter, Suzanne M

    2010-01-01

    GnRH neurons are the final central pathway controlling fertility. Kisspeptin potently activates GnRH release via G protein-coupled receptor 54 (GPR54). GnRH neurons express GPR54, and kisspeptin can act directly; however, GPR54 is broadly expressed, suggesting indirect actions are possible. Transsynaptic mechanisms are involved in estradiol-induced potentiation of GnRH neuron response to kisspeptin. To investigate these mechanisms, separate whole-cell voltage-clamp recordings were performed of gamma-aminobutyric acid (GABA)-ergic and glutamatergic transmission to GnRH neurons in brain slices before and during kisspeptin treatment. To determine whether estradiol alters the effect of kisspeptin on synaptic transmission, mice were ovariectomized and either left with no further treatment (OVX) or treated with estradiol implants (OVX+E). Cells were first studied in the morning when estradiol exerts negative feedback. Kisspeptin increased frequency and amplitude of GABAergic postsynaptic currents (PSCs) in GnRH neurons from OVX+E mice. Blocking action potentials eliminated the effect on frequency, indicating presynaptic actions. Amplitude changes were due to postsynaptic actions. Kisspeptin also increased frequency of glutamatergic excitatory PSCs in cells from OVX+E animals. Kisspeptin did not affect either GABAergic or glutamatergic transmission to GnRH neurons in cells from OVX mice, indicating effects on transmission are estradiol dependent. In contrast to stimulatory effects on GABAergic PSC frequency during negative feedback, kisspeptin had no effect during positive feedback. These data suggest estradiol enables kisspeptin-mediated increases in GABA and glutamate transmission to GnRH neurons. Furthermore, the occlusion of the response during positive feedback implies one consequence of estradiol positive feedback is an increase in transmission to GnRH neurons mediated by endogenous kisspeptin.

  11. An imprinted signature helps isolate ESC-equivalent iPSCs

    Institute of Scientific and Technical Information of China (English)

    Emesto Lujan; Marius Wernig

    2010-01-01

    @@ Since the demonstration of direct reprogramming of differentiated cells such as fibroblasts, to a malleable, pluri-potent state by defined transcription fac-tors great effort has been given to isolate induced pluripotent stem cells (iPSCs) with the same developmental potential as embryonic stem cells (ESCs) derived from blastocysts. Various selection and morphological criteria have led to the isolation of iPSCs with differential pluripotent capacity, but without the addition of small molecules very few lines have been able to undergo the most stringent pluripotent test - generate vi-able "all iPS cell mice" by tetraploid complementation. In a recent elegant study, Stadtfeld and colleagues have proposed that silencing of the imprinted Dlkl-Dio3 gene cluster is responsible for this variability of pluripotency po-tential in iPSCs, and the key to isolating ESC equivalent iPSCs [1].

  12. Generation of Viable Mice from Induced Pluripotent Stem Cells (iPSCs) Through Tetraploid Complementation.

    Science.gov (United States)

    Kang, Lan; Gao, Shaorong

    2015-01-01

    Tetraploid complementation assay is the most rigorous criteria for pluripotency characterization of pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Pluripotent stem cells could complement the developmental deficiency of tetraploid embryos and thus support the full-term mice development. Here we describe the protocol for tetraploid complementation using iPSCs to produce viable all-iPSC mice.

  13. Human Gingival Integration-Free iPSCs; a Source for MSC-Like Cells

    Directory of Open Access Journals (Sweden)

    Yasuyuki Umezaki

    2015-06-01

    Full Text Available Mesenchymal stem cells (MSCs are considered a potential autologous therapy for tissue engineering. The available procedures for MSC retrieval from patients are invasive, and their limited in vitro proliferation restricts their use in the treatment of damaged tissues. Therefore, it is important to establish an alternative and safe source of MSCs. The objective of this study was to demonstrate induced pluripotent stem cell (iPSC generation from a combination of an accessible source tissue and an integration-free method; we also attempted the differentiation of iPSCs into MSC-like cells (MSLCs for future autologous tissue engineering. iPSCs were derived from human gingival tissues, which are easily accessible in the field of dentistry, via the use of non-integrating episomal plasmids. Established iPSCs expressed embryonic stem (ES cell-specific markers, as assessed by gene analysis and immunocytochemistry. Embryoid bodies and teratoma formation were formed from iPSCs, showing their capacity to differentiate into three germ layers. Furthermore, we were successful in differentiating iPSCs into MSLCs. They tested positively for their capacity of trilineage differentiation. Our results demonstrate that human gingival integration-free iPSCs, readily accessible stem cells generated using episomal plasmid vectors, are a promising source of MSLCs, which can be used in tissue regeneration.

  14. Metabotropic glutamatergic receptors and their ligands in drug addiction.

    Science.gov (United States)

    Pomierny-Chamioło, Lucyna; Rup, Kinga; Pomierny, Bartosz; Niedzielska, Ewa; Kalivas, Peter W; Filip, Małgorzata

    2014-06-01

    Glutamatergic excitatory transmission is implicated in physiological and pathological conditions like learning, memory, neuronal plasticity and emotions, while glutamatergic abnormalities are reported in numerous neurological and psychiatric disorders, including neurodegenerative diseases, epilepsy, stroke, traumatic brain injury, depression, anxiety, schizophrenia and pain. Also, several lines of evidence have accumulated indicating a pivotal role for glutamatergic neurotransmission in mediating addictive behaviors. Among the proteins regulating glutamatergic transmission, the metabotropic glutamate receptors (mGluR) are being developed as pharmacological targets for treating many neuropsychiatric disorders, including drug addiction. In this review we describe the molecular structure of mGluRs and their distribution, physiology and pharmacology in the central nervous system, as well as their use as targets in preclinical studies of drug addiction.

  15. The effects of stress on glutamatergic transmission in the brain.

    Science.gov (United States)

    Yuan, Ti-Fei; Hou, Gonglin

    2015-01-01

    Stress leads to detrimental effects on brain functions and results in various diseases. Recent studies highlight the involvement of glutamatergic transmission in pathogenesis of depressive behaviors and fears. Acute stress generates different impacts on the excitatory transmission compared to chronic stress. Different neuromodulators and epigenetic factors also participate in the alteration of synaptic transmission and the regulation of synaptic plasticity. Restoration of the glutamatergic transmission in stress-affected brain areas therefore provides novel directions of therapeutic interventions against stress.

  16. Paradoxical upregulation of glutamatergic presynaptic boutons during mild cognitive impairment.

    Science.gov (United States)

    Bell, Karen F S; Bennett, David A; Cuello, A Claudio

    2007-10-03

    Synaptic integrity is now recognized as a central component of Alzheimer's disease. Surprisingly, however, the structural status of glutamatergic synapses in Alzheimer's disease is unclear, despite the fact that glutamate is the major excitatory transmitter of the CNS and has key roles in excitotoxicity and long-term potentiation. The identification of specific markers of glutamatergic neurons now allows an assessment of the structural involvement of the glutamatergic system across progressive stages of the Alzheimer's pathology, an opportunity not afforded by previously used neurochemical approaches. Glutamatergic presynaptic bouton density and dystrophic neurite abundance were quantified in midfrontal gyrus brain tissue from subjects with no cognitive impairment, mild cognitive impairment, or mild- or severe-stage Alzheimer's disease. Our study demonstrates a striking pathology-dependent pattern of glutamatergic synaptic remodeling with disease progression. Subjects with mild cognitive impairment display a paradoxical elevation in glutamatergic presynaptic bouton density, a situation akin to that observed in the cholinergic system, which then depletes and drops with disease progression. This pattern of synaptic remodeling mirrors our previous findings in transgenic animal models and is of major relevance to current transmitter-based therapeutics.

  17. Lrp4 in astrocytes modulates glutamatergic transmission.

    Science.gov (United States)

    Sun, Xiang-Dong; Li, Lei; Liu, Fang; Huang, Zhi-Hui; Bean, Jonathan C; Jiao, Hui-Feng; Barik, Arnab; Kim, Seon-Myung; Wu, Haitao; Shen, Chengyong; Tian, Yun; Lin, Thiri W; Bates, Ryan; Sathyamurthy, Anupama; Chen, Yong-Jun; Yin, Dong-Min; Xiong, Lei; Lin, Hui-Ping; Hu, Jin-Xia; Li, Bao-Ming; Gao, Tian-Ming; Xiong, Wen-Cheng; Mei, Lin

    2016-08-01

    Neurotransmission requires precise control of neurotransmitter release from axon terminals. This process is regulated by glial cells; however, the underlying mechanisms are not fully understood. We found that glutamate release in the brain was impaired in mice lacking low-density lipoprotein receptor-related protein 4 (Lrp4), a protein that is critical for neuromuscular junction formation. Electrophysiological studies revealed compromised release probability in astrocyte-specific Lrp4 knockout mice. Lrp4 mutant astrocytes suppressed glutamatergic transmission by enhancing the release of ATP, whose level was elevated in the hippocampus of Lrp4 mutant mice. Consequently, the mutant mice were impaired in locomotor activity and spatial memory and were resistant to seizure induction. These impairments could be ameliorated by blocking the adenosine A1 receptor. The results reveal a critical role for Lrp4, in response to agrin, in modulating astrocytic ATP release and synaptic transmission. Our findings provide insight into the interaction between neurons and astrocytes for synaptic homeostasis and/or plasticity.

  18. Blocking lhh Signaling Pathway Inhibits the Proliferation and Pro-motes the Apoptosis of PSCs

    Institute of Scientific and Technical Information of China (English)

    Kai XU; Fengjing GUO; Shuwei ZHANG; Cheng LIU; Feixiong WANG; Zhiguo ZHOU; Anmin CHEN

    2009-01-01

    The roles of Indian hedgehog (Ihh) signaling pathway in the proliferation and apoptosis of precartilaginous stem cells (PSCs) were investigated.PSCs,labeled with fibroblast growth factor receptor 3 (FGFR-3),were isolated from neonatal rats by immanomagnetic separation.After identifi-cation with FGFR-3 and Col Ⅱ,the cells were incubated with different concentrations of cyclopamine (cyclo),the specific inhibitor of lhh signaling pathway.The morphologic changes of the cells were observed under the inverted phase contrast microscope.The mRNA expression levels of Ibh,para-thyroid hormonerelated peptide (PTHrP),protein Patched (Ptch),Bcl-2 and p21 were detected by RT-PCR.The protein expression levels of Ihh and Ptch were measured by Western blot.MTT assay was used to examine the effects of cyclo on proliferation of PSCs.Apoptosis rate of PSCs was exam-ined by Annexin V/PI assay of flow cytometric analyses.After PSCs were incubated with cyclo,ob-vious morphologic changes were observed as compared with the control group.The mRNA expres-sion levels of PTHrP,Ptch and Bcl-2 were decreased to varying degrees in a cyclo dose-dependent manner.However,the expression levels of lhh and p21 mRNA were increased.The protein expres-sion of Ptch and Ihh had the same change as the mRNA expression.Meanwhile,cyclo could obvi-ously inhibit the proliferation and promote the apoptosis of PSCs.The results indicated that Ihh sig-naling pathway plays an important role in regulating the proliferation and apoptosis of PSCs,which is probably mediated by Bcl-2 and p21.

  19. The effect of the non-NMDA receptor antagonist GYKI 52466 and NBQX and the competitive NMDA receptor antagonist D-CPPene on the development of amygdala kindling and on amygdala-kindled seizures.

    Science.gov (United States)

    Dürmüller, N; Craggs, M; Meldrum, B S

    1994-02-01

    A competitive (NBQX) and a non-competitive (GYKI 52466) AMPA antagonist, and a competitive NMDA antagonist (D-CPPene) were tested against the development of kindling and against fully kindled seizures in amygdala-kindled rats. GYKI 52466, 10 mg/kg given i.p. 5 min prior to electrical stimulation in fully kindled animals, reduces both the cortical after-discharge duration and the behavioural seizure score. GYKI 52466, 20 mg/kg, reduces seizure score and after-discharge duration significantly (after 5-30 min) but the animals show severe motor side effects and an irregular cortical and hippocampal EEG. Administration of GYKI 52466, 10 mg/kg, prior to kindling stimulation on days 3-8, does not slow the development of kindling. NBQX, 20 mg/kg or 40 mg/kg i.p., 30 min prior to stimulation, significantly reduces the seizure score in fully kindled animals. NBQX 20 mg/kg i.p. has no effect on the development of kindling. D-CPPene, 8 mg/kg or 12 mg/kg, 120 min prior to stimulation reduces the behavioural seizure score in fully kindled animals. D-CPPene, 8 mg/kg on days 3-8, delays the development of kindling. NMDA receptors play a key role in the kindling process. Expression of kindled seizures involves non-NMDA and NMDA receptors.

  20. Manipulation of KLF4 Expression Generates iPSCs Paused at Successive Stages of Reprogramming

    Directory of Open Access Journals (Sweden)

    Ken Nishimura

    2014-11-01

    Full Text Available The detailed mechanism of reprogramming somatic cells into induced pluripotent stem cells (iPSCs remains largely unknown. Partially reprogrammed iPSCs are informative and useful for understanding the mechanism of reprogramming but remain technically difficult to generate in a predictable and reproducible manner. Using replication-defective and persistent Sendai virus (SeVdp vectors, we analyzed the effect of decreasing the expression levels of OCT4, SOX2, KLF4, and c-MYC and found that low KLF4 expression reproducibly gives rise to a homogeneous population of partially reprogrammed iPSCs. Upregulation of KLF4 allows these cells to resume reprogramming, indicating that they are paused iPSCs that remain on the path toward pluripotency. Paused iPSCs with different KLF4 expression levels remain at distinct intermediate stages of reprogramming. This SeVdp-based stage-specific reprogramming system (3S reprogramming system is applicable for both mouse and human somatic cells and will facilitate the mechanistic analysis of reprogramming.

  1. Simple Derivation of Spinal Motor Neurons from ESCs/iPSCs Using Sendai Virus Vectors

    Directory of Open Access Journals (Sweden)

    Kazuya Goto

    2017-03-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive and fatal degenerative disorder of motor neurons (MNs. Embryonic stem cells (ESCs/induced pluripotent stem cells (iPSCs now help us to understand the pathomechanisms of ALS via disease modeling. Various methods to differentiate ESCs/iPSCs into MNs by the addition of signaling molecules have been reported. However, classical methods require multiple steps, and newer simple methods using the transduction of transcription factors run the risk of genomic integration of the vector genes. Heterogeneity of the expression levels of the transcription factors also remains an issue. Here we describe a novel approach for differentiating human and mouse ESCs/iPSCs into MNs using a single Sendai virus vector encoding three transcription factors, LIM/homeobox protein 3, neurogenin 2, and islet-1, which are integration free. This single-vector method, generating HB9-positive cells on day 2 from human iPSCs, increases the ratio of MNs to neurons compared to the use of three separate Sendai virus vectors. In addition, the MNs derived via this method from iPSCs of ALS patients and model mice display disease phenotypes. This simple approach significantly reduces the efforts required to generate MNs, and it provides a useful tool for disease modeling.

  2. Stepping back to move forward: a current review of iPSCs in the fight against Alzheimer’s disease

    OpenAIRE

    Devineni, Aditya; Tohme, Scarlett; Kody, Michael T; Cowley, R Adams; Harris, Brent T.

    2016-01-01

    The successful generation of the first iPSCs about ten years ago has provided deeper insight into previously unknown disease mechanisms and therapeutic opportunities for many diseases. In particular, iPSCs are becoming an important tool in advancing modeling and therapeutic intervention for Alzheimer’s disease. In this manuscript, we assess the research climate surrounding the application of iPSCs to familial and sporadic Alzheimer’s disease, including the generation and isolation of individu...

  3. iPSCs: A Minireview from Bench to Bed, including Organoids and the CRISPR System

    Directory of Open Access Journals (Sweden)

    Andrés Javier Orqueda

    2016-01-01

    Full Text Available When Dolly the sheep was born, the first probe into an adult mammalian genome traveling back in time and generating a whole new animal appeared. Ten years later, the reprogramming process became a defined method of producing induced pluripotent stem cells (iPSCs through the overexpression of four transcription factors. iPSCs are capable of originating virtually all types of cells and tissues, including a whole new animal. The reprogramming strategies based on patient-derived cells should make the development of clinical applications of cell based therapy much more straightforward. Here, we analyze the current state, opportunities, and challenges of iPSCs from bench to bed, including organoids and the CRISPR system.

  4. Efficient and allele-specific genome editing of disease loci in human iPSCs.

    Science.gov (United States)

    Smith, Cory; Abalde-Atristain, Leire; He, Chaoxia; Brodsky, Brett R; Braunstein, Evan M; Chaudhari, Pooja; Jang, Yoon-Young; Cheng, Linzhao; Ye, Zhaohui

    2015-03-01

    Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

  5. CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

    DEFF Research Database (Denmark)

    Mandegar, Mohammad A.; Huebsch, Nathaniel; Frolov, Ekaterina B.

    2016-01-01

    repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range...

  6. The Epigenetic Reprogramming Roadmap in Generation of iPSCs from Somatic Cells

    DEFF Research Database (Denmark)

    Brix, Jacob; Zhou, Yan; Luo, Yonglun

    2015-01-01

    Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprograming, pluripotency, and differentiation cap...

  7. Metabolic correction of congenital erythropoietic porphyria with iPSCs free of reprogramming factors.

    Science.gov (United States)

    Bedel, Aurélie; Taillepierre, Miguel; Guyonnet-Duperat, Véronique; Lippert, Eric; Dubus, Pierre; Dabernat, Sandrine; Mautuit, Thibaud; Cardinaud, Bruno; Pain, Catherine; Rousseau, Benoît; Lalanne, Magalie; Ged, Cécile; Duchartre, Yann; Richard, Emmanuel; de Verneuil, Hubert; Moreau-Gaudry, François

    2012-07-13

    Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs.

  8. 76 FR 49650 - Private Security Contractors (PSCs) Operating in Contingency Operations, Combat Operations or...

    Science.gov (United States)

    2011-08-11

    ... States and elsewhere. We agree that the behavior of PSCs may affect the national security goals of the U.... Comment: USAID involvement is not evident Response: USAID has been actively involved in various working... more; a major increase in costs or prices for consumers, individual industries, Federal, State,...

  9. Allosteric modulators for the treatment of schizophrenia: targeting glutamatergic networks.

    Science.gov (United States)

    Menniti, Frank S; Lindsley, Craig W; Conn, P Jeffrey; Pandit, Jayvardhan; Zagouras, Panayiotis; Volkmann, Robert A

    2013-01-01

    Schizophrenia is a highly debilitating mental disorder which afflicts approximately 1% of the global population. Cognitive and negative deficits account for the lifelong disability associated with schizophrenia, whose symptoms are not effectively addressed by current treatments. New medicines are needed to treat these aspects of the disease. Neurodevelopmental, neuropathological, genetic, and behavioral pharmacological data indicate that schizophrenia stems from a dysfunction of glutamate synaptic transmission, particularly in frontal cortical networks. A number of novel pre- and postsynaptic mechanisms affecting glutamatergic synaptic transmission have emerged as viable targets for schizophrenia. While developing orthosteric glutamatergic agents for these targets has proven extremely difficult, targeting allosteric sites of these targets has emerged as a promising alternative. From a medicinal chemistry perspective, allosteric sites provide an opportunity of finding agents with better drug-like properties and greater target specificity. Furthermore, allosteric modulators are better suited to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission. Herein, we review neuropathological and genomic/genetic evidence underscoring the importance of glutamate synaptic dysfunction in the etiology of schizophrenia and make a case for allosteric targets for therapeutic intervention. We review progress in identifying allosteric modulators of AMPA receptors, NMDA receptors, and metabotropic glutamate receptors, all with the aim of restoring physiological glutamatergic synaptic transmission. Challenges remain given the complexity of schizophrenia and the difficulty in studying cognition in animals and humans. Nonetheless, important compounds have emerged from these efforts and promising preclinical and variable clinical validation has been achieved.

  10. Glutamatergic Signaling at the Vestibular Hair Cell Calyx Synapse

    NARCIS (Netherlands)

    Sadeghi, Soroush G.; Pyott, Sonja J.; Yu, Zhou; Glowatzki, Elisabeth

    2014-01-01

    In the vestibular periphery a unique postsynaptic terminal, the calyx, completely covers the basolateral walls of type I hair cells and receives input from multiple ribbon synapses. To date, the functional role of this specialized synapse remains elusive. There is limited data supporting glutamaterg

  11. Serotonin modulates glutamatergic transmission to neurons in the lateral habenula.

    Science.gov (United States)

    Xie, Guiqin; Zuo, Wanhong; Wu, Liangzhi; Li, Wenting; Wu, Wei; Bekker, Alex; Ye, Jiang-Hong

    2016-04-01

    The lateral habenula (LHb) is bilaterally connected with serotoninergic raphe nuclei, and expresses high density of serotonin receptors. However, actions of serotonin on the excitatory synaptic transmission to LHb neurons have not been thoroughly investigated. The LHb contains two anatomically and functionally distinct regions: lateral (LHbl) and medial (LHbm) divisions. We compared serotonin's effects on glutamatergic transmission across the LHb in rat brains. Serotonin bi-directionally and differentially modulated glutamatergic transmission. Serotonin inhibited glutamatergic transmission in higher percentage of LHbl neurons but potentiated in higher percentage of LHbm neurons. Magnitude of potentiation was greater in LHbm than in LHbl. Type 2 and 3 serotonin receptor antagonists attenuated serotonin's potentiation. The serotonin reuptake blocker, and the type 2 and 3 receptor agonists facilitated glutamatergic transmission in both LHbl and LHbm neurons. Thus, serotonin via activating its type 2, 3 receptors, increased glutamate release at nerve terminals in some LHb neurons. Our data demonstrated that serotonin affects both LHbm and LHbl. Serotonin might play an important role in processing information between the LHb and its downstream-targeted structures during decision-making. It may also contribute to a homeostatic balance underlying the neural circuitry between the LHb and raphe nuclei.

  12. Energy substrates to support glutamatergic and GABAergic synaptic function

    DEFF Research Database (Denmark)

    Schousboe, Arne; Bak, Lasse K; Sickmann, Helle M;

    2007-01-01

    Maintenance of glutamatergic and GABAergic activity requires a continuous supply of energy since the exocytotic processes as well as high affinity glutamate and GABA uptake and subsequent metabolism of glutamate to glutamine are energy demanding processes. The main energy substrate for the brain ...

  13. Glutamatergic synaptic plasticity in the mesocorticolimbic system in addiction

    Science.gov (United States)

    van Huijstee, Aile N.; Mansvelder, Huibert D.

    2015-01-01

    Addictive drugs remodel the brain’s reward circuitry, the mesocorticolimbic dopamine (DA) system, by inducing widespread adaptations of glutamatergic synapses. This drug-induced synaptic plasticity is thought to contribute to both the development and the persistence of addiction. This review highlights the synaptic modifications that are induced by in vivo exposure to addictive drugs and describes how these drug-induced synaptic changes may contribute to the different components of addictive behavior, such as compulsive drug use despite negative consequences and relapse. Initially, exposure to an addictive drug induces synaptic changes in the ventral tegmental area (VTA). This drug-induced synaptic potentiation in the VTA subsequently triggers synaptic changes in downstream areas of the mesocorticolimbic system, such as the nucleus accumbens (NAc) and the prefrontal cortex (PFC), with further drug exposure. These glutamatergic synaptic alterations are then thought to mediate many of the behavioral symptoms that characterize addiction. The later stages of glutamatergic synaptic plasticity in the NAc and in particular in the PFC play a role in maintaining addiction and drive relapse to drug-taking induced by drug-associated cues. Remodeling of PFC glutamatergic circuits can persist into adulthood, causing a lasting vulnerability to relapse. We will discuss how these neurobiological changes produced by drugs of abuse may provide novel targets for potential treatment strategies for addiction. PMID:25653591

  14. Developmental patterning of glutamatergic synapses onto retinal ganglion cells

    Directory of Open Access Journals (Sweden)

    Schubert Timm

    2008-03-01

    Full Text Available Abstract Background Neurons receive excitatory synaptic inputs that are distributed across their dendritic arbors at densities and with spatial patterns that influence their output. How specific synaptic distributions are attained during development is not well understood. The distribution of glutamatergic inputs across the dendritic arbors of mammalian retinal ganglion cells (RGCs has long been correlated to the spatial receptive field profiles of these neurons. Thus, determining how glutamatergic inputs are patterned onto RGC dendritic arbors during development could provide insight into the cellular mechanisms that shape their functional receptive fields. Results We transfected developing and mature mouse RGCs with plasmids encoding fluorescent proteins that label their dendrites and glutamatergic postsynaptic sites. We found that as dendritic density (dendritic length per unit area of dendritic field decreases with maturation, the density of synapses along the dendrites increases. These changes appear coordinated such that RGCs attain the mature average density of postsynaptic sites per unit area (areal density by the time synaptic function emerges. Furthermore, stereotypic centro-peripheral gradients in the areal density of synapses across the arbor of RGCs are established at an early developmental stage. Conclusion The spatial pattern of glutamatergic inputs onto RGCs arises early in synaptogenesis despite ensuing reorganization of dendritic structure. We raise the possibility that these early patterns of synaptic distributions may arise from constraints placed on the number of contacts presynaptic neurons are able to make with the RGCs.

  15. Potential therapeutic applications of differentiated induced pluripotent stem cells (iPSCs) in the treatment of neurodegenerative diseases.

    Science.gov (United States)

    Gao, Aijing; Peng, Yuhua; Deng, Yulin; Qing, Hong

    2013-01-01

    Difficulties in realizing persistent neurogenesis, inabilities in modeling pathogenesis of most cases, and a shortage of disease material for screening therapeutic agents restrict our progress to overcome challenges presented by neurodegenerative diseases. We propose that reprogramming primary somatic cells of patients into induced pluripotent stem cells (iPSCs) provides a new avenue to overcome these impediments. Their abilities in self-renewal and differentiation into various cell types will enable disease investigation and drug development. In this review, we introduce efficient approaches to generate iPSCs and distinct iPSCs differentiation stages, and critically discuss paradigms of iPSCs technology application to investigate neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Although iPSCs technology is in its infancy and faces many obstacles, it has great potential in helping to identify therapeutic targets for treating neurodegenerative diseases.

  16. Induced pluripotent stem cells (iPSCs)--a new era of reprogramming.

    Science.gov (United States)

    Kang, Lan; Kou, Zhaohui; Zhang, Yu; Gao, Shaorong

    2010-07-01

    Embryonic stem cells (ESCs) derived from the early embryos possess two important characteristics: self-renewal and pluripotency, which make ESCs ideal seed cells that could be potentially utilized for curing a number of degenerative and genetic diseases clinically. However, ethical concerns and immune rejection after cell transplantation limited the clinical application of ESCs. Fortunately, the recent advances in induced pluripotent stem cell (iPSC) research have clearly shown that differentiated somatic cells from various species could be reprogrammed into pluripotent state by ectopically expressing a combination of several transcription factors, which are highly enriched in ESCs. This ground-breaking achievement could circumvent most of the limitations that ESCs faced. However, it remains challenging if the iPS cell lines, especially the human iPSCs lines, available are fully pluripotent. Therefore, it is prerequisite to establish a molecular standard to distinguish the better quality iPSCs from the inferior ones.

  17. Clinical translation of TALENS: Treating SCID-X1 by gene editing in iPSCs.

    Science.gov (United States)

    Biffi, Alessandra

    2015-04-02

    Mutations causing X-linked severe combined immunodeficiency (SCID-X1) reduce immune cell populations and function and may be amenable to targeted gene correction strategies. Now in Cell Stem Cell, Menon et al. (2015) correct SCID-X1-related blood differentiation defects by TALEN-mediated genome editing in patient-derived iPSCs, suggesting a possible strategy for autologous cell therapy of SCID-X1.

  18. Potential applications of induced pluripotent stem cells (iPSCs) in hepatology research.

    Science.gov (United States)

    Sun, Chao; Wilson, George S; Fan, Jian-Gao; Qiao, Liang

    2015-01-01

    Induced pluripotent stem cells (iPSCs) share many characteristics with embryonic stem cells (ESCs), but circumvent most of the ethical issues surrounding ESCs. The use of iPSCs to treat liver diseases is gaining increasing interest. Recent studies show these iPSCs can be differentiated into the hepatic lineage and provide an accurate model for liver diseases, drug screening and drug toxicity testing. Recently, the potential application of iPSC-derived hepatocytes to be used in cell-based therapies has been explored as a novel strategy to treat human liver disease. However, the successful use of these iPSC-derived hepatocytes hinges on overcoming the inherent problems of using iPSC in cell-based therapies. Given these problems are addressed in the future, these iPSC derived hepatocytes provide a limitless supply of cells that could be used to treat liver diseases not only in screening and toxicity testing but also in cell-based therapies.

  19. iPSCs and small molecules: a reciprocal effort towards better approaches for drug discovery

    Institute of Scientific and Technical Information of China (English)

    Ru ZHANG; Li-hong ZHANG; Xin XIE

    2013-01-01

    The revolutionary induced pluripotent stem cell (iPSC) technology provides a new path for cell replacement therapies and drug screening.Patient-specific iPSCs and subsequent differentiated cells manifesting disease phenotypes will finally position human disease pathology at the core of drug discovery.Cells used to test the toxic effects of drugs can also be generated from normal iPSCs and provide a much more accurate and cost-effective system than many animal models.Here,we highlight the recent progress in iPSC-based cell therapy,disease modeling and drug evaluations.In addition,we discuss the use of small molecule drugs to improve the generation of iPSCs and understand the reprogramming mechanism.It is foreseeable that the interplay between iPSC technology and small molecule compounds will push forward the applications of iPSC-based therapy and screening systems in the real world and eventually revolutionize the methods used to treat diseases.

  20. Nos3-/- iPSCs model concordant signatures of in utero cardiac pathogenesis.

    Science.gov (United States)

    Campbell, Katherine A; Li, Xing; Biendarra, Sherri M; Terzic, Andre; Nelson, Timothy J

    2015-10-01

    Through genome-wide transcriptional comparisons, this study interrogates the capacity of in vitro differentiation of induced pluripotent stem cells (iPSCs) to accurately model pathogenic signatures of developmental cardiac defects. Herein, we studied the molecular etiology of cardiac defects in Nos3(-/-) mice via transcriptional analysis of stage-matched embryonic tissues and iPSC-derived cells. In vitro comparisons of differentiated cells were calibrated to in utero benchmarks of health and disease. Integrated systems biology analysis of WT and Nos3(-/-) transcriptional profiles revealed 50% concordant expression patterns between in utero embryonic tissues and ex vivo iPSC-derived cells. In particular, up-regulation of glucose metabolism (p-value=3.95×10(-12)) and down-regulation of fatty acid metabolism (p-value=6.71×10(-12)) highlight a bioenergetic signature of early Nos3 deficiency during cardiogenesis that can be recapitulated in iPSC-derived differentiated cells. The in vitro concordance of early Nos3(-/-) disease signatures supports the utility of iPSCs as a cellular model of developmental heart defects. Moreover, this study supports the use of iPSCs as a platform to pinpoint initial stages of congenital cardiac pathogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Signaling by postsynaptic AMPA receptors in glutamatergic synapse maturation

    OpenAIRE

    2010-01-01

    Excitatory transmission in the brain is largely mediated by synapses containing the neurotransmitter glutamate. Neuronal circuitry is first established early in brain development requiring the formation of vast numbers of glutamatergic synapses at individual sites of contact made between presynaptic axons and postsynaptic dendrites. Despite mounting efforts in the last decade to identify the complex molecular mechanisms underlying initial synaptogenesis and the subsequent steps of synapse m...

  2. Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission

    DEFF Research Database (Denmark)

    Schousboe, A; Sarup, A; Bak, L K

    2004-01-01

    The fine tuning of both glutamatergic and GABAergic neurotransmission is to a large extent dependent upon optimal function of astrocytic transport processes. Thus, glutamate transport in astrocytes is mandatory to maintain extrasynaptic glutamate levels sufficiently low to prevent excitotoxic...... neuronal damage. In GABA synapses hyperactivity of astroglial GABA uptake may lead to diminished GABAergic inhibitory activity resulting in seizures. As a consequence of this the expression and functional activity of astrocytic glutamate and GABA transport is regulated in a number of ways...

  3. Activation of 5-HT6 receptors inhibits corticostriatal glutamatergic transmission.

    Science.gov (United States)

    Tassone, Annalisa; Madeo, Graziella; Schirinzi, Tommaso; Vita, Daniela; Puglisi, Francesca; Ponterio, Giulia; Borsini, Franco; Pisani, Antonio; Bonsi, Paola

    2011-09-01

    We investigated the effect of 5-HT6 receptor subtype activation on glutamatergic transmission by means of whole-cell patch-clamp electrophysiological recordings from medium spiny neurons of the striatum and layer V pyramidal neurons of the prefrontal cortex. To this aim, we took advantage of a novel ligand, ST1936, showing nM affinity and agonist activity at the 5-HT6 receptor subtype. Our data show that 5-HT6 receptor activation by ST1936 reduces the frequency of spontaneous excitatory postsynaptic currents, with an IC50 of 1.3 μM. Moreover, 5-HT6 receptor activation also reduced the amplitude of spontaneous excitatory postsynaptic currents recorded from medium spiny neurons, suggesting a mechanism of action involving postsynaptic 5-HT6 receptors, as further confirmed by the paired-pulse analysis on evoked excitatory postsynaptic currents and by recordings of miniature glutamatergic events. The inhibitory effect of ST1936 on glutamatergic transmission was prevented by the selective 5-HT6 receptor antagonist SB258585 and mimicked by a different agonist, WAY-181187. Conversely, in the cortex ST1936 reduced the frequency, but not the amplitude, of spontaneous excitatory postsynaptic currents suggesting a presynaptic or indirect effect of the 5-HT6 receptor.

  4. Ultradian corticosterone pulses balance glutamatergic transmission and synaptic plasticity.

    Science.gov (United States)

    Sarabdjitsingh, Ratna Angela; Jezequel, Julie; Pasricha, Natasha; Mikasova, Lenka; Kerkhofs, Amber; Karst, Henk; Groc, Laurent; Joëls, Marian

    2014-09-30

    The rodent adrenal hormone corticosterone (CORT) reaches the brain in hourly ultradian pulses, with a steep rise in amplitude before awakening. The impact of a single CORT pulse on glutamatergic transmission is well documented, but it remains poorly understood how consecutive pulses impact on glutamate receptor trafficking and synaptic plasticity. By using high-resolution imaging and electrophysiological approaches, we report that a single pulse of CORT to hippocampal networks causes synaptic enrichment of glutamate receptors and increased responses to spontaneously released glutamatergic vesicles, collectively abrogating the ability to subsequently induce synaptic long-term potentiation. Strikingly, a second pulse of CORT one hour after the first--mimicking ultradian pulses--completely normalizes all aspects of glutamate transmission investigated, restoring the plastic range of the synapse. The effect of the second pulse is precisely timed and depends on a nongenomic glucocorticoid receptor-dependent pathway. This normalizing effect through a sequence of CORT pulses--as seen around awakening--may ensure that hippocampal glutamatergic synapses remain fully responsive and able to encode new stress-related information when daily activities start.

  5. Variable behavior of iPSCs derived from CML patients for response to TKI and hematopoietic differentiation.

    Directory of Open Access Journals (Sweden)

    Aurélie Bedel

    Full Text Available Chronic myeloid leukemia disease (CML found effective therapy by treating patients with tyrosine kinase inhibitors (TKI, which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs derived from CD34⁺ blood cells isolated from CML patients (CML-iPSCs as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.

  6. [Glutamatergic neurotransmitter system in regulation of the gastrointestinal tract motor activity].

    Science.gov (United States)

    Alekseeva, E V; Popova, T S; Sal'nikov, P S

    2015-01-01

    The review include actual facts, demonstrating high probability of glutamatergic neurotransmitter system role in the regulation of the gastrointestinal tract motor activity. These facts suggest significant role of the glutamatergic neurotransmitter system dysfunction in forming motor activity disorders of the digestive tract, including in patients in critical condition. The analysis is based on results of multiple experimental and clinical researches of glutamic acid and other components of the glutamatergic neurotransmitter system in central nervous system and autonomic nervous system (with the accent on the enteral nervous system) in normal conditions and with functioning changes of the glutamatergic neurotransmitter system in case of inflammation, hupoxia, stress and in critical condition.

  7. iPSCs, a Future Tool for Therapeutic Intervention in Mitochondrial Disorders: Pros and Cons.

    Science.gov (United States)

    Galera, Teresa; Zurita-Díaz, Francisco; Garesse, Rafael; Gallardo, M Esther

    2016-11-01

    Mitochondrial disorders, although individually are rare, taken together constitute a big group of diseases that share a defect in the oxidative phosphorylation system. Up to now, the development of therapies for these diseases is very slow and ineffective due in part to the lack of appropriate disease models. Therefore, there is an urgent need for the discovery of new therapeutic interventions. Regarding this, the generation of induced pluripotent stem cells (iPSCs) has opened new expectations in the regenerative medicine field. However, special cares and considerations must be taken into account previous to a replacement therapy. J. Cell. Physiol. 231: 2317-2318, 2016. © 2016 Wiley Periodicals, Inc.

  8. The Epigenetic Reprogramming Roadmap in Generation of iPSCs from Somatic Cells.

    Science.gov (United States)

    Brix, Jacob; Zhou, Yan; Luo, Yonglun

    2015-12-20

    Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprogramming, pluripotency, and differentiation capacity. Here, we review the epigenetic changes with a focus on histone modification (methylation and acetylation) and DNA modification (methylation) during iPSC induction. We look at changes in specific epigenetic signatures, aberrations and epigenetic memory during reprogramming and small molecules influencing the epigenetic reprogramming of somatic cells. Finally, we discuss how to improve iPSC generation and pluripotency through epigenetic manipulations.

  9. Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human iPSCs

    Directory of Open Access Journals (Sweden)

    Akihiro Yamashita

    2015-03-01

    Full Text Available Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs. We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor β1 (TGF-β1, and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects.

  10. Lymphoid regeneration from gene-corrected SCID-X1 subject-derived iPSCs.

    Science.gov (United States)

    Menon, Tushar; Firth, Amy L; Scripture-Adams, Deirdre D; Galic, Zoran; Qualls, Susan J; Gilmore, William B; Ke, Eugene; Singer, Oded; Anderson, Leif S; Bornzin, Alexander R; Alexander, Ian E; Zack, Jerome A; Verma, Inder M

    2015-04-02

    X-linked Severe Combined Immunodeficiency (SCID-X1) is a genetic disease that leaves newborns at high risk of serious infection and a predicted life span of less than 1 year in the absence of a matched bone marrow donor. The disease pathogenesis is due to mutations in the gene encoding the Interleukin-2 receptor gamma chain (IL-2Rγ), leading to a lack of functional lymphocytes. With the leukemogenic concerns of viral gene therapy there is a need to explore alternative therapeutic options. We have utilized induced pluripotent stem cell (iPSC) technology and genome editing mediated by TALENs to generate isogenic subject-specific mutant and gene-corrected iPSC lines. While the subject-derived mutant iPSCs have the capacity to generate hematopoietic precursors and myeloid cells, only wild-type and gene-corrected iPSCs can additionally generate mature NK cells and T cell precursors expressing the correctly spliced IL-2Rγ. This study highlights the potential for the development of autologous cell therapy for SCID-X1 subjects.

  11. Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs.

    Science.gov (United States)

    Yamashita, Akihiro; Morioka, Miho; Yahara, Yasuhito; Okada, Minoru; Kobayashi, Tomohito; Kuriyama, Shinichi; Matsuda, Shuichi; Tsumaki, Noriyuki

    2015-03-10

    Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs). We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor β1 (TGF-β1), and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Normal Collagen and Bone Production by Gene-targeted Human Osteogenesis Imperfecta iPSCs

    Science.gov (United States)

    Deyle, David R; Khan, Iram F; Ren, Gaoying; Wang, Pei-Rong; Kho, Jordan; Schwarze, Ulrike; Russell, David W

    2012-01-01

    Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease. PMID:22031238

  13. A pressurized nitrogen counterbalance to cortical glutamatergic pathway stimulation.

    Science.gov (United States)

    Vallee, Nicolas; Rostain, Jean-Claude; Risso, Jean-Jacques

    2010-05-01

    Previous microdialysis studies performed in rats have revealed a decrease of striatal dopamine and glutamate induced by nitrogen narcosis. We sought to establish the hypothetical role of the glutamatergic corticostriatal pathway because of the glutamate deficiency which occurs in the basal ganglia in this hyperbaric syndrome. Retrodialysis with 1 mM of Saclofen and 100 mM of KCl in the prefrontal cortex under normobaric conditions led to an increase in striatal levels of glutamate by 95.2% and no changes in dopamine levels. Under 3 MPa of nitrogen and with the infusion, the rate of striatal glutamate decreased by 51.3%, to a greater extent than under pressurised nitrogen alone (-23.8%). The rate of dopamine decreased, which also occurred under pressurised nitrogen (-36.9 and -31.4%, respectively). In conclusion, the function of the corticostriatal pathway is affected by nitrogen under pressure. This suggests that the nitrogen-induced break point seems to be located at the glutamatergic striatopetal neurons.

  14. Induced pluripotent stem cells (iPSCs) derived from different cell sources and their potential for regenerative and personalized medicine.

    Science.gov (United States)

    Shtrichman, R; Germanguz, I; Itskovitz-Eldor, J

    2013-06-01

    Human induced pluripotent stem cells (hiPSCs) have great potential as a robust source of progenitors for regenerative medicine. The novel technology also enables the derivation of patient-specific cells for applications to personalized medicine, such as for personal drug screening and toxicology. However, the biological characteristics of iPSCs are not yet fully understood and their similarity to human embryonic stem cells (hESCs) is still unresolved. Variations among iPSCs, resulting from their original tissue or cell source, and from the experimental protocols used for their derivation, significantly affect epigenetic properties and differentiation potential. Here we review the potential of iPSCs for regenerative and personalized medicine, and assess their expression pattern, epigenetic memory and differentiation capabilities in relation to their parental tissue source. We also summarize the patient-specific iPSCs that have been derived for applications in biological research and drug discovery; and review risks that must be overcome in order to use iPSC technology for clinical applications.

  15. Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs.

    Science.gov (United States)

    Kang, Eunju; Wang, Xinjian; Tippner-Hedges, Rebecca; Ma, Hong; Folmes, Clifford D L; Gutierrez, Nuria Marti; Lee, Yeonmi; Van Dyken, Crystal; Ahmed, Riffat; Li, Ying; Koski, Amy; Hayama, Tomonari; Luo, Shiyu; Harding, Cary O; Amato, Paula; Jensen, Jeffrey; Battaglia, David; Lee, David; Wu, Diana; Terzic, Andre; Wolf, Don P; Huang, Taosheng; Mitalipov, Shoukhrat

    2016-05-05

    The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.

  16. Doxycycline supplementation allows for the culture of human ESCs/iPSCs with media changes at 3-day intervals.

    Science.gov (United States)

    Chang, Mi-Yoon; Oh, Boram; Rhee, Yong-Hee; Lee, Sang-Hun

    2015-11-01

    Culturing human embryonic stem and induced pluripotent stem cells (hESCs/iPSCs) is one of the most costly and labor-intensive tissue cultures, as media containing expensive factors/cytokines should be changed every day to maintain and propagate undifferentiated hESCs/iPSCs in vitro. We recently reported that doxycycline, an anti-bacterial agent, had dramatic effects on hESC/iPSC survival and promoted self-renewal. In this study, we extended the effects of doxycycline to a more practical issue to save cost and labor in hESC/iPSC cultures. Regardless of cultured cell conditions, hESCs/iPSCs in doxycycline-supplemented media were viable and proliferating for at least 3 days without media change, while none or few viable cells were detected in the absence of doxycycline in the same conditions. Thus, hESCs/iPSCs supplemented with doxycycline can be cultured for a long period of time with media changes at 3-day intervals without altering their self-renewal and pluripotent properties, indicating that doxycycline supplementation can reduce the frequency of media changes and the amount of media required by 1/3. These findings strongly encourage the use of doxycycline to save cost and labor in culturing hESCs/iPSCs.

  17. Escape Mutations, Ganciclovir Resistance, and Teratoma Formation in Human iPSCs Expressing an HSVtk Suicide Gene

    Directory of Open Access Journals (Sweden)

    Andriana G Kotini

    2016-01-01

    Full Text Available Human pluripotent stem cells (hPSCs hold great promise for cell therapy. However, a major concern is the risk of tumor formation by residual undifferentiated cells contaminating the hPSC-derived cell product. Suicide genes could safeguard against such adverse events by enabling elimination of cells gone astray, but the efficacy of this approach has not yet been thoroughly tested. Here, we engineered a lentivirally encoded herpes simplex virus thymidine kinase (HSVtk with expression restricted to undifferentiated hPSCs through regulation by the let7 family of miRNAs. We show that induced pluripotent stem cells (iPSCs expressing a let7-regulated HSVtk transgene are selectively killed by ganciclovir (GCV, whereas differentiated cells are fully protected. However, in contrast to previous studies, we find that in vivo GCV administration results in longer latency but does not prevent teratoma formation by iPSCs expressing either a constitutive or a let7-regulated HSVtk, without evidence of silencing of the HSVtk. Clonal analyses of iPSCs expressing HSVtk revealed frequent emergence of GCV resistance which, at least in some cases, could be attributed to preexisting inactivating mutations in the HSVtk coding sequence, selected for upon GCV treatment. Our findings have important consequences for the future use of suicide genes in hPSC-based cell therapies.

  18. Stress-induced impairment of glutamatergic terminals ultrastructure: High vulnerability of medial prefrontal cortex and preventing action of desipramine

    DEFF Research Database (Denmark)

    Nava, N.; Popoli, M.; Musazzi, L.

    2013-01-01

    volume quantified [2]. Distribution of glutamatergic synaptic terminals was evaluated with immunohistochemistry. Asymmetric-glutamatergic synapses were identified and the size of readilyreleasable pool and reserve-pool of vesicles estimated, through serial section electron microscopy. Extension of post...

  19. More sensitivity of cortical GABAergic neurons than glutamatergic neurons in response to acidosis.

    Science.gov (United States)

    Liu, Hua; Li, Fang; Wang, Chunyan; Su, Zhiqiang

    2016-05-25

    Acidosis impairs brain functions. Neuron-specific mechanisms underlying acidosis-induced brain dysfunction remain elusive. We studied the sensitivity of cortical GABAergic neurons and glutamatergic neurons to acidosis by whole-cell recording in brain slices. The acidification to the neurons was induced by perfusing artificial cerebral spinal fluid with lower pH. This acidification impairs excitability and synaptic transmission in the glutamatergic and GABAergic neurons. Acidosis impairs spiking capacity in the GABAergic neurons more than in the glutamatergic neurons. Acidosis also strengthens glutamatergic synaptic transmission and attenuates GABAergic synaptic transmission on the GABAergic neurons more than the glutamatergic neurons, which results in the functional impairment of these GABAergic neurons. This acidosis-induced dysfunction predominantly in the cortical GABAergic neurons drives the homeostasis of neuronal networks toward overexcitation and exacerbates neuronal impairment.

  20. REGULATING PRIVATE SECURITY COMPANIES (PSCs AND PRIVATE MILITARY COMPANIES (PMCs UNDER THE LAW OF TIMOR­LESTE

    Directory of Open Access Journals (Sweden)

    Salvador Soares

    2015-02-01

    Full Text Available Use  of  Private  Security  Companies  (PSCs  and  Private  Military  Companies  (PMCs  in  conflict  and post-conflict  countries  has  been  the  subject  of  ongoing  critical  discussion  among  scholars  and  media for many years. This paper assesses the legal status and responsibilities of PSCs and PMCs, and their operation in Timor-Leste where they are not properly regulated. It examines key legal issues, such as their definition and roles, scope and limits of operations, approved and prohibited activities, accountability, and monitoring. The paper also examines the impact of PSCs and PMCs on Timor-Leste law and society and offers astatutory framework for their management and regulation. Implementasi Private Security Companies (PSCs dan Private Military Companies (PMCs dalam negara yang sedang berkonflik dan pasca berkonflik telah menjadi subjek pembahasan penting diantara akademisi dan media massa selama bertahun-tahun. Penulisan ini akan menilai status hukum dan kewajiban PSCs dan PMCs, dan pelaksanaan kerja kedua lembaga tersebut di Timor-Leste dimana belum ada pengaturan yang mumpuni. Penulisan ini menganalisa isu-isu krusial mengenai beberapa pengaturan, seperti definisi dan peran, batasan dan ruang lingkup kerja, aktivitas yang diperbolehkan dan yang dilarang, akuntabilitas, serta pengawasan. Penulisan ini juga menganalisa implikasi dari PSCs dan PMCs di tatanan hukum dan masyarakat Timor-Leste, serta menawarkan sebuah kerangka undang-undang untuk manajerial dan pengaturan kedua lembaga tersebut.

  1. Generation of iPSCs from mouse fibroblasts with a single gene,Oct4,and small molecules

    Institute of Scientific and Technical Information of China (English)

    Yanqin Li; Xu Zhang; Yetao Wu; Honggang Li; Kang Liu; Chen Wu; Zhihua Song; Yang Zhao; Yan Shi; Hongkui Deng; Qiang Zhang; Xiaolei Yin; Weifeng Yang; Yuanyuan Du; Pingping Hou; Jian Ge; Chun Liu; Weiqi Zhang

    2011-01-01

    The introduction of four transcription factors Oct4,Klf4,Sox2 and c-Myc by viral transduction can induce reprogramming of somatic cells into induced pluripotent stem cells(iPSCs),but the use of iPSCs is hindered by the use of viral delivery systems.Chemical-induced reprogramming offers a novel approach to generating iPSCs without any viral vector-based genetic modification.Previous reports showed that several small molecules could replace some of the reprogramming factors although at least two transcription factors,Oct4 and Klf4,are still required to generate iPSCs from mouse embryonic fibroblasts.Here,we identify a specific chemical combination,which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor,Oct4,within 20 days,replacing Sox2,Klf4 and c-Myc.The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile,epigenetic status and pluripotency both in vitro and in vivo.We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules,which suggests that reprogramming was initiated within the first 8 days and was independent of continuous exogenous Oct4 expression.These discoveries will aid in the future generation of iPSCs without genetic modification,as well as elucidating the molecular mechanisms that underlie the reprogramming process.

  2. Peripheral blood derived induced pluripotent stem cells (iPSCs from a female with familial hypertrophic cardiomyopathy

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    Samantha Barratt Ross

    2017-04-01

    Full Text Available Induced pluripotent stem cells (iPSCs were generated from peripheral blood mononuclear cells (PBMCs obtained from a 62-year-old female with familial hypertrophic cardiomyopathy (HCM. PBMCs were reprogrammed to a pluripotent state following transfection with non-integrative episomal vectors carrying reprogramming factors OCT4, SOX2, LIN28, KLF4 and L-MYC. iPSCs were shown to express pluripotency markers, possess trilineage differentiation potential, carry rare variants identified in DNA isolated directly from the patient's whole blood, have a normal karyotype and no longer carry episomal vectors for reprogramming. This line is a useful resource for identifying unknown genetic causes of HCM.

  3. Generation, Expansion, and Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) Derived From the Umbilical Cords of Newborns.

    Science.gov (United States)

    Song, Richard S; Carroll, Jeanne M; Acevedo, Lisette; Wu, Dongmei; Liu, Yang; Snyder, Evan Y

    2014-05-16

    The umbilical cord is tissue that is normally discarded after the delivery of the infant, but it has been shown to be a rich source of stem cells from the cord blood, Wharton's jelly, and umbilical endothelial cells. Patient-specific human induced pluripotent stem cells (hiPSCs) reprogrammed from patient specific human umbilical vein endothelial cells in the neonatal intensive care unit (NICU) population (specifically, premature neonates) have not been shown in the literature. This unit describes a protocol for the generation and expansion of hiPSCs originating from umbilical cords collected from patients in the NICU.

  4. iPSCs-based anti-aging therapies: Recent discoveries and future challenges.

    Science.gov (United States)

    Pareja-Galeano, Helios; Sanchis-Gomar, Fabián; Pérez, Laura M; Emanuele, Enzo; Lucia, Alejandro; Gálvez, Beatriz G; Gallardo, María Esther

    2016-05-01

    The main biological hallmarks of the aging process include stem cell exhaustion and cellular senescence. Consequently, research efforts to treat age-related diseases as well as anti-aging therapies in general have recently focused on potential 'reprogramming' regenerative therapies. These new approaches are based on induced pluripotent stem cells (iPSCs), including potential in vivo reprogramming for tissue repair. Another possibility is targeting pathways of cellular senescence, e.g., through modulation of p16INK4a signaling and especially inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Here, we reviewed and discussed these recent developments together with their possible usefulness for future treatments against sarcopenia, a major age-related condition.

  5. Human neural progenitors derived from integration-free iPSCs for SCI therapy

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    Ying Liu

    2017-03-01

    Full Text Available As a potentially unlimited autologous cell source, patient induced pluripotent stem cells (iPSCs provide great capability for tissue regeneration, particularly in spinal cord injury (SCI. However, despite significant progress made in translation of iPSC-derived neural progenitor cells (NPCs to clinical settings, a few hurdles remain. Among them, non-invasive approach to obtain source cells in a timely manner, safer integration-free delivery of reprogramming factors, and purification of NPCs before transplantation are top priorities to overcome. In this study, we developed a safe and cost-effective pipeline to generate clinically relevant NPCs. We first isolated cells from patients' urine and reprogrammed them into iPSCs by non-integrating Sendai viral vectors, and carried out experiments on neural differentiation. NPCs were purified by A2B5, an antibody specifically recognizing a glycoganglioside on the cell surface of neural lineage cells, via fluorescence activated cell sorting. Upon further in vitro induction, NPCs were able to give rise to neurons, oligodendrocytes and astrocytes. To test the functionality of the A2B5+ NPCs, we grafted them into the contused mouse thoracic spinal cord. Eight weeks after transplantation, the grafted cells survived, integrated into the injured spinal cord, and differentiated into neurons and glia. Our specific focus on cell source, reprogramming, differentiation and purification method purposely addresses timing and safety issues of transplantation to SCI models. It is our belief that this work takes one step closer on using human iPSC derivatives to SCI clinical settings.

  6. Functional significance of brain glycogen in sustaining glutamatergic neurotransmission

    DEFF Research Database (Denmark)

    Sickmann, Helle M; Walls, Anne B; Schousboe, Arne

    2009-01-01

    The involvement of brain glycogen in sustaining neuronal activity has previously been demonstrated. However, to what extent energy derived from glycogen is consumed by astrocytes themselves or is transferred to the neurons in the form of lactate for oxidative metabolism to proceed is at present...... in co-cultures of cerebellar neurons and astrocytes. In the astrocytes it was shown that uptake of the glutamate analogue D-[3H]aspartate was impaired when glycogen degradation was inhibited irrespective of the presence of glucose, signifying that energy derived from glycogen degradation is important...... for the astrocytic compartment. By inhibiting glycogen degradation in co-cultures it was evident that glycogen provides energy to sustain glutamatergic neurotransmission, i.e. release and uptake of glutamate. The relocation of glycogen derived lactate to the neuronal compartment was investigated by employing d...

  7. Genetic deletion of NR3A accelerates glutamatergic synapse maturation.

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    Maile A Henson

    Full Text Available Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs; however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD. NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth.

  8. Investigation of synapse formation and function in a glutamatergic-GABAergic two-neuron microcircuit.

    Science.gov (United States)

    Chang, Chia-Ling; Trimbuch, Thorsten; Chao, Hsiao-Tuan; Jordan, Julia-Christine; Herman, Melissa A; Rosenmund, Christian

    2014-01-15

    Neural circuits are composed of mainly glutamatergic and GABAergic neurons, which communicate through synaptic connections. Many factors instruct the formation and function of these synapses; however, it is difficult to dissect the contribution of intrinsic cell programs from that of extrinsic environmental effects in an intact network. Here, we perform paired recordings from two-neuron microculture preparations of mouse hippocampal glutamatergic and GABAergic neurons to investigate how synaptic input and output of these two principal cells develop. In our reduced preparation, we found that glutamatergic neurons showed no change in synaptic output or input regardless of partner neuron cell type or neuronal activity level. In contrast, we found that glutamatergic input caused the GABAergic neuron to modify its output by way of an increase in synapse formation and a decrease in synaptic release efficiency. These findings are consistent with aspects of GABAergic synapse maturation observed in many brain regions. In addition, changes in GABAergic output are cell wide and not target-cell specific. We also found that glutamatergic neuronal activity determined the AMPA receptor properties of synapses on the partner GABAergic neuron. All modifications of GABAergic input and output required activity of the glutamatergic neuron. Because our system has reduced extrinsic factors, the changes we saw in the GABAergic neuron due to glutamatergic input may reflect initiation of maturation programs that underlie the formation and function of in vivo neural circuits.

  9. PSCs and GLP-1R: occurrence in normal pancreas, acute/chronic pancreatitis and effect of their activation by a GLP-1R agonist.

    Science.gov (United States)

    Nakamura, Taichi; Ito, Tetsuhide; Uchida, Masahiko; Hijioka, Masayuki; Igarashi, Hisato; Oono, Takamasa; Kato, Masaki; Nakamura, Kazuhiko; Suzuki, Koichi; Jensen, Robert T; Takayanagi, Ryoichi

    2014-01-01

    There is increasing concern about the development of pancreatitis in patients with diabetes mellitus who received long-term glucagon-like peptide-1 (GLP-1) analog treatment. Its pathogenesis is unknown. The effects of GLP-1 agonists on pancreatic endocrine cells are well studied; however, there is little information on effects on other pancreatic tissues that might be involved in inflammatory processes. Pancreatic stellate cells (PSCs) can have an important role in pancreatitis, secreting various inflammatory cytokines/chemokines, as well as collagen. In this study, we investigated GLP-1R occurrence in normal pancreas, acute pancreatitis (AP)/chronic pancreatitis (CP), and the effects of GLP-1 analog on normal PSCs, their ability to stimulate inflammatory mediator secretion or proliferation. GLP-1 receptor (GLP-1R) expression/localization in normal pancreas and pancreatitis (AP/CP) tissues were evaluated with histological/immunohistochemical analysis. PSCs were isolated from male Wistar rats. GLP-1R expression and effects of GLP-1 analog on activated PSCs was examined with real-time PCR, MTS assays and western blotting. In normal pancreas, pancreatic β cells expressed GLP-1R, with only low expression in acinar cells, whereas in AP or CP, acinar cells, ductal cells and activated PSCs expressed GLP-1R. With activation of normal PSCs, GLP-1R is markedly increased, as is multiple other incretin-related receptors. The GLP-1 analog, liraglutide, did not induce inflammatory genes expression in activated PSCs, but induced proliferation. Liraglutide activated multiple signaling cascades in PSCs, and the extracellular signal-regulated kinase pathway mediated the PSCs proliferation. GLP-1Rs are expressed in normal pancreas and there is marked enhanced expression in AP/CP. GLP-1-agonist induced cell proliferation of activated PSCs without increasing release of inflammatory mediators. These results suggest chronic treatment with GLP-1R agonists could lead to proliferation

  10. Gene Correction of iPSCs from a Wiskott-Aldrich Syndrome Patient Normalizes the Lymphoid Developmental and Functional Defects

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    Tamara J. Laskowski

    2016-08-01

    Full Text Available Wiskott-Aldrich syndrome (WAS is an X-linked primary immunodeficiency disease caused by mutations in the gene encoding the WAS protein (WASp. Here, induced pluripotent stem cells (iPSCs were derived from a WAS patient (WAS-iPSC and the endogenous chromosomal WAS locus was targeted with a wtWAS-2A-eGFP transgene using zinc finger nucleases (ZFNs to generate corrected WAS-iPSC (cWAS-iPSC. WASp and GFP were first expressed in the earliest CD34+CD43+CD45− hematopoietic precursor cells and later in all hematopoietic lineages examined. Whereas differentiation to non-lymphoid lineages was readily obtained from WAS-iPSCs, in vitro T lymphopoiesis from WAS-iPSC was deficient with few CD4+CD8+ double-positive and mature CD3+ T cells obtained. T cell differentiation was restored for cWAS-iPSCs. Similarly, defects in natural killer cell differentiation and function were restored on targeted correction of the WAS locus. These results demonstrate that the defects exhibited by WAS-iPSC-derived lymphoid cells were fully corrected and suggests the potential therapeutic use of gene-corrected WAS-iPSCs.

  11. Fate of iPSCs Derived from Azoospermic and Fertile Men following Xenotransplantation to Murine Seminiferous Tubules

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    Cyril Ramathal

    2014-05-01

    Full Text Available Historically, spontaneous deletions and insertions have provided means to probe germline developmental genetics in Drosophila, mouse and other species. Here, induced pluripotent stem cell (iPSC lines were derived from infertile men with deletions that encompass three Y chromosome azoospermia factor (AZF regions and are associated with production of few or no sperm but normal somatic development. AZF-deleted iPSC lines were compromised in germ cell development in vitro. Undifferentiated iPSCs transplanted directly into murine seminiferous tubules differentiated extensively to germ-cell-like cells (GCLCs that localized near the basement membrane, demonstrated morphology indistinguishable from fetal germ cells, and expressed germ-cell-specific proteins diagnostic of primordial germ cells. Alternatively, all iPSCs that exited tubules formed primitive tumors. iPSCs with AZF deletions produced significantly fewer GCLCs in vivo with distinct defects in gene expression. Findings indicate that xenotransplantation of human iPSCs directs germ cell differentiation in a manner dependent on donor genetic status.

  12. Resilience dysregulation in major depressive disorder: focus on glutamatergic imbalance and microglial activation.

    Science.gov (United States)

    Réus, Gislaine Z; de Moura, Airam B; Silva, Ritele H; Resende, Wilson R; Quevedo, João

    2017-06-30

    Many studies have been shown an important role of glutamatergic system as well microglial activation in the pathophysiology of major depressive disorder (MDD). Experimental and clinical data suggest that attenuation of N-methyl-D-aspartate (NMDA) receptor function exerts antidepressant effects. Glutamatergic system is involved with memory establishment and function, and it regulates plasticity in the brain. Microglial cells play pivotal role to the brain functions; however, under chronic inflammation status microglial could be turn activated and increase the pro-inflammatory cytokines. In humans most resistant to the development of psychiatric disorders, including MDD, are observed a greater degree of resilience resulting from stress. Less resilience is associated with neuroendocrine and neuroinflammatory markers, as well as with glutamatergic system dysregulation. Thus, this review we highlighted findings from literature identifying the function of glutamatergic system, microglial activation and inflammation in resilience. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Cholinergic modulation of primary afferent glutamatergic transmission in rat medullary dorsal horn neurons.

    Science.gov (United States)

    Jeong, Seok-Gwon; Choi, In-Sun; Cho, Jin-Hwa; Jang, Il-Sung

    2013-12-01

    Although muscarinic acetylcholine (mACh) receptors are expressed in trigeminal ganglia, it is still unknown whether mACh receptors modulate glutamatergic transmission from primary afferents onto medullary dorsal horn neurons. In this study, we have addressed the cholinergic modulation of primary afferent glutamatergic transmission using a conventional whole cell patch clamp technique. Glutamatergic excitatory postsynaptic currents (EPSCs) were evoked from primary afferents by electrical stimulation of trigeminal tract and monosynaptic EPSCs were recorded from medullary dorsal horn neurons of rat horizontal brain stem slices. Muscarine and ACh reversibly and concentration-dependently decreased the amplitude of glutamatergic EPSCs and increased the paired-pulse ratio. In addition, muscarine reduced the frequency of miniature EPSCs without affecting the current amplitude, suggesting that muscarine acts presynaptically to decrease the probability of glutamate release onto medullary dorsal horn neurons. The muscarine-induced decrease of glutamatergic EPSCs was significantly occluded by methoctramine or AF-DX116, M2 receptor antagonists, but not pirenzepine, J104129 and MT-3, selective M1, M3 and M4 receptor antagonists. The muscarine-induced decrease of glutamatergic EPSCs was highly dependent on the extracellular Ca2+ concentration. Physostigmine and clinically available acetylcholinesterase inhibitors, such as rivastigmine and donepezil, significantly shifted the concentration-inhibition relationship of ACh for glutamatergic EPSCs. These results suggest that muscarine acts on presynaptic M2 receptors to inhibit glutamatergic transmission by reducing the Ca2+ influx into primary afferent terminals, and that M2 receptor agonists and acetylcholinesterase inhibitors could be, at least, potential targets to reduce nociceptive transmission from orofacial tissues.

  14. Modulation of the glutamatergic transmission by Dopamine: a focus on Parkinson, Huntington and Addiction diseases.

    Science.gov (United States)

    Gardoni, Fabrizio; Bellone, Camilla

    2015-01-01

    Dopamine (DA) plays a major role in motor and cognitive functions as well as in reward processing by regulating glutamatergic inputs. In particular in the striatum the release of DA rapidly influences synaptic transmission modulating both AMPA and NMDA receptors. Several neurodegenerative and neuropsychiatric disorders, including Parkinson, Huntington and addiction-related diseases, manifest a dysregulation of glutamate and DA signaling. Here, we will focus our attention on the mechanisms underlying the modulation of the glutamatergic transmission by DA in striatal circuits.

  15. Amygdalar glutamatergic neuronal systems play a key role on the hibernating state of hamsters

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    Facciolo Rosa

    2011-01-01

    Full Text Available Abstract Background Excitatory transmitting mechanisms are proving to play a critical role on neuronal homeostasis conditions of facultative hibernators such as the Syrian golden hamster. Indeed works have shown that the glutamatergic system of the main olfactory brain station (amygdala is capable of controlling thermoregulatory responses, which are considered vital for the different hibernating states. In the present study the role of amygdalar glutamatergic circuits on non-hibernating (NHIB and hibernating (HIB hamsters were assessed on drinking stimuli and subsequently compared to expression variations of some glutamatergic subtype mRNA levels in limbic areas. For this study the two major glutamatergic antagonists and namely that of N-methyl-D-aspartate receptor (NMDAR, 3-(+-2-carboxypiperazin-4-yl-propyl-1-phosphonate (CPP plus that of the acid α-amine-3-hydroxy-5-metil-4-isoxazol-propionic receptor (AMPAR site, cyano-7-nitro-quinoxaline-2,3-dione (CNQX were infused into the basolateral amygdala nucleus. Attempts were made to establish the type of effects evoked by amygdalar glutamatergic cross-talking processes during drinking stimuli, a response that may corroborate their major role at least during some stages of this physiological activity in hibernators. Results From the behavioral results it appears that the two glutamatergic compounds exerted distinct effects. In the first case local infusion of basolateral complexes (BLA with NMDAR antagonist caused very great (p Conclusion We conclude that predominant drinking events evoked by glutamatergic mechanisms, in the presence of prevalently down regulated levels of NR1/2A of some telencephalic and hypothalamic areas appear to constitute an important neuronal switch at least during arousal stage of hibernation. The establishment of the type of glutamatergic subtypes that are linked to successful hibernating states, via drinking stimuli, may have useful bearings toward sleeping disorders.

  16. Modulation of excitatory neurotransmission by neuronal/glial signalling molecules: interplay between purinergic and glutamatergic systems.

    Science.gov (United States)

    Köles, László; Kató, Erzsébet; Hanuska, Adrienn; Zádori, Zoltán S; Al-Khrasani, Mahmoud; Zelles, Tibor; Rubini, Patrizia; Illes, Peter

    2016-03-01

    Glutamate is the main excitatory neurotransmitter of the central nervous system (CNS), released both from neurons and glial cells. Acting via ionotropic (NMDA, AMPA, kainate) and metabotropic glutamate receptors, it is critically involved in essential regulatory functions. Disturbances of glutamatergic neurotransmission can be detected in cognitive and neurodegenerative disorders. This paper summarizes the present knowledge on the modulation of glutamate-mediated responses in the CNS. Emphasis will be put on NMDA receptor channels, which are essential executive and integrative elements of the glutamatergic system. This receptor is crucial for proper functioning of neuronal circuits; its hypofunction or overactivation can result in neuronal disturbances and neurotoxicity. Somewhat surprisingly, NMDA receptors are not widely targeted by pharmacotherapy in clinics; their robust activation or inhibition seems to be desirable only in exceptional cases. However, their fine-tuning might provide a promising manipulation to optimize the activity of the glutamatergic system and to restore proper CNS function. This orchestration utilizes several neuromodulators. Besides the classical ones such as dopamine, novel candidates emerged in the last two decades. The purinergic system is a promising possibility to optimize the activity of the glutamatergic system. It exerts not only direct and indirect influences on NMDA receptors but, by modulating glutamatergic transmission, also plays an important role in glia-neuron communication. These purinergic functions will be illustrated mostly by depicting the modulatory role of the purinergic system on glutamatergic transmission in the prefrontal cortex, a CNS area important for attention, memory and learning.

  17. VTA glutamatergic inputs to nucleus accumbens drive aversion by acting on GABAergic interneurons

    Science.gov (United States)

    Qi, Jia; Zhang, Shiliang; Wang, Hui-Ling; Barker, David J.; Miranda-Barrientos, Jorge; Morales, Marisela

    2016-01-01

    The ventral tegmental area (VTA) is best known for its dopamine neurons, some of which project to nucleus accumbens (nAcc). However, the VTA also has glutamatergic neurons that project to nAcc. The function of the mesoaccumbens-glutamatergic pathway remains unknown. Here, we report that nAcc photoactivation of mesoaccumbens-glutamatergic fibers promotes aversion. Although we found that these mesoaccumbens-glutamate-fibers lack GABA, the aversion evoked by their photoactivation depends on glutamate and GABA receptor signaling, and not on dopamine receptor signaling. We found that mesoaccumbens-glutamatergic-fibers establish multiple asymmetric synapses on single parvalbumin-GABAergic interneurons, and that nAcc photoactivation of these fibers drives AMPA-mediated cellular firing of parvalbumin-GABAergic interneurons. These parvalbumin-GABAergic-interneurons, in turn, inhibit nAcc medium spiny output neurons, as such, controlling inhibitory neurotransmission within nAcc. The mesoaccumbens-glutamatergic pathway is the first glutamatergic input to nAcc shown to mediate aversion, instead of reward, and the first pathway shown to establish excitatory synapses on nAcc parvalbumin-GABAergic interneurons. PMID:27019014

  18. CALIPSO Observations of PSCs and Cirrus During the 2015-2016 Arctic Winter

    Science.gov (United States)

    Pitts, Michael; Poole, Lamont

    2016-04-01

    The POLSTRACC (POlar STRAtosphere in a Changing Climate) field campaign was conducted in the Arctic during December 2015 - March 2016 to investigate the chemical, microphysical, and dynamical processes of the Arctic lowermost stratosphere and upper troposphere. The primary measurement platform for POLSTRACC was the German HALO (High Altitude LOng range) research aircraft carrying a large suite of in situ and remote sensing instruments to measure key chemical species, tracers, as well as aerosol and cloud particles and meteorological parameters. Two primary science objectives of POLSTRACC are to improve our understanding of polar stratospheric cloud (PSC) particle characteristics and formation processes and investigate the impact of Arctic cirrus clouds on radiative forcing and chlorine activation. To complement the more focused measurements from the POLSTRACC field campaign, we have used spaceborne lidar measurements from CALIPSO to characterize PSC occurrence and composition, as well as the occurrence of Arctic cirrus during the 2015-2016 season on vortex-wide scales. In this paper, we present a general overview of the 2015-2016 winter, examine in detail the evolution of PSCs and cirrus during the season, and explore the unique aspects of this season in attempt to understand the underlying physical mechanisms.

  19. Vulnerability of Purkinje Cells Generated from Spinocerebellar Ataxia Type 6 Patient-Derived iPSCs

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    Yoshihito Ishida

    2016-11-01

    Full Text Available Spinocerebellar ataxia type 6 (SCA6 is a dominantly inherited neurodegenerative disease characterized by loss of Purkinje cells in the cerebellum. SCA6 is caused by CAG trinucleotide repeat expansion in CACNA1A, which encodes Cav2.1, α1A subunit of P/Q-type calcium channel. However, the pathogenic mechanism and effective therapeutic treatments are still unknown. Here, we have succeeded in generating differentiated Purkinje cells that carry patient genes by combining disease-specific iPSCs and self-organizing culture technologies. Patient-derived Purkinje cells exhibit increased levels of full-length Cav2.1 protein but decreased levels of its C-terminal fragment and downregulation of the transcriptional targets TAF1 and BTG1. We further demonstrate that SCA6 Purkinje cells exhibit thyroid hormone depletion-dependent degeneration, which can be suppressed by two compounds, thyroid releasing hormone and Riluzole. Thus, we have constructed an in vitro disease model recapitulating both ontogenesis and pathogenesis. This model may be useful for pathogenic investigation and drug screening.

