Sample records for cortical gabaergic neurons

  1. Cholinergic Neurons Excite Cortically Projecting Basal Forebrain GABAergic Neurons

    Yang, Chun; McKenna, James T.; Zant, Janneke C.; Winston, Stuart; Basheer, Radhika


    The basal forebrain (BF) plays an important role in the control of cortical activation and attention. Understanding the modulation of BF neuronal activity is a prerequisite to treat disorders of cortical activation involving BF dysfunction, such as Alzheimer's disease. Here we reveal the interaction between cholinergic neurons and cortically projecting BF GABAergic neurons using immunohistochemistry and whole-cell recordings in vitro. In GAD67-GFP knock-in mice, BF cholinergic (choline acetyltransferase-positive) neurons were intermingled with GABAergic (GFP+) neurons. Immunohistochemistry for the vesicular acetylcholine transporter showed that cholinergic fibers apposed putative cortically projecting GABAergic neurons containing parvalbumin (PV). In coronal BF slices from GAD67-GFP knock-in or PV-tdTomato mice, pharmacological activation of cholinergic receptors with bath application of carbachol increased the firing rate of large (>20 μm diameter) BF GFP+ and PV (tdTomato+) neurons, which exhibited the intrinsic membrane properties of cortically projecting neurons. The excitatory effect of carbachol was blocked by antagonists of M1 and M3 muscarinic receptors in two subpopulations of BF GABAergic neurons [large hyperpolarization-activated cation current (Ih) and small Ih, respectively]. Ion substitution experiments and reversal potential measurements suggested that the carbachol-induced inward current was mediated mainly by sodium-permeable cation channels. Carbachol also increased the frequency of spontaneous excitatory and inhibitory synaptic currents. Furthermore, optogenetic stimulation of cholinergic neurons/fibers caused a mecamylamine- and atropine-sensitive inward current in putative GABAergic neurons. Thus, cortically projecting, BF GABAergic/PV neurons are excited by neighboring BF and/or brainstem cholinergic neurons. Loss of cholinergic neurons in Alzheimer's disease may impair cortical activation, in part, through disfacilitation of BF cortically

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

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


    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.

  3. Acidosis-Induced Dysfunction of Cortical GABAergic Neurons through Astrocyte-Related Excitotoxicity.

    Huang, Li; Zhao, Shidi; Lu, Wei; Guan, Sudong; Zhu, Yan; Wang, Jin-Hui


    Acidosis impairs cognitions and behaviors presumably by acidification-induced changes in neuronal metabolism. Cortical GABAergic neurons are vulnerable to pathological factors and their injury leads to brain dysfunction. How acidosis induces GABAergic neuron injury remains elusive. As the glia cells and neurons interact each other, we intend to examine the role of the astrocytes in acidosis-induced GABAergic neuron injury. Experiments were done at GABAergic cells and astrocytes in mouse cortical slices. To identify astrocytic involvement in acidosis-induced impairment, we induced the acidification in single GABAergic neuron by infusing proton intracellularly or in both neurons and astrocytes by using proton extracellularly. Compared the effects of intracellular acidification and extracellular acidification on GABAergic neurons, we found that their active intrinsic properties and synaptic outputs appeared more severely impaired in extracellular acidosis than intracellular acidosis. Meanwhile, extracellular acidosis deteriorated glutamate transporter currents on the astrocytes and upregulated excitatory synaptic transmission on the GABAergic neurons. Moreover, the antagonists of glutamate NMDA-/AMPA-receptors partially reverse extracellular acidosis-induced injury in the GABAergic neurons. Our studies suggest that acidosis leads to the dysfunction of cortical GABAergic neurons by astrocyte-mediated excitotoxicity, in addition to their metabolic changes as indicated previously.

  4. Cortical GABAergic neurons are more severely impaired by alkalosis than acidosis


    Background Acid–base imbalance in various metabolic disturbances leads to human brain dysfunction. Compared with acidosis, the patients suffered from alkalosis demonstrate more severe neurological signs that are difficultly corrected. We hypothesize a causative process that the nerve cells in the brain are more vulnerable to alkalosis than acidosis. Methods The vulnerability of GABAergic neurons to alkalosis versus acidosis was compared by analyzing their functional changes in response to the extracellular high pH and low pH. The neuronal and synaptic functions were recorded by whole-cell recordings in the cortical slices. Results The elevation or attenuation of extracellular pH impaired these GABAergic neurons in terms of their capability to produce spikes, their responsiveness to excitatory synaptic inputs and their outputs via inhibitory synapses. Importantly, the dysfunction of these active properties appeared severer in alkalosis than acidosis. Conclusions The severer impairment of cortical GABAergic neurons in alkalosis patients leads to more critical neural excitotoxicity, so that alkalosis-induced brain dysfunction is difficultly corrected, compared to acidosis. The vulnerability of cortical GABAergic neurons to high pH is likely a basis of severe clinical outcomes in alkalosis versus acidosis. PMID:24314112

  5. Development of Cortical GABAergic Neurons: Interplay of progenitor diversity and environmental factors on fate specification

    Juliana Alves Brandão


    Full Text Available Cortical GABAergic interneurons constitute an extremely diverse population of cells organized in a well-defined topology of precisely interconnected cells. They play a crucial role regulating inhibitory-excitatory balance in brain circuits, gating sensory perception and regulating spike timing to brain oscillations during distinct behaviors. Dysfunctions in the establishment of proper inhibitory circuits have been associated to several brain disorders such as autism, epilepsy and schizophrenia. In the rodent adult cortex, inhibitory neurons are generated during the second gestational week from distinct progenitor lineages located in restricted domains of the ventral telencephalon. However, only recently, studies have revealed some of the mechanisms generating the heterogeneity of neuronal subtypes and their modes of integration in brain networks. Here we will discuss some the events involved in the production of cortical GABAergic neuron diversity with focus on the interaction between intrinsically driven genetic programs and environmental signals during development.