  20. Glutamatergic neurons are present in the rat ventral tegmental area

    Science.gov (United States)

    Yamaguchi, Tsuyoshi; Sheen, Whitney; Morales, Marisela

    2010-01-01

    The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or γ-amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, we have proposed that additional cell types, such as those that express the neurotransmitter glutamate may also be present in this area. Thus, by using in situ hybridization histochemistry we investigated whether transcripts encoded by genes for the two vesicular glutamate transporters, VGluT1 or VGluT2, were expressed in the VTA. We found that VGluT2 mRNA but not VGluT1 mRNA is expressed in the VTA. Neurons expressing VGluT2 mRNA were differentially distributed throughout the rostro-caudal and medio-lateral aspects of the VTA, with the highest concentration detected in rostro-medial areas. Phenotypic characterization with double in situ hybridization of these neurons indicated that they rarely co–expressed mRNAs for tyrosine hydroxylase (TH, marker for DAergic neurons) or glutamic acid decarboxylase (GAD, marker for GABAergic neurons). Based on the results described here, we concluded that the VTA contains glutamatergic neurons that in their vast majority are clearly non-DAergic and non-GABAergic. PMID:17241272

  1. D-serine and serine racemase are associated with PSD-95 and glutamatergic synapse stability

    Directory of Open Access Journals (Sweden)

    Hong eLin

    2016-02-01

    Full Text Available D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs, synthesized by serine racemase (SR through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking alpha7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1, in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5 and 7-chlorokynurenic acid (7-CK, a specific antagonist at the glycine site of NMDARs

  2. CALIPSO observations of wave-induced PSCs with near-unity optical depth over Antarctica in 2006-2007

    CERN Document Server

    Noel, Vincent; Chepfer, Hélène

    2009-01-01

    Ground-based and satellite observations have hinted at the existence of polar stratospheric clouds (PSCs) with relatively high optical depths, even if optical depth values are hard to come by. This study documents a type II PSC observed from spaceborne lidar, with visible optical depths up to 0.8. Comparisons with multiple temperature fields, including reanalyses and results from mesoscale simulations, suggest that intense small-scale temperature fluctuations due to gravity waves play an important role in its formation, while nearby observations show the presence of a potentially related type Ia PSC farther downstream inside the polar vortex. Following this first case, the geographic distribution and microphysical properties of PSCs with optical depths above 0.3 are explored over Antarctica during the 2006 and 2007 austral winters. These clouds are rare (less than 1% of profiles) and concentrated over areas where strong winds hit steep ground slopes in the Western Hemisphere, especially over the peninsula. Su...

  3. Induced pluripotent stem cells (iPSCs derived from cerebrotendinous xanthomatosis (CTX patient's fibroblasts carrying a R395S mutation

    Directory of Open Access Journals (Sweden)

    Philip Höflinger

    2016-09-01

    Full Text Available Induced pluripotent stem cells (iPSCs were generated from dermal fibroblasts from a 60-year-old cerebrotendinous xanthomatosis (CTX patient, carrying a homozygous mutation c. [1183C>A]; p. R395S in CYP27A1. Episomal plasmids encoding the pluripotency genes OCT4, SOX2, KLF4, L-MYC and LIN28 were introduced via electroporation. The generated line iPS-CTX-R395S has no sign of plasmid integration or chromosomal aberration and retained the mutation site in CYP27A1. Furthermore, iPSCs express pluripotency markers and are able to differentiate in all germ layers in vitro. The generated line may be a useful tool for disease modelling of CTX.

  4. Peripheral blood mononuclear cell-converted induced pluripotent stem cells (iPSCs) from an early onset Alzheimer's patient.

    Science.gov (United States)

    Lee, Han-Kyu; Morin, Peter; Xia, Weiming

    2016-03-01

    Improvement in transduction efficiency makes it possible to convert blood cells into induced pluripotent stem cells (iPSC). In this study, we generated an iPSC line from peripheral blood mononuclear cells (PBMC) donated by a patient who exhibited memory deficit at age 59; outcome of positron emission tomography scan is consistent with a diagnosis of Alzheimer's disease. Integration-free CytoTune-iPS Sendai Reprogramming factors which include Sendai virus particles of the four Yamanaka factors Oct4, Sox2, Klf4, and c-Myc were introduced to PBMC to convert them to iPSCs without retention of virus. Three germ layer differentiation was induced to demonstrate the pluripotency of these iPSCs. Published by Elsevier B.V.

  5. Peripheral blood mononuclear cell-converted induced pluripotent stem cells (iPSCs from an early onset Alzheimer's patient

    Directory of Open Access Journals (Sweden)

    Han-Kyu Lee

    2016-03-01

    Full Text Available Improvement in transduction efficiency makes it possible to convert blood cells into induced pluripotent stem cells (iPSC. In this study, we generated an iPSC line from peripheral blood mononuclear cells (PBMC donated by a patient who exhibited memory deficit at age 59; outcome of positron emission tomography scan is consistent with a diagnosis of Alzheimer's disease. Integration-free CytoTune-iPS Sendai Reprogramming factors which include Sendai virus particles of the four Yamanaka factors Oct4, Sox2, Klf4, and c-Myc were introduced to PBMC to convert them to iPSCs without retention of virus. Three germ layer differentiation was induced to demonstrate the pluripotency of these iPSCs.

  6. Derivation of Parathyroid Gland Cells and Their Progenitors fromInduced Pluripotent Stem Cells (iPSCs) for Personalized Therapy

    Science.gov (United States)

    2016-09-01

    Award Number: W81XWH-15-1-0261 TITLE: Derivation of Parathyroid Gland Cells and Their Progenitors from Induced Pluripotent Stem Cells (iPSCs) for...REPORT TYPE Annual 3. DATES COVERED 1 Aug 2015 - 31 Jul 2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Derivation of Parathyroid Gland Cells and... parathyroid gland (PTG) dysfunction and a disruption of calcium homeostasis and metabolism. Hypoparathyroidism is particularly devastating and is becoming

  7. Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM)

    DEFF Research Database (Denmark)

    Xiong, Kai; Zhou, Yan; Hyttel, Poul

    2016-01-01

    Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4...... a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes....

  8. Role of nucleus accumbens glutamatergic plasticity in drug addiction.

    Science.gov (United States)

    Quintero, Gabriel C

    2013-01-01

    . Antagonism of the CP-AMPARs reduces cravings. It is necessary to pursue further exploration of the AMPA receptor subunit composition and variations at the level of the NAc for a better understanding of glutamatergic plastic changes. It is known that cocaine and morphine are able to induce changes in dendritic spine morphology by modifying actin cycling. These changes include an initial increase in spine head diameter and increases in AMPA receptor expression, followed by a second stage of spine head diameter retraction and reduction of the AMPA receptors' expression in spines. Besides glutamate and dopamine, other factors, like brain-derived neurotrophic factor (BDNF), can influence NAc activity and induce changes in dendritic spine density. BDNF also induces drug-related behaviors like self-administration and relapse. Neither apoptosis nor neurogenesis plays a relevant role in the neurobiological processes subjacent to cocaine addiction in adults (rodent or human). Different therapeutic drugs like N-acetylcysteine (NAC), modafinil, acamprosate, and topiramate have been tested in preclinical and/or clinical models for alleviating drug relapse. Moreover, these therapeutic drugs target the glutamatergic circuitry between the PFC and the NAc. NAC and acamprosate have shown inconsistent results in clinical trials. Modafinil and topiramate have shown some success, but more clinical trials are necessary. Based on the current review findings, it could be recommendable to explore therapeutic approaches that include synergism between different drugs and neurotransmitter systems. The discrepancy in the results of some therapeutic drugs between preclinical versus clinical trials for alleviating relapse or drug dependence could be linked to the scarce exploration of preclinical models that mimic polydrug abuse patterns, for example, cocaine plus alcohol. At the clinical level, the pattern of polydrug consumption is a phenomenon of considerable frequency. Finally, as a complement at the

  9. Formation of solid particles in synoptic-scale Arctic PSCs in early winter 2002/2003

    Directory of Open Access Journals (Sweden)

    N. Larsen

    2004-05-01

    Full Text Available Polar stratospheric clouds (PSC have been observed in early winter (December 2002 during the SOLVE II/Vintresol campaign, both from balloons carrying comprehensive instrumentation for measurements of chemical composition, size distributions, and optical properties of the particles, as well as from individual backscatter soundings from Esrange and Sodankylä. The observations are unique in the sense that the PSC particles seem to have formed in the early winter under synoptic temperature conditions and not being influenced by mountain lee waves. A sequence of measurements during a 5-days period shows a gradual change between liquid and solid type PSCs with the development of a well-known sandwich structure. It appears that all PSC observations show the presence of a background population of solid particles, occasionally mixed in with more dominating liquid particles. The measurements have been compared with results from a detailed microphysical and optical simulation of the formation processes. Calculated extinction(indices are in good agreement with SAGE-III measurements from the same period. Apparently the solid particles are controlled by the synoptic temperature history while the presence of liquid particles is controlled by the local temperatures at the time of observation. The temperature histories indicate that the solid particles are nucleated above the ice frost point, and a surface freezing mechanism for this is included in the model. Reducing the calculated freezing rates by a factor 10–20, the model is able to simulate the observed particle size distributions and reproduce observed HNO3 gas phase concentrations.

  10. Formation of solid particles in synoptic-scale Arctic PSCs in early winter 2002/2003

    Directory of Open Access Journals (Sweden)

    N. Larsen

    2004-01-01

    Full Text Available Polar stratospheric clouds (PSC have been observed in early winter (December 2002 during the SOLVE II/Vintersol campaign, both from balloons carrying comprehensive instrumentation for measurements of chemical composition, size distributions, and optical properties of the particles, as well as from individual backscatter soundings from Esrange and Sodankylä. The observations are unique in the sense that the PSC particles seem to have formed in the early winter under synoptic temperature conditions and not being influenced by mountain lee waves. A sequence of measurements during a 5-days period shows a gradual change between liquid and solid type PSCs with the development of a well-known sandwich structure. It appears that all PSC observations show the presence of a background population of solid particles, occasionally mixed in with more optically dominating liquid particles. The measurements have been compared with results from a detailed microphysical and optical simulation of the formation processes. Calculated extinctions are in good agreement with SAGE-III measurements from the same period. Apparently the solid particles are controlled by the synoptic temperature history while the presence of liquid particles is controlled by the local temperatures at the time of observation. The temperature histories indicate that the solid particles are nucleated above the ice frost point, and a surface freezing mechanism for this is included in the model. Reducing the calculated freezing rates by a factor 10-20, the model is able to simulate the observed particle size distributions and reproduce observed HNO3 gas phase concentrations.

  11. Glutamatergic Neurotransmission Links Sensitivity to Volatile Anesthetics with Mitochondrial Function.

    Science.gov (United States)

    Zimin, Pavel I; Woods, Christian B; Quintana, Albert; Ramirez, Jan-Marino; Morgan, Philip G; Sedensky, Margaret M

    2016-08-22

    An enigma of modern medicine has persisted for over 150 years. The mechanisms by which volatile anesthetics (VAs) produce their effects (loss of consciousness, analgesia, amnesia, and immobility) remain an unsolved mystery. Many attractive putative molecular targets have failed to produce a significant effect when genetically tested in whole-animal models [1-3]. However, mitochondrial defects increase VA sensitivity in diverse organisms from nematodes to humans [4-6]. Ndufs4 knockout (KO) mice lack a subunit of mitochondrial complex I and are strikingly hypersensitive to VAs yet resistant to the intravenous anesthetic ketamine [7]. The change in VA sensitivity is the largest reported for a mammal. Limiting NDUFS4 loss to a subset of glutamatergic neurons recapitulates the VA hypersensitivity of Ndufs4(KO) mice, while loss in GABAergic or cholinergic neurons does not. Baseline electrophysiologic function of CA1 pyramidal neurons does not differ between Ndufs4(KO) and control mice. Isoflurane concentrations that anesthetize only Ndufs4(KO) mice (0.6%) decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) only in Ndufs4(KO) CA1 neurons, while concentrations effective in control mice (1.2%) decreased sEPSC frequencies in both control and Ndufs4(KO) CA1 pyramidal cells. Spontaneous inhibitory postsynaptic currents (sIPSCs) were not differentially affected between genotypes. The effects of isoflurane were similar on evoked field excitatory postsynaptic potentials (fEPSPs) and paired pulse facilitation (PPF) in KO and control hippocampal slices. We propose that CA1 presynaptic excitatory neurotransmission is hypersensitive to isoflurane in Ndufs4(KO) mice due to the inhibition of pre-existing reduced complex I function, reaching a critical reduction that can no longer meet metabolic demands.

  12. In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs.

    Science.gov (United States)

    Wu, Yong; Hu, Zhiqing; Li, Zhuo; Pang, Jialun; Feng, Mai; Hu, Xuyun; Wang, Xiaolin; Lin-Peng, Siyuan; Liu, Bo; Chen, Fangping; Wu, Lingqian; Liang, Desheng

    2016-01-08

    Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5' part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23-26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition.

  13. Corticotropin releasing factor and catecholamines enhance glutamatergic neurotransmission in the lateral subdivision of the central amygdala.

    Science.gov (United States)

    Silberman, Yuval; Winder, Danny G

    2013-07-01

    Glutamatergic neurotransmission in the central nucleus of the amygdala (CeA) plays an important role in many behaviors including anxiety, memory consolidation and cardiovascular responses. While these behaviors can be modulated by corticotropin releasing factor (CRF) and catecholamine signaling, the mechanism(s) by which these signals modify CeA glutamatergic neurotransmission remains unclear. Utilizing whole-cell patch-clamp electrophysiology recordings from neurons in the lateral subdivision of the CeA (CeAL), we show that CRF, dopamine (DA) and the β-adrenergic receptor agonist isoproterenol (ISO) all enhance the frequency of spontaneous excitatory postsynaptic currents (sEPSC) without altering sEPSC kinetics, suggesting they increase presynaptic glutamate release. The effect of CRF on sEPSCs was mediated by a combination of CRFR1 and CRFR2 receptors. While previous work from our lab suggests that CRFRs mediate the effect of catecholamines on excitatory transmission in other subregions of the extended amygdala, blockade of CRFRs in the CeAL failed to significantly alter effects of DA and ISO on glutamatergic transmission. These findings suggest that catecholamine and CRF enhancement of glutamatergic transmission onto CeAL neurons occurs via distinct mechanisms. While CRF increased spontaneous glutamate release in the CeAL, CRF caused no significant changes to optogenetically evoked glutamate release in this region. The dissociable effects of CRF on different types of glutamatergic neurotransmission suggest that CRF may specifically regulate spontaneous excitatory transmission. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Interplay between glutamatergic and GABAergic neurotransmission alterations in cognitive and motor impairment in minimal hepatic encephalopathy.

    Science.gov (United States)

    Llansola, Marta; Montoliu, Carmina; Agusti, Ana; Hernandez-Rabaza, Vicente; Cabrera-Pastor, Andrea; Gomez-Gimenez, Belen; Malaguarnera, Michele; Dadsetan, Sherry; Belghiti, Majedeline; Garcia-Garcia, Raquel; Balzano, Tiziano; Taoro, Lucas; Felipo, Vicente

    2015-09-01

    The cognitive and motor alterations in hepatic encephalopathy (HE) are the final result of altered neurotransmission and communication between neurons in neuronal networks and circuits. Different neurotransmitter systems cooperate to modulate cognitive and motor function, with a main role for glutamatergic and GABAergic neurotransmission in different brain areas and neuronal circuits. There is an interplay between glutamatergic and GABAergic neurotransmission alterations in cognitive and motor impairment in HE. This interplay may occur: (a) in different brain areas involved in specific neuronal circuits; (b) in the same brain area through cross-modulation of glutamatergic and GABAergic neurotransmission. We will summarize some examples of the (1) interplay between glutamatergic and GABAergic neurotransmission alterations in different areas in the basal ganglia-thalamus-cortex circuit in the motor alterations in minimal hepatic encephalopathy (MHE); (2) interplay between glutamatergic and GABAergic neurotransmission alterations in cerebellum in the impairment of cognitive function in MHE through altered function of the glutamate-nitric oxide-cGMP pathway. We will also comment the therapeutic implications of the above studies and the utility of modulators of glutamate and GABA receptors to restore cognitive and motor function in rats with hyperammonemia and hepatic encephalopathy.

  15. Morphological changes of glutamatergic synapses in animal models of Parkinson’s disease

    Directory of Open Access Journals (Sweden)

    Rosa M Villalba

    2015-09-01

    Full Text Available The striatum and the subthalamic nucleus are the main entry doors for extrinsic inputs to reach the basal ganglia circuitry. The cerebral cortex, thalamus and brainstem are the key sources of glutamatergic inputs to these nuclei. There is functional and neurochemical evidence that glutamatergic neurotransmission is altered in the striatum and subthalamic nucleus of animal models of Parkinson’s disease, and that these changes may contribute to aberrant network neuronal activity in the basal ganglia-thalamocortical circuitry. Postmortem studies of animal models and PD patients have revealed significant pathology of glutamatergic synapses, dendritic spines and microcircuits in the striatum of parkinsonians. More recent findings have also demonstrated a significant breakdown of the glutamatergic corticosubthalamic system in parkinsonian monkeys. In this review, we will discuss evidence for synaptic glutamatergic dysfunction and pathology of cortical and thalamic inputs to the striatum and subthalamic nucleus in models of Parkinson’s disease. The potential functional implication of these alterations on synaptic integration, processing and transmission of extrinsic information through the basal ganglia circuits will be considered. Finally, the significance of these pathological changes in the pathophysiology of motor and non-motor symptoms in Parkinson’s disease will be examined.

  16. Cholinergic interneurons mediate fast VGluT3-dependent glutamatergic transmission in the striatum.

    Science.gov (United States)

    Higley, Michael J; Gittis, Aryn H; Oldenburg, Ian A; Balthasar, Nina; Seal, Rebecca P; Edwards, Robert H; Lowell, Bradford B; Kreitzer, Anatol C; Sabatini, Bernardo L

    2011-04-22

    The neurotransmitter glutamate is released by excitatory projection neurons throughout the brain. However, non-glutamatergic cells, including cholinergic and monoaminergic neurons, express markers that suggest that they are also capable of vesicular glutamate release. Striatal cholinergic interneurons (CINs) express the Type-3 vesicular glutamate transporter (VGluT3), although whether they form functional glutamatergic synapses is unclear. To examine this possibility, we utilized mice expressing Cre-recombinase under control of the endogenous choline acetyltransferase locus and conditionally expressed light-activated Channelrhodopsin2 in CINs. Optical stimulation evoked action potentials in CINs and produced postsynaptic responses in medium spiny neurons that were blocked by glutamate receptor antagonists. CIN-mediated glutamatergic responses exhibited a large contribution of NMDA-type glutamate receptors, distinguishing them from corticostriatal inputs. CIN-mediated glutamatergic responses were insensitive to antagonists of acetylcholine receptors and were not seen in mice lacking VGluT3. Our results indicate that CINs are capable of mediating fast glutamatergic transmission, suggesting a new role for these cells in regulating striatal activity.

  17. Cholinergic interneurons mediate fast VGluT3-dependent glutamatergic transmission in the striatum.

    Directory of Open Access Journals (Sweden)

    Michael J Higley

    Full Text Available The neurotransmitter glutamate is released by excitatory projection neurons throughout the brain. However, non-glutamatergic cells, including cholinergic and monoaminergic neurons, express markers that suggest that they are also capable of vesicular glutamate release. Striatal cholinergic interneurons (CINs express the Type-3 vesicular glutamate transporter (VGluT3, although whether they form functional glutamatergic synapses is unclear. To examine this possibility, we utilized mice expressing Cre-recombinase under control of the endogenous choline acetyltransferase locus and conditionally expressed light-activated Channelrhodopsin2 in CINs. Optical stimulation evoked action potentials in CINs and produced postsynaptic responses in medium spiny neurons that were blocked by glutamate receptor antagonists. CIN-mediated glutamatergic responses exhibited a large contribution of NMDA-type glutamate receptors, distinguishing them from corticostriatal inputs. CIN-mediated glutamatergic responses were insensitive to antagonists of acetylcholine receptors and were not seen in mice lacking VGluT3. Our results indicate that CINs are capable of mediating fast glutamatergic transmission, suggesting a new role for these cells in regulating striatal activity.

  18. Autocrine glutamatergic transmission for the regulation of embryonal carcinoma stem cells.

    Science.gov (United States)

    Teng, Lin; Lei, Hui-Min; Sun, Fan; An, Shi-Min; Tang, Ya-Bin; Meng, Shuang; Wang, Cong-Hui; Shen, Ying; Chen, Hong-Zhuan; Zhu, Liang

    2016-08-02

    Glutamate behaves as the principal excitatory neurotransmitter in the vertebrate central nervous system and recently demonstrates intercellular signaling activities in periphery cancer cells. How the glutamatergic transmission is organized and operated in cancer stem cells remains undefined. We have identified a glutamatergic transmission circuit in embryonal carcinoma stem cells. The circuit is organized and operated in an autocrine mechanism and suppresses the cell proliferation and motility. Biological analyses determined a repertoire of glutamatergic transmission components, glutaminase, vesicular glutamate transporter, glutamate NMDA receptor, and cell membrane excitatory amino-acid transporter, for glutamate biosynthesis, package for secretion, reaction, and reuptake in mouse and human embryonal carcinoma stem cells. The glutamatergic components were also identified in mouse transplanted teratocarcinoma and in human primary teratocarcinoma tissues. Released glutamate acting as the signal was directly quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Genetic and pharmacological abolishment of the endogenously released glutamate-induced tonic activation of the NMDA receptors increased the cell proliferation and motility. The finding suggests that embryonal carcinoma stem cells can be actively regulated by establishing a glutamatergic autocrine/paracrine niche via releasing and responding to the transmitter.

  19. Morphological changes of glutamatergic synapses in animal models of Parkinson's disease.

    Science.gov (United States)

    Villalba, Rosa M; Mathai, Abraham; Smith, Yoland

    2015-01-01

    The striatum and the subthalamic nucleus (STN) are the main entry doors for extrinsic inputs to reach the basal ganglia (BG) circuitry. The cerebral cortex, thalamus and brainstem are the key sources of glutamatergic inputs to these nuclei. There is anatomical, functional and neurochemical evidence that glutamatergic neurotransmission is altered in the striatum and STN of animal models of Parkinson's disease (PD) and that these changes may contribute to aberrant network neuronal activity in the BG-thalamocortical circuitry. Postmortem studies of animal models and PD patients have revealed significant pathology of glutamatergic synapses, dendritic spines and microcircuits in the striatum of parkinsonians. More recent findings have also demonstrated a significant breakdown of the glutamatergic corticosubthalamic system in parkinsonian monkeys. In this review, we will discuss evidence for synaptic glutamatergic dysfunction and pathology of cortical and thalamic inputs to the striatum and STN in models of PD. The potential functional implication of these alterations on synaptic integration, processing and transmission of extrinsic information through the BG circuits will be considered. Finally, the significance of these pathological changes in the pathophysiology of motor and non-motor symptoms in PD will be examined.

  20. Sequential generation of olfactory bulb glutamatergic neurons by Neurog2-expressing precursor cells

    Directory of Open Access Journals (Sweden)

    Brill Monika S

    2011-04-01

    Full Text Available Abstract Background While the diversity and spatio-temporal origin of olfactory bulb (OB GABAergic interneurons has been studied in detail, much less is known about the subtypes of glutamatergic OB interneurons. Results We studied the temporal generation and diversity of Neurog2-positive precursor progeny using an inducible genetic fate mapping approach. We show that all subtypes of glutamatergic neurons derive from Neurog2 positive progenitors during development of the OB. Projection neurons, that is, mitral and tufted cells, are produced at early embryonic stages, while a heterogeneous population of glutamatergic juxtaglomerular neurons are generated at later embryonic as well as at perinatal stages. While most juxtaglomerular neurons express the T-Box protein Tbr2, those generated later also express Tbr1. Based on morphological features, these juxtaglomerular cells can be identified as tufted interneurons and short axon cells, respectively. Finally, targeted electroporation experiments provide evidence that while the majority of OB glutamatergic neurons are generated from intrabulbar progenitors, a small portion of them originate from extrabulbar regions at perinatal ages. Conclusions We provide the first comprehensive analysis of the temporal and spatial generation of OB glutamatergic neurons and identify distinct populations of juxtaglomerular interneurons that differ in their antigenic properties and time of origin.

  1. Divergent Modulation of Nociception by Glutamatergic and GABAergic Neuronal Subpopulations in the Periaqueductal Gray

    Science.gov (United States)

    Grajales-Reyes, Jose G.; Copits, Bryan A.; O’Brien, Daniel E.; Trigg, Sarah L.; Gomez, Adrian M.

    2017-01-01

    Abstract The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pronociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here, we demonstrate the different contributions of genetically defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception. PMID:28374016

  2. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a L150P mutation in PSEN-1.

    Science.gov (United States)

    Tubsuwan, Alisa; Pires, Carlota; Rasmussen, Mikkel A; Schmid, Benjamin; Nielsen, Jørgen E; Hjermind, Lena E; Hall, Vanessa; Nielsen, Troels T; Waldemar, Gunhild; Hyttel, Poul; Clausen, Christian; Kitiyanant, Narisorn; Freude, Kristine K; Holst, Bjørn

    2016-01-01

    Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts isolated from a 58-year old male with a L150P mutation in the presenilin 1 (PSEN-1) gene, which is responsible for the majority of familial cases of Alzheimer's disease (AD). The iPSCs were established by co-electroporation with episomal plasmids containing hOCT4, hSOX2, hL-MYC, hKLF4, hNANOG, hLIN28, and short hairpin RNA against TP53. The iPSCs contained the specific heterozygous mutation c.449C>T, had normal karyotype, expressed the expected pluripotency genes and displayed in vitro differentiation potential to the three germ layers. The iPSCs may be useful for studying familial AD pathology in vitro. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors.

    Science.gov (United States)

    Ban, Hiroshi; Nishishita, Naoki; Fusaki, Noemi; Tabata, Toshiaki; Saeki, Koichi; Shikamura, Masayuki; Takada, Nozomi; Inoue, Makoto; Hasegawa, Mamoru; Kawamata, Shin; Nishikawa, Shin-Ichi

    2011-08-23

    After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34(+) cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.

  4. Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamatergic synapses.

    Science.gov (United States)

    Wang, Xulong; Lippi, Giordano; Carlson, David M; Berg, Darwin K

    2013-12-01

    Astrocytes, an abundant form of glia, are known to promote and modulate synaptic signaling between neurons. They also express α7-containing nicotinic acetylcholine receptors (α7-nAChRs), but the functional relevance of these receptors is unknown. We show here that stimulation of α7-nAChRs on astrocytes releases components that induce hippocampal neurons to acquire more α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors post-synaptically at glutamatergic synapses. The increase is specific in that no change is seen in synaptic NMDA receptor clusters or other markers for glutamatergic synapses, or in markers for GABAergic synapses. Moreover, the increases in AMPA receptors on the neuron surface are accompanied by increases in the frequency of spontaneous miniature synaptic currents mediated by the receptors and increases in the ratio of evoked synaptic currents mediated by AMPA versus NMDA receptors. This suggests that stimulating α7-nAChRs on astrocytes can convert 'silent' glutamatergic synapses to functional status. Astrocyte-derived thrombospondin is necessary but not sufficient for the effect, while tumor necrosis factor-α is sufficient but not necessary. The results identify astrocyte α7-nAChRs as a novel pathway through which nicotinic cholinergic signaling can promote the development of glutamatergic networks, recruiting AMPA receptors to post-synaptic sites and rendering the synapses more functional. We find that activation of nicotinic receptors on astrocytes releases a component that specifically recruits AMPA receptors to glutamatergic synapses. The recruitment appears to occur preferentially at what may be 'silent synapses', that is, synapses that have all the components required for glutamatergic transmission (including NMDA receptors) but lack sufficient AMPA receptors to generate a response. The results are unexpected and open up new possibilities for mechanisms underlying network formation and synaptic plasticity.

  5. Role of nucleus accumbens glutamatergic plasticity in drug addiction

    Directory of Open Access Journals (Sweden)

    Quintero GC

    2013-09-01

    -isoxazolepropionic acid (AMPA receptors at the level of the NAc. Also, cocaine self-administration and withdrawal induce the formation of subunit glutamate receptor 2 (GluA2, lacking the Ca2+-permeable AMPA receptors (CP-AMPARs at the level of the NAc. Antagonism of the CP-AMPARs reduces cravings. It is necessary to pursue further exploration of the AMPA receptor subunit composition and variations at the level of the NAc for a better understanding of glutamatergic plastic changes. It is known that cocaine and morphine are able to induce changes in dendritic spine morphology by modifying actin cycling. These changes include an initial increase in spine head diameter and increases in AMPA receptor expression, followed by a second stage of spine head diameter retraction and reduction of the AMPA receptors’ expression in spines. Besides glutamate and dopamine, other factors, like brain-derived neurotrophic factor (BDNF, can influence NAc activity and induce changes in dendritic spine density. BDNF also induces drug-related behaviors like self-administration and relapse. Neither apoptosis nor neurogenesis plays a relevant role in the neurobiological processes subjacent to cocaine addiction in adults (rodent or human. Different therapeutic drugs like N-acetylcysteine (NAC, modafinil, acamprosate, and topiramate have been tested in preclinical and/or clinical models for alleviating drug relapse. Moreover, these therapeutic drugs target the glutamatergic circuitry between the PFC and the NAc. NAC and acamprosate have shown inconsistent results in clinical trials. Modafinil and topiramate have shown some success, but more clinical trials are necessary. Based on the current review findings, it could be recommendable to explore therapeutic approaches that include synergism between different drugs and neurotransmitter systems. The discrepancy in the results of some therapeutic drugs between preclinical versus clinical trials for alleviating relapse or drug dependence could be linked to the

  6. Innervation by a GABAergic neuron depresses spontaneous release in glutamatergic neurons and unveils the clamping phenotype of synaptotagmin-1

    DEFF Research Database (Denmark)

    Wierda, Keimpe D B; Sørensen, Jakob Balslev

    2014-01-01

    The role of spontaneously occurring release events in glutamatergic and GABAergic neurons and their regulation is intensely debated. To study the interdependence of glutamatergic and GABAergic spontaneous release, we compared reciprocally connected "mixed" glutamatergic/GABAergic neuronal pairs...... from mice cultured on astrocyte islands with "homotypic" glutamatergic or GABAergic pairs and autaptic neurons. We measured mEPSC and mIPSC frequencies simultaneously from both neurons. Neuronal pairs formed both interneuronal synaptic and autaptic connections indiscriminately. We find that whereas m......EPSC and mIPSC frequencies did not deviate between autaptic and synaptic connections, the frequency of mEPSCs in mixed pairs was strongly depressed compared with either autaptic neurons or glutamatergic pairs. Simultaneous imaging of synapses, or comparison to evoked release amplitudes, showed...

  7. Genetic inhibition of neurotransmission reveals role of glutamatergic input to dopamine neurons in high-effort behavior.

    Science.gov (United States)

    Hutchison, M A; Gu, X; Adrover, M F; Lee, M R; Hnasko, T S; Alvarez, V A; Lu, W

    2017-02-14

    Midbrain dopamine neurons are crucial for many behavioral and cognitive functions. As the major excitatory input, glutamatergic afferents are important for control of the activity and plasticity of dopamine neurons. However, the role of glutamatergic input as a whole onto dopamine neurons remains unclear. Here we developed a mouse line in which glutamatergic inputs onto dopamine neurons are specifically impaired, and utilized this genetic model to directly test the role of glutamatergic inputs in dopamine-related functions. We found that while motor coordination and reward learning were largely unchanged, these animals showed prominent deficits in effort-related behavioral tasks. These results provide genetic evidence that glutamatergic transmission onto dopaminergic neurons underlies incentive motivation, a willingness to exert high levels of effort to obtain reinforcers, and have important implications for understanding the normal function of the midbrain dopamine system.Molecular Psychiatry advance online publication, 14 February 2017; doi:10.1038/mp.2017.7.

  8. In search for peripheral markers for epilepsy and ALS - focus on glutamatergic signaling in blood cells

    NARCIS (Netherlands)

    Bos, Willemina Minke

    2005-01-01

    This thesis describes the research that was undertaken to find peripheral markers for epilepsy and ALS. Changes in the glutamatergic system and excitotoxicity are suggested to play a role in the pathogenesis of epilepsy and amyotrophic lateral sclerosis (ALS) (chapter 1) and therefore research was f

  9. GABA(A) receptor-mediated presynaptic inhibition on glutamatergic transmission.

    Science.gov (United States)

    Yamamoto, Sokatsu; Yoshimura, Megumu; Shin, Min-Chul; Wakita, Masahito; Nonaka, Kiku; Akaike, Norio

    2011-01-15

    We investigated the functional roles of presynaptic GABA(A) receptors on excitatory nerve terminals in contributing to spontaneous and action potential-evoked glutamatergic transmission to rat hippocampal CA3 pyramidal neurons. Single CA3 neurons were mechanically isolated with adherent nerve terminals, namely the 'synaptic bouton preparation', and spontaneous glutamatergic excitatory synaptic potentials (sEPSCs) and EPSCs evoked by focal electrical stimuli of a single presynaptic glutamatergic boutons (eEPSCs) were recorded using conventional whole-cell patch recordings. Selective activation of presynaptic GABA(A) receptors on these excitatory nerve terminals by muscimol, markedly facilitated sEPSCs frequency but inhibited eEPSC amplitude. The facilitation of sEPSC frequency was completely occluded by GABA(A) receptor-Cl⁻ channel blockers bicuculline or penicillin (PN). PN itself concentration-dependently inhibited the GABA(A) receptor response induced by bath application of muscimol, but had no effect on the glutamate receptor response. In addition, pretreatment with a blocker of the Na(+), K(+), 2Cl⁻ co-transporter type 1 (NKCC-1), bumetanide, prevented the muscimol-induced inhibition of eEPSCs. The results indicate that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic excitatory nerve terminals and thereby differentially modulates sEPSCs and eEPSCs.

  10. Effects of 3 weeks GMP oral administration on glutamatergic parameters in mice neocortex.

    Science.gov (United States)

    Ganzella, Marcelo; Moreira, Julia Dubois; Almeida, Roberto Farina; Böhmer, Ana Elisa; Saute, Jonas Alex Morales; Holmseth, Silvia; Souza, Diogo Onofre

    2012-03-01

    Overstimulation of the glutamatergic system (excitotoxicity) is involved in various acute and chronic brain diseases. Several studies support the hypothesis that guanosine-5'-monophosphate (GMP) can modulate glutamatergic neurotransmission. The aim of this study was to evaluate the effects of chronically administered GMP on brain cortical glutamatergic parameters in mice. Additionally, we investigated the neuroprotective potential of the GMP treatment submitting cortical brain slices to oxygen and glucose deprivation (OGD). Moreover, measurements of the cerebrospinal fluid (CSF) purine levels were performed after the treatment. Mice received an oral administration of saline or GMP during 3 weeks. GMP significantly decreases the cortical brain glutamate binding and uptake. Accordingly, GMP reduced the immunocontent of the glutamate receptors subunits, NR2A/B and GluR1 (NMDA and AMPA receptors, respectively) and glutamate transporters EAAC1 and GLT1. GMP treatment significantly reduced the immunocontent of PSD-95 while did not affect the content of Snap 25, GLAST and GFAP. Moreover, GMP treatment increased the resistance of neocortex to OGD insult. The chronic GMP administration increased the CSF levels of GMP and its metabolites. Altogether, these findings suggest a potential modulatory role of GMP on neocortex glutamatergic system by promoting functional and plastic changes associated to more resistance of mice neocortex against an in vitro excitotoxicity event.

  11. Kölliker-Fuse GABAergic and glutamatergic neurons project to distinct targets.

    Science.gov (United States)

    Geerling, Joel C; Yokota, Shigefumi; Rukhadze, Irma; Roe, Dan; Chamberlin, Nancy L

    2017-06-01

    The Kölliker-Fuse nucleus (KF) is known primarily for its respiratory function as the "pneumotaxic center" or "pontine respiratory group." Considered part of the parabrachial (PB) complex, KF contains glutamatergic neurons that project to respiratory-related targets in the medulla and spinal cord (Yokota, Oka, Tsumori, Nakamura, & Yasui, 2007). Here we describe an unexpected population of neurons in the caudal KF and adjacent lateral crescent subnucleus (PBlc), which are γ-aminobutyric acid (GABA)ergic and have an entirely different pattern of projections than glutamatergic KF neurons. First, immunofluorescence, in situ hybridization, and Cre-reporter labeling revealed that many of these GABAergic neurons express FoxP2 in both rats and mice. Next, using Cre-dependent axonal tracing in Vgat-IRES-Cre and Vglut2-IRES-Cre mice, we identified different projection patterns from GABAergic and glutamatergic neurons in this region. GABAergic neurons in KF and PBlc project heavily and almost exclusively to trigeminal sensory nuclei, with minimal projections to cardiorespiratory nuclei in the brainstem, and none to the spinal cord. In contrast, glutamatergic KF neurons project heavily to the autonomic, respiratory, and motor regions of the medulla and spinal cord previously identified as efferent targets mediating KF cardiorespiratory effects. These findings identify a novel, GABAergic subpopulation of KF/PB neurons with a distinct efferent projection pattern targeting the brainstem trigeminal sensory system. Rather than regulating breathing, we propose that these neurons influence vibrissal sensorimotor function. © 2017 Wiley Periodicals, Inc.

  12. A critical role for p38MAPK signalling pathway during reprogramming of human fibroblasts to iPSCs

    Science.gov (United States)

    Neganova, Irina; Chichagova, Valeria; Armstrong, Lyle; Lako, Majlinda

    2017-01-01

    Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) holds enormous promise for regenerative medicine. Reprogramming is a stepwise process with well-defined stages of initiation, maturation and stabilisation which are critically dependent on interactions between key pluripotency transcription factors, epigenetic regulators and signalling pathways. In this manuscript we have investigated the role of p38 MAPK signalling pathway and have shown a subpopulation- and phase-specific pattern of activation occurring during the initiation and maturation stage of reprogramming in partially and fully reprogrammed cells respectively. Downregulation of p38 MAPK activity via RNA interference or small molecule inhibitor led to cell accumulation in G1 phase of the cell cycle and reduced expression of cell cycle regulators during the initiation stage of reprogramming. This was associated with a significant downregulation of key pluripotency marker expression, disruption of mesenchymal to epithelial transition (MET), increased expression of differentiation markers and presence of partially reprogrammed cells which retained a typical gene expression profile of mesendodermal cells and were unable to progress to fully reprogrammed phenotype. Together our data indicate an important role for p38 MAPK activity in proliferation, MET progression and establishment of pluripotent phenotype, which are necessary steps for the development of human iPSCs. PMID:28155868

  13. Potential of Induced Pluripotent Stem Cells (iPSCs) for Treating Age-Related Macular Degeneration (AMD).

    Science.gov (United States)

    Fields, Mark; Cai, Hui; Gong, Jie; Del Priore, Lucian

    2016-12-08

    The field of stem cell biology has rapidly evolved in the last few decades. In the area of regenerative medicine, clinical applications using stem cells hold the potential to be a powerful tool in the treatment of a wide variety of diseases, in particular, disorders of the eye. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are promising technologies that can potentially provide an unlimited source of cells for cell replacement therapy in the treatment of retinal degenerative disorders such as age-related macular degeneration (AMD), Stargardt disease, and other disorders. ESCs and iPSCs have been used to generate retinal pigment epithelium (RPE) cells and their functional behavior has been tested in vitro and in vivo in animal models. Additionally, iPSC-derived RPE cells provide an autologous source of cells for therapeutic use, as well as allow for novel approaches in disease modeling and drug development platforms. Clinical trials are currently testing the safety and efficacy of these cells in patients with AMD. In this review, the current status of iPSC disease modeling of AMD is discussed, as well as the challenges and potential of this technology as a viable option for cell replacement therapy in retinal degeneration.

  14. Highly efficient gene targeting of expressed and silent genes in human ESCs and iPSCs using zinc finger nucleases

    Science.gov (United States)

    Hockemeyer, Dirk; Soldner, Frank; Beard, Caroline; Gao, Qing; Mitalipova, Maisam; DeKelver, Russell C.; Katibah, George E.; Amora, Ranier; Boydston, Elizabeth A.; Zeitler, Bryan; Meng, Xiangdong; Miller, Jeffrey C.; Zhang, Lei; Rebar, Edward J.; Gregory, Philip D.; Urnov, Fyodor D.; Jaenisch, Rudolf

    2014-01-01

    Human embryonic stem cells and induced pluripotent stem cells (hESCs and hiPSCs) are powerful tools for biomedical research. Realizing the full potential of these cells requires efficient genetic modification. However, techniques to generate cell type specific lineage reporters as well as reliable tools to disrupt, repair or overexpress genes by gene targeting are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc finger nuclease (ZFN) mediated genome editing. First, using ZFNs specific for the OCT4 locus we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Secondly, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. PMID:19680244

  15. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.

    Science.gov (United States)

    Hockemeyer, Dirk; Soldner, Frank; Beard, Caroline; Gao, Qing; Mitalipova, Maisam; DeKelver, Russell C; Katibah, George E; Amora, Ranier; Boydston, Elizabeth A; Zeitler, Bryan; Meng, Xiangdong; Miller, Jeffrey C; Zhang, Lei; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Jaenisch, Rudolf

    2009-09-01

    Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.

  16. Hipótese glutamatérgica da esquizofrenia Glutamatergic hypothesis of schizophrenia

    Directory of Open Access Journals (Sweden)

    Rodrigo A Bressan

    2003-09-01

    Full Text Available A esquizofrenia é um transtorno psiquiátrico devastador cuja fisiopatologia ainda está para ser esclarecida. Apesar de uma disfunção dopaminérgica estar bem estabelecida na esquizofrenia, há uma série de evidências sugerindo o envolvimento do sistema glutamatérgico na fisiopatologia do transtorno. Este artigo faz uma breve revisão de alguns aspectos básicos do funcionamento dos receptores glutamatérgicos com ênfase nos receptores N-metil-D-aspartato (NMDA. Apresenta evidências científicas sugerindo uma disfunção do sistema glutamatérgico na esquizofrenia (hipofunção de receptores NMDA. E discute as interações entre os sistemas dopaminérgico e glutamatérgico; mais especificamente como os estados hiperdopaminérgicos encontrados na esquizofrenia podem estar associados a uma alteração glutamatérgica.Schizophrenia is a devastating psychiatric disorder whose pathophysiology has not been fully clarified yet. Although dopamine dysfunction in schizophrenia is unequivocal, there are many evidences suggesting the involvement of the glutamatergic system. This paper briefly describes some basic knowledge regarding the functioning of the glutamatergic receptors with emphasis on the N-methyl-D-aspartate (NMDA receptors. Presents evidence for glutamatergic dysfunction in schizophrenia, more specifically NMDA receptor hypofunction. Finaly the paper discusses the interaction between the dopaminergic and the glutamatergic systems; in special how hyperdopaminergic state found in schizophrenia can be associated to glutamatergic dysfunctions.

  17. Multiple roles for mammalian target of rapamycin signaling in both glutamatergic and GABAergic synaptic transmission.

    Science.gov (United States)

    Weston, Matthew C; Chen, Hongmei; Swann, John W

    2012-08-15

    The mammalian target of rapamycin (mTOR) signaling pathway in neurons integrates a variety of extracellular signals to produce appropriate translational responses. mTOR signaling is hyperactive in neurological syndromes in both humans and mouse models that are characterized by epilepsy, autism, and cognitive disturbances. In addition, rapamycin, a clinically important immunosuppressant, is a specific and potent inhibitor of mTOR signaling. While mTOR is known to regulate growth and synaptic plasticity of glutamatergic neurons, its effects on basic parameters of synaptic transmission are less well studied, and its role in regulating GABAergic transmission is unexplored. We therefore performed an electrophysiological and morphological comparison of glutamatergic and GABAergic neurons in which mTOR signaling was either increased by loss of the repressor Pten or decreased by treatment with rapamycin. We found that hyperactive mTOR signaling increased evoked synaptic responses in both glutamatergic and GABAergic neurons by ∼50%, due to an increase in the number of synaptic vesicles available for release, the number of synapses formed, and the miniature event size. Prolonged (72 h) rapamycin treatment prevented these abnormalities and also decreased synaptic transmission in wild-type glutamatergic, but not GABAergic, neurons. Further analyses suggested that hyperactivation of the mTOR pathway also impairs presynaptic function, possibly by interfering with vesicle fusion. Despite this presynaptic impairment, the net effect of Pten loss is enhanced synaptic transmission in both GABAergic and glutamatergic neurons, which has numerous implications, depending on where in the brain mutations of an mTOR suppressor gene occur.

  18. Targeting of the human F8 at the multicopy rDNA locus in Hemophilia A patient-derived iPSCs using TALENickases.

    Science.gov (United States)

    Pang, Jialun; Wu, Yong; Li, Zhuo; Hu, Zhiqing; Wang, Xiaolin; Hu, Xuyun; Wang, Xiaoyan; Liu, Xionghao; Zhou, Miaojin; Liu, Bo; Wang, Yanchi; Feng, Mai; Liang, Desheng

    2016-03-25

    Hemophilia A (HA) is a monogenic disease due to lack of the clotting factor VIII (FVIII). This deficiency may lead to spontaneous joint hemorrhages or life-threatening bleeding but there is no cure for HA until very recently. In this study, we derived induced pluripotent stem cells (iPSCs) from patients with severe HA and used transcription activator-like effector nickases (TALENickases) to target the factor VIII gene (F8) at the multicopy ribosomal DNA (rDNA) locus in HA-iPSCs, aiming to rescue the shortage of FVIII protein. The results revealed that more than one copy of the exogenous F8 could be integrated into the rDNA locus. Importantly, we detected exogenous F8 mRNA and FVIII protein in targeted HA-iPSCs. After they were differentiated into endothelial cells (ECs), the exogenous FVIII protein was still detectable. Thus, it is showed that the multicopy rDNA locus could be utilized as an effective target site in patient-derived iPSCs for gene therapy. This strategy provides a novel iPSCs-based therapeutic option for HA and other monogenic diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Innervation by a GABAergic neuron depresses spontaneous release in glutamatergic neurons and unveils the clamping phenotype of synaptotagmin-1.

    Science.gov (United States)

    Wierda, Keimpe D B; Sørensen, Jakob B

    2014-02-01

    The role of spontaneously occurring release events in glutamatergic and GABAergic neurons and their regulation is intensely debated. To study the interdependence of glutamatergic and GABAergic spontaneous release, we compared reciprocally connected "mixed" glutamatergic/GABAergic neuronal pairs from mice cultured on astrocyte islands with "homotypic" glutamatergic or GABAergic pairs and autaptic neurons. We measured mEPSC and mIPSC frequencies simultaneously from both neurons. Neuronal pairs formed both interneuronal synaptic and autaptic connections indiscriminately. We find that whereas mEPSC and mIPSC frequencies did not deviate between autaptic and synaptic connections, the frequency of mEPSCs in mixed pairs was strongly depressed compared with either autaptic neurons or glutamatergic pairs. Simultaneous imaging of synapses, or comparison to evoked release amplitudes, showed that this decrease was not caused by fewer active synapses. The mEPSC frequency was negatively correlated with the mIPSC frequency, indicating interdependence. Moreover, the reduction in mEPSC frequency was abolished when established pairs were exposed to bicuculline for 3 d, but not by long-term incubation with tetrodotoxin, indicating that spontaneous GABA release downregulates mEPSC frequency. Further investigations showed that knockout of synaptotagmin-1 did not affect mEPSC frequencies in either glutamatergic autaptic neurons or in glutamatergic pairs. However, in mixed glutamatergic/GABAergic pairs, mEPSC frequencies were increased by a factor of four in the synaptotagmin-1-null neurons, which is in line with data obtained from mixed cultures. The effect persisted after incubation with BAPTA-AM. We conclude that spontaneous GABA release exerts control over mEPSC release, and GABAergic innervation of glutamatergic neurons unveils the unclamping phenotype of the synaptotagmin-1-null neurons.

  20. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a M146I mutation in PSEN1.

    Science.gov (United States)

    Li, Tong; Pires, Carlota; Nielsen, Troels T; Waldemar, Gunhild; Hjermind, Lena E; Nielsen, Jørgen E; Dinnyes, Andras; Holst, Bjørn; Hyttel, Poul; Freude, Kristine K

    2016-03-01

    Skin fibroblasts were obtained from a 46-year-old symptomatic man carrying a M146I mutation in the presenilin 1 gene (PSEN1), responsible for causing Alzheimer's disease (AD). Induced pluripotent stem cells (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28 and shTP53 genes. M146I-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported M146I-iPSCs line may be a useful resource for in vitro modeling of familial AD. Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

  1. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying an A79V mutation in PSEN1.

    Science.gov (United States)

    Li, Tong; Pires, Carlota; Nielsen, Troels T; Waldemar, Gunhild; Hjermind, Lena E; Nielsen, Jørgen E; Dinnyes, Andras; Hyttel, Poul; Freude, Kristine K

    2016-03-01

    Skin fibroblasts were obtained from a 48-year-old presymptomatic woman carrying a A79V mutation in the presenilin 1 gene (PSEN1), causing Alzheimer's disease (AD). Induced pluripotent stem cell (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28 and shTP53 genes. A79V-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported A79V-iPSCs line may be a useful resource for in vitro modeling of familial AD. Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

  2. Generation of induced pluripotent stem cells (iPSCs from an Alzheimer's disease patient carrying a M146I mutation in PSEN1

    Directory of Open Access Journals (Sweden)

    Tong Li

    2016-03-01

    Full Text Available Skin fibroblasts were obtained from a 46-year-old symptomatic man carrying a M146I mutation in the presenilin 1 gene (PSEN1, responsible for causing Alzheimer's disease (AD. Induced pluripotent stem cells (iPSCs were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28 and shTP53 genes. M146I-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported M146I-iPSCs line may be a useful resource for in vitro modeling of familial AD.

  3. Generation of induced pluripotent stem cells (iPSCs from an Alzheimer's disease patient carrying an A79V mutation in PSEN1

    Directory of Open Access Journals (Sweden)

    Tong Li

    2016-03-01

    Full Text Available Skin fibroblasts were obtained from a 48-year-old presymptomatic woman carrying a A79V mutation in the presenilin 1 gene (PSEN1, causing Alzheimer's disease (AD. Induced pluripotent stem cell (iPSCs were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28 and shTP53 genes. A79V-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported A79V-iPSCs line may be a useful resource for in vitro modeling of familial AD.

  4. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a L150P mutation in PSEN-1

    DEFF Research Database (Denmark)

    Tubsuwan, Alisa; Pires, Carlota; Rasmussen, Mikkel A.

    2016-01-01

    Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts isolated from a 58-year old male with a L150P mutation in the presenilin 1 (PSEN-1) gene, which is responsible for the majority of familial cases of Alzheimer's disease (AD). The iPSC swere established by co-electroporati......Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts isolated from a 58-year old male with a L150P mutation in the presenilin 1 (PSEN-1) gene, which is responsible for the majority of familial cases of Alzheimer's disease (AD). The iPSC swere established by co...

  5. Chromosome therapy. Correction of large chromosomal aberrations by inducing ring chromosomes in induced pluripotent stem cells (iPSCs).

    Science.gov (United States)

    Kim, Taehyun; Bershteyn, Marina; Wynshaw-Boris, Anthony

    2014-01-01

    The fusion of the short (p) and long (q) arms of a chromosome is referred to as a "ring chromosome." Ring chromosome disorders occur in approximately 1 in 50,000-100,000 patients. Ring chromosomes can result in birth defects, mental disabilities, and growth retardation if additional genes are deleted during the formation of the ring. Due to the severity of these large-scale aberrations affecting multiple contiguous genes, no possible therapeutic strategies for ring chromosome disorders have so far been proposed. Our recent study (Bershteyn et al.) using patient-derived fibroblast lines containing ring chromosomes, found that cellular reprogramming of these fibroblasts into induced pluripotent stem cells (iPSCs) resulted in the cell-autonomous correction of the ring chromosomal aberration via compensatory uniparental disomy (UPD). These observations have important implications for studying the mechanism of chromosomal number control and may lead to the development of effective therapies for other, more common, chromosomal aberrations.

  6. Fronto-striatal glutamatergic compounds in compulsive and impulsive syndromes: A review of magnetic resonance spectroscopy studies

    NARCIS (Netherlands)

    Naaijen, J.; Lithgoe, D.J.; Amiri, H.; Buitelaar, J.; Glennon, J.C.

    2015-01-01

    Compulsivity and impulsivity are cross-disorder traits observed in autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD) and obsessive compulsive disorder (OCD). Aberrant fronto-striatal glutamatergic signalling is core to the understanding of compulsive and impulsive diso

  7. Inositol 1,4,5-Triphosphate Drives Glutamatergic and Cholinergic Inhibition Selectively in Spiny Projection Neurons in the Striatum

    OpenAIRE

    Clements, Michael A; Swapna, Immani; Morikawa, Hitoshi

    2013-01-01

    The striatum is critically involved in the selection of appropriate actions in a constantly changing environment. The spiking activity of striatal spiny projection neurons (SPNs), driven by extrinsic glutamatergic inputs, is shaped by local GABAergic and cholinergic networks. For example, it is well established that different types of GABAergic interneurons, activated by extrinsic glutamatergic and local cholinergic inputs, mediate powerful feedforward inhibition of SPN activity. In this stud...

  8. Prenatal betamethasone does not affect glutamatergic or GABAergic neurogenesis in preterm newborns.

    Science.gov (United States)

    Vose, L R; Vinukonda, G; Diamond, D; Korumilli, R; Hu, F; Zia, M T K; Hevner, R; Ballabh, P

    2014-06-13

    Prenatal glucocorticoids (GCs) are routinely used for pregnant women in preterm labor to prevent respiratory distress syndrome and intraventricular hemorrhage in premature infants. However, the effect of antenatal GCs on neurogenesis in preterm neonates remains elusive. Herein, we hypothesized that prenatal GCs might suppress both glutamatergic and GABAergic neurogenesis in preterm rabbits and that this treatment would induce distinct changes in the expression of transcription factors regulating these developmental events. To test our hypotheses, we treated pregnant rabbits with betamethasone at E27 and E28, delivered the pups at E29 (term=32d), and assessed neurogenesis at birth and postnatal day 3. We quantified radial glia (Sox2(+)) and intermediate progenitor cells (Tbr2(+)) in the dorsal cortical subventricular zone to assess glutamatergic neuronal progenitors, and counted Nkx2.1(+) and Dlx2(+) cells in the ganglionic eminence to evaluate GABAergic neurogenesis. In addition, we assayed transcription factors regulating neurogenesis. We found that prenatal GCs did not affect the densities of radial glia and intermediate progenitors of glutamatergic or GABAergic neurons. The number of GABA(+) interneurons in the ganglionic eminence was similar between the prenatal GC-treated pups compared to untreated controls. Moreover, the mRNA expression of transcription factors, including Pax6, Ngn1/2, Emx1/2, Insm1, Dlx1, Nkx2.1, and Gsh2, were comparable between the two groups. However, there was a transient elevation in Mash1 protein in betamethasone-treated pups relative to controls at birth. These data suggest that prenatal GC treatment does not significantly impact the balance of glutamatergic and GABAergic neurogenesis in premature infants.

  9. High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Thomas eAndreska

    2014-04-01

    Full Text Available In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic synapses and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM. Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.

  10. Salsolinol facilitates glutamatergic transmission to dopamine neurons in the posterior ventral tegmental area of rats.

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    Guiqin Xie

    Full Text Available Although in vivo evidence indicates that salsolinol, the condensation product of acetaldehyde and dopamine, has properties that may contribute to alcohol abuse, the underlying mechanisms have not been fully elucidated. We have reported previously that salsolinol stimulates dopamine neurons in the posterior ventral tegmental area (p-VTA partly by reducing inhibitory GABAergic transmission, and that ethanol increases glutamatergic transmission to VTA-dopamine neurons via the activation of dopamine D(1 receptors (D(1Rs. In this study, we tested the hypothesis that salsolinol stimulates dopamine neurons involving activation of D(1Rs. By using whole-cell recordings on p-VTA-dopamine neurons in acute brain slices of rats, we found that salsolinol-induced increase in spike frequency of dopamine neurons was substantially attenuated by DL-2-amino-5-phosphono-valeric acid and 6, 7-dinitroquinoxaline-2, 3-dione, the antagonists of glutamatergic N-Methyl-D-aspartic acid and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Moreover, salsolinol increased the amplitude of evoked excitatory postsynaptic currents (EPSCs and the frequency but not the amplitude of spontaneous EPSCs. Additionally, SKF83566, a D(1R antagonist attenuated the salsolinol-induced facilitation of EPSCs and of spontaneous firing of dopamine neurons. Our data reveal that salsolinol enhances glutamatergic transmission onto dopamine neurons via activation of D(1Rs at the glutamatergic afferents in dopamine neurons, which contributes to salsolinol's stimulating effect on p-VTA dopamine neurons. This appears to be a novel mechanism which contributes toward rewarding properties of salsolinol.

  11. Salsolinol facilitates glutamatergic transmission to dopamine neurons in the posterior ventral tegmental area of rats.

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    Xie, Guiqin; Ye, Jiang-Hong

    2012-01-01

    Although in vivo evidence indicates that salsolinol, the condensation product of acetaldehyde and dopamine, has properties that may contribute to alcohol abuse, the underlying mechanisms have not been fully elucidated. We have reported previously that salsolinol stimulates dopamine neurons in the posterior ventral tegmental area (p-VTA) partly by reducing inhibitory GABAergic transmission, and that ethanol increases glutamatergic transmission to VTA-dopamine neurons via the activation of dopamine D(1) receptors (D(1)Rs). In this study, we tested the hypothesis that salsolinol stimulates dopamine neurons involving activation of D(1)Rs. By using whole-cell recordings on p-VTA-dopamine neurons in acute brain slices of rats, we found that salsolinol-induced increase in spike frequency of dopamine neurons was substantially attenuated by DL-2-amino-5-phosphono-valeric acid and 6, 7-dinitroquinoxaline-2, 3-dione, the antagonists of glutamatergic N-Methyl-D-aspartic acid and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Moreover, salsolinol increased the amplitude of evoked excitatory postsynaptic currents (EPSCs) and the frequency but not the amplitude of spontaneous EPSCs. Additionally, SKF83566, a D(1)R antagonist attenuated the salsolinol-induced facilitation of EPSCs and of spontaneous firing of dopamine neurons. Our data reveal that salsolinol enhances glutamatergic transmission onto dopamine neurons via activation of D(1)Rs at the glutamatergic afferents in dopamine neurons, which contributes to salsolinol's stimulating effect on p-VTA dopamine neurons. This appears to be a novel mechanism which contributes toward rewarding properties of salsolinol.

  12. Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors.

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    Martín-García, Elena; Bourgoin, Lucie; Cathala, Adeline; Kasanetz, Fernando; Mondesir, Miguel; Gutiérrez-Rodriguez, Ana; Reguero, Leire; Fiancette, Jean-François; Grandes, Pedro; Spampinato, Umberto; Maldonado, Rafael; Piazza, Pier Vincenzo; Marsicano, Giovanni; Deroche-Gamonet, Véronique

    2016-08-01

    The type 1 cannabinoid receptor (CB1) modulates numerous neurobehavioral processes and is therefore explored as a target for the treatment of several mental and neurological diseases. However, previous studies have investigated CB1 by targeting it globally, regardless of its two main neuronal localizations on glutamatergic and GABAergic neurons. In the context of cocaine addiction this lack of selectivity is critical since glutamatergic and GABAergic neuronal transmission is involved in different aspects of the disease. To determine whether CB1 exerts different control on cocaine seeking according to its two main neuronal localizations, we used mutant mice with deleted CB1 in cortical glutamatergic neurons (Glu-CB1) or in forebrain GABAergic neurons (GABA-CB1). In Glu-CB1, gene deletion concerns the dorsal telencephalon, including neocortex, paleocortex, archicortex, hippocampal formation and the cortical portions of the amygdala. In GABA-CB1, it concerns several cortical and non-cortical areas including the dorsal striatum, nucleus accumbens, thalamic, and hypothalamic nuclei. We tested complementary components of cocaine self-administration, separating the influence of primary and conditioned effects. Mechanisms underlying each phenotype were explored using in vivo microdialysis and ex vivo electrophysiology. We show that CB1 expression in forebrain GABAergic neurons controls mouse sensitivity to cocaine, while CB1 expression in cortical glutamatergic neurons controls associative learning processes. In accordance, in the nucleus accumbens, GABA-CB1 receptors control cocaine-induced dopamine release and Glu-CB1 receptors control AMPAR/NMDAR ratio; a marker of synaptic plasticity. Our findings demonstrate a critical distinction of the altered balance of Glu-CB1 and GABA-CB1 activity that could participate in the vulnerability to cocaine abuse and addiction. Moreover, these novel insights advance our understanding of CB1 neuropathophysiology.

  13. Hypoxia-induced hypothermia mediated by the glutamatergic transmission in the lateral preoptic area.

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    Osaka, T

    2012-12-13

    Hypoxia evokes a regulated decrease in the body core temperature, which response is mediated, at least in part, by noradrenaline (NA) and nitric oxide (NO) in the rostromedial preoptic area (POA) of the hypothalamus. In the accompanying paper, it was shown that glutamatergic activation of the lateral POA also evokes hypothermic responses. Here, I tested the hypothesis that the glutamatergic transmission in the lateral POA is critically involved in the neural mechanism of hypoxia-induced hypothermia. Hypoxic ventilation (10% O(2)-90% N(2), 5 min) as well as a single microinjection of NA (50 pmol) or the NO donor sodium nitroprusside (8.4 nmol) into the rostromedial POA evoked an increase in the tail skin temperature and a decrease in the colonic temperature in urethane-chloralose-anesthetized, neuromuscularly blocked, artificially ventilated rats. All of these responses were greatly attenuated by pretreatment with multiple microinjections of kynurenic acid (10 nmol, four locations), a nonselective glutamate receptor antagonist, but not by those with saline solution, in the bilateral rostral and central parts of the lateral POA. These results suggest that the NA- and NO-sensitive structure in the rostromedial POA activated the glutamatergic transmission in the lateral POA to mediate hypoxia-induced hypothermia.

  14. The role of the glutamatergic system in the patogenesis and treatment of alcohol dependence

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    Karina Łukasik

    2010-10-01

    Full Text Available The lack of satisfactory results of alcohol dependence treatment has necessitated the search for new directions of studies. One of them is connected with glutamatergic transmission. The influence of alcohol on this transmission is very complex and relates to changes including at the molecular level. However, the diversity of glutamatergic receptors creates a new possibility of modulation of its activity. It leads to decrease of alcohol reward abilities, prolongs abstinence time and reduces the incidence of acute alcohol intoxication in alcohol addicts. The use of acamprosate – a glutamatergic transmission modulator drug – and naltrexone (an opioid receptor antagonist improves therapy effectiveness of acamprosate alone. Satisfactory results were achieved in the studies of topiramate – an antagonist of AMPA and KA receptors. Its effectiveness was proved in clinical studies. Topiramate reduced alcohol craving and prolonged abstinence time, which decreased the probability of relapse. There are promising preclinical results of groups I and II metabotropic receptor antagonists. However, further studies are necessary to elucidate precisely their role in alcohol dependence.

  15. Plasticity-Related Gene 1 Affects Mouse Barrel Cortex Function via Strengthening of Glutamatergic Thalamocortical Transmission.

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    Unichenko, Petr; Kirischuk, Sergei; Yang, Jenq-Wei; Baumgart, Jan; Roskoden, Thomas; Schneider, Patrick; Sommer, Angela; Horta, Guilherme; Radyushkin, Konstantin; Nitsch, Robert; Vogt, Johannes; Luhmann, Heiko J

    2016-07-01

    Plasticity-related gene-1 (PRG-1) is a brain-specific protein that modulates glutamatergic synaptic transmission. Here we investigated the functional role of PRG-1 in adolescent and adult mouse barrel cortex both in vitro and in vivo. Compared with wild-type (WT) animals, PRG-1-deficient (KO) mice showed specific behavioral deficits in tests assessing sensorimotor integration and whisker-based sensory discrimination as shown in the beam balance/walking test and sandpaper tactile discrimination test, respectively. At P25-31, spontaneous network activity in the barrel cortex in vivo was higher in KO mice compared with WT littermates, but not at P16-19. At P16-19, sensory evoked cortical responses in vivo elicited by single whisker stimulation were comparable in KO and WT mice. In contrast, at P25-31 evoked responses were smaller in amplitude and longer in duration in WT animals, whereas KO mice revealed no such developmental changes. In thalamocortical slices from KO mice, spontaneous activity was increased already at P16-19, and glutamatergic thalamocortical inputs to Layer 4 spiny stellate neurons were potentiated. We conclude that genetic ablation of PRG-1 modulates already at P16-19 spontaneous and evoked excitability of the barrel cortex, including enhancement of thalamocortical glutamatergic inputs to Layer 4, which distorts sensory processing in adulthood.

  16. M4 mAChR-mediated modulation of glutamatergic transmission at corticostriatal synapses.

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    Pancani, Tristano; Bolarinwa, Caroline; Smith, Yoland; Lindsley, Craig W; Conn, P Jeffrey; Xiang, Zixiu

    2014-04-16

    The striatum is the main input station of the basal ganglia and is extensively involved in the modulation of motivated behavior. The information conveyed to this subcortical structure through glutamatergic projections from the cerebral cortex and thalamus is processed by the activity of several striatal neuromodulatory systems including the cholinergic system. Acetylcholine potently modulates glutamate signaling in the striatum via activation of muscarinic receptors (mAChRs). It is, however, unclear which mAChR subtype is responsible for this modulatory effect. Here, by using electrophysiological, optogenetic, and immunoelectron microscopic approaches in conjunction with a novel, highly selective M4 positive allosteric modulator VU0152100 (ML108) and M4 knockout mice, we show that M4 is a major mAChR subtype mediating the cholinergic inhibition of corticostriatal glutamatergic input on both striatonigral and striatopallidal medium spiny neurons (MSNs). This effect is due to activation of presynaptic M4 receptors, which, in turn, leads to a decrease in glutamate release from corticostriatal terminals. The findings of the present study raise the interesting possibility that M4 mAChR could be a novel therapeutic target for the treatment of neurological and neuropsychiatric disorders involving hyper-glutamatergic transmission at corticostriatal synapses.

  17. Upregulation of glutamatergic transmission in anterior cingulate cortex in the diabetic rats with neuropathic pain.

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    Li, Weifang; Wang, Peng; Li, Hua

    2014-05-07

    Peripheral neuropathic pain is a common complication in the diabetic patients, and the underlying central mechanism remains unclear. Forebrain anterior cingulate cortex (ACC) is critically involved in the supraspinal perception of physical and affective components of noxious stimulus and pain modulation. Excitatory glutamatergic transmission in the ACC extensively contributed to the maintenance of negative affective component of chronic pain. The present study examined the adaptation of glutamatergic transmission in the ACC in rats with diabetic neuropathic pain. Injection with streptozotocin (STZ) induced hyperglycemia, thermal hyperalgesia and mechanical allodynia in the rats. In these rats, significant enhanced basal glutamatergic transmission was observed in the ACC neurons. The increased presynaptic glutamate release and enhanced conductance of postsynaptic glutamate receptors were also observed in the ACC neurons of these modeled rats. Increased phosphorylation of PKMζ, but not the expression of total PKMζ, was also observed in the ACC. Microinjection of PKMζ inhibitor ZIP into ACC attenuated the upregulation of glutamate transmission and painful behaviors in STZ-injected rats. These results revealed a substantial central sensitization in the ACC neurons in the rodents with diabetic neuropathic pain, which may partially underlie the negative affective components of patients with diabetic neuropathic pain.

  18. Effects of Etomidate on GABAergic and Glutamatergic Transmission in Rat Thalamocortical Slices.

    Science.gov (United States)

    Fu, Bao; Wang, Yuan; Yang, Hao; Yu, Tian

    2016-12-01

    Although accumulative evidence indicates that the thalamocortical system is an important target for general anesthetics, the underlying mechanisms of anesthetic action on thalamocortical neurotransmission are not fully understood. The aim of the study is to explore the action of etomidate on glutamatergic and GABAergic transmission in rat thalamocortical slices by using whole cell patch-clamp recording. We found that etomidate mainly prolonged the decay time of spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs), without changing the frequency. Furthermore, etomidate not only prolonged the decay time of miniature inhibitory postsynaptic currents (mIPSCs) but also increased the amplitude. On the other hand, etomidate significantly decreased the frequency of spontaneous glutamatergic excitatory postsynaptic currents (sEPSCs), without altering the amplitude or decay time in the absence of bicuculline. When GABAA receptors were blocked using bicuculline, the effects of etomidate on sEPSCs were mostly eliminated. These results suggest that etomidate enhances GABAergic transmission mainly through postsynaptic mechanism in thalamocortical neuronal network. Etomidate attenuates glutamatergic transmission predominantly through presynaptic action and requires presynaptic GABAA receptors involvement.

  19. Baicalein ameliorated the upregulation of striatal glutamatergic transmission in the mice model of Parkinson's disease.

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    Xue, Xinhong; Liu, Hong; Qi, Lifeng; Li, Xueli; Guo, Cunju; Gong, Dianrong; Qu, Huaiqian

    2014-04-01

    Parkinson's disease (PD) is a common neurodegenerative disorder, which is characterized by a loss of projecting dopaminergic neurons in the substantia nigra and diminished dopamine level in the striatum. Dopaminergic deficit consequently leads to the alterations of striatal basal glutamatergic synaptic transmission and plasticity in the medium spiny neurons. The cytokines and neurotoxins released from the reactive immune cells induced the loss of the projecting dopaminergic neurons in the substantia nigra, which triggering the pathogenesis of PD. The present study investigated the effect of treatment with baicalein (5,6,7-trihydroxyflavone) on the central cytokine synthesis, striatal glutamatergic transmission, and behavioral performance in the rotarod task in the mice injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Treatment with baicalein significantly attenuated the upregulation of striatal basal glutamatergic strength by decreasing the presynaptic glutamate release and recovering the insertion of postsynaptic glutamate receptor subunit GluR1 induced by MPTP. It also significantly improved the behavioral performance in the rotarod task in the mice injected with MPTP. Treatment with baicalein decreased the upregulation of cytokines (tumor necrosis factor-α and interleukin-1β) in the substantia nigra and striatum in the mice injected with MPTP. These results indicated that baicalein might serve as novel approach for the treatment of the patients with PD.

  20. Estrogen protects against the detrimental effects of repeated stress on glutamatergic transmission and cognition.

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    Wei, J; Yuen, E Y; Liu, W; Li, X; Zhong, P; Karatsoreos, I N; McEwen, B S; Yan, Z

    2014-05-01

    Converging evidence suggests that females and males show different responses to stress; however, little is known about the mechanism underlying the sexually dimorphic effects of stress. In this study, we found that young female rats exposed to 1 week of repeated restraint stress show no negative effects on temporal order recognition memory (TORM), a cognitive process controlled by the prefrontal cortex (PFC), which was contrary to the impairment in TORM observed in stressed males. Concomitantly, normal glutamatergic transmission and glutamate receptor surface expression in PFC pyramidal neurons were found in repeatedly stressed females, in contrast to the significant reduction seen in stressed males. The detrimental effects of repeated stress on TORM and glutamate receptors were unmasked in stressed females when estrogen receptors were inhibited or knocked down in PFC, and were prevented in stressed males with the administration of estradiol. Blocking aromatase, the enzyme for the biosynthesis of estrogen, revealed the stress-induced glutamatergic deficits and memory impairment in females, and the level of aromatase was significantly higher in the PFC of females than in males. These results suggest that estrogen protects against the detrimental effects of repeated stress on glutamatergic transmission and PFC-dependent cognition, which may underlie the stress resilience of females.

  1. Apoptosis of glutamatergic neurons fails to trigger a neurogenic response in the adult neocortex.

    Science.gov (United States)

    Diaz, Frank; McKeehan, Nicholas; Kang, Wenfei; Hébert, Jean M

    2013-04-10

    Adult neurogenesis is actively studied in part because of the potential to manipulate endogenous neural stem and progenitor cells for tissue repair. Although constitutive generation of neurons in the adult rodent olfactory bulb and hippocampal dentate gyrus is widely accepted and stroke-induced generation of striatal inhibitory neurons consistently observed, evidence supporting the generation of neurons in the neocortex after neuronal loss remains slim. Nevertheless, a few studies suggested that targeted apoptosis of neocortical glutamatergic neurons could trigger the generation of new ones in the adult brain. In light of such studies, we tested whether apoptosis of glutamatergic cortical neurons using two novel transgenic approaches in mice, an inducible Caspase-8 protein and an inducible diphtheria toxin gene, results in new neurons. After a thorough analysis, no new neurons were detected in the neocortex. Interestingly, an increase in the expression of the neuroblast marker DCX was observed in both models, in some cases in cells with morphologies previously associated with poststroke neuroblasts, but DCX(+) cells coexpressed the oligodendrocyte precursor marker Olig2, suggesting caution when using DCX as a marker for neuroblasts after injury. Given that the adult neocortex lacks an innate potential to regenerate lost glutamatergic neurons, future strategies should concentrate on manipulating the differentiation potential of endogenous or exogenous precursor cells.

  2. Disrupted-in-schizophrenia (DISC1 functions presynaptically at glutamatergic synapses.

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    Brady J Maher

    Full Text Available The pathophysiology of schizophrenia is believed to involve defects in synaptic transmission, and the function of many schizophrenia-associated genes, including DISC1, have been linked to synaptic function at glutamatergic synapses. Here we develop a rodent model via in utero electroporation to assay the presynaptic function of DISC1 at glutamatergic synapses. We used a combination of mosaic transgene expression, RNAi knockdown and optogenetics to restrict both genetic manipulation and synaptic stimulation of glutamatergic neurons presynaptic to other layer 2/3 neocortical pyramidal neurons that were then targeted for whole-cell patch-clamp recording. We show that expression of the DISC1 c-terminal truncation variant that is associated with Schizophrenia alters the frequency of mEPSCs and the kinetics of evoked glutamate release. In addition, we show that expression level of DISC1 is correlated with the probability of glutamate release such that increased DISC1 expression results in paired-pulse depression and RNAi knockdown of DISC1 produces paired-pulse facilitation. Overall, our results support a direct presynaptic function for the schizophrenia-associated gene, DISC1.

  3. Morphine treatment enhances glutamatergic input onto neurons of the nucleus accumbens via both disinhibitory and stimulating effect.

    Science.gov (United States)

    Yuan, Kejing; Sheng, Huan; Song, Jiaojiao; Yang, Li; Cui, Dongyang; Ma, Qianqian; Zhang, Wen; Lai, Bin; Chen, Ming; Zheng, Ping

    2016-08-22

    Drug addiction is a chronic brain disorder characterized by the compulsive repeated use of drugs. The reinforcing effect of repeated use of drugs on reward plays an important role in morphine-induced addictive behaviors. The nucleus accumbens (NAc) is an important site where morphine treatment produces its reinforcing effect on reward. However, how morphine treatment produces its reinforcing effect on reward in the NAc remains to be clarified. In the present study, we studied the influence of morphine treatment on the effects of DA and observed whether morphine treatment could directly change glutamatergic synaptic transmission in the NAc. We also explored the functional significance of morphine-induced potentiation of glutamatergic synaptic transmission in the NAc at behavioral level. Our results show that (1) morphine treatment removes the inhibitory effect of DA on glutamatergic input onto NAc neurons; (2) morphine treatment potentiates glutamatergic input onto NAc neurons, especially the one from the basolateral amygdala (BLA) to the NAc; (3) blockade of glutamatergic synaptic transmission in the NAc or ablation of projection neurons from BLA to NAc significantly decreases morphine treatment-induced increase in locomotor activity. These results suggest that morphine treatment enhances glutamatergic input onto neurons of the NAc via both disinhibitory and stimulating effect and therefore increases locomotor activity.

  4. Synaptic plasticity in glutamatergic and GABAergic neurotransmission following chronic memantine treatment in an in vitro model of limbic epileptogenesis.

    Science.gov (United States)

    He, Shuijin; Bausch, Suzanne B

    2014-02-01

    Chronic N-methyl-D-aspartate receptor (NMDAR) blockade with high affinity competitive and uncompetitive antagonists can lead to seizure exacerbation, presumably due to an imbalance in glutamatergic and GABAergic transmission. Acute administration of the moderate affinity NMDAR antagonist memantine in vivo has been associated with pro- and anticonvulsive properties. Chronic treatment with memantine can exacerbate seizures. Therefore, we hypothesized that chronic memantine treatment would increase glutamatergic and decrease GABAergic transmission, similar to high affinity competitive and uncompetitive antagonists. To test this hypothesis, organotypic hippocampal slice culture were treated for 17-21 days with memantine and then subjected to electrophysiological recordings. Whole-cell recordings from dentate granule cells revealed that chronic memantine treatment slightly, but significantly increased sEPSC frequency, mEPSC amplitude and mEPSC charge transfer, consistent with minimally increased glutamatergic transmission. Chronic memantine treatment also increased both sIPSC and mIPSC frequency and amplitude, suggestive of increased GABAergic transmission. Results suggest that a simple imbalance between glutamatergic and GABAergic neurotransmission may not underlie memantine's ictogenic properties. That said, glutamatergic and GABAergic transmission were assayed independently of one another in the current study. More complex interactions between glutamatergic and GABAergic transmission may prevail under conditions of intact circuitry.

  5. Enhanced ability of TRPV1 channels in regulating glutamatergic transmission after repeated morphine exposure in the nucleus accumbens of rat.

    Science.gov (United States)

    Zhang, Haitao; Jia, Dong; Wang, Yuan; Qu, Liang; Wang, Xuelian; Song, Jian; Heng, Lijun; Gao, Guodong

    2017-04-01

    Glutamatergic projections to nucleus accumbens (NAc) drive drug-seeking behaviors during opioids withdrawal. Modulating glutamatergic neurotransmission provides a novel pharmacotherapeutic avenue for treatment of opioids dependence. Great deals of researches have verified that transient receptor potential vanilloid 1 (TRPV1) channels alters synaptic transmitter release and regulate neural plasticity. In the present study, whole-cell patch clamp recordings were adopted to examine the activity of TRPV1 Channels in regulating glutamate-mediated excitatory postsynaptic currents (EPSCs) in NAc of rat during morphine withdrawal for 3days and 3weeks. The data showed that the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and the amplitudes of evoked excitatory postsynaptic currents (eEPSCs) were increased during morphine withdrawal after applied with capsaicin (TRPV1 agonist). Capsaicin decreased the paired pulse ratio (PPR) and increased sEPSCs frequency but not their amplitudes suggesting a presynaptic locus of action during morphine withdrawal. All these effects were fully blocked by the TRPV1 antagonist Capsazepine. Additionally, In the presence of AM251 (CB1 receptor antagonist), depolarization-induced release of endogenous cannabinoids activated TRPV1 channels to enhance glutamatergic neurotransmission during morphine withdrawal. The functional enhancement of TRPV1 Channels in facilitating glutamatergic transmission was not recorded in dorsal striatum. Our findings demonstrate the ability of TRPV1 in regulating excitatory glutamatergic transmission is enhanced during morphine withdrawal in NAc, which would deepen our understanding of glutamatergic modulation during opioids withdrawal.

  6. Differentiation of control and ALS mutant human iPSCs into functional skeletal muscle cells, a tool for the study of neuromuscolar diseases

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    Jessica Lenzi

    2016-07-01

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a severe and fatal neurodegenerative disease characterized by progressive loss of motoneurons, muscle atrophy and paralysis. Recent evidence suggests that ALS should be considered as a multi-systemic disease, in which several cell types contribute to motoneuron degeneration. In this view, mutations in ALS linked genes in other neural and non-neural cell types may exert non-cell autonomous effects on motoneuron survival and function. Induced Pluripotent Stem Cells (iPSCs have been recently derived from several patients with ALS mutations and it has been shown that they can generate motoneurons in vitro, providing a valuable tool to study ALS. However, the potential of iPSCs could be further valorized by generating other cell types that may be relevant to the pathology. In this paper, by taking advantage of a novel inducible system for MyoD expression, we show that both control iPSCs and iPSCs carrying mutations in ALS genes can generate skeletal muscle cells. We provide evidence that both control and mutant iPSC-derived myotubes are functionally active. This in vitro system will be instrumental to dissect the molecular and cellular pathways impairing the complex motoneuron microenvironment in ALS.

  7. Microarray-Based Comparisons of Ion Channel Expression Patterns: Human Keratinocytes to Reprogrammed hiPSCs to Differentiated Neuronal and Cardiac Progeny

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    Leonhard Linta

    2013-01-01

    Full Text Available Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of, for example, ion selectivity, gating mechanism, composition, or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs and their somatic cell source, keratinocytes from plucked human hair. This comparison revealed that 26% of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6% were downregulated. Additionally, iPSCs express a much higher number of ion channels compared to keratinocytes. Further, to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny, namely, neurons and cardiomyocytes derived from iPS cells. To conclude, hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms.

  8. Origin-Dependent Neural Cell Identities in Differentiated Human iPSCs In Vitro and after Transplantation into the Mouse Brain

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    Gunnar Hargus

    2014-09-01

    Full Text Available The differentiation capability of induced pluripotent stem cells (iPSCs toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs, dermal fibroblasts, or cord blood CD34+ hematopoietic progenitor cells. Neural progenitor cells (NPCs and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.

  9. Induced pluripotent stem cells (iPSCs) derived from a patient with frontotemporal dementia caused by a R406W mutation in microtubule-associated protein tau (MAPT)

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel A.; Hjermind, Lena E.; Hasholt, Lis F.

    2016-01-01

    Skin fibroblasts were obtained from a 59-year-old woman diagnosed with frontotemporal dementia. The disease is caused by a R406W mutation in microtubule-associated protein tau (MAPT). Induced pluripotent stem cells (iPSCs) were established by electroporation with episomal plasmids containing hOCT4...

  10. Induced pluripotent stem cells (iPSCs) derived from a patient with frontotemporal dementia caused by a P301L mutation in microtubule-associated protein tau (MAPT)

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel A.; Hjermind, Lena Elisabeth; Hasholt, Lis Frydenreich

    2016-01-01

    Skin fibroblasts were obtained froma 57-year-old woman diagnosed with frontotemporal dementia. The diseaseis caused by a P301L mutation in microtubule-associated protein tau (MAPT). Induced pluripotent stem cells (iPSCs) were established by electroporation with episomal plasmids containing hOCT4, h...

  11. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying an A79V mutation in PSEN1

    DEFF Research Database (Denmark)

    Li, Tong; Pires, Carlota; Nielsen, Troels T.

    2016-01-01

    Skin fibroblasts were obtained from a 48-year-old presymptomatic woman carrying a A79V mutation in the presenilin 1 gene (PSEN1), causing Alzheimer's disease (AD). Induced pluripotent stem cell (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28...

  12. Reprogramming of endometrial adult stromal cells in the presence of a ROCK inhibitor, thiazovivin, could obtain more efficient iPSCs.

    Science.gov (United States)

    Mohseni, Rashin; Shoae-Hassani, Alireza; Verdi, Javad

    2015-05-01

    Today, there is a need for a platform to efficiently generate and maintain a feeder free culture of pluripotent stem cells by small molecules or pharmacological agents. Induced pluripotent stem cell (iPSC) is considered a promising resource for restorative cell therapy in clinical areas. While fully reprogrammed iPSCs are similar to embryonic stem cells, iPSCs could be derived from the patient's own cells (autologous), which avoids the immune rejection activities. Recent advances have demonstrated that iPSCs could be generated from human fibroblasts using only four transcription factors: OCT4, SOX2, CMYC, and KLF4. However, the limitations of reprogramming technologies include low efficiency, slow kinetics, transgene integration and residual expression. Surprisingly, adult stem cells from human endometrium (endometrial stem cells; EnSCs) express OCT4 and KLF4 pluripotency factors. On the other hand, small molecule inhibitors of specific signaling pathways such as thiazovivin have been used in various aspects of iPSC generation and maintenance. Thiazovivin is a selective small molecule that directly targets Rho-associated kinase (ROCK) and increases expression of pluripotency factors. The process using thiazovivin could be easier, faster and more cost effective than transgene integration into somatic cells. So reprogramming of OCT4 and KLF4 expressing EnSCs by a ROCK inhibitor, thiazovivin, could result in producing more efficient iPSCs compared with fibroblasts or conventional somatic cells without integration any transgene and retroviral vector. © 2015 International Federation for Cell Biology.

  13. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a L150P mutation in PSEN-1

    DEFF Research Database (Denmark)

    Tubsuwan, Alisa; Pires, Carlota; Rasmussen, Mikkel A.;

    2016-01-01

    Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts isolated from a 58-year old male with a L150P mutation in the presenilin 1 (PSEN-1) gene, which is responsible for the majority of familial cases of Alzheimer's disease (AD). The iPSC swere established by co-electroporation...

  14. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a M146I mutation in PSEN1

    DEFF Research Database (Denmark)

    Li, Tong; Pires, Carlota; Nielsen, Troels Tolstrup

    2016-01-01

    Skin fibroblasts were obtained from a 46-year-old symptomatic man carrying a M146I mutation in the presenilin 1 gene (PSEN1), responsible for causing Alzheimer's disease (AD). Induced pluripotent stem cells (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, ...

  15. An Exploratory Study of Spectroscopic Glutamatergic Correlates of Cortical Excitability in Depressed Adolescents

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    Charles P. Lewis

    2016-11-01

    Full Text Available Introduction: Transcranial magnetic stimulation (TMS research has suggested dysfunction in cortical glutamatergic systems in depression, while proton magnetic resonance spectroscopy (1H-MRS studies have demonstrated deficits in concentrations of glutamatergic metabolites in depressed individuals in several cortical regions, including the anterior cingulate cortex (ACC. However, few studies have combined TMS and MRS methods to examine relationships between glutamatergic neurochemistry and excitatory and inhibitory neural functions, and none have utilized TMS-MRS methodology in clinical populations or in youth. This exploratory study aimed to examine relationships between TMS measures of cortical excitability and inhibition and concentrations of glutamatergic metabolites as measured by 1H-MRS in depressed adolescents. Methods: Twenty-four children and adolescents (aged 11-18 years with depressive symptoms underwent TMS testing, which included measures of the resting motor threshold (RMT, cortical silent period (CSP, short-interval intracortical inhibition (SICI, and intracortical facilitation (ICF. Fourteen participants from the same sample also completed 1H-MRS in a 3 T MRI scanner after TMS testing. Glutamate + glutamine (Glx concentrations were measured in medial ACC and left primary motor cortex voxels with a TE-optimized PRESS sequence. Metabolite concentrations were corrected for cerebrospinal fluid after tissue segmentation. Pearson product-moment and Spearman rank-order correlations were calculated to assess relationships between TMS measures and Glx. Results: In the left primary motor cortex voxel, Glx had a significant positive correlation with the RMT. In the medial ACC voxel, Glx had significant positive correlations with ICF at the 10-ms and 20-ms ISIs.Conclusions: These preliminary data implicate glutamate in cortical excitatory processes measured by TMS. Limitations included small sample size, lack of healthy control comparators

  16. Glutamatergic transmission in the nucleus tractus solitarii: from server to peripherals in the cardiovascular information superhighway

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    Talman W.T.

    1997-01-01

    Full Text Available Afferent nerves carrying signals from mechanoreceptors in the aortic arch and carotid sinus terminate predominantly in the nucleus tractus solitarii (NTS. Signal transduction and neurotransmission in the NTS are critical for central cardiovascular reflex control, but little was known about either until the late 1970's. None of the numerous neuroactive chemicals found in the NTS had met strict criteria as a neurotransmitter in the baroreflex arc until data suggested that the excitatory amino acid L-glutamate (GLU might be released from baroreceptor afferent terminals in the NTS. In anesthetized animals microinjection into the NTS of GLU, which can be demonstrated in terminals in the NTS, produces cardiovascular responses like those seen with activation of the baroreceptor reflex. Similar responses occur in awake animals if the chemoreceptor reflex is eliminated; otherwise, in conscious animals responses mimic those of chemoreceptor reflex activation. GLU is released in the NTS upon selective activation of the baroreceptor, and possibly the chemoreceptor, reflex. Responses to selective agonists as well as baroreflex responses are eliminated by GLU antagonists microinjected into the NTS. Non-NMDA (N-methyl-D-aspartic acid receptors seem to predominate at primary baroreceptor synapses in the NTS while NMDA receptors may be involved at later synapses. Although inhibition of soluble guanylate cyclase attenuates responses to ionotropic glutamate agonists in the NTS, nitric oxide does not seem to play a role in glutamate transmission in the NTS. GLU may also participate in transmission at cardiovascular neurons beyond the NTS. For example, a role has been suggested for GLU in the ventrolateral medulla and spinal cord. Work continues concerning GLU signal transduction and mechanisms that modulate that transduction both at the NTS and at other cardiovascular nuclei

  17. Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway.

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    Szlachcic, Wojciech J; Switonski, Pawel M; Krzyzosiak, Wlodzimierz J; Figlerowicz, Marek; Figiel, Maciej

    2015-09-01

    Huntington disease (HD) is a brain disorder characterized by the late onset of motor and cognitive symptoms, even though the neurons in the brain begin to suffer dysfunction and degeneration long before symptoms appear. There is currently no cure. Several molecular and developmental effects of HD have been identified using neural stem cells (NSCs) and differentiated cells, such as neurons and astrocytes. Still, little is known regarding the molecular pathogenesis of HD in pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Therefore, we examined putative signaling pathways and processes involved in HD pathogenesis in pluripotent cells. We tested naïve mouse HD YAC128 iPSCs and two types of human HD iPSC that were generated from HD and juvenile-HD patients. Surprisingly, we found that a number of changes affecting cellular processes in HD were also present in undifferentiated pluripotent HD iPSCs, including the dysregulation of the MAPK and Wnt signaling pathways and the dysregulation of the expression of genes related to oxidative stress, such as Sod1. Interestingly, a common protein interactor of the huntingtin protein and the proteins in the above pathways is p53, and the expression of p53 was dysregulated in HD YAC128 iPSCs and human HD iPSCs. In summary, our findings demonstrate that multiple molecular pathways that are characteristically dysregulated in HD are already altered in undifferentiated pluripotent cells and that the pathogenesis of HD might begin during the early stages of life.

  18. Integration-free reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) without viral vectors, recombinant DNA, and genetic modification.

    Science.gov (United States)

    Heng, Boon Chin; Fussenegger, Martin

    2014-01-01

    Stem cells are envisaged to be integral components of multicellular systems engineered for therapeutic applications. The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) via recombinant expression of a limited number of transcription factors, which was first achieved by Yamanaka and colleagues in 2007, heralded a major breakthrough in the stem cell field. Since then, there has been rapid progress in the field of iPSC generation, including the identification of various small molecules that can enhance reprogramming efficiency and reduce the number of different transcription factors required for reprogramming. Nevertheless, the major obstacles facing clinical applications of iPSCs are safety concerns associated with the use of viral vectors and recombinant DNA for expressing the appropriate transcription factors during reprogramming. In particular, permanent genetic modifications to newly reprogrammed iPSCs have to be avoided in order to meet stringent safety requirements for clinical therapy. These safety challenges can be overcome by new technology platforms that enable cellular reprogramming to iPSCs without the need to utilize either recombinant DNA or viral vectors. The use of recombinant cell-penetrating peptides and direct transfection of synthetic mRNA encoding appropriate transcription factors have both been shown to successfully reprogram somatic cells to iPSCs. It has also been shown more recently that the direct transfection of certain miRNA species can reprogram somatic cells to pluripotency without the need for any of the transcription factors commonly utilized for iPSC generation. This chapter describes protocols for iPSC generation with these new techniques, which would obviate the use of recombinant DNA and viral vectors in cellular reprogramming, thus avoiding permanent genetic modification to the reprogrammed cells.

  19. Reprogramming of HUVECs into Induced Pluripotent Stem Cells (HiPSCs), Generation and Characterization of HiPSC-Derived Neurons and Astrocytes

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    Boakye, Paul A.; Baker, Glen; Smith, Peter A.; Murray, Allan G.; Giuliani, Fabrizio; Jahroudi, Nadia

    2015-01-01

    Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for “patient-matched” cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their

  20. VAMP-2, SNAP-25A/B and syntaxin-1 in glutamatergic and GABAergic synapses of the rat cerebellar cortex

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    Benagiano Vincenzo

    2011-11-01

    Full Text Available Abstract Background The aim of this study was to assess the distribution of key SNARE proteins in glutamatergic and GABAergic synapses of the adult rat cerebellar cortex using light microscopy immunohistochemical techniques. Analysis was made of co-localizations of vGluT-1 and vGluT-2, vesicular transporters of glutamate and markers of glutamatergic synapses, or GAD, the GABA synthetic enzyme and marker of GABAergic synapses, with VAMP-2, SNAP-25A/B and syntaxin-1. Results The examined SNARE proteins were found to be diffusely expressed in glutamatergic synapses, whereas they were rarely observed in GABAergic synapses. However, among glutamatergic synapses, subpopulations which did not contain VAMP-2, SNAP-25A/B and syntaxin-1 were detected. They included virtually all the synapses established by terminals of climbing fibres (immunoreactive for vGluT-2 and some synapses established by terminals of parallel and mossy fibres (immunoreactive for vGluT-1, and for vGluT-1 and 2, respectively. The only GABA synapses expressing the SNARE proteins studied were the synapses established by axon terminals of basket neurons. Conclusion The present study supplies a detailed morphological description of VAMP-2, SNAP-25A/B and syntaxin-1 in the different types of glutamatergic and GABAergic synapses of the rat cerebellar cortex. The examined SNARE proteins characterize most of glutamatergic synapses and only one type of GABAergic synapses. In the subpopulations of glutamatergic and GABAergic synapses lacking the SNARE protein isoforms examined, alternative mechanisms for regulating trafficking of synaptic vesicles may be hypothesized, possibly mediated by different isoforms or homologous proteins.

  1. cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness.

    Science.gov (United States)

    Wiley, Luke A; Burnight, Erin R; DeLuca, Adam P; Anfinson, Kristin R; Cranston, Cathryn M; Kaalberg, Emily E; Penticoff, Jessica A; Affatigato, Louisa M; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-07-29

    Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

  2. Glutamatergic deficits and parvalbumin-containing inhibitory neurons in the prefrontal cortex in schizophrenia

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    Kelley JF

    2009-11-01

    Full Text Available Abstract Background We have previously reported that the expression of the messenger ribonucleic acid (mRNA for the NR2A subunit of the N-methyl-D-aspartate (NMDA class of glutamate receptor was decreased in a subset of inhibitory interneurons in the cerebral cortex in schizophrenia. In this study, we sought to determine whether a deficit in the expression of NR2A mRNA was present in the subset of interneurons that contain the calcium buffer parvalbumin (PV and whether this deficit was associated with a reduction in glutamatergic inputs in the prefrontal cortex (PFC in schizophrenia. Methods We examined the expression of NR2A mRNA, labeled with a 35S-tagged riboprobe, in neurons that expressed PV mRNA, visualized with a digoxigenin-labeled riboprobe via an immunoperoxidase reaction, in twenty schizophrenia and twenty matched normal control subjects. We also immunohistochemically labeled the glutamatergic axon terminals with an antibody against vGluT1. Results The density of the PV neurons that expressed NR2A mRNA was significantly decreased by 48-50% in layers 3 and 4 in the subjects with schizophrenia, but the cellular expression of NR2A mRNA in the PV neurons that exhibited a detectable level of this transcript was unchanged. In addition, the density of vGluT1-immunoreactive boutons was significantly decreased by 79% in layer 3, but was unchanged in layer 5 of the PFC in schizophrenia. Conclusion These findings suggest that glutamatergic neurotransmission via NR2A-containing NMDA receptors on PV neurons in the PFC may be deficient in schizophrenia. This may disinhibit the postsynaptic excitatory circuits, contributing to neuronal injury, aberrant information flow and PFC functional deficits in schizophrenia.

  3. Functional recovery after cervical spinal cord injury: Role of neurotrophin and glutamatergic signaling in phrenic motoneurons.

    Science.gov (United States)

    Gill, Luther C; Gransee, Heather M; Sieck, Gary C; Mantilla, Carlos B

    2016-06-01

    Cervical spinal cord injury (SCI) interrupts descending neural drive to phrenic motoneurons causing diaphragm muscle (DIAm) paralysis. Recent studies using a well-established model of SCI, unilateral spinal hemisection of the C2 segment of the cervical spinal cord (SH), provide novel information regarding the molecular and cellular mechanisms of functional recovery after SCI. Over time post-SH, gradual recovery of rhythmic ipsilateral DIAm activity occurs. Recovery of ipsilateral DIAm electromyogram (EMG) activity following SH is enhanced by increasing brain-derived neurotrophic factor (BDNF) in the region of the phrenic motoneuron pool. Delivery of exogenous BDNF either via intrathecal infusion or via mesenchymal stem cells engineered to release BDNF similarly enhance recovery. Conversely, recovery after SH is blunted by quenching endogenous BDNF with the fusion-protein TrkB-Fc in the region of the phrenic motoneuron pool or by selective inhibition of TrkB kinase activity using a chemical-genetic approach in TrkB(F616A) mice. Furthermore, the importance of BDNF signaling via TrkB receptors at phrenic motoneurons is highlighted by the blunting of recovery by siRNA-mediated downregulation of TrkB receptor expression in phrenic motoneurons and by the enhancement of recovery evident following virally-induced increases in TrkB expression specifically in phrenic motoneurons. BDNF/TrkB signaling regulates synaptic plasticity in various neuronal systems, including glutamatergic pathways. Glutamatergic neurotransmission constitutes the main inspiratory-related, excitatory drive to motoneurons, and following SH, spontaneous neuroplasticity is associated with increased expression of ionotropic N-methyl-d-aspartate (NMDA) receptors in phrenic motoneurons. Evidence for the role of BDNF/TrkB and glutamatergic signaling in recovery of DIAm activity following cervical SCI is reviewed.

  4. M-type potassium channels modulate Schaffer collateral-CA1 glutamatergic synaptic transmission.

    Science.gov (United States)

    Sun, Jianli; Kapur, Jaideep

    2012-08-15

    Previous studies have suggested that muscarinic receptor activation modulates glutamatergic transmission. M-type potassium channels mediate the effects of muscarinic activation in the hippocampus, and it has been proposed that they modulate glutamatergic synaptic transmission. We tested whether M1 muscarinic receptor activation enhances glutamatergic synaptic transmission via the inhibition of the M-type potassium channels that are present in Schaffer collateral axons and terminals. Miniature excitatory postsynaptic currents (mEPSCs) were recorded from CA1 pyramidal neurons. The M1 receptor agonist, NcN-A-343, increased the frequency of mEPSCs, but did not alter their amplitude. The M-channel blocker XE991 and its analogue linopirdine also increased the frequency of mEPSCs. Flupirtine, which opens M-channels, had the opposite effect. XE991 did not enhance mEPSCs frequency in a calcium-free external medium. Blocking P/Q- and N-type calcium channels abolished the effect of XE991 on mEPSCs. These data suggested that the inhibition of M-channels increases presynaptic calcium-dependent glutamate release in CA1 pyramidal neurons. The effects of these agents on the membrane potentials of presynaptic CA3 pyramidal neurons were studied using current clamp recordings; activation of M1 receptors and blocking M-channels depolarized neurons and increased burst firing. The input resistance of CA3 neurons was increased by the application of McN-A-343 and XE991; these effects were consistent with the closure of M-channels. Muscarinic activation inhibits M-channels in CA3 pyramidal neurons and its efferents – Schaffer collateral, which causes the depolarization, activates voltage-gated calcium channels, and ultimately elevates the intracellular calcium concentration to increase the release of glutamate on CA1 pyramidal neurons.

  5. Multi-walled carbon nanotube inhibits CA1 glutamatergic synaptic transmission in rat's hippocampal slices.

    Science.gov (United States)

    Chen, Ting; Yang, Jiajia; Zhang, Hui; Ren, Guogang; Yang, Zhuo; Zhang, Tao

    2014-09-17

    The purpose of the study was to investigate the neurotoxic effect of multi-walled carbon nanotubes (MWCNTs) on the properties of glutamatergic synaptic transmission in rat's hippocampal slices using whole-cell patch clamp technique. The amplitude and frequency of excitatory postsynaptic current (EPSC) were accessed on the hippocampal pyramidal neurons. The alterations of glutamatergic synaptic transmission in CA3-CA1 were examined by measuring both the amplitude of evoked excitatory postsynaptic current (eEPSC) and paired-pulse ratio (PPR). The data showed that the amplitude of either spontaneous excitatory postsynaptic current (sEPSC) or miniature excitatory postsynaptic current (mEPSC) was significantly inhibited by 1 μg/mL MWCNTs. However, it was found that there was a trend of different change on the frequency index. When 1 μg/mL MWCNTs was applied, there were a decreased frequency of mEPSC and an increased frequency of sEPSC, which might be due to the effect of action potential. Furthermore, the amplitudes of eEPSC at CA3-CA1 synapses were remarkably decreased. And the mean amplitude of AMPAR-mediated eEPSC was significantly reduced as well. Meanwhile, a majority of PPRs data were greater than one. There were no significant differences of PPRs between control and MWCNTs states, but an increased trend of paired-pulse facilitation was found. These results suggested that MWCNT markedly inhibited hippocampal CA1 glutamatergic synaptic transmission in vitro, which provided new insights into the MWCNT toxicology on CNS at cellular level.

  6. Prenatal exposure of ethanol induces increased glutamatergic neuronal differentiation of neural progenitor cells

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    Han Seol-Heui

    2010-11-01

    Full Text Available Abstract Background Prenatal ethanol exposure during pregnancy induces a spectrum of mental and physical disorders called fetal alcohol spectrum disorder (FASD. The central nervous system is the main organ influenced by FASD, and neurological symptoms include mental retardation, learning abnormalities, hyperactivity and seizure susceptibility in childhood along with the microcephaly. In this study, we examined whether ethanol exposure adversely affects the proliferation of NPC and de-regulates the normal ratio between glutamatergic and GABAergic neuronal differentiation using primary neural progenitor culture (NPC and in vivo FASD models. Methods Neural progenitor cells were cultured from E14 embryo brain of Sprague-Dawley rat. Pregnant mice and rats were treated with ethanol (2 or 4 g/kg/day diluted with normal saline from E7 to E16 for in vivo FASD animal models. Expression level of proteins was investigated by western blot analysis and immunocytochemical assays. MTT was used for cell viability. Proliferative activity of NPCs was identified by BrdU incorporation, immunocytochemistry and FACS analysis. Results Reduced proliferation of NPCs by ethanol was demonstrated using BrdU incorporation, immunocytochemistry and FACS analysis. In addition, ethanol induced the imbalance between glutamatergic and GABAergic neuronal differentiation via transient increase in the expression of Pax6, Ngn2 and NeuroD with concomitant decrease in the expression of Mash1. Similar pattern of expression of those transcription factors was observed using an in vivo model of FASD as well as the increased expression of PSD-95 and decreased expression of GAD67. Conclusions These results suggest that ethanol induces hyper-differentiation of glutamatergic neuron through Pax6 pathway, which may underlie the hyper-excitability phenotype such as hyperactivity or seizure susceptibility in FASD patients.

  7. Induced pluripotent stem cells (iPSCs) derived from a patient with frontotemporal dementia caused by a P301L mutation in microtubule-associated protein tau (MAPT)

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel A.; Hjermind, Lena Elisabeth; Hasholt, Lis Frydenreich;

    2016-01-01

    Skin fibroblasts were obtained froma 57-year-old woman diagnosed with frontotemporal dementia. The diseaseis caused by a P301L mutation in microtubule-associated protein tau (MAPT). Induced pluripotent stem cells (iPSCs) were established by electroporation with episomal plasmids containing hOCT4, h......SOX2, hKLF2, hL-MYC,hLIN-28 and shP53. iPSCs were free of genomically integrated reprogramming genes, contained the expected c.902C>T substitution in exon 10 of the MAPT gene, expressed the expected pluripotency markers, displayed in vitro differentiation potential to the three germ layers and had...... normal karyotype. The iPSC line may be useful for studying hereditary frontotemporal dementia and TAU pathology in vitro....

  8. Omega-3 polyunsaturated fatty acids and chronic stress-induced modulations of glutamatergic neurotransmission in the hippocampus.

    Science.gov (United States)

    Hennebelle, Marie; Champeil-Potokar, Gaëlle; Lavialle, Monique; Vancassel, Sylvie; Denis, Isabelle

    2014-02-01

    Chronic stress causes the release of glucocorticoids, which greatly influence cerebral function, especially glutamatergic transmission. These stress-induced changes in neurotransmission could be counteracted by increasing the dietary intake of omega-3 polyunsaturated fatty acids (n-3 PUFAs). Numerous studies have described the capacity of n-3 PUFAs to help protect glutamatergic neurotransmission from damage induced by stress and glucocorticoids, possibly preventing the development of stress-related disorders such as depression or anxiety. The hippocampus contains glucocorticoid receptors and is involved in learning and memory. This makes it particularly sensitive to stress, which alters certain aspects of hippocampal function. In this review, the various ways in which n-3 PUFAs may prevent the harmful effects of chronic stress, particularly the alteration of glutamatergic synapses in the hippocampus, are summarized.

  9. Pax6-dependent cortical glutamatergic neuronal differentiation regulates autism-like behavior in prenatally valproic acid-exposed rat offspring.

    Science.gov (United States)

    Kim, Ki Chan; Lee, Dong-Keun; Go, Hyo Sang; Kim, Pitna; Choi, Chang Soon; Kim, Ji-Woon; Jeon, Se Jin; Song, Mi-Ryoung; Shin, Chan Young

    2014-02-01

    Imbalance in excitatory/inhibitory signal in the brain has been proposed as one of the main pathological features in autism spectrum disorders, although the underlying cellular and molecular mechanism is unclear yet. Because excitatory/inhibitory imbalance can be induced by aberration in glutamatergic/GABAergic neuronal differentiation, we investigated the mechanism of dysregulated neuronal differentiation between excitatory and inhibitory neurons in the embryonic and postnatal brain of prenatally valproic acid-exposed rat offspring, which is often used as an animal model of autism spectrum disorders. Transcription factor Pax6, implicated in glutamatergic neuronal differentiation, was transiently increased in embryonic cortex by valproate exposure, which resulted in the increased expression of glutamatergic proteins in postnatal brain of offspring. Chromatin immunoprecipitation showed increased acetylated histone binding on Pax6 promoter region, which may underlie the transcriptional up-regulation of Pax6. Other histone deacetylase (HDAC) inhibitors including TSA and SB but not valpromide, which is devoid of HDAC inhibitor activity, induced Pax6 up-regulation. Silencing Pax6 expression in cultured rat primary neural progenitor cells demonstrated that up-regulation of Pax6 plays an essential role in valproate-induced glutamatergic differentiation. Blocking glutamatergic transmission with MK-801 or memantine treatment, and to a lesser extent with MPEP treatment, reversed the impaired social behaviors and seizure susceptibility of prenatally valproate-exposed offspring. Together, environmental factors may contribute to the imbalance in excitatory/inhibitory neuronal activity in autistic brain by altering expression of transcription factors governing glutamatergic/GABAergic differentiation during fetal neural development, in conjunction with the genetic preload.

  10. Glutamatergic and HPA-axis pathway genes in bipolar disorder comorbid with alcohol- and substance use disorders.

    Science.gov (United States)

    Dalvie, Shareefa; Fabbri, Chiara; Ramesar, Raj; Serretti, Alessandro; Stein, Dan J

    2016-02-01

    Glutamatergic neurotransmission has been shown to be dysregulated in bipolar disorder (BD), alcohol use disorder (AUD) and substance use disorder (SUD). Similarly, disruption in the hypothalamic-pituitary-adrenal (HPA)-axis has also been observed in these conditions. BD is often comorbid with AUD and SUD. The effects of the glutamatergic and HPA systems have not been extensively examined in individuals with BD-AUD and BD-SUD comorbidity. The aim of this investigation was to determine whether variants in the glutamatergic pathway and HPA-axis are associated with BD-AUD and BD-SUD comorbidity. The research cohort consisted of 498 individuals with BD type I from the Systematic Treatment Enhancement Program for Bipolar Disorder (STEP-BD). A subset of the cohort had comorbid current AUD and current SUD. A total of 1935 SNPs from both the glutamatergic and HPA pathways were selected from the STEP-BD genome-wide dataset. To identify population stratification, IBS clustering was performed using the program Plink 1.07. Single SNP association and gene-based association testing were conducted using logistic regression. A pathway analysis of glutamatergic and HPA genes was performed, after imputation using IMPUTE2. No single SNP was associated with BD-AUD or BD-SUD comorbidity after correction for multiple testing. However, from the gene-based analysis, the gene PRKCI was significantly associated with BD-AUD. The pathway analysis provided overall negative findings, although several genes including GRIN2B showed high percentage of associated SNPs for BD-AUD. Even though the glutamatergic and HPA pathways may not be involved in BD-AUD and BD-SUD comorbidity, PRKCI deserves further investigation in BD-AUD.

  11. Cholinergic, Glutamatergic, and GABAergic Neurons of the Pedunculopontine Tegmental Nucleus Have Distinct Effects on Sleep/Wake Behavior in Mice.

    Science.gov (United States)

    Kroeger, Daniel; Ferrari, Loris L; Petit, Gaetan; Mahoney, Carrie E; Fuller, Patrick M; Arrigoni, Elda; Scammell, Thomas E

    2017-02-01

    The pedunculopontine tegmental (PPT) nucleus has long been implicated in the regulation of cortical activity and behavioral states, including rapid eye-movement (REM) sleep. For example, electrical stimulation of the PPT region during sleep leads to rapid awakening, whereas lesions of the PPT in cats reduce REM sleep. Though these effects have been linked with the activity of cholinergic PPT neurons, the PPT also includes intermingled glutamatergic and GABAergic cell populations, and the precise roles of cholinergic, glutamatergic, and GABAergic PPT cell groups in regulating cortical activity and behavioral state remain unknown. Using a chemogenetic approach in three Cre-driver mouse lines, we found that selective activation of glutamatergic PPT neurons induced prolonged cortical activation and behavioral wakefulness, whereas inhibition reduced wakefulness and increased non-REM (NREM) sleep. Activation of cholinergic PPT neurons suppressed lower-frequency electroencephalogram rhythms during NREM sleep. Last, activation of GABAergic PPT neurons slightly reduced REM sleep. These findings reveal that glutamatergic, cholinergic, and GABAergic PPT neurons differentially influence cortical activity and sleep/wake states. More than 40 million Americans suffer from chronic sleep disruption, and the development of effective treatments requires a more detailed understanding of the neuronal mechanisms controlling sleep and arousal. The pedunculopontine tegmental (PPT) nucleus has long been considered a key site for regulating wakefulness and REM sleep. This is mainly because of the cholinergic neurons contained in the PPT nucleus. However, the PPT nucleus also contains glutamatergic and GABAergic neurons that likely contribute to the regulation of cortical activity and sleep-wake states. The chemogenetic experiments in the present study reveal that cholinergic, glutamatergic, and GABAergic PPT neurons each have distinct effects on sleep/wake behavior, improving our

  12. Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines.

    Science.gov (United States)

    Ghasemi-Dehkordi, Payam; Allahbakhshian-Farsani, Mehdi; Abdian, Narges; Mirzaeian, Amin; Saffari-Chaleshtori, Javad; Heybati, Fatemeh; Mardani, Gashtasb; Karimi-Taghanaki, Alireza; Doosti, Abbas; Jami, Mohammad-Saeid; Abolhasani, Marziyeh; Hashemzadeh-Chaleshtori, Morteza

    2015-09-01

    Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore

  13. Development of a protein marker panel for characterization of human induced pluripotent stem cells (hiPSCs using global quantitative proteome analysis

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    Natalia S. Pripuzova

    2015-05-01

    Full Text Available The emergence of new methods for reprogramming of adult somatic cells into induced pluripotent stem cells (iPSC led to the development of new approaches in drug discovery and regenerative medicine. Investigation of the molecular mechanisms underlying the self-renewal, expansion and differentiation of human iPSC (hiPSC should lead to improvements in the manufacture of safe and reliable cell therapy products. The goal of our study was qualitative and quantitative proteomic characterizations of hiPSC by means of electrospray ionization (ESI-MSe and MALDI-TOF/TOF mass spectrometry (MS. Proteomes of hiPSCs of different somatic origins: fibroblasts and peripheral blood CD34+ cells, reprogrammed by the same technique, were compared with the original somatic cells and hESC. Quantitative proteomic comparison revealed approximately 220 proteins commonly up-regulated in all three pluripotent stem cell lines compared to the primary cells. Expression of 21 proteins previously reported as pluripotency markers was up-regulated in both hiPSCs (8 were confirmed by Western blot. A number of novel candidate marker proteins with the highest fold-change difference between hiPSCs/hESC and somatic cells discovered by MS were confirmed by Western blot. A panel of 22 candidate marker proteins of hiPSC was developed and expression of these proteins was confirmed in 8 additional hiPSC lines.

  14. Glutamatergic motoneurons in the stomatogastric ganglion of the mantis shrimp Squilla oratoria.

    Science.gov (United States)

    Chiba, C; Tazaki, K

    1992-07-01

    1. Transmitters of motoneurons in the stomatogastric ganglion (STG) of Squilla were identified by analyzing the excitatory neuromuscular properties of muscles in the posterior cardiac plate (pcp) and pyloric regions. 2. Bath and iontophoretic applications of glutamate produce depolarizations in these muscles. The pharmacological experiments and desensitization of the junctional receptors elucidate the glutamatergic nature of the excitatory junctional potentials (EJPs) evoked in the constrictor and dilator muscles. The reversal potentials for the excitatory junctional current (EJC) and for the glutamate-induced current are almost the same. 3. Some types of dilator muscle show sensitivity to both glutamate and acetylcholine (ACh) exogenously applied. The pharmacological evidence and desensitization of the junctional receptors indicate the glutamatergic nature of neuromuscular junctions in these dually sensitive muscles. The reversal potentials for the EJC and for the ACh-induced current are not identical. 4. Glutamate is a candidate as an excitatory neuro-transmitter at the neuromuscular junctions which the STG motoneurons named PCP, PY, PD, LA and VC make with the identified muscles. Kainic and quisqualic acids which act on glutamate receptors are potent excitants of these muscles. Extrajunctional receptors to ACh are present in two types of the muscle innervated by LA and VC. 5. Neurotransmitters used by the STG motoneurons of stomatopods are compared to those of decapods.

  15. Targeting the Glutamatergic System to Treat Pathological Gambling: Current Evidence and Future Perspectives

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    Mauro Pettorruso

    2014-01-01

    Full Text Available Pathological gambling or gambling disorder has been defined by the DSM-5 as a behavioral addiction. To date, its pathophysiology is not completely understood and there is no FDA-approved treatment for gambling disorders. Glutamate is the principal excitatory neurotransmitter in the nervous system and it has been recently involved in the pathophysiology of addictive behaviors. In this paper, we review the current literature on a class of drugs that act as modulating glutamate system in PG. A total of 19 studies have been included, according to inclusion and exclusion criteria. Clinical trial and case series using glutamatergic drugs (N-acetylcysteine, memantine, amantadine, topiramate, acamprosate, baclofen, gabapentin, pregabalin, and modafinil will be presented to elucidate the effectiveness on gambling behaviors and on the related clinical dimensions (craving, withdrawal, and cognitive symptoms in PG patients. The results have been discussed to gain more insight in the pathophysiology and treatment of PG. In conclusion, manipulation of glutamatergic neurotransmission appears to be promising in developing improved therapeutic agents for the treatment of gambling disorders. Further studies are required. Finally, we propose future directions and challenges in this research area.

  16. The effect of α7 nicotinic receptor activation on glutamatergic transmission in the hippocampus.

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    Cheng, Qing; Yakel, Jerrel L

    2015-10-15

    Nicotinic acetylcholine receptors (nAChRs) are expressed widely in the CNS, and mediate both synaptic and perisynaptic activities of endogenous cholinergic inputs and pharmacological actions of exogenous compounds (e.g., nicotine and choline). Behavioral studies indicate that nicotine improves such cognitive functions as learning and memory, however the cellular mechanism of these actions remains elusive. With help from newly developed biosensors and optogenetic tools, recent studies provide new insights on signaling mechanisms involved in the activation of nAChRs. Here we will review α7 nAChR's action in the tri-synaptic pathway in the hippocampus. The effects of α7 nAChR activation via either exogenous compounds or endogenous cholinergic innervation are detailed for spontaneous and evoked glutamatergic synaptic transmission and synaptic plasticity, as well as the underlying signaling mechanisms. In summary, α7 nAChRs trigger intracellular calcium rise and calcium-dependent signaling pathways to enhance glutamate release and induce glutamatergic synaptic plasticity.

  17. Melatonin receptor activation increases glutamatergic synaptic transmission in the rat medial lateral habenula.

    Science.gov (United States)

    Evely, Katherine M; Hudson, Randall L; Dubocovich, Margarita L; Haj-Dahmane, Samir

    2016-05-01

    Melatonin (MLT) is secreted from the pineal gland and mediates its physiological effects through activation of two G protein-coupled receptors, MT1 and MT2 . These receptors are expressed in several brain areas, including the habenular complex, a pair of nuclei that relay information from forebrain to midbrain and modulate a plethora of behaviors, including sleep, mood, and pain. However, so far, the precise mechanisms by which MLT control the function of habenula neurons remain unknown. Using whole cell recordings from male rat brain slices, we examined the effects of MLT on the excitability of medial lateral habenula (MLHb) neurons. We found that MLT had no significant effects on the intrinsic excitability of MLHb neurons, but profoundly increased the amplitude of glutamate-mediated evoked excitatory post-synaptic currents (EPSC). The increase in strength of glutamate synapses onto MLHb neurons was mediated by an increase in glutamate release. The MLT-induced increase in glutamatergic synaptic transmission was blocked by the competitive MT1 /MT2 receptor antagonist luzindole (LUZ). These results unravel a potential cellular mechanism by which MLT receptor activation enhances the excitability of MLHb neurons. The MLT-mediated control of glutamatergic inputs to the MLHb may play a key role in the modulation of various behaviors controlled by the habenular complex.

  18. Impaired glutamatergic and GABAergic transmission by amitraz in primary hippocampal cells.

    Science.gov (United States)

    Del Pino, Javier; Frejo, María Teresa; Baselga, María José Anadon; Moyano, Paula; Díaz, María Jesús

    2015-01-01

    Amitraz is a formamidine pesticide that has been reported to be a neurotoxic compound that induces convulsions among other effects. Excitatory and inhibitory neurotransmission is mediated mainly by glutamate and GABA, respectively, so their alteration could be responsible for induction of seizures. In this regard, amitraz α2 adrenergic agonist action, which has been suggested as likely responsible for this effect, could alter these neurotransmitter systems and lead to seizure induction. Moreover, other amitraz mechanisms such as histamine H1 receptor inhibition could be involved. To confirm if amitraz disrupts glutamatergic/GABAergic transmission by these mechanisms, we evaluated, in primary hippocampal neurons, the effect of amitraz (0.01 μM to 100 μM) with or without the α2 adrenergic antagonist idazoxan (1 μM) and/or the H1 receptor agonist n-methylhistaprodifen (30 μM) co-treatment on 4-aminobutyrate aminotransferase, glutamate decarboxylase 65 (GAD 65), succinate-semialdehyde dehydrogenase and glutaminase gene expression and on glutamate and GABA levels after 24h treatment. We observed that amitraz disrupts glutaminase and GAD 65 gene expression, altering glutamatergic and GABAergic transmission. These effects were mediated partially by H1 and α2 receptors suggesting that other mechanisms could be involved. These data could help explain the mechanism by which amitraz induces seizures and provide a therapeutic strategy to protect against this effect in case of poisoning.

  19. The role of glutamatergic and GABAergic systems on serotonin- induced feeding behavior in chicken.

    Science.gov (United States)

    Mortezaei, Sepideh Seyedali; Zendehdel, Morteza; Babapour, Vahab; Hasani, Keyvan

    2013-12-01

    It has been reported that serotonin can modulate glutamate and GABA release in central nervous system (CNS). The present study was designed to examine the role of glutamatergic and GABAergic systems on serotonin- induced feeding behavior in chickens. In Experiment 1 intracerebroventricular (ICV) injection of MK- 801(NMDA receptor antagonist, 15 nmol) performed followed by serotonin (10 μg). In experiments 2, 3, 4, 5, 6 and 7 prior to serotonin injection, chickens received CNQX (AMPA/kainate receptor antagonist, 390 nmol), AIDA (mGluR1 antagonist, 2 nmol), LY341495 (mGluR2 antagonist, 150 nmol), UBP1112 (mGluR3 antagonist, 2 nmol), picrotoxin (GABA A receptor antagonist, 0.5 μg), CGP54626 (GABAB receptor antagonist, 20 ng) respectively. Cumulative food intake was determined at 3 h post injection. The results of this study showed that the hypophagic effect of serotonin was significantly attenuated by pretreatment with MK- 801 and CNQX (p 0.05). Also, the inhibitory effect of serotonin on food intake was amplified by picrotoxin (p 0.05). These results suggest that serotonin as a modulator probably interacts with glutamatergic (via NMDA and AMPA/Kainate receptors) and GABAergic (via GABAA receptor) systems on feeding behavior in chicken.

  20. Deletion of Shank1 has minimal effects on the molecular composition and function of glutamatergic afferent postsynapses in the mouse inner ear

    NARCIS (Netherlands)

    Braude, Jeremy P.; Vijayakumar, Sarath; Baumgarner, Katherine; Laurine, Rebecca; Jones, Timothy A.; Jones, Sherri M.; Pyott, Sonja J.

    2015-01-01

    Shank proteins (1-3) are considered the master organizers of glutamatergic postsynaptic densities in the central nervous system, and the genetic deletion of either Shank1, 2, or 3 results in altered composition, form, and strength of glutamatergic postsynapses. To investigate the contribution of Sha

  1. Amygdala EphB2 Signaling Regulates Glutamatergic Neuron Maturation and Innate Fear.

    Science.gov (United States)

    Zhu, Xiao-Na; Liu, Xian-Dong; Zhuang, Hanyi; Henkemeyer, Mark; Yang, Jing-Yu; Xu, Nan-Jie

    2016-09-28

    The amygdala serves as emotional center to mediate innate fear behaviors that are reflected through neuronal responses to environmental aversive cues. However, the molecular mechanism underlying the initial neuron responses is poorly understood. In this study, we monitored the innate defensive responses to aversive stimuli of either elevated plus maze or predator odor in juvenile mice and found that glutamatergic neurons were activated in amygdala. Loss of EphB2, a receptor tyrosine kinase expressed in amygdala neurons, suppressed the reactions and led to defects in spine morphogenesis and fear behaviors. We further found a coupling of spinogenesis with these threat cues induced neuron activation in developing amygdala that was controlled by EphB2. A constitutively active form of EphB2 was sufficient to rescue the behavioral and morphological defects caused by ablation of ephrin-B3, a brain-enriched ligand to EphB2. These data suggest that kinase-dependent EphB2 intracellular signaling plays a major role for innate fear responses during the critical developing period, in which spinogenesis in amygdala glutamatergic neurons was involved. Generation of innate fear responses to threat as an evolutionally conserved brain feature relies on development of functional neural circuit in amygdala, but the molecular mechanism remains largely unknown. We here identify that EphB2 receptor tyrosine kinase, which is specifically expressed in glutamatergic neurons, is required for the innate fear responses in the neonatal brain. We further reveal that EphB2 mediates coordination of spinogenesis and neuron activation in amygdala during the critical period for the innate fear. EphB2 catalytic activity plays a major role for the behavior upon EphB-ephrin-B3 binding and transnucleus neuronal connections. Our work thus indicates an essential synaptic molecular signaling within amygdala that controls synapse development and helps bring about innate fear emotions in the postnatal

  2. Development of Glutamatergic Proteins in Human Visual Cortex across the Lifespan.

    Science.gov (United States)

    Siu, Caitlin R; Beshara, Simon P; Jones, David G; Murphy, Kathryn M

    2017-06-21

    Traditionally, human primary visual cortex (V1) has been thought to mature within the first few years of life, based on anatomical studies of synapse formation, and establishment of intracortical and intercortical connections. Human vision, however, develops well beyond the first few years. Previously, we found prolonged development of some GABAergic proteins in human V1 (Pinto et al., 2010). Yet as >80% of synapses in V1 are excitatory, it remains unanswered whether the majority of synapses regulating experience-dependent plasticity and receptive field properties develop late, like their inhibitory counterparts. To address this question, we used Western blotting of postmortem tissue from human V1 (12 female, 18 male) covering a range of ages. Then we quantified a set of postsynaptic glutamatergic proteins (PSD-95, GluA2, GluN1, GluN2A, GluN2B), calculated indices for functional pairs that are developmentally regulated (GluA2:GluN1; GluN2A:GluN2B), and determined interindividual variability. We found early loss of GluN1, prolonged development of PSD-95 and GluA2 into late childhood, protracted development of GluN2A until ∼40 years, and dramatic loss of GluN2A in aging. The GluA2:GluN1 index switched at ∼1 year, but the GluN2A:GluN2B index continued to shift until ∼40 year before changing back to GluN2B in aging. We also identified young childhood as a stage of heightened interindividual variability. The changes show that human V1 develops gradually through a series of five orchestrated stages, making it likely that V1 participates in visual development and plasticity across the lifespan.SIGNIFICANCE STATEMENT Anatomical structure of human V1 appears to mature early, but vision changes across the lifespan. This discrepancy has fostered two hypotheses: either other aspects of V1 continue changing, or later changes in visual perception depend on extrastriate areas. Previously, we showed that some GABAergic synaptic proteins change across the lifespan, but most

  3. High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research

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    Hubbard Kyle S

    2012-10-01

    Full Text Available Abstract Background Recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. Derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. However, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available. Methods With the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell (ESC lines (R1, C57BL/6 and D3 adapted to feeder-independent suspension culture. Neurogenesis and neuronal maturation were characterized as a function of time in culture using immunological, genomic, morphological and functional metrics. The functional responses of ESNs to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin (LTX, and botulinum neurotoxin (BoNT, were also evaluated. Results Suspension-adapted ESCs expressed markers of pluripotency through at least 30 passages, and differentiation produced 97×106 neural progenitor cells (NPCs per 10-cm dish. Greater than 99% of embryonic stem cell-derived neurons (ESNs expressed neuron-specific markers by 96 h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. Expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that ESN populations were predominantly glutamatergic. Furthermore, ESNs were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons

  4. Counting Vesicular Release Events Reveals Binomial Release Statistics at Single Glutamatergic Synapses.

    Science.gov (United States)

    Malagon, Gerardo; Miki, Takafumi; Llano, Isabel; Neher, Erwin; Marty, Alain

    2016-04-06

    Many central glutamatergic synapses contain a single presynaptic active zone and a single postsynaptic density. However, the basic functional properties of such "simple synapses" remain unclear. One important step toward understanding simple synapse function is to analyze the number of synaptic vesicles released in such structures per action potential, but this goal has remained elusive until now. Here, we describe procedures that allow reliable vesicular release counting at simple synapses between parallel fibers and molecular layer interneurons of rat cerebellar slices. Our analysis involves local extracellular stimulation of single parallel fibers and deconvolution of resulting EPSCs using quantal signals as template. We observed a reduction of quantal amplitudes (amplitude occlusion) in pairs of consecutive EPSCs due to receptor saturation. This effect is larger (62%) than previously reported and primarily reflects receptor activation rather than desensitization. In addition to activation-driven amplitude occlusion, each EPSC reduces amplitudes of subsequent events by an estimated 3% due to cumulative desensitization. Vesicular release counts at simple synapses follow binomial statistics with a maximum that varies from 2 to 10 among experiments. This maximum presumably reflects the number of docking sites at a given synapse. These results show striking similarities, as well as significant quantitative differences, with respect to previous results at simple GABAergic synapses. It is generally accepted that the output signal of individual central synapses saturates at high release probability, but it remains unclear whether the source of saturation is presynaptic, postsynaptic, or both presynaptic and postsynaptic. To clarify this and other issues concerning the function of synapses, we have developed new recording and analysis methods at single central glutamatergic synapses. We find that individual release events engage a high proportion of postsynaptic

  5. Optogenetic stimulation of glutamatergic neuronal activity in the striatum enhances neurogenesis in the subventricular zone of normal and stroke mice.

    Science.gov (United States)

    Song, Mingke; Yu, Shan Ping; Mohamad, Osama; Cao, Wenyuan; Wei, Zheng Zachory; Gu, Xiaohuan; Jiang, Michael Qize; Wei, Ling

    2017-02-01

    Neurogenesis in the subventricular zone (SVZ) of the adult brain may contribute to tissue repair after brain injuries. Whether SVZ neurogenesis can be upregulated by specific neuronal activity in vivo and promote functional recovery after stroke is largely unknown. Using the spatial and cell type specific optogenetic technique combined with multiple approaches of in vitro, ex vivo and in vivo examinations, we tested the hypothesis that glutamatergic activation in the striatum could upregulate SVZ neurogenesis in the normal and ischemic brain. In transgenic mice expressing the light-gated channelrhodopsin-2 (ChR2) channel in glutamatergic neurons, optogenetic stimulation of the glutamatergic activity in the striatum triggered glutamate release into SVZ region, evoked membrane currents, Ca(2+) influx and increased proliferation of SVZ neuroblasts, mediated by AMPA receptor activation. In ChR2 transgenic mice subjected to focal ischemic stroke, optogenetic stimuli to the striatum started 5days after stroke for 8days not only promoted cell proliferation but also the migration of SVZ neuroblasts into the peri-infarct cortex with increased neuronal differentiation and improved long-term functional recovery. These data provide the first morphological and functional evidence showing a unique striatum-SVZ neuronal regulation via a semi-phasic synaptic mechanism that can boost neurogenic cascades and stroke recovery. The benefits from stimulating endogenous glutamatergic activity suggest a novel regenerative strategy after ischemic stroke and other brain injuries.

  6. Metabotropic glutamate and GABA receptors modulate cellular excitability and glutamatergic transmission in chicken cochlear nucleus angularis neurons.

    Science.gov (United States)

    Shi, Wei; Lu, Yong

    2017-03-01

    Neurons in the avian cochlear nucleus angularis (NA) receive glutamatergic input from the auditory nerve, and GABAergic input from the superior olivary nucleus. Physiologically heterogeneous, NA neurons perform multiple functions including encoding sound intensity information. Using in vitro whole-cell patch recordings from acute brain slices and immunohistochemistry staining, we investigated neuromodulation mediated by metabotropic glutamate and GABA receptors (mGluRs and GABABRs) in NA neurons. Based on their intrinsic firing patterns in response to somatic current injections, NA neurons were classified into onset, damped, and tonic cells. Pharmacological activation of group II mGluRs, group III mGluRs, and GABABRs, by their respective agonists, suppressed the cellular excitability of non-onset firing NA neurons. Each of these agonists inhibited the glutamatergic transmission in NA neurons, in a cell type-independent manner. The frequency but not the amplitude of spontaneous release of glutamate was reduced by each of these agonists, suggesting that the modulation of the glutamatergic transmission was via presynaptic actions. Interestingly, activation of group I mGluRs increased cellular excitability and suppressed glutamatergic transmission in non-onset neurons. These results elaborate that auditory processing in NA neurons is subject to neuromodulation mediated by metabotropic receptors activated by native neurotransmitters released at NA.

  7. Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles

    NARCIS (Netherlands)

    Banks, R.W.; Cahusac, P.M.; Graca, A.; Kain, N.; Shenton, F.; Singh, P.; Nja, A.; Simon, A.; Watson, S.; Slater, C.R.; Bewick, G.S.

    2013-01-01

    Abstract Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending w

  8. Activation of both Group I and Group II metabotropic glutamatergic receptors suppress retinogeniculate transmission.

    Science.gov (United States)

    Lam, Y-W; Sherman, S M

    2013-07-09

    Relay cells of dorsal lateral geniculate nucleus (LGN) receive a Class 1 glutamatergic input from the retina and a Class 2 input from cortical layer 6. Among the properties of Class 2 synapses is the ability to activate metabotropic glutamate receptors (mGluRs), and mGluR activation is known to affect thalamocortical transmission via regulating retinogeniculate and thalamocortical synapses. Using brain slices, we studied the effects of Group I (dihydroxyphenylglycine) and Group II ((2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine) mGluR agonists on retinogeniculate synapses. We showed that both agonists inhibit retinogeniculate excitatory postsynaptic currents (EPSCs) through presynaptic mechanisms, and their effects are additive and independent. We also found high-frequency stimulation of the layer 6 corticothalamic input produced a similar suppression of retinogeniculate EPSCs, suggesting layer 6 projection to LGN as a plausible source of activating these presynaptic mGluRs.

  9. Glutamatergic and GABAergic gene sets in attention-deficit/hyperactivity disorder

    DEFF Research Database (Denmark)

    Naaijen, J; Bralten, J; Poelmans, G

    2017-01-01

    Attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorders (ASD) often co-occur. Both are highly heritable; however, it has been difficult to discover genetic risk variants. Glutamate and GABA are main excitatory and inhibitory neurotransmitters in the brain; their balance...... within glutamatergic and GABAergic genes were investigated using the MAGMA software in an ADHD case-only sample (n=931), in which we assessed ASD symptoms and response inhibition on a Stop task. Gene set analysis for ADHD symptom severity, divided into inattention and hyperactivity/impulsivity symptoms......, autism symptom severity and inhibition were performed using principal component regression analyses. Subsequently, gene-wide association analyses were performed. The glutamate gene set showed an association with severity of hyperactivity/impulsivity (P=0.009), which was robust to correcting for genome...

  10. GDNF control of the glutamatergic cortico-striatal pathway requires tonic activation of adenosine A2A Receptors

    Science.gov (United States)

    Gomes, Catarina A.R.V.; Simões, Patrícia F.; Canas, Paula M.; Quiroz, César; Sebastião, Ana M.; Ferré, Sergi; Cunha, Rodrigo A.; Ribeiro, Joaquim A.

    2009-01-01

    Glial cell line-derived neurotrophic factor (GDNF) affords neuroprotection in Parkinson’s disease in accordance with its ability to bolster nigrostriatal innervation. We previously found that GDNF facilitates dopamine release in a manner dependent on adenosine A2A receptor activation. Since motor dysfunction also involves modifications of striatal glutamatergic innervation, we now tested if GDNF and its receptor system, Ret (rearranged during transfection) and GFRα1 (GDNF family receptor alpha 1) controlled the cortico-striatal glutamatergic pathway in an A2A receptor-dependent manner. GDNF (10 ng/ml) enhanced (by ≈13%) glutamate release from rat striatal nerve endings, an effect potentiated (up to ≈ 30%) by the A2A receptor agonist CGS 21680 (10 nM) and prevented by the A2A receptor antagonist, SCH 58261 (50 nM). Triple immunocytochemical studies revealed that Ret and GFRα1 were located in 50% of rat striatal glutamatergic terminals (immunopositive for vesicular glutamate transporters-1/2), where they were found to be co-located with A2A receptors. Activation of the glutamatergic system upon in vivo electrical stimulation of the rat cortico-striatal input induced striatal Ret phosphoprylation that was prevented by pre-treatment with the A2A receptor antagonist, MSX-3 (3 mg/kg). The results provide the first functional and morphological evidence that GDNF controls cortico-striatal glutamatergic pathways in a manner largely dependent on the co-activation of adenosine A2A receptors. PMID:19141075

  11. Mice deficient of glutamatergic signaling from intrinsically photosensitive retinal ganglion cells exhibit abnormal circadian photoentrainment.

    Science.gov (United States)

    Purrier, Nicole; Engeland, William C; Kofuji, Paulo

    2014-01-01

    Several aspects of behavior and physiology, such as sleep and wakefulness, blood pressure, body temperature, and hormone secretion exhibit daily oscillations known as circadian rhythms. These circadian rhythms are orchestrated by an intrinsic biological clock in the suprachiasmatic nuclei (SCN) of the hypothalamus which is adjusted to the daily environmental cycles of day and night by the process of photoentrainment. In mammals, the neuronal signal for photoentrainment arises from a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that send a direct projection to the SCN. ipRGCs also mediate other non-image-forming (NIF) visual responses such as negative masking of locomotor activity by light, and the pupillary light reflex (PLR) via co-release of neurotransmitters glutamate and pituitary adenylate cyclase-activating peptide (PACAP) from their synaptic terminals. The relative contribution of each neurotransmitter system for the circadian photoentrainment and other NIF visual responses is still unresolved. We investigated the role of glutamatergic neurotransmission for circadian photoentrainment and NIF behaviors by selective ablation of ipRGC glutamatergic synaptic transmission in mice. Mutant mice displayed delayed re-entrainment to a 6 h phase shift (advance or delay) in the light cycle and incomplete photoentrainment in a symmetrical skeleton photoperiod regimen (1 h light pulses between 11 h dark periods). Circadian rhythmicity in constant darkness also was reduced in some mutant mice. Other NIF responses such as the PLR and negative masking responses to light were also partially attenuated. Overall, these results suggest that glutamate from ipRGCs drives circadian photoentrainment and negative masking responses to light.

  12. Glutamatergic synaptic currents of nigral dopaminergic neurons follow a postnatal developmental sequence

    Directory of Open Access Journals (Sweden)

    Edouard ePearlstein

    2015-05-01

    Full Text Available The spontaneous activity pattern of adult dopaminergic (DA neurons of the substantia nigra pars compacta (SNc results from interactions between intrinsic membrane conductances and afferent inputs. In adult SNc DA neurons, low-frequency tonic background activity is generated by intrinsic pacemaker mechanisms, whereas burst generation depends on intact synaptic inputs in particular the glutamatergic ones. Tonic DA release in the striatum during pacemaking is required to maintain motor activity, and burst firing evokes phasic DA release, necessary for cue-dependent learning tasks. However, it is still unknown how the firing properties of SNc DA neurons mature during postnatal development before reaching the adult state. We studied the postnatal developmental profile of spontaneous and evoked AMPA and NMDA receptor-mediated excitatory postsynaptic currents (EPSCs in SNc DA neurons in brain slices from immature (postnatal days P4-10 and young adult (P30-50 tyrosine hydroxylase (TH-GFP mice. We found that somato-dendritic fields of SNc DA neurons are already mature at P4-10. In contrast, spontaneous glutamatergic EPSCs show a developmental sequence. Spontaneous NMDA EPSCs in particular are larger and more frequent in immature SNc DA neurons than in young adult ones and have a bursty pattern. They are mediated by GluN2B and GluN2D subunit-containing NMDA receptors. The latter generate long-lasting, DQP1105-sensitive, spontaneous EPSCs, which are transiently recorded during this early period. Due to high NMDA activity, immature SNc DA neurons generate large and long lasting NMDA receptor-dependent (APV-sensitive bursts in response to the stimulation of the subthalamic nucleus. We conclude that the transient high NMDA activity allows calcium influx into the dendrites of developing SNc DA neurons.

  13. Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons.

    Science.gov (United States)

    Zhang, Shuzhuo; Jin, Yuelei; Liu, Xiaoyan; Yang, Lujia; Ge, Zhi juan; Wang, Hui; Li, Jin; Zheng, Jianquan

    2014-09-25

    Methamphetamine (METH) is a psychostimulant drug. Abuse of METH produces long-term behavioral changes including behavioral, sensitization, tolerance, and dependence. It induces neurotoxic effects in several areas of the brain via enhancing dopamine (DA) level abnormally, which may cause a secondary release of glutamate (GLU). However, repeated administration of METH still increases release of GLU even when dopamine content in tissue is significantly depleted. It implies that some other mechanisms are likely to involve in METH-induced GLU release. The goal of this study was to observe METH affected glutamatergic synaptic transmission in rat primary cultured hippocampal neurons and to explore the mechanism of METH modulated GLU release. Using whole-cell patch-clamp recordings, we found that METH (0.1-50.0μM) increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). However, METH decreased the frequency of sEPSCs and mEPSCs at high concentration of 100μM. The postsynaptic NMDA receptor currents and P/Q-type calcium channel were not affected by the use of METH (10,100μM). METH did not present visible effect on N-type Ca(2+) channel current at the concentration lower than 50.0μM, but it was inhibited by use of METH at a 100μM. The effect of METH on glutamatergic synaptic transmission was not revered by pretreated with DA receptor antagonist SCH23390. These results suggest that METH directly modulated presynaptic GLU release at a different concentration, while dopaminergic system was not involved in METH modulated release of GLU in rat primary cultured hippocampal neurons.

  14. Presynaptic muscarinic M(2) receptors modulate glutamatergic transmission in the bed nucleus of the stria terminalis.

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    Guo, Ji-Dong; Hazra, Rimi; Dabrowska, Joanna; Muly, E Chris; Wess, Jürgen; Rainnie, Donald G

    2012-03-01

    The anterolateral cell group of the bed nucleus of the stria terminalis (BNST(ALG)) serves as an important relay station in stress circuitry. Limbic inputs to the BNST(ALG) are primarily glutamatergic and activity-dependent changes in this input have been implicated in abnormal behaviors associated with chronic stress and addiction. Significantly, local infusion of acetylcholine (ACh) receptor agonists into the BNST trigger stress-like cardiovascular responses, however, little is known about the effects of these agents on glutamatergic transmission in the BNST(ALG). Here, we show that glutamate- and ACh-containing fibers are found in close association in the BNST(ALG). Moreover, in the presence of the acetylcholinesterase inhibitor, eserine, endogenous ACh release evoked a long-lasting reduction of the amplitude of stimulus-evoked EPSCs. This effect was mimicked by exogenous application of the ACh analog, carbachol, which caused a reversible, dose-dependent, reduction of the evoked EPSC amplitude, and an increase in both the paired-pulse ratio and coefficient of variation, suggesting a presynaptic site of action. Uncoupling of postsynaptic G-proteins with intracellular GDP-β-S, or application of the nicotinic receptor antagonist, tubocurarine, failed to block the carbachol effect. In contrast, the carbachol effect was blocked by prior application of atropine or M(2) receptor-preferring antagonists, and was absent in M(2)/M(4) receptor knockout mice, suggesting that presynaptic M(2) receptors mediate the effect of ACh. Immunoelectron microscopy studies further revealed the presence of M(2) receptors on axon terminals that formed asymmetric synapses with BNST neurons. Our findings suggest that presynaptic M(2) receptors might be an important modulator of the stress circuit and hence a novel target for drug development.

  15. Glutamatergic transmission aberration: a major cause of behavioral deficits in a murine model of Down's syndrome.

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    Kaur, Gurjinder; Sharma, Ajay; Xu, Wenjin; Gerum, Scott; Alldred, Melissa J; Subbanna, Shivakumar; Basavarajappa, Balapal S; Pawlik, Monika; Ohno, Masuo; Ginsberg, Stephen D; Wilson, Donald A; Guilfoyle, David N; Levy, Efrat

    2014-04-09

    Trisomy 21, or Down's syndrome (DS), is the most common genetic cause of intellectual disability. Altered neurotransmission in the brains of DS patients leads to hippocampus-dependent learning and memory deficiency. Although genetic mouse models have provided important insights into the genes and mechanisms responsible for DS-specific changes, the molecular mechanisms leading to memory deficits are not clear. We investigated whether the segmental trisomy model of DS, Ts[Rb(12.1716)]2Cje (Ts2), exhibits hippocampal glutamatergic transmission abnormalities and whether these alterations cause behavioral deficits. Behavioral assays demonstrated that Ts2 mice display a deficit in nest building behavior, a measure of hippocampus-dependent nonlearned behavior, as well as dysfunctional hippocampus-dependent spatial memory tested in the object-placement and the Y-maze spontaneous alternation tasks. Magnetic resonance spectra measured in the hippocampi revealed a significantly lower glutamate concentration in Ts2 as compared with normal disomic (2N) littermates. The glutamate deficit accompanied hippocampal NMDA receptor1 (NMDA-R1) mRNA and protein expression level downregulation in Ts2 compared with 2N mice. In concert with these alterations, paired-pulse analyses suggested enhanced synaptic inhibition and/or lack of facilitation in the dentate gyrus of Ts2 compared with 2N mice. Ts2 mice also exhibited disrupted synaptic plasticity in slice recordings of the hippocampal CA1 region. Collectively, these findings imply that deficits in glutamate and NMDA-R1 may be responsible for impairments in synaptic plasticity in the hippocampus associated with behavioral dysfunctions in Ts2 mice. Thus, these findings suggest that glutamatergic deficits have a significant role in causing intellectual disabilities in DS.

  16. Effect of VGLUT inhibitors on glutamatergic synaptic transmission in the rodent hippocampus and prefrontal cortex.

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    Neale, S A; Copeland, C S; Salt, T E

    2014-07-01

    Vesicular glutamate transporters (VGLUTs) are known to be important in the uptake of glutamate into vesicles in the presynaptic terminal; thereby playing a role in synaptic function. VGLUT dysfunction has also been suggested in neurological and psychiatric disorders such as epilepsy and schizophrenia. A number of compounds have been identified as VGLUT inhibitors; however, little is known as to how these compounds affect synaptic transmission. We therefore investigated the effects of structurally unrelated VGLUT inhibitors on synaptic transmission in the rodent hippocampus and prefrontal cortex. In the CA1 and dentate gyrus regions of the in vitro slice preparation of mouse hippocampus, AMPA receptor-mediated field excitatory postsynaptic potentials (fEPSPs) were evoked in response to Schaffer collateral/commissural pathway stimulation. Application of the VGLUT inhibitors Rose Bengal (RB), Congo Red (CR) or Chicago Sky Blue 6B (CB) resulted in a concentration-related reduction of fEPSP amplitudes. RB (30μM) or CB (300μM) also depressed NMDA receptor-mediated responses in the CA1 region. The naturally occurring kynurenine Xanthurenic Acid (XA) is reported to be a VGLUT inhibitor. We found XA attenuated both AMPA and NMDA receptor-mediated synaptic transmission. The potency order of the VGLUT inhibitors was consistent with literature Ki values for VGLUT inhibition. Impaired glutamatergic neurotransmission is believed to contribute to schizophrenia, and VGLUTs have also been implicated in this disease. We therefore investigated the effect of VGLUT inhibition in the prefrontal cortex. Application of the VGLUT inhibitors RB or CB resulted in a concentration-dependent reduction in the amplitude of glutamate receptor-mediated fEPSPs recorded in layer V/VI in response to stimulation in the forceps minor. We conclude that VGLUT inhibitors can modulate glutamatergic synaptic transmission in the PFC and hippocampus. This could be important in the pathophysiology of nervous

  17. Mice deficient of glutamatergic signaling from intrinsically photosensitive retinal ganglion cells exhibit abnormal circadian photoentrainment.

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    Nicole Purrier

    Full Text Available Several aspects of behavior and physiology, such as sleep and wakefulness, blood pressure, body temperature, and hormone secretion exhibit daily oscillations known as circadian rhythms. These circadian rhythms are orchestrated by an intrinsic biological clock in the suprachiasmatic nuclei (SCN of the hypothalamus which is adjusted to the daily environmental cycles of day and night by the process of photoentrainment. In mammals, the neuronal signal for photoentrainment arises from a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs that send a direct projection to the SCN. ipRGCs also mediate other non-image-forming (NIF visual responses such as negative masking of locomotor activity by light, and the pupillary light reflex (PLR via co-release of neurotransmitters glutamate and pituitary adenylate cyclase-activating peptide (PACAP from their synaptic terminals. The relative contribution of each neurotransmitter system for the circadian photoentrainment and other NIF visual responses is still unresolved. We investigated the role of glutamatergic neurotransmission for circadian photoentrainment and NIF behaviors by selective ablation of ipRGC glutamatergic synaptic transmission in mice. Mutant mice displayed delayed re-entrainment to a 6 h phase shift (advance or delay in the light cycle and incomplete photoentrainment in a symmetrical skeleton photoperiod regimen (1 h light pulses between 11 h dark periods. Circadian rhythmicity in constant darkness also was reduced in some mutant mice. Other NIF responses such as the PLR and negative masking responses to light were also partially attenuated. Overall, these results suggest that glutamate from ipRGCs drives circadian photoentrainment and negative masking responses to light.