  6. BDNF-modulated spatial organization of Cajal-Retzius and GABAergic neurons in the marginal zone plays a role in the development of cortical organization.

    Alcántara, Soledad; Pozas, Esther; Ibañez, Carlos F; Soriano, Eduardo


    The present study utilizes nestin-BDNF transgenic mice, which offer a model for early increased brain-derived neurotrophic factor (BDNF) signalling, to examine the role of BDNF in the development of cortical architecture. Our results demonstrate that the premature and homogeneous expression of BDNF, while preserving tangential migration from the ganglionic eminence to the cortex, impairs the final radial migration of GABAergic neurons, as well as their integration in the appropriate cortical layers. Moreover, Cajal-Retzius (CR) cells and GABAergic neurons segregate in the cortical marginal zone (MZ) in response to BDNF signalling, leading to an alternating pattern and a columnar cortical organization, within which the migration of different neuronal populations is specifically affected. These results suggest that both CR and GABAergic neurons play a role in directing the radial migration of late-generated cortical neurons, and that the spatial distribution of these cells in the MZ is critical for the development of correct cortical organization. In addition, reelin secreted by CR cells in the MZ is not sufficient to direct the migration of late-born neurons to the upper cortical layers, which most likely requires the presence of reelin-secreting interneurons in layers V-VI. We propose that in addition to modulating reelin expression, BDNF regulates the patched distribution of CR and GABAergic neurons in the MZ, and that this spatial distribution is involved in the formation of anatomical and/or functional columns and convoluted structures.

  7. Diminished perisomatic GABAergic terminals on cortical neurons adjacent to amyloid plaques

    Virginia Garcia-Marin


    Full Text Available One of the main pathological hallmarks of Alzheimer’s disease (AD is the accumulation of plaques in the cerebral cortex, which may appear either in the neuropil or in direct association with neuronal somata. Since different axonal systems innervate the dendritic (mostly glutamatergic and perisomatic (mostly GABAergic regions of neurons, the accumulation of plaques in the neuropil or associated with the soma might produce different alterations to synaptic circuits. We have used a variety of conventional light, confocal and electron microscopy techniques to study their relationship with neuronal somata in the cerebral cortex from AD patients and APP/PS1 transgenic mice. The main finding was that the membrane surfaces of neurons (mainly pyramidal cells in contact with plaques lack GABAergic perisomatic synapses. Since these perisomatic synapses are thought to exert a strong influence on the output of pyramidal cells, their loss may lead to the hyperactivity of the neurons in contact with plaques. These results suggest that plaques modify circuits in a more selective manner than previously thought.

  8. Autaptic self-inhibition of cortical GABAergic neurons: synaptic narcissism or useful introspection?

    Deleuze, Charlotte; Pazienti, Antonio; Bacci, Alberto


    Fast synaptic inhibition sculpts all forms of cortical activity by means of a specialized connectivity pattern between highly heterogeneous inhibitory interneurons and principal excitatory cells. Importantly, inhibitory neurons connect also to each other extensively, following a detailed blueprint, and, indeed, specific forms of disinhibition affect important behavioral functions. Here we discuss a peculiar form of cortical disinhibition: the massive autaptic self-inhibition of parvalbumin-(PV) positive basket cells. Despite being described long ago, autaptic inhibition onto PV basket cells is rarely included in cortical circuit diagrams, perhaps because of its still elusive function. We propose here a potential dual role of autaptic feedback inhibition in temporally coordinating PV basket cells during cortical network activity.

  9. Corticofugal GABAergic projection neurons in the mouse frontal cortex

    Ryohei eTomioka


    Full Text Available Cortical projection neurons are classified by hodology in corticocortical, commissural and corticofugal subtypes. Although cortical projection neurons had been regarded as only glutamatergic neurons, recently corticocortical GABAergic projection neurons has been also reported in several species. Here we demonstrate corticofugal GABAergic projection neurons in the mouse frontal cortex. We employed viral-vector-mediated anterograde tracing, classical retrograde tracing, and immunohistochemistry to characterize neocortical GABAergic projection neurons. Injections of the Cre-dependent adeno-associated virus into glutamate decarboxylase 67-Cre knock-in mice revealed neocortical GABAergic projections widely to the forebrain, including the cerebral cortices, caudate putamen, ventral pallidum, lateral globus pallidus, nucleus accumbens, and olfactory tubercle. Minor GABAergic projections were also found in the mediodorsal thalamic nucleus, diagonal band of Broca, medial globus pallidus, substantial nigra, and dorsal raphe nucleus. Retrograde tracing studies also demonstrated corticofugal GABAergic projection neurons in the mouse frontal cortex. Further immunohistochemical screening with neurochemical markers revealed the majority of corticostriatal GABAergic projection neurons were positive for somatostatin-immunoreactivity. In contrast, corticothalamic GABAergic projection neurons were not identified by representative neurochemical markers for GABAergic neurons. These findings suggest that corticofugal GABAergic projection neurons are heterogeneous in terms of their neurochemical properties and target nuclei, and provide axonal innervations mainly to the nuclei in the basal ganglia.