  18. Cannabinoid Type 1 Receptors Transiently Silence Glutamatergic Nerve Terminals of Cultured Cerebellar Granule Cells

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    Ramírez-Franco, Jorge; Bartolomé-Martín, David; Alonso, Beatris; Torres, Magdalena; Sánchez-Prieto, José

    2014-01-01

    Cannabinoid receptors are the most abundant G protein-coupled receptors in the brain and they mediate retrograde short-term inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at many excitatory synapses. The induction of presynaptically silent synapses is a means of modulating synaptic strength, which is important for synaptic plasticity. Persistent activation of cannabinoid type 1 receptors (CB1Rs) mutes GABAergic terminals, although it is unclear if CB1Rs can also induce silencing at glutamatergic synapses. Cerebellar granule cells were transfected with VGLUT1-pHluorin to visualise the exo-endocytotic cycle. We found that prolonged stimulation (10 min) of cannabinoid receptors with the agonist HU-210 induces the silencing of previously active synapses. However, the presynaptic silencing induced by HU-210 is transient as it reverses after 20 min. cAMP with forskolin prevented CB1R-induced synaptic silencing, via activation of the Exchange Protein directly Activated by cAMP (Epac). Furthermore, Epac activation accelerated awakening of already silent boutons. Electron microscopy revealed that silencing was associated with synaptic vesicle (SV) redistribution within the nerve terminal, which diminished the number of vesicles close to the active zone of the plasma membrane. Finally, by combining functional and immunocytochemical approaches, we observed a strong correlation between the release capacity of the nerve terminals and RIM1α protein content, but not that of Munc13-1 protein. These results suggest that prolonged stimulation of cannabinoid receptors can transiently silence glutamatergic nerve terminals. PMID:24533119

  19. Glutamatergic mechanisms associated with stress-induced amygdala excitability and anxiety-related behavior.

    Science.gov (United States)

    Masneuf, Sophie; Lowery-Gionta, Emily; Colacicco, Giovanni; Pleil, Kristen E; Li, Chia; Crowley, Nicole; Flynn, Shaun; Holmes, Andrew; Kash, Thomas

    2014-10-01

    The neural factors underlying individual differences in susceptibility to chronic stress remain poorly understood. Preclinical studies demonstrate that mouse strains vary greatly in anxiety-related responses to chronic stress in a manner paralleled by differential stress-induced changes in glutamatergic signaling in the basolateral amygdala (BLA). Previous work has also shown that alterations in the amygdala gene expression of the GluN1 NMDA and the GluK1 kainate receptors are associated with stress-induced alterations in anxiety-like behavior in the C57BL/6J mouse strain. Using in vivo behavioral pharmacological and ex vivo physiological approaches, the aim of the current study was to further elucidate changes in glutamate neurotransmission in the BLA caused by stress and to test the functional roles of GluN1 and GluK1 in mediating stress-related changes in behavior. Results showed that stress-induced alterations in anxiety-like behavior (light/dark exploration test) were absent following bilateral infusion of the GluK1 agonist ATPA into the BLA. Intra-BLA infusion of the competitive NMDA antagonist AP5 produced a generalized behavioral disinhibition/locomotor hyperactivity, irrespective of stress. Slice electrophysiological recordings showed that ATPA augmented BLA GABAergic neurotransmission and that stress increased the amplitude of network-dependent spontaneous excitatory postsynaptic currents and amplitude of GABAergic miniature inhibitory postsynaptic currents in BLA. These findings could indicate stress-induced BLA glutamatergic neuronal network hyperexcitability and a compensatory increase in GABAergic neurotransmission, suggesting that GluK1 agonism augmented GABAergic inhibition to prevent behavioral sequelae of stress. Current data could have implications for developing novel therapeutic approaches, including GluK1 agonists, for stress-related anxiety disorders.

  20. Differential presynaptic actions of pyrethroid insecticides on glutamatergic and GABAergic neurons in the hippocampus.

    Science.gov (United States)

    Hossain, Muhammad Mubarak; Suzuki, Tadahiko; Unno, Toshihiro; Komori, Seiichi; Kobayashi, Haruo

    2008-01-14

    This study was designed to investigate the effects of several pyrethroids on the extracellular level of glutamate and gamma-aminobutyric acid (GABA) in the hippocampus of rats measured using microdialysis following systemic (i.p.) administration. Pyrethroids, allethrin (type I), cyhalothrin (type II) and deltamethrin (type II), were found to have differential effects on glutamatergic and GABAergic neurons in the hippocampus. Allethrin had an interesting dual effect, increasing glutamate release with low doses (10 and 20mg/kg) to about 175-150% and decreasing glutamate release with high dose (60 mg/kg) to about 50% of baseline. Cyhalothrin (10, 20 and 60 mg/kg) inhibited the release of glutamate dose-dependently to about 60-30% of baseline. The extracellular level of GABA was decreased to about 50% of baseline by 10 and 20mg/kg allethrin. The high dose of allethrin (60 mg/kg) and all doses of cyhalothrin (10, 20 and 60 mg/kg) increased the extracellular level of GABA while decreasing the level of glutamate. Deltamethrin dose-dependently increased extracellular glutamate levels to about 190-275% of baseline while decreasing the level of GABA. Local infusion of TTX (1 microM), a Na(+) channel blocker, completely prevented the effect of allethrin (10, 20 and 60 mg/kg), cyhalothrin (20 and 60 mg/kg) and deltamethrin (20mg/kg) on glutamate and GABA release, but only partially blocked the effects of 60 mg/kg deltamethrin. The effect of deltamethrin (60 mg/kg) on glutamate release was completely prevented by local infusion of nimodipine (10 microM), an L-type Ca(2+) channel blocker. Collectively, results from this study suggest that the excitatory glutamatergic neurons in the hippocampus are modulated by inhibitory GABA-releasing interneurons and that other mechanisms, beside sodium channels, may be involved with the neurotoxic action of pyrethroids.

  1. Toluene inhalation in adolescent rats reduces flexible behaviour in adulthood and alters glutamatergic and GABAergic signalling.

    Science.gov (United States)

    Furlong, Teri M; Duncan, Jhodie R; Corbit, Laura H; Rae, Caroline D; Rowlands, Benjamin D; Maher, Anthony D; Nasrallah, Fatima A; Milligan, Carol J; Petrou, Steven; Lawrence, Andrew J; Balleine, Bernard W

    2016-12-01

    Toluene is a commonly abused inhalant that is easily accessible to adolescents. Despite the increasing incidence of use, our understanding of its long-term impact remains limited. Here, we used a range of techniques to examine the acute and chronic effects of toluene exposure on glutameteric and GABAergic function, and on indices of psychological function in adult rats after adolescent exposure. Metabolomics conducted on cortical tissue established that acute exposure to toluene produces alterations in cellular metabolism indicative of a glutamatergic and GABAergic profile. Similarly, in vitro electrophysiology in Xenopus oocytes found that acute toluene exposure reduced NMDA receptor signalling. Finally, in an adolescent rodent model of chronic intermittent exposure to toluene (10 000 ppm), we found that, while toluene exposure did not affect initial learning, it induced a deficit in updating that learning when response-outcome relationships were reversed or degraded in an instrumental conditioning paradigm. There were also group differences when more effort was required to obtain the reward; toluene-exposed animals were less sensitive to progressive ratio schedules and to delayed discounting. These behavioural deficits were accompanied by changes in subunit expression of both NMDA and GABA receptors in adulthood, up to 10 weeks after the final exposure to toluene in the hippocampus, prefrontal cortex and ventromedial striatum; regions with recognized roles in behavioural flexibility and decision-making. Collectively, our data suggest that exposure to toluene is sufficient to induce adaptive changes in glutamatergic and GABAergic systems and in adaptive behaviour that may underlie the deficits observed following adolescent inhalant abuse, including susceptibility to further drug-use. © 2016 International Society for Neurochemistry.

  2. Interaction of PGHS-2 and glutamatergic mechanisms controlling the ovine fetal hypothalamus-pituitary-adrenal axis.

    Science.gov (United States)

    Knutson, Nathan; Wood, Charles E

    2010-07-01

    Prostaglandins, generated within the fetal brain, are integral components of the mechanism controlling the fetal hypothalamus-pituitary-adrenal (HPA) axis. Previous studies in this laboratory demonstrated that prostaglandin G/H synthase isozyme 2 (PGHS-2) inhibition reduces the fetal HPA axis response to cerebral hypoperfusion, blocks the preparturient rise in fetal plasma ACTH concentration, and delays parturition. We also discovered that blockade of N-methyl-d-aspartate (NMDA) receptors reduces the fetal ACTH response to cerebral hypoperfusion. The present study was designed to test the hypothesis that PGHS-2 action and the downstream effect of HPA axis stimulation are stimulated by NMDA-mediated glutamatergic neurotransmission. Chronically catheterized late-gestation fetal sheep (n = 8) were injected with NMDA (1 mg iv). All responded with increases in fetal plasma ACTH and cortisol concentrations. Pretreatment with resveratrol (100 mg iv, n = 5), a specific inhibitor of PGHS-1, did not alter the magnitude of the HPA axis response to NMDA. Pretreatment with nimesulide (10 mg iv, n = 6), a specific inhibitor of PGHS-2, significantly reduced the HPA axis response to NMDA. To further explore this interaction, we injected NMDA in six chronically catheterized fetal sheep that were chronically infused with nimesulide (n = 6) at a rate of 1 mg/day into the lateral cerebral ventricle for 5-7 days. In this group, there was no significant ACTH response to NMDA. Finally, we tested whether the HPA axis response to prostaglandin E(2) (PGE(2)) is mediated by NMDA receptors. Seven chronically catheterized late-gestation fetal sheep were injected with 100 ng of PGE(2), which significantly increased fetal plasma ACTH and cortisol concentrations. Pretreatment with ketamine (10 mg iv), an NMDA antagonist, did not alter the ACTH or cortisol response to PGE(2). We conclude that generation of prostanoids via the action of PGHS-2 in the fetal brain augments the fetal HPA axis response

  3. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

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    Rafael Rodríguez-Muñoz

    Full Text Available The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f, during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV. By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60% and multipolar Glutamatergic (≤40% neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC: dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively, in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  4. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Science.gov (United States)

    Rodríguez-Muñoz, Rafael; Cárdenas-Aguayo, María Del Carmen; Alemán, Víctor; Osorio, Beatriz; Chávez-González, Oscar; Rendon, Alvaro; Martínez-Rojas, Dalila; Meraz-Ríos, Marco Antonio

    2015-01-01

    The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  5. Paracrine SDF-1α signaling mediates the effects of PSCs on GEM chemoresistance through an IL-6 autocrine loop in pancreatic cancer cells.

    Science.gov (United States)

    Zhang, Hui; Wu, Huanwen; Guan, Jian; Wang, Li; Ren, Xinyu; Shi, Xiaohua; Liang, Zhiyong; Liu, Tonghua

    2015-02-20

    Pancreatic cancer exhibits the poorest prognosis among all tumors and is characterized by high resistance to the currently available chemotherapeutic agents. Our previous studies have suggested that stromal components could promote the chemoresistance of pancreatic cancer cells (PCCs). Here, we explored the roles of pancreatic stellate cells (PSCs) and the SDF-1α/CXCR4 axis in pancreatic cancer chemoresitance. Our results showed that primary PSCs typically expressed SDF-1α, whereas its receptor CXCR4 was highly expressed in PCCs. PSC-conditioned medium (PSC-CM) inhibited Gemcitabine (GEM)-induced cytotoxicity and apoptosis in the human PCC line Panc-1, which was antagonized by an SDF-1α neutralizing Ab. Recombinant human SDF-1α (rhSDF-1α) increased IL-6 expression and secretion in Panc-1 cells in a time and dose-dependent manner, and this effect was suppressed by the CXCR4 antagonist AMD3100. rhSDF-1α protected Panc-1 cells from GEM-induced apoptosis, and the protective effect was significantly reduced by blocking IL-6 using a neutralizing antibody. Moreover, rhSDF-1α increased FAK, ERK1/2, AKT and P38 phosphorylation in Panc-1 cells, and either FAK or ERK1/2 inhibition suppressed SDF-1α-upregulated IL-6 expression. SDF-1α-induced AKT activation was almost completely blocked by FAK inhibition. In conclusion, we demonstrate for the first time that PSCs promote the chemoresistance of PCCs to GEM, and this effect is mediated by paracrine SDF-1α/CXCR4 signaling-induced activation of the intracellular FAK-AKT and ERK1/2 signaling pathways and a subsequent IL-6 autocrine loop in PCCs. Our findings indicate that blocking the PSC-PCC interaction by inhibiting SDF-1α/CXCR4 signaling may be a promising therapeutic strategy for overcoming chemoresistance in pancreatic cancer.

  6. Adenosine A₂A receptors in striatal glutamatergic terminals and GABAergic neurons oppositely modulate psychostimulant action and DARPP-32 phosphorylation.

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    Hai-Ying Shen

    Full Text Available Adenosine A2A receptors (A2AR are located postsynaptically in striatopallidal GABAergic neurons, antagonizing dopamine D2 receptor functions, and are also located presynaptically at corticostriatal terminals, facilitating glutamate release. To address the hypothesis that these two A2AR populations differently control the action of psychostimulants, we characterized A2AR modulation of cocaine-induced effects at the level of DARPP-32 phosphorylation at Thr-34 and Thr-75, c-Fos expression, and psychomotor activity using two lines of cell-type selective A2AR knockout (KO mice with selective A2AR deletion in GABAergic neurons (striatum-A2AR-KO mice, or with A2AR deletion in both striatal GABAergic neurons and projecting cortical glutamatergic neurons (forebrain-A2AR-KO mice. We demonstrated that striatum-A2AR KO mice lacked A2ARs exclusively in striatal GABAergic terminals whereas forebrain-A2AR KO mice lacked A2ARs in both striatal GABAergic and glutamatergic terminals leading to a blunted A2AR-mediated facilitation of synaptosomal glutamate release. The inactivation of A2ARs in GABAergic neurons reduced striatal DARPP-32 phosphorylation at Thr-34 and increased its phosphorylation at Thr-75. Conversely, the additional deletion of corticostriatal glutamatergic A2ARs produced opposite effects on DARPP-32 phosphorylation at Thr-34 and Thr-75. This distinct modulation of DARPP-32 phosphorylation was associated with opposite responses to cocaine-induced striatal c-Fos expression and psychomotor activity in striatum-A2AR KO (enhanced and forebrain-A2AR KO mice (reduced. Thus, A2ARs in glutamatergic corticostriatal terminals and in GABAergic striatal neurons modulate the action of psychostimulants and DARPP-32 phosphorylation in opposite ways. We conclude that A2ARs in glutamatergic terminals prominently control the action of psychostimulants and define a novel mechanism by which A2ARs fine-tune striatal activity by integrating GABAergic, dopaminergic and

  7. The Planar Cell Polarity Transmembrane Protein Vangl2 Promotes Dendrite, Spine and Glutamatergic Synapse Formation in the Mammalian Forebrain.

    Science.gov (United States)

    Okerlund, Nathan D; Stanley, Robert E; Cheyette, Benjamin N R

    2016-07-01

    The transmembrane protein Vangl2, a key regulator of the Wnt/planar cell polarity (PCP) pathway, is involved in dendrite arbor elaboration, dendritic spine formation and glutamatergic synapse formation in mammalian central nervous system neurons. Cultured forebrain neurons from Vangl2 knockout mice have simpler dendrite arbors, fewer total spines, less mature spines and fewer glutamatergic synapse inputs on their dendrites than control neurons. Neurons from mice heterozygous for a semidominant Vangl2 mutation have similar but not identical phenotypes, and these phenotypes are also observed in Golgi-stained brain tissue from adult mutant mice. Given increasing evidence linking psychiatric pathophysiology to these subneuronal sites and structures, our findings underscore the relevance of core PCP proteins including Vangl2 to the underlying biology of major mental illnesses and their treatment.

  8. Inositol 1,4,5-triphosphate drives glutamatergic and cholinergic inhibition selectively in spiny projection neurons in the striatum.

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    Clements, Michael A; Swapna, Immani; Morikawa, Hitoshi

    2013-02-06

    The striatum is critically involved in the selection of appropriate actions in a constantly changing environment. The spiking activity of striatal spiny projection neurons (SPNs), driven by extrinsic glutamatergic inputs, is shaped by local GABAergic and cholinergic networks. For example, it is well established that different types of GABAergic interneurons, activated by extrinsic glutamatergic and local cholinergic inputs, mediate powerful feedforward inhibition of SPN activity. In this study, using mouse striatal slices, we show that glutamatergic and cholinergic inputs exert direct inhibitory regulation of SPN activity via activation of metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors. While pressure ejection of the group I mGluR (mGluR1/5) agonist DHPG [(S)-3,5-dihydroxyphenylglycine] equally engages both mGluR1 and mGluR5 subtypes, the mGluR-dependent component of IPSCs elicited by intrastriatal electrical stimulation is almost exclusively mediated by the mGluR1 subtype. Ca(2+) release from intracellular stores specifically through inositol 1,4,5-triphospahte receptors (IP(3)Rs) and not ryanodine receptors (RyRs) mediates this form of inhibition by gating two types of Ca(2+)-activated K(+) channels (i.e., small-conductance SK channels and large-conductance BK channels). Conversely, spike-evoked Ca(2+) influx triggers Ca(2+) release solely through RyRs to generate SK-dependent slow afterhyperpolarizations, demonstrating functional segregation of IP(3)Rs and RyRs. Finally, IP(3)-induced Ca(2+) release is uniquely observed in SPNs and not in different types of interneurons in the striatum. These results demonstrate that IP(3)-mediated activation of SK and BK channels provides a robust mechanism for glutamatergic and cholinergic inputs to selectively suppress striatal output neuron activity.

  9. Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles

    OpenAIRE

    Robert W Banks; Cahusac, Peter M. B.; Graca, Anna; Kain, Nakul; Shenton, Fiona; Singh, Paramjeet; Njå, Arild; Simon, Anna; Watson, Sonia; Slater, Clarke R; Bewick, Guy S.

    2013-01-01

    Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending we have described previously. Both types of endings are formed by the peripheral sensory terminals of primary mechanosensory dorsal root ganglion cells, so the presence of such a system in the lanceolate end...

  10. Unbalance of CB1 receptors expressed in GABAergic and glutamatergic neurons in a transgenic mouse model of Huntington's disease.

    Science.gov (United States)

    Chiodi, Valentina; Uchigashima, Motokazu; Beggiato, Sarah; Ferrante, Antonella; Armida, Monica; Martire, Alberto; Potenza, Rosa Luisa; Ferraro, Luca; Tanganelli, Sergio; Watanabe, Masahiko; Domenici, Maria Rosaria; Popoli, Patrizia

    2012-03-01

    Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntington's disease (HD). However, the functional meaning of this reduction, if any, is still unclear. Here, the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN) were investigated on striatal synaptic transmission and on glutamate and GABA release in symptomatic R6/2 mice, a genetic model of HD. The expression levels of CB1Rs in glutamatergic and GABAergic synapses were also evaluated. We found that in R6/2 mice, WIN effects on synaptic transmission and glutamate release were significantly increased with respect to wild type mice. On the contrary, a decrease in WIN-induced reduction of GABA release was found in R6/2 versus WT mice. The expression of CB1Rs in GABAergic neurons was drastically reduced, while CB1Rs levels in glutamatergic neurons were unchanged. These results demonstrate that the expression and functionality of CB1Rs are differentially affected in GABAergic and glutamatergic neurons in R6/2 mice. As a result, the balance between CB1Rs expressed by the two neuronal populations and, thus, the net effect of CB1R stimulation, is profoundly altered in HD mice.

  11. Activation of steroid-sensitive TRPM3 channels potentiates glutamatergic transmission at cerebellar Purkinje neurons from developing rats.

    Science.gov (United States)

    Zamudio-Bulcock, Paula A; Everett, Julie; Harteneck, Christian; Valenzuela, C Fernando

    2011-11-01

    The functional implications of transient receptor potential melastatin 3 (TRPM3) activation, the most recently described member of the melastatin subfamily of cation permeable TRP channels, have begun to be elucidated in recent years. The discovery of TRPM3 activation by the steroid pregnenolone sulfate (PregS) has shed new light on the physiological role of this channel. For example, TRPM3 activation enhances insulin secretion from β pancreatic cells, induces contraction of vascular smooth muscle, and is also involved in the detection of noxious heat. Although TRPM3 expression has been detected in several regions of the developing and mature brain, little is known about the roles of TRPM3 in brain physiology. In this study, we demonstrate the abundant expression of TRPM3 steroid-sensitive channels in the developing cerebellar cortex. We also show that TRPM3-like channels are expressed at glutamatergic synapses in neonatal Purkinje cells. We recently showed that PregS potentiates spontaneous glutamate release onto neonatal Purkinje cells during a period of active glutamatergic synapse formation; we now show that this effect of PregS is mediated by TRPM3-like channels. Mefenamic acid, a recently discovered TRPM3 antagonist, blocked the effect of PregS on glutamate release. The PregS effect on glutamate release was mimicked by other TRPM3 agonists (nifedipine and epipregnanolone sulfate) but not by a TRMP3-inactive steroid (progesterone). Our findings identify TRPM3 channels as novel modulators of glutamatergic transmission in the developing brain.

  12. Early sequential formation of functional GABA(A) and glutamatergic synapses on CA1 interneurons of the rat foetal hippocampus.

    Science.gov (United States)

    Hennou, Sonia; Khalilov, Ilgam; Diabira, Diabé; Ben-Ari, Yehezkel; Gozlan, Henri

    2002-07-01

    During postnatal development of CA1 pyramidal neurons, GABAergic synapses are excitatory and established prior to glutamatergic synapses. As interneurons are generated before pyramidal cells, we have tested the hypothesis that the GABAergic interneuronal network is operative before glutamate pyramidal neurons and provides the initial patterns of activity. We patch-clamp recorded interneurons in foetal (69 neurons) and neonatal P0 (162 neurons) hippocampal slices and performed a morphofunctional analysis of biocytin-filled neurons. At P0, three types of interneurons were found: (i) non-innervated "silent" interneurons (5%) with no spontaneous or evoked synaptic currents; (ii) G interneurons (17%) with GABA(A) synapses only; and (iii) GG interneurons with GABA and glutamatergic synapses (78%). Relying on the neuronal capacitance, cell body size and arborization of dendrites and axons, the three types of interneurons correspond to three stages of development with non-innervated neurons and interneurons with GABA(A) and glutamatergic synapses being, respectively, the least and the most developed. Recordings from both pyramidal neurons and interneurons in foetuses (E18-20) revealed that the majority of interneurons (65%) had functional synapses whereas nearly 90% of pyramidal neurons were quiescent. Therefore, interneurons follow the same GABA-glutamate sequence of synapse formation but earlier than the principal cells. Interneurons are the source and the target of the first synapses formed in the hippocampus and are thus in a position to modulate the development of the hippocampus in the foetal stage.

  13. In vivo effects of antibodies from patients with anti-NMDA receptor encephalitis: further evidence of synaptic glutamatergic dysfunction.

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    Manto, Mario; Dalmau, Josep; Didelot, Adrien; Rogemond, Véronique; Honnorat, Jérôme

    2010-11-26

    A severe encephalitis that associates with auto-antibodies to the NR1 subunit of the NMDA receptor (NMDA-R) was recently reported. Patients' antibodies cause a decrease of the density of NMDA-R and synaptic mediated currents, but the in vivo effects on the extracellular glutamate and glutamatergic transmission are unknown. We investigated the acute metabolic effects of patients' CSF and purified IgG injected in vivo. Injections were performed in CA1 area of Ammon's horn and in premotor cortex in rats. Patient's CSF increased the concentrations of glutamate in the extracellular space. The increase was dose-dependent and was dramatic with purified IgG. Patients' CSF impaired both the NMDA- and the AMPA-mediated synaptic regulation of glutamate, and did not affect the glial transport of glutamate. Blockade of GABA-A receptors was associated with a marked elevation of extra-cellular levels of glutamate following a pretreatment with patients' CSF. These results support a direct role of NMDA-R antibodies upon altering glutamatergic transmission. Furthermore, we provide additional evidence in vivo that NMDA-R antibodies deregulate the glutamatergic pathways and that the encephalitis associated with these antibodies is an auto-immune synaptic disorder.

  14. In vivo effects of antibodies from patients with anti-NMDA receptor encephalitis: further evidence of synaptic glutamatergic dysfunction

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    Manto Mario

    2010-11-01

    Full Text Available Abstract Background A severe encephalitis that associates with auto-antibodies to the NR1 subunit of the NMDA receptor (NMDA-R was recently reported. Patients' antibodies cause a decrease of the density of NMDA-R and synaptic mediated currents, but the in vivo effects on the extracellular glutamate and glutamatergic transmission are unknown. Methods We investigated the acute metabolic effects of patients' CSF and purified IgG injected in vivo. Injections were performed in CA1 area of Ammon's horn and in premotor cortex in rats. Results Patient's CSF increased the concentrations of glutamate in the extracellular space. The increase was dose-dependent and was dramatic with purified IgG. Patients' CSF impaired both the NMDA- and the AMPA-mediated synaptic regulation of glutamate, and did not affect the glial transport of glutamate. Blockade of GABA-A receptors was associated with a marked elevation of extra-cellular levels of glutamate following a pretreatment with patients' CSF. Conclusion These results support a direct role of NMDA-R antibodies upon altering glutamatergic transmission. Furthermore, we provide additional evidence in vivo that NMDA-R antibodies deregulate the glutamatergic pathways and that the encephalitis associated with these antibodies is an auto-immune synaptic disorder.

  15. Abundance of gap junctions at glutamatergic mixed synapses in adult Mosquitofish spinal cord neurons

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    Jose L Serrano-Velez

    2014-06-01

    Full Text Available Dye-coupling, whole-mount immunohistochemistry for gap junction channel protein connexin 35 (Cx35, and freeze-fracture replica immunogold labeling (FRIL reveal an abundance of electrical synapses/gap junctions at glutamatergic mixed synapses in the 14th spinal segment that innervates the adult male gonopodium of Western Mosquitofish, Gambusia affinis (Mosquitofish.To study gap junctions’ role in fast motor behavior, we used a minimally-invasive neural-tract-tracing technique to introduce gap junction-permeant or -impermeant dyes into deep muscles controlling the gonopodium of the adult male Mosquitofish, a teleost fish that rapidly transfers (complete in 50 of the 62 gap junctions at mixed synapses are in the 14th spinal segment.Our results support and extend studies showing gap junctions at mixed synapses in spinal cord segments involved in control of genital reflexes in rodents, and they suggest a link between mixed synapses and fast motor behavior. The findings provide a basis for studies of specific roles of spinal neurons in the generation/regulation of sex-specific behavior and for studies of gap junctions’ role in regulating fast motor behavior. Finally, the CoPA IN provides a novel candidate neuron for future studies of gap junctions and neural control of fast motor behaviors.

  16. Key role of the dopamine D4 receptor in the modulation of corticostriatal glutamatergic neurotransmission

    Science.gov (United States)

    Bonaventura, Jordi; Quiroz, César; Cai, Ning-Sheng; Rubinstein, Marcelo; Tanda, Gianluigi; Ferré, Sergi

    2017-01-01

    Polymorphic variants of the dopamine D4 receptor gene (DRD4) have been repeatedly associated with numerous neuropsychiatric disorders. Yet, the functional role of the D4 receptor and the functional differences of the products of DRD4 polymorphic variants remained enigmatic. Immunohistochemical and optogenetic-microdialysis experiments were performed in knock-in mice expressing a D4 receptor with the long intracellular domain of a human DRD4 polymorphic variant associated with attention deficit hyperactivity disorder (ADHD). When compared with the wild-type mouse D4 receptor, the expanded intracellular domain of the humanized D4 receptor conferred a gain of function, blunting methamphetamine-induced cortical activation and optogenetic and methamphetamine-induced corticostriatal glutamate release. The results demonstrate a key role of the D4 receptor in the modulation of corticostriatal glutamatergic neurotransmission. Furthermore, these data imply that enhanced D4 receptor–mediated dopaminergic control of corticostriatal transmission constitutes a vulnerability factor of ADHD and other neuropsychiatric disorders. PMID:28097219

  17. Morphine disinhibits glutamatergic input to VTA dopamine neurons and promotes dopamine neuron excitation.

    Science.gov (United States)

    Chen, Ming; Zhao, Yanfang; Yang, Hualan; Luan, Wenjie; Song, Jiaojiao; Cui, Dongyang; Dong, Yi; Lai, Bin; Ma, Lan; Zheng, Ping

    2015-07-24

    One reported mechanism for morphine activation of dopamine (DA) neurons of the ventral tegmental area (VTA) is the disinhibition model of VTA-DA neurons. Morphine inhibits GABA inhibitory neurons, which shifts the balance between inhibitory and excitatory input to VTA-DA neurons in favor of excitation and then leads to VTA-DA neuron excitation. However, it is not known whether morphine has an additional strengthening effect on excitatory input. Our results suggest that glutamatergic input to VTA-DA neurons is inhibited by GABAergic interneurons via GABAB receptors and that morphine promotes presynaptic glutamate release by removing this inhibition. We also studied the contribution of the morphine-induced disinhibitory effect on the presynaptic glutamate release to the overall excitatory effect of morphine on VTA-DA neurons and related behavior. Our results suggest that the disinhibitory action of morphine on presynaptic glutamate release might be the main mechanism for morphine-induced increase in VTA-DA neuron firing and related behaviors.

  18. Activity-dependent switch of GABAergic inhibition into glutamatergic excitation in astrocyte-neuron networks

    Science.gov (United States)

    Perea, Gertrudis; Gómez, Ricardo; Mederos, Sara; Covelo, Ana; Ballesteros, Jesús J; Schlosser, Laura; Hernández-Vivanco, Alicia; Martín-Fernández, Mario; Quintana, Ruth; Rayan, Abdelrahman; Díez, Adolfo; Fuenzalida, Marco; Agarwal, Amit; Bergles, Dwight E; Bettler, Bernhard; Manahan-Vaughan, Denise; Martín, Eduardo D; Kirchhoff, Frank; Araque, Alfonso

    2016-01-01

    Interneurons are critical for proper neural network function and can activate Ca2+ signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, we found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and determined the sign (inhibition vs potentiation) of the GABA-mediated effects. While synaptic inhibition was mediated by GABAA receptors, potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1) knockout mice, we confirmed the glial source of the interneuron-induced potentiation, and demonstrated the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay. DOI: http://dx.doi.org/10.7554/eLife.20362.001 PMID:28012274

  19. Synergistic regulation of glutamatergic transmission by serotonin and norepinephrine reuptake inhibitors in prefrontal cortical neurons.

    Science.gov (United States)

    Yuen, Eunice Y; Qin, Luye; Wei, Jing; Liu, Wenhua; Liu, Aiyi; Yan, Zhen

    2014-09-05

    The monoamine system in the prefrontal cortex has been implicated in various mental disorders and has been the major target of anxiolytics and antidepressants. Clinical studies show that serotonin and norepinephrine reuptake inhibitors (SNRIs) produce better therapeutic effects than single selective reuptake inhibitors, but the underlying mechanisms are largely unknown. Here, we found that low dose SNRIs, by acting on 5-HT(1A) and α2-adrenergic receptors, synergistically reduced AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents and AMPAR surface expression in prefrontal cortex pyramidal neurons via a mechanism involving Rab5/dynamin-mediated endocytosis of AMPARs. The synergistic effect of SNRIs on AMPARs was blocked by inhibition of activator of G protein signaling 3, a G protein modulator that prevents reassociation of G(i) protein α subunit and prolongs the βγ-mediated signaling pathway. Moreover, the depression of AMPAR-mediated excitatory postsynaptic currents by SNRIs required p38 kinase activity, which was increased by 5-HT(1A) and α2-adrenergic receptor co-activation in an activator of G protein signaling 3-dependent manner. These results have revealed a potential mechanism for the synergy between the serotonin and norepinephrine systems in the regulation of glutamatergic transmission in cortical neurons.

  20. Transmembrane protein 108 is required for glutamatergic transmission in dentate gyrus.

    Science.gov (United States)

    Jiao, Hui-Feng; Sun, Xiang-Dong; Bates, Ryan; Xiong, Lei; Zhang, Lei; Liu, Fang; Li, Lei; Zhang, Hong-Sheng; Wang, Shun-Qi; Xiong, Ming-Tao; Patel, Mihir; Stranahan, Alexis M; Xiong, Wen-Cheng; Li, Bao-Ming; Mei, Lin

    2017-01-31

    Neurotransmission in dentate gyrus (DG) is critical for spatial coding, learning memory, and emotion processing. Although DG dysfunction is implicated in psychiatric disorders, including schizophrenia, underlying pathological mechanisms remain unclear. Here we report that transmembrane protein 108 (Tmem108), a novel schizophrenia susceptibility gene, is highly enriched in DG granule neurons and its expression increased at the postnatal period critical for DG development. Tmem108 is specifically expressed in the nervous system and enriched in the postsynaptic density fraction. Tmem108-deficient neurons form fewer and smaller spines, suggesting that Tmem108 is required for spine formation and maturation. In agreement, excitatory postsynaptic currents of DG granule neurons were decreased in Tmem108 mutant mice, indicating a hypofunction of glutamatergic activity. Further cell biological studies indicate that Tmem108 is necessary for surface expression of AMPA receptors. Tmem108-deficient mice display compromised sensorimotor gating and cognitive function. Together, these observations indicate that Tmem108 plays a critical role in regulating spine development and excitatory transmission in DG granule neurons. When Tmem108 is mutated, mice displayed excitatory/inhibitory imbalance and behavioral deficits relevant to schizophrenia, revealing potential pathophysiological mechanisms of schizophrenia.

  1. Restoration of glutamatergic transmission by dopamine D4 receptors in stressed animals.

    Science.gov (United States)

    Yuen, Eunice Y; Zhong, Ping; Li, Xiangning; Wei, Jing; Yan, Zhen

    2013-09-06

    The prefrontal cortex (PFC), a key brain region for cognitive and emotional processes, is highly regulated by dopaminergic inputs. The dopamine D4 receptor, which is enriched in PFC, has been implicated in mental disorders, such as attention deficit-hyperactivity disorder and schizophrenia. Recently we have found homeostatic regulation of AMPA receptor-mediated synaptic transmission in PFC pyramidal neurons by the D4 receptor, providing a potential mechanism for D4 in stabilizing cortical excitability. Because stress is tightly linked to adaptive and maladaptive changes associated with mental health and disorders, we examined the synaptic actions of D4 in stressed rats. We found that neural excitability was elevated by acute stress and dampened by repeated stress. D4 activation produced a potent reduction of excitatory transmission in acutely stressed animals and a marked increase of excitatory transmission in repeatedly stressed animals. These effects of D4 targeted GluA2-lacking AMPA receptors and relied on the bi-directional regulation of calcium/calmodulin kinase II activity. The restoration of PFC glutamatergic transmission in stress conditions may enable D4 receptors to serve as a synaptic stabilizer in normal and pathological conditions.

  2. Notch1 regulates hippocampal plasticity through interaction with the Reelin pathway, glutamatergic transmission and CREB signaling

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    Emanuele eBrai

    2015-11-01

    Full Text Available Notch signaling plays a crucial role in adult brain function such as synaptic plasticity, memory and olfaction. Several reports suggest an involvement of this pathway in neurodegenerative dementia. Yet, to date, the mechanism underlying Notch activity in mature neurons remains unresolved. In this work, we investigate how Notch regulates synaptic potentiation and contributes to the establishment of memory in mice. We observe that Notch1 is a postsynaptic receptor with functional interactions with the Reelin receptor, ApoER2, and the ionotropic receptor, NMDAR. Targeted loss of Notch1 in the hippocampal CA fields affects Reelin signaling by influencing Dab1 expression and impairs the synaptic potentiation achieved through Reelin stimulation. Further analysis indicates that loss of Notch1 affects the expression and composition of the NMDAR but not AMPAR. Glutamatergic signaling is further compromised through downregulation of CamKII and its secondary and tertiary messengers resulting in reduced CREB signaling. Our results identify Notch1 as an important regulator of mechanisms involved in synaptic plasticity and memory formation. These findings emphasize the possible involvement of this signaling receptor in dementia.

  3. Plasticity in Single Axon Glutamatergic Connection to GABAergic Interneurons Regulates Complex Events in the Human Neocortex.

    Science.gov (United States)

    Szegedi, Viktor; Paizs, Melinda; Csakvari, Eszter; Molnar, Gabor; Barzo, Pal; Tamas, Gabor; Lamsa, Karri

    2016-11-01

    In the human neocortex, single excitatory pyramidal cells can elicit very large glutamatergic EPSPs (VLEs) in inhibitory GABAergic interneurons capable of triggering their firing with short (3-5 ms) delay. Similar strong excitatory connections between two individual neurons have not been found in nonhuman cortices, suggesting that these synapses are specific to human interneurons. The VLEs are crucial for generating neocortical complex events, observed as single pyramidal cell spike-evoked discharge of cell assemblies in the frontal and temporal cortices. However, long-term plasticity of the VLE connections and how the plasticity modulates neocortical complex events has not been studied. Using triple and dual whole-cell recordings from synaptically connected human neocortical layers 2-3 neurons, we show that VLEs in fast-spiking GABAergic interneurons exhibit robust activity-induced long-term depression (LTD). The LTD by single pyramidal cell 40 Hz spike bursts is specific to connections with VLEs, requires group I metabotropic glutamate receptors, and has a presynaptic mechanism. The LTD of VLE connections alters suprathreshold activation of interneurons in the complex events suppressing the discharge of fast-spiking GABAergic cells. The VLEs triggering the complex events may contribute to cognitive processes in the human neocortex, and their long-term plasticity can alter the discharging cortical cell assemblies by learning.

  4. Nicotinic modulation of glutamatergic synaptic transmission in region CA3 of the hippocampus.

    Science.gov (United States)

    Giocomo, Lisa M; Hasselmo, Michael E

    2005-09-01

    Cholinergic modulation of synaptic transmission in the hippocampus appears to be involved in learning, memory and attentional processes. In brain slice preparations of hippocampal region CA3, we have explored the effect of nicotine on the afferent connections of stratum lacunosum moleculare (SLM) vs. the intrinsic connections of stratum radiatum (SR). Nicotine application had a lamina-selective effect, causing changes in synaptic transmission only in SLM. The nicotinic effect in SLM was characterized by a transient decrease in synaptic potential size followed by a longer period of enhancement of synaptic transmission. The effect was blocked by gamma-aminobutyric acid (GABA)ergic antagonists, indicating the role of GABAergic interneurons in the observed nicotinic effect. The biphasic nature of the nicotinic effect could be due to a difference in receptor subtypes, as supported by the effects of the nicotinic antagonists mecamylamine and methyllycaconitine. Nicotinic modulation of glutamatergic synaptic transmission could complement muscarinic suppression of intrinsic connections, amplifying incoming information and providing a physiological mechanism for the memory-enhancing effect of nicotine.

  5. Tangential migration of glutamatergic neurons and cortical patterning during development: Lessons from Cajal-Retzius cells.

    Science.gov (United States)

    Barber, Melissa; Pierani, Alessandra

    2016-08-01

    Tangential migration is a mode of cell movement, which in the developing cerebral cortex, is defined by displacement parallel to the ventricular surface and orthogonal to the radial glial fibers. This mode of long-range migration is a strategy by which distinct neuronal classes generated from spatially and molecularly distinct origins can integrate to form appropriate neural circuits within the cortical plate. While it was previously believed that only GABAergic cortical interneurons migrate tangentially from their origins in the subpallial ganglionic eminences to integrate in the cortical plate, it is now known that transient populations of glutamatergic neurons also adopt this mode of migration. These include Cajal-Retzius cells (CRs), subplate neurons (SPs), and cortical plate transient neurons (CPTs), which have crucial roles in orchestrating the radial and tangential development of the embryonic cerebral cortex in a noncell-autonomous manner. While CRs have been extensively studied, it is only in the last decade that the molecular mechanisms governing their tangential migration have begun to be elucidated. To date, the mechanisms of SPs and CPTs tangential migration remain unknown. We therefore review the known signaling pathways, which regulate parameters of CRs migration including their motility, contact-redistribution and adhesion to the pial surface, and discuss this in the context of how CR migration may regulate their signaling activity in a spatial and temporal manner. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 847-881, 2016.

  6. Glutamatergic Mechanisms of Comorbidity Between Acute Stress and Cocaine Self-administration

    Science.gov (United States)

    Garcia-Keller, Constanza; Kupchik, Yonatan; Gipson, Cassandra D; Brown, Robyn M; Spencer, Sade; Bollati, Flavia; Esparza, Maria A; Roberts-Wolfe, Doug; Heinsbroek, Jasper; Bobadilla, Ana-Clara; Cancela, Liliana M; Kalivas, Peter W

    2015-01-01

    There is substantial comorbidity between stress disorders and substance use disorders (SUDs), and acute stress augments the locomotor stimulant effect of cocaine in animal models. Here we endeavor to understand the neural underpinnings of comorbid stress disorders and drug use by determining if the glutamatergic neuroadaptations that characterize cocaine self-administration are induced by acute stress. Rats were exposed to acute (2 h) immobilization stress and 3 weeks later the nucleus accumbens core was examined for changes in glutamate transport, glutamate mediated synaptic currents, and dendritic spine morphology. We also determined if acute stress potentiated the acquisition of cocaine self-administration. Acute stress produced an enduring reduction in glutamate transport, and potentiated excitatory synapses on medium spiny neurons. Acute stress also augmented the acquisition of cocaine self-administration. Importantly, by restoring glutamate transport in the accumbens core with ceftriaxone the capacity of acute stress to augment the acquisition of cocaine self-administration was abolished. Similarly, ceftriaxone treatment prevented stress-induced potentiation of cocaine-induced locomotor activity. However, ceftriaxone did not reverse stress-induced synaptic potentiation, indicating that this effect of stress exposure did not underpin the increased acquisition of cocaine self-administration. Reversing acute stress-induced vulnerability to self-administer cocaine by normalizing glutamate transport poses a novel treatment possibility for reducing comorbid SUDs in stress disorders. PMID:26821978

  7. Key role of the dopamine D4 receptor in the modulation of corticostriatal glutamatergic neurotransmission.

    Science.gov (United States)

    Bonaventura, Jordi; Quiroz, César; Cai, Ning-Sheng; Rubinstein, Marcelo; Tanda, Gianluigi; Ferré, Sergi

    2017-01-01

    Polymorphic variants of the dopamine D4 receptor gene (DRD4) have been repeatedly associated with numerous neuropsychiatric disorders. Yet, the functional role of the D4 receptor and the functional differences of the products of DRD4 polymorphic variants remained enigmatic. Immunohistochemical and optogenetic-microdialysis experiments were performed in knock-in mice expressing a D4 receptor with the long intracellular domain of a human DRD4 polymorphic variant associated with attention deficit hyperactivity disorder (ADHD). When compared with the wild-type mouse D4 receptor, the expanded intracellular domain of the humanized D4 receptor conferred a gain of function, blunting methamphetamine-induced cortical activation and optogenetic and methamphetamine-induced corticostriatal glutamate release. The results demonstrate a key role of the D4 receptor in the modulation of corticostriatal glutamatergic neurotransmission. Furthermore, these data imply that enhanced D4 receptor-mediated dopaminergic control of corticostriatal transmission constitutes a vulnerability factor of ADHD and other neuropsychiatric disorders.

  8. Activity-dependent switch of GABAergic inhibition into glutamatergic excitation in astrocyte-neuron networks.

    Science.gov (United States)

    Perea, Gertrudis; Gómez, Ricardo; Mederos, Sara; Covelo, Ana; Ballesteros, Jesús J; Schlosser, Laura; Hernández-Vivanco, Alicia; Martín-Fernández, Mario; Quintana, Ruth; Rayan, Abdelrahman; Díez, Adolfo; Fuenzalida, Marco; Agarwal, Amit; Bergles, Dwight E; Bettler, Bernhard; Manahan-Vaughan, Denise; Martín, Eduardo D; Kirchhoff, Frank; Araque, Alfonso

    2016-12-24

    Interneurons are critical for proper neural network function and can activate Ca(2+) signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, we found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and determined the sign (inhibition vs potentiation) of the GABA-mediated effects. While synaptic inhibition was mediated by GABAA receptors, potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1) knockout mice, we confirmed the glial source of the interneuron-induced potentiation, and demonstrated the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay.

  9. Release probability of hippocampal glutamatergic terminals scales with the size of the active zone.

    Science.gov (United States)

    Holderith, Noemi; Lorincz, Andrea; Katona, Gergely; Rózsa, Balázs; Kulik, Akos; Watanabe, Masahiko; Nusser, Zoltan

    2012-06-10

    Cortical synapses have structural, molecular and functional heterogeneity; our knowledge regarding the relationship between their ultrastructural and functional parameters is still fragmented. Here we asked how the neurotransmitter release probability and presynaptic [Ca(2+)] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca(2+) imaging-derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active-zone area. Peak amplitude of [Ca(2+)] transients in single boutons also positively correlated with the active-zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated calcium channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active-zone area. Gold particles labeling Cav2.1 were nonrandomly distributed in the active zones. Our results demonstrate that the numbers of several active-zone proteins, including presynaptic calcium channels, as well as the number of docked vesicles and the release probability, scale linearly with the active-zone area.

  10. The NG2 Protein Is Not Required for Glutamatergic Neuron-NG2 Cell Synaptic Signaling.

    Science.gov (United States)

    Passlick, Stefan; Trotter, Jacqueline; Seifert, Gerald; Steinhäuser, Christian; Jabs, Ronald

    2016-01-01

    NG2 glial cells (as from now NG2 cells) are unique in receiving synaptic input from neurons. However, the components regulating formation and maintenance of these neuron-glia synapses remain elusive. The transmembrane protein NG2 has been considered a potential mediator of synapse formation and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering, because it contains 2 extracellular Laminin G/Neurexin/Sex Hormone-Binding Globulin domains, which in neurons are crucial for formation of transsynaptic neuroligin-neurexin complexes. NG2 is connected via Glutamate Receptor-Interacting Protein with GluA2/3-containing AMPARs, thereby possibly mediating receptor clustering in glial postsynaptic density. To elucidate the role of NG2 in neuron-glia communication, we investigated glutamatergic synaptic transmission in juvenile and aged hippocampal NG2 cells of heterozygous and homozygous NG2 knockout mice. Neuron-NG2 cell synapses readily formed in the absence of NG2. Short-term plasticity, synaptic connectivity, postsynaptic AMPAR current kinetics, and density were not affected by NG2 deletion. During development, an NG2-independent acceleration of AMPAR current kinetics and decreased synaptic connectivity were observed. Our results indicate that the lack of NG2 does not interfere with genesis and basic properties of neuron-glia synapses. In addition, we demonstrate frequent expression of neuroligins 1-3 in juvenile and aged NG2 cells, suggesting a role of these molecules in synapse formation between NG2 glia and neurons.

  11. Specification of Region-Specific Neurons Including Forebrain Glutamatergic Neurons from Human Induced Pluripotent Stem Cells

    Science.gov (United States)

    Martins-Taylor, Kristen; Wang, Xiaofang; Zhang, Zheng; Park, Jung Woo; Zhan, Shuning; Kronenberg, Mark S.; Lichtler, Alexander; Liu, Hui-Xia; Chen, Fang-Ping; Yue, Lixia; Li, Xue-Jun; Xu, Ren-He

    2010-01-01

    Background Directed differentiation of human induced pluripotent stem cells (hiPSC) into functional, region-specific neural cells is a key step to realizing their therapeutic promise to treat various neural disorders, which awaits detailed elucidation. Methodology/Principal Findings We analyzed neural differentiation from various hiPSC lines generated by others and ourselves. Although heterogeneity in efficiency of neuroepithelial (NE) cell differentiation was observed among different hiPSC lines, the NE differentiation process resembles that from human embryonic stem cells (hESC) in morphology, timing, transcriptional profile, and requirement for FGF signaling. NE cells differentiated from hiPSC, like those from hESC, can also form rostral phenotypes by default, and form the midbrain or spinal progenitors upon caudalization by morphogens. The rostrocaudal neural progenitors can further mature to develop forebrain glutamatergic projection neurons, midbrain dopaminergic neurons, and spinal motor neurons, respectively. Typical ion channels and action potentials were recorded in the hiPSC-derived neurons. Conclusions/Significance Our results demonstrate that hiPSC, regardless of how they were derived, can differentiate into a spectrum of rostrocaudal neurons with functionality, which supports the considerable value of hiPSC for study and treatment of patient-specific neural disorders. PMID:20686615

  12. Nicotinic, glutamatergic and dopaminergic synaptic transmission and plasticity in the mesocorticolimbic system: focus on nicotine effects.

    Science.gov (United States)

    Pistillo, Francesco; Clementi, Francesco; Zoli, Michele; Gotti, Cecilia

    2015-01-01

    Cigarette smoking is currently the leading cause of preventable deaths and disability throughout the world, being responsible for about five million premature deaths/year. Unfortunately, fewer than 10% of tobacco users who try to stop smoking actually manage to do so. The main addictive agent delivered by cigarette smoke is nicotine, which induces psychostimulation and reward, and reduces stress and anxiety. The use of new technologies (including optogenetics) and the development of mouse models characterised by cell-specific deletions of receptor subtype genes or the expression of gain-of-function nAChR subunits has greatly increased our understanding of the molecular mechanisms and neural substrates of nicotine addiction first revealed by classic electrophysiological, neurochemical and behavioural approaches. It is now becoming clear that various aspects of nicotine dependence are mediated by close interactions of the glutamatergic, dopaminergic and γ-aminobutyric acidergic systems in the mesocorticolimbic system. This review is divided into two parts. The first provides an updated overview of the circuitry of the ventral tegmental area, ventral striatum and prefrontal cortex, the neurotransmitter receptor subtypes expressed in these areas, and their physiological role in the mesocorticolimbic system. The second will focus on the molecular, functional and behavioural mechanisms involved in the acute and chronic effects of nicotine on the mesocorticolimbic system.

  13. Specification of region-specific neurons including forebrain glutamatergic neurons from human induced pluripotent stem cells.

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    Hui Zeng

    Full Text Available BACKGROUND: Directed differentiation of human induced pluripotent stem cells (hiPSC into functional, region-specific neural cells is a key step to realizing their therapeutic promise to treat various neural disorders, which awaits detailed elucidation. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed neural differentiation from various hiPSC lines generated by others and ourselves. Although heterogeneity in efficiency of neuroepithelial (NE cell differentiation was observed among different hiPSC lines, the NE differentiation process resembles that from human embryonic stem cells (hESC in morphology, timing, transcriptional profile, and requirement for FGF signaling. NE cells differentiated from hiPSC, like those from hESC, can also form rostral phenotypes by default, and form the midbrain or spinal progenitors upon caudalization by morphogens. The rostrocaudal neural progenitors can further mature to develop forebrain glutamatergic projection neurons, midbrain dopaminergic neurons, and spinal motor neurons, respectively. Typical ion channels and action potentials were recorded in the hiPSC-derived neurons. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that hiPSC, regardless of how they were derived, can differentiate into a spectrum of rostrocaudal neurons with functionality, which supports the considerable value of hiPSC for study and treatment of patient-specific neural disorders.

  14. Induced pluripotent stem cells (iPSCs) derived from a symptomatic carrier of a S305I mutation in the microtubule-associated protein tau (MAPT)-gene causing frontotemporal dementia

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    Nimsanor, Natakarn; Jørring, Ida; Rasmussen, Mikkel A.

    2016-01-01

    Frontotemporal dementia with parkinsonism linked to chromosome 17q21.2 (FTDP-17) is an autosomal-dominant neurodegenerative disorder. Mutations in the gene coding the microtubule-associated protein tau (MAPT) can cause FTDP-17 but the underlying mechanisms of the disease are still unknown. Induce...... pluripotent stem cells (iPSCs) hold great promise to model FTDP-17 as such cells can be differentiated in vitro to the required neuronal cell type. Here, we report the generation of iPSCs from a 44-year-old symptomatic woman carrying a S305I mutation in the MAPT-gene....

  15. Dynamics of glutamatergic signaling in the mushroom body of young adult Drosophila

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    Grau Yves

    2010-04-01

    Full Text Available Abstract Background The mushroom bodies (MBs are paired brain centers located in the insect protocerebrum involved in olfactory learning and memory and other associative functions. Processes from the Kenyon cells (KCs, their intrinsic neurons, form the bulk of the MB's calyx, pedunculus and lobes. In young adult Drosophila, the last-born KCs extend their processes in the α/β lobes as a thin core (α/β cores that is embedded in the surrounding matrix of other mature KC processes. A high level of L-glutamate (Glu immunoreactivity is present in the α/β cores (α/βc of recently eclosed adult flies. In a Drosophila model of fragile X syndrome, the main cause of inherited mental retardation, treatment with metabotropic Glu receptor (mGluR antagonists can rescue memory deficits and MB structural defects. Results To address the role of Glu signaling in the development and maturation of the MB, we have compared the time course of Glu immunoreactivity with the expression of various glutamatergic markers at various times, that is, 1 hour, 1 day and 10 days after adult eclosion. We observed that last-born α/βc KCs in young adult as well as developing KCs in late larva and at various pupal stages transiently express high level of Glu immunoreactivity in Drosophila. One day after eclosion, the Glu level was already markedly reduced in the α/βc neurons. Glial cell processes expressing glutamine synthetase and the Glu transporter dEAAT1 were found to surround the Glu-expressing KCs in very young adults, subsequently enwrapping the α/β lobes to become distributed equally over the entire MB neuropil. The vesicular Glu transporter DVGluT was detected by immunostaining in processes that project within the MB lobes and pedunculus, but this transporter is apparently never expressed by the KCs themselves. The NMDA receptor subunit dNR1 is widely expressed in the MB neuropil just after eclosion, but was not detected in the α/βc neurons. In contrast, we

  16. Peptide and lipid modulation of glutamatergic afferent synaptic transmission in the solitary tract nucleus

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    Michael C. Andresen

    2013-01-01

    Full Text Available The brainstem nucleus of the solitary tract (NTS holds the first central neurons in major homeostatic reflex pathways. These homeostatic reflexes regulate and coordinate multiple organ systems from gastrointestinal to cardiopulmonary functions. The core of many of these pathways arise from cranial visceral afferent neurons that enter the brain as the solitary tract (ST with more than two-thirds arising from the gastrointestinal system. About one quarter of ST afferents have myelinated axons but the majority are classed as unmyelinated C-fibers. All ST afferents release the fast neurotransmitter glutamate with remarkably similar, high-probability release characteristics. Second order NTS neurons receive surprisingly limited primary afferent information with one or two individual inputs converging on single second order NTS neurons. A- and C-fiber afferents never mix at NTS second order neurons. Many transmitters modify the basic glutamatergic excitatory postsynaptic current (EPSC often by reducing glutamate release or interrupting terminal depolarization. Thus, a distinguishing feature of ST transmission is presynaptic expression of G-protein coupled receptors for peptides common to peripheral or forebrain (e.g. hypothalamus neuron sources. Presynaptic receptors for angiotensin (AT1, vasopressin (V1a, oxytocin (OT, opioid (MOR, ghrelin (GHSR1 and cholecystokinin (CCK differentially control glutamate release on particular subsets of neurons with most other ST afferents unaffected. Lastly, lipid-like signals are transduced by two key ST presynaptic receptors, the transient receptor potential vanilloid type 1 (TRPV1 and the cannabinoid receptor (CB1 that oppositely control glutamate release. Increasing evidence suggests that peripheral nervous signaling mechanisms are repurposed at central terminals to control excitation and are major sites of signal integration of peripheral and central inputs particularly from the hypothalamus.

  17. Effects of propofol on GABAergic and glutamatergic transmission in isolated hippocampal single nerve-synapse preparations.

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    Wakita, Masahito; Kotani, Naoki; Nonaka, Kiku; Shin, Min-Chul; Akaike, Norio

    2013-10-15

    We evaluated the effects of propofol on synaptic transmission using a mechanically dissociated preparation of rat hippocampal CA3 neurons to allow assays of single bouton responses evoked from retained functional native nerve endings. We studied synaptic and extrasynaptic GABAA and glutamate receptor responses in a preparation in which experimental solutions rapidly accessed synaptic terminals. Whole-cell responses were evoked by bath application of GABA and glutamate. Synaptic inhibitory and excitatory postsynaptic currents (IPSC and EPSC) were measured as spontaneous and evoked postsynaptic responses. Evoked currents were elicited by focal electrical stimulation. Propofol (1-100 μM) enhanced extrasynaptic GABAA-receptor mediated responses but the increase at clinically relevant concentrations (1 μM) were minor. In contrast, 1 μM propofol significantly increased both the amplitude and frequency of spontaneous IPSCs (sIPSCs) and increased the amplitudes of evoked IPSCs (eIPSCs) while decreasing failure rates (Rf) and paired-pulse ratios (PPR). Decay times of sIPSCs and eIPSCs were significantly prolonged. Although propofol had no effect on extrasynaptic glutamate responses, only supra-clinical propofol concentrations (≥ 10 µM) increased the spontaneous EPSCs (sEPSCs, amplitudes and frequencies) but suppressed evoked EPSCs (eEPSCs decreased amplitudes with increased Rf and PPR). The decay phases of sEPSCs and eEPSCs were not changed. The propofol-induced changes in sEPSCs and eEPSCs resulted from presynaptic GABAA receptor-mediated depolarization, because these actions were blocked by bicuculline. These results suggest that propofol acts at presynaptic and postsynaptic GABAA receptors within GABAergic synapses, but also increases extrasynaptic GABA responses. Our results expand the locus of propofol actions to GABAergic and glutamatergic synapses.

  18. Simulation of postsynaptic glutamate receptors reveals critical features of glutamatergic transmission.

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    Renaud Greget

    Full Text Available Activation of several subtypes of glutamate receptors contributes to changes in postsynaptic calcium concentration at hippocampal synapses, resulting in various types of changes in synaptic strength. Thus, while activation of NMDA receptors has been shown to be critical for long-term potentiation (LTP and long term depression (LTD of synaptic transmission, activation of metabotropic glutamate receptors (mGluRs has been linked to either LTP or LTD. While it is generally admitted that dynamic changes in postsynaptic calcium concentration represent the critical elements to determine the direction and amplitude of the changes in synaptic strength, it has been difficult to quantitatively estimate the relative contribution of the different types of glutamate receptors to these changes under different experimental conditions. Here we present a detailed model of a postsynaptic glutamatergic synapse that incorporates ionotropic and mGluR type I receptors, and we use this model to determine the role of the different receptors to the dynamics of postsynaptic calcium with different patterns of presynaptic activation. Our modeling framework includes glutamate vesicular release and diffusion in the cleft and a glutamate transporter that modulates extracellular glutamate concentration. Our results indicate that the contribution of mGluRs to changes in postsynaptic calcium concentration is minimal under basal stimulation conditions and becomes apparent only at high frequency of stimulation. Furthermore, the location of mGluRs in the postsynaptic membrane is also a critical factor, as activation of distant receptors contributes significantly less to calcium dynamics than more centrally located ones. These results confirm the important role of glutamate transporters and of the localization of mGluRs in postsynaptic sites in their signaling properties, and further strengthen the notion that mGluR activation significantly contributes to postsynaptic calcium

  19. Blockade of glutamatergic transmission in the primate basolateral amygdala suppresses active behavior without altering social interaction.

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    Forcelli, Patrick A; Wellman, Laurie L; Malkova, Ludise

    2017-04-01

    The amygdala is an integrator of affective processing, and a key component of a network regulating social behavior. While decades of lesion studies in nonhuman primates have shown alterations in social interactions after amygdala damage, acute manipulations of the amygdala in primates have been underexplored. We recently reported (Wellman, Forcelli, Aguilar, & Malkova, 2016) that acute pharmacological inhibition of the basolateral complex of the amygdala (BLA) or the central nucleus of the amygdala increased affiliative social interactions in experimental dyads of macaques; this was achieved through microinjection of a GABA-A receptor agonist. Prior studies in rodents have shown similar effects achieved by blocking NMDA receptors or AMPA receptors within the BLA. Here, we sought to determine the role of these receptor systems in the primate BLA in the context of social behavior. In familiar dyads, we microinjected the NMDA receptor antagonist 2-amino-7-phosphonoheptanoic acid (AP7) or the AMPA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) and observed behaviors and social interactions in the immediate postinjection period. In striking contrast with our prior report using GABA agonists, and in contrast with prior reports in rodents using glutamate antagonists, we found that neither NMDA nor AMPA blockade increase social interaction. Both treatments, however, were associated with decreases in locomotion and manipulation and increases in passive behavior. These data suggest that local blockade of glutamatergic neurotransmission in BLA is not the functional equivalent of local activation of GABAergic signaling, and raise interesting questions regarding the functional microcircuitry of the nonhuman primate amygdala in the context of social behavior. (PsycINFO Database Record

  20. Acute stress enhances the glutamatergic transmission onto basoamygdala neurons embedded in distinct microcircuits.

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    Song, Chen; Zhang, Wen-Hua; Wang, Xue-Hui; Zhang, Jun-Yu; Tian, Xiao-Li; Yin, Xiao-Ping; Pan, Bing-Xing

    2017-01-09

    Amygdala activation is known to be critical for the processing of stressful events in brain. Recent studies have shown that the projection neurons (PNs) in amygdala, although architecturally intermingled, are integrated into distinct microcircuits and thus play divergent roles in amygdala-related behaviors. It remains unknown how stress regulates the individual amygdala PNs embedded in distinct microcircuits. Here, by using retrograde tracing and electrophysiological recording in in vitro slices, we explored the modulation of acute immobilization stress (AIS) on the basoamygdala (BA) PNs projecting either to medial prefrontal cortex (mPFC) or elsewhere, which we designated as BA-mPFC and non-BA-mPFC PNs respectively. The results showed that in the control mice, both the excitatory and inhibitory postsynaptic currents (sEPSCs/sIPSCs) were comparable between these two subsets of BA PNs. The influences of AIS on sEPSCs and sIPSCs were overall similar between the two neuronal populations. It markedly increased the sEPSCs amplitude but left unaltered their frequency as well as the sIPSCs amplitude and frequency. Despite this, several differences emerged between the effects of AIS on the distribution of sEPSCs/sIPSCs frequency in these two groups of BA PNs. Similar changes were also observed in the sEPSCs/sIPSCs of the two PN populations from mice experiencing forced swimming stress. Their intrinsic excitability, on the other hand, was nearly unaltered following AIS. Our results thus suggest that acute stress recruit both BA-mPFC and non-BA-mPFC PNs mainly through enhancing the glutamatergic transmission they receive.

  1. Developmental changes in the acute ethanol sensitivity of glutamatergic and GABAergic transmission in the BNST.

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    Wills, T A; Kash, T L; Winder, D G

    2013-11-01

    Glutamatergic and GABAergic transmission undergo significant changes during adolescence. Receptors for both of these transmitters (NMDAR, and GABAA) are known to be key targets for the acute effects of ethanol in adults. The current study set out to investigate the acute effects of ethanol on both NMDAR-mediated excitatory transmission and GABAergic inhibitory transmission within the bed nucleus of the stria terminalis (BNST) across age. The BNST is an area of the brain implicated in the negative reinforcing properties associated with alcohol dependence, and the BNST plays a critical role in stress-induced relapse. Therefore, assessing the developmental regulation of ethanol sensitivity in this key brain region is important to understanding the progression of ethanol dependence. To do this, whole-cell recordings of isolated NMDAR-evoked excitatory postsynaptic currents (eEPSCs) or evoked GABAergic inhibitory postsynaptic currents (eIPSCs) were performed on BNST neurons in slices from 4- or 8-week-old male C57BL/6J mice. Ethanol (50 mm) produced greater inhibition of NMDAR-eEPSCs in adolescent mice than in adult mice. This enhanced sensitivity in adolescence was not a result of shifts in function of the GluN2B subunit of the NMDAR, measured by Ro25-6981 inhibition and decay kinetics measured across age. Adolescent mice also exhibited greater ethanol sensitivity of GABAergic transmission, as ethanol (50 mm) enhanced eIPSCs in the BNST of adolescent but not adult mice. Collectively, this work illustrates that a moderate dose of ethanol produces greater inhibition of transmission in the BNST (through greater excitatory inhibition and enhancement of inhibitory transmission) in adolescents compared to adults. Given the role of the BNST in alcohol dependence, these developmental changes in acute ethanol sensitivity could accelerate neuroadaptations that result from chronic ethanol use during the critical period of adolescence.

  2. Dissociation of μ- and δ-opioid inhibition of glutamatergic synaptic transmission in superficial dorsal horn

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    Vaughan Christopher W

    2010-10-01

    Full Text Available Abstract Background There is anatomical and behavioural evidence that μ- and δ-opioid receptors modulate distinct nociceptive modalities within the superficial dorsal horn. The aim of the present study was to examine whether μ- and δ-opioid receptor activation differentially modulates TRP sensitive inputs to neurons within the superficial dorsal horn. To do this, whole cell patch clamp recordings were made from lamina I - II neurons in rat spinal cord slices in vitro to examine the effect of opioids on TRP agonist-enhanced glutamatergic spontaneous miniature excitatory postsynaptic currents (EPSCs. Results Under basal conditions the μ-opioid agonist DAMGO (3 μM reduced the rate of miniature EPSCs in 68% of neurons, while the δ- and κ-opioid agonists deltorphin-II (300 nM and U69593 (300 nM did so in 13 - 17% of neurons tested. The TRP agonists menthol (400 μM and icilin (100 μM both produced a Ca2+-dependent increase in miniature EPSC rate which was unaffected by the voltage dependent calcium channel (VDCC blocker Cd2+. The proportion of neurons in which deltorphin-II reduced the miniature EPSC rate was enhanced in the presence of icilin (83%, but not menthol (0%. By contrast, the proportion of DAMGO and U69593 responders was unaltered in the presence of menthol (57%, 0%, or icilin (57%, 17%. Conclusions These findings demonstrate that δ-opioid receptor activation selectively inhibits inputs activated by icilin, whereas μ-opioid receptor activation has a more widespread effect on synaptic inputs to neurons in the superficial dorsal horn. These findings suggest that δ-opioids may provide a novel analgesic approach for specific, TRPA1-like mediated pain modalities.

  3. Enhancement by citral of glutamatergic spontaneous excitatory transmission in adult rat substantia gelatinosa neurons.

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    Zhu, Lan; Fujita, Tsugumi; Jiang, Chang-Yu; Kumamoto, Eiichi

    2016-02-10

    Although citral, which is abundantly present in lemongrass, has various actions including antinociception, how citral affects synaptic transmission has not been examined as yet. Citral activates in heterologous cells transient receptor potential vanilloid-1, ankyrin-1, and melastatin-8 (TRPV1, TRPA1, and TRPM8, respectively) channels, the activation of which in the spinal lamina II [substantia gelatinosa (SG)] increases the spontaneous release of L-glutamate from nerve terminals. It remains to be examined what types of transient receptor potential channel in native neurons are activated by citral. With a focus on transient receptor potential activation, we examined the effect of citral on glutamatergic spontaneous excitatory transmission using the whole-cell patch-clamp technique to SG neurons in adult rat spinal cord slices. Bath-applied citral for 3 min increased the frequency of spontaneous excitatory postsynaptic current in a concentration-dependent manner (half-maximal effective concentration=0.58 mM), with a small increase in its amplitude. The spontaneous excitatory postsynaptic current frequency increase produced by citral was repeated at a time interval of 30 min, albeit this action recovered with a slow time course after washout. The presynaptic effect of citral was inhibited by TRPA1 antagonist HC-030031, but not by voltage-gated Na-channel blocker tetrodotoxin, TRPV1 antagonist capsazepine, and TRPM8 antagonist BCTC. It is concluded that citral increases spontaneous L-glutamate release in SG neurons by activating TRPA1 channels. Considering that the SG plays a pivotal role in modulating nociceptive transmission from the periphery, the citral activity could contribute toward at least a part of the modulation.

  4. Presynaptic α7 nicotinic acetylcholine receptors enhance hippocampal mossy fiber glutamatergic transmission via PKA activation.

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    Cheng, Qing; Yakel, Jerrel L

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) are expressed widely in the CNS, and mediate both synaptic and perisynaptic activities of endogenous cholinergic inputs and pharmacological actions of exogenous compounds (e.g., nicotine and choline). Behavioral studies indicate that nicotine improves such cognitive functions as learning and memory. However, the mechanism of nicotine's action on cognitive function remains elusive. We performed patch-clamp recordings from hippocampal CA3 pyramidal neurons to determine the effect of nicotine on mossy fiber glutamatergic synaptic transmission. We found that nicotine in combination with NS1738, an α7 nAChR-positive allosteric modulator, strongly potentiated the amplitude of evoked EPSCs (eEPSCs), and reduced the EPSC paired-pulse ratio. The action of nicotine and NS1738 was mimicked by PNU-282987 (an α7 nAChR agonist), and was absent in α7 nAChR knock-out mice. These data indicate that activation of α7 nAChRs was both necessary and sufficient to enhance the amplitude of eEPSCs. BAPTA applied postsynaptically failed to block the action of nicotine and NS1738, suggesting again a presynaptic action of the α7 nAChRs. We also observed α7 nAChR-mediated calcium rises at mossy fiber giant terminals, indicating the presence of functional α7 nAChRs at presynaptic terminals. Furthermore, the addition of PNU-282987 enhanced action potential-dependent calcium transient at these terminals. Last, the potentiating effect of PNU-282987 on eEPSCs was abolished by inhibition of protein kinase A (PKA). Our findings indicate that activation of α7 nAChRs at presynaptic sites, via a mechanism involving PKA, plays a critical role in enhancing synaptic efficiency of hippocampal mossy fiber transmission.

  5. In vivo and in vitro effects of multiple sclerosis immunomodulatory therapeutics on glutamatergic excitotoxicity.

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    Luchtman, Dirk; Gollan, René; Ellwardt, Erik; Birkenstock, Jérôme; Robohm, Kerstin; Siffrin, Volker; Zipp, Frauke

    2016-03-01

    In multiple sclerosis (MS), a candidate downstream mechanism for neuronal injury is glutamate (Glu)-induced excitotoxicity, leading to toxic increases in intraneuronal Ca(2+) . Here, we used in vivo two-photon imaging in the brain of TN-XXL transgenic Ca(2+) reporter mice to test whether promising oral MS therapeutics, namely fingolimod, dimethyl fumarate, and their respective metabolites fingolimod-phosphate and monomethyl fumarate, can protect neurons against acute glutamatergic excitotoxic damage. We also assessed whether these drugs can protect against excitotoxicity in vitro using primary cortical neurons, and whether they can directly inhibit Glu release from pathogenic T-helper 17 lymphocytes. In vivo, direct and acute (1 h) administration of 100 mM Glu to the brainstem resulted in a rapid and significant up-regulation in neuronal Ca(2+) signaling as well as morphological excitotoxic changes that were attenuated by the NMDA-receptor antagonist MK801. Direct CNS administration of MS drugs prior to Glu significantly delayed or reduced, but did not prevent the neuronal Ca(2+) increase or morphological changes. In vitro, prolonged (24 h) treatment of primary neurons with the fumarates significantly protected against neurotoxicity induced by Glu as well as NMDA, similar to MK801. Furthermore, monomethyl fumerate significantly reduced Glu release from pathogenic T-helper 17 lymphocytes. Overall, these data suggest that MS drugs may mediate neuroprotection via excitotoxicity modulating effects. Evidence suggests MS pathogenesis may involve neuronal excitotoxicity, induced by local release of glutamate. However, current MS drugs, including dimethyl fumerate (DMF) and fingolimod (FTY720) are largely anti-inflammatory and not yet fully tested for their neuroprotective potential. Here, we show that the drugs, in particular DMF metabolite monomethyl fumerate (MMF), protect neurons by excitotoxicity modulating effects. Th17, T-helper 17.

  6. Glutamatergic and GABAergic modulations of ultrasonic vocalizations during maternal separation distress in mouse pups

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    Takahashi, Aki.; Yap, Jasmine. J.; Bohager, Dawnya Zitzman; Faccidomo, Sara; Clayton, Terry; Cook, James. M.

    2009-01-01

    Introduction Dysregulation of GABAergic inhibition and glutamatergic excitation has been implicated in exaggerated anxiety. Mouse pups emit distress-like ultrasonic vocalizations (USVs) when they are separated from their dam/siblings, and this behavior is reduced by benzodiazepines (BZs) which modulate GABAergic inhibition. The roles of glutamate receptors on USVs remain to be investigated. Materials and methods We examined the roles of glutamate receptor subtypes on mouse pup USVs using N-methyl-D-aspartate (NMDA) receptor antagonists with different affinities [dizocilpine (MK-801), memantine, and neramexane] and group II metabotropic glutamate receptor agonist (LY-379268) and antagonist (LY-341495). These effects were compared with classic BZs: flunitrazepam, bromazepam, and chlordiazepoxide. To assess the role of GABAA receptor subunits on USVs, drugs that have preferential actions at different GABAA-α subunits (L-838417 and QH-ii-066) were tested. Seven-day-old CFW mouse pups were separated from their dam and littermates and placed individually on a 19°C test platform for 4 min. Grid crossings and body rolls were measured in addition to USVs. Results Dizocilpine dose-dependently reduced USVs, whereas memantine and neramexane showed biphasic effects and enhanced USVs at low to moderate doses. The NMDA receptor antagonists increased locomotion. LY-379268 reduced USVs but also suppressed locomotion. All BZs reduced USVs and increased motor incoordination. Neither L-838417 nor QH-ii-066 changed USVs, but both induced motor incoordination. Conclusion Low-affinity NMDA receptor antagonists, but not the high-affinity antagonist, enhanced mouse pup distress calls, which may be reflective of an anxiety-like state. BZs reduced USVs but also induced motor incoordination, possibly mediated by the α5 subunit containing GABAA receptors. PMID:19099296

  7. Global brain gene expression analysis links glutamatergic and GABAergic alterations to suicide and major depression.

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    Adolfo Sequeira

    Full Text Available BACKGROUND: Most studies investigating the neurobiology of depression and suicide have focused on the serotonergic system. While it seems clear that serotonergic alterations play a role in the pathogenesis of these major public health problems, dysfunction in additional neurotransmitter systems and other molecular alterations may also be implicated. Microarray expression studies are excellent screening tools to generate hypotheses about additional molecular processes that may be at play. In this study we investigated brain regions that are known to be implicated in the neurobiology of suicide and major depression are likely to represent valid global molecular alterations. METHODOLOGY/PRINCIPAL FINDINGS: We performed gene expression analysis using the HG-U133AB chipset in 17 cortical and subcortical brain regions from suicides with and without major depression and controls. Total mRNA for microarray analysis was obtained from 663 brain samples isolated from 39 male subjects, including 26 suicide cases and 13 controls diagnosed by means of psychological autopsies. Independent brain samples from 34 subjects and animal studies were used to control for the potential confounding effects of comorbidity with alcohol. Using a Gene Ontology analysis as our starting point, we identified molecular pathways that may be involved in depression and suicide, and performed follow-up analyses on these possible targets. Methodology included gene expression measures from microarrays, Gene Score Resampling for global ontological profiling, and semi-quantitative RT-PCR. We observed the highest number of suicide specific alterations in prefrontal cortical areas and hippocampus. Our results revealed alterations of synaptic neurotransmission and intracellular signaling. Among these, Glutamatergic (GLU and GABAergic related genes were globally altered. Semi-quantitative RT-PCR results investigating expression of GLU and GABA receptor subunit genes were consistent with

  8. Activation of mu opioid receptor inhibits the excitatory glutamatergic transmission in the anterior cingulate cortex of the rats with peripheral inflammation.

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    Zheng, Weihong

    2010-02-25

    Emerging evidence recently indicates that the anterior cingulate cortex is critically involved in the central processing and modulation of noxious stimulus, although the neuroadaptation in the anterior cingulate cortex has not been well documented in the conditions of chronic pain. Meanwhile, the cellular mechanism underlying opiate analgesia in the anterior cingulate cortex remains unclear. To address these issues, the present study was undertaken to explore the adaptation of excitatory glutamatergic transmission and mu opioid receptor-mediated modulation of glutamatergic transmission in the anterior cingulate cortex slices from the complete Freund's adjuvant (CFA)-inflamed rats. The results demonstrated that glutamatergic paired-pulse facilitation was decreased in the anterior cingulate cortex neurons from the CFA-inflamed rats, indicating an enhanced presynaptic glutamate release. In addition, activation of mu opioid receptor significantly inhibited the glutamatergic excitatory postsynaptic currents (EPSCs) in the anterior cingulate cortex neurons, which was attained through the suppression of presynaptic glutamate release. Taken together, these findings provided the evidence for the functional adaptation of central glutamatergic transmission induced by peripheral inflammation, and elucidated the cellular mechanism underlying opiate analgesia in the anterior cingulate cortex.

  9. Induced pluripotent stem cells (iPSCs) derived from af pre-symptomatic carrier of a R406W mutation in microtubule-associated protein tau (MAPT) causing frontotemporal dementia

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel A.; Hjermind, Lena Elisabeth; Hasholt, Lis Frydenreich

    2016-01-01

    Skin fibroblasts were obtained from a 28-year-old pre-symptomatic woman carrying a R406W mutation in microtubule-associated protein tau (MAPT), known to cause frontotemporal dementia. Induced pluripotent stem cell (iPSCs) were established by electroporation with episomal plasmids containing hOCT4...

  10. Sonic hedgehog and retinoic Acid induce bone marrow-derived stem cells to differentiate into glutamatergic neural cells.

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    Yu, Zhenhai; Wu, Shixing; Liu, Zhen; Lin, Haiyan; Chen, Lei; Yuan, Xinli; Zhang, Zhiying; Liu, Fang; Zhang, Chuansen

    2015-01-01

    Studies have showed that transplanted stem cells in the inner ear won't regenerate to replace the damaged sensory hair cells. They can spontaneously differentiate into mesenchymal cells and fibrocytes in the damaged inner ear. Only mature sensory cells of MSCs-derived possess the great potency for cell transplantation in the treatment of sensorineural hearing loss. So, we try to establish an efficient generation of the glutamatergic sensory neural phenotype for the cell transplantation of the hearing loss. We isolated MSCs from femoral and tibial bones according to their adherence to culture dishes. After purification, proliferation, and passaged, cells became homogeneous in appearance, showing more uniformity and grew in a monolayer with a typical spindle-shape morphology. The cell surface markers were assessed using FACS to characterize the isolated cells. For neural induction to harvest the glutamatergic sensory neurons, passage 3 MSCs were incubated with preinduced medium for 24 hr, and neural-induced medium for an additional 14 days. The cells exhibit a typical neural shape. RT-PCR analysis indicated that the mRNA levels of the neural cell marker nestin, Tau, MAP-2, β-tubulin III, GluR-3, and GluR-4 were higher compared with primary MSCs. Immunohistochemistry and western-blotting proofed that nestin, MAP-2, β-tubulin III, and GluR-4 proteins indeed exhibit their expression difference in the induced cells compared to the MSCs. We show an efficient protocol by the combined applications of Sonic Hedgehog (Shh) and Retinoic Acid (RA) to induce MSCs to differentiate into the glutamatergic sensory neuron which were identified from the morphological, biochemical, and molecular characteristics.

  11. Perifornical hypothalamic pathway to the adrenal gland: Role for glutamatergic transmission in the glucose counter-regulatory response.

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    Sabetghadam, A; Korim, W S; Verberne, A J M

    2017-03-01

    Adrenaline is an important counter-regulatory hormone that helps restore glucose homeostasis during hypoglycaemia. However, the neurocircuitry that connects the brain glucose sensors and the adrenal sympathetic outflow to the chromaffin cells is poorly understood. We used electrical microstimulation of the perifornical hypothalamus (PeH) and the rostral ventrolateral medulla (RVLM) combined with adrenal sympathetic nerve activity (ASNA) recording to examine the relationship between the RVLM, the PeH and ASNA. In urethane-anaesthetised male Sprague-Dawley rats, intermittent single pulse electrical stimulation of the rostroventrolateral medulla (RVLM) elicited an evoked ASNA response that consisted of early (60±3ms) and late peaks (135±4ms) of preganglionic and postganglionic activity. In contrast, RVLM stimulation evoked responses in lumbar sympathetic nerve activity that were almost entirely postganglionic. PeH stimulation also produced an evoked excitatory response consisting of both preganglionic and postganglionic excitatory peaks in ASNA. Both peaks in ASNA following RVLM stimulation were reduced by intrathecal kynurenic acid (KYN) injection. In addition, the ASNA response to systemic neuroglucoprivation induced by 2-deoxy-d-glucose was abolished by bilateral microinjection of KYN into the RVLM. This suggests that a glutamatergic pathway from the perifornical hypothalamus (PeH) relays in the RVLM to activate the adrenal SPN and so modulate ASNA. The main findings of this study are that (i) adrenal premotor neurons in the RVLM may be, at least in part, glutamatergic and (ii) that the input to these neurons that is activated during neuroglucoprivation is also glutamatergic.

  12. Melamine Alters Glutamatergic Synaptic Transmission of CA3-CA1 Synapses Presynaptically Through Autophagy Activation in the Rat Hippocampus.

    Science.gov (United States)

    Zhang, Hui; Wang, Hui; Xiao, Xi; Zhang, Tao

    2016-01-01

    Melamine is an industrial chemical that can cause central nervous system disorders including excitotoxicity and cognitive impairment. Its illegal use in powdered baby formula was the focus of a milk scandal in China in 2008. One of our previous studies showed that melamine impaired glutamatergic transmission in rat hippocampal CA1 pyramidal cells. However, the underlying mechanism of action of melamine is unclear, and it is unknown if the CA3-CA1 pathway is directly involved. In the present study, a whole-cell patch-clamp technique was employed to investigate the effect of melamine on the hippocampal CA3-CA1 pathway in vitro. Both the evoked excitatory postsynaptic current (eEPSC) and the paired-pulse ratio (PPR) were recorded. Furthermore, we examined whether autophagy was involved in glutamatergic transmission alterations induced by melamine. Our data showed that melamine significantly increased the amplitude of eEPSCs in a dose-dependent manner. Inhibition of the N-methyl-D-aspartic acid receptor did not prevent the increase in eEPSC amplitude. In addition, the PPR was remarkably decreased by a melamine concentration of 5 × 10(-5) g/mL. It was found that autophagy could be activated by melamine and an autophagy inhibitor, 3-MA, prevented the melamine-induced increase in eEPSC amplitude. Overall, our results show that melamine presynaptically alters glutamatergic synaptic transmission of hippocampal CA3-CA1 synapses in vitro and this is likely associated with autophagy alteration.

  13. Biphasic Effects of Cannabinoids in Anxiety Responses: CB1 and GABAB Receptors in the Balance of GABAergic and Glutamatergic Neurotransmission

    Science.gov (United States)

    Rey, Alejandro Aparisi; Purrio, Martin; Viveros, Maria-Paz; Lutz, Beat

    2012-01-01

    Biphasic effects of cannabinoids have been shown in processes such as feeding behavior, motor activity, motivational processes and anxiety responses. Using two different tests for the characterization of anxiety-related behavior (elevated plus-maze and holeboard), we first identified in wild-type C57BL/6N mice, two doses of the synthetic CB1 cannabinoid receptor agonist CP-55,940 with anxiolytic (1 μg/kg) and anxiogenic properties (50 μg/kg), respectively. To clarify the role of CB1 receptors in this biphasic effect, both doses were applied to two different conditional CB1 receptor knockout (KO) mouse lines, GABA-CB1-KO (CB1 receptor inactivation in forebrain GABAergic neurons) and Glu-CB1-KO (CB1 receptor inactivation in cortical glutamatergic neurons). We found that the anxiolytic-like effects of the low dose of cannabinoids are mediated via the CB1 receptor on cortical glutamatergic terminals, because this anxiolytic-like response was abrogated only in Glu-CB1-KO mice. On the contrary, the CB1 receptor on the GABAergic terminals is required to induce an anxiogenic-like effect under a high-dose treatment because of the fact that this effect was abolished specifically in GABA-CB1-KO mice. These experiments were carried out in both sexes, and no differences occurred with the doses tested in the mutant mice. Interestingly, the positive allosteric modulation of GABAB receptor with GS-39783 was found to largely abrogate the anxiogenic-like effect of the high dose of CP-55,940. Our results shed new light in further understanding the biphasic effects of cannabinoids at the molecular level and, importantly, pave the way for the development of novel anxiolytic cannabinoid drugs, which may have favorable effect profiles targeting the CB1 receptor on glutamatergic terminals. PMID:22850737

  14. Spatial relationships between GABAergic and glutamatergic synapses on the dendrites of distinct types of mouse retinal ganglion cells across development.

    Directory of Open Access Journals (Sweden)

    Adam Bleckert

    Full Text Available Neuronal output requires a concerted balance between excitatory and inhibitory (I/E input. Like other circuits, inhibitory synaptogenesis in the retina precedes excitatory synaptogenesis. How then do neurons attain their mature balance of I/E ratios despite temporal offset in synaptogenesis? To directly compare the development of glutamatergic and GABAergic synapses onto the same cell, we biolistically transfected retinal ganglion cells (RGCs with PSD95CFP, a marker of glutamatergic postsynaptic sites, in transgenic Thy1-YFPγ2 mice in which GABAA receptors are fluorescently tagged. We mapped YFPγ2 and PSD95CFP puncta distributions on three RGC types at postnatal day P12, shortly before eye opening, and at P21 when robust light responses in RGCs are present. The mature IGABA/E ratios varied among ON-Sustained (S A-type, OFF-S A-type, and bistratified direction selective (DS RGCs. These ratios were attained at different rates, before eye-opening for ON-S and OFF-S A-type, and after eye-opening for DS RGCs. At both ages examined, the IGABA/E ratio was uniform across the arbors of the three RGC types. Furthermore, measurements of the distances between neighboring PSD95CFP and YFPγ2 puncta on RGC dendrites indicate that their local relationship is established early in development, and cannot be predicted by random organization. These close spatial associations between glutamatergic and GABAergic postsynaptic sites appear to represent local synaptic arrangements revealed by correlative light and EM reconstructions of a single RGC's dendrites. Thus, although RGC types have different IGABA/E ratios and establish these ratios at separate rates, the local relationship between excitatory and inhibitory inputs appear similarly constrained across the RGC types studied.

  15. Extrinsic and local glutamatergic inputs of the rat hippocampal CA1 area differentially innervate pyramidal cells and interneurons.

    Science.gov (United States)

    Takács, Virág T; Klausberger, Thomas; Somogyi, Peter; Freund, Tamás F; Gulyás, Attila I

    2012-06-01

    The two main glutamatergic pathways to the CA1 area, the Schaffer collateral/commissural input and the entorhinal fibers, as well as the local axons of CA1 pyramidal cells innervate both pyramidal cells and interneurons. To determine whether these inputs differ in their weights of activating GABAergic circuits, we have studied the relative proportion of pyramidal cells and interneurons among their postsynaptic targets in serial electron microscopic sections. Local axons of CA1 pyramidal cells, intracellularly labeled in vitro or in vivo, innervated a relatively high proportion of interneuronal postsynaptic targets (65.9 and 53.8%, in vitro and in vivo, respectively) in stratum (str.) oriens and alveus. In contrast, axons of in vitro labeled CA3 pyramidal cells in str. oriens and str. radiatum of the CA1 area made synaptic junctions predominantly with pyramidal cell spines (92.9%). The postsynaptic targets of anterogradely labeled medial entorhinal cortical boutons in CA1 str. lacunosum-moleculare were primarily pyramidal neuron dendritic spines and shafts (90.8%). The alvear group of the entorhinal afferents, traversing str. oriens, str. pyramidale, and str. radiatum showed a higher preference for innervating GABAergic cells (21.3%), particularly in str. oriens/alveus. These data demonstrate that different glutamatergic pathways innervate CA1 GABAergic cells to different extents. The results suggest that the numerically smaller CA1 local axonal inputs together with the alvear part of the entorhinal input preferentially act on GABAergic interneurons in contrast to the CA3, or the entorhinal input in str. lacunosum-moleculare. The results highlight differences in the postsynaptic target selection of the feed-forward versus recurrent glutamatergic inputs to the CA1 and CA3 areas.

  16. Mechanisms involved in systemic nicotine-induced glutamatergic synaptic plasticity on dopamine neurons in the ventral tegmental area.

    Science.gov (United States)

    Gao, Ming; Jin, Yu; Yang, Kechun; Zhang, Die; Lukas, Ronald J; Wu, Jie

    2010-10-13

    Systemic exposure to nicotine induces glutamatergic synaptic plasticity on dopamine (DA) neurons in the ventral tegmental area (VTA), but mechanisms are largely unknown. Here, we report that single, systemic exposure in rats to nicotine (0.17 mg/kg free base) increases the ratio of DA neuronal currents mediated by AMPA relative to NMDA receptors (AMPA/NMDA ratio) assessed 24 h later, based on slice-patch recording. The AMPA/NMDA ratio increase is evident within 1 h and lasts for at least 72 h after nicotine exposure (and up to 8 d after repeated nicotine administration). This effect cannot be prevented by systemic injection of either α7-nAChR (nicotinic ACh receptor)-selective [methyllycaconitine (MLA)] or β2*-nAChR-selective [mecamylamine (MEC)] antagonists but is prevented by coinjection of MLA and MEC. In either nAChR α7 or β2 subunit knock-out mice, systemic exposure to nicotine still increases the AMPA/NMDA ratio. Preinjection in rats of a NMDA receptor antagonist MK-801((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), but neither DA receptor antagonists [SCH-23390 (R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) plus haloperidol] nor a calcineurin inhibitor (cyclosporine), prevents the nicotine-induced increase in AMPA/NMDA ratio. After systemic exposure to nicotine, glutamatergic (but not GABAergic) transmission onto rat VTA DA neuronal inputs is enhanced. Correspondingly, DA neuronal firing measured 24 h after nicotine exposure using extracellular single-unit recording in vivo is significantly faster, and there is conversion of silent to active DA neurons. Collectively, these findings demonstrate that systemic nicotine acting via either α7- or β2*-nAChRs increases presynaptic and postsynaptic glutamatergic function, and consequently initiates glutamatergic synaptic plasticity, which may be an important, early neuronal adaptation in nicotine reward and reinforcement.

  17. Prefrontal beta2 subunit-containing and alpha7 nicotinic acetylcholine receptors differentially control glutamatergic and cholinergic signaling.

    Science.gov (United States)

    Parikh, Vinay; Ji, Jinzhao; Decker, Michael W; Sarter, Martin

    2010-03-03

    One-second-long increases in prefrontal cholinergic activity ("transients") were demonstrated previously to be necessary for the incorporation of cues into ongoing cognitive processes ("cue detection"). Nicotine and, more robustly, selective agonists at alpha4beta2* nicotinic acetylcholine receptors (nAChRs) enhance cue detection and attentional performance by augmenting prefrontal cholinergic activity. The present experiments determined the role of beta2-containing and alpha7 nAChRs in the generation of prefrontal cholinergic and glutamatergic transients in vivo. Transients were evoked by nicotine, the alpha4beta2* nAChR agonist ABT-089 [2-methyl-3-(2-(S)-pyrrolindinylmethoxy) pyridine dihydrochloride], or the alpha7 nAChR agonist A-582941 [2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole]. Transients were recorded in mice lacking beta2 or alpha7 nAChRs and in rats after removal of thalamic glutamatergic or midbrain dopaminergic inputs to prefrontal cortex. The main results indicate that stimulation of alpha4beta2* nAChRs evokes glutamate release and that the presence of thalamic afferents is necessary for the generation of cholinergic transients. ABT-089-evoked transients were completely abolished in mice lacking beta2* nAChRs. The amplitude, but not the decay rate, of nicotine-evoked transients was reduced by beta2* knock-out. Conversely, in mice lacking the alpha7 nAChR, the decay rate, but not the amplitude, of nicotine-evoked cholinergic and glutamatergic transients was attenuated. Substantiating the role of alpha7 nAChR in controlling the duration of release events, stimulation of alpha7 nAChR produced cholinergic transients that lasted 10- to 15-fold longer than those evoked by nicotine. alpha7 nAChR-evoked cholinergic transients are mediated in part by dopaminergic activity. Prefrontal alpha4beta2* nAChRs play a key role in evoking and facilitating the transient glutamatergic-cholinergic interactions that are necessary for cue detection

  18. Stress-induced impairment of glutamatergic terminals ultrastructure: High vulnerability of medial prefrontal cortex and preventing action of desipramine

    DEFF Research Database (Denmark)

    Nava, N.; Popoli, M.; Musazzi, L.

    2013-01-01

    mediators, glucocorticoids, on brain volume and dendritic remodeling, in both humans and rodents. Nevertheless, few is still known on the structural changes exerted by behavioral stress on the features of glutamatergic synapses as sites of neuronal communication. Indeed, in excitatory synapses synaptic...... communication is driven by neurotransmitter which is stored, within the presynaptic terminal, in morphologically distinct pools of vesicles, namely the readily-releasable pool of vesicles (RRP), docked to the active zone and ready for release, and the reserve pool of vesicles. When neurotransmitter is released...

  19. Gene expression related to serotonergic and glutamatergic neurotransmission is altered in the flinders sensitive line rat model of depression: Effect of ketamine.

    Science.gov (United States)

    Du Jardin, Kristian Gaarn; Müller, Heidi Kaastrup; Sanchez, Connie; Wegener, Gregers; Elfving, Betina

    2017-01-01

    Major depressive disorder (MDD) is associated with dysfunctional serotonergic and glutamatergic neurotransmission, and the genetic animal model of depression Flinders Sensitive Line (FSL) rats display alterations in these systems relatively to their control strain Flinders Resistant Line (FRL). However, changes on transcript level related to serotonergic and glutamatergic signaling have only been sparsely studied in this model. The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine has fast-onset antidepressant properties, and recent data implicate serotonergic neurotransmission in ketamine's antidepressant-like activities in rodents. Here, we investigated the transcript levels of 40 genes involved in serotonergic and glutamatergic neurotransmission in FSL and FRL rats in response to a single dose of ketamine (15 mg/kg; 90 min prior to euthanization). Using real-time quantitative polymerase chain reaction, we studied the effect of ketamine in the hippocampus, whereas strain differences were investigated in both hippocampus and frontal cortex. The expression of genes involved in serotonergic and glutamatergic neurotransmission were unaffected by a single dose of ketamine in the hippocampus. Relative to FRL rats, FSL rats displayed enhanced hippocampal transcript levels of 5-ht2c , and P11, whereas the expression was reduced for 5-ht2a , Nr2a, and Mglur2. In the frontal cortex, we found higher transcript levels of 5-ht2c and Mglur2, whereas the expression of 5-ht2a was reduced in FSL rats. Thus, ketamine is not associated with hippocampal alterations in serotonergic or glutamatergic genes at 90 min after an antidepressant dose. Furthermore, FSL rats display serotonergic and glutamatergic abnormalities on gene expression level that partly may resemble findings in MDD patients. © 2016 Wiley Periodicals, Inc.

  20. Discharge Profiles across the Sleep–Waking Cycle of Identified Cholinergic, GABAergic, and Glutamatergic Neurons in the Pontomesencephalic Tegmentum of the Rat

    Science.gov (United States)

    Boucetta, Soufiane; Cissé, Youssouf; Mainville, Lynda; Morales, Marisela

    2014-01-01

    Distributed within the laterodorsal tegmental and pedunculopontine tegmental nuclei (LDT and PPT), cholinergic neurons in the pontomesencephalic tegmentum have long been thought to play a critical role in stimulating cortical activation during waking (W) and paradoxical sleep (PS, also called REM sleep), yet also in promoting PS with muscle atonia. However, the discharge profile and thus precise roles of the cholinergic neurons have remained uncertain because they lie intermingled with GABAergic and glutamatergic neurons, which might also assume these roles. By applying juxtacellular recording and labeling in naturally sleeping–waking, head-fixed rats, we investigated the discharge profiles of histochemically identified cholinergic, GABAergic, and glutamatergic neurons in the LDT, SubLDT, and adjoining medial part of the PPT (MPPT) in relation to sleep–wake states, cortical activity, and muscle tone. We found that all cholinergic neurons were maximally active during W and PS in positive correlation with fast (γ) cortical activity, as “W/PS-max active neurons.” Like cholinergic neurons, many GABAergic and glutamatergic neurons were also “W/PS-max active.” Other GABAergic and glutamatergic neurons were “PS-max active,” being minimally active during W and maximally active during PS in negative correlation with muscle tone. Conversely, some glutamatergic neurons were “W-max active,” being maximally active during W and minimally active during PS in positive correlation with muscle tone. Through different discharge profiles, the cholinergic, GABAergic, and glutamatergic neurons of the LDT, SubLDT, and MPPT thus appear to play distinct roles in promoting W and PS with cortical activation, PS with muscle atonia, or W with muscle tone. PMID:24672016

  1. Neuroimaging markers of glutamatergic and GABAergic systems in drug addiction: relationships to resting-state functional connectivity

    Science.gov (United States)

    Moeller, Scott J.; London, Edythe D.; Northoff, Georg

    2015-01-01

    Drug addiction is characterized by widespread abnormalities in brain function and neurochemistry, including drug-associated effects on concentrations of the excitatory and inhibitory neurotransmitters glutamate and gamma-aminobutyric acid (GABA), respectively. In healthy individuals, these neurotransmitters drive the resting state, a default condition of brain function also disrupted in addiction. Here, our primary goal was to review in vivo magnetic resonance spectroscopy and positron emission tomography studies that examined markers of glutamate and GABA abnormalities in human drug addiction. Addicted individuals tended to show decreases in these markers compared with healthy controls, but findings also varied by individual characteristics (e.g., abstinence length). Interestingly, select corticolimbic brain regions showing glutamatergic and/or GABAergic abnormalities have been similarly implicated in resting-state functional connectivity deficits in drug addiction. Thus, our secondary goals were to provide a brief review of this resting-state literature, and an initial rationale for the hypothesis that abnormalities in glutamatergic and/or GABAergic neurotransmission may underlie resting-state functional deficits in drug addiction. In doing so, we suggest future research directions and possible treatment implications. PMID:26657968

  2. Sensory Input-Dependent Changes in Glutamatergic Neurotransmission- Related Genes and Proteins in the Adult Rat Trigeminal Ganglion

    Science.gov (United States)

    Fernández-Montoya, Julia; Buendia, Izaskun; Martin, Yasmina B.; Egea, Javier; Negredo, Pilar; Avendaño, Carlos

    2016-01-01

    Experience-dependent plasticity induces lasting changes in the structure of synapses, dendrites, and axons at both molecular and anatomical levels. Whilst relatively well studied in the cortex, little is known about the molecular changes underlying experience-dependent plasticity at peripheral levels of the sensory pathways. Given the importance of glutamatergic neurotransmission in the somatosensory system and its involvement in plasticity, in the present study, we investigated gene and protein expression of glutamate receptor subunits and associated molecules in the trigeminal ganglion (TG) of young adult rats. Microarray analysis of naïve rat TG revealed significant differences in the expression of genes, coding for various glutamate receptor subunits and proteins involved in clustering and stabilization of AMPA receptors, between left and right ganglion. Long-term exposure to sensory-enriched environment increased this left–right asymmetry in gene expression. Conversely, unilateral whisker trimming on the right side almost eliminated the mentioned asymmetries. The above manipulations also induced side-specific changes in the protein levels of glutamate receptor subunits. Our results show that sustained changes in sensory input induce modifications in glutamatergic transmission-related gene expression in the TG, thus supporting a role for this early sensory-processing node in experience-dependent plasticity. PMID:27965535

  3. Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission.

    Science.gov (United States)

    Martire, Alberto; Tebano, Maria Teresa; Chiodi, Valentina; Ferreira, Samira G; Cunha, Rodrigo A; Köfalvi, Attila; Popoli, Patrizia

    2011-01-01

    An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.

  4. Effects of Fluoxetine and Visual Experience on Glutamatergic and GABAergic Synaptic Proteins in Adult Rat Visual Cortex.

    Science.gov (United States)

    Beshara, Simon; Beston, Brett R; Pinto, Joshua G A; Murphy, Kathryn M

    2015-01-01

    Fluoxetine has emerged as a novel treatment for persistent amblyopia because in adult animals it reinstates critical period-like ocular dominance plasticity and promotes recovery of visual acuity. Translation of these results from animal models to the clinic, however, has been challenging because of the lack of understanding of how this selective serotonin reuptake inhibitor affects glutamatergic and GABAergic synaptic mechanisms that are essential for experience-dependent plasticity. An appealing hypothesis is that fluoxetine recreates a critical period (CP)-like state by shifting synaptic mechanisms to be more juvenile. To test this we studied the effect of fluoxetine treatment in adult rats, alone or in combination with visual deprivation [monocular deprivation (MD)], on a set of highly conserved presynaptic and postsynaptic proteins (synapsin, synaptophysin, VGLUT1, VGAT, PSD-95, gephyrin, GluN1, GluA2, GluN2B, GluN2A, GABAAα1, GABAAα3). We did not find evidence that fluoxetine shifted the protein amounts or balances to a CP-like state. Instead, it drove the balances in favor of the more mature subunits (GluN2A, GABAAα1). In addition, when fluoxetine was paired with MD it created a neuroprotective-like environment by normalizing the glutamatergic gain found in adult MDs. Together, our results suggest that fluoxetine treatment creates a novel synaptic environment dominated by GluN2A- and GABAAα1-dependent plasticity.

  5. [Evidence on the key role of the metabotrobic glutamatergic receptors in the pathogenesis of schizophrenia: a "breakthrough" in pharmacological treatment].

    Science.gov (United States)

    Pannese, Rossella; Minichino, Amedeo; Pignatelli, Marco; Delle Chiaie, Roberto; Biondi, Massimo; Nicoletti, Ferdinando

    2012-01-01

    The metabotropic glutamate receptors (mGluRs) are expressed pre- and post synaptically throughout the nervous system where they serve as modulators of synaptic transmission and neuronal excitability. The glutamatergic system is involved in a wide range of physiological processes in the brain, and its dysfunction plays an important role in the etiology and pathophysiology of psychiatric disorders, including schizophrenia. This paper reviews the neurodevelopmental origin and genetic susceptibility of schizophrenia relevant to NMDA receptor neurotransmission, and discusses the relationship between NMDA hypofunction and different domains of symptom in schizophrenia as well as putative treatment modality for the disorder. mGlu receptors have been hypothesizes as attractive therapeutic targets for the development of novel interventions for psychiatric disorders. Group II of mGlu receptors are of particular interest because of their unique distribution and the regulatory roles they have in neurotransmission. The glutamate hypothesis of schizophrenia predicts that agents that restore the balance in glutamatergic neurotransmission will ameliorate the symptomatology associated with this illness. Development of potent, efficacious, systemically active drugs will help to address the antipsychotic potential of these novel therapeutics. This review will discuss recent progress in elucidating the pharmacology and function of group II receptors in the context of current hypotheses on the pathophysiology of schizophrenia and the need for new and better antipsychotics.

  6. Ongoing intrinsic synchronous activity is required for the functional maturation of CA3-CA1 glutamatergic synapses.

    Science.gov (United States)

    Huupponen, Johanna; Molchanova, Svetlana M; Lauri, Sari E; Taira, Tomi

    2013-11-01

    Fine-tuning of synaptic connectivity during development is guided by intrinsic activity of the immature networks characteristically consisting of intermittent bursts of synchronous activity. However, the role of synchronous versus asynchronous activity in synapse maturation in the brain is unclear. Here, we have pharmacologically prevented generation of synchronous activity in the immature rat CA3-CA1 circuitry in a manner that preserves unitary activity. Long-term desynchronization of the network resulted in weakening of AMPA-receptor-mediated glutamatergic transmission in CA1 pyramidal cells. This weakening was dependent on protein phosphatases and mGluR activity, associated with an increase in the proportion of silent synapses and a decrease in the protein levels of GluA4 suggesting postsynaptic mechanisms of expression. The findings demonstrate that synchronous activity in the immature CA3-CA1 circuitry is critical for the induction and maintenance of glutamatergic synapses and underscores the importance of temporal activity patterns in shaping the synaptic circuitry during development.

  7. VGLUT2-dependent glutamatergic transmission in primary afferents is required for intact nociception in both acute and persistent pain modalities.

    Science.gov (United States)

    Rogoz, Katarzyna; Lagerström, Malin C; Dufour, Sylvie; Kullander, Klas

    2012-07-01

    Glutamate is an essential transmitter in pain pathways. However, its broad usage in the central and peripheral nervous system prevents us from designing efficient glutamate-based pain therapies without causing harmful side effects. The discovery of vesicular glutamate transporters (VGLUT1-3) has been a crucial step in describing specific glutamatergic neuronal subpopulations and glutamate-dependent pain pathways. To assess the role of VGLUT2-mediated glutamatergic contribution to pain transmission from the entire primary sensory population, we crossed our Vglut2(f/f) line with the Ht-Pa-Cre line. Such Vglut2-deficient mice showed significantly decreased, but not completely absent, acute nociceptive responses. The animals were less prone to develop an inflammatory-related state of pain and were, in the partial sciatic nerve ligation chronic pain model, much less hypersensitive to mechanical stimuli and did not develop cold allodynia or heat hyperalgesia. To take advantage of this neuropathic pain-resistant model, we analyzed Vglut2-dependent transcriptional changes in the dorsal spinal cord after nerve injury, which revealed several novel candidate target genes potentially relevant for the development of neuropathic pain therapeutics. Taken together, we conclude that VGLUT2 is a major mediator of nociception in primary afferents, implying that glutamate is the key somatosensory neurotransmitter.

  8. Glutamatergic inputs to the CVLM independent of the NTS promote tonic inhibition of sympathetic vasomotor tone in rats.

    Science.gov (United States)

    Mandel, Daniel A; Schreihofer, Ann M

    2008-10-01

    GABAergic neurons in the caudal ventrolateral medulla (CVLM) are driven by baroreceptor inputs relayed via the nucleus tractus solitarius (NTS), and they inhibit neurons in rostral ventrolateral medulla to reduce sympathetic nerve activity (SNA) and arterial pressure (AP). After arterial baroreceptor denervation or lesions of the NTS, inhibition of the CVLM continues to increase AP, suggesting additional inputs also tonically activate the CVLM. This study examined whether the NTS contributes to baroreceptor-independent drive to the CVLM and whether glutamate promotes baroreceptor- and NTS-independent activation of the CVLM to tonically reduce SNA. In addition, we evaluated whether altering central respiratory drive, a baroreceptor-independent regulator of CVLM neurons, influences glutamatergic inputs to the CVLM. Splanchnic SNA and AP were measured in chloralose-anesthetized, ventilated, paralyzed rats. The infusion of nitroprusside decreased AP below threshold for baroreceptor afferent firing (NTS by microinjection of the GABA(A) agonist muscimol did not further increase SNA. In contrast, after inhibition of the NTS, blockade of glutamatergic inputs to CVLM by microinjection of kynurenate increased SNA (274+/-54%; PNTS-mediated excitation of the CVLM. Furthermore, glutamate tonically activates the CVLM to reduce SNA independent of the NTS, and this excitatory input appears to be affected by the strength of central respiratory drive.

  9. Neuroimaging markers of glutamatergic and GABAergic systems in drug addiction: Relationships to resting-state functional connectivity.

    Science.gov (United States)

    Moeller, Scott J; London, Edythe D; Northoff, Georg

    2016-02-01

    Drug addiction is characterized by widespread abnormalities in brain function and neurochemistry, including drug-associated effects on concentrations of the excitatory and inhibitory neurotransmitters glutamate and gamma-aminobutyric acid (GABA), respectively. In healthy individuals, these neurotransmitters drive the resting state, a default condition of brain function also disrupted in addiction. Here, our primary goal was to review in vivo magnetic resonance spectroscopy and positron emission tomography studies that examined markers of glutamate and GABA abnormalities in human drug addiction. Addicted individuals tended to show decreases in these markers compared with healthy controls, but findings also varied by individual characteristics (e.g., abstinence length). Interestingly, select corticolimbic brain regions showing glutamatergic and/or GABAergic abnormalities have been similarly implicated in resting-state functional connectivity deficits in drug addiction. Thus, our secondary goals were to provide a brief review of this resting-state literature, and an initial rationale for the hypothesis that abnormalities in glutamatergic and/or GABAergic neurotransmission may underlie resting-state functional deficits in drug addiction. In doing so, we suggest future research directions and possible treatment implications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Glutamatergic and GABAergic disturbances as markers of choice-of-treatment – part of Pan European Collaboration on Antipsychotic Naïve Schizophrenia II (PECANS II)

    DEFF Research Database (Denmark)

    Bojesen, Kirsten Borup; Jessen, Kasper; Rostrup, Egill;

    may be related to persistent high levels of glutamate, the main excitatory neurotransmitter in the brain. However, the relationship of glutamatergic disturbances to psychopathology and level of functioning is largely unknown. Aim The aim is to clarify the relationship between glutamatergic...... disturbances in the two interconnected brain areas anterior cingulate cortex and thalamus to psychopathology and level of functioning in 40 anti-psychotic naïve patients with schizophrenia before and after 6 weeks treatment with aripiprazol. Method Design: Prospective 6 week follow-up study of 40 antipsychotic...

  11. NMDA and non-NMDA receptor gene expression following global brain ischemia in rats: effect of NMDA and non-NMDA receptor antagonists.

    Science.gov (United States)

    Pellegrini-Giampietro, D E; Pulsinelli, W A; Zukin, R S

    1994-03-01

    Transient forebrain or global ischemia in rats induces selective and delayed damage of hippocampal CA1 neurons. In a previous study, we have shown that expression of GluR2, the kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that governs Ca2+ permeability, is preferentially reduced in CA1 at a time point preceding neuronal degeneration. Postischemic administration of the selective AMPA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), protects CA1 neurons against delayed death. In this study we examined the effects of NBQX (at a neuroprotective dose) and of MK-801 (a selective NMDA receptor antagonist, not protective in this model) on kainate/AMPA receptor gene expression changes after global ischemia. We also examined the effects of transient forebrain ischemia on expression of the NMDA receptor subunit NMDAR1. In ischemic rats treated with saline, GluR2 and GluR3 mRNAs were markedly reduced in CA1 but were unchanged in CA3 or dentate gyrus. GluR1 and NMDAR1 mRNAs were not significantly changed in any region examined. Administration of NBQX or MK-801 did not alter the ischemia-induced changes in kainate/AMPA receptor gene expression. These findings suggest that NBQX affords neuroprotection by a direct blockade of kainate/AMPA receptors, rather than by a modification of GluR2 expression changes.

  12. Efficient and rapid induction of human iPSCs/ESCs into nephrogenic intermediate mesoderm using small molecule-based differentiation methods.

    Science.gov (United States)

    Araoka, Toshikazu; Mae, Shin-ichi; Kurose, Yuko; Uesugi, Motonari; Ohta, Akira; Yamanaka, Shinya; Osafune, Kenji

    2014-01-01

    The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.

  13. Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors

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    Wei Wen

    2016-06-01

    Full Text Available We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB with an improved episomal vector (EV system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B. Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1 equimolar expression of OCT4 (O and SOX2 (S, by using a 2A linker; (2 a higher and gradual increase in the MYC (M to KLF4 (K ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.

  14. Inclusion of mountain wave-induced cooling for the formation of PSCs over the Antarctic Peninsula in a chemistry–climate model

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    A. Orr

    2014-07-01

    Full Text Available An important source of polar stratospheric clouds (PSCs, which play a crucial role in controlling polar stratospheric ozone depletion, is from the temperature fluctuations induced by mountain waves. However, this formation mechanism is usually missing in chemistry–climate models because these temperature fluctuations are neither resolved nor parameterised. Here, we investigate the representation of stratospheric mountain wave-induced temperature fluctuations by the UK Met Office Unified Model (UM at high and low spatial resolution against Atmospheric Infrared Sounder satellite observations for three case studies over the Antarctic Peninsula. At a high horizontal resolution (4 km the mesoscale configuration of the UM correctly simulates the magnitude, timing, and location of the measured temperature fluctuations. By comparison, at a low horizontal resolution (2.5° × 3.75° the climate configuration fails to resolve such disturbances. However, it is demonstrated that the temperature fluctuations computed by a mountain wave parameterisation scheme inserted into the climate configuration (which computes the temperature fluctuations due to unresolved mountain waves are in excellent agreement with the mesoscale configuration responses. The parameterisation was subsequently used to compute the local mountain wave-induced cooling phases in the chemistry–climate configuration of the UM. This increased stratospheric cooling was passed to the PSC scheme of the chemistry–climate model, and caused a 30–50% increase in PSC surface area density over the Antarctic Peninsula compared to a 30 year control simulation.

  15. Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.

    Science.gov (United States)

    Wen, Wei; Zhang, Jian-Ping; Xu, Jing; Su, Ruijun Jeanna; Neises, Amanda; Ji, Guang-Zhen; Yuan, Weiping; Cheng, Tao; Zhang, Xiao-Bing

    2016-06-14

    We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B) is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.

  16. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

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    Laura Ordovás

    2015-11-01

    Full Text Available Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs. We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

  17. Efficient and rapid induction of human iPSCs/ESCs into nephrogenic intermediate mesoderm using small molecule-based differentiation methods.

    Directory of Open Access Journals (Sweden)

    Toshikazu Araoka

    Full Text Available The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM, an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days and efficient (80% induction rate IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA, and bone morphogenetic protein (BMP pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.

  18. Genome Editing of the CYP1A1 Locus in iPSCs as a Platform to Map AHR Expression throughout Human Development

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    Brenden W. Smith

    2016-01-01

    Full Text Available The aryl hydrocarbon receptor (AHR is a ligand activated transcription factor that increases the expression of detoxifying enzymes upon ligand stimulation. Recent studies now suggest that novel endogenous roles of the AHR exist throughout development. In an effort to create an optimized model system for the study of AHR signaling in several cellular lineages, we have employed a CRISPR/CAS9 genome editing strategy in induced pluripotent stem cells (iPSCs to incorporate a reporter cassette at the transcription start site of one of its canonical targets, cytochrome P450 1A1 (CYP1A1. This cell line faithfully reports on CYP1A1 expression, with luciferase levels as its functional readout, when treated with an endogenous AHR ligand (FICZ at escalating doses. iPSC-derived fibroblast-like cells respond to acute exposure to environmental and endogenous AHR ligands, and iPSC-derived hepatocytes increase CYP1A1 in a similar manner to primary hepatocytes. This cell line is an important innovation that can be used to map AHR activity in discrete cellular subsets throughout developmental ontogeny. As further endogenous ligands are proposed, this line can be used to screen for safety and efficacy and can report on the ability of small molecules to regulate critical cellular processes by modulating the activity of the AHR.

  19. Local Control of Extracellular Dopamine Levels in the Medial Nucleus Accumbens by a Glutamatergic Projection from the Infralimbic Cortex.

    Science.gov (United States)

    Quiroz, César; Orrú, Marco; Rea, William; Ciudad-Roberts, Andrés; Yepes, Gabriel; Britt, Jonathan P; Ferré, Sergi

    2016-01-20

    It is generally assumed that infralimbic cortex (ILC) and prelimbic cortex, two adjacent areas of the medial prefrontal cortex (mPFC) in rodents, provide selective excitatory glutamatergic inputs to the nucleus accumbens (NAc) shell and core, respectively. It is also generally believed that mPFC influences the extracellular levels of dopamine in the NAc primarily by an excitatory collateral to the ventral tegmental area (VTA). In the present study, we first established the existence of a selective functional connection between ILC and the posteromedial portions of the VTA (pmVTA) and the mNAc shell (pmNAc shell), by measuring striatal neuronal activation (immunohistochemical analysis of ERK1/2 phosphorylation) and glutamate release (in vivo microdialysis) upon ILC electrical stimulation. A novel optogenetic-microdialysis approach allowed the measurement of extracellular concentrations of glutamate and dopamine in the pmNAc shell upon local light-induced stimulation of glutamatergic terminals from ILC. Cortical electrical and local optogenetic stimulation produced significant increases in the extracellular concentrations of glutamate and dopamine in the pmNAc shell. Local blockade of glutamate release by perfusion of an adenosine A2A receptor antagonist in the pmNAc shell blocked the dopamine release induced by local optogenetic stimulation but only partially antagonized dopamine release induced by cortical electrical stimulation. The results demonstrate that ILC excitatory afferents directly modulate the extracellular concentration of dopamine in the pmNAc shell, but also support the involvement of an indirect mechanism of dopamine control, through a concomitant ILC-mediated activation of the pmVTA. Significance statement: We established the existence of a functional connection between the infralimbic cortex (ILC) and the posteromedial portions of the ventral tegmental area (pmVTA) and the medial nucleus acumbens shell (pmNAc shell). A novel optogenetic

  20. Distinct Localization of SNAP47 Protein in GABAergic and Glutamatergic Neurons in the Mouse and the Rat Hippocampus.

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    Münster-Wandowski, Agnieszka; Heilmann, Heike; Bolduan, Felix; Trimbuch, Thorsten; Yanagawa, Yuchio; Vida, Imre

    2017-01-01

    Synaptosomal-associated protein of 47 kDa (SNAP47) isoform is an atypical member of the SNAP family, which does not contribute directly to exocytosis and synaptic vesicle (SV) recycling. Initial characterization of SNAP47 revealed a widespread expression in nervous tissue, but little is known about its cellular and subcellular localization in hippocampal neurons. Therefore, in the present study we applied multiple-immunofluorescence labeling, immuno-electron microscopy and in situ hybridization (ISH) and analyzed the localization of SNAP47 in pre- and postsynaptic compartments of glutamatergic and GABAergic neurons in the mouse and rat hippocampus. While the immunofluorescence signal for SNAP47 showed a widespread distribution in both mouse and rat, the labeling pattern was complementary in the two species: in the mouse the immunolabeling was higher over the CA3 stratum radiatum, oriens and cell body layer. In contrast, in the rat the labeling was stronger over the CA1 neuropil and in the CA3 stratum lucidum. Furthermore, in the mouse high somatic labeling for SNAP47 was observed in GABAergic interneurons (INs). On the contrary, in the rat, while most INs were positive, they blended in with the high neuropil labeling. ISH confirmed the high expression of SNAP47 RNA in INs in the mouse. Co-staining for SNAP47 and pre- and postsynaptic markers in the rat revealed a strong co-localization postsynaptically with PSD95 in dendritic spines of pyramidal cells and, to a lesser extent, presynaptically, with ZnT3 and vesicular glutamate transporter 1 (VGLUT1) in glutamatergic terminals such as mossy fiber (MF) boutons. Ultrastructural analysis confirmed the pre- and postsynaptic localization at glutamatergic synapses. Furthermore, in the mouse hippocampus SNAP47 was found to be localized at low levels to dendritic shafts and axon terminals of putative INs forming symmetric synapses, indicating that this protein could be trafficked to both post- and presynaptic sites in both

  1. Oestradiol Regulates Neuropeptide Y Release and Gene Coupling with the GABAergic and Glutamatergic Synapses in the Adult Female Rat Dentate Gyrus.

    Science.gov (United States)

    Velíšková, J; Iacobas, D; Iacobas, S; Sidyelyeva, G; Chachua, T; Velíšek, L

    2015-12-01

    Neuropeptide Y (NPY) is an endogenous modulator of neuronal activity affecting both GABAergic and glutamatergic transmission. Previously, we found that oestradiol modifies the number of NPY immunoreactive neurones in the hippocampal dentate gyrus. In the present study, we investigated which oestrogen receptor type is responsible for these changes in the number of NPY-positive neurones. Furthermore, we determined the effects of oestrogen receptor activation on NPY release. Finally, we examined the contribution of oestrogen toward the remodelling of the GABAergic and glutamatergic gene networks in terms of coupling with Npy gene expression in ovariectomised rats. We found that activation of either oestrogen receptor type (ERα or ERβ) increases the number of NPY-immunopositive neurones and enhances NPY release in the dentate gyrus. We also found that, compared to oestrogen-lacking ovariectomised rats, oestrogen replacement increases the probability of synergistic/antagonistic coupling between the Npy and GABAergic synapse genes, whereas the glutamatergic synapse genes are less likely to be coupled with Npy under similar conditions. The data together suggest that oestrogens play a critical role in the regulation of NPY system activity and are also involved in the coupling/uncoupling of the Npy gene with the GABAergic and glutamatergic synapses in the female rat dentate gyrus. © 2015 British Society for Neuroendocrinology.

  2. The Origin, Development and Molecular Diversity of Rodent Olfactory Bulb Glutamatergic Neurons Distinguished by Expression of Transcription Factor NeuroD1.

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    Laurent Roybon

    Full Text Available Production of olfactory bulb neurons occurs continuously in the rodent brain. Little is known, however, about cellular diversity in the glutamatergic neuron subpopulation. In the central nervous system, the basic helix-loop-helix transcription factor NeuroD1 (ND1 is commonly associated with glutamatergic neuron development. In this study, we utilized ND1 to identify the different subpopulations of olfactory bulb glutamategic neurons and their progenitors, both in the embryo and postnatally. Using knock-in mice, transgenic mice and retroviral transgene delivery, we demonstrate the existence of several different populations of glutamatergic olfactory bulb neurons, the progenitors of which are ND1+ and ND1- lineage-restricted, and are temporally and regionally separated. We show that the first olfactory bulb glutamatergic neurons produced - the mitral cells - can be divided into molecularly diverse subpopulations. Our findings illustrate the complexity of neuronal diversity in the olfactory bulb and that seemingly homogenous neuronal populations can consist of multiple subpopulations with unique molecular signatures of transcription factors and expressing neuronal subtype-specific markers.

  3. Cholinergic and glutamatergic transmission at synapses between pedunculopotine tegmental nucleus axonal terminals and A7 catecholamine cell group noradrenergic neurons in the rat.

    Science.gov (United States)

    Li, Meng-Jiyuan; Chang, Tien-Wei; Hung, Wei-Chen; Wu, Chieh-Yi; Luo, Yu-Cheng; Chang, Ting-Hsuan; Lin, Chingju; Yang, Chi-Sheng; Yang, Hsiu-Wen; Min, Ming-Yuan

    2016-11-01

    We characterized transmission from the pedunculopotine tegmental nucleus (PPTg), which contains cholinergic and glutamatergic neurons, at synapses with noradrenergic (NAergic) A7 neurons. Injection of an anterograde neuronal tracer, biotinylated-dextran amine, into the PPTg resulted in labeling of axonal terminals making synaptic connection with NAergic A7 neurons. Consistent with this, extracellular stimulation using a train of 10 pulses at 100 Hz evoked both fast and slow excitatory synaptic currents (EPSCs) that were blocked, respectively, by DNQX, a non-N-methyl-d-aspartate receptor blocker, or atropine, a cholinergic muscarinic receptor (mAChR) blocker. Interestingly, many spontaneous-like, but stimulation-dependent, EPSCs, were seen for up to one second after the end of stimulation and were blocked by DNQX and decreased by EGTA-AM, a membrane permeable form of EGTA, showing they are glutamatergic EPSCs causing by asynchronous release of vesicular quanta. Moreover, application of atropine or carbachol, an mAChR agonist, caused, respectively, an increase in the number of asynchronous EPSCs or a decrease in the frequency of miniature EPSCs, showing that mAChRs mediated presynaptic inhibition of glutamatergic transmission of the PPTg onto NAergic A7 neurons. In conclusion, our data show direct synaptic transmission of PPTg afferents onto pontine NAergic neurons that involves cooperation of cholinergic and glutamatergic transmission. This dual-transmitter transmission drives the firing rate of NAergic neurons, which may correlate with axonal and somatic/dendritic release of NA.

  4. Non-Invasive Evaluation of the GABAergic/Glutamatergic System in Autistic Patients Observed by MEGA-Editing Proton MR Spectroscopy Using a Clinical 3 Tesla Instrument

    Science.gov (United States)

    Harada, Masafumi; Taki, Masako M.; Nose, Ayumi; Kubo, Hitoshi; Mori, Kenji; Nishitani, Hiromu; Matsuda, Tsuyoshi

    2011-01-01

    Amino acids related to neurotransmitters and the GABAergic/glutamatergic system were measured using a 3 T-MRI instrument in 12 patients with autism and 10 normal controls. All measurements were performed in the frontal lobe (FL) and lenticular nuclei (LN) using a conventional sequence for n-acetyl aspartate (NAA) and glutamate (Glu), and the…

  5. Corticolimbic hyper-response to emotion and glutamatergic function in people with high schizotypy : a multimodal fMRI-MRS study

    NARCIS (Netherlands)

    Modinos, G; McLaughlin, A; Egerton, A; McMullen, K; Kumari, V; Barker, G J; Keysers, C; Williams, S C R

    2017-01-01

    Animal models and human neuroimaging studies suggest that altered levels of glutamatergic metabolites within a corticolimbic circuit have a major role in the pathophysiology of schizophrenia. Rodent models propose that prefrontal glutamate dysfunction could lead to amygdala hyper-response to

  6. Mice lacking brain/kidney phosphate-activated glutaminase have impaired glutamatergic synaptic transmission, altered breathing, disorganized goal-directed behavior and die shortly after birth.

    Science.gov (United States)

    Masson, Justine; Darmon, Michèle; Conjard, Agnès; Chuhma, Nao; Ropert, Nicole; Thoby-Brisson, Muriel; Foutz, Arthur S; Parrot, Sandrine; Miller, Gretchen M; Jorisch, Renée; Polan, Jonathan; Hamon, Michel; Hen, René; Rayport, Stephen

    2006-04-26

    Neurotransmitter glutamate has been thought to derive mainly from glutamine via the action of glutaminase type 1 (GLS1). To address the importance of this pathway in glutamatergic transmission, we knocked out GLS1 in mice. The insertion of a STOP cassette by homologous recombination produced a null allele that blocked transcription, encoded no immunoreactive protein, and abolished GLS1 enzymatic activity. Null mutants were slightly smaller, were deficient in goal-directed behavior, hypoventilated, and died in the first postnatal day. No gross or microscopic defects were detected in peripheral organs or in the CNS. In cultured neurons from the null mutants, miniature EPSC amplitude and duration were normal; however, the amplitude of evoked EPSCs decayed more rapidly with sustained 10 Hz stimulation, consistent with an observed reduction in depolarization-evoked glutamate release. Because of this activity-dependent impairment in glutamatergic transmission, we surmised that respiratory networks, which require temporal summation of synaptic input, would be particularly affected. We found that the amplitude of inspirations was decreased in vivo, chemosensitivity to CO2 was severely altered, and the frequency of pacemaker activity recorded in the respiratory generator in the pre-Bötzinger complex, a glutamatergic brainstem network that can be isolated in vitro, was increased. Our results show that although alternate pathways to GLS1 glutamate synthesis support baseline glutamatergic transmission, the GLS1 pathway is essential for maintaining the function of active synapses, and thus the mutation is associated with impaired respiratory function, abnormal goal-directed behavior, and neonatal demise.

  7. Non-Invasive Evaluation of the GABAergic/Glutamatergic System in Autistic Patients Observed by MEGA-Editing Proton MR Spectroscopy Using a Clinical 3 Tesla Instrument

    Science.gov (United States)

    Harada, Masafumi; Taki, Masako M.; Nose, Ayumi; Kubo, Hitoshi; Mori, Kenji; Nishitani, Hiromu; Matsuda, Tsuyoshi

    2011-01-01

    Amino acids related to neurotransmitters and the GABAergic/glutamatergic system were measured using a 3 T-MRI instrument in 12 patients with autism and 10 normal controls. All measurements were performed in the frontal lobe (FL) and lenticular nuclei (LN) using a conventional sequence for n-acetyl aspartate (NAA) and glutamate (Glu), and the…

  8. Generation of induced pluripotent stem cells (iPSCs from a hypertrophic cardiomyopathy patient with the pathogenic variant p.Val698Ala in beta-myosin heavy chain (MYH7 gene

    Directory of Open Access Journals (Sweden)

    Samantha Barratt Ross

    2017-04-01

    Full Text Available Induced pluripotent stem cells (iPSCs were generated from peripheral blood mononuclear cells (PBMCs isolated from the whole blood of a 43-year-old male with hypertrophic cardiomyopathy (HCM who carries the pathogenic variant p.Val698Ala in beta-myosin heavy chain (MYH7. Patient-derived PBMCs were reprogrammed using non-integrative episomal vectors containing reprogramming factors OCT4, SOX2, LIN28, KLF4 and L-MYC. iPSCs were shown to express pluripotent markers, have trilineage differentiation potential, carry the pathogenic MYH7 variant p.Val698Ala, have a normal karyotype and no longer carry the episomal reprogramming vector. This line is useful for studying the link between variants in MYH7 and the pathogenesis of HCM.

  9. The Drosophila larval neuromuscular junction as a model for scaffold complexes at glutamatergic synapses: benefits and limitations.

    Science.gov (United States)

    Thomas, Ulrich; Kobler, Oliver; Gundelfinger, Eckart D

    2010-09-01

    Based on unbeatable genetic accessibility and relative simplicity, the Drosophila larval neuromuscular junction has become a widely used model system for studying functional and structural aspects of excitatory glutamatergic synapses. Membrane-associated guanylate kinase-like proteins (MAGUKs) are first-order scaffolding molecules enriched at many cellular junctions, including synapses, where they coordinate multiple binding partners, including cell adhesion molecules and ion channels. The enrichment of the prototypic MAGUK Discs-Large at larval NMJs apparently parallels the high abundance of its homologs at excitatory synapses in the mammalian central nervous system. Here, the authors review selected aspects of the long-standing work on Dlg at fly neuromuscular junctions, thereby scrutinizing its subcellular localization, function, and regulation with regard to corresponding aspects of MAGUKs in vertebrate neurons.

  10. Specification of spatial identities of cerebellar neuron progenitors by ptf1a and atoh1 for proper production of GABAergic and glutamatergic neurons.

    Science.gov (United States)

    Yamada, Mayumi; Seto, Yusuke; Taya, Shinichiro; Owa, Tomoo; Inoue, Yukiko U; Inoue, Takayoshi; Kawaguchi, Yoshiya; Nabeshima, Yo-Ichi; Hoshino, Mikio

    2014-04-01

    In the cerebellum, the bHLH transcription factors Ptf1a and Atoh1 are expressed in distinct neuroepithelial regions, the ventricular zone (VZ) and the rhombic lip (RL), and are required for producing GABAergic and glutamatergic neurons, respectively. However, it is unclear whether Ptf1a or Atoh1 is sufficient for specifying GABAergic or glutamatergic neuronal fates. To test this, we generated two novel knock-in mouse lines, Ptf1a(Atoh1) and Atoh1(Ptf1a), that are designed to express Atoh1 and Ptf1a ectopically in the VZ and RL, respectively. In Ptf1a(Atoh1) embryos, ectopically Atoh1-expressing VZ cells produced glutamatergic neurons, including granule cells and deep cerebellar nuclei neurons. Correspondingly, in Atoh1(Ptf1a) animals, ectopically Ptf1a-expressing RL cells produced GABAergic populations, such as Purkinje cells and GABAergic interneurons. Consistent results were also obtained from in utero electroporation of Ptf1a or Atoh1 into embryonic cerebella, suggesting that Ptf1a and Atoh1 are essential and sufficient for GABAergic versus glutamatergic specification in the neuroepithelium. Furthermore, birthdating analyses with BrdU in the knock-in mice or with electroporation studies showed that ectopically produced fate-changed neuronal types were generated at temporal schedules closely simulating those of the wild-type RL and VZ, suggesting that the VZ and RL share common temporal information. Observations of knock-in brains as well as electroporated brains revealed that Ptf1a and Atoh1 mutually negatively regulate their expression, probably contributing to formation of non-overlapping neuroepithelial domains. These findings suggest that Ptf1a and Atoh1 specify spatial identities of cerebellar neuron progenitors in the neuroepithelium, leading to appropriate production of GABAergic and glutamatergic neurons, respectively.

  11. Distribution of type 1 cannabinoid receptor-expressing neurons in the septal-hypothalamic region of the mouse: colocalization with GABAergic and glutamatergic markers.

    Science.gov (United States)

    Hrabovszky, Erik; Wittmann, Gábor; Kalló, Imre; Füzesi, Tamás; Fekete, Csaba; Liposits, Zsolt

    2012-04-01

    Type 1 cannabinoid receptor (CB1) is the principal mediator of retrograde endocannabinoid signaling in the brain. In this study, we addressed the topographic distribution and amino acid neurotransmitter phenotype of endocannabinoid-sensitive hypothalamic neurons in mice. The in situ hybridization detection of CB1 mRNA revealed high levels of expression in the medial septum (MS) and the diagonal band of Broca (DBB), moderate levels in the preoptic area and the hypothalamic lateroanterior (LA), paraventricular (Pa), ventromedial (VMH), lateral mammillary (LM), and ventral premammillary (PMV) nuclei, and low levels in many other hypothalamic regions including the suprachiasmatic (SCh) and arcuate (Arc) nuclei. This regional distribution pattern was compared with location of γ-aminobutyric acid (GABA)ergic and glutamatergic cell groups, as identified by the expression of glutamic acid decarboxylase 65 (GAD65) and type 2 vesicular glutamate transporter (VGLUT2) mRNAs, respectively. The MS, DBB, and preoptic area showed overlaps between GABAergic and CB1-expressing neurons, whereas hypothalamic sites with moderate CB1 signals, including the LA, Pa, VMH, LM, and PMV, were dominated by glutamatergic neurons. Low CB1 mRNA levels were also present in other glutamatergic and GABAergic regions. Dual-label in situ hybridization experiments confirmed the cellular co-expression of CB1 with both glutamatergic and GABAergic markers. In this report we provide a detailed anatomical map of hypothalamic glutamatergic and GABAergic systems whose neurotransmitter release is controlled by retrograde endocannabinoid signaling from hypothalamic and extrahypothalamic target neurons. This neuroanatomical information contributes to an understanding of the role that the endocannabinoid system plays in the regulation of endocrine and metabolic functions.

  12. Subregional Expression of Hippocampal Glutamatergic and GABAergic Genes in F344 Rats with Social Isolation after Weaning.

    Science.gov (United States)

    Iwata, Hisaya; Yamamuro, Yutaka

    2016-02-01

    Many studies have shown that postweaning social isolation (pwSI) alters various behavioral phenotypes, including hippocampusdependent tasks. Here, we report the comprehensive analysis of the expression of glutamatergic and GABAergic neurotransmissionrelated genes in the distinct hippocampal subregions of pwSI rats. Male F344 rats (age, 4 wk) experienced either pwSI or group housing (controls). At 7 wk of age, the hippocampus of each rat was removed and laser-microdissected into the CA1 and CA3 layers of pyramidal cells and the granule cell layer of the dentate gyrus. Subsequently, the expression of glutamatergic- and GABAergic- related genes was analyzed by quantitative RT-PCR. In the CA1 and CA3 pyramidal cell layers, 18 of 24 glutamate receptor subunit genes were at least 1.5-fold increased in expression after pwSI. In particular, the expression of several N-methyl-D-aspartate and kainate receptors (for example, Grin2a in CA1, Grik4 in CA3) was significantly increased after pwSI. In contrast, pwSI tended to decrease the expression of GABAA receptor subunit genes, and Gabra1, Gabra2, Gabra4, Gabra5, Gabrb2, Gabrg1, and Gabrg2 were all significantly decreased in expression compared with the levels in the group-housed rats. These results indicate a subregion- specific increase of glutamate receptors and reduction of GABAA receptors, suggesting that the hippocampal circuits of pwSI rats may be in more excitable states than those of group-housed rats.

  13. Regulation of the ERK pathway in the dentate gyrus by in vivo dopamine D1 receptor stimulation requires glutamatergic transmission.

    Science.gov (United States)

    Gangarossa, Giuseppe; Valjent, Emmanuel

    2012-11-01

    Acute systemic administration of the dopamine D1/D5 receptors (D1Rs) agonist, SKF81297, activates the extracellular signal-regulated protein kinases (ERK) pathway selectively in the granule cells of the dentate gyrus. In this study, we examined the mechanisms involved in this regulation and investigated the molecular components that could promote ERK-dependent transcription and translation. SKF81297 induced phosphorylation of ERK and histone H3 required intact glutamatergic transmission. Blockade of glutamate release achieved by the mGluR2/3 agonist, LY354740 or the selective adenosine A1R agonist, CCPA as well as neurotoxic lesions of lateral entorhinal cortex reduced the ability of SKF81297 to induce ERK activation in the dentate gyrus. This activation required the combined stimulation of NR2B-containing NMDARs, mGluR1 and mGluR5. SKF81297 evoked phosphorylation of the ribosomal protein S6 (rpS6) selectively at the Ser235/236 site while the Ser240/244 site remains unchanged. The SKF81297 induced increased phosphorylation of rpS6 was dependent on PKC and ERK/p90RSK activation. Surprisingly, administration of D1Rs agonist suppressed mTORC1/p70S6K pathway suggesting an mTOR-independent regulation of rpS6 phosphorylation. Taken together, our results show that intact glutamatergic transmission plays a major role in the regulation of ERK-dependent phosphorylation of histone H3 and rpS6 observed in the mouse dentate gyrus after systemic administration of SKF81297.

  14. Expression of the System N transporter (SNAT5/SN2) during development indicates its plausible role in glutamatergic neurotransmission.

    Science.gov (United States)

    Rodríguez, Angelina; Ortega, Arturo; Berumen, Laura C; García-Alcocer, María G; Giménez, Cecilio; Zafra, Francisco

    2014-07-01

    Solute neutral amino acid transporter 5 (SNAT5/SN2) is a member of the System N family, expressed in glial cells in the adult brain, able to transport glutamine, histidine or glycine among other substrates. Its tight association with synapses and its electroneutral mode of operation that allows the bidirectional movement of substrates, supports the idea that this transporter participates in the function of the glutamine-glutamate cycle between neurons and glia. Moreover, SNAT5/SN2 might contribute to the regulation of glycine concentration in glutamatergic synapses and, therefore, to the functioning of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors. Ontogenic maturation of these synapses occurs postnatally through the coordinate expression of a large number of receptors, transporters, structural and regulatory proteins that ensure the correct operation of the excitatory pathways in the central nervous system. Since the temporal pattern of expression of SNAT5/SN2 is unknown, we analyzed it by immunoblot and immunohistochemical techniques. Results indicate that the expression of SNAT5/SN2 is triggered between the second and third postnatal week in the cerebral cortex, in parallel to the expression of the vesicular glutamate transporter vGLUT1 and the glial glutamate transporter GLT1/EAAT2. In the cerebellum, this process occurs about one week later than in the cerebral cortex. Immunohistochemical staining of cortical sections shows that from postnatal day 14 to adulthood the transporter was expressed exclusively in glial cells. Our results are consistent with the idea that SNAT5/SN2 expression is coordinated with that of other proteins necessary for the operation of glutamatergic synapses and reinforce the existence of a regulatory cross-talk between neurons and glia that orchestrates the building up of these synapses.

  15. Mixed Electrical-Chemical Synapses in Adult Rat Hippocampus are Primarily Glutamatergic and Coupled by Connexin-36.

    Science.gov (United States)

    Hamzei-Sichani, Farid; Davidson, Kimberly G V; Yasumura, Thomas; Janssen, William G M; Wearne, Susan L; Hof, Patrick R; Traub, Roger D; Gutiérrez, Rafael; Ottersen, Ole P; Rash, John E

    2012-01-01

    Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for "mixed" (electrical/chemical) synapses on both principal cells and interneurons in adult rat hippocampus. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF) terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr), apparently forming glutamatergic mixed synapses. Lucifer Yellow injected into weakly fixed CA3pyr was detected in MF axons that contacted four injected CA3pyr, supporting gap junction-mediated coupling between those two types of principal cells. Freeze-fracture replica immunogold labeling revealed diverse sizes and morphologies of connexin-36-containing gap junctions throughout hippocampus. Of 20 immunogold-labeled gap junctions, seven were large (328-1140 connexons), three of which were consistent with electrical synapses between interneurons; but nine were at axon terminal synapses, three of which were immediately adjacent to distinctive glutamate receptor-containing postsynaptic densities, forming mixed glutamatergic synapses. Four others were adjacent to small clusters of immunogold-labeled 10-nm E-face intramembrane particles, apparently representing extrasynaptic glutamate receptor particles. Gap junctions also were on spines in stratum lucidum, stratum oriens, dentate gyrus, and hilus, on both interneurons and unidentified neurons. In addition, one putative GABAergic mixed synapse was found in thin-section images of a CA3pyr, but none were found by immunogold labeling, suggesting the rarity of GABAergic mixed synapses. Cx36-containing gap junctions throughout hippocampus suggest the possibility of reciprocal modulation of electrical and chemical signals in diverse hippocampal neurons.

  16. Mixed electrical-chemical synapses in adult rat hippocampus are primarily glutamatergic and coupled by connexin-36

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    Farid eHamzei-Sichani

    2012-05-01

    Full Text Available Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in the mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for mixed (electrical/chemical synapses in adult rat hippocampus on both principal cells and interneurons. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr, apparently forming glutamatergic mixed synapses. Lucifer Yellow injected into four weakly-fixed CA3pyr was detected in MF axons that contacted the injected CA3pyr, supporting gap junction-mediated coupling between those two types of principal cells. Freeze-fracture replica immunogold-labeling revealed diverse sizes and morphologies of connexin36-containing gap junctions throughout hippocampus. Of 20 immunogold-labeled gap junctions, seven were large (328-1140 connexons, three of which were consistent with electrical synapses between interneurons; but nine were at axon terminal synapses, three of which were immediately adjacent to distinctive glutamate receptor-containing postsynaptic densities, forming mixed glutamatergic synapses. Four others were adjacent to small clusters of immunogold-labeled 10-nm E-face intramembrane particles, apparently representing extrasynaptic glutamate receptor particles. Gap junctions also were on spines in stratum lucidum, stratum oriens, dentate gyrus, and hilus, on both interneurons and unidentified neurons. In addition, one putative GABAergic mixed synapse was found in thin section images of a CA3pyr, but none found by immunogold-labeling were at GABAergic mixed synapses, suggesting their rarity. Cx36-containing gap junctions throughout hippocampus suggest the possibility of reciprocal modulation of electrical and chemical signals in diverse hippocampal

  17. PSD-Zip70 Deficiency Causes Prefrontal Hypofunction Associated with Glutamatergic Synapse Maturation Defects by Dysregulation of Rap2 Activity.

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    Mayanagi, Taira; Yasuda, Hiroki; Sobue, Kenji

    2015-10-21

    Dysregulation of synapse formation and plasticity is closely related to the pathophysiology of psychiatric and neurodevelopmental disorders. The prefrontal cortex (PFC) is particularly important for executive functions such as working memory, cognition, and emotional control, which are impaired in the disorders. PSD-Zip70 (Lzts1/FEZ1) is a postsynaptic density (PSD) protein predominantly expressed in the frontal cortex, olfactory bulb, striatum, and hippocampus. Here we found that PSD-Zip70 knock-out (PSD-Zip70KO) mice exhibit working memory and cognitive defects, and enhanced anxiety-like behaviors. These abnormal behaviors are caused by impaired glutamatergic synapse transmission accompanied by tiny-headed immature dendritic spines in the PFC, due to aberrant Rap2 activation, which has roles in synapse formation and plasticity. PSD-Zip70 modulates the Rap2 activity by interacting with SPAR (spine-associated RapGAP) and PDZ-GEF1 (RapGEF) in the postsynapse. Furthermore, suppression of the aberrant Rap2 activation in the PFC rescued the behavioral defects in PSD-Zip70KO mice. Our data demonstrate a critical role for PSD-Zip70 in Rap2-dependent spine synapse development in the PFC and underscore the importance of this regulation in PFC-dependent behaviors. PSD-Zip70 deficiency causes behavioral defects in working memory and cognition, and enhanced anxiety due to prefrontal hypofunction. This study revealed that PSD-Zip70 plays essential roles in glutamatergic synapse maturation via modulation of the Rap2 activity in the PFC. PSD-Zip70 interacts with both SPAR (spine-associated RapGAP) and PDZ-GEF1 (RapGEF) and modulates the Rap2 activity in postsynaptic sites. Our results provide a novel Rap2-specific regulatory mechanism in synaptic maturation involving PSD-Zip70. Copyright © 2015 the authors 0270-6474/15/3514327-14$15.00/0.

  18. Eugenol reduces acute pain in mice by modulating the glutamatergic and tumor necrosis factor alpha (TNF-α) pathways.

    Science.gov (United States)

    Dal Bó, Wladmir; Luiz, Ana Paula; Martins, Daniel F; Mazzardo-Martins, Leidiane; Santos, Adair R S

    2013-10-01

    Eugenol is utilized together with zinc oxide in odontological clinical for the cementation of temporary prostheses and the temporary restoration of teeth and cavities. This work explored the antinociceptive effects of the eugenol in different models of acute pain in mice and investigated its possible modulation of the inhibitory (opioid) and excitatory (glutamatergic and pro-inflammatory cytokines) pathways of nociceptive signaling. The administration of eugenol (3-300 mg/kg, p.o., 60 min or i.p., 30 min) inhibited 82 ± 10% and 90 ± 6% of the acetic acid-induced nociception, with ID₅₀ values of 51.3 and 50.2 mg/kg, respectively. In the glutamate test, eugenol (0.3-100 mg/kg, i.p.) reduced the response behavior by 62 ± 5% with an ID₅₀ of 5.6 mg/kg. In addition, the antinociceptive effect of eugenol (10 mg/kg, i.p.) in the glutamate test was prevented by the i.p. treatment for mice with naloxone. The pretreatment of mice with eugenol (10 mg/kg, i.p.) was able to inhibit the nociception induced by the intrathecal (i.t.) injection of glutamate (37 ± 9%), kainic (acid kainite) (41 ± 12%), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (55 ± 5%), and substance P (SP) (39 ± 8%). Furthermore, eugenol (10 mg/kg, i.p.) also inhibited biting induced by tumor necrosis factor alpha (TNF-α, 65 ± 8%). These results extend our current knowledge of eugenol and confirm that it promotes significant antinociception against different mouse models of acute pain. The mechanism of action appears to involve the modulation of the opioid system and glutamatergic receptors (i.e., kainate and AMPA), and the inhibition of TNF-α. Thus, eugenol could represent an important compound in the treatment for acute pain.

  19. Satellite NG2 progenitor cells share common glutamatergic inputs with associated interneurons in the mouse dentate gyrus.

    Science.gov (United States)

    Mangin, Jean-Marie; Kunze, Albrecht; Chittajallu, Ramesh; Gallo, Vittorio

    2008-07-23

    Several studies have provided evidence that NG2-expressing (NG2(+)) progenitor cells are anatomically associated to neurons in gray matter areas. By analyzing the spatial distribution of NG2(+) cells in the hilus of the mouse dentate gyrus, we demonstrate that NG2(+) cells are indeed closely associated to interneurons. To define whether this anatomical proximity reflected a specific physiological interaction, we performed patch-clamp recordings on hilar NG2(+) cells and interneurons between 3 and 21 postnatal days. We first observed that hilar NG2(+) cells exhibit spontaneous glutamatergic EPSCs (sEPSCs) whose frequency and amplitude increase during the first 3 postnatal weeks. At the same time, the rise time and decay time of sEPSCs significantly decreased, suggesting that glutamatergic synapses in NG2(+) cells undergo a maturation process that is reminiscent of what has been reported in neurons during the same time period. We also observed that hilar interneurons and associated NG2(+) cells are similarly integrated into the local network, receiving excitatory inputs from both granule cells and CA3 pyramidal neurons. By performing pair recordings, we found that bursts of activity induced by GABAergic antagonists were strongly synchronized between both cell types and that the amplitude of these bursts was positively correlated. Finally, by applying carbachol to increase EPSC activity, we observed that closely apposed cells were more likely to exhibit synchronized EPSCs than cells separated by >200 microm. The finding that NG2(+) cells are sensing patterns of activity arising in closely associated neurons suggests that NG2(+) cell function is finely regulated by the local network.