  10. Assortment of GABAergic plasticity in the cortical interneuron melting pot.

    Méndez, Pablo; Bacci, Alberto


    Cortical structures of the adult mammalian brain are characterized by a spectacular diversity of inhibitory interneurons, which use GABA as neurotransmitter. GABAergic neurotransmission is fundamental for integrating and filtering incoming information and dictating postsynaptic neuronal spike timing, therefore providing a tight temporal code used by each neuron, or ensemble of neurons, to perform sophisticated computational operations. However, the heterogeneity of cortical GABAergic cells is associated to equally diverse properties governing intrinsic excitability as well as strength, dynamic range, spatial extent, anatomical localization, and molecular components of inhibitory synaptic connections that they form with pyramidal neurons. Recent studies showed that similarly to their excitatory (glutamatergic) counterparts, also inhibitory synapses can undergo activity-dependent changes in their strength. Here, some aspects related to plasticity and modulation of adult cortical and hippocampal GABAergic synaptic transmission will be reviewed, aiming at providing a fresh perspective towards the elucidation of the role played by specific cellular elements of cortical microcircuits during both physiological and pathological operations.

  11. Subtypes of GABAergic neurons project axons in the neocortex

    Shigeyoshi Higo


    Full Text Available γ-aminobutyric acid (GABAergic neurons in the neocortex have been regarded as interneurons and speculated to modulate the activity of neurons locally. Recently, however, several experiments revealed that neuronal nitric oxide synthase (nNOS-positive GABAergic neurons project cortico-cortically with long axons. In this study, we illustrate Golgi-like images of the nNOS-positive GABAergic neurons using a nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d reaction and follow the emanating axon branches in cat brain sections. These axon branches projected cortico-cortically with other non-labeled arcuate fibers, contra-laterally via the corpus callosum and anterior commissure. The labeled fibers were not limited to the neocortex but found also in the fimbria of the hippocampus. In order to have additional information on these GABAergic neuron projections, we investigated green fluorescent protein (GFP-labeled GABAergic neurons in GAD67-Cre knock-in / GFP Cre-reporter mice. GFP-labeled axons emanate densely, especially in the fimbria, a small number in the anterior commissure, and very sparsely in the corpus callosum. These two different approaches confirm that not only nNOS-positive GABAergic neurons but also other subtypes of GABAergic neurons project long axons in the cerebral cortex and are in a position to be involved in information processing.

  12. Localization of the brainstem GABAergic neurons controlling paradoxical (REM sleep.

    Emilie Sapin

    Full Text Available Paradoxical sleep (PS is a state characterized by cortical activation, rapid eye movements and muscle atonia. Fifty years after its discovery, the neuronal network responsible for the genesis of PS has been only partially identified. We recently proposed that GABAergic neurons would have a pivotal role in that network. To localize these GABAergic neurons, we combined immunohistochemical detection of Fos with non-radioactive in situ hybridization of GAD67 mRNA (GABA synthesis enzyme in control rats, rats deprived of PS for 72 h and rats allowed to recover after such deprivation. Here we show that GABAergic neurons gating PS (PS-off neurons are principally located in the ventrolateral periaqueductal gray (vlPAG and the dorsal part of the deep mesencephalic reticular nucleus immediately ventral to it (dDpMe. Furthermore, iontophoretic application of muscimol for 20 min in this area in head-restrained rats induced a strong and significant increase in PS quantities compared to saline. In addition, we found a large number of GABAergic PS-on neurons in the vlPAG/dDPMe region and the medullary reticular nuclei known to generate muscle atonia during PS. Finally, we showed that PS-on neurons triggering PS localized in the SLD are not GABAergic. Altogether, our results indicate that multiple populations of PS-on GABAergic neurons are distributed in the brainstem while only one population of PS-off GABAergic neurons localized in the vlPAG/dDpMe region exist. From these results, we propose a revised model for PS control in which GABAergic PS-on and PS-off neurons localized in the vlPAG/dDPMe region play leading roles.

  13. An Overview of the Mechanisms of Abnormal GABAergic Interneuronal Cortical Migration Associated with Prenatal Ethanol Exposure.

    Shenoda, Botros B


    GABAergic Interneuronal migration constitutes an essential process during corticogenesis. Derived from progenitor cells located in the proliferative zones of the ventral telencephalon, newly generated GABAergic Interneuron migrate to their cortical destinations. Cortical dysfunction associated with defects in neuronal migration results in severe developmental consequences. There is growing evidence linking prenatal ethanol exposure to abnormal GABAergic interneuronal migration and subsequent cortical dysfunction. Investigating the pathophysiological mechanisms behind disrupted GABAergic interneuronal migration encountered with prenatal alcohol exposure is crucial for understanding and managing fetal alcohol spectrum disorders. This review explores the molecular pathways regulating GABAergic interneuronal cortical migration that might be altered by prenatal ethanol exposure thus opening new avenues for further research in this topic.

  14. Role of the NR2A/2B subunits of the N-methyl-D-aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro.

    Monnerie, H; Hsu, F-C; Coulter, D A; Le Roux, P D


    The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)'s expression in surviving DIV 11 cortical GABAergic neurons in vitro [Monnerie and Le Roux, (2007) Exp Neurol 205:367-382, (2008) Exp Neurol 213:145-153]. An N-methyl-D-aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamate's effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamate's effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered

  15. Response Patterns of GABAergic Neurons in the Anterior Piriform Cortex of Awake Mice.

    Hu, Rongfeng; Zhang, Juen; Luo, Minmin; Hu, Ji


    Local inhibition by γ-amino butyric acid (GABA)-containing neurons is of vital importance for the operation of sensory cortices. However, the physiological response patterns of cortical GABAergic neurons are poorly understood, especially in the awake condition. Here, we utilized the recently developed optical tagging technique to specifically record GABAergic neurons in the anterior piriform cortex (aPC) in awake mice. The identified aPC GABAergic neurons were stimulated with robotic delivery of 32 distinct odorants, which covered a broad range of functional groups. We found that aPC GABAergic neurons could be divided into 4 types based on their response patterns. Type I, type II, and type III neurons displayed broad excitatory responses to test odorants with different dynamics. Type I neurons were constantly activated during odorant stimulation, whereas type II neurons were only transiently activated at the onset of odorant delivery. In addition, type III neurons displayed transient excitatory responses both at the onset and termination of odorant presentation. Interestingly, type IV neurons were broadly inhibited by most of the odorants. Taken together, aPC GABAergic neurons adopt different strategies to affect the cortical circuitry. Our results will allow for better understanding of the role of cortical GABAergic interneurons in sensory information processing.

  16. Functional diversity of supragranular GABAergic neurons in the barrel cortex

    Luc J Gentet


    Full Text Available Although the neocortex forms a distributed system comprised of several functional areas, its vertical columnar organization is largely conserved across areas and species, suggesting the existence of a canonical neocortical microcircuit. In order to elucidate the principles governing the organization of such a cortical diagram, a detailed understanding of the dynamics binding different types of cortical neurons into a coherent algorithm is essential. Within this complex circuitry, GABAergic interneurons, while forming approximately only 15-20% of all cortical neurons, appear critical in maintaining a dynamic balance between excitation and inhibition. Despite their importance, cortical GABAergic neurons have not been extensively studied in vivo and their precise role in shaping the local microcircuit sensory response still remains to be determined. Their paucity, combined with their molecular, anatomical and physiological diversity, has made it difficult to even establish a consensual nomenclature.However, recent technological advances in microscopy and mouse genetics have fostered a renewed interest in neocortical interneurons by putting them within visible reach of experimenters. The anatomically well-defined whisker-to-barrel pathway of the rodent is particularly amenable to studies attempting to link cortical circuit dynamics to behavior. To each whisker corresponds a discrete cortical unit equivalent to a single column, specialized in the encoding and processing of the sensory information it receives. In this review, we will focus on the functional role that each subtype of supragranular GABAergic neuron embedded within such a single neocortical unit may play in shaping the dynamics of the local circuit during somatosensory integration.

  17. Local connections of layer 5 GABAergic interneurons to corticospinal neurons

    Yasuyo H Tanaka


    Full Text Available In the local circuit of the cerebral cortex, GABAergic inhibitory interneurons are considered to work in collaboration with excitatory neurons. Although many interneuron subgroups have been described in the cortex, local inhibitory connections of each interneuron subgroup are only partially understood with respect to the functional neuron groups that receive these inhibitory connections. In the present study, we morphologically examined local inhibitory inputs to corticospinal neurons (CSNs in motor areas using transgenic rats in which GABAergic neurons expressed fluorescent protein Venus. By analysis of biocytin-filled axons obtained with whole-cell recording/staining in cortical slices, we classified fast-spiking (FS neurons in layer (L 5 into two types, FS1 and FS2, by their high and low densities of axonal arborization, respectively. We then investigated the connections of FS1, FS2, somatostatin-immunopositive (SOM and other (non-FS/non-SOM interneurons to CSNs that were retrogradely labeled in a Golgi-like manner in motor areas. When close appositions between the axon boutons of the intracellularly labeled interneurons and the somata/dendrites of the retrogradely labeled CSNs were examined electron-microscopically, 74% of these appositions made symmetric synaptic contacts. The axon boutons of single FS1 neurons were 2–4-fold more frequent in appositions to the somata/dendrites of CSNs than those of FS2, SOM and non-FS/non-SOM neurons. Axosomatic appositions were most frequently formed with axon boutons of FS1 and FS2 neurons (approximately 30% and least frequently formed with those of SOM neurons (7%. In contrast, SOM neurons most extensively sent axon boutons to the apical dendrites of CSNs. These results might suggest that motor outputs are controlled differentially by the subgroups of L5 GABAergic interneurons in cortical motor areas. 

  18. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine

    Leke, Renata; Bak, Lasse Kristoffer; Anker, Malene


    in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure...... enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important......Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme...

  19. Transient epileptiform signaling during neuronal network development: regulation by external stimulation and bimodal GABAergic activity.