  20. Investigating glutamatergic mechanism in attention and impulse control using rats in a modified 5-choice serial reaction time task.

    Directory of Open Access Journals (Sweden)

    Abigail Benn

    Full Text Available The 5-choice serial reaction time task (5CSRTT has been widely used to study attention and impulse control in rodents. In order to mimic cognitive impairments in psychiatry, one approach has been to use acute administration of NMDA antagonists. This disruption in glutamatergic transmission leads to impairments in accuracy, omissions, and premature responses although findings have been inconsistent. In this study, we further investigated glutamatergic mechanisms using a novel version of the 5CSRTT, which we have previously shown to be more sensitive to cognitive enhancers. We first investigated the effects of systemic treatment with NMDA antagonists. We also carried out a preliminary investigation using targeted medial prefrontal cortex infusions of a NMDA antagonist (MK801, mGluR2/3 antagonist (LY341495, and mGluR7 negative allosteric modulator (MMPIP. Acute systemic administration of the different NMDA antagonists had no specific effects on accuracy. At higher doses PCP, ketamine, and memantine, increased omissions and affected other measures suggesting a general disruption in task performance. Only MK801 increased premature responses, and reduced omissions at lower doses suggesting stimulant like effects. None of the NMDA antagonists affected accuracy or any other measures when tested using a short stimulus challenge. Infusions of MK801 had no effect on accuracy but increased premature responses following infralimbic, but not prelimbic infusion. LY341495 had no effects in either brain region but a decrease in accuracy was observed following prelimbic infusion of MMPIP. Contrary to our hypothesis, disruptions to glutamate transmission using NMDA antagonists did not induce any clear deficits in accuracy in this modified version of the 5CSRTT. We also found that the profile of effects for MK801 differed from those observed with PCP, ketamine, and memantine. The effects of MK801 in the infralimbic cortex add to the literature indicating this brain

  1. Late postnatal shifts of parvalbumin and nitric oxide synthase expression within the GABAergic and glutamatergic phenotypes of inferior colliculus neurons.

    Science.gov (United States)

    Fujimoto, Hisataka; Konno, Kotaro; Watanabe, Masahiko; Jinno, Shozo

    2017-03-01

    The inferior colliculus (IC) is partitioned into three subdivisions: the dorsal and lateral cortices (DC and LC) and the central nucleus (ICC), and serves as an integration center of auditory information. Recent studies indicate that a certain population of IC neurons may represent the non-GABAergic phenotype, while they express well-established cortical/hippocampal GABAergic neuron markers. In this study we used the optical disector to investigate the phenotype of IC neurons expressing parvalbumin (PV) and/or nitric oxide synthase (NOS) in C57BL/6J mice during the late postnatal period. Four major types of IC neurons were defined by the presence (+) or absence (-) of PV, NOS, and glutamic acid decarboxylase 67 (GAD67): PV(+) /NOS(-) /GAD67(+) , PV(+) /NOS(+) /GAD67(+) , PV(+) /NOS(-) /GAD67(-) , and PV(-) /NOS(+) /GAD67(-) . Fluorescent in situ hybridization for vesicular glutamate transporter 2 mRNA indicated that almost all GAD67(-) IC neurons represented the glutamatergic phenotype. The numerical densities (NDs) of total GAD67(+) IC neurons remained unchanged in all subdivisions. The NDs of PV(+) /NOS(-) /GAD67(+) neurons and PV(-) /NOS(+) /GAD67(-) neurons were reduced with age in the ICC, while they remained unchanged in the DC and LC. By contrast, the NDs of PV(+) /NOS(+) /GAD67(+) neurons and PV(+) /NOS(-) /GAD67(-) neurons were increased with age in the ICC, although there were no changes in the DC and LC. The cell body size of GAD67(+) IC neurons did not vary according to the expression of PV with or without NOS. The present findings indicate that the expression of PV and NOS may shift with age within the GABAergic and glutamatergic phenotypes of IC neurons during the late postnatal period. J. Comp. Neurol. 525:868-884, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Ethanol produces corticotropin releasing factor receptor-dependent enhancement of spontaneous glutamatergic transmission in the mouse central amygdala

    Science.gov (United States)

    Silberman, Yuval; Fetterly, Tracy L.; Awad, Elias K.; Milano, Elana J.; Usdin, Ted B.; Winder, Danny G.

    2015-01-01

    Background Ethanol modulation of Central Amygdala (CeA) neurocircuitry plays a key role in the development of alcoholism via activation of the corticotropin releasing factor (CRF) receptor system. Previous work has predominantly focused on ethanol/CRF interactions on the CeA GABA circuitry; however our lab recently showed that CRF enhances CeA glutamatergic transmission. Therefore, this study sought to determine if ethanol modulates CeA glutamate transmission via activation of CRF signaling. Methods The effects of ethanol on spontaneous excitatory postsynaptic currents (sEPSCs) and basal resting membrane potentials were examined via standard electrophysiology methods in adult male C57BL/6J mice. Local ablation of CeA CRF neurons (CRFCeAhDTR) was achieved by targeting the human diphtheria toxin receptor (hDTR) to CeA CRF neurons with an adeno-associated virus. Ablation was quantified post-hoc with confocal microscopy. Genetic targeting of the diphtheria toxin active subunit to CRF neurons (CRFDTA mice) ablated CRF neurons throughout the CNS, as assessed by qRT-PCR quantification of CRF mRNA. Results Acute bath application of ethanol significantly increased sEPSC frequency in a concentration dependent manner in CeA neurons, and this effect was blocked by pretreatment of co-applied CRF receptor 1 and CRF receptor 2 antagonists. In experiments utilizing a CRF-tomato reporter mouse, ethanol did not significantly alter the basal membrane potential of CeA CRF neurons. The ability of ethanol to enhance CeA sEPSC frequency was not altered in CRFCeAhDTR mice despite a ~78% reduction in CeA CRF cell counts. The ability of ethanol to enhance CeA sEPSC frequency was also not altered in the CRFDTA mice despite a three-fold reduction in CRF mRNA levels. Conclusion These findings demonstrate that ethanol enhances spontaneous glutamatergic transmission in the CeA via a CRF receptor dependent mechanism. Surprisingly, our data suggest that this action may not require endogenous CRF

  3. Nicotinic α7 receptor activation selectively potentiates the function of NMDA receptors in glutamatergic terminals of the nucleus accumbens

    Directory of Open Access Journals (Sweden)

    Stefania eZappettini

    2014-10-01

    Full Text Available We here provide functional and immunocytochemical evidence supporting the co-localization and functional interaction between nicotinic acetylcholine receptors (nAChRs and N-methyl-D-aspartic acid receptors (NMDARs in glutamatergic terminals of the nucleus accumbens (NAc. Immunocytochemical studies showed that a significant percentage of NAc terminals were glutamatergic and possessed GluN1 and α7-containing nAChR. A short-term pre-exposure of synaptosomes to nicotine (30 µM or choline (1 mM caused a significant potentiation of the 100 µM NMDA-evoked [3H]D-aspartate ([3H]D-Asp outflow, which was prevented by α-bungarotoxin (100 nM. The pre-exposure to nicotine (100 µM or choline (1 mM also enhanced the NMDA-induced cytosolic free calcium levels, as measured by FURA-2 fluorescence imaging in individual NAc terminals, an effect also prevented by α-bungarotoxin. Pre-exposure to the α4-nAChR agonists 5IA85380 (10 nM or RJR2429 (1 µM did not modify NMDA-evoked ([3H]D-Asp outflow and calcium transients. The NMDA-evoked ([3H]D-Asp overflow was partially antagonized by the NMDAR antagonists MK801, D-AP5, 5,7-DCKA and R(-CPP and unaffected by the GluN2B-NMDAR antagonists Ro256981 and ifenprodil. Notably, pre-treatment with choline increased GluN2A biotin-tagged proteins. In conclusion, our results show that the GluN2A-NMDA receptor function can be positively regulated in NAc terminals in response to a brief incubation with α7 but not α4 nAChRs agonists. This might be a general feature in different brain areas since a similar nAChR-mediated bolstering of NMDA-induced ([3H]D-Aspoverflow was also observed in hippocampal synaptosomes.

  4. Presynaptic effects of anandamide and WIN55,212-2 on glutamatergic nerve endings isolated from rat hippocampus.

    Science.gov (United States)

    Cannizzaro, C; D'Amico, M; Preziosi, P; Martire, M

    2006-02-01

    We examined the effects of the endocannabinoide-anandamide (AEA), the synthetic cannabinoid, WIN55,212-2, and the active phorbol ester, 4-beta-phorbol 12-myristate 13-acetate (4-beta-PMA), on the release of [(3)H]d-Aspartate ([(3)H]d-ASP) from rat hippocampal synaptosomes. Release was evoked with three different stimuli: (1) KCl-induced membrane depolarization, which activates voltage-dependent Ca(2+) channels and causes limited neurotransmitter exocytosis, presumably from ready-releasable vesicles docked in the active zone; (2) exposure to the Ca(2+) ionophore-A23187, which causes more extensive transmitter release, presumably from intracellular reserve vesicles; and (3) K(+) channel blockade by 4-aminopyridine (4-AP), which generates repetitive depolarization that stimulates release from both ready-releasable and reserve vesicles. AEA produced concentration-dependent inhibition of [(3)H]d-ASP release stimulated with 15 mM KCl (E(max)=47.4+/-2.8; EC(50)=0.8 microM) but potentiated the release induced by 4-AP (1mM) (+22.0+/-1.3% at 1 microM) and by A23187 (1 microM) (+98.0+/-5.9% at 1 microM). AEA's enhancement of the [(3)H]d-ASP release induced by the Ca(2+) ionophore was mimicked by 4-beta-PMA, which is known to activate protein kinase C (PKC), and the increases produced by both compounds were completely reversed by synaptosome treatment with staurosporine (1 microM), a potent PKC blocker. In contrast, WIN55,212-2 inhibited the release of [(3)H]d-ASP evoked by KCl (E(max)=47.1+/-2.8; EC(50)=0.9 microM) and that produced by 4-AP (-26.0+/-1.5% at 1 microM) and had no significant effect of the release induced by Ca(2+) ionophore treatment. AEA thus appears to exert a dual effect on hippocampal glutamatergic nerve terminals. It inhibits release from ready-releasable vesicles and potentiates the release observed during high-frequency stimulation, which also involves the reserve vesicles. The latter effect is mediated by PKC. These findings reveal novel effects of AEA

  5. Role of a hippocampal SRC-family kinase-mediated glutamatergic mechanism in drug context-induced cocaine seeking.

    Science.gov (United States)

    Xie, Xiaohu; Arguello, Amy A; Wells, Audrey M; Reittinger, Andrew M; Fuchs, Rita A

    2013-12-01

    Glutamatergic neurotransmission in the dorsal hippocampus (DH) is necessary for drug context-induced reinstatement of cocaine-seeking behavior in an animal model of drug relapse. Furthermore, in vitro studies suggest that the Src family of tyrosine kinases critically regulates glutamatergic cellular functions within the DH. Thus, Src-family kinases in the DH may similarly control contextual cocaine-seeking behavior. To test this hypothesis, rats were trained to lever press for un-signaled cocaine infusions in a distinct context followed by extinction training in a different context. Cocaine-seeking behavior (non-reinforced active lever pressing) was then assessed in the previously cocaine-paired and extinction contexts after AP5 (N-methyl-D-aspartate glutamate (NMDA) receptor (NMDAR) antagonist; 0.25 or 2.5 μg/0.5 μl/hemisphere), PP2 (Src-family kinase inhibitor; 6.25 or 62.5 ng/0.5 μl/hemisphere), Ro25-6981 (NR2B subunit-containing NMDAR antagonist; 0.2 or 2 μg/0.5 μl/hemisphere), or vehicle administration into the DH. Administration of AP5, PP2, or Ro25-6981 into the DH dose-dependently impaired drug context-induced reinstatement of cocaine-seeking behavior relative to vehicle, without altering instrumental behavior in the extinction context or food-reinforced instrumental responding and general motor activity in control experiments. Cocaine-seeking behavior during the first 20 min of the test session in the cocaine-paired context was associated with an increase in NR2B subunit activation, and intra-DH PP2 pretreatment disrupted this relationship. Together, these findings suggest that Src-family kinase activation, NMDAR stimulation, and likely Src-family kinase-mediated NR2B subunit-containing NMDAR activation in the DH are necessary for incentive motivational and/or memory processes that promote contextual cocaine-seeking behavior.

  6. Excitotoxic effects of non-NMDA receptor agonists in organotypic corticostriatal slice cultures

    DEFF Research Database (Denmark)

    Kristensen, B W; Noraberg, J; Jakobsen, B

    1999-01-01

    of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium and loss of glutamic acid decarboxylase (GAD) activity in the tissue. Histological sections were also stained by the fluorescent dye Fluoro-Jade (FJ), for degenerating neurons and by immunocytochemical staining for gamma-aminobutyric acid......The excitotoxic effects of the glutamate receptor agonists kainic acid (KA) and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and the corresponding neuroprotective effects of the AMPA/KA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) were examined...... rats and grown on semiporous membranes in serum-free medium for 3-4 weeks before exposure to KA or AMPA for 48 h. The uptake by injured cells of the fluorescent dye propidium iodide (PI) added to the culture medium was used as a quantifiable measure for neuronal degeneration and compared with efflux...

  7. Stereoselective effects of AMOA on non-NMDA receptors expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Wahl, P; Nielsen, B; Krogsgaard-Larsen, P

    1992-01-01

    a nearly parallel shift to the right of the dose-response curve for kainate-induced currents. AMOA was found to have two different effects on AMPA receptors: 1) currents elicited by low concentrations of AMPA (6 microM) were inhibited by AMOA with an IC50 value of 160 +/- 19 microM and 2) currents elicited...

  8. Excitotoxic effects of non-NMDA receptor agonists in organotypic corticostriatal slice cultures

    DEFF Research Database (Denmark)

    Kristensen, B W; Noraberg, J; Jakobsen, B;

    1999-01-01

    of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium and loss of glutamic acid decarboxylase (GAD) activity in the tissue. Histological sections were also stained by the fluorescent dye Fluoro-Jade (FJ), for degenerating neurons and by immunocytochemical staining for gamma-aminobutyric acid...... effect on striatum and cortex at low doses. NBQX was thus more protective against KA in the cortex than in the striatum, while the opposite was seen in relation to AMPA. Regarding neurodegenerative markers, PI uptake was significantly correlated with (1) LDH release into the culture medium, (2) optical...

  9. Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat

    OpenAIRE

    Nair-Roberts, R.G.; Chatelain-Badie, S.D.; Benson, E.; White-Cooper, H; BOLAM, J. P.; Ungless, M.A.

    2008-01-01

    Midbrain dopamine neurons in the ventral tegmental area, substantia nigra and retrorubral field play key roles in reward processing, learning and memory, and movement. Within these midbrain regions and admixed with the dopamine neurons, are also substantial populations of GABAergic neurons that regulate dopamine neuron activity and have projection targets similar to those of dopamine neurons. Additionally, there is a small group of putative glutamatergic neurons within the ventral tegmental a...

  10. Differential expression of vesicular glutamate transporters 1 and 2 may identify distinct modes of glutamatergic transmission in the macaque visual system.

    Science.gov (United States)

    Balaram, Pooja; Hackett, Troy A; Kaas, Jon H

    2013-05-01

    Glutamate is the primary neurotransmitter utilized by the mammalian visual system for excitatory neurotransmission. The sequestration of glutamate into synaptic vesicles, and the subsequent transport of filled vesicles to the presynaptic terminal membrane, is regulated by a family of proteins known as vesicular glutamate transporters (VGLUTs). Two VGLUT proteins, VGLUT1 and VGLUT2, characterize distinct sets of glutamatergic projections between visual structures in rodents and prosimian primates, yet little is known about their distributions in the visual system of anthropoid primates. We have examined the mRNA and protein expression patterns of VGLUT1 and VGLUT2 in the visual system of macaque monkeys, an Old World anthropoid primate, in order to determine their relative distributions in the superior colliculus, lateral geniculate nucleus, pulvinar complex, V1 and V2. Distinct expression patterns for both VGLUT1 and VGLUT2 identified architectonic boundaries in all structures, as well as anatomical subdivisions of the superior colliculus, pulvinar complex, and V1. These results suggest that VGLUT1 and VGLUT2 clearly identify regions of glutamatergic input in visual structures, and may identify common architectonic features of visual areas and nuclei across the primate radiation. Additionally, we find that VGLUT1 and VGLUT2 characterize distinct subsets of glutamatergic projections in the macaque visual system; VGLUT2 predominates in driving or feedforward projections from lower order to higher order visual structures while VGLUT1 predominates in modulatory or feedback projections from higher order to lower order visual structures. The distribution of these two proteins suggests that VGLUT1 and VGLUT2 may identify class 1 and class 2 type glutamatergic projections within the primate visual system (Sherman and Guillery, 2006).

  11. Adenosinergic depression of glutamatergic transmission in the entorhinal cortex of juvenile rats via reduction of glutamate release probability and the number of releasable vesicles.

    Science.gov (United States)

    Wang, Shouping; Kurada, Lalitha; Cilz, Nicholas I; Chen, Xiaotong; Xiao, Zhaoyang; Dong, Hailong; Lei, Saobo

    2013-01-01

    Adenosine is an inhibitory neuromodulator that exerts antiepileptic effects in the brain and the entorhinal cortex (EC) is an essential structure involved in temporal lobe epilepsy. Whereas microinjection of adenosine into the EC has been shown to exert powerful antiepileptic effects, the underlying cellular and molecular mechanisms in the EC have not been determined yet. We tested the hypothesis that adenosine-mediated modulation of synaptic transmission contributes to its antiepileptic effects in the EC. Our results demonstrate that adenosine reversibly inhibited glutamatergic transmission via activation of adenosine A1 receptors without effects on GABAergic transmission in layer III pyramidal neurons in the EC. Adenosine-induced depression of glutamatergic transmission was mediated by inhibiting presynaptic glutamate release probability and decreasing the number of readily releasable vesicles. Bath application of adenosine also reduced the frequency of the miniature EPSCs recorded in the presence of TTX suggesting that adenosine may interact with the exocytosis processes downstream of Ca(2+) influx. Both Gαi/o proteins and the protein kinase A pathway were required for adenosine-induced depression of glutamatergic transmission. We further showed that bath application of picrotoxin to the EC slices induced stable epileptiform activity and bath application of adenosine dose-dependently inhibited the epileptiform activity in this seizure model. Adenosine-mediated depression of epileptiform activity was mediated by activation of adenosine A1 receptors and required the functions of Gαi/o proteins and protein kinase A pathway. Our results suggest that the depression of glutamatergic transmission induced by adenosine contributes to its antiepileptic effects in the EC.

  12. Adenosinergic depression of glutamatergic transmission in the entorhinal cortex of juvenile rats via reduction of glutamate release probability and the number of releasable vesicles.

    Directory of Open Access Journals (Sweden)

    Shouping Wang

    Full Text Available Adenosine is an inhibitory neuromodulator that exerts antiepileptic effects in the brain and the entorhinal cortex (EC is an essential structure involved in temporal lobe epilepsy. Whereas microinjection of adenosine into the EC has been shown to exert powerful antiepileptic effects, the underlying cellular and molecular mechanisms in the EC have not been determined yet. We tested the hypothesis that adenosine-mediated modulation of synaptic transmission contributes to its antiepileptic effects in the EC. Our results demonstrate that adenosine reversibly inhibited glutamatergic transmission via activation of adenosine A1 receptors without effects on GABAergic transmission in layer III pyramidal neurons in the EC. Adenosine-induced depression of glutamatergic transmission was mediated by inhibiting presynaptic glutamate release probability and decreasing the number of readily releasable vesicles. Bath application of adenosine also reduced the frequency of the miniature EPSCs recorded in the presence of TTX suggesting that adenosine may interact with the exocytosis processes downstream of Ca(2+ influx. Both Gαi/o proteins and the protein kinase A pathway were required for adenosine-induced depression of glutamatergic transmission. We further showed that bath application of picrotoxin to the EC slices induced stable epileptiform activity and bath application of adenosine dose-dependently inhibited the epileptiform activity in this seizure model. Adenosine-mediated depression of epileptiform activity was mediated by activation of adenosine A1 receptors and required the functions of Gαi/o proteins and protein kinase A pathway. Our results suggest that the depression of glutamatergic transmission induced by adenosine contributes to its antiepileptic effects in the EC.

  13. Subpopulations of neurokinin 1 receptor-expressing neurons in the rat lateral amygdala display a differential pattern of innervation from distinct glutamatergic afferents

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    Sreepathi, H.K.; Ferraguti, F.

    2012-01-01

    Substance P by acting on its preferred receptor neurokinin 1 (NK1) in the amygdala appears to be critically involved in the modulation of fear and anxiety. The present study was undertaken to identify neurochemically specific subpopulations of neuron expressing NK1 receptors in the lateral amygdaloid nucleus (LA), a key site for regulating these behaviors. We also analyzed the sources of glutamatergic inputs to these neurons. Immunofluorescence analysis of the co-expression of NK1 with calciu...

  14. Activation of group II metabotropic glutamate receptors inhibits glutamatergic transmission in the rat entorhinal cortex via reduction of glutamate release probability.

    Science.gov (United States)

    Wang, Shouping; Chen, Xiaotong; Kurada, Lalitha; Huang, Zitong; Lei, Saobo

    2012-03-01

    Glutamate interacts with ionotropic and metabotropic glutamate receptors (mGluRs). Whereas the entorhinal cortex (EC) is a principal structure involved in learning and memory, the roles of mGluRs in synaptic transmission in the EC have not been completely determined. Here, we show that activation of group II mGluRs (mGluR II) induced robust depression of glutamatergic transmission in the EC. The mGluR II-induced depression was due to a selective reduction of presynaptic release probability without alterations of the quantal size and the number of release sites. The mechanisms underlying mGluR II-mediated suppression of glutamate release included the inhibition of presynaptic release machinery and the depression of presynaptic P/Q-type Ca(2+) channels. Whereas mGluR II-induced depression required the function of Gα(i/o) proteins, protein kinase A (PKA) pathway was only involved in mGluR II-mediated inhibition of release machinery and thereby partially required for mGluR II-induced inhibition of glutamate release. Presynaptic stimulation at 5 Hz for 10 min also induced depression of glutamatergic transmission via activation of presynaptic mGluR II suggesting an endogenous role for mGluR II in modulating glutamatergic transmission.

  15. Aβ induces acute depression of excitatory glutamatergic synaptic transmission through distinct phosphatase-dependent mechanisms in rat CA1 pyramidal neurons.

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    Yao, Wen; Zou, Hao-Jun; Sun, Da; Ren, Si-Qiang

    2013-06-17

    Beta-amyloid peptide (Aβ) has a causal role in the pathophysiology of Alzheimer's disease (AD). Recent studies indicate that Aβ can disrupt excitatory glutamatergic synaptic function at synaptic level. However, the underlying mechanisms remain obscure. In this study, we recorded evoked and spontaneous EPSCs in hippocampal CA1 pyramidal neurons via whole-cell voltage-clamping methods and found that 1 μM Aβ can induce acute depression of basal glutamatergic synaptic transmission through both presynaptic and postsynaptic dysfunction. Moreover, we also found that Aβ-induced both presynaptic and postsynaptic dysfunction can be reversed by the inhibitor of protein phosphatase 2B (PP2B), FK506, whereas only postsynaptic disruption can be ameliorated by the inhibitor of PP1/PP2A, Okadaic acid (OA). These results indicate that PP1/PP2A and PP2B have overlapping but not identical functions in Aβ-induced acute depression of excitatory glutamatergic synaptic transmission of hippocampal CA1 pyramidal neurons.

  16. Enhanced Glutamatergic Synaptic Plasticity in the Hippocampal CA1 Field of Food-Restricted Rats: Involvement of CB1 Receptors.

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    Talani, Giuseppe; Licheri, Valentina; Biggio, Francesca; Locci, Valentina; Mostallino, Maria Cristina; Secci, Pietro Paolo; Melis, Valentina; Dazzi, Laura; Carta, Gianfranca; Banni, Sebastiano; Biggio, Giovanni; Sanna, Enrico

    2016-04-01

    The endogenous endocannabinoid system has a crucial role in regulating appetite and feeding behavior in mammals, as well as working memory and reward mechanisms. In order to elucidate the possible role of cannabinoid type-1 receptors (CB1Rs) in the regulation of hippocampal plasticity in animals exposed to food restriction (FR), we limited the availability of food to a 2-h daily period for 3 weeks in Sprague-Dawley rats. FR rats showed a higher long-term potentiation at hippocampal CA1 excitatory synapses with a parallel increase in glutamate release when compared with animals fed ad libitum. FR rats showed a significant increase in the long-term spatial memory determined by Barnes maze. FR was also associated with a decreased inhibitory effect of the CB1R agonist win55,212-2 on glutamatergic field excitatory postsynaptic potentials, together with a decrease in hippocampal CB1R protein expression. In addition, hippocampal brain-derived neurotrophic factor protein levels and mushroom dendritic spine density were significantly enhanced in FR rats. Altogether, our data suggest that alterations of hippocampal CB1R expression and function in FR rats are associated with dendritic spine remodeling and functional potentiation of CA1 excitatory synapses, and these findings are consistent with increasing evidence supporting the idea that FR may improve cognitive functions.

  17. Ketamine as the prototype glutamatergic antidepressant: pharmacodynamic actions, and a systematic review and meta-analysis of efficacy

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    Caddy, Caroline; Giaroli, Giovanni; White, Thomas P.; Shergill, Sukhwinder S.

    2014-01-01

    The burden of depressive disorders and the frequent inadequacy of their current pharmacological treatments are well established. The anaesthetic and hallucinogenic drug ketamine has provoked much interest over the past decade or so as an extremely rapidly acting antidepressant that does not modify ‘classical’ monoaminergic receptors. Current evidence has shown several ways through which it might exert therapeutic antidepressant actions: blockade of glutamatergic NMDA receptors and relative upregulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtypes may alter cortical connectivity patterns; through intracellular changes in protein expression, including the proteins mammalian target of rapamycin (mTOR) and brain-derived neurotrophic factor (BDNF); and alteration of intracellular signalling cascades. The clinical evidence demonstrates rapid improvements in mood and suicidal thinking in most participants, although study numbers have generally been small and many trials are unblinded and methodologically weak. There is a small body of work to suggest ketamine might also augment electroconvulsive therapy and potentially have a role as a surgical anaesthetic in depressed patients. A major problem is that the effects of ketamine appear temporary, disappearing after days to weeks (although longer benefits have been sustained in some), and attempts to circumvent this through pharmacological augmentation have been disappointing thus far. These exciting data are providing new insights into neurobiological models of depression, and potentially opening up a new class of antidepressants, but there are significant practical and ethical issues about any future mainstream clinical role it might have. PMID:24688759

  18. Third trimester-equivalent ethanol exposure increases anxiety-like behavior and glutamatergic transmission in the basolateral amygdala.

    Science.gov (United States)

    Baculis, Brian C; Diaz, Marvin R; Valenzuela, C Fernando

    2015-10-01

    Ethanol consumption during pregnancy produces a wide range of morphological and behavioral alterations known as fetal alcohol spectrum disorder (FASD). Among the behavioral deficits associated with FASD is an increased probability of developing anxiety disorders. Studies with animal models of FASD have demonstrated that ethanol exposure during the equivalent to the 1(st) and 2(nd) trimesters of human pregnancy increases anxiety-like behavior. Here, we examined the impact on this type of behavior of exposure to high doses of ethanol in vapor inhalation chambers during the rat equivalent to the human 3rd trimester of pregnancy (i.e., neonatal period in these animals). We evaluated anxiety-like behavior with the elevated plus maze. Using whole-cell patch-clamp electrophysiological techniques in brain slices, we also characterized glutamatergic and GABAergic synaptic transmission in the basolateral amygdala, a brain region that has been implicated to play a role in emotional behavior. We found that ethanol-exposed adolescent offspring preferred the closed arms over the open arms in the elevated plus maze and displayed lower head dipping activity than controls. Electrophysiological measurements showed an increase in the frequency of spontaneous and miniature excitatory postsynaptic currents in pyramidal neurons from the ethanol group. These findings suggest that high-dose ethanol exposure during the equivalent to the last trimester of human pregnancy can persistently increase excitatory synaptic inputs to principal neurons in the basolateral amygdala, leading to an increase in anxiety-like behaviors.

  19. Effects of the kainate receptor agonist ATPA on glutamatergic synaptic transmission and plasticity during early postnatal development.

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    Sallert, Marko; Malkki, Hemi; Segerstråle, Mikael; Taira, Tomi; Lauri, Sari E

    2007-05-01

    Kainate type of glutamate receptors (KARs) modulate synaptic transmission in a developmentally regulated manner at several synapses in the brain. Previous studies have shown that KARs depress glutamatergic transmission at CA3-CA1 synapses in the hippocampus and these receptors are tonically active during early postnatal development. Here we use the GluR5 subunit specific agonist ATPA to further characterize the role of KARs in the modulation of synaptic transmission and plasticity in area CA1 during the first two weeks of life. We find that the depressant effect of ATPA on evoked fEPSPs/EPSCs is smaller in the neonate (P3-P6) than in the juvenile (P14-P18) rat CA1, due to endogenous activity of KAR in the neonate. Further, in the neonate but not juvenile CA1, ATPA downregulates action-potential independent transmission (mEPSCs) and its effects are dependent on protein kinase C activity. ATPA-induced depression of fEPSPs in the neonate occludes the presynaptic component of long-term depression (LTD). In contrast, at P14-P18, ATPA prevents LTD indirectly via GABAergic mechanisms. These data show that GluR5 signaling mechanisms are developmentally regulated and suggest distinct functional role for KARs in the modulation of synaptic transmission and plasticity at different stages of development.

  20. In vivo comparison of harmine efficacy against psychostimulants: preferential inhibition of the cocaine response through a glutamatergic mechanism.

    Science.gov (United States)

    Owaisat, Suzan; Raffa, Robert B; Rawls, Scott M

    2012-09-06

    Harmine is a β-carboline compound that targets glutamatergic, monoaminergic, and GABAergic pathways underlying drug addiction. We compared the efficacy of harmine against different psychoactive drugs using an invertebrate (planarian) assay designed to quantify 'C-shape' responses. Harmine itself (0.01-10 μM) did not produce C-shapes. However, when applied over the same concentration range, harmine significantly inhibited C-shapes elicited by cocaine, with a concentration of 0.1 μM producing almost 90% inhibition. Consistent with its putative actions, harmine produced a similar, though less efficacious, inhibition of C-shapes elicited by the substituted amphetamines methamphetamine and mephedrone (4-methylmethcathinone) but was much less effective against nicotine. When tested in the presence of the glutamate transporter inhibitor dihydrokainate (DHK) (0.1, 1 μM), harmine (0.1 μM) efficacy against cocaine-induced C-shapes was significantly reduced. Harmine also attenuated C-shapes elicited by N-methyl-d-aspartate (NMDA) and by glutamate itself. The present data suggest that harmine displays preferential efficacy against different addictive substances (cocaine>amphetamines>nicotine) and, at least for cocaine, is dependent on the glutamate system.

  1. Alterations in pharmacological sensitivity of GABAergic but not dopaminergic and glutamatergic systems during ontogenesis in dystonic mutant hamsters.

    Science.gov (United States)

    Richter, A; Löscher, W

    1993-01-26

    Attacks of sustained dystonia of the limbs and trunk can be initiated by handling or mild environmental stimuli (e.g. new cage) in mutant (gene symbol dtsz) Syrian golden hamsters. The severity of the dystonic syndrome in these mutant hamsters is age-dependent, with a peak at weaning (21 days of age) and a second peak at about 30-40 days of age. Spontaneous remission occurs at an age of about 70 days. The syndrome in hamsters is thus similar to transient paroxysmal dystonia in children. In the present experiments, it was examined whether dystonic hamsters exhibit age-dependent differences in susceptibility to drugs which affect GABA (gamma-aminobutyrate)ergic, glutamatergic or dopaminergic functions. After acute administration, the GABA-elevating drug aminooxyacetic acid was significantly less potent in attenuating the severity of dystonic attacks at 21 days than at 31 days of age. Similar but less marked age-dependent differences in antidystonic activity were found for phenobarbital and diazepam. In contrast to these GABAmimetic drugs, the NMDA receptor antagonist CGP 37849 (DL-[E]-2-amino-4-methyl-5-phosphono-3-pentenoic acid) or the dopamine receptor antagonist haloperidol had about the same antidystonic potency at both 21 and 31 days of age. Chronic treatment of dystonic hamsters with aminooxyacetic acid, starting at 21 days of age, did not alter the time course or the severity of dystonia.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Intranasal exposure to manganese disrupts neurotransmitter release from glutamatergic synapses in the central nervous system in vivo.

    Science.gov (United States)

    Moberly, Andrew H; Czarnecki, Lindsey A; Pottackal, Joseph; Rubinstein, Tom; Turkel, Daniel J; Kass, Marley D; McGann, John P

    2012-10-01

    Chronic exposure to aerosolized manganese induces a neurological disorder that includes extrapyramidal motor symptoms and cognitive impairment. Inhaled manganese can bypass the blood-brain barrier and reach the central nervous system by transport down the olfactory nerve to the brain's olfactory bulb. However, the mechanism by which Mn disrupts neural function remains unclear. Here we used optical imaging techniques to visualize exocytosis in olfactory nerve terminals in vivo in the mouse olfactory bulb. Acute Mn exposure via intranasal instillation of 2-200 μg MnCl(2) solution caused a dose-dependent reduction in odorant-evoked neurotransmitter release, with significant effects at as little as 2 μg MnCl(2) and a 90% reduction compared to vehicle controls with a 200 μg exposure. This reduction was also observed in response to direct electrical stimulation of the olfactory nerve layer in the olfactory bulb, demonstrating that Mn's action is occurring centrally, not peripherally. This is the first direct evidence that Mn intoxication can disrupt neurotransmitter release, and is consistent with previous work suggesting that chronic Mn exposure limits amphetamine-induced dopamine increases in the basal ganglia despite normal levels of dopamine synthesis (Guilarte et al., J Neurochem 2008). The commonality of Mn's action between glutamatergic neurons in the olfactory bulb and dopaminergic neurons in the basal ganglia suggests that a disruption of neurotransmitter release may be a general consequence wherever Mn accumulates in the brain and could underlie its pleiotropic effects.

  3. Glutamatergic signaling and low prodynorphin expression are associated with intact memory and reduced anxiety in rat models of healthy aging

    Directory of Open Access Journals (Sweden)

    Caroline eMenard

    2014-05-01

    Full Text Available The LOU/C/Jall (LOU rat strain is considered a model of healthy aging due to its increased longevity, maintenance of stable body weight (BW throughout life and low incidence of age-related diseases. However, aging LOU rat cognitive and anxiety status has yet to be investigated. In the present study, male and female LOU rat cognitive performances (6-42 months were assessed using novel object recognition and Morris Water Maze tasks. Recognition memory remained intact in all LOU rats up to 42 months of age. As for spatial memory, old LOU rat performed similarly as young animals for learning acquisition, reversal learning and retention. While LOU rat BW remained stable despite aging, 20-month-old ad-libitum-fed (OAL male Sprague Dawley rats become obese. We determined if long-term caloric restriction (LTCR prevents age-related BW increase and cognitive deficits in this rat strain, as observed in the obesity-resistant LOU rats. Compared to young animals, recognition memory was impaired in OAL but intact in 20-month-old calorie-restricted (OCR rats. Similarly, OAL spatial learning acquisition was impaired but LTCR prevented the deficits. Exacerbated stress responses may favor age-related cognitive decline. In the elevated plus maze and open field tasks, LOU and OCR rats exhibited high levels of exploratory activity whereas OAL rats displayed anxious behaviors. Expression of prodynorphin (Pdyn, an endogenous peptide involved in stress-related memory impairments, was increased in the hippocampus of OAL rats. Group 1 metabotropic glutamate receptor 5 and immediate early genes Homer 1a and Arc expression, both associated with successful cognitive aging, were unaltered in aging LOU rats but lower in OAL than OCR rats. Altogether, our results, supported by principal component analysis and correlation matrix, suggest that intact memory and low anxiety are associated with glutamatergic signaling and low Pdyn expression in the hippocampus of non obese aging

  4. Enhancement of inorganic Martian dust simulant with carbon component and its effects on key characteristics of glutamatergic neurotransmission

    Science.gov (United States)

    Borisova, Tatiana; Krisanova, Natalia; Nazarova, Anastasiya; Borysov, Arseniy; Pastukhov, Artem; Pozdnyakova, Natalia; Dudarenko, Marina

    2016-07-01

    Evidence on the past existence of subsurface organic-bearing fluids on Mars was recently achieved basing on the investigation of organic carbon from the Tissint Martian meteorite (Lin et al., 2014). Tremendous amount of meteorites containing abundant carbon and carbon-enriched dust particles have reached the Earth daily (Pizzarello and Shock 2010). National Institute of Environmental Health Sciences/National Institute of Health panel of research scientists revealed recently that accumulating evidences suggest that nano-sized air pollution may have a significant impact on central nervous system in health and disease (Block et al., Neurotoxicology, 2012). During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and can be transported to the central nervous system (Oberdorster et al., 2004). Based on above facts, the aims of this study were: 1) to upgrade inorganic Martian dust stimulant derived from volcanic ash (JSC-1a/JSC, ORBITEC Orbital Technologies Corporation, Madison, Wisconsin) by the addition of carbon components, that is, nanodiamonds; 2) to analyse acute effects of upgraded stimulant on the key characteristic of synaptic neurotransmission and to compare its effects with those of inorganic dust and carbon components per se. Acute administration of carbon-containing Martian dust analogue resulted in a significant decrease in Na+-dependent uptake of L-[14C]glutamate that is the major excitatory neurotransmitter in the central nervous system (CNS). The ambient level of the neurotransmitter in the preparation of isolated rat brain nerve terminals increased in the presence of carbon-contained Martian dust analogue. This fact indicated that carbon component of native Martian dust can have deleterious effects on extracellular glutamate homeostasis in the CNS, and so glutamatergic neurtransmission.

  5. Glutamatergic regulation of brain histamine neurons: In vivo microdialysis and electrophysiology studies in the rat.

    Science.gov (United States)

    Fell, Matthew J; Flik, Gunnar; Dijkman, Ulrike; Folgering, Joost H A; Perry, Kenneth W; Johnson, Bryan J; Westerink, Ben H C; Svensson, Kjell A

    2015-12-01

    The interactions between the glutamatergic and the histaminergic systems in the brain are not fully understood. Here we studied histamine release in the medial prefrontal cortex and the posterior hypothalamus-tuberomamillary nucleus (PH-TMN) using in vivo microdialysis and electrophysiological recordings of histaminergc neurons in the PH-TMN in vivo to further address the mechanistic details of these interactions. We demonstrated that histaminergic activity was regulated by group II metabotropic glutamate receptors (mGluR 2 and 3) using systemic dosing with mGluR 2/3 agonist and antagonists and an mGluR 2 positive allosteric modulator. These interactions likely occur via direct modulation of glutamate release in the PH-TMN. The importance of circadian rhythm for histamine release was also shown using microdialysis studies with mGluR 2/3 compounds under light and dark conditions. Based on histamine release studies with NMDA and ketamine, we propose the existence of two sub-populations of NMDA receptors where one subtype is located on histaminergic cell bodies in the PH-TMN and the second on GABA-ergic neurons projecting to the PH-TMN. These subpopulations could be distinguished based on function, notably opposing actions were seen on histamine release in the medial prefrontal cortex of the rat. In summary, this paper provides evidence that the histaminergic system is closely regulated by glutamate neurons in multiple ways. In addition, this interaction depends to a great extent on the activity state of the subject.

  6. It's MORe exciting than mu: crosstalk between mu opioid receptors and glutamatergic transmission in the mesolimbic dopamine system.

    Science.gov (United States)

    Chartoff, Elena H; Connery, Hilary S

    2014-01-01

    Opioids selective for the G protein-coupled mu opioid receptor (MOR) produce potent analgesia and euphoria. Heroin, a synthetic opioid, is considered one of the most addictive substances, and the recent exponential rise in opioid addiction and overdose deaths has made treatment development a national public health priority. Existing medications (methadone, buprenorphine, and naltrexone), when combined with psychosocial therapies, have proven efficacy in reducing aspects of opioid addiction. Unfortunately, these medications have critical limitations including those associated with opioid agonist therapies (e.g., sustained physiological dependence and opioid withdrawal leading to high relapse rates upon discontinuation), non-adherence to daily dosing, and non-renewal of monthly injection with extended-release naltrexone. Furthermore, current medications fail to ameliorate key aspects of addiction such as powerful conditioned associations that trigger relapse (e.g., cues, stress, the drug itself). Thus, there is a need for developing novel treatments that target neural processes corrupted with chronic opioid use. This requires a basic understanding of molecular and cellular mechanisms underlying effects of opioids on synaptic transmission and plasticity within reward-related neural circuits. The focus of this review is to discuss how crosstalk between MOR-associated G protein signaling and glutamatergic neurotransmission leads to immediate and long-term effects on emotional states (e.g., euphoria, depression) and motivated behavior (e.g., drug-seeking, relapse). Our goal is to integrate findings on how opioids modulate synaptic release of glutamate and postsynaptic transmission via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors in the nucleus accumbens and ventral tegmental area with the clinical (neurobehavioral) progression of opioid dependence, as well as to identify gaps in knowledge that can be addressed in future studies.

  7. Presynaptic facilitation by tetracaine of glutamatergic spontaneous excitatory transmission in the rat spinal substantia gelatinosa - Involvement of TRPA1 channels.

    Science.gov (United States)

    Piao, Lian-Hua; Fujita, Tsugumi; Yu, Ting; Kumamoto, Eiichi

    2017-02-15

    The amide-type local anesthetic (LA) lidocaine activates transient receptor potential (TRP) ankyrin-1 (TRPA1) channels to facilitate spontaneous l-glutamate release onto spinal substantia gelatinosa (SG) neurons, which play a crucial role in regulating nociceptive transmission. In contrast, the ester-type LA procaine reduces the spontaneous release of l-glutamate in SG neurons. In order to determine whether TRPA1 activation by LAs is specific to amide-types, we examined the actions of tetracaine, another ester-type LA, and other amide-type LAs on glutamatergic spontaneous excitatory transmission in SG neurons by focusing on TRP activation. Whole-cell patch-clamp recordings were performed on SG neurons of adult rat spinal cord slices at a holding potential of -70mV. Bath-applied tetracaine increased spontaneous excitatory postsynaptic current (sEPSC) frequency in a concentration-dependent manner. Tetracaine activity was resistant to the voltage-gated Na(+)-channel blocker tetrodotoxin, the TRP vanilloid-1 antagonist capsazepine, and the TRP melastatin-8 antagonist BCTC, but was inhibited by the non-selective TRP antagonist ruthenium red and the TRPA1 antagonist HC-030031. With respect to amide-type LAs, prilocaine had a tendency to increase sEPSC frequency, while ropivacaine and levobupivacaine reduced the frequency. In conclusion, tetracaine facilitated spontaneous l-glutamate release from nerve terminals by activating TRPA1 channels in the SG, resulting in an increase in the excitability of SG neurons. TRPA1 activation was not specific to amide-type or ester-type LAs. The facilitatory action of LAs may be involved in pain occurring after recovery from spinal anesthesia.

  8. Astrocyte activation in the anterior cingulate cortex and altered glutamatergic gene expression during paclitaxel-induced neuropathic pain in mice

    Directory of Open Access Journals (Sweden)

    Willias Masocha

    2015-10-01

    Full Text Available Spinal astrocyte activation contributes to the pathogenesis of paclitaxel-induced neuropathic pain (PINP in animal models. We examined glial fibrillary acidic protein (GFAP; an astrocyte marker immunoreactivity and gene expression of GFAP, glutamate transporters and receptor subunits by real time PCR in the anterior cingulate cortex (ACC at 7 days post first administration of paclitaxel, a time point when mice had developed thermal hyperalgesia. The ACC, an area in the brain involved in pain perception and modulation, was chosen because changes in this area might contribute to the pathophysiology of PINP. GFAP transcripts levels were elevated by more than fivefold and GFAP immunoreactivity increased in the ACC of paclitaxel-treated mice. The 6 glutamate transporters (GLAST, GLT-1 EAAC1, EAAT4, VGLUT-1 and VGLUT-2 quantified were not significantly altered by paclitaxel treatment. Of the 12 ionotropic glutamate receptor subunits transcripts analysed 6 (GLuA1, GLuA3, GLuK2, GLuK3, GLuK5 and GLuN1 were significantly up-regulated, whereas GLuA2, GLuK1, GLuK4, GLuN2A and GLuN2B were not significantly altered and GLuA4 was lowly expressed. Amongst the 8 metabotropic receptor subunits analysed only mGLuR8 was significantly elevated. In conclusion, during PINP there is astrocyte activation, with no change in glutamate transporter expression and differential up-regulation of glutamate receptor subunits in the ACC. Thus, targeting astrocyte activation and the glutamatergic system might be another therapeutic avenue for management of PINP.

  9. Whiplash-like facet joint loading initiates glutamatergic responses in the DRG and spinal cord associated with behavioral hypersensitivity

    Science.gov (United States)

    Dong, Ling; Quindlen, Julia C.; Lipschutz, Daniel E.; Winkelstein, Beth A.

    2012-01-01

    The cervical facet joint and its capsule are a common source of neck pain from whiplash. Mechanical hyperalgesia elicited by painful facet joint distraction is associated with spinal neuronal hyperexcitability that can be induced by transmitter/receptor systems that potentiate the synaptic activation of neurons. This study investigated the temporal response of a glutamate receptor and transporters in the dorsal root ganglia (DRG) and spinal cord. Bilateral C6/C7 facet joint distractions were imposed in the rat either to produce behavioral sensitivity or without inducing any sensitivity. Neuronal metabotropic glutamate receptor-5 (mGluR5) and protein kinase C-epsilon (PKCε) expression in the DRG and spinal cord were evaluated on days 1 and 7. Spinal expression of a glutamate transporter, excitatory amino acid carrier 1 (EAAC1), was also quantified at both time points. Painful distraction produced immediate behavioral hypersensitivity that was sustained for 7 days. Increased expression of mGluR5 and PKCε in the DRG was not evident until day 7 and only following painful distraction; this increase was observed in small-diameter neurons. Only painful facet joint distraction produced a significant increase (p<0.001) in neuronal mGluR5 over time, and this increase also was significantly elevated (p ≤ 0.05) over responses in the other groups at day 7. However, there were no differences in spinal PKCε expression on either day or between groups. Spinal EAAC1 expression was significantly increased (p<0.03) only in the nonpainful groups on day 7. Results from this study suggest spinal glutamatergic plasticity is selectively modulated in association with facet-mediated pain. PMID:22578356

  10. Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles.

    Science.gov (United States)

    Banks, Robert W; Cahusac, Peter M B; Graca, Anna; Kain, Nakul; Shenton, Fiona; Singh, Paramjeet; Njå, Arild; Simon, Anna; Watson, Sonia; Slater, Clarke R; Bewick, Guy S

    2013-05-15

    Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending we have described previously. Both types of endings are formed by the peripheral sensory terminals of primary mechanosensory dorsal root ganglion cells, so the presence of such a system in the lanceolate ending would provide support for our hypothesis that it is a general property of fundamental importance to the regulation of the responsiveness of the broad class of primary mechanosensory endings. We show not only that an SLV-based system is present in lanceolate endings, but also that there are clear parallels between its operation in the two types of mechanosensory endings. In particular, we demonstrate that, as in the muscle spindle: (i) FM1-43 labels the sensory terminals of the lanceolate ending, rather than the closely associated accessory (glial) cells; (ii) the dye enters and leaves the terminals primarily by SLV recycling; (iii) the dye does not block the electrical response to mechanical stimulation, in contrast to its effect on the hair cell and dorsal root ganglion cells in culture; (iv) SLV recycling is Ca(2+) sensitive; and (v) the sensory terminals are enriched in glutamate. Thus, in the lanceolate sensory ending SLV recycling is itself regulated, at least in part, by glutamate acting through a phospholipase D-coupled metabotropic glutamate receptor.

  11. Potentiation of the glutamatergic synaptic input to rat locus coeruleus neurons by P2X7 receptors.

    Science.gov (United States)

    Khakpay, Roghayeh; Polster, Daniel; Köles, Laszlo; Skorinkin, Andrey; Szabo, Bela; Wirkner, Kerstin; Illes, Peter

    2010-09-01

    Locus coeruleus (LC) neurons in a rat brain slice preparation were superfused with a Mg(2+)-free and bicuculline-containing external medium. Under these conditions, glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) were recorded by means of the whole-cell patch-clamp method. ATP, as well as its structural analogue 2-methylthio ATP (2-MeSATP), both caused transient inward currents, which were outlasted by an increase in the frequency but not the amplitude of the sEPSCs. PPADS, but not suramin or reactive blue 2 counteracted both effects of 2-MeSATP. By contrast, α,β-methylene ATP (α,β-meATP), UTP and BzATP did not cause an inward current response. Of these latter agonists, only BzATP slightly facilitated the sEPSC amplitude and strongly potentiated its frequency. PPADS and Brilliant Blue G, as well as fluorocitric acid and aminoadipic acid prevented the activity of BzATP. Furthermore, BzATP caused a similar facilitation of the miniature (m)EPSC (recorded in the presence of tetrodotoxin) and sEPSC frequencies (recorded in its absence). Eventually, capsaicin augmented the frequency of the sEPSCs in a capsazepine-, but not PPADS-antagonizable, manner. In conclusion, the stimulation of astrocytic P2X7 receptors appears to lead to the outflow of a signalling molecule, which presynaptically increases the spontaneous release of glutamate onto LC neurons from their afferent fibre tracts. It is suggested, that the two algogenic compounds ATP and capsaicin utilise separate receptor systems to potentiate the release of glutamate and in consequence to increase the excitability of LC neurons.

  12. Reduction of glutamatergic neurotransmission by prolonged exposure to dieldrin involves NMDA receptor internalization and metabotropic glutamate receptor 5 downregulation.

    Science.gov (United States)

    Briz, Victor; Galofré, Mireia; Suñol, Cristina

    2010-01-01

    Dieldrin was previously used as a pesticide. Although its use has been discontinued, humans are still exposed to it due to its high environmental persistence and because it accumulates in the adipose tissue of animals. Acute exposure to dieldrin provokes convulsions due to its antagonism on the gamma-aminobutyric acid-A (GABA(A)) receptor. However, little is known about the effects of low chronic exposure to this pollutant. In the present work, we use primary cultures of cortical neurons to study the mechanisms involved in the toxic action of dieldrin. We found that 2 and 6 days in vitro (DIV) exposure to a subcytotoxic concentration (60nM) of dieldrin reduced the increase in intracellular calcium concentration ([Ca(2+)](i)) and the excitotoxicity caused by glutamate. Exposure to dieldrin for 6 DIV induced N-methyl-D-aspartate receptor (NMDAR) internalization and reduced metabotropic glutamate receptor 5 (mGLUR5) levels. Double immunostaining for NMDAR and mGLUR5 showed that these receptors lose colocalization on the cell membrane in neurons treated with dieldrin. No changes were observed in receptor functionalities or receptor levels after 2 DIV of exposure to dieldrin. However, the increase in [Ca(2+)](i) induced by coactivation of NMDAR and mGLUR5 was significantly reduced. Thus, a functional interaction between the two receptors seems to play an important role in glutamate-induced excitotoxicity. We confirm that permanent blockade of the GABA(A) receptor by this persistent pesticide triggers adaptive neuronal changes consisting of a reduction of glutamatergic neurotransmission. This might explain the cognitive and learning deficits observed in animals after chronic treatment with dieldrin.

  13. Multi-locus genome-wide association analysis supports the role of glutamatergic synaptic transmission in the etiology of major depressive disorder.

    Science.gov (United States)

    Lee, P H; Perlis, R H; Jung, J-Y; Byrne, E M; Rueckert, E; Siburian, R; Haddad, S; Mayerfeld, C E; Heath, A C; Pergadia, M L; Madden, P A F; Boomsma, D I; Penninx, B W; Sklar, P; Martin, N G; Wray, N R; Purcell, S M; Smoller, J W

    2012-11-13

    Major depressive disorder (MDD) is a common psychiatric illness characterized by low mood and loss of interest in pleasurable activities. Despite years of effort, recent genome-wide association studies (GWAS) have identified few susceptibility variants or genes that are robustly associated with MDD. Standard single-SNP (single nucleotide polymorphism)-based GWAS analysis typically has limited power to deal with the extensive heterogeneity and substantial polygenic contribution of individually weak genetic effects underlying the pathogenesis of MDD. Here, we report an alternative, gene-set-based association analysis of MDD in an effort to identify groups of biologically related genetic variants that are involved in the same molecular function or cellular processes and exhibit a significant level of aggregated association with MDD. In particular, we used a text-mining-based data analysis to prioritize candidate gene sets implicated in MDD and conducted a multi-locus association analysis to look for enriched signals of nominally associated MDD susceptibility loci within each of the gene sets. Our primary analysis is based on the meta-analysis of three large MDD GWAS data sets (total N=4346 cases and 4430 controls). After correction for multiple testing, we found that genes involved in glutamatergic synaptic neurotransmission were significantly associated with MDD (set-based association P=6.9 × 10(-4)). This result is consistent with previous studies that support a role of the glutamatergic system in synaptic plasticity and MDD and support the potential utility of targeting glutamatergic neurotransmission in the treatment of MDD.

  14. Glutamatergic transmission in the central nucleus of the amygdala is selectively altered in Marchigian Sardinian alcohol-preferring rats: Alcohol and CRF effects.

    Science.gov (United States)

    Herman, Melissa A; Varodayan, Florence P; Oleata, Christopher S; Luu, George; Kirson, Dean; Heilig, Markus; Ciccocioppo, Roberto; Roberto, Marisa

    2016-03-01

    The CRF system of the central nucleus of the amygdala (CeA) is important for the processing of anxiety, stress, and effects of acute and chronic ethanol. We previously reported that ethanol decreases evoked glutamate transmission in the CeA of Sprague Dawley rats and that ethanol dependence alters glutamate release in the CeA. Here, we examined the effects of ethanol, CRF and a CRF1 receptor antagonist on spontaneous and evoked glutamatergic transmission in CeA neurons from Wistar and Marchigian Sardinian Preferring (msP) rats, a rodent line genetically selected for excessive alcohol drinking and characterized by heightened activity of the CRF1 system. Basal spontaneous and evoked glutamate transmission in CeA neurons from msP rats was increased compared to Wistar rats. Ethanol had divergent effects, either increasing or decreasing spontaneous glutamate release in the CeA of Wistar rats. This bidirectional effect was retained in msP rats, but the magnitude of the ethanol-induced increase in glutamate release was significantly smaller. The inhibitory effect of ethanol on evoked glutamatergic transmission was similar in both strains. CRF also either increased or decreased spontaneous glutamate release in CeA neurons of Wistar rats, however, in msP rats CRF only increased glutamate release. The inhibitory effect of CRF on evoked glutamatergic transmission was also lost in neurons from msP rats. A CRF1 antagonist produced only minor effects on spontaneous glutamate transmission, which were consistent across strains, and no effects on evoked glutamate transmission. These results demonstrate that the genetically altered CRF system of msP rats results in alterations in spontaneous and stimulated glutamate signaling in the CeA that may contribute to both the anxiety and drinking behavioral phenotypes.

  15. Dysfunctional astrocytic and synaptic regulation of hypothalamic glutamatergic transmission in a mouse model of early-life adversity: relevance to neurosteroids and programming of the stress response.

    Science.gov (United States)

    Gunn, Benjamin G; Cunningham, Linda; Cooper, Michelle A; Corteen, Nicole L; Seifi, Mohsen; Swinny, Jerome D; Lambert, Jeremy J; Belelli, Delia

    2013-12-11

    Adverse early-life experiences, such as poor maternal care, program an abnormal stress response that may involve an altered balance between excitatory and inhibitory signals. Here, we explored how early-life stress (ELS) affects excitatory and inhibitory transmission in corticotrophin-releasing factor (CRF)-expressing dorsal-medial (mpd) neurons of the neonatal mouse hypothalamus. We report that ELS associates with enhanced excitatory glutamatergic transmission that is manifested as an increased frequency of synaptic events and increased extrasynaptic conductance, with the latter associated with dysfunctional astrocytic regulation of glutamate levels. The neurosteroid 5α-pregnan-3α-ol-20-one (5α3α-THPROG) is an endogenous, positive modulator of GABAA receptors (GABAARs) that is abundant during brain development and rises rapidly during acute stress, thereby enhancing inhibition to curtail stress-induced activation of the hypothalamic-pituitary-adrenocortical axis. In control mpd neurons, 5α3α-THPROG potently suppressed neuronal discharge, but this action was greatly compromised by prior ELS exposure. This neurosteroid insensitivity did not primarily result from perturbations of GABAergic inhibition, but rather arose functionally from the increased excitatory drive onto mpd neurons. Previous reports indicated that mice (dams) lacking the GABAAR δ subunit (δ(0/0)) exhibit altered maternal behavior. Intriguingly, δ(0/0) offspring showed some hallmarks of abnormal maternal care that were further exacerbated by ELS. Moreover, in common with ELS, mpd neurons of δ(0/0) pups exhibited increased synaptic and extrasynaptic glutamatergic transmission and consequently a blunted neurosteroid suppression of neuronal firing. This study reveals that increased synaptic and tonic glutamatergic transmission may be a common maladaptation to ELS, leading to enhanced excitation of CRF-releasing neurons, and identifies neurosteroids as putative early regulators of the stress

  16. Biphasic effects of cannabinoids in anxiety responses: CB1 and GABA(B) receptors in the balance of GABAergic and glutamatergic neurotransmission.

    Science.gov (United States)

    Rey, Alejandro Aparisi; Purrio, Martin; Viveros, Maria-Paz; Lutz, Beat

    2012-11-01

    Biphasic effects of cannabinoids have been shown in processes such as feeding behavior, motor activity, motivational processes and anxiety responses. Using two different tests for the characterization of anxiety-related behavior (elevated plus-maze and holeboard), we first identified in wild-type C57BL/6N mice, two doses of the synthetic CB1 cannabinoid receptor agonist CP-55,940 with anxiolytic (1 μg/kg) and anxiogenic properties (50 μg/kg), respectively. To clarify the role of CB1 receptors in this biphasic effect, both doses were applied to two different conditional CB1 receptor knockout (KO) mouse lines, GABA-CB1-KO (CB1 receptor inactivation in forebrain GABAergic neurons) and Glu-CB1-KO (CB1 receptor inactivation in cortical glutamatergic neurons). We found that the anxiolytic-like effects of the low dose of cannabinoids are mediated via the CB1 receptor on cortical glutamatergic terminals, because this anxiolytic-like response was abrogated only in Glu-CB1-KO mice. On the contrary, the CB1 receptor on the GABAergic terminals is required to induce an anxiogenic-like effect under a high-dose treatment because of the fact that this effect was abolished specifically in GABA-CB1-KO mice. These experiments were carried out in both sexes, and no differences occurred with the doses tested in the mutant mice. Interestingly, the positive allosteric modulation of GABA(B) receptor with GS-39783 was found to largely abrogate the anxiogenic-like effect of the high dose of CP-55,940. Our results shed new light in further understanding the biphasic effects of cannabinoids at the molecular level and, importantly, pave the way for the development of novel anxiolytic cannabinoid drugs, which may have favorable effect profiles targeting the CB1 receptor on glutamatergic terminals.

  17. CB1 receptor activation in the rat paraventricular nucleus induces bi-directional cardiovascular effects via modification of glutamatergic and GABAergic neurotransmission.

    Science.gov (United States)

    Grzęda, Emilia; Schlicker, Eberhard; Toczek, Marek; Zalewska, Iwona; Baranowska-Kuczko, Marta; Malinowska, Barbara

    2017-01-01

    We have shown previously that the cannabinoid receptor agonist CP55940 microinjected into the paraventricular nucleus of the hypothalamus (PVN) of urethane-anaesthetized rats induces depressor and pressor cardiovascular effects in the absence and presence of the CB1 antagonist AM251, respectively. The aim of our study was to examine whether the hypotension and/or hypertension induced by CP55940 given into the PVN results from its influence on glutamatergic and GABAergic neurotransmission. CP55940 was microinjected into the PVN of urethane-anaesthetized rats twice (S1 and S2, 20 min apart). Antagonists of the following receptors, NMDA (MK801), β2-adrenergic (ICI118551), thromboxane A2-TP (SQ29548), angiotensin II-AT1 (losartan) or GABAA (bicuculline), or the NO synthase inhibitor L-NAME were administered intravenously 5 min before S2 alone or together with AM251. The CP55940-induced hypotension was reversed into a pressor response by AM251, bicuculline and L-NAME, but not by the other antagonists. The CP55940-induced pressor effect examined in the presence of AM251 was completely reversed by losartan, reduced by about 50-60 % by MK801, ICI118551 and SQ29548, prevented by bilateral adrenalectomy but not modified by bicuculline and L-NAME. Parallel, but smaller, changes in heart rate accompanied the changes in blood pressure. The bi-directional CB1 receptor-mediated cardiovascular effects of cannabinoids microinjected into the PVN of anaesthetized rats depend on stimulatory glutamatergic and inhibitory GABAergic inputs to the sympathetic tone; the glutamatergic input is related to AT1, TP and β2-adrenergic receptors and catecholamine release from the adrenal medulla whereas the GABAergic input is reinforced by NO.

  18. Subpopulations of neurokinin 1 receptor-expressing neurons in the rat lateral amygdala display a differential pattern of innervation from distinct glutamatergic afferents

    Science.gov (United States)

    Sreepathi, H.K.; Ferraguti, F.

    2012-01-01

    Substance P by acting on its preferred receptor neurokinin 1 (NK1) in the amygdala appears to be critically involved in the modulation of fear and anxiety. The present study was undertaken to identify neurochemically specific subpopulations of neuron expressing NK1 receptors in the lateral amygdaloid nucleus (LA), a key site for regulating these behaviors. We also analyzed the sources of glutamatergic inputs to these neurons. Immunofluorescence analysis of the co-expression of NK1 with calcium binding proteins in LA revealed that ∼35% of NK1-containing neurons co-expressed parvalbumin (PV), whereas no co-localization was detected in the basal amygdaloid nucleus. We also show that neurons expressing NK1 receptors in LA did not contain detectable levels of calcium/calmodulin kinase IIα, thus suggesting that NK1 receptors are expressed by interneurons. By using a dual immunoperoxidase/immunogold-silver procedure at the ultrastructural level, we found that in LA ∼75% of glutamatergic synapses onto NK1-expressing neurons were labeled for the vesicular glutamate transporter 1 indicating that they most likely are of cortical, hippocampal, or intrinsic origin. The remaining ∼25% were immunoreactive for the vesicular glutamate transporter 2 (VGluT2), and may then originate from subcortical areas. On the other hand, we could not detect VGluT2-containing inputs onto NK1/PV immunopositive neurons. Our data add to previous localization studies by describing an unexpected variation between LA and basal nucleus of the amygdala (BA) in the neurochemical phenotype of NK1-expressing neurons and reveal the relative source of glutamatergic inputs that may activate these neurons, which in turn regulate fear and anxiety responses. PMID:22210508

  19. Effects of hypoxia on glutamatergic and GABAergic synaptic transmission%缺氧对谷氨酸能和GABA能突触传递的影响

    Institute of Scientific and Technical Information of China (English)

    李晶; 杜永平; 张月萍

    2013-01-01

    Neurons in the mammalian central nervous sysytem (CNS) are highly sensitive to the availability of oxygen. Hypoxia alters synaptic transmission in a few minutes. Both glutamatergic and γ-aminobutyric acid (GABA)ergic synaptic transmissions respond to hypoxic exposure with prominent modification. Glutamate receptors, GABA receptors, adenosine receptor, and some endogenous neuromodulators are involved in the preservation of neuron function. Since the neuroprotection in all hypoxic tolerant species examined so far relies on significant increase in GABA and decrease in glutamate , it may be an important strategy to make a moderate balance of glutamate/GAB A synaptic transmission against hypoxic insults.

  20. Selective potentiation of (α4)3(β2)2 nicotinic acetylcholine receptors augments amplitudes of prefrontal acetylcholine- and nicotine-evoked glutamatergic transients in rats.

    Science.gov (United States)

    Grupe, Morten; Paolone, Giovanna; Jensen, Anders A; Sandager-Nielsen, Karin; Sarter, Martin; Grunnet, Morten

    2013-11-15

    Prefrontal glutamate release evoked through activation of α4β2* nicotinic acetylcholine receptors (nAChRs) situated on thalamic glutamatergic afferents mediates cue detection processes and thus contributes to attentional performance. However, little is known about the respective contributions of the high sensitivity and low sensitivity (LS) stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, to these processes. In the present study we employed glutamate-sensitive microelectrodes and the (α4)3(β2)2-selective positive allosteric modulator (PAM) NS9283 to investigate the importance of the LS α4β2 nAChR for glutamate release in the rat medial prefrontal cortex (mPFC). Firstly, the signaling evoked by physiologically relevant ACh concentrations through the (α4)3(β2)2 nAChR in HEK293 cells was potentiated by NS9283, consistent with the classification of NS9283 as a PAM. In urethane-anesthetized rats, intra-prefrontal pressure ejections of NS9283 evoked glutamatergic transients. Importantly, this glutamate release was attenuated by removal of cholinergic projections to the recording area. This finding indicates that the effects of NS9283 depend on endogenous ACh, again consistent with effects of a PAM. We then conducted microdialysis to demonstrate the presence of extracellular ACh in urethane-anesthetized control rats. While detectable, those levels were significantly lower than in awake rats. Finally, the amplitudes of glutamatergic transients evoked by local pressure ejections of a low concentration of nicotine were significantly augmented following systemic administration of NS9283 (3.0mg/kg). In conclusion, our results indicate that a LS α4β2 nAChR PAM such as NS9283 may enhance the cholinergic modulation of glutamatergic neurotransmission in the cortex, thereby perhaps alleviating the attentional impairments common to a range of brain disorders. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Hypoxic preconditioning differentially affects GABAergic and glutamatergic neuronal cells in the injured cerebellum of the neonatal rat.

    Directory of Open Access Journals (Sweden)

    Sergio G Benitez

    Full Text Available In this study we examined cerebellar alterations in a neonatal rat model of hypoxic-ischemic brain injury with or without hypoxic preconditioning (Pc. Between postnatal days 7 and 15, the cerebellum is still undergoing intense cellular proliferation, differentiation and migration, dendritogenesis and synaptogenesis. The expression of glutamate decarboxylase 1 (GAD67 and the differentiation factor NeuroD1 were examined as markers of Purkinje and granule cells, respectively. We applied quantitative immunohistochemistry to sagittal cerebellar slices, and Western blot analysis of whole cerebella obtained from control (C rats and rats submitted to Pc, hypoxia-ischemia (L and a combination of both treatments (PcL. We found that either hypoxia-ischemia or Pc perturbed the granule cells in the posterior lobes, affecting their migration and final placement in the internal granular layer. These effects were partially attenuated when the Pc was delivered prior to the hypoxia-ischemia. Interestingly, whole nuclear NeuroD1 levels in Pc animals were comparable to those in the C rats. However, a subset of Purkinje cells that were severely affected by the hypoxic-ischemic insult--showing signs of neuronal distress at the levels of the nucleus, cytoplasm and dendritic arborization--were not protected by Pc. A monoclonal antibody specific for GAD67 revealed a three-band pattern in cytoplasmic extracts from whole P15 cerebella. A ∼110 kDa band, interpreted as a potential homodimer of a truncated form of GAD67, was reduced in Pc and L groups while its levels were close to the control animals in PcL rats. Additionally we demonstrated differential glial responses depending on the treatment, including astrogliosis in hypoxiated cerebella and a selective effect of hypoxia-ischemia on the vimentin-immunolabeled intermediate filaments of the Bergmann glia. Thus, while both glutamatergic and GABAergic cerebellar neurons are compromised by the hypoxic-ischemic insult

  2. Short-term repeated corticosterone administration enhances glutamatergic but not GABAergic transmission in the rat motor cortex.

    Science.gov (United States)

    Kula, Joanna; Blasiak, Anna; Czerw, Anna; Tylko, Grzegorz; Sowa, Joanna; Hess, Grzegorz

    2016-04-01

    It has been demonstrated that stress impairs performance of skilled reaching and walking tasks in rats due to the action of glucocorticoids involved in the stress response. Skilled reaching and walking are controlled by the primary motor cortex (M1); however, it is not known whether stress-related impairments in skilled motor tasks are related to functional and/or structural alterations within the M1. We studied the effects of single and repeated injections of corticosterone (twice daily for 7 days) on spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) recorded from layer II/III pyramidal neurons in ex vivo slices of the M1, prepared 2 days after the last administration of the hormone. We also measured the density of dendritic spines on pyramidal cells and the protein levels of selected subunits of AMPA, NMDA, and GABAA receptors after repeated corticosterone administration. Repeatedly administered corticosterone induced an increase in the frequency but not in the amplitude of sEPSCs, while a single administration had no effect on the recorded excitatory currents. The frequency and amplitude of sIPSCs as well as the excitability of pyramidal cells were changed neither after single nor after repeated corticosterone administration. Treatment with corticosterone for 7 days did not modify the density of dendritic spines on pyramidal neurons. Corticosterone influenced neither the protein levels of GluA1, GluA2, GluN1, GluN2A, and GluN2B subunits of glutamate receptors nor those of α1, β2, and γ2 subunits of the GABAA receptor. The increase in sEPSCs frequency induced by repeated corticosterone administration faded out within 7 days. These data indicate that prolonged administration of exogenous corticosterone selectively and reversibly enhances glutamatergic, but not GABAergic transmission in the rat motor cortex. Our results suggest that corticosterone treatment results in an enhancement of spontaneous glutamate release from presynaptic

  3. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter.

    Science.gov (United States)

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P; Geller, Alfred I

    2007-05-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine

  4. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter

    Science.gov (United States)

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P.; Geller, Alfred I.

    2009-01-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an ∼9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported ∼90 % glutamatergic neuron-specific expression. The GAD67 promoter supported ∼90 % GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic

  5. Midbrain Gene Screening Identifies a New Mesoaccumbal Glutamatergic Pathway and a Marker for Dopamine Cells Neuroprotected in Parkinson’s Disease

    Science.gov (United States)

    Viereckel, Thomas; Dumas, Sylvie; Smith-Anttila, Casey J. A.; Vlcek, Bianca; Bimpisidis, Zisis; Lagerström, Malin C.; Konradsson-Geuken, Åsa; Wallén-Mackenzie, Åsa

    2016-01-01

    The ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) of the midbrain are associated with Parkinson’s disease (PD), schizophrenia, mood disorders and addiction. Based on the recently unraveled heterogeneity within the VTA and SNc, where glutamate, GABA and co-releasing neurons have been found to co-exist with the classical dopamine neurons, there is a compelling need for identification of gene expression patterns that represent this heterogeneity and that are of value for development of human therapies. Here, several unique gene expression patterns were identified in the mouse midbrain of which NeuroD6 and Grp were expressed within different dopaminergic subpopulations of the VTA, and TrpV1 within a small heterogeneous population. Optogenetics-coupled in vivo amperometry revealed a previously unknown glutamatergic mesoaccumbal pathway characterized by TrpV1-Cre-expression. Human GRP was strongly detected in non-melanized dopaminergic neurons within the SNc of both control and PD brains, suggesting GRP as a marker for neuroprotected neurons in PD. This study thus unravels markers for distinct subpopulations of neurons within the mouse and human midbrain, defines unique anatomical subregions within the VTA and exposes an entirely new glutamatergic pathway. Finally, both TRPV1 and GRP are implied in midbrain physiology of importance to neurological and neuropsychiatric disorders. PMID:27762319

  6. Cell type-specific long-term plasticity at glutamatergic synapses onto hippocampal interneurons expressing either parvalbumin or CB1 cannabinoid receptor.

    Science.gov (United States)

    Nissen, Wiebke; Szabo, Andras; Somogyi, Jozsef; Somogyi, Peter; Lamsa, Karri P

    2010-01-27

    Different GABAergic interneuron types have specific roles in hippocampal function, and anatomical as well as physiological features vary greatly between interneuron classes. Long-term plasticity of interneurons has mostly been studied in unidentified GABAergic cells and is known to be very heterogeneous. Here we tested whether cell type-specific plasticity properties in distinct GABAergic interneuron types might underlie this heterogeneity. We show that long-term potentiation (LTP) and depression (LTD), two common forms of synaptic plasticity, are expressed in a highly cell type-specific manner at glutamatergic synapses onto hippocampal GABAergic neurons. Both LTP and LTD are generated in interneurons expressing parvalbumin (PV+), whereas interneurons with similar axon distributions but expressing cannabinoid receptor-1 show no lasting plasticity in response to the same protocol. In addition, LTP or LTD occurs in PV+ interneurons with different efferent target domains. Perisomatic-targeting PV+ basket and axo-axonic interneurons express LTP, whereas glutamatergic synapses onto PV+ bistratified cells display LTD. Both LTP and LTD are pathway specific, independent of NMDA receptors, and occur at synapses with calcium-permeable (CP) AMPA receptors. Plasticity in interneurons with CP-AMPA receptors strongly modulates disynaptic GABAergic transmission onto CA1 pyramidal cells. We propose that long-term plasticity adjusts the synaptic strength between pyramidal cells and interneurons in a cell type-specific manner and, in the defined CA1 interneurons, shifts the spatial pattern of inhibitory weight from pyramidal cell dendrites to the perisomatic region.