    Zemianek, Jill M; Shultz, Abraham M; Lee, Sangmook; Guaraldi, Mary; Yanco, Holly A; Shea, Thomas B


    A predominance of excitatory activity, with protracted appearance of inhibitory activity, accompanies cortical neuronal development. It is unclear whether or not inhibitory neuronal activity is solicited exclusively by excitatory neurons or whether the transient excitatory activity displayed by developing GABAergic neurons contributes to an excitatory threshold that fosters their conversion to inhibitory activity. We addressed this possibility by culturing murine embryonic neurons on multi-electrode arrays. A wave of individual 0.2-0.4 mV signals ("spikes") appeared between approx. 20-30 days in culture, then declined. A transient wave of high amplitude (>0.5 mV) epileptiform activity coincided with the developmental decline in spikes. Bursts (clusters of ≥3 low-amplitude spikes within 0.7s prior to returning to baseline) persisted following this decline. Addition of the GABAergic antagonist bicuculline initially had no effect on signaling, consistent with delayed development of GABAergic synapses. This was followed by a period in which bicuculline inhibited overall signaling, confirming that GABAergic neurons initially display excitatory activity in ex vivo networks. Following the transient developmental wave of epileptiform signaling, bicuculline induced a resurgence of epileptiform signaling, indicating that GABAergic neurons at this point displayed inhibitory activity. The appearance of transition after the developmental and decline of epileptiform activity, rather than immediately after the developmental decline in lower-amplitude spikes, suggests that the initial excitatory activity of GABAergic neurons contributes to their transition into inhibitory neurons, and that inhibitory GABAergic activity is essential for network development. Prior studies indicate that a minority (25%) of neurons in these cultures were GABAergic, suggesting that inhibitory neurons regulate multiple excitatory neurons. A similar robust increase in signaling following cessation of

  20. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine.

    Leke, Renata; Bak, Lasse K; Anker, Malene; Melø, Torun M; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Portela, Luis V; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S


    Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

  1. GABAergic synaptic inhibition is reduced before seizure onset in a genetic model of cortical malformation.

    Trotter, Stacey A; Kapur, Jaideep; Anzivino, Matthew J; Lee, Kevin S


    Malformations of the neocortex are a common cause of human epilepsy; however, the critical issue of how disturbances in cortical organization render neurons epileptogenic remains controversial. The present study addressed this issue by studying inhibitory structure and function before seizure onset in the telencephalic internal structural heterotopia (tish) rat, which is a genetic model of heightened seizure susceptibility associated with a prominent neocortical malformation. Both normally positioned (normotopic) and misplaced (heterotopic) pyramidal neurons in the tish neocortex exhibited lower resting membrane potentials and a tendency toward higher input resistance compared with pyramidal neurons from control brains. GABAergic synaptic transmission was attenuated in the tish cortex, characterized by significant reductions in the frequency of spontaneous IPSCs (sIPSCs) and miniature IPSCs recorded from pyramidal neurons. In addition, the amplitudes of sIPSCs were reduced in the tish neocortex, an effect that was more profound in the normotopic cells. Immunohistochemical assessment of presynaptic GABAergic terminals showed a reduction in terminals surrounding pyramidal cell somata in normotopic and heterotopic tish neocortex. The attenuation of inhibitory innervation was more prominent for normotopic neurons and was associated with a reduction in a subset of GABAergic interneurons expressing the calcium-binding protein parvalbumin. Together, these findings indicate that key facets of inhibitory GABAergic neurotransmission are disturbed before seizure onset in a brain predisposed to developing seizures. Such alterations represent a rational substrate for reduced seizure thresholds associated with certain cortical malformations.

  2. Dopamine-deprived striatal GABAergic interneurons burst and generate repetitive gigantic IPSCs in medium spiny neurons.

    Dehorter, Nathalie; Guigoni, Celine; Lopez, Catherine; Hirsch, June; Eusebio, Alexandre; Ben-Ari, Yehezkel; Hammond, Constance


    Striatal GABAergic microcircuits modulate cortical responses and movement execution in part by controlling the activity of medium spiny neurons (MSNs). How this is altered by chronic dopamine depletion, such as in Parkinson's disease, is not presently understood. We now report that, in dopamine-depleted slices of the striatum, MSNs generate giant spontaneous postsynaptic GABAergic currents (single or in bursts at 60 Hz) interspersed with silent episodes, rather than the continuous, low-frequency GABAergic drive (5 Hz) observed in control MSNs. This shift was observed in one-half of the MSN population, including both "D(1)-negative" and "D(1)-positive" MSNs. Single GABA and NMDA channel recordings revealed that the resting membrane potential and reversal potential of GABA were similar in control and dopamine-depleted MSNs, and depolarizing, but not excitatory, actions of GABA were observed. Glutamatergic and cholinergic antagonists did not block the GABAergic oscillations, suggesting that they were generated by GABAergic neurons. In support of this, cell-attached recordings revealed that a subpopulation of intrastriatal GABAergic interneurons generated bursts of spikes in dopamine-deprived conditions. This subpopulation included low-threshold spike interneurons but not fast-spiking interneurons, cholinergic interneurons, or MSNs. Therefore, a population of local GABAergic interneurons shifts from tonic to oscillatory mode when dopamine deprived and gives rise to spontaneous repetitive giant GABAergic currents in one-half the MSNs. We suggest that this may in turn alter integration of cortical signals by MSNs.

  3. Nerve Growth Factor is Primarily Produced by GABAergic Neurons of the Rat Neocortex

    Jeremy eBiane


    Full Text Available Within the cortex, nerve growth factor (NGF mediates the innervation of cholinergic neurons during development, maintains cholinergic corticopetal projections during adulthood and modulates cholinergic function through phenotypic control of the cholinergic gene locus. Recent studies suggest NGF may also play an important role in cortical plasticity in adulthood. Previously, NGF-producing cells have been shown to colocalize with GABAergic cell markers within the hippocampus, striatum, and basal forebrain. Classification of cells producing NGF in the cortex is lacking, however, and cholinergic corticopetal projections have been shown to innervate both pyramidal and GABAergic neurons in the cortex. In order to clarify potential trophic interactions between cortical neurons and cholinergic projections, we used double-fluorescent immunohistochemistry to classify NGF-expressing cells in several cortical regions, including the prefrontal cortex, primary motor cortex, parietal cortex and temporal cortex. Our results show that NGF colocalizes extensively with GABAergic cell markers in all cortical regions examined, with >91% of NGF-labeled cells coexpressing GAD65/67. Conversely, NGF-labeled cells exhibit very little co-localization with the excitatory cell marker CaMKIIα (less than 5% of cells expressing NGF. NGF expression was present in 56% of GAD-labeled cells, suggesting that production is confined to a specific subset of GABAergic neurons. These findings demonstrate that GABAergic cells are the primary source of NGF production in the cortex, and likely support the maintenance and function of basal forebrain cholinergic projections in adulthood.

  4. New insights into the classification and nomenclature of cortical GABAergic interneurons

    DeFelipe, Javier; López-Cruz, Pedro L.; Benavides-Piccione, Ruth; Bielza, Concha; Larrañaga, Pedro; Anderson, Stewart; Burkhalter, Andreas; Cauli, Bruno; Fairén, Alfonso; Feldmeyer, Dirk; Fishell, Gord; Fitzpatrick, David; Freund, Tamás F.; González-Burgos, Guillermo; Hestrin, Shaul; Hill, Sean; Hof, Patrick R.; Huang, Josh; Jones, Edward G.; Kawaguchi, Yasuo; Kisvárday, Zoltán; Kubota, Yoshiyuki; Lewis, David A.; Marín, Oscar; Markram, Henry; McBain, Chris J.; Meyer, Hanno S.; Monyer, Hannah; Nelson, Sacha B.; Rockland, Kathleen; Rossier, Jean; Rubenstein, John L. R.; Rudy, Bernardo; Scanziani, Massimo; Shepherd, Gordon M.; Sherwood, Chet C.; Staiger, Jochen F.; Tamás, Gábor; Thomson, Alex; Wang, Yun; Yuste, Rafael; Ascoli, Giorgio A.


    A systematic classification and accepted nomenclature of neuron types is much needed but is currently lacking. This article describes a possible taxonomical solution for classifying GABAergic interneurons of the cerebral cortex based on a novel, web-based interactive system that allows experts to classify neurons with pre-determined criteria. Using Bayesian analysis and clustering algorithms on the resulting data, we investigated the suitability of several anatomical terms and neuron names for cortical GABAergic interneurons. Moreover, we show that supervised classification models could automatically categorize interneurons in agreement with experts’ assignments. These results demonstrate a practical and objective approach to the naming, characterization and classification of neurons based on community consensus. PMID:23385869

  5. Neuronal diversity in GABAergic long-range projections from the hippocampus.

    Jinno, Shozo; Klausberger, Thomas; Marton, Laszlo F; Dalezios, Yannis; Roberts, J David B; Fuentealba, Pablo; Bushong, Eric A; Henze, Darrell; Buzsáki, György; Somogyi, Peter


    The formation and recall of sensory, motor, and cognitive representations require coordinated fast communication among multiple cortical areas. Interareal projections are mainly mediated by glutamatergic pyramidal cell projections; only few long-range GABAergic connections have been reported. Using in vivo recording and labeling of single cells and retrograde axonal tracing, we demonstrate novel long-range GABAergic projection neurons in the rat hippocampus: (1) somatostatin- and predominantly mGluR1alpha-positive neurons in stratum oriens project to the subiculum, other cortical areas, and the medial septum; (2) neurons in stratum oriens, including somatostatin-negative ones; and (3) trilaminar cells project to the subiculum and/or other cortical areas but not the septum. These three populations strongly increase their firing during sharp wave-associated ripple oscillations, communicating this network state to the septotemporal system. Finally, a large population of somatostatin-negative GABAergic cells in stratum radiatum project to the molecular layers of the subiculum, presubiculum, retrosplenial cortex, and indusium griseum and fire rhythmically at high rates during theta oscillations but do not increase their firing during ripples. The GABAergic projection axons have a larger diameter and thicker myelin sheet than those of CA1 pyramidal cells. Therefore, rhythmic IPSCs are likely to precede the arrival of excitation in cortical areas (e.g., subiculum) that receive both glutamatergic and GABAergic projections from the CA1 area. Other areas, including the retrosplenial cortex, receive only rhythmic GABAergic CA1 input. We conclude that direct GABAergic projections from the hippocampus to other cortical areas and the septum contribute to coordinating oscillatory timing across structures.