  7. Effects of oxytocin on methamphetamine-induced conditioned place preference and the possible role of glutamatergic neurotransmission in the medial prefrontal cortex of mice in reinstatement.

    Science.gov (United States)

    Qi, Jia; Yang, Jing-Yu; Wang, Fang; Zhao, Ya-Nan; Song, Ming; Wu, Chun-Fu

    2009-04-01

    Accumulating evidence has shown the neuroactive properties of oxytocin (OT), a neurohypophyseal neuropeptide, and its ability to reduce the abuse potential of drugs. The present study investigated the effects of OT on the conditioned place preference (CPP) induced by methamphetamine (MAP, 2.0 mg/kg, i.p.) in mice and the possible role of glutamatergic neurotransmission in the reinstatement of CPP. The results showed that OT (0.1, 0.5, 2.5 microg, i.c.v.) significantly inhibited the acquisition and facilitated the extinction of MAP-induced CPP and abolished the reinstatement of CPP induced by restraint stress. This effect of OT could be attenuated by atosiban (Ato, 2.0 microg, i.c.v.), a selective OT-receptor antagonist. OT failed to block the expression and the reinstatement of CPP induced by MAP challenge. Extracellular glutamate (Glu) levels in the medial prefrontal cortex (mPFC) were determined using microdialysis coupled to a high-performance liquid chromatography (HPLC) with a fluorescence detection system. The results indicated that OT markedly inhibited extracellular Glu levels induced by restraint stress in CPP mice, but not those induced by MAP priming. Ato also attenuated the effects of OT on the changes in Glu levels. Therefore, these findings suggest that OT inhibits drug reward-related behaviors induced by MAP via the OT receptor, and OT blocks the reinstatement of CPP, at least partially, by interfering with the glutamatergic system in the mPFC.

  8. Differential requirement for NMDAR activity in SAP97β-mediated regulation of the number and strength of glutamatergic AMPAR-containing synapses.

    Science.gov (United States)

    Liu, Mingna; Lewis, Laura D; Shi, Rebecca; Brown, Emery N; Xu, Weifeng

    2014-02-01

    PSD-95-like, disc-large (DLG) family membrane-associated guanylate kinase proteins (PSD/DLG-MAGUKs) are essential for regulating synaptic AMPA receptor (AMPAR) function and activity-dependent trafficking of AMPARs. Using a molecular replacement strategy to replace endogenous PSD-95 with SAP97β, we show that the prototypic β-isoform of the PSD-MAGUKs, SAP97β, has distinct NMDA receptor (NMDAR)-dependent roles in regulating basic properties of AMPAR-containing synapses. SAP97β enhances the number of AMPAR-containing synapses in an NMDAR-dependent manner, whereas its effect on the size of unitary synaptic response is not fully dependent on NMDAR activity. These effects contrast with those of PSD-95α, which increases both the number of AMPAR-containing synapses and the size of unitary synaptic responses, with or without NMDAR activity. Our results suggest that SAP97β regulates synaptic AMPAR content by increasing surface expression of GluA1-containing AMPARs, whereas PSD-95α enhances synaptic AMPAR content presumably by increasing the synaptic scaffold capacity for synaptic AMPARs. Our approach delineates discrete effects of different PSD-MAGUKs on principal properties of glutamatergic synaptic transmission. Our results suggest that the molecular diversity of PSD-MAGUKs can provide rich molecular substrates for differential regulation of glutamatergic synapses in the brain.

  9. Maturation of glutamatergic transmission in the vestibulo-olivary pathway impacts on the registration of head rotational signals in the brainstem of rats.

    Science.gov (United States)

    Lai, Chun-Hong; Ma, Chun-Wai; Lai, Suk-King; Han, Lei; Wong, Hoi-Man; Yeung, Kelvin Wai-Kwok; Shum, Daisy Kwok-Yan; Chan, Ying-Shing

    2016-01-01

    The recognition of head orientation in the adult involves multi-level integration of inputs within the central vestibular circuitry. How the different inputs are recruited during postnatal development remains unclear. We hypothesize that glutamatergic transmission at the vestibular nucleus contributes to developmental registration of head orientations along the vestibulo-olivary pathway. To investigate the maturation profile by which head rotational signals are registered in the brainstem, we used sinusoidal rotations on the orthogonal planes of the three pairs of semicircular canals. Fos expression was used as readout of neurons responsive to the rotational stimulus. Neurons in the vestibular nucleus and prepositus hypoglossal nucleus responded to all rotations as early as P4 and reached adult numbers by P21. In the reticular formation and inferior olive, neurons also responded to horizontal rotations as early as P4 but to vertical rotations not until P21 and P25, respectively. Neuronal subpopulations that distinguish between rotations activating the orthogonally oriented vertical canals were identifiable in the medial and spinal vestibular nuclei by P14 and in the inferior olivary subnuclei IOβ and IOK by P25. Neonatal perturbation of glutamate transmission in the vestibular nucleus was sufficient to derange formation of this distribution in the inferior olive. This is the first demonstration that developmental refinement of glutamatergic synapses in the central vestibular circuitry is essential for developmental registration of head rotational signals in the brainstem.

  10. DIXDC1 contributes to psychiatric susceptibility by regulating dendritic spine and glutamatergic synapse density via GSK3 and Wnt/β-catenin signaling.

    Science.gov (United States)

    Martin, P-M; Stanley, R E; Ross, A P; Freitas, A E; Moyer, C E; Brumback, A C; Iafrati, J; Stapornwongkul, K S; Dominguez, S; Kivimäe, S; Mulligan, K A; Pirooznia, M; McCombie, W R; Potash, J B; Zandi, P P; Purcell, S M; Sanders, S J; Zuo, Y; Sohal, V S; Cheyette, B N R

    2016-10-18

    Mice lacking DIX domain containing-1 (DIXDC1), an intracellular Wnt/β-catenin signal pathway protein, have abnormal measures of anxiety, depression and social behavior. Pyramidal neurons in these animals' brains have reduced dendritic spines and glutamatergic synapses. Treatment with lithium or a glycogen synthase kinase-3 (GSK3) inhibitor corrects behavioral and neurodevelopmental phenotypes in these animals. Analysis of DIXDC1 in over 9000 cases of autism, bipolar disorder and schizophrenia reveals higher rates of rare inherited sequence-disrupting single-nucleotide variants (SNVs) in these individuals compared with psychiatrically unaffected controls. Many of these SNVs alter Wnt/β-catenin signaling activity of the neurally predominant DIXDC1 isoform; a subset that hyperactivate this pathway cause dominant neurodevelopmental effects. We propose that rare missense SNVs in DIXDC1 contribute to psychiatric pathogenesis by reducing spine and glutamatergic synapse density downstream of GSK3 in the Wnt/β-catenin pathway.Molecular Psychiatry advance online publication, 18 October 2016; doi:10.1038/mp.2016.184.

  11. Dissociation of CA3 pyramidal cells with attached, functional, identified mossy fiber and interneuronal boutons for studying glutamatergic and GABAergic synaptic transmission.

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    Beltrán, Jesús Q; Reyes, Sebastián; Pérez-Guzmán, José A; Elías-Viñas, David; Gutiérrez, Rafael

    2012-07-15

    Pyramidal cells of CA3 area receive glutamatergic signals from the mossy fibers (MFs), perforant path and collaterals of other pyramidal cells, as well as GABAergic inputs from interneurons. In hippocampal slices, an extracellular stimulation electrode is often used to activate the MFs, with the disadvantage of possibly activating fibers other than MFs. We set-up a preparation that allows the analysis of the glutamatergic input from identified, giant MF boutons as well as of GABAergic inputs from boutons of interneurons on single CA3 pyramidal cells. Mossy fiber boutons were labeled by exposing hippocampal slices to a zinc-reactive fluorescent dye, or by injecting a fluorescent dye in the granule cell layer and allowing its transport along the MFs to their terminals in CA3 area. After conducting an enzyme-free, mechanical dissociation of CA3 area, we obtained pyramidal cells containing fluorescent, giant MF boutons attached to their apical dendrites, as well as boutons of interneuronal origin. Whole cell recordings were then performed, whereby synaptic responses could be evoked by selective stimulation of the identified boutons. The synaptic currents evoked by stimulation of MF boutons, unlike those evoked by stimulation of interneuronal boutons, underwent strong frequency potentiation and were depressed by activation of metabotropic glutamate receptors, which are characteristics of transmission of MF origin. Combination of fluorophores can be used to label different tracts/boutons allowing the study of the different characteristics of neurotransmitter release from a variety of sources on single target cells.

  12. Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition

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    Schlichter Rémy

    2008-05-01

    Full Text Available Abstract Background Recent evidence suggests that oxytocin (OT, secreted in the superficial spinal cord dorsal horn by descending axons of paraventricular hypothalamic nucleus (PVN neurons, produces antinociception and analgesia. The spinal mechanism of OT is, however, still unclear and requires further investigation. We have used patch clamp recording of lamina II neurons in spinal cord slices and immunocytochemistry in order to identify PVN-activated neurons in the superficial layers of the spinal cord and attempted to determine how this neuronal population may lead to OT-mediated antinociception. Results We show that OT released during PVN stimulation specifically activates a subpopulation of lamina II glutamatergic interneurons which are localized in the most superficial layers of the dorsal horn of the spinal cord (lamina I-II. This OT-specific stimulation of glutamatergic neurons allows the recruitment of all GABAergic interneurons in lamina II which produces a generalized elevation of local inhibition, a phenomenon which might explain the reduction of incoming Aδ and C primary afferent-mediated sensory messages. Conclusion Our results obtained in lamina II of the spinal cord provide the first clear evidence of a specific local neuronal network that is activated by OT release to induce antinociception. This OT-specific pathway might represent a novel and interesting therapeutic target for the management of neuropathic and inflammatory pain.

  13. Synaptic vesicle cycling is not impaired in a glutamatergic and a cholinergic synapse that exhibit deficits in acidification and filling

    Directory of Open Access Journals (Sweden)

    Bento João Abreu

    2012-03-01

    Full Text Available The purpose of the present work was to investigate synaptic vesicle trafficking when vesicles exhibit alterations in filling and acidification in two different synapses: a cholinergic frog neuromuscular junction and a glutamatergic ribbon-type nerve terminal in the retina. These synapses display remarkable structural and functional differences, and the mechanisms regulating synaptic vesicle cycling might also differ between them. The lipophilic styryl dye FM1-43 was used to monitor vesicle trafficking. Both preparations were exposed to pharmacological agents that collapse ΔpH (NH4Cl and methylamine or the whole ΔµH+ (bafilomycin, a necessary situation to provide the driving force for neurotransmitter accumulation into synaptic vesicles. The results showed that FM1-43 loading and unloading in neuromuscular junctions did not differ statistically between control and experimental conditions (P > 0.05. Also, FM1-43 labeling in bipolar cell terminals proved highly similar under all conditions tested. Despite remarkable differences in both experimental models, the present findings show that acidification and filling are not required for normal vesicle trafficking in either synapse.O objetivo do presente trabalho foi investigar o tráfego de vesículas sinápticas quando estas apresentam alterações no armazenamento de neurotransmissores e acidificação em duas distintas sinapses: a junção neuromuscular colinérgica de rãs versus o terminal nervoso glutamatérgico do tipo ribbon em céulas bipolares da retina. Essas sinapses exibem notáveis diferenças estruturais e funcionais e os mecanismos de regulação de ciclo das vesículas sinápticas podem ser diferentes entre eles. Para monitorar o tráfego de vesícula, foi utilizado o marcador lipofílico FM1-43. Ambas as preparações foram expostas a agentes farmacológicos que provocam o colapso de ΔpH (NH4Cl e metilamina ou de todo ΔµH+ (bafilomicina, gradientes necessários para o ac

  14. Electrical Stimulation of Low-Threshold Proprioceptive Fibers in the Adult Rat Increases Density of Glutamatergic and Cholinergic Terminals on Ankle Extensor α-Motoneurons.

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    Gajewska-Woźniak, Olga; Grycz, Kamil; Czarkowska-Bauch, Julita; Skup, Małgorzata

    2016-01-01

    The effects of stimulation of low-threshold proprioceptive afferents in the tibial nerve on two types of excitatory inputs to α-motoneurons were tested. The first input is formed by glutamatergic Ia sensory afferents contacting monosynaptically α-motoneurons. The second one is the cholinergic input originating from V0c-interneurons, located in lamina X of the spinal cord, modulating activity of α-motoneurons via C-terminals. Our aim was to clarify whether enhancement of signaling to ankle extensor α-motoneurons, via direct electrical stimulation addressed predominantly to low-threshold proprioceptive fibers in the tibial nerve of awake rats, will affect Ia glutamatergic and cholinergic innervation of α-motoneurons of lateral gastrocnemius (LG). LG motoneurons were identified with True Blue tracer injected intramuscularly. Tibial nerve was stimulated for 7 days with continuous bursts of three pulses applied in four 20 min sessions daily. The Hoffmann reflex and motor responses recorded from the soleus muscle, LG synergist, allowed controlling stimulation. Ia terminals and C-terminals abutting on LG-labeled α-motoneurons were detected by immunofluorescence (IF) using input-specific anti- VGLUT1 and anti-VAChT antibodies, respectively. Quantitative analysis of confocal images revealed that the number of VGLUT1 IF and VAChT IF terminals contacting the soma of LG α-motoneurons increased after stimulation by 35% and by 26%, respectively, comparing to the sham-stimulated side. The aggregate volume of VGLUT1 IF and VAChT IF terminals increased by 35% and by 30%, respectively. Labeling intensity of boutons was also increased, suggesting an increase of signaling to LG α-motoneurons after stimulation. To conclude, one week of continuous burst stimulation of proprioceptive input to LG α-motoneurons is effective in enrichment of their direct glutamatergic but also indirect cholinergic inputs. The effectiveness of such and longer stimulation in models of injury is a

  15. GABA transporter subtype 1 and GABA transporter subtype 3 modulate glutamatergic transmission via activation of presynaptic GABA(B) receptors in the rat globus pallidus.

    Science.gov (United States)

    Jin, Xiao-Tao; Paré, Jean-Francois; Smith, Yoland

    2012-08-01

    The intra-pallidal application of γ-aminobutyric acid (GABA) transporter subtype 1 (GAT-1) or GABA transporter subtype 3 (GAT-3) transporter blockers [1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF 89976A) or 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid (SNAP 5114)] reduces the activity of pallidal neurons in monkey. This effect could be mediated through the activation of presynaptic GABA(B) heteroreceptors in glutamatergic terminals by GABA spillover following GABA transporter (GAT) blockade. To test this hypothesis, we applied the whole-cell recording technique to study the effects of SKF 89976A and SNAP 5114 on evoked excitatory postsynaptic currents (eEPSCs) in the presence of gabazine, a GABA(A) receptor antagonist, in rat globus pallidus slice preparations. Under the condition of postsynaptic GABA(B) receptor blockade by the intra-cellular application of N-(2,6-dimethylphenylcarbamoylmethyl)-triethylammonium bromide (OX314), bath application of SKF 89976A (10 μM) or SNAP 5114 (10 μM) decreased the amplitude of eEPSCs, without a significant effect on its holding current and whole cell input resistance. The inhibitory effect of GAT blockade on eEPSCs was blocked by (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid, a GABA(B) receptor antagonist. The paired-pulse ratio of eEPSCs was increased, whereas the frequency, but not the amplitude, of miniature excitatory postsynaptic currents was reduced in the presence of either GAT blocker, demonstrating a presynaptic effect. These results suggest that synaptically released GABA can inhibit glutamatergic transmission through the activation of presynaptic GABA(B) heteroreceptors following GAT-1 or GAT-3 blockade. In conclusion, our findings demonstrate that presynaptic GABA(B) heteroreceptors in putative glutamatergic subthalamic afferents to the globus pallidus are sensitive to increases in extracellular GABA induced

  16. Electrical Stimulation of Low-Threshold Proprioceptive Fibers in the Adult Rat Increases Density of Glutamatergic and Cholinergic Terminals on Ankle Extensor α-Motoneurons

    Science.gov (United States)

    Gajewska-Woźniak, Olga; Grycz, Kamil; Czarkowska-Bauch, Julita; Skup, Małgorzata

    2016-01-01

    The effects of stimulation of low-threshold proprioceptive afferents in the tibial nerve on two types of excitatory inputs to α-motoneurons were tested. The first input is formed by glutamatergic Ia sensory afferents contacting monosynaptically α-motoneurons. The second one is the cholinergic input originating from V0c—interneurons, located in lamina X of the spinal cord, modulating activity of α-motoneurons via C-terminals. Our aim was to clarify whether enhancement of signaling to ankle extensor α-motoneurons, via direct electrical stimulation addressed predominantly to low-threshold proprioceptive fibers in the tibial nerve of awake rats, will affect Ia glutamatergic and cholinergic innervation of α-motoneurons of lateral gastrocnemius (LG). LG motoneurons were identified with True Blue tracer injected intramuscularly. Tibial nerve was stimulated for 7 days with continuous bursts of three pulses applied in four 20 min sessions daily. The Hoffmann reflex and motor responses recorded from the soleus muscle, LG synergist, allowed controlling stimulation. Ia terminals and C-terminals abutting on LG-labeled α-motoneurons were detected by immunofluorescence (IF) using input-specific anti- VGLUT1 and anti-VAChT antibodies, respectively. Quantitative analysis of confocal images revealed that the number of VGLUT1 IF and VAChT IF terminals contacting the soma of LG α-motoneurons increased after stimulation by 35% and by 26%, respectively, comparing to the sham-stimulated side. The aggregate volume of VGLUT1 IF and VAChT IF terminals increased by 35% and by 30%, respectively. Labeling intensity of boutons was also increased, suggesting an increase of signaling to LG α-motoneurons after stimulation. To conclude, one week of continuous burst stimulation of proprioceptive input to LG α-motoneurons is effective in enrichment of their direct glutamatergic but also indirect cholinergic inputs. The effectiveness of such and longer stimulation in models of injury is a

  17. Characterization of energy and neurotransmitter metabolism in cortical glutamatergic neurons derived from human induced pluripotent stem cells: A novel approach to study metabolism in human neurons.

    Science.gov (United States)

    Aldana, Blanca I; Zhang, Yu; Lihme, Maria Fog; Bak, Lasse K; Nielsen, Jørgen E; Holst, Bjørn; Hyttel, Poul; Freude, Kristine K; Waagepetersen, Helle S

    2017-02-24

    Alterations in the cellular metabolic machinery of the brain are associated with neurodegenerative disorders such as Alzheimer's disease. Novel human cellular disease models are essential in order to study underlying disease mechanisms. In the present study, we characterized major metabolic pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC-derived neurons were incubated with [U-(13)C]glucose, [U-(13)C]glutamate or [U-(13)C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass spectrometry, and cellular amino acid content was quantified by high-performance liquid chromatography. Additionally, we evaluated mitochondrial function using real-time assessment of oxygen consumption via the Seahorse XF(e)96 Analyzer. Moreover, in order to validate the hiPSC-derived neurons as a model system, a metabolic profiling was performed in parallel in primary neuronal cultures of mouse cerebral cortex and cerebellum. These serve as well-established models of GABAergic and glutamatergic neurons, respectively. The hiPSC-derived neurons were previously characterized as being forebrain-specific cortical glutamatergic neurons. However, a comparable preparation of predominantly mouse cortical glutamatergic neurons is not available. We found a higher glycolytic capacity in hiPSC-derived neurons compared to mouse neurons and a substantial oxidative metabolism through the mitochondrial tricarboxylic acid (TCA) cycle. This finding is supported by the extracellular acidification and oxygen consumption rates measured in the cultured human neurons. [U-(13)C]Glutamate and [U-(13)C]glutamine were found to be efficient energy substrates for the neuronal cultures originating from both mice and humans. Interestingly, isotopic labeling in metabolites from [U-(13)C]glutamate was higher than that from [U-(13)C]glutamine. Although the metabolic profile of hiPSC-derived neurons in vitro was

  18. A possible role of the non-GAT1 GABA transporters in transfer of GABA from GABAergic to glutamatergic neurons in mouse cerebellar neuronal cultures.

    Science.gov (United States)

    Suñol, C; Babot, Z; Cristòfol, R; Sonnewald, U; Waagepetersen, H S; Schousboe, A

    2010-09-01

    Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, beta-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC(50) 142 microM), beta-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC(50) 0.8 microM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 microM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of beta-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also

  19. Effects of age and acute ethanol on glutamatergic neurotransmission in the medial prefrontal cortex of freely moving rats using enzyme-based microelectrode amperometry.

    Science.gov (United States)

    Mishra, Devesh; Harrison, Nicholas R; Gonzales, Carolina B; Schilström, Björn; Konradsson-Geuken, Åsa

    2015-01-01

    Ethanol abuse during adolescence may significantly alter development of the prefrontal cortex which continues to undergo structural remodeling into adulthood. Glutamatergic neurotransmission plays an important role during these brain maturation processes and is modulated by ethanol. In this study, we investigated glutamate dynamics in the medial prefrontal cortex of freely moving rats, using enzyme-based microelectrode amperometry. We analyzed the effects of an intraperitoneal ethanol injection (1 g/kg) on cortical glutamate levels in adolescent and adult rats. Notably, basal glutamate levels decreased with age and these levels were found to be significantly different between postnatal day (PND) 28-38 vs PND 44-55 (pprefrontal cortex and suggest that acute ethanol injections have both inhibitory and excitatory effects in adolescent rats. These effects of ethanol on the prefrontal cortex may disturb its maturation and possibly limiting individuals´ control over addictive behaviors.

  20. Enhancing glutamatergic transmission during adolescence reverses early-life stress-induced deficits in the rewarding effects of cocaine in rats.

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    O'Connor, Richard M; Moloney, Rachel D; Glennon, Jeffrey; Vlachou, Styliani; Cryan, John F

    2015-12-01

    Adolescence marks a critical time when the brain is highly susceptible to pathological insult yet also uniquely amenable to therapeutic intervention. It is during adolescence that the onset of the majority of psychiatric disorders, including substance use disorder (SUDs), occurs. It has been well established that stress, particularly during early development, can contribute to the pathological changes which contribute to the development of SUDs. Glutamate as the main excitatory neurotransmitter in the mammalian CNS plays a key role in various physiological processes, including reward function, and in mediating the effects of psychological stress. We hypothesised impairing glutamatergic signalling during the key adolescent period would attenuate early-life stress induced impaired reward function. To test this, we induced early-life stress in male rats using the maternal-separation procedure. During the critical adolescent period (PND25-46) animals were treated with the glutamate transporter activator, riluzole, or the NMDA receptor antagonist, memantine. Adult reward function was assessed using voluntary cocaine intake measured via intravenous self-administration. We found that early-life stress in the form of maternal-separation impaired reward function, reducing the number of successful cocaine-infusions achieved during the intravenous self-administration procedure as well impairing drug-induced reinstatement of cocaine-taking behaviour. Interestingly, riluzole and memantine treatment reversed this stress-induced impairment. These data suggest that reducing glutamatergic signalling may be a viable therapeutic strategy for treating vulnerable individuals at risk of developing SUDs including certain adolescent populations, particularly those which may have experienced trauma during early-life.

  1. Modulation of glutamatergic transmission by presynaptic N-methyl-D-aspartate mechanisms in second-order neurons of the rat nucleus tractus solitarius.

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    Ohi, Yoshiaki; Kimura, Satoko; Haji, Akira

    2015-02-05

    The present study investigated the physiological function of presynaptic N-methyl-d aspartate (NMDA) mechanisms in glutamatergic transmission in the rat nucleus tractus solitarius (NTS). Membrane currents were recorded from the NTS second-order neurons by using whole-cell patch pipettes including MK-801 to block postsynaptic NMDA receptors. All experiments were performed under blockade of inhibitory synaptic transmission. Co-application of NMDA and d-serine decreased the tractus solitarius (TS)-evoked excitatory postsynaptic currents (eEPSCs) in 7/12 (58%) of neurons, and increased the paired pulse ratio. The remaining neurons were insensitive to NMDA and d-serine. Application of an NMDA antagonist D-AP5 had no effect on eEPSCs in all 8 neurons tested. Action potential-independent EPSCs (miniature EPSCs; mEPSCs) were recorded in the presence of tetrodotoxin. Co-application of NMDA and d-serine increased the mEPSC frequency but had no significant effect on the amplitude in 5/28 (18%) of neurons. D-AP5 decreased the mEPSC frequency without effect on the amplitude in 6/18 (33%) of neurons. This study demonstrated that (1) NMDA receptors were presynaptically distributed in a subset of NTS second-order neurons and that (2) the presynaptic NMDA receptors played an inhibitory role in TS-mediated release of glutamate and a facilitatory role in spontaneous release of glutamate. The present results suggest that the activation of presynaptic NMDA receptors modulates glutamatergic transmissions in the rat NTS second-order neurons.

  2. Modulation of glutamatergic transmission by metabotropic glutamate receptor activation in second-order neurons of the guinea pig nucleus tractus solitarius.

    Science.gov (United States)

    Ohi, Yoshiaki; Kimura, Satoko; Haji, Akira

    2014-09-18

    Activity of second-order relay neurons in the nucleus tractus solitarius (NTS) is regulated by peripheral and intrinsic synaptic inputs, and modulation of those inputs by metabotropic glutamate receptors (mGluRs) has been proposed. This study investigated effects of mGluR activation on glutamatergic transmission in the NTS second-order neurons of guinea pigs. Whole-cell patch-clamp recordings from the brainstem slices revealed that activation of mGluRs exerted its effects on the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) but not on the amplitude. The sEPSC frequency was increased by an agonist of group I mGluRs, and it was decreased by an mGluR1 antagonist but not by an mGluR5 antagonist. The agonists of group II and III mGluRs decreased the sEPSC frequency, while their antagonists alone had no effect. Perfusion of cystine or TBOA, either of which elevates extracellular glutamate concentration, resulted in an increase in the sEPSC frequency, leaving the amplitude unchanged. The increased frequency of sEPSCs was returned to control by an mGluR1 antagonist. The tractus solitarius-evoked EPSCs were not altered by an agonist of group I mGluRs, whereas they were decreased along with an increase in paired-pulse ratio by agonists of group II and III mGluRs. These results suggest that mGluRs are present at the presynaptic sites in the NTS second-order neurons in guinea pigs. The mGluR1s function to facilitate the release of glutamate from axon terminals of intrinsic interneurons and the group II and III mGluRs play an inhibitory role in glutamatergic transmission.

  3. The novel antipsychotic drug brexpiprazole, alone and in combination with escitalopram, facilitates prefrontal glutamatergic transmission via a dopamine D1 receptor-dependent mechanism.

    Science.gov (United States)

    Björkholm, Carl; Marcus, Monica M; Konradsson-Geuken, Åsa; Jardemark, Kent; Svensson, Torgny H

    2017-02-09

    Brexpiprazole (Rexulti(®)), a novel D2/3 receptor (R) partial agonist, was recently approved as monotherapy for schizophrenia, demonstrating effectiveness against both positive and negative symptoms, and also approved as add-on treatment to antidepressant drugs, inducing a potent antidepressant effect with a faster onset compared to an antidepressant given alone. Moreover, brexpiprazole has demonstrated pro-cognitive effects in preclinical studies. To explore whether the observed effects may be mediated via modulation of prefrontal glutamatergic transmission, we investigated the effect of brexpiprazole, alone and in combination with the SSRI escitalopram, on prefrontal glutamatergic transmission using in vitro electrophysiological intracellular recordings of deep layer pyramidal cells of the rat medial prefrontal cortex (mPFC). Nanomolar concentrations of brexpiprazole potentiated NMDAR-induced currents and electrically evoked EPSPs via activation of dopamine D1Rs, in similarity with the effect of the atypical antipsychotic drug clozapine. The effect of an ineffective concentration of brexpiprazole was significantly potentiated by the addition of escitalopram. When combined with escitalopram, brexpiprazole also potentiated AMPAR-mediated transmission, in similarity with the clinically rapid acting antidepressant drug ketamine. The effect on the AMPAR-mediated currents was also D1R dependent. In conclusion, our data propose that brexpiprazole exerts a clozapine-like potentiation of NMDAR-mediated currents in the mPFC, which can explain its efficacy on negative symptoms of schizophrenia and the pro-cognitive effects observed preclinically. Moreover, add-on brexpiprazole to escitalopram also potentiated AMPAR-mediated transmission, which may provide a neurobiological explanation to the faster antidepressant effect of add-on brexpiprazole in major depression.

  4. Glutamatergic synapse protein composition of wild-type mice is sensitive to in utero MTHFR genotype and the timing of neonatal vigabatrin exposure.

    Science.gov (United States)

    Zuckerman, Chava; Blumkin, Elinor; Melamed, Osnat; Golan, Hava M

    2015-10-01

    The enzyme methylenetetrahydrofolate-reductase (MTHFR) is part of the homocysteine and folate metabolic pathways. In utero, Mthfr-deficient environment has been reported as a risk factor for neurodevelopmental disorders such as autism and neural tube defects. Neonatal disruption of the GABAergic system is also associated with behavioral outcomes. The interaction between Mthfr deficiency and neonatal exposure to the GABA-potentiating drug vigabatrin (GVG) in mice alters anxiety, memory, and social behavior in a gender-dependent manner. In addition, a gender-dependent enhancement of proteins implicated in excitatory synapse plasticity in the cerebral cortex was shown. Here we show that in utero MTHFR deficiency is sufficient to alter the levels of glutamate receptor subunits GluR1, GluR2, and NR2B in the cerebral cortex and hippocampus of adult offspring with a WT genotype. In addition, FMRP1, CAMKII α and γ, and NLG1 levels in WT offspring were vulnerable to the in utero genotype. These effects depend on brain region and the cellular compartment tested. The effect of in utero MTHFR deficiency varies with the age of neonatal GVG exposure to modify GluR1, NR2A, reelin, CAMKII α, and NLG1 levels. These changes in molecular composition of the glutamatergic synapse were associated with increased anxiety-like behavior. Complex, multifactorial disorders of the nervous system show significant association with several genetic and environmental factors. Our data exemplify the contribution of an in utero MTHFR-deficient environment and early exposure to an antiepileptic drug to the basal composition of the glutamatergic synapses. The robust effect is expected to alter synapse function and plasticity and the cortico-hippocampal circuitry. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.

  5. The effect of Schisandra chinensis extracts on depression by noradrenergic, dopaminergic, GABAergic and glutamatergic systems in the forced swim test in mice.

    Science.gov (United States)

    Yan, Tingxu; Xu, Mengjie; Wu, Bo; Liao, Zhengzheng; Liu, Zhi; Zhao, Xu; Bi, Kaishun; Jia, Ying

    2016-06-15

    Schisandra chinensis (Turcz.) Baill., as a Chinese functional food, has been widely used in neurological disorders including insomnia and Alzheimer's disease. The treatment of classical neuropsychiatric disorder depression is to be developed from Schisandra chinensis. The antidepressant-like effects of the Schisandra chinensis extracts (SCE), and their probable involvement in the serotonergic, noradrenergic, dopaminergic, GABAergic and glutamatergic systems were investigated by the forced swim test (FST). Acute administration of SCE (600 mg kg(-1), i.g.), a combination of SCE (300 mg kg(-1), i.g.) and reboxetine (a noradrenalin reuptake inhibitor, 2.5 mg kg(-1), i.p.) or imipramine (a TCA, 2 mg kg(-1), i.p.) reduced the immobility time in the FST. Pretreatment with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4, a selective noradrenergic neurotoxin, 50 mg kg(-1), i.p., 4 days), haloperidol (a non-selective D2 receptor antagonist, 0.2 mg kg(-1), i.p.), SCH 23390 (a selective D1 receptor antagonist, 0.03 mg kg(-1), i.p.), bicuculline (a competitive GABA antagonist, 4 mg kg(-1), i.p.) and N-methyl-d-aspartic acid (NMDA, an agonist at the glutamate site, 75 mg kg(-1), i.p.) effectively reversed the antidepressant-like effect of SCE (600 mg kg(-1), i.g.). However, p-chlorophenylalanine (pCPA, an inhibitor of 5-HT synthesis, 100 mg kg(-1), i.p., 4 days,) did not eliminate the reduced immobility time induced by SCE (600 mg kg(-1), i.g.). Moreover, the treatments did not change the locomotor activity. Altogether, these results indicated that SCE produced antidepressant-like activity, which might be mediated by the modification of noradrenergic, dopaminergic, GABAergic and glutamatergic systems.

  6. Development of "Pinceaux" formations and dendritic translocation of climbing fibers during the acquisition of the balance between glutamatergic and gamma-aminobutyric acidergic inputs in developing Purkinje cells.

    Science.gov (United States)

    Sotelo, Constantino

    2008-01-10

    The acquisition of the dynamic balance between excitation and inhibition in developing Purkinje cells, necessary for their proper function, is analyzed. Newborn (P0) mouse cerebellum contains glutamatergic (VGLUT2-IR) and gamma-aminobutyric acid (GABA)-ergic (VIAAT-IR) axons. The former prevail and belong to climbing fibers, whereas the latter neither colabel with calbindin-expressing fibers nor belong to axons of the cortical GABAergic interneurons. During the first postnatal week, VIAAT-IR axons in the Purkinje cell neighborhood remains very low, and the first synapses with basket fibers are formed at P7, when climbing fibers have already established dense pericellular nets. The descending basket fibers reach the Purkinje cell axon initial segment by P9, immediately establishing axoaxonic synapses. The pinceaux appear as primitive vortex-like arrangements by P12, and by P20 interbasket fiber septate-like junctions, typical of fully mature pinceaux, are still missing. The climbing fiber's somatodendritic translocation occurs later than expected, after the regression of the multiple innervation, and follows the ascending collaterals of the basket axons, which are apparently the optimal substrate for the proper subcellular targeting of the climbing fibers. These results emphasize that chemical transmission in the axon initial segment precedes the electrical inhibition generated by field effects. In addition, GABAergic Purkinje cells, as opposed to glutamatergic projection neurons in other cortical structures, do not begin to receive their excitation to inhibition balance until the end of the first postnatal week, despite the early presence of potentially functional GABAergic axons that possess the required vesicular transport system.

  7. Refeeding-activated glutamatergic neurons in the hypothalamic paraventricular nucleus (PVN) mediate effects of melanocortin signaling in the nucleus tractus solitarius (NTS).

    Science.gov (United States)

    Singru, Praful S; Wittmann, Gábor; Farkas, Erzsébet; Zséli, Györgyi; Fekete, Csaba; Lechan, Ronald M

    2012-08-01

    We previously demonstrated that refeeding after a prolonged fast activates a subset of neurons in the ventral parvocellular subdivision of the paraventricular nucleus (PVNv) as a result of increased melanocortin signaling. To determine whether these neurons contribute to satiety by projecting to the nucleus tractus solitarius (NTS), the retrogradely transported marker substance, cholera toxin-β (CTB), was injected into the dorsal vagal complex of rats that were subsequently fasted and refed for 2 h. By double-labeling immunohistochemistry, CTB accumulation was found in the cytoplasm of the majority of refeeding-activated c-Fos neurons in the ventral parvocellular subdivision of the hypothalamic paraventricular nucleus (PVNv). In addition, a large number of refeeding-activated c-Fos-expressing neurons were observed in the lateral parvocellular subdivision (PVNl) that also contained CTB and were innervated by axon terminals of proopiomelanocortin neurons. To visualize the location of neuronal activation within the NTS by melanocortin-activated PVN neurons, α-MSH was focally injected into the PVN, resulting in an increased number of c-Fos-containing neurons in the PVN and in the NTS, primarily in the medial and commissural parts. All refeeding-activated neurons in the PVNv and PVNl expressed the mRNA of the glutamatergic marker, type 2 vesicular glutamate transporter (VGLUT2), indicating their glutamatergic phenotype, but only rare neurons contained oxytocin. These data suggest that melanocortin-activated neurons in the PVNv and PVNl may contribute to refeeding-induced satiety through effects on the NTS and may alter the sensitivity of NTS neurons to vagal satiety inputs via glutamate excitation.

  8. Bisphenol A impairs the memory function and glutamatergic homeostasis in a sex-dependent manner in mice: Beneficial effects of diphenyl diselenide.

    Science.gov (United States)

    Jardim, Natália S; Sartori, Glaúbia; Sari, Marcel H M; Müller, Sabrina G; Nogueira, Cristina W

    2017-08-15

    Bisphenol A (BPA) is a compound integrated in commodities, which consequently increases the human exposure to this toxicant. The deleterious effects of BPA exposure during periods of brain development have been documented mainly concerning the impairment in memory functions. Diphenyl diselenide (PhSe)2, an organoselenium compound, shows protective/restorative effects against memory deficits in experimental models. Thus, this study investigated the effects of (PhSe)2 on the memory impairments induced by BPA exposure to male and female mice and the possible involvement of glutamatergic system in these effects. Three-week-old male and female Swiss mice received BPA (5mg/kg), intragastrically, from 21st to 60th postnatal day. After, the animals were intragastrically treated with (PhSe)2 (1mg/kg) during seven days. The mice performed the behavioral memory tests and the [(3)H] glutamate uptake and NMDA receptor subunits (2A and 2B) analyses were carried out in the hippocampus and cerebral cortex of mice. The results demonstrated that the BPA exposure induced impairment of object recognition memory in both sexes. However, it caused impairments in spatial memory in female and in the passive avoidance memory in male mice. Besides, BPA caused a decrease in the [(3)H] glutamate uptake and NMDA receptor subunit levels in the cortical and hippocampal regions depending on the sex. Treatment with (PhSe)2 reversed in a sex-independent manner the behavioral impairments and molecular alterations. In conclusion, BPA had a negative effect in different memory types as well as in the glutamatergic parameters in a sex-dependent manner and (PhSe)2 treatment was effective against these alterations. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Characterizing PCDH19 in human induced pluripotent stem cells (iPSCs) and iPSC-derived developing neurons: emerging role of a protein involved in controlling polarity during neurogenesis.

    Science.gov (United States)

    Compagnucci, Claudia; Petrini, Stefania; Higuraschi, Norimichi; Trivisano, Marina; Specchio, Nicola; Hirose, Shinichi; Bertini, Enrico; Terracciano, Alessandra

    2015-09-29

    PCDH19 (Protocadherin 19), a member of the cadherin superfamily, is involved in the pathogenic mechanism of an X-linked model of neurological disease. The biological function of PCHD19 in human neurons and during neurogenesis is currently unknown. Therefore, we decided to use the model of the induced pluripotent stem cells (iPSCs) to characterize the location and timing of expression of PCDH19 during cortical neuronal differentiation. Our data show that PCDH19 is expressed in pluripotent cells before differentiation in a homogeneous pattern, despite its localization is often limited to one pole of the cell. During neuronal differentiation, positional information on the progenitor cells assumes an important role in acquiring polarization. The proper control of the cell orientation ensures a fine balancing between symmetric (giving rise to two progenitor sister cells) versus asymmetric (giving rise to one progenitor cell and one newborn neuron) division. This process results in the polar organization of the neural tube with a lumen indicating the basal part of the polarized neuronal progenitor cell; in the iPSC model the cells are organized in the 'neural rosette' and interestingly, PCDH19 is located at the center of the rosette, with other well-known markers of the lumen (N-cadherin and ZO-1). These data suggest that PCDH19 has a role in instructing the apico-basal polarity of the progenitor cells, thus regulating the development of a properly organized human brain.

  10. Synergistic effects of carboxymethyl-hexanoyl chitosan, cationic polyurethane-short branch PEI in miR122 gene delivery: accelerated differentiation of iPSCs into mature hepatocyte-like cells and improved stem cell therapy in a hepatic failure model.

    Science.gov (United States)

    Chien, Yueh; Chang, Yuh-Lih; Li, Hsin-Yang; Larsson, Mikael; Wu, Wai-Wah; Chien, Chian-Shiu; Wang, Chien-Ying; Chu, Pen-Yuan; Chen, Kuan-Hsuan; Lo, Wen-Liang; Chiou, Shih-Hwa; Lan, Yuan-Tzu; Huo, Teh-Ia; Lee, Shou-Dong; Huang, Pin-I

    2015-02-01

    MicroRNA122 (miR122), a liver-specific microRNA, plays critical roles in homeostatic regulation and hepatic-specific differentiation. Induced pluripotent stem cells (iPSCs) have promising potential in regenerative medicine, but it remains unknown whether non-viral vector-mediated miR122 delivery can enhance the differentiation of iPSCs into hepatocyte-like cells (iPSC-Heps) and rescue thioacetamide-induced acute hepatic failure (AHF) in vivo. In this study, we demonstrated that embedment of miR122 complexed with polyurethane-graft-short-branch polyethylenimine copolymer (PU-PEI) in nanostructured amphiphatic carboxymethyl-hexanoyl chitosan (CHC) led to dramatically enhanced miR122 delivery into human dental pulp-derived iPSCs (DP-iPSCs) and facilitated these DP-iPSCs to differentiate into iPSC-Heps (miR122-iPSC-Heps) with mature hepatocyte functions. Microarray and bioinformatics analysis further indicated that CHC/PU-PEI-miR122 promoted the gene-signature pattern of DP-iPSCs to shift into a liver-specific pattern. Furthermore, intrahepatic delivery of miR122-iPSC-Heps, but not miR-Scr-iPSC-Heps, improved liver functions and rescued recipient survival, and CHC-mediated delivery showed a better efficacy than that using phosphate buffered saline as a delivery vehicle. In addition, these transplanted miR122-iPSC-Heps remained viable and could produce circulatory albumin for 4 months. Taken together, our findings demonstrate that non-viral delivery of miR122 shortens the time of iPSC differentiation into hepatocytes and the delivery of miR122-iPSC-Heps using CHC as a vehicle exhibited promising hepatoprotective efficacy in vivo. miR122-iPSC-Heps may represent a feasible cell source and provide an efficient and alternative strategy for hepatic regeneration in AHF.

  11. Tumor necrosis factor-mediated downregulation of spinal astrocytic connexin43 leads to increased glutamatergic neurotransmission and neuropathic pain in mice.

    Science.gov (United States)

    Morioka, Norimitsu; Zhang, Fang Fang; Nakamura, Yoki; Kitamura, Tomoya; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

    2015-10-01

    Spinal cord astrocytes are critical in the maintenance of neuropathic pain. Connexin 43 (Cx43) expressed on spinal dorsal horn astrocytes modulates synaptic neurotransmission, but its role in nociceptive transduction has yet to be fully elaborated. In mice, Cx43 is mainly expressed in astrocytes, not neurons or microglia, in the spinal dorsal horn. Hind paw mechanical hypersensitivity was observed beginning 3days after partial sciatic nerve ligation (PSNL), but a persistent downregulation of astrocytic Cx43 in ipsilateral lumbar spinal dorsal horn was not observed until 7days post-PSNL, suggesting that Cx43 downregulation mediates the maintenance and not the initiation of nerve injury-induced hypersensitivity. Downregulation of Cx43 expression by intrathecal treatment with Cx43 siRNA also induced mechanical hypersensitivity. Conversely, restoring Cx43 by an adenovirus vector expressing Cx43 (Ad-Cx43) ameliorated PSNL-induced mechanical hypersensitivity. The sensitized state following PSNL is likely maintained by dysfunctional glutamatergic neurotransmission, as Cx43 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of glutamate receptor antagonists MK801 and CNQX, but not neurokinin-1 receptor antagonist CP96345 or the Ca(2+) channel subunit α2δ1 blocker gabapentin. The source of this dysfunctional glutamatergic neurotransmission is likely decreased clearance of glutamate from the synapse rather than increased glutamate release into the synapse. Astrocytic expression of glutamate transporter GLT-1, but not GLAST, and activity of glutamate transport were markedly decreased in mice intrathecally injected with Cx43-targeting siRNA but not non-targeting siRNA. Glutamate release from spinal synaptosomes prepared from mice treated with either Cx43-targeting siRNA or non-targeting siRNA was unchanged. Intrathecal injection of Ad-Cx43 in PSNL mice restored astrocytic GLT-1 expression. The cytokine tumor necrosis factor (TNF) has been

  12. Prazosin blocks the glutamatergic effects of N-methyl-D-aspartic acid on lordosis behavior and luteinizing hormone secretion in the estrogen-primed female rat

    Directory of Open Access Journals (Sweden)

    Landa A.I.

    2006-01-01

    Full Text Available We have observed that intracerebroventricular (icv injection of selective N-methyl-D-aspartic acid (NMDA-type glutamatergic receptor antagonists inhibits lordosis in ovariectomized (OVX, estrogen-primed rats receiving progesterone or luteinizing hormone-releasing hormone (LHRH. When NMDA was injected into OVX estrogen-primed rats, it induced a significant increase in lordosis. The interaction between LHRH and glutamate was previously explored by us and another groups. The noradrenergic systems have a functional role in the regulation of LHRH release. The purpose of the present study was to explore the interaction between glutamatergic and noradrenergic transmission. The action of prazosin, an alpha1- and alpha2b-noradrenergic antagonist, was studied here by injecting it icv (1.75 and 3.5 µg/6 µL prior to NMDA administration (1 µg/2 µL in OVX estrogen-primed Sprague-Dawley rats (240-270 g. Rats manually restrained were injected over a period of 2 min, and tested 1.5 h later. The enhancing effect induced by NMDA on the lordosis/mount ratio at high doses (67.06 ± 3.28, N = 28 when compared to saline controls (6 and 2 µL, 16.59 ± 3.20, N = 27 was abolished by prazosin administration (17.04 ± 5.52, N = 17, and 9.33 ± 3.21, N = 20, P < 0.001 for both doses. Plasma LH levels decreased significantly only with the higher dose of prazosin (1.99 ± 0.24 ng/mL, N = 18, compared to saline-NMDA effect, 5.96 ± 2.01 ng/mL, N = 13, P < 0.05. Behavioral effects seem to be more sensitive to the alpha-blockade than hormonal effects. These findings strongly suggest that the facilitatory effects of NMDA on both lordosis and LH secretion in this model are mediated by alpha-noradrenergic transmission.

  13. Time-dependent impact of glutamatergic modulators on the promnesiant effect of 5-HT6R blockade on mice recognition memory.

    Science.gov (United States)

    Asselot, Rachel; Simon-O'Brien, Emmanuelle; Lebourgeois, Sophie; Nee, Gérald; Delaunay, Virgile; Duchatelle, Pascal; Bouet, Valentine; Dauphin, François

    2017-04-01

    Selective antagonists at serotonin 5-HT6 receptors (5-HT6R) improve memory performance in rodents and are currently under clinical investigations. If blockade of 5-HT6R is known to increase glutamate release, only two studies have so far demonstrated an interaction between 5-HT6R and glutamate transmission, but both, using the non-competitive NMDA antagonist MK-801, insensitive to variations of glutamate concentrations. In a place recognition task, we investigated here in mice the role of glutamate transmission in the beneficial effects of 5-HT6R blockade (SB-271046). Through the use of increasing intervals (2, 4 and 6h) between acquisition and retrieval, we investigated the time-dependent impact of two different glutamatergic modulators. NMDAR-dependant glutamate transmission (NMDA Receptors) was either blocked by the competitive antagonist at NMDAR, CGS 19755, or potentiated by the glycine transporter type 1 (GlyT1) inhibitor, NFPS. Results showed that neither SB-271046, nor CGS 19755, nor NFPS, alter behavioural performances after short intervals, i.e. when control mice displayed significant memory performances (2h and 4h) (respectively 10, 3, and 0.625mg.kg(-1)). Conversely, with the 6h-interval, a situation in which spontaneous forgetting is observed in control mice, SB-271046 improved recognition memory performances. This beneficial effect was prevented when co-administered with either CGS 19755 or NFPS, which themselves had no effect. Interestingly, a dose-dependent effect was observed with NFPS, with promnesic effect observed at lower dose (0.156mg.kg(-1)) when administrated alone, whereas it did no modify promnesic effect of SB-271046. These results demonstrate that promnesiant effect induced by 5-HT6R blockade is sensitive to the competitive blockade of NMDAR and underline the need of a fine adjustment of the inhibition of GlyT1. Overall, our findings support the idea of a complex crosstalk between serotonergic and glutamatergic systems in the promnesic

  14. Glutamatergic and central cholinergic dysfunction in the CA1, CA2 and CA3 fields on spatial learning and memory in chronic cerebral ischemia-Induced vascular dementia of rats.

    Science.gov (United States)

    Cao, Yanjing; Gou, Zengmei; Du, Yifeng; Fan, Yongjun; Liang, Lizhen; Yan, Yongxing; Lin, Ping; Jin, Mudan; Du, Yifenf

    2016-05-04

    Chronic cerebral ischemia (CCI) is associated with cognitive decline in aging, vascular dementia and Alzheimer's disease. Substantial evidence has shown that chronic cerebral ischemia may cause cognitive impairment, but the underlying neurobiological mechanism is poorly understood so far. In the present study, we used a rat model of chronic cerebral ischemia by permanent bilateral common carotid artery occlusion (BCCAO) to investigate the alterations of glutamatergic and central cholinergic dysfunction, and their causal relationship with the cognitive deficits induced by chronic cerebral ischemia. We found that BCCAO rats exhibited spatial learning and memory impairments dysfunction 3 month after BCCAO. Meanwhile, vGluT levels as well as glutamatergic and central cholinergic positive neurons in the hippocampus CA1-3 field significantly decreased. The protection of glutamergic and cholinergic neurons or regulating glutamate and central cholinergic levels in hippocampal subregion may have beneficial effects on cognitive impairments associated with the possible mechanism in CCI-induced vascular dementia.

  15. Supplementation of antipsychotic treatment with sarcosine – GlyT1 inhibitor – causes changes of glutamatergic (1)NMR spectroscopy parameters in the left hippocampus in patients with stable schizophrenia.

    Science.gov (United States)

    Strzelecki, Dominik; Podgórski, Michał; Kałużyńska, Olga; Gawlik-Kotelnicka, Oliwia; Stefańczyk, Ludomir; Kotlicka-Antczak, Magdalena; Gmitrowicz, Agnieszka; Grzelak, Piotr

    2015-10-01

    Glutamatergic system, the main stimulating system of the brain, plays an important role in the pathogenesis of schizophrenia. Hippocampus, a structure crucial for memory and cognitive functions and rich in glutamatergic neurons, is a natural object of interest in studies on psychoses. Sarcosine, a glycine transporter (GlyT-1) inhibitor influences the function of NMDA receptor and glutamate-dependent transmission. The aim of the study was to assess the effects of sarcosine on metabolism parameters in the left hippocampus in patients with schizophrenia. Assessments were performed using proton nuclear magnetic resonance ((1)H NMR) spectroscopy (1.5T). Fifty patients diagnosed with schizophrenia (DSM-IV-TR), with dominant negative symptoms, in stable clinical condition and stable antipsychotics doses were treated either with sarcosine (n=25) or placebo (n=25). Spectroscopic parameters were evaluated within groups and between two groups before and after 6-month intervention. All patients were also assessed with the Positive and Negative Syndrome Scale (PANSS). In the sarcosine group, after 6-month treatment, we found significant decrease in hippocampal Glx/Cr (Glx-complex of glutamate, glutamine and GABA, Cr-creatine) and Glx/Cho (Cho-choline), while N-acetylaspartate (NAA), myo-inositol (mI), Cr and Cho parameters remained stable along the study and also did not differ significantly between both groups. This is the first study showing that a pharmacological intervention in schizophrenia, particularly augmentation of the antypsychotic treatment with sarcosine, may reverse the pathological increase in glutamatergic transmission in the hippocampus. The results confirm involvement of glutamatergic system in the pathogenesis of schizophrenia and demonstrate beneficial effects of GlyT-1 inhibitor on the metabolism in the hippocampus and symptoms of schizophrenia.

  16. The Prdm13 histone methyltransferase encoding gene is a Ptf1a-Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube.

    Science.gov (United States)

    Hanotel, Julie; Bessodes, Nathalie; Thélie, Aurore; Hedderich, Marie; Parain, Karine; Van Driessche, Benoit; Brandão, Karina De Oliveira; Kricha, Sadia; Jorgensen, Mette C; Grapin-Botton, Anne; Serup, Palle; Van Lint, Carine; Perron, Muriel; Pieler, Tomas; Henningfeld, Kristine A; Bellefroid, Eric J

    2014-02-15

    The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a and likely constitutes a direct transcriptional target. We also showed that this regulation requires the formation of the Ptf1a-Rbp-j complex. Prdm13 knockdown in Xenopus embryos and in Ptf1a overexpressing ectodermal explants lead to an upregulation of Tlx3/Hox11L2, which specifies a glutamatergic lineage and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural tube confirm that Prdm13 suppresses Tlx3(+)/glutamatergic and induces Pax2(+)/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube.

  17. The Prdm13 histone methyltransferase encoding gene is a Ptf1a-Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube

    DEFF Research Database (Denmark)

    Hanotel, Julie; Bessodes, Nathalie; Thélie, Aurore

    2014-01-01

    The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing gene...... and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube....... and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural...... tube confirm that Prdm13 suppresses Tlx3(+)/glutamatergic and induces Pax2(+)/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic...

  18. Witnessing stressful events induces glutamatergic synapse pathway alterations and gene set enrichment of positive EPSP regulation within the VTA of adult mice: An ontology based approach

    Science.gov (United States)

    Brewer, Jacob S.

    It is well known that exposure to severe stress increases the risk for developing mood disorders. Currently, the neurobiological and genetic mechanisms underlying the functional effects of psychological stress are poorly understood. Presenting a major obstacle to the study of psychological stress is the inability of current animal models of stress to distinguish between physical and psychological stressors. A novel paradigm recently developed by Warren et al., is able to tease apart the effects of physical and psychological stress in adult mice by allowing these mice to "witness," the social defeat of another mouse thus removing confounding variables associated with physical stressors. Using this 'witness' model of stress and RNA-Seq technology, the current study aims to study the genetic effects of psychological stress. After, witnessing the social defeat of another mouse, VTA tissue was extracted, sequenced, and analyzed for differential expression. Since genes often work together in complex networks, a pathway and gene ontology (GO) analysis was performed using data from the differential expression analysis. The pathway and GO analyzes revealed a perturbation of the glutamatergic synapse pathway and an enrichment of positive excitatory post-synaptic potential regulation. This is consistent with the excitatory synapse theory of depression. Together these findings demonstrate a dysregulation of the mesolimbic reward pathway at the gene level as a result of psychological stress potentially contributing to depressive like behaviors.

  19. Gastrin-releasing peptide facilitates glutamatergic transmission in the hippocampus and effectively prevents vascular dementia induced cognitive and synaptic plasticity deficits.

    Science.gov (United States)

    Yang, Jiajia; Yao, Yang; Wang, Ling; Yang, Chunxiao; Wang, Faqi; Guo, Jie; Wang, Zhiyun; Yang, Zhuo; Ming, Dong

    2017-01-01

    Neuronal gastrin-releasing peptide (GRP) has been proved to be an important neuromodulator in the brain and involved in a variety of neurological diseases. Whether GRP could attenuate cognition impairment induced by vascular dementia (VD) in rats, and the mechanism of synaptic plasticity and GRP's action on synaptic efficiency are still poorly understood. In this study, we first investigated the effects of GRP on glutamatergic transmission with patch-clamp recording. We found that acute application of GRP enhanced the excitatory synaptic transmission in hippocampal CA1 neurons via GRPR in a presynaptic mechanism. Secondly, we examined whether exogenous GRP or its analogue neuromedin B (NMB) could prevent VD-induced cognitive deficits and the mechanism of synaptic plasticity. By using Morris water maze, long-term potentiation (LTP) recording, western blot assay and immunofluorescent staining, we verified for the first time that GRP or NMB substantially improved the spatial learning and memory abilities in VD rats, restored the impaired synaptic plasticity and was able to elevate the expression of synaptic proteins, synaptophysin (SYP) and CaMKII, which play pivotal roles in synaptic plasticity. These results suggest that the facilitatory effects of GRP on glutamate release may contribute to its long-term action on synaptic efficacy which is essential in cognitive function. Our findings present a new entry point for a better understanding of physiological function of GRP and raise the possibility that GRPR agonists might ameliorate cognitive deficits associated with neurological diseases.

  20. Estrogen receptor GPR30 exerts anxiolytic effects by maintaining the balance between GABAergic and glutamatergic transmission in the basolateral amygdala of ovariectomized mice after stress.

    Science.gov (United States)

    Tian, Zhen; Wang, Yu; Zhang, Nan; Guo, Yan-Yan; Feng, Bin; Liu, Shui-Bing; Zhao, Ming-Gao

    2013-10-01

    G-protein-coupled receptor 30 (GPR30)/G-protein-coupled estrogen receptor is a novel estrogen membrane receptor that localizes to the cell membrane and endoplasmic reticulum. GPR30 is widely distributed and has numerous physiological functions in the central nervous system. We found that GPR30 is highly expressed in the basolateral amygdala (BLA). Additionally, GPR30 expression in the amygdala of ovariectomized (OVX) mice significantly increased after acute stress and was accompanied by anxiety-like behaviors. These effects, however, were reversed by local infusion of the GPR30 agonist (G1) in the BLA. Protein assessments revealed that G1 attenuated the up-regulation of the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and NR2A-containing N-methyl-d-aspartate receptors (NMDARs) in the BLA of OVX mice using an acute stress paradigm. In the same model, we found that the agonist also blocked the down-regulation of γ-aminobutyric acid A (GABAA) receptors and NR2B-containing NMDARs. Electrophysiological recording showed that the activation of GPR30 increased the inhibitory synaptic transmission in the BLA. Overall, our results indicate that estradiol reduces anxiety-like behaviors induced by acute stress at least partially through GPR30 signaling, maintaining the balance between GABAergic and glutamatergic transmission in the BLA of OVX-stressed mice.

  1. GPR30 disrupts the balance of GABAergic and glutamatergic transmission in the spinal cord driving to the development of bone cancer pain.

    Science.gov (United States)

    Luo, Jie; Huang, Xiaoxia; Li, Yali; Li, Yang; Xu, Xueqin; Gao, Yan; Shi, Ruoshi; Yao, Wanjun; Liu, Juying; Ke, Changbin

    2016-11-08

    Cancer induced bone pain is a very complicated clinical pain states that has proven difficult to be treated effectively due to poorly understand of underlying mechanism, but bone cancer pain (BCP) seems to be enhanced by a state of spinal sensitization. In the present study, we showed that carcinoma tibia implantation induced notable pain sensitization and up-regulation of G-protein-coupled estrogen receptor (GPR30) in the spinal cord of rats which was reversed by GPR30 knockdown. Further studies indicated that upregulation of GPR30 induced by cancer implantation resulted in a select loss of γ-aminobutyric acid-ergic (GABAergic) neurons and functionally diminished the inhibitory transmission due to reduce expression of the vesicular GABA transporter (VGAT). GPR30 contributed to spinal cord disinhibition by diminishing the inhibitory transmission via upregulation of α1 subunit and downregulation of γ2 subunits. GPR30 also facilitated excitatory transmission by promoting functional up-regulation of the calcium/calmodulin-dependent protein kinase II α (CaMKII α) in glutamatergic neurons and increasing the clustering of the glutamate receptor subunit 1 (GluR1) subunit to excitatory synapse.Taken together, GPR30 contributed to the development of BCP by both facilitating excitatory transmission and inhibiting inhibitory transmission in the spinal cord. Our findings provide the new spinal disinhibition and sensitivity mechanisms underlying the development of bone cancer pain.

  2. IL1RAPL1 associated with mental retardation and autism regulates the formation and stabilization of glutamatergic synapses of cortical neurons through RhoA signaling pathway.

    Directory of Open Access Journals (Sweden)

    Takashi Hayashi

    Full Text Available Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1 is associated with X-linked mental retardation and autism spectrum disorder. We found that IL1RAPL1 regulates synapse formation of cortical neurons. To investigate how IL1RAPL1 controls synapse formation, we here screened IL1RAPL1-interacting proteins by affinity chromatography and mass spectroscopy. IL1RAPL1 interacted with Mcf2-like (Mcf2l, a Rho guanine nucleotide exchange factor, through the cytoplasmic Toll/IL-1 receptor domain. Knockdown of endogenous Mcf2l and treatment with an inhibitor of Rho-associated protein kinase (ROCK, the downstream kinase of RhoA, suppressed IL1RAPL1-induced excitatory synapse formation of cortical neurons. Furthermore, we found that the expression of IL1RAPL1 affected the turnover of AMPA receptor subunits. Insertion of GluA1-containing AMPA receptors to the cell surface was decreased, whereas that of AMPA receptors composed of GluA2/3 was enhanced. Mcf2l knockdown and ROCK inhibitor treatment diminished the IL1RAPL1-induced changes of AMPA receptor subunit insertions. Our results suggest that Mcf2l-RhoA-ROCK signaling pathway mediates IL1RAPL1-dependent formation and stabilization of glutamatergic synapses of cortical neurons.

  3. Nuclear respiratory factor 1 co-regulates AMPA glutamate receptor subunit 2 and cytochrome c oxidase: tight coupling of glutamatergic transmission and energy metabolism in neurons.

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    Dhar, Shilpa S; Liang, Huan Ling; Wong-Riley, Margaret T T

    2009-03-01

    Neuronal activity, especially of the excitatory glutamatergic type, is highly dependent on energy from the oxidative pathway. We hypothesized that the coupling existed at the transcriptional level by having the same transcription factor to regulate a marker of energy metabolism, cytochrome c oxidase (COX) and an important subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors, GluR2 (Gria2). Nuclear respiratory factor 1 (NRF-1) was a viable candidate because it regulates all COX subunits and potentially activates Gria2. By means of in silico analysis, electrophoretic mobility shift and supershift, chromatin immunoprecipitation, and promoter mutational assays, we found that NRF-1 functionally bound to Gria2 promoter. Silencing of NRF-1 with small interference RNA prevented the depolarization-stimulated up-regulation of Gria2 and COX, and over-expression of NRF-1 rescued neurons from tetrodotoxin-induced down-regulation of Gria2 and COX transcripts. Thus, neuronal activity and energy metabolism are tightly coupled at the molecular level, and NRF-1 is a critical agent in this process.

  4. Characterization of the influence of age on GABAA and glutamatergic mediated functions in the dorsolateral prefrontal cortex using paired-pulse TMS-EEG.

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    Noda, Yoshihiro; Zomorrodi, Reza; Cash, Robin F H; Barr, Mera S; Farzan, Faranak; Rajji, Tarek K; Chen, Robert; Daskalakis, Zafiris J; Blumberger, Daniel M

    2017-02-16

    Gamma-aminobutyric acid (GABA)ergic and glutamatergic neurotransmissions in the prefrontal cortex decreases with age. Further, cognitive function mediated through the dorsolateral prefrontal cortex (DLPFC) also declines with age. Although neuroimaging studies have demonstrated decreased levels of these substances, direct neurophysiological data investigating the effect of aging in the DLPFC in human subjects is lacking. The advent of transcranial magnetic stimulation (TMS) combined with electroencephalography (EEG) has allowed for the assessment of functional neurotransmission in vivo. In the present study, we examined short interval intracortical inhibition (SICI) and intracortical facilitation (ICF) in a group of older adults (> 60 yrs) to evaluate the strength of GABAA and glutamate-mediated neurotransmission in the DLPFC, compared to younger adults (18-59 yrs). Older adults showed an increase of amplitude of N100 by the SICI paradigm, while N45 amplitude was increased and N100 amplitude was decreased by ICF. Moreover, these modulations significantly correlated with age. Our findings provide evidence for age-related alterations of excitatory and inhibitory functions in the prefrontal cortex in healthy adults. Future studies may aim to explore these neurophysiological relationships in the DLPFC in pathological forms of aging that affect cortical functioning such as mild cognitive impairment and Alzheimer's disease.

  5. Effects of intracerebroventricular NMDA and non-NMDA receptor agonists or antagonists on general anesthesia of propofol in mice

    Institute of Scientific and Technical Information of China (English)

    XU Aijun; DUAN Shiming; TIAN Yuke

    2007-01-01

    The efiects of intracerebroventricular(icv)agonists and antagonists of N-methyl-D-aspartate(NMDA)and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors on the general anesthesia of propofol were studied.A tohal of 144 Kunming mice,male and female with body mass of(22±3)g,were used.Part One of the Experiment:a total of 104 Kunming mice,male and female,were randomly divided into 13 groups.Intracerebroventricular artificial cerebral fluid (aCSF)or different doses of NMDA,AMPA,MK-801 or NBOX was iniected immediately after intravenously administered propofol 25 mg/kg and the recovery time following the loss of righting reflex (LORR)was recorded.Part Two of the Experiment:a total of 40 Kunming female mice were divided randomly into 5 groups and iniected with icv aCSF or NMDA.AMPA.MK-801 or NBQX after intraperitoneally administered propofol 50 mg/kg.The pain threshold of the mice was then investigated by hot-plate test(HPPT).NMDA(0.05 or 0.075μg,icv)or AMPA(0.05 μg,icv)exhibited no effects on the LORR,but NMDA(0.1 μg,icv)or AMPA(0.075 or 0.1 μg,icv)prolonged the LORR significantly compared with the aCSF group(P<0.05,P<0.01).The LORR of the 2 μg MK-801 group had no changes,while those of the 4 or 8 μg MK-801 groups were prolonged significantly.The LORR of the 0.5,2 or 4 μg NBQX groups were all prolonged significantly.NMDA 0.05 μg or AMPA 0.05 μg decreased the pain threshold slightly but did not differ in effect compared with the aCSF group;2 μg MK-801 or 0.5 μg NBQX both increased the pain threshold significantly.Our results indicate that propofol produces general anesthesia partly through an interaction with brain NMDA and AMPA receptors in mice.

  6. Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells in the hippocampal neurogenesis involving myelin vacuolation of cholinergic and glutamatergic inputs in mice.

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    Kato, Mizuho; Abe, Hajime; Itahashi, Megu; Kikuchihara, Yoh; Kimura, Masayuki; Mizukami, Sayaka; Yoshida, Toshinori; Shibutani, Makoto

    2016-02-01

    Hexachlorophene (HCP) has been shown to induce myelin vacuolation due to intramyelinic edema of the nerve fibers in animal neural tissue. We investigated the maternal exposure effect of HCP on hippocampal neurogenesis in the offspring of pregnant mice supplemented with 0 (control), 33 or 100 ppm HCP in diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, offspring as examined in males exhibited decreased granule cell lineage populations expressing paired box 6, sex-determining region Y-box 2 and eomesodermin in the hippocampal subgranular zone (SGZ) accompanied by myelin vacuolation involving white matter tracts of the hippocampal fimbria at ≥ 33 ppm. However, SGZ cellular populations expressing brain lipid binding protein and doublecortin were unchanged at any dose. Transcript expression of cholinergic receptor genes, Chrna4 and Chrnb2, and glutamate receptor genes, Grm1 and Grin2d, examined at 100 ppm, decreased in the dentate gyrus. HCP exposure did not alter the number of proliferating or apoptotic cells in the SGZ, or reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)ergic interneurons in the dentate hilus, on PND 21 and PND 77. All neurogenesis-related changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77, suggesting that maternal HCP exposure at ≥ 33 ppm reversibly decreased type 2 intermediate-stage progenitor cells in the hippocampal neurogenesis. Myelin vacuolation might be responsible for changes in neurogenesis possibly by reducing nerve conduction velocity of cholinergic inputs from the septal-hippocampal pathway to granule cell lineages and/or GABAergic interneurons, and of glutamatergic inputs to granule cell lineages.

  7. Long-lasting alterations in membrane properties, K+ currents and glutamatergic synaptic currents of nucleus accumbens medium spiny neurons in a rat model of alcohol dependence

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    Igor eSpigelman

    2012-06-01

    Full Text Available Chronic alcohol exposure causes marked changes in reinforcement mechanisms and motivational state that are thought to contribute to the development of cravings and relapse during protracted withdrawal. The nucleus accumbens (NAcc is a key structure of the mesolimbic dopaminergic reward system. Although the NAcc plays an important role in mediating alcohol-seeking behaviors, little is known about the molecular mechanisms underlying alcohol-induced neuroadaptive changes in NAcc function. The aim of this study was to investigate the effects of chronic intermittent ethanol (CIE treatment, a rat model of alcohol withdrawal and dependence, on intrinsic electrical membrane properties and glutamatergic synaptic transmission of medium spiny neurons (MSNs in the NAcc core during protracted withdrawal. We show that CIE treatment followed by prolonged withdrawal increased the inward rectification of MSNs observed at hyperpolarized potentials. In addition, MSNs from CIE-treated animals displayed a lower input resistance, faster action potentials (APs and larger fast afterhyperpolarizations (fAHPs than MSNs from vehicle-treated animals, all suggestive of increases in K+-channel conductances. Significant increases in the Cs+-sensitive inwardly-rectifying K+-current accounted for the increased input resistance, while increases in the A-type K+-current accounted for the faster APs and increased fAHPs in MSNs from CIE rats. We also show that the amplitude and the conductance of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR-mediated mEPSCs were enhanced in CIE-treated animals due to an increase in a small fraction of functional postsynaptic GluA2-lacking AMPARs. These long-lasting modifications of excitability and excitatory synaptic receptor function of MSNs in the NAcc core could play a critical role in the neuroadaptive changes underlying alcohol withdrawal and dependence.

  8. Intracellular accumulation of amyloid-β (Aβ) protein plays a major role in Aβ-induced alterations of glutamatergic synaptic transmission and plasticity.

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    Ripoli, Cristian; Cocco, Sara; Li Puma, Domenica D; Piacentini, Roberto; Mastrodonato, Alessia; Scala, Federico; Puzzo, Daniela; D'Ascenzo, Marcello; Grassi, Claudio

    2014-09-17

    Intracellular accumulation of amyloid-β (Aβ) protein has been proposed as an early event in AD pathogenesis. In patients with mild cognitive impairment, intraneuronal Aβ immunoreactivity was found especially in brain regions critically involved in the cognitive deficits of AD. Although a large body of evidence demonstrates that Aβ42 accumulates intraneuronally ((in)Aβ), the action and the role of Aβ42 buildup on synaptic function have been poorly investigated. Here, we demonstrate that basal synaptic transmission and LTP were markedly depressed following Aβ42 injection into the neuron through the patch pipette. Control experiments performed with the reverse peptide (Aβ42-1) allowed us to exclude that the effects of (in)Aβ depended on changes in oncotic pressure. To further investigate (in)Aβ synaptotoxicity we used an Aβ variant harboring oxidized methionine in position 35 that does not cross the neuronal plasma membrane and is not uploaded from the extracellular space. This Aβ42 variant had no effects on synaptic transmission and plasticity when applied extracellularly, but induced synaptic depression and LTP inhibition after patch-pipette dialysis. Finally, the injection of an antibody raised against human Aβ42 (6E10) in CA1 pyramidal neurons of mouse hippocampal brain slices and autaptic microcultures did not, per se, significantly affect LTP and basal synaptic transmission, but it protected against the toxic effects of extracellular Aβ42. Collectively, these findings suggest that Aβ42-induced impairment of glutamatergic synaptic function depends on its internalization and intracellular accumulation thus paving the way to a systemic proteomic analysis of intracellular targets/partners of Aβ42.

  9. Reversal of novelty-induced hippocampal c-Fos expression in GluA1 subunit-deficient mice by chronic treatment targeting glutamatergic transmission.

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    Maksimovic, Milica; Aitta-aho, Teemu; Korpi, Esa R

    2014-12-15

    Malfunction of glutamate transmission is implicated in several neuropsychiatric disorders. Gria1-/- mouse line with knocked-out GluA1 subunits of ionotropic AMPA glutamate receptor displays several behavioural features of schizoaffective disorder. Typically, these mice show hyperactivity provoked by environmental novelty, which is attenuated after 4-week treatment with the standard mood-stabilisers lithium and valproate and the mood-stabilising anticonvulsants topiramate and lamotrigine (Maksimovic, M., Vekovischeva, O.Y., Aitta-Aho, T., Korpi, E.R., 2014. Chronic treatment with mood-stabilizers attenuates abnormal hyperlocomotion of GluA1-subunit deficient mice. PloS One. 9, e100188). Here, we complement our study by treating these mice chronically with perampanel, a novel non-competitive antagonist of AMPA receptors, for 4 weeks at the dose of 60 mg/kg diet, and found reduced locomotor hyperactivity in the Gria1-/- animals, while not affecting the wild-type littermates. To study the cellular mechanism by which chronic treatments with glutamate-modulating mood-stabilizing drugs alleviate this hyperactivity, we used the immediate early gene c-Fos protein expression as a marker of neuronal activity in the brain. Chronic lithium, valproate and topiramate blunted the c-Fos expression especially in the dorsal hippocampus of the Gria1-/- mice, with all of them reducing the number of c-Fos-positive cells in the CA3 region and valproate and topiramate also in the dentate gyrus (DG). Lamotrigine and perampanel treatments had the same effect in the all CA1, CA3 and DG subfields of the dorsal hippocampus of Gria1-/- mice. The results suggest that abnormal (hippocampal) glutamatergic transmission underlies the hyperactive phenotype of the Gria1-/- mice in a novel environment, and based on the efficacies of the present chronic drug treatments, this mouse model may serve as a predictive tool for studying novel mood-stabilisers.

  10. A subnanomolar concentration of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) pre-synaptically modulates glutamatergic transmission in the rat hippocampus acting through acetylcholine.