  6. Glutamate alteration of glutamic acid decarboxylase (GAD) in GABAergic neurons: the role of cysteine proteases.

    Monnerie, Hubert; Le Roux, Peter D


    Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation

  7. Adenosine Inhibits the Excitatory Synaptic Inputs to Basal Forebrain Cholinergic, GABAergic and Parvalbumin Neurons in mice

    Chun eYang


    Full Text Available Coffee and tea contain the stimulants caffeine and theophylline. These compounds act as antagonists of adenosine receptors. Adenosine promotes sleep and its extracellular concentration rises in association with prolonged wakefulness, particularly in the basal forebrain (BF region involved in activating the cerebral cortex. However, the effect of adenosine on identified BF neurons, especially non-cholinergic neurons, is incompletely understood. Here we used whole-cell patch-clamp recordings in mouse brain slices prepared from two validated transgenic mouse lines with fluorescent proteins expressed in GABAergic or parvalbumin (PV neurons to determine the effect of adenosine. Whole-cell recordings were made BF cholinergic neurons and from BF GABAergic & PV neurons with the size (>20 µm and intrinsic membrane properties (prominent H-currents corresponding to cortically projecting neurons. A brief (2 min bath application of adenosine (100 μM decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents in all groups of BF cholinergic, GABAergic and PV neurons we recorded. In addition, adenosine decreased the frequency of miniature EPSCs in BF cholinergic neurons. Adenosine had no effect on the frequency of spontaneous inhibitory postsynaptic currents in cholinergic neurons or GABAergic neurons with large H-currents but reduced them in a group of GABAergic neurons with smaller H-currents. All effects of adenosine were blocked by a selective, adenosine A1 receptor antagonist, cyclopentyltheophylline (CPT, 1 μM. Adenosine had no postsynaptic effects. Taken together, our work suggests that adenosine promotes sleep by an A1-receptor mediated inhibition of glutamatergic inputs to cortically-projecting cholinergic and GABA/PV neurons. Conversely, caffeine and theophylline promote attentive wakefulness by inhibiting these A1 receptors in BF thereby promoting the high-frequency oscillations in the cortex required for

  8. Chronic stress affects the number of GABAergic neurons in the orbitofrontal cortex of rats.

    Varga, Zsófia; Csabai, Dávid; Miseta, Attila; Wiborg, Ove; Czéh, Boldizsár


    Cortical GABAergic dysfunctions have been documented by clinical studies in major depression. We used here an animal model for depression and investigated whether long-term stress exposure can affect the number of GABAergic neurons in the orbitofrontal cortex (OFC). Adult male rats were subjected to 7-weeks of daily stress exposure and behaviorally phenotyped as anhedonic or stress-resilient animals. GABAergic interneurons were identified by immunohistochemistry and systematically quantified. We analyzed calbindin-(CB), calretinin-(CR), cholecystokinin-(CCK), parvalbumin-(PV), neuropeptide Y-(NPY) and somatostatin-positive (SST+) neurons in the following specific subareas of the OFC: medial orbital (MO), ventral orbital (VO), lateral orbital (LO) and dorsolateral orbital (DLO) cortex. For comparison, we also analyzed the primary motor cortex (M1) as a non-limbic cortical area. Stress had a pronounced effect on CB+ neurons and reduced their densities by 40-50% in the MO, VO and DLO. Stress had no effect on CCK+, CR+, PV+, NPY+ and SST+ neurons in any cortical areas. None of the investigated GABAergic neurons were affected by stress in the primary motor cortex. Interestingly, in the stress-resilient animals, we observed a significantly increased density of CCK+ neurons in the VO. NPY+ neuron densities were also significantly different between the anhedonic and stress-resilient rats, but only in the LO. Our present data demonstrate that chronic stress can specifically reduce the density of calbindin-positive GABAergic neurons in the orbitofrontal cortex and suggest that NPY and CCK expression in the OFC may relate to the stress resilience of the animals. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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


    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

  10. Identification of rat ventral tegmental area GABAergic neurons.

    Elyssa B Margolis

    Full Text Available The canonical two neuron model of opioid reward posits that mu opioid receptor (MOR activation produces reward by disinhibiting midbrain ventral tegmental area (VTA dopamine neurons through inhibition of local GABAergic interneurons. Although indirect evidence supports the neural circuit postulated by this model, its validity has been called into question by growing evidence for VTA neuronal heterogeneity and the recent demonstration that MOR agonists inhibit GABAergic terminals in the VTA arising from extrinsic neurons. In addition, VTA MOR reward can be dopamine-independent. To directly test the assumption that MOR activation directly inhibits local GABAergic neurons, we investigated the properties of rat VTA GABA neurons directly identified with either immunocytochemistry for GABA or GAD65/67, or in situ hybridization for GAD65/67 mRNA. Utilizing co-labeling with an antibody for the neural marker NeuN and in situ hybridization against GAD65/67, we found that 23±3% of VTA neurons are GAD65/67(+. In contrast to the assumptions of the two neuron model, VTA GABAergic neurons are heterogeneous, both physiologically and pharmacologically. Importantly, only 7/13 confirmed VTA GABA neurons were inhibited by the MOR selective agonist DAMGO. Interestingly, all confirmed VTA GABA neurons were insensitive to the GABA(B receptor agonist baclofen (0/6 inhibited, while all confirmed dopamine neurons were inhibited (19/19. The heterogeneity of opioid responses we found in VTA GABAergic neurons, and the fact that GABA terminals arising from neurons outside the VTA are inhibited by MOR agonists, make further studies essential to determine the local circuit mechanisms underlying VTA MOR reward.