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    Pecoraro, Valeria; Sardone, Lara Maria; Chisari, Mariangela; Licata, Flora; Li Volsi, Guido; Perciavalle, Vincenzo; Ciranna, Lucia; Costa, Lara

    2017-01-06

    The neuropeptide PACAP modulates synaptic transmission in the hippocampus exerting multiple effects through different receptor subtypes: the underlying mechanisms have not yet been completely elucidated. The neurotransmitter acetylcholine (ACh) also exerts a well-documented modulation of hippocampal synaptic transmission and plasticity. Since PACAP was shown to stimulate ACh release in the hippocampus, we tested whether PACAP acting through ACh might indirectly modulate glutamate-mediated synaptic transmission at a pre- and/or at a post-synaptic level. Using patch clamp on rat hippocampal slices, we tested PACAP effects on stimulation-evoked AMPA receptor-mediated excitatory post-synaptic currents (EPSCsAMPA) in the CA3-CA1 synapse and on spontaneous miniature EPSCs (mEPSCs) in CA1 pyramidal neurons. A subnanomolar dose of PACAP (0.5nM) decreased EPSCsAMPA amplitude, enhanced EPSC paired-pulse facilitation (PPF) and reduced mEPSC frequency, indicating a pre-synaptic decrease of glutamate release probability: these effects were abolished by simultaneous blockade of muscarinic and nicotinic ACh receptors, indicating the involvement of endogenous ACh. The effect of subnanomolar PACAP was abolished by a PAC1 receptor antagonist but not by a VPAC receptor blocker. At a higher concentration (10nM), PACAP inhibited EPSCsAMPA: this effect persisted in the presence of ACh receptor antagonists and did not involve any change in PPF or in mEPSC frequency, thus was not mediated by ACh and was exerted post- synaptically on CA1 pyramidal neurons. We suggest that a high-affinity PAC1 receptor pre-synaptically modulates hippocampal glutamatergic transmission acting through ACh. Therefore, administration of PACAP at very low doses might be envisaged in cognitive diseases with reduced cholinergic transmission.

  11. Zingerone enhances glutamatergic spontaneous excitatory transmission by activating TRPA1 but not TRPV1 channels in the adult rat substantia gelatinosa.

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    Yue, Hai-Yuan; Jiang, Chang-Yu; Fujita, Tsugumi; Kumamoto, Eiichi

    2013-08-01

    Transient receptor potential (TRP) channels are thought to play a role in regulating nociceptive transmission to spinal substantia gelatinosa (SG) neurons. It remains to be unveiled whether the TRP channels in the central nervous system are different in property from those involved in receiving nociceptive stimuli in the peripheral nervous system. We examined the effect of the vanilloid compound zingerone, which activates TRPV1 channels in the cell body of a primary afferent neuron, on glutamatergic excitatory transmission in the SG neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. Bath-applied zingerone reversibly and concentration-dependently increased spontaneous excitatory postsynaptic current (EPSC) frequency. This effect was accompanied by an inward current at -70 mV that was resistant to glutamate receptor antagonists. These zingerone effects were repeated and persisted in Na(+)-channel blocker tetrodotoxin-, La(3+)-, or IP3-induced Ca(2+)-release inhibitor 2-aminoethoxydiphenyl borate-containing or Ca(2+)-free Krebs solution. Zingerone activity was resistant to the selective TRPV1 antagonist capsazepine but sensitive to the nonselective TRP antagonist ruthenium red, the TRPA1 antagonist HC-030031, and the Ca(2+)-induced Ca(2+)-release inhibitor dantrolene. TRPA1 agonist allyl isothiocyanate but not capsaicin inhibited the facilitatory effect of zingerone. On the other hand, zingerone reduced monosynaptically evoked EPSC amplitudes, as did TRPA1 agonists. Like allyl isothiocyanate, zingerone enhanced GABAergic spontaneous inhibitory transmission in a manner sensitive to tetrodotoxin. We conclude that zingerone presynaptically facilitates spontaneous excitatory transmission, probably through Ca(2+)-induced Ca(2+)-release mechanisms, and produces a membrane depolarization in SG neurons by activating TRPA1 but not TRPV1 channels.

  12. Activation of CRH receptor type 1 expressed on glutamatergic neurons increases excitability of CA1 pyramidal neurons by the modulation of voltage-gated ion channels

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    Stephan eKratzer

    2013-07-01

    Full Text Available Corticotropin-releasing hormone (CRH plays an important role in a substantial number of patients with stress-related mental disorders, such as anxiety disorders and depression. CRH has been shown to increase neuronal excitability in the hippocampus, but the underlying mechanisms are poorly understood. The effects of CRH on neuronal excitability were investigated in acute hippocampal brain slices. Population spikes (PS and field excitatory postsynaptic potentials (fEPSP were evoked by stimulating Schaffer-collaterals and recorded simultaneously from the somatic and dendritic region of CA1 pyramidal neurons. CRH was found to increase PS amplitudes (mean  Standard error of the mean; 231.8  31.2% of control; n=10 while neither affecting fEPSPs (104.3 ± 4.2%; n=10 nor long-term potentiation (LTP. However, when Schaffer-collaterals were excited via action potentials (APs generated by stimulation of CA3 pyramidal neurons, CRH increased fEPSP amplitudes (119.8 ± 3.6%; n=8 and the magnitude of LTP in the CA1 region. Experiments in slices from transgenic mice revealed that the effect on PS amplitude is mediated exclusively by CRH receptor 1 (CRHR1 expressed on glutamatergic neurons. The effects of CRH on PS were dependent on phosphatase-2B, L- and T-type calcium channels and voltage-gated potassium channels but independent on intracellular Ca2+-elevation. In patch-clamp experiments, CRH increased the frequency and decay times of APs and decreased currents through A-type and delayed-rectifier potassium channels. These results suggest that CRH does not affect synaptic transmission per se, but modulates voltage-gated ion currents important for the generation of APs and hence elevates by this route overall neuronal activity.

  13. Atorvastatin Prevents Cognitive Deficits Induced by Intracerebroventricular Amyloid-β1-40 Administration in Mice: Involvement of Glutamatergic and Antioxidant Systems.

    Science.gov (United States)

    Martins, Wagner C; dos Santos, Vanessa Valgas; dos Santos, Alessandra Antunes; Vandresen-Filho, Samuel; Dal-Cim, Tharine A; de Oliveira, Karen A; Mendes-de-Aguiar, Claudia B N; Farina, Marcelo; Prediger, Rui Daniel; Viola, Giordano Gubert; Tasca, Carla I

    2015-07-01

    Deposition of amyloid-β (Aβ) peptides into specific encephalic structures has been pointed as an important event related to Alzheimer's disease pathogenesis and associated with activation of glial cells, neuroinflammation, oxidative responses, and cognitive deficits. Aβ-induced pro-oxidative damage may regulate the activity of glutamate transporters, leading to reduced glutamate uptake and, as a consequence, excitotoxic events. Herein, we evaluated the effects of the pretreatment of atorvastatin, a HMG-CoA reductase inhibitor, on behavioral and biochemical alterations induced by a single intracerebroventricular (i.c.v.) injection of aggregated Aβ1-40 in mice. Atorvastatin (10 mg/kg/day, p.o.) was administered through seven consecutive days before Aβ1-40 administration. Aβ1-40 caused significant cognitive impairment in the object-place recognition task (2 weeks after the i.c.v. injection) and this phenomenon was abolished by atorvastatin pretreatment. Ex vivo evaluation of glutamate uptake into hippocampal and cerebral cortices slices showed atorvastatin, and Aβ1-40 decreased hippocampal and cortical Na(+)-dependent glutamate uptake. However, Aβ1-40 increased Na(+)-independent glutamate uptake and it was prevented by atorvastatin in prefrontal cortex slices. Moreover, Aβ1-40 treatment significantly increased the cerebrocortical activities of glutathione reductase and glutathione peroxidase and these events were blunted by atorvastatin pretreatment. Reduced or oxidized glutathione levels were not altered by Aβ1-40 and/or atorvastatin treatment. These results extend the notion of the protective action of atorvastatin against neuronal toxicity induced by Aβ1-40 demonstrating that a pretreatment with atorvastatin prevents the spatial learning and memory deficits induced by Aβ in rodents and promotes changes in glutamatergic and antioxidant systems mainly in prefrontal cortex.

  14. Chronic Treatment with a Clinically Relevant Dose of Methylphenidate Increases Glutamate Levels in Cerebrospinal Fluid and Impairs Glutamatergic Homeostasis in Prefrontal Cortex of Juvenile Rats.

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    Schmitz, Felipe; Pierozan, Paula; Rodrigues, André F; Biasibetti, Helena; Coelho, Daniella M; Mussulini, Ben Hur; Pereira, Mery S L; Parisi, Mariana M; Barbé-Tuana, Florencia; de Oliveira, Diogo L; Vargas, Carmen R; Wyse, Angela T S

    2016-05-01

    The understanding of the consequences of chronic treatment with methylphenidate is very important since this psychostimulant is extensively prescribed to preschool age children, and little is known about the mechanisms underlying the persistent changes in behavior and neuronal function related with the use of methylphenidate. In this study, we initially investigate the effect of early chronic treatment with methylphenidate on amino acids profile in cerebrospinal fluid and prefrontal cortex of juvenile rats, as well as on glutamatergic homeostasis, Na(+),K(+)-ATPase function, and balance redox in prefrontal cortex of rats. Wistar rats at early age received intraperitoneal injections of methylphenidate (2.0 mg/kg) or an equivalent volume of 0.9% saline solution (controls), once a day, from the 15th to the 45th day of age. Twenty-four hours after the last injection, the animals were decapitated and the cerebrospinal fluid and prefrontal cortex were obtained. Results showed that methylphenidate altered amino acid profile in cerebrospinal fluid, increasing the levels of glutamate. Glutamate uptake was decreased by methylphenidate administration, but GLAST and GLT-1 were not altered by this treatment. In addition, the astrocyte marker GFAP was not altered by MPH. The activity and immunocontent of catalytic subunits (α1, α2, and α3) of Na(+),K(+)-ATPase were decreased in prefrontal cortex of rats subjected to methylphenidate treatment, as well as changes in α1 and α2 gene expression of catalytic α subunits of Na(+),K(+)-ATPase were also observed. CAT activity was increased and SOD/CAT ratio and sulfhydryl content were decreased in rat prefrontal cortex. Taken together, our results suggest that chronic treatment with methylphenidate at early age induces excitotoxicity, at least in part, due to inhibition of glutamate uptake probably caused by disturbances in the Na(+),K(+)-ATPase function and/or in protein damage observed in the prefrontal cortex.

  15. Role of glutamatergic receptors located in the nucleus raphe magnus on antinociceptive effect of morphine microinjected into the nucleus cuneiformis of rat.

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    Haghparast, Abbas; Soltani-Hekmat, Ava; Khani, Abbas; Komaki, Alireza

    2007-10-29

    Neurons in the nucleus cuneiformis (CnF), located just ventrolateral to the periaqueductal gray, project to medullary nucleus raphe magnus (NRM), which is a key medullary relay for descending pain modulation and is critically involved in opioid-induced analgesia. Previous studies have shown that antinociceptive response of CnF-microinjected morphine can be modulated by the specific subtypes of glutamatergic receptors within the CnF. In this study, we evaluated the role of NMDA and kainate/AMPA receptors that are widely distributed within the NRM on morphine-induced antinociception elicited from the CnF. Hundred and five male Wistar rats weighing 250-300 g were used. Morphine (10, 20 and 40 microg) and NMDA receptor antagonist, MK-801 (10 microg) or kainate/AMPA receptor antagonist, DNQX (0.5 microg) in 0.5 microl saline were stereotaxically microinjected into the CnF and NRM, respectively. The latency of tail-flick response was measured at set intervals (2, 7, 12, 17, 22, 27 min after microinjection) by using an automated tail-flick analgesiometer. The results showed that morphine microinjection into the CnF dose-dependently causes increase in tail-flick latency (TFL). MK-801 microinjected into the NRM, just 1 min before morphine injection into the CnF, significantly attenuated antinociceptive effects of morphine. On the other hand, DNQX microinjected into the NRM, significantly increased TFL after local application of morphine into the CnF. We suggest that morphine related antinociceptive effect elicited from the CnF is mediated, in part, by NMDA receptor at the level of the NRM whereas kainite/AMPA receptor has a net inhibitory influence at the same pathway.

  16. Impact of subanesthetic doses of ketamine on AMPA-mediated responses in rats: An in vivo electrophysiological study on monoaminergic and glutamatergic neurons

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    El Iskandrani, Kareem S; Oosterhof, Chris A; Blier, Pierre

    2015-01-01

    The rapid antidepressant action of a subanesthetic dose of ketamine in treatment-resistant patients represents the most striking recent breakthrough in the understanding of the antidepressant response. Evidence demonstrates tight interactions between the glutamatergic and monoaminergic systems. It is thus hypothesized that monoamine systems may play a role in the immediate/rapid effects of ketamine. In vivo electrophysiological recordings were carried in male rats following ketamine administration (10 and 25 mg/kg, i.p.) to first assess its effects on monoaminergic neuron firing. In a second series of experiments, the effects of ketamine administration on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and N-methyl-D-aspartate receptor (NMDA)-evoked responses in hippocampus CA3 pyramidal neurons were also investigated using micro-iontophoretic applications. Although acute (~2 hours) ketamine administration did not affect the mean firing activity of dorsal raphe serotonin and ventral tegmental area dopamine neurons, it did increase that of locus coeruleus norepinephrine neurons. In the latter brain region, while ketamine also enhanced bursting activity, it did increase population activity of dopamine neurons in the ventral tegmental area. These effects of ketamine were prevented by the prior administration of the AMPA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide. An increase in AMPA-evoked response of CA3 pyramidal neurons was also observed 30 minutes following acute ketamine administration. The present findings suggest that acute ketamine administration produces a rapid enhancement of catecholaminergic neurons firing activity through an amplification of AMPA transmission. These effects may play a crucial role in the antidepressant effects of ketamine observed shortly following its infusion in depressed patients. PMID:25759403

  17. Severe depression is associated with increased microglial quinolinic acid in subregions of the anterior cingulate gyrus: Evidence for an immune-modulated glutamatergic neurotransmission?

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    Mawrin Christian

    2011-08-01

    Full Text Available Abstract Background Immune dysfunction, including monocytosis and increased blood levels of interleukin-1, interleukin-6 and tumour necrosis factor α has been observed during acute episodes of major depression. These peripheral immune processes may be accompanied by microglial activation in subregions of the anterior cingulate cortex where depression-associated alterations of glutamatergic neurotransmission have been described. Methods Microglial immunoreactivity of the N-methyl-D-aspartate (NMDA glutamate receptor agonist quinolinic acid (QUIN in the subgenual anterior cingulate cortex (sACC, anterior midcingulate cortex (aMCC and pregenual anterior cingulate cortex (pACC of 12 acutely depressed suicidal patients (major depressive disorder/MDD, n = 7; bipolar disorder/BD, n = 5 was analyzed using immunohistochemistry and compared with its expression in 10 healthy control subjects. Results Depressed patients had a significantly increased density of QUIN-positive cells in the sACC (P = 0.003 and the aMCC (P = 0.015 compared to controls. In contrast, counts of QUIN-positive cells in the pACC did not differ between the groups (P = 0.558. Post-hoc tests showed that significant findings were attributed to MDD and were absent in BD. Conclusions These results add a novel link to the immune hypothesis of depression by providing evidence for an upregulation of microglial QUIN in brain regions known to be responsive to infusion of NMDA antagonists such as ketamine. Further work in this area could lead to a greater understanding of the pathophysiology of depressive disorders and pave the way for novel NMDA receptor therapies or immune-modulating strategies.

  18. Optical mapping reveals developmental dynamics of Mg2+-/APV-sensitive components of glossopharyngeal glutamatergic EPSPs in the embryonic chick NTS.

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    Sato, Katsushige; Momose-Sato, Yoko

    2004-10-01

    To examine whether there are any differences in functional organization between the glossopharyngeal nerve (N. IX)- and vagus nerve (N. X)-projecting areas in the nucleus of the tractus solitarius (NTS), we performed optical recording of neural responses evoked by N. IX stimulation in 5- to 9-day-old embryonic chick brain stem preparations and compared the results with those in our previous studies concerning the N. X-related NTS. First, we investigated DL-2-amino-5-phosphonovaleric acid (APV)/Mg2+ sensitivity of the glutamatergic excitatory postsynaptic potentials (EPSPs) in the N. IX-related NTS. In 7- to 9-day-old preparations, we found regional differences in the degree of both the APV-induced reduction and Mg2+-free-induced enhancement of the EPSPs. We constructed developmental maps of spatial patterns of the APV- and Mg2+-sensitive components and showed that functional expression of the N-methyl-D-aspartate (NMDA) receptor dynamically changed during development. Second, we studied initial expression of synaptic functions in the N. IX-related NTS. In 6-day-old preparations, although action potentials alone were usually detected in normal Ringer solution, small EPSPs were elicited in a Mg2+-free solution. This result suggests that the NMDA receptor-mediated synaptic function is latently generated in the N. IX-related NTS at the 6-day-old embryonic stage and that external Mg2+ regulates the onset of synaptic functions. Developmental patterns of APV/Mg2+ sensitivity and the stage of initial expression of the glossopharyngeal EPSP were similar to those of the N. X, suggesting that the developmental sequence of the synaptic function in the NTS is the same for the N. IX- and N. X-related NTS.

  19. Changes in sensitivity of reward and motor behavior to dopaminergic, glutamatergic, and cholinergic drugs in a mouse model of fragile X syndrome.

    Directory of Open Access Journals (Sweden)

    Eric W Fish

    Full Text Available Fragile X syndrome (FXS is a leading cause of intellectual disability. FXS is caused by loss of function of the FMR1 gene, and mice in which Fmr1 has been inactivated have been used extensively as a preclinical model for FXS. We investigated the behavioral pharmacology of drugs acting through dopaminergic, glutamatergic, and cholinergic systems in fragile X (Fmr1 (-/Y mice with intracranial self-stimulation (ICSS and locomotor activity measurements. We also measured brain expression of tyrosine hydroxylase (TH, the rate-limiting enzyme in dopamine biosynthesis. Fmr1 (-/Y mice were more sensitive than wild type mice to the rewarding effects of cocaine, but less sensitive to its locomotor stimulating effects. Anhedonic but not motor depressant effects of the atypical neuroleptic, aripiprazole, were reduced in Fmr1 (-/Y mice. The mGluR5-selective antagonist, 6-methyl-2-(phenylethynylpyridine (MPEP, was more rewarding and the preferential M1 antagonist, trihexyphenidyl, was less rewarding in Fmr1 (-/Y than wild type mice. Motor stimulation by MPEP was unchanged, but stimulation by trihexyphenidyl was markedly increased, in Fmr1 (-/Y mice. Numbers of midbrain TH+ neurons in the ventral tegmental area were unchanged, but were lower in the substantia nigra of Fmr1 (-/Y mice, although no changes in TH levels were found in their forebrain targets. The data are discussed in the context of known changes in the synaptic physiology and pharmacology of limbic motor systems in the Fmr1 (-/Y mouse model. Preclinical findings suggest that drugs acting through multiple neurotransmitter systems may be necessary to fully address abnormal behaviors in individuals with FXS.

  20. Transcriptional evidence for the role of chronic venlafaxine treatment in neurotrophic signaling and neuroplasticity including also Glutamatergic [corrected] - and insulin-mediated neuronal processes.

    Directory of Open Access Journals (Sweden)

    Viola Tamási

    Full Text Available Venlafaxine (VLX, a serotonine-noradrenaline reuptake inhibitor, is one of the most commonly used antidepressant drugs in clinical practice for the treatment of major depressive disorder (MDD. Despite being more potent than its predecessors, similarly to them, the therapeutical effect of VLX is visible only 3-4 weeks after the beginning of treatment. Furthermore, recent papers show that antidepressants, including also VLX, enhance the motor recovery after stroke even in non depressed persons. In the present, transcriptomic-based study we looked for changes in gene expressions after a long-term VLX administration.Osmotic minipumps were implanted subcutaneously into Dark Agouti rats providing a continuous (40 mg/kg/day VLX delivery for three weeks. Frontal regions of the cerebral cortex were isolated and analyzed using Illumina bead arrays to detect genes showing significant chances in expression. Gene set enrichment analysis was performed to identify specific regulatory networks significantly affected by long term VLX treatment.Chronic VLX administration may have an effect on neurotransmitter release via the regulation of genes involved in vesicular exocytosis and receptor endocytosis (such as Kif proteins, Myo5a, Sv2b, Syn2 or Synj2. Simultaneously, VLX activated the expression of genes involved in neurotrophic signaling (Ntrk2, Ntrk3, glutamatergic transmission (Gria3, Grin2b and Grin2a, neuroplasticity (Camk2g/b, Cd47, synaptogenesis (Epha5a, Gad2 and cognitive processes (Clstn2. Interestingly, VLX increased the expression of genes involved in mitochondrial antioxidant activity (Bcl2 and Prdx1. Additionally, VLX administration also modulated genes related to insulin signaling pathway (Negr1, Ppp3r1, Slc2a4 and Enpp1, a mechanism that has recently been linked to neuroprotection, learning and memory.Our results strongly suggest that chronic VLX treatment improves functional reorganization and brain plasticity by influencing gene expression in

  1. SLC6A15, a novel stress vulnerability candidate, modulates anxiety and depressive-like behavior: involvement of the glutamatergic system.

    Science.gov (United States)

    Santarelli, S; Wagner, K V; Labermaier, C; Uribe, A; Dournes, C; Balsevich, G; Hartmann, J; Masana, M; Holsboer, F; Chen, A; Müller, M B; Schmidt, M V

    2016-01-01

    Major depression is a multifactorial disease, involving both environmental and genetic risk factors. Recently, SLC6A15 - a neutral amino acid transporter mainly expressed in neurons - was proposed as a new candidate gene for major depression and stress vulnerability. Risk allele carriers for a single nucleotide polymorphism (SNP) in a SLC6A15 regulatory region display altered hippocampal volume, glutamate levels, and hypothalamus-pituitary-adrenal axis activity, all markers associated with major depression. Despite this genetic link between SLC6A15 and depression, its functional role with regard to the development and maintenance of depressive disorder is still unclear. The aim of the current study was therefore to characterize the role of mouse slc6a15 in modulating brain function and behavior, especially in relation to stress as a key risk factor for the development of mood disorders. We investigated the effects of slc6a15 manipulation using two mouse models, a conventional slc6a15 knock-out mouse line (SLC-KO) and a virus-mediated hippocampal slc6a15 overexpression (SLC-OE) model. Mice were tested under basal conditions and following chronic social stress. We found that SLC-KO animals displayed a similar behavioral profile to wild-type littermates (SLC-WT) under basal conditions. Interestingly, following chronic social stress SLC-KO animals showed lower levels of anxiety- and depressive-like behavior compared to stressed WT littermates. In support of these findings, SLC-OE animals displayed increased anxiety-like behavior already under basal condition. We also provide evidence that GluR1 expression in the dentate gyrus, but not GluR2 or NR1, are regulated by slc6a15 expression, and may contribute to the difference in stress responsiveness observed between SLC-KO and SLC-WT animals. Taken together, our data demonstrate that slc6a15 plays a role in modulating emotional behavior, possibly mediated by its impact on glutamatergic neurotransmission.

  2. Plasticity in glutamatergic NTS neurotransmission.

    Science.gov (United States)

    Kline, David D

    2008-12-10

    Changes in the physiological state of an animal or human can result in alterations in the cardiovascular and respiratory system in order to maintain homeostasis. Accordingly, the cardiovascular and respiratory systems are not static but readily adapt under a variety of circumstances. The same can be said for the brainstem circuits that control these systems. The nucleus tractus solitarius (NTS) is the central integration site of baroreceptor and chemoreceptor sensory afferent fibers. This central nucleus, and in particular the synapse between the sensory afferent and second-order NTS cell, possesses a remarkable degree of plasticity in response to a variety of stimuli, both acute and chronic. This brief review is intended to describe the plasticity observed in the NTS as well as the locus and mechanisms as they are currently understood. The functional consequence of NTS plasticity is also discussed.

  3. Knockdown of Myo-Inositol Transporter SMIT1 Normalizes Cholinergic and Glutamatergic Function in an Immortalized Cell Line Established from the Cerebral Cortex of a Trisomy 16 Fetal Mouse, an Animal Model of Human Trisomy 21 (Down Syndrome).

    Science.gov (United States)

    Cárdenas, Ana María; Fernández-Olivares, Paola; Díaz-Franulic, Ignacio; González-Jamett, Arlek M; Shimahara, Takeshi; Segura-Aguilar, Juan; Caviedes, Raúl; Caviedes, Pablo

    2017-07-10

    The Na(+)/myo-inositol cotransporter (SMIT1) is overexpressed in human Down syndrome (DS) and in trisomy 16 fetal mice (Ts16), an animal model of the human condition. SMIT1 overexpression determines increased levels of intracellular myo-inositol, a precursor of phophoinositide synthesis. SMIT1 is overexpressed in CTb cells, an immortalized cell line established from the cerebral cortex of a Ts16 mouse fetus. CTb cells exhibit impaired cytosolic Ca(2+) signals in response to glutamatergic and cholinergic stimuli (increased amplitude and delayed time-dependent kinetics in the decay post-stimulation), compared to our CNh cell line, derived from the cerebral cortex of a euploid animal. Considering the role of myo-inositol in intracellular signaling, we normalized SMIT1 expression in CTb cells using specific mRNA antisenses. Forty-eight hours post-transfection, SMIT1 levels in CTb cells reached values comparable to those of CNh cells. At this time, decay kinetics of Ca(2+) signals induced by either glutamate, nicotine, or muscarine were accelerated in transfected CTb cells, to values similar to those of CNh cells. The amplitude of glutamate-induced cytosolic Ca(2+) signals in CTb cells was also normalized. The results suggest that SMIT1 overexpression contributes to abnormal cholinergic and glutamatergic Ca(2+) signals in the trisomic condition, and knockdown of DS-related genes in our Ts16-derived cell line could constitute a relevant tool to study DS-related neuronal dysfunction.

  4. Longitudinal RNA sequencing of the deep transcriptome during neurogenesis of cortical glutamatergic neurons from murine ESCs [v1; ref status: indexed, http://f1000r.es/w2

    Directory of Open Access Journals (Sweden)

    Kyle S Hubbard

    2013-02-01

    Full Text Available Using paired-end RNA sequencing, we have quantified the deep transcriptional changes that occur during differentiation of murine embryonic stem cells into a highly enriched population of glutamatergic cortical neurons. These data provide a detailed and nuanced account of longitudinal changes in the transcriptome during neurogenesis and neuronal maturation, starting from mouse embryonic stem cells and progressing through neuroepithelial stem cell induction, radial glial cell formation, neurogenesis, neuronal maturation and cortical patterning. Understanding the transcriptional mechanisms underlying the differentiation of stem cells into mature, glutamatergic neurons of cortical identity has myriad applications, including the elucidation of mechanisms of cortical patterning; identification of neurogenic processes; modeling of disease states; detailing of the host cell response to neurotoxic stimuli; and determination of potential therapeutic targets. In future work we anticipate correlating changes in longitudinal gene expression to other cell parameters, including neuronal function as well as characterizations of the proteome and metabolome. In this data article, we describe the methods used to produce the data and present the raw sequence read data in FASTQ files, sequencing run statistics and a summary flatfile of raw counts for 22,164 genes across 31 samples, representing 3-5 biological replicates at each timepoint. We propose that this data will be a valuable contribution to diverse research efforts in bioinformatics, stem cell research and developmental neuroscience studies.

  5. NMDA and AMPA/kainate glutamatergic receptors in the prelimbic medial prefrontal cortex modulate the elaborated defensive behavior and innate fear-induced antinociception elicited by GABAA receptor blockade in the medial hypothalamus.

    Science.gov (United States)

    de Freitas, Renato Leonardo; Salgado-Rohner, Carlos José; Biagioni, Audrey Francisco; Medeiros, Priscila; Hallak, Jaime Eduardo Cecílio; Crippa, José Alexandre S; Coimbra, Norberto Cysne

    2014-06-01

    The aim of the present study was to investigate the involvement of N-methyl-d-aspartate (NMDA) and amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptors of the prelimbic (PL) division of the medial prefrontal cortex (MPFC) on the panic attack-like reactions evoked by γ-aminobutyric acid-A receptor blockade in the medial hypothalamus (MH). Rats were pretreated with NaCl 0.9%, LY235959 (NMDA receptor antagonist), and NBQX (AMPA/kainate receptor antagonist) in the PL at 3 different concentrations. Ten minutes later, the MH was treated with bicuculline, and the defensive responses were recorded for 10 min. The antagonism of NMDA receptors in the PL decreased the frequency and duration of all defensive behaviors evoked by the stimulation of the MH and reduced the innate fear-induced antinociception. However, the pretreatment of the PL cortex with NBQX was able to decrease only part of defensive responses and innate fear-induced antinociception. The present findings suggest that the NMDA-glutamatergic system of the PL is critically involved in panic-like responses and innate fear-induced antinociception and those AMPA/kainate receptors are also recruited during the elaboration of fear-induced antinociception and in panic attack-related response. The activation of the glutamatergic neurotransmission of PL division of the MPFC during the elaboration of oriented behavioral reactions elicited by the chemical stimulation of the MH recruits mainly NMDA receptors in comparison with AMPA/kainate receptors.

  6. Glucagon-like peptide-1 excites firing and increases GABAergic miniature postsynaptic currents (mPSCs in gonadotropin-releasing hormone (GnRH neurons of the male mice via activation of nitric oxide (NO and suppression of endocannabinoid signaling pathways

    Directory of Open Access Journals (Sweden)

    Imre Farkas

    2016-09-01

    Full Text Available Glucagon-like peptide-1 (GLP-1, a metabolic signal molecule, regulates reproduction, although, the involved molecular mechanisms have not been elucidated, yet. Therefore, responsiveness of gonadotropin-releasing hormone (GnRH neurons to the GLP-1 analog Exendin-4 and elucidation of molecular pathways acting downstream to the GLP-1 receptor (GLP-1R have been challenged. Loose patch-clamp recordings revealed that Exendin-4 (100 nM–5 μM elevated firing rate in hypothalamic GnRH-GFP neurons of male mice via activation of GLP-1R. Whole-cell patch-clamp measurements demonstrated increased excitatory GABAergic miniature postsynaptic currents (mPSCs frequency after Exendin-4 administration, which was eliminated by the GLP-1R antagonist Exendin-3(9-39 (1 μM. Intracellular application of the G-protein inhibitor GDP-beta-S (2 mM impeded action of Exendin-4 on mPSCs, suggesting direct excitatory action of GLP-1 on GnRH neurons. Blockade of nitric-oxide (NO synthesis by L-NAME (100 μM or NPLA (1 μM or intracellular scavenging of NO by CPTIO (1 mM partially attenuated the excitatory effect of Exendin-4. Similar partial inhibition was achieved by hindering endocannabinoid pathway using CB1 inverse-agonist AM251 (1 μM. Simultaneous blockade of NO and endocannabinoid signaling mechanisms eliminated action of Exendin-4 suggesting involvement of both retrograde machineries. Intracellular application of the TRPV1-antagonist AMG9810 (10 μM or the FAAH-inhibitor PF3845 (5 μM impeded the GLP-1-triggered endocannabinoid pathway indicating an anandamide-TRPV1-sensitive control of 2-AG production. Furthermore, GLP-1 immunoreactive axons innervated GnRH neurons in the hypothalamus suggesting that GLP-1 of both peripheral and neuronal sources can modulate GnRH neurons. RT-qPCR study confirmed the expression of GLP-1R and nNOS mRNAs in GnRH-GFP neurons. Immuno-electron microscopic analysis revealed the presence of neuronal nitric oxide synthase (nNOS protein in Gn

  7. 基于多能干细胞的Alport综合征的microRNA差异性表达与碱基编辑功能分析%Identification of different expression microRNA and base edit function in Alport syndrome base on iPSCs

    Institute of Scientific and Technical Information of China (English)

    陈文标; 彭武建; 林小聪; 戴勇

    2014-01-01

    目的 分析Alport综合征(AS)的诱导多能干细胞(iPSCs)全基因微小核糖核酸(microRNA)的表达谱,筛选出具有差异性表达的microRNA与发生碱基突变的microRNA.方法 收集1例AS患者与1例健康人的尿液,从尿液中分离尿肾脏管细胞,尿肾脏管细胞分化诱导成iPSCs.运用高通量测序方法对iPSCs的microRNA进行测序与表达量测定,比较分析两人microRNA的表达差异,寻找具有特异性的microRNA靶点.同时,将mi-croRNA序列与miRBase数据库(http://www.mirbase.org/)中已知成熟microRNA序列进行比对,比较两标本mi-croRNA碱基编辑数量,找出发生了碱基突变的microRNA.结果 在两人标本中发现30个microRNA具有显著的差异性表达,其中19个microRNAs表达上调,11个microRNA表达下调.has-miR-3117-3p的倍比值(log2 Ratio)为7.79,在表达上调的microRNA最具有特异性;has-miR-544b的倍比值为-9.09,在表达下调的microRNA最具有特异性.同时在两标本中共有208个microRNA发生了碱基突变,其中104个microRNA,AS患者发生碱基编辑概率比健康人大;77个microRNA,健康人发生碱基编辑概率比AS患者大.结论 AS患者与健康人肾脏组织中的microRNA存在差异性,AS发生碱基编辑引起基因突变的microRNA比健康人更常见.这些差异性表达的microRNAs与microRNA碱基突变的概率可能在AS发病机制中起重要作用.

  8. Synaptic and molecular mechanisms of glutamatergic synapses in pain and memory%谷氨酸性突触在痛觉和记忆中的突触和分子机制

    Institute of Scientific and Technical Information of China (English)

    卓敏

    2003-01-01

    谷氨酸是哺乳动物脑中的兴奋性递质.中枢神经系统的谷氨酸性突触广泛参与痛觉传递, 突触可塑性和递质的调节.谷氨酸的NMDA受体参与前脑相关的学习及功能.在这篇综述中, 我们提出前脑的NMDA受体通过增强谷氨酸性突触传递导致长期性的炎痛.具有增强NMDA受体功能的小鼠会产生更多的慢性痛.NMDA NR2B受体抑制剂在未来可能被用来控制人类的慢性痛.%Glutamate is a fast excitatory transmitter in mammalian brains. Glutamatergic synapses are found in central regions related to pain transmission, plasticity and modulation. Glutamate NMDA receptors in forebrain structures are well known to contribute to the formation and storage of information. Here we propose the hypothesis that forebrain NMDA receptors play an important role in persistent inflammatory pain by re-enforcing glutamate sensory transmission in the brain. Mice with enhanced function of forebrain NMDA receptors demonstrate selective enhancement of persistent pain and allodynia. Drugs targeting forebrain NMDA NR2B receptors may serve as a new class of medicine to control persistent pain in humans.

  9. Prenatal exposure to the CB1 and CB2 cannabinoid receptor agonist WIN 55,212-2 alters migration of early-born glutamatergic neurons and GABAergic interneurons in the rat cerebral cortex.

    Science.gov (United States)

    Saez, Trinidad M M; Aronne, María P; Caltana, Laura; Brusco, Alicia H

    2014-05-01

    The endocannabinoid system, composed of cannabinoid receptors, endocannabinoids, and synthesis and degradation enzymes, is present since early stages of brain development. During this period, the endocannabinoid system is involved in the regulation of neural progenitor proliferation and specification as well as the migration and differentiation of pyramidal neurons and interneurons. Marijuana consumption during pregnancy represents a serious risk in relation to the fetal brain development since Δ(9) -tetrahidrocannabinol, the main active compound of cannabis, can reach the fetus through placenta and hemato-encephalic barrier. Cohort studies performed on children and adolescents of mothers who consumed marijuana during pregnancy reported cognitive and comportamental abnormalities. In the present study, we examined the expression of the cannabinoid receptor CB1 R during corticogenesis in radially and tangentially migrating post-mitotic neurons. We found that prenatal exposure to WIN impaired tangential and radial migration of post-mitotic neurons in the dorsal pallium. In addition, we described alterations of two transcription factors associated with proliferating and newly post-mitotic glutamatergic cells in the dorsal pallium, Tbr1 and Tbr2, and disruption in the number of Cajal-Retzius cells. The present results contribute to the knowledge of neurobiological substrates that determine neuro-comportamental changes that will persist through post-natal life.

  10. Glucagon-Like Peptide-1 Excites Firing and Increases GABAergic Miniature Postsynaptic Currents (mPSCs) in Gonadotropin-Releasing Hormone (GnRH) Neurons of the Male Mice via Activation of Nitric Oxide (NO) and Suppression of Endocannabinoid Signaling Pathways

    Science.gov (United States)

    Farkas, Imre; Vastagh, Csaba; Farkas, Erzsébet; Bálint, Flóra; Skrapits, Katalin; Hrabovszky, Erik; Fekete, Csaba; Liposits, Zsolt

    2016-01-01

    Glucagon-like peptide-1 (GLP-1), a metabolic signal molecule, regulates reproduction, although, the involved molecular mechanisms have not been elucidated, yet. Therefore, responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the GLP-1 analog Exendin-4 and elucidation of molecular pathways acting downstream to the GLP-1 receptor (GLP-1R) have been challenged. Loose patch-clamp recordings revealed that Exendin-4 (100 nM–5 μM) elevated firing rate in hypothalamic GnRH-GFP neurons of male mice via activation of GLP-1R. Whole-cell patch-clamp measurements demonstrated increased excitatory GABAergic miniature postsynaptic currents (mPSCs) frequency after Exendin-4 administration, which was eliminated by the GLP-1R antagonist Exendin-3(9–39) (1 μM). Intracellular application of the G-protein inhibitor GDP-β-S (2 mM) impeded action of Exendin-4 on mPSCs, suggesting direct excitatory action of GLP-1 on GnRH neurons. Blockade of nitric-oxide (NO) synthesis by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 100 μM) or N5-[Imino(propylamino)methyl]-L-ornithine hydrochloride (NPLA; 1 μM) or intracellular scavenging of NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO; 1 mM) partially attenuated the excitatory effect of Exendin-4. Similar partial inhibition was achieved by hindering endocannabinoid pathway using cannabinoid receptor type-1 (CB1) inverse-agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl) pyrazole-3-carboxamide (AM251; 1 μM). Simultaneous blockade of NO and endocannabinoid signaling mechanisms eliminated action of Exendin-4 suggesting involvement of both retrograde machineries. Intracellular application of the transient receptor potential vanilloid 1 (TRPV1)-antagonist 2E-N-(2, 3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1, 1-dimethylethyl)phenyl]-2-Propenamide (AMG9810; 10 μM) or the fatty acid amide hydrolase (FAAH)-inhibitor PF3845 (5 μM) impeded the GLP-1-triggered endocannabinoid

  11. 琥珀酸对幼龄大鼠小脑谷氨酸能突触传递的抑制作用%Inhibition of succinic acid on cerebellar glutamatergic synaptic transmission in neonatal rats

    Institute of Scientific and Technical Information of China (English)

    何海燕; 陈静; 晋芙丽; 李凌; 杜永平; 张月萍

    2016-01-01

    目的:探讨琥珀酸( succinic acid,SA)对幼龄大鼠小脑谷氨酸能突触传递的影响。方法采用全细胞膜片钳记录法,在矢状位小脑脑片上记录浦肯野细胞( Purkinje cells,PCs)自发性微小兴奋性突触后电流( miniture excitatory postsynaptic current,mEPSC)和刺激平行纤维( parallel fibre,PF)诱发的PCs兴奋性突触后电位( excitatory postsynaptic potential,EPSP),比较琥珀酸处理前后mEPSC和PF-PC EPSP的变化。结果琥珀酸处理后,幼鼠小脑PCs的自发性mEPSCs幅值显著减小,由给药前的(24.85±2.78)pA减小至给药后的(13.14±0.84)pA,频率也由给药前的(5.04±1.07)Hz降至给药后的(2.77±0.79)Hz,差异均有统计学意义(P<0.01);琥珀酸显著抑制了PF-PC EPSPs的幅值,使其降低至用药前的(37.76±1.10)%(P<0.01),并使EPSP双脉冲(paired-pulse facilitation,PPF)增强的比率较用药前增加了(40.26±2.9)%(P<0.01),差异均有统计学意义。结论琥珀酸对幼龄大鼠小脑谷氨酸能突触传递有显著的抑制作用。%Objective To investigate the effects of succinic acid( SA) on the glutamatergic synaptic transmission in the neonatal rat cerebellum. Methods The whole-cell patch-clamp technique was carried out in Purkinje cells( PCs) of sagittal cerebellar slices to record the spontaneous miniture excitatory postsynaptic current(mEPSC) and the excitatory postsynaptic potential(EPSP) induced by parallel fiber( PF) stimulation. The changes of the mEPSC and the PF-PC EPSP upon SA were analyzed before and after SA perfusion. Results SA significantly reduced the amplitude[from(24.85 ±2.78)pA to(13.14 ±0.84)pA,P<0.01]and the frequency[from (5.04 ±1.07)Hz to (2.77 ±0.79)Hz,P<0.01] of the spontaneous mEPSCs. SA also significantly inhibited PF-PC EPSPs ampli-tude to (37. 76 ± 1. 10)% of the control(P<0. 01) and enhanced the EPSP paired-pulse facilitation(PPF) by(40. 26 ± 2. 9)%(P<0. 01). Conclusion SA may provide an inhibitory effect on cerebellar

  12. Prenatal immune challenge in rats: altered responses to dopaminergic and glutamatergic agents, prepulse inhibition of acoustic startle, and reduced route-based learning as a function of maternal body weight gain after prenatal exposure to poly IC.

    Science.gov (United States)

    Vorhees, Charles V; Graham, Devon L; Braun, Amanda A; Schaefer, Tori L; Skelton, Matthew R; Richtand, Neil M; Williams, Michael T

    2012-08-01

    Prenatal maternal immune activation has been used to test the neurodevelopmental hypothesis of schizophrenia. Most of the data are in mouse models; far less is available for rats. We previously showed that maternal weight change in response to the immune activator polyinosinic-polycytidylic acid (Poly IC) in rats differentially affects offspring. Therefore, we treated gravid Harlan Sprague-Dawley rats i.p. on embryonic day 14 with 8 mg/kg of Poly IC or Saline. The Poly IC group was divided into those that lost or gained the least weight, Poly IC (L), versus those that gained the most weight, Poly IC (H), following treatment. The study design controlled for litter size, litter sampling, sex distribution, and test experience. We found no effects of Poly IC on elevated zero maze, open-field activity, object burying, light-dark test, straight channel swimming, Morris water maze spatial acquisition, reversal, or shift navigation or spatial working or reference memory, or conditioned contextual or cued fear or latent inhibition. The Poly IC (H) group showed a significant decrease in the rate of route-based learning when visible cues were unavailable in the Cincinnati water maze and reduced prepulse inhibition of acoustic startle in females, but not males. The Poly IC (L) group exhibited altered responses to acute pharmacological challenges: exaggerated hyperactivity in response to (+)-amphetamine and an attenuated hyperactivity in response to MK-801. This model did not exhibit the cognitive, or latent inhibition deficits reported in Poly IC-treated rats but showed changes in response to drugs acting on neurotransmitter systems implicated in the pathophysiology of schizophrenia (dopaminergic hyperfunction and glutamatergic hypofunction).

  13. The role of α1 adrenaline receptors on GABAergic and glutamatergic synapse in spinal dorsal horn%α1-肾上腺素受体参与调控脊髓背角神经元突触传递的作用机制

    Institute of Scientific and Technical Information of China (English)

    袁维秀; 郭英; 徐娟; 张宏

    2012-01-01

    Objective To investigate the role of α1-adrenaline receptors in GABAergic and glutamatergic synapses via GABAA receptors in spinal dorsal horn. Methods Using whole -cell voltage-clamp recordings on acute spinal cord slice, the effect of blockade of α1-adrenaline receptors on GABAergic and glutamatergic synaptic transmission was studied. Results Selective al -adrenline receptors agonist phenylephrine cold significantly increase the frequency of GABAergic spontaneous IPSCs in a concentration dependent manner, and this effect was abolished by the α1-adrenaline receptor antagonist WB4101. Phenylephrine also significantly reduced the amplitude of monosynaptic and polysynaptic EPSCs evoked from primary afferents. The inhibitory effect of phenylephrine on evoked monosynaptic glutamatergic EPSCs was largely blocked by the GABAA receptor antagonist picrotoxin. Conclusion Activation of α1- adrenoceptors in the spinal cord attenuates glutamatergic input from primary afferents mainly through GABAA receptors, indicating that presynaptic inhibition in the spinal cord may be involved in the regulation of nociception by the descending noradrenergic system.%目的 研究α1-肾上腺素受体(α1- AR)调控脊髓背角感觉神经元谷氨酸能突触传递的作用机制.方法 在急性切取的腰段脊髓切片上,利用全细胞膜片钳法记录α1- AR激动剂苯肾上腺素对脊髓背角浅层神经元抑制性和兴奋性突触后电流(IPSCs和eEPSCs)的影响.结果 苯肾上腺素对IPSCs频率产生剂量依赖性兴奋作用,此作用可被α1- AR特异性拮抗剂WB4101完全拮抗.苯肾上腺素对eEPSCs振幅的抑制作用可以部分被GABAA受体拮抗剂印防己毒素(picrotoxin,PTX)拮抗.结论 位于脊髓背角神经元的α1-AR,促进初级传入纤维在脊髓背角释放γ-氨基丁酸(GABA),进而主要通过GABAA受体抑制初级传入纤维兴奋性谷氨酸能神经冲动的传入.下行肾上腺素能系统可能通过GABAA受体机制参

  14. Avian Imc-tectal projection is mediated by acetylcholine and glutamate.

    Science.gov (United States)

    Wang, S R; Wu, G Y; Felix, D

    1995-03-27

    In the bird, biochemical and histochemical data suggest that the neurotransmitter between nucleus isthmi pars magnocellularis (Imc) and tectum is either acetylcholine or glutamate. There are, however, discrepancies regarding the functional role of acetylcholine. In the present study we investigated the action of acetylcholine and glutamate and their specific antagonists on excitatory isthmo-tectal synaptic transmission using electrophysiological and microiontophoretic techniques. The results show two different population of cells: (1) excitatory cholinergic input, blocked by atropine sulphate but not by glutamate antagonist; (2) excitatory glutamatergic input of NMDA or non-NMDA receptor type, which is blocked or reduced by CPP or CNQX but not by atropine sulphate.

  15. Ambient GABA modulates glutamatergic synapse in sacral dorsal commissural nucleus by presynaptic GABAB receptors%周边γ-氨基丁酸通过GABAB受体调控骶髓后联合核神经元谷氨酸能突触

    Institute of Scientific and Technical Information of China (English)

    马红雨; 林凯; 罗丹; 吕建晓; 杨鲲

    2011-01-01

    Aim To investigate the role of ambient γ-aminobutyric acid ( CABA ) on glutamatergic synapse via CABAB receptors in sacral dorsal commissural nucleus ( SDCN ). Method Using whole-cell voltageclamp recordings on acute spinal cord slice, effect of blockade of CABAB receptors on glutamatergic synaptic transmission was studied. Results Blockade of GABAB receptors by a selective antagonist CGP52432 increased evoked excitatory postsynaptic currents ( eEPSCs ) , decreased paired-pulse ration ( PPR ). switched " presynaptically" silent synapse to active one, indicating a presynaptic GABAB receptor-mediated modulation by ambient GABA. Conclusions Ambient GABA modulates glutamate release by GABAB receptors on presynaptic glutamatergic terminals to SDCN neurons;this action may contribute to nociception transmission at spinal cord level.%目的 研究突触周边γ-氨基丁酸(ambient GABA)通过GABAB受体调控骶髓后联合核(SDCN)神经元谷氨酸能突触的机制.方法 在急性切取的骶段脊髓薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元谷氨酸能兴奋性突触后电流(EPSCs),将GABAB受体用其特异性受体拮抗剂CGP52432阻断,观察谷氨酸突触终末上的GABAB受体被周边GABA作用的影响.结果 在突触后GABAB受体被从胞内阻断的条件下,再灌流CGP52432阻断谷氨酸能突触前GABAB受体,可增加刺激引发的EPSCs (eEPSCs) 幅度; 改变配对刺激的两个EPSC比率 (paired-pulse ratio,PPR),并激发沉默突触 (silent synapse).但CGP52432对微小兴奋性突触后电流(mEPSCs)无影响.结论 位于SDCN神经元谷氨酸能突触前的GABAB受体受周边GABA调控.这种影响参与调节谷氨酸释放并可能参与痛觉信息在脊髓水平的传递.

  16. The role of α1-adrenoceptors on glutamatergic synapse via GABAB receptors in spinal dorsal horn%α1-肾上腺素受体通过GABAB受体调控脊髓背角神经元谷氨酸能突触传递

    Institute of Scientific and Technical Information of China (English)

    袁维秀; 郭英; 徐娟; 张宏

    2013-01-01

    目的 研究下行去甲肾上腺素系统α1-肾上腺素受体通过GABAB受体调控脊髓背角感觉神经元谷氨酸能突触传递的机制.方法 在急性切取的腰段脊髓切片上,利用全细胞膜片钳法记录α1-肾上腺素受体激动剂苯肾上腺素刺激诱发的脊髓背角浅层神经元谷氨酸能兴奋性突触后电流(eEPSCs),给予GABAB受体特异性拮抗剂CGP55845,进一步观察GABAB受体在苯肾上腺素对突触终末eEPSCs调节过程中的作用.结果 苯肾上腺素显著降低初级传入末梢单突触和多突触eEPSCs幅度,在突触后GABAB受体被从胞内阻断的条件下,再灌流CGP55845,阻断谷氨酸能突触前GABAB受体,可部分拮抗苯肾上腺素对刺激引发的EPSCs (eEPSCs)幅度的抑制作用.结论 位于脊髓背角神经元α1-肾上腺素受体,通过GABAB受体抑制初级传入纤维兴奋性谷氨酸能神经冲动的传入,这种突触前对谷氨酸释放的调节可能是下行肾上腺素能系统对伤害性刺激调控的作用机制.%Objective To investigate the role of α 1-adrenoceptors on glutamatergic synapse via GABAB receptors in spinal dorsal horn. Methods Using whole-cell voltage-clamp recordings on acute spinal cord slice,effect of blockade of α1 -adrenoceptors on glutamatergic synaptic transmission was studied. Results Selective α1 -adrenoceptors agonist phenylephrine significantly reduced the amplitude of monosynaptic and polysynaptic EPSCs evoked from primary afferents. The inhibitory effect of phenylephrine on evoked monosynaptic and polysynaptic glutamatergic EPSCs was largely blocked by the GABAB receptor antagonist CGP55845. Conclusion Activation of α1- adrenoceptors in the spinal cord attenuates glutamatergic input from primary afferents mainly through GABAB receptors, indicating a presynaptic inhibition in the spinal cord may be involved in the regulation of nociception by the descending noradrenergic system.

  17. Effect of diabetes on baclofen-induced inhibition of mEPSCs in spinal glutamatergic neurons in rats with neuropathic pain%糖尿病因素对巴氯芬抑制神经痛大鼠脊髓谷氨酸能神经元突触后电流的影响

    Institute of Scientific and Technical Information of China (English)

    白惠萍; 彭云水; 王倩; 吴川; 刘飞飞; 刘朋; 郭跃先; 王秀丽

    2014-01-01

    Objective To evaluate the effect of diabetes on baclofen-induced inhibition of miniature excitatory postsynaptic currents (mEPSCs) in spinal glutamatergic neurons in rats with neuropathic pain.Methods Thirty male Sprague-Dawley rats,aged 4 weeks,weighing 150-170 g,were randomly divided into 2 groups (n=15 each group):control group (C group) and diabetic neuropathic pain group (D group).Diabetic neuropathic pain was induced by intraperitoneal injection of streptozotocin 50 mg/kg and confirmed 28 days later by blood glucose > 16.7 mmol/L and pain threshold < 4 g in group D,while the rats received the equal volume of normal saline in C group.The animals were anesthetized with intraperitoneal 10% chloral hydrate 50 mg/kg.The rats were then sacrificed and lumbar segments (L1.5) of the spinal cord were removed for slice preparations.Glutamatergic mEPSCs in lamina Ⅱ neurons were recorded by using whole-cell patch-clamp technique and 20 neurons located in lamina Ⅱ of the spinal cord were recorded in each group.The cells stabilized for 30 min after sealing,and then baclofen with the final concentrations of 1,10,20,50 μmol/L was added to the perfusion solution at 5 min intervals followed by washout.The frequency and intensity of glutamatergic mEPSCs were recorded immediately before and after administration and at 5 min after washout.The inhibitory effect of baclofen on glutamatergic mEPSCs was measured and the inhibitory rate was calculated.Results Compared with group C,the frequency of glutamatergic mEPSCs was significantly increased and the inhibitory rate was decreased under each concentration (P < 0.05),and no significant change was found in the intensity of mEPSCs under each concentration in D group (P > 0.05).Conclusion Diabetes decreases baclofen-induced analgesic effect in rats with neuropathic pain,which is related to inhibition of mEPSCs in spinal glutamatergic neurons.%目的 评价糖尿病因素对巴氯芬抑制神经痛大鼠脊髓谷氨酸

  18. Comparative study on glutamatergic synaptic connections in rat striatum with laser scanning confocal microscopy and electron microscopy%激光共聚焦扫描显微镜和透射电镜观察大鼠纹状体内谷氨酸能突触连接的对比观察

    Institute of Scientific and Technical Information of China (English)

    蔡青; 姬曼; 张进禄; 赵君朋

    2011-01-01

    Objective To evaluate the advantage of the use of laser scanning confocal microscopy (LSCM) for observing the glutamatergic striatal synaptic connections in comparison with the observation with transmission electron microscopy (TEM). Methods 12 normal adult rats were divided into two groups: 6 rats for LSCM or TEM each. With CM-Dil and VGluT1 immunofluorescence labeling,the distribution of the asymmetrical glutamatergic synaptic connections on the dendrites of striatal neurons was observed, and 3-D reconstruction was done. The 6 rats were studied with TEM. Results There is no significant difference in the distribution characters of the glutamatergic striatal synaptic connections obtained with either LSCM or TEM. However, the whole view of the synapses and dendrites and the three-dimensional reconstruction of synaptic connections between neurons can be obtained with LSCM but not TEM.Conclusion LSCM is an effective and quantitative technique to investigate the glutamate synaptic connections of striatal neurons.%目的 探讨使用激光共聚焦扫描显微镜 (Laser scanning confocal microscope,LSCM)观察大鼠纹状体内谷氨酸能突触连接的方法的可行性.方法 12只正常大鼠分为两组,6只大鼠进行纹状体中等棘刺神经元的CM-DiI 单细胞标记,然后Ⅰ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 1,VGluT1 )免疫荧光标记,LSCM层扫后三维重建,观察VGluT1阳性位点在中等棘刺神经元树突上的分布.另外6只大鼠用TEM观察不对称性突触在纹状体神经元树突上的分布.对两种方法的结果进行比较.结果 用LSCM 和TEM方法观察到的纹状体神经元上谷氨酸能突触连接分布情况一致,没有统计学差异.但LSCM更具优越性的是,可以对图像进行三维重构,从而有利于对神经元之间突触连接的空间分布观察和定量分析.结论 神经细胞荧光标记技术结合LSCM观察是考察纹状体神经元上谷氨酸能突触连接的有效方法.

  19. Endogenous interleukin-1β in neuropathic rats enhances glutamate release from the primary afferents in the spinal dorsal horn through coupling with presynaptic N-methyl-D-aspartic acid receptors.

    Science.gov (United States)

    Yan, Xisheng; Weng, Han-Rong

    2013-10-18

    Excessive activation of glutamate receptors and overproduction of proinflammatory cytokines, including interleukin-1β (IL-1β) in the spinal dorsal horn, are key mechanisms underlying the development and maintenance of neuropathic pain. In this study, we investigated the mechanisms by which endogenous IL-1β alters glutamatergic synaptic transmission in the spinal dorsal horn in rats with neuropathic pain induced by ligation of the L5 spinal nerve. We demonstrated that endogenous IL-1β in neuropathic rats enhances glutamate release from the primary afferent terminals and non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Myeloid differentiation primary response protein 88 (MyD88) is a mediator used by IL-1β to enhance non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Presynaptic NMDA receptors are effector receptors used by the endogenous IL-1β to enhance glutamate release from the primary afferents in neuropathic rats. This is further supported by the fact that NMDA currents recorded from small neurons in the dorsal root ganglion of normal rats are potentiated by exogenous IL-1β. Furthermore, we provided evidence that functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is mediated by the neutral sphingomyelinase/ceramide signaling pathway. Hence, functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals is a crucial mechanism leading to enhanced glutamate release and activation of non-NMDA receptors in the spinal dorsal horn neurons in neuropathic pain conditions. Interruption of such functional coupling could be an effective approach for the treatment of neuropathic pain.

  20. 毒蕈碱乙酰胆碱M2/M4受体亚型在调节脊髓背角神经元谷氨酸能递质释放中的作用%Role of muscarinic cholinergic receptor subtypes in regulating glutamatergic synaptic transmission in rat spinal dorsal horn

    Institute of Scientific and Technical Information of China (English)

    杜威; 郭英; 袁维秀

    2013-01-01

    Objective To investigate the role of muscarinic cholinergic receptor (mAChR) subtypes in the regulation of glutamatergic input to the spinal dorsal horn neurons and the possible mechanism.Methods Whole-cell voltage-clamp recordings on acute spinal slice was utilized to investigate the effect of activation of mAChRs and blockade of M2/M4 subtypes on glutamatergic synaptic transmission in rat spinal dorsal horn neurons.Results The nonselective mAChRs agonist oxotremorine-M concentration-dependently decreased the amplitude of monosynaptic and polysynaptic evoked glutamate-mediated excitatory postsynaptic currents (eEPSCs) in most of the neurons.The M2/M4 antagonist himbacine completely blocked the inhibitory effect of oxotremorine-M in 92.3% of monosynaptic and 75% of polysynaptic neurons in the spinal cord slices.In the remaining 16% neurons,himbacine partially blocked the inhibitory effect of oxotremorine-M.Conclusions Activation of mAChRs in the spinal cord attenuates synaptic glutamate release to the dorsal horn neurons mainly through M2 and M4 receptor subtypes,indicating that a presynaptic inhibition in the spinal cord may be involved in the regulation of nociception by the cholinergic system and mAChRs.%目的 研究毒蕈碱胆碱能受体(mAChRs)亚型对脊髓背角感觉神经元谷氨酸能突触传递的调节机制.方法 在急性切取的腰段脊髓切片上,利用全细胞膜片钳法记录mAChRs非特异性激动剂氢化震颤素M(Oxo-M)对脊髓背角浅层神经元谷氨酸能兴奋性突触后电流(eEPSCs)的影响,给予M2/M4受体特异性拮抗剂喜巴辛,观察mAChRs在脊髓背角浅层神经元谷氨酸能递质释放调节过程中的作用.结果 不同浓度Oxo-M使脊髓背角神经元单突触和多突触eEPSCs的幅度显著降低,其抑制强度呈浓度依赖性,喜巴辛可以拮抗Oxo-M对刺激诱发eEPSCs幅度的抑制作用,在记录的25个细胞中,92.3%的单突触细胞和75%的多突触细胞表现为Oxo-M

  1. Repeated morphine pretreatment reduces glutamatergic synaptic potentiation in the nucleus accumbens induced by acute morphine exposure%慢性吗啡预处理减弱急性吗啡对伏隔核谷氨酸能突触传递的影响

    Institute of Scientific and Technical Information of China (English)

    吴晓杰; 张静; 魏春玲; 刘志强; 任维

    2012-01-01

    Repeated exposure to morphine leads to the addiction, which influences its clinical application seriously. The glutamatergic projection from prefrontal cortex (PFC) to the nucleus accumbens (Nac) plays an important role in rewarding effects. It is still unknown whether morphine exposure changes PFC-Nac synaptic transmission. To address this question, in vivo field excitatory post-synaptic potentials (fEPSPs) induced by electric stimulating PFC-Nac projection fibers were recorded to evaluate the effect of acute morphine exposure (10 mg/kg, s.c.) on glutamatergic synaptic transmission in Nac shell of repeated saline/morphine pretreated rats. It was showed that acute morphine exposure enhanced fEPSP amplitude and reduced paired-pulse ratio (PPR) in saline pretreated rats, which could be reversed by following naloxone injection (1 mg/kg, I.p.), an opiate receptor antagonist. However, repeated morphine pretreatment significantly inhibited both the enhancement of fEPSP amplitude and reduction of PPR induced by acute morphine exposure. Those results indicate that the initial morphine exposure enhances PFC-Nac synaptic transmission by pre-synaptic mechanisms, whereas morphine pretreatment occludes this effect.%吗啡长期作用后会产生成瘾(addiction),严重影响其临床应用.前额叶(prefrontal cortex,PFC)投射至伏隔核(nucleus accumbens,NAc)的谷氨酸能突触对奖赏效应有重要的调节作用,但该突触在吗啡成瘾中的具体作用尚不完全清楚.为探讨PFC至NAc的谷氨酸能突触在成瘾形成过程中的具体作用及其机制,本研究利用成年大鼠在体记录的方式,记录电刺激PFC至NAc谷氨酸能传入纤维引起的NAc壳区场兴奋性突触后电位(filed excitatory postsynaptic potential,fEPSP),观察慢性吗啡/盐水预处理后依次急性皮下注射吗啡及腹腔注射纳络酮对fEPSP幅值和配对脉冲比率(paired-pulse ratio,PPR)的影响.结果显示,与基础fEPSP相比,慢性盐水预处理组急

  2. Effect of excitatory amino acids on serum TSH and thyroid hormone levels in freely moving rats.

    Science.gov (United States)

    Alfonso, M; Durán, R; Arufe, M C

    2000-01-01

    The actions of glutamate (L-Glu), and glutamate receptor agonists on serum thyroid hormones (T4 and T3) and TSH levels have been studied in conscious and freely moving adult male rats. The excitatory amino acids (EAA), L-Glu, N-methyl-D-aspartate (NMDA), kainic acid (KA) and domoic acid (Dom) were administered intraperitoneally. Blood samples were collected through a cannula implanted in the rats jugular 0--60 min after injection. Thyroid hormone concentrations were measured by enzyme immunoassay, and thyrotrophin (TSH) concentrations were determined by radioimmunoassay. The results showed that L-Glu (20 and 25 mg/kg) and NMDA (25 mg/kg) increased serum thyroxine (T4), triiodothyronine (T3) and TSH concentrations. Serum thyroid hormone levels increased 30 min after treatment, while serum TSH levels increased 5 min after i.p. administration, in both cases serum levels remained elevated during one hour. Injection of the non-NMDA glutamatergic agonists KA (30 mg/kg) and Dom (1 mg/kg) produced an increase in serum thyroid hormones and TSH levels. These results suggest the importance of EAAs in the regulation of hormone secretion from the pituitary-thyroid axis, as well as the importance of the NMDA and non-NMDA receptors in this stimulatory effect.

  3. Electrophysiology of a nonmyelinated glutamatergic axon in rat hippocampus

    OpenAIRE

    Alle, Henrik

    2012-01-01

    The common theme of the presented work on the nonmyelinated hippocampal mossy fiber (the axon of the granule cell in the dentate gyrus) is the generation of subthreshold and suprathreshold electrical signals. Subthreshold depolarizations in the axon can occur due to passive propagation of excitatory postsynaptic potentials, which are generated in the somato-dendritic domain. The remote passive propagation of these comparatively slow but transient signals is due to a space constant...

  4. Glutamatergic and Dopaminergic Neurons in the Mouse Ventral Tegmental Area

    OpenAIRE

    Yamaguchi, Tsuyoshi; Qi, Jia; Wang, Hui-Ling; Zhang, Shiliang; Morales, Marisela

    2015-01-01

    The ventral tegmental area (VTA) comprises dopamine (DA), GABA and glutamate (Glu) neurons. Some rat VTA Glu neurons, expressing vesicular glutamate transporter 2 (VGluT2), co-express tyrosine hydroxylase (TH). While transgenic mice are now being used in attempts to determine the role of VGluT2/TH neurons in reward and neuronal signaling, such neurons have not been characterized in mouse tissue. By cellular detection of VGluT2-mRNA and TH-immunoreactivity (TH-IR), we determined the cellular e...

  5. Glutamatergic neurons are present in the rat ventral tegmental area

    OpenAIRE

    Yamaguchi, Tsuyoshi; Sheen, Whitney; Morales, Marisela

    2007-01-01

    The ventral tegmental area (VTA) is thought to play an important role in reward function. Two populations of neurons, containing either dopamine (DA) or γ-amino butyric acid (GABA), have been extensively characterized in this area. However, recent electrophysiological studies are consistent with the notion that neurons that utilize neurotransmitters other than DA or GABA are likely to be present in the VTA. Given the pronounced phenotypic diversity of neurons in this region, we have proposed ...

  6. Corticosteroid effects on glutamatergic transmission and fear memory

    NARCIS (Netherlands)

    Xiong, Hui

    2016-01-01

    In our daily life we are regularly exposed to situations which we experience as stressful. In response to these events our body increases the release of corticosteroid hormones from the adrenal glands. These hormones promote behavioural adaptation to stressful experiences by enhancing the storage of

  7. Hippocampal Fast Glutamatergic Transmission Is Transiently Regulated by Corticosterone Pulsatility

    NARCIS (Netherlands)

    Sarabdjitsingh, R Angela; Pasricha, Natasha; Smeets, Johanna A S; Kerkhofs, Amber; Mikasova, Lenka; Karst, Henk; Groc, Laurent; Joëls, Marian

    2016-01-01

    In recent years it has become clear that corticosteroid hormones (such as corticosterone) are released in ultradian pulses as a natural consequence of pituitary-adrenal interactions. All organs, including the brain, are thus exposed to pulsatile changes in corticosteroid hormone level, important to

  8. Glutamatergic metabolites are associated with visual plasticity in humans.

    Science.gov (United States)

    Wijtenburg, S Andrea; West, Jeffrey; Korenic, Stephanie A; Kuhney, Franchesca; Gaston, Frank E; Chen, Hongji; Roberts, Meredith; Kochunov, Peter; Hong, L Elliot; Rowland, Laura M

    2017-02-10

    Long-term potentiation (LTP) is a basic cellular mechanism underlying learning and memory. LTP-like plasticity in the visual cortex can be induced by high frequency visual stimulation in rodents and humans. Since glutamate plays a fundamental role in LTP, this study investigated if visual cortical glutamate and glutamine levels, measured by proton magnetic resonance spectroscopy (MRS), relate to visual plasticity in humans. Since plasticity requires a delicate excitation and inhibition balance, GABA was also explored. Eighteen healthy participants completed MRS and a visual fMRI paradigm. Results revealed enhanced fMRI activations after high frequency visual stimulation, suggesting visual plasticity occurred. Higher activations were associated with higher resting glutamine levels after family wise error-correction. Exploratory analyses revealed that higher resting glutamate and GABA levels were associated with visual plasticity, suggesting there may be a critical excitation-inhibition balance necessary for experience dependent plasticity. This is the first empirical evidence that resting glutamine levels and potentially glutamate and GABA levels are associated with visual plasticity in humans.

  9. Glutamatergic signaling maintains the epithelial phenotype of proximal tubular cells

    NARCIS (Netherlands)

    Bozic, M.; de Rooij, J.; Parisi, E.; Ortega, M.R.; Fernandez, E.; Valdivielso, J.M.

    2011-01-01

    Epithelial-mesenchymal transition (EMT) contributes to the progression of renal tubulointerstitial fibrosis. The N-methyl-d-aspartate receptor (NMDAR), which is present in proximal tubular epithelium, is a glutamate receptor that acts as a calcium channel. Activation of NMDAR induces actin rearrange

  10. Glutamatergic transmission in drug reward: implications for drug addiction.

    Science.gov (United States)

    D'Souza, Manoranjan S

    2015-01-01

    Individuals addicted to drugs of abuse such as alcohol, nicotine, cocaine, and heroin are a significant burden on healthcare systems all over the world. The positive reinforcing (rewarding) effects of the above mentioned drugs play a major role in the initiation and maintenance of the drug-taking habit. Thus, understanding the neurochemical mechanisms underlying the reinforcing effects of drugs of abuse is critical to reducing the burden of drug addiction in society. Over the last two decades, there has been an increasing focus on the role of the excitatory neurotransmitter glutamate in drug addiction. In this review, pharmacological and genetic evidence supporting the role of glutamate in mediating the rewarding effects of the above described drugs of abuse will be discussed. Further, the review will discuss the role of glutamate transmission in two complex heterogeneous brain regions, namely the nucleus accumbens (NAcc) and the ventral tegmental area (VTA), which mediate the rewarding effects of drugs of abuse. In addition, several medications approved by the Food and Drug Administration that act by blocking glutamate transmission will be discussed in the context of drug reward. Finally, this review will discuss future studies needed to address currently unanswered gaps in knowledge, which will further elucidate the role of glutamate in the rewarding effects of drugs of abuse.

  11. Glutamatergic transmission in drug reward: Implications for drug addiction

    Directory of Open Access Journals (Sweden)

    Manoranjan S Dsouza

    2015-11-01

    Full Text Available Individuals addicted to drugs of abuse such as alcohol, nicotine, cocaine, and heroin are a significant burden on healthcare systems all over the world. The positive reinforcing (rewarding effects of the above mentioned drugs play a major role in the initiation and maintenance of the drug-taking habit. Thus, understanding the neurochemical mechanisms underlying the reinforcing effects of drugs of abuse is critical to reducing the burden of drug addiction in society. Over the last two decades, there has been an increasing focus on the role of the excitatory neurotransmitter glutamate in drug addiction. In this review, pharmacological and genetic evidence supporting the role of glutamate in mediating the rewarding effects of the above described drugs of abuse will be discussed. Further, the review will discuss the role of glutamate transmission in two complex heterogeneous brain regions, namely the nucleus accumbens (NAcc and the ventral tegmental area (VTA, which mediate the rewarding effects of drugs of abuse. In addition, several medications approved by the Food and Drug Administration that act by blocking glutamate transmission will be discussed in the context of drug reward. Finally, this review will discuss future studies needed to address currently unanswered gaps in knowledge, which will further elucidate the role of glutamate in the rewarding effects of drugs of abuse.

  12. Generation of Hermansky Pudlak syndrome type 2 (HPS2 induced pluripotent stem cells (iPSCs

    Directory of Open Access Journals (Sweden)

    Jean Ann Maguire

    2016-03-01

    Full Text Available Hermansky–Pudlak syndrome type 2 (HPS2 is a rare autosomal recessive disorder resulting from functional mutations in the adaptor-related protein complex 3, beta 1 subunit (AP3B1 gene. This gene plays a role in organelle biogenesis associated with melanosomes, platelet dense granules, and lysosomes. Here we describe the generation of an HPS2 iPS cell line (CHOPHPS2 using a Cre-excisable polycistronic STEMCCA lentivirus. This line was derived from human fibroblasts isolated from a patient carrying two mutations in the AP3B1 gene. The patient presented with severe neutropenia, ocular albinism, interstitial pulmonary fibrosis, hemorrhagic diathesis, and an absence of platelet-dense granules.

  13. Revisiting the flight of Icarus: making human organs from PSCs with large animal chimeras.

    Science.gov (United States)

    Rashid, Tamir; Kobayashi, Toshihiro; Nakauchi, Hiromitsu

    2014-10-02

    While cell therapies hold great potential for treating many conditions, their utility for treating patients that require whole organ replacement is unclear. To address this challenge, we propose using genetically engineered "organ niches" in large animals to generate human organs from pluripotent stem cells and discuss the hurdles facing such strategies.

  14. iPSCs Offer a New Look at GATA1-Trisomy 21 Cooperation.

    Science.gov (United States)

    McNulty, Maureen; Crispino, John D

    2016-05-05

    GATA1 mutations and trisomy 21 are inextricably linked in the neonatal leukemia of children with Down syndrome (DS). A recent report by Banno et al. (2016) sheds new light on the mechanism of the synergy between these genetic alterations by modeling hematopoietic abnormalities with patient-derived induced pluripotent stem cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Generation of poikiloderma with neutropenia (PN) induced pluripotent stem cells (iPSCs).

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    Mills, Jason A; Hudock, Kristin M; Sullivan, Spencer K; Herrera, Pamela; Sullivan, Lisa M; Gadue, Paul; French, Deborah L

    2015-11-01

    Poikiloderma with neutropenia (PN, Clericuzio-type poikiloderma with neutropenia) is a rare autosomal recessive disorder caused by biallelic mutations in the USB1 gene (Alias C16orf57 and MPN1). To date, there have been only 37 reported cases worldwide of this disorder that presents with neutropenia, early onset poikiloderma, respiratory infections, palmo-plantar hyperkeratosis, and skeletal defects. Here we described the generation of human induced pluripotent stem cell lines (PN1 and PN2) from the peripheral blood of a 1-year-old patient using the dox-inducible STEMCCA vector. This patient presented with bacteremia, pneumonia, and neutropenia. Analysis of bone marrow demonstrated normal cellularity with trilineage hematopoiesis and neutropenia.

  16. Generation of poikiloderma with neutropenia (PN induced pluripotent stem cells (iPSCs

    Directory of Open Access Journals (Sweden)

    Jason A. Mills

    2015-11-01

    Full Text Available Poikiloderma with neutropenia (PN, Clericuzio-type poikiloderma with neutropenia is a rare autosomal recessive disorder caused by biallelic mutations in the USB1 gene (Alias C16orf57 and MPN1. To date, there have been only 37 reported cases worldwide of this disorder that presents with neutropenia, early onset poikiloderma, respiratory infections, palmo-plantar hyperkeratosis, and skeletal defects. Here we described the generation of human induced pluripotent stem cell lines (PN1 and PN2 from the peripheral blood of a 1-year-old patient using the dox-inducible STEMCCA vector. This patient presented with bacteremia, pneumonia, and neutropenia. Analysis of bone marrow demonstrated normal cellularity with trilineage hematopoiesis and neutropenia.

  17. Genomic DISC1 Disruption in hiPSCs Alters Wnt Signaling and Neural Cell Fate

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