  11. Control of REM sleep by ventral medulla GABAergic neurons.

    Weber, Franz; Chung, Shinjae; Beier, Kevin T; Xu, Min; Luo, Liqun; Dan, Yang


    Rapid eye movement (REM) sleep is a distinct brain state characterized by activated electroencephalogram and complete skeletal muscle paralysis, and is associated with vivid dreams. Transection studies by Jouvet first demonstrated that the brainstem is both necessary and sufficient for REM sleep generation, and the neural circuits in the pons have since been studied extensively. The medulla also contains neurons that are active during REM sleep, but whether they play a causal role in REM sleep generation remains unclear. Here we show that a GABAergic (γ-aminobutyric-acid-releasing) pathway originating from the ventral medulla powerfully promotes REM sleep in mice. Optogenetic activation of ventral medulla GABAergic neurons rapidly and reliably initiated REM sleep episodes and prolonged their durations, whereas inactivating these neurons had the opposite effects. Optrode recordings from channelrhodopsin-2-tagged ventral medulla GABAergic neurons showed that they were most active during REM sleep (REMmax), and during wakefulness they were preferentially active during eating and grooming. Furthermore, dual retrograde tracing showed that the rostral projections to the pons and midbrain and caudal projections to the spinal cord originate from separate ventral medulla neuron populations. Activating the rostral GABAergic projections was sufficient for both the induction and maintenance of REM sleep, which are probably mediated in part by inhibition of REM-suppressing GABAergic neurons in the ventrolateral periaqueductal grey. These results identify a key component of the pontomedullary network controlling REM sleep. The capability to induce REM sleep on command may offer a powerful tool for investigating its functions.

  12. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons.

    Stefanie Besser

    Full Text Available GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65. TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.

  13. Lineage-specific laminar organization of cortical GABAergic interneurons.

    Ciceri, Gabriele; Dehorter, Nathalie; Sols, Ignasi; Huang, Z Josh; Maravall, Miguel; Marín, Oscar


    In the cerebral cortex, pyramidal cells and interneurons are generated in distant germinal zones, and so the mechanisms that control their precise assembly into specific microcircuits remain an enigma. Here we report that cortical interneurons labeled at the clonal level do not distribute randomly but rather have a strong tendency to cluster in the mouse neocortex. This behavior is common to different classes of interneurons, independently of their origin. Interneuron clusters are typically contained within one or two adjacent cortical layers, are largely formed by isochronically generated neurons and populate specific layers, as revealed by unbiased hierarchical clustering methods. Our results suggest that different progenitor cells give rise to interneurons populating infra- and supragranular cortical layers, which challenges current views of cortical neurogenesis. Thus, specific lineages of cortical interneurons seem to be produced to primarily mirror the laminar structure of the cerebral cortex, rather than its columnar organization.

  14. Loss of MeCP2 From Forebrain Excitatory Neurons Leads to Cortical Hyperexcitation and Seizures

    Zhang, Wen; Peterson, Matthew; Beyer, Barbara; Frankel, Wayne N.


    Mutations of MECP2 cause Rett syndrome (RTT), a neurodevelopmental disorder leading to loss of motor and cognitive functions, impaired social interactions, and seizure at young ages. Defects of neuronal circuit development and function are thought to be responsible for the symptoms of RTT. The majority of RTT patients show recurrent seizures, indicating that neuronal hyperexcitation is a common feature of RTT. However, mechanisms underlying hyperexcitation in RTT are poorly understood. Here we show that deletion of Mecp2 from cortical excitatory neurons but not forebrain inhibitory neurons in the mouse leads to spontaneous seizures. Selective deletion of Mecp2 from excitatory but not inhibitory neurons in the forebrain reduces GABAergic transmission in layer 5 pyramidal neurons in the prefrontal and somatosensory cortices. Loss of MeCP2 from cortical excitatory neurons reduces the number of GABAergic synapses in the cortex, and enhances the excitability of layer 5 pyramidal neurons. Using single-cell deletion of Mecp2 in layer 2/3 pyramidal neurons, we show that GABAergic transmission is reduced in neurons without MeCP2, but is normal in neighboring neurons with MeCP2. Together, these results suggest that MeCP2 in cortical excitatory neurons plays a critical role in the regulation of GABAergic transmission and cortical excitability. PMID:24523563

  15. Properties of persistent postnatal cortical subplate neurons.

    Torres-Reveron, Juan; Friedlander, Michael J


    Subplate (SP) neurons are important for the proper development of thalamocortical innervation. They are necessary for formation of ocular dominance and orientation columns in visual cortex. During the perinatal period, many SP neurons die. The surviving cohort forms interstitial cells in the white matter (WM) and a band of horizontally oriented cells below layer VI (layer VIb, layer VII, or subplate cells). Although the function of embryonic SP neurons has been well established, the functional roles of WM and postnatal SP cells are not known. We used a combination of anatomical, immunohistochemical, and electrophysiological techniques to explore the dendritic morphology, neurotransmitter phenotype, intrinsic electrophysiological, and synaptic input properties of these surviving cells in the rat visual cortex. The density of SP and WM cells significantly decreases during the first month of life. Both populations express neuronal markers and have extensive dendritic arborizations within the SP, WM, and to the overlying visual cortex. Some intrinsic electrophysiological properties of SP and WM cells are similar: each generates high-frequency slowly adapting trains of action potentials in response to a sustained depolarization. However, SP cells exhibit greater frequency-dependent action potential broadening than WM neurons. Both cell types receive predominantly AMPA/kainate receptor-mediated excitatory synaptic input that undergoes paired-pulse facilitation as well as NMDA receptor and GABAergic input. Synaptic inputs to these cells can also undergo long-term synaptic plasticity. Thus, surviving SP and WM cells are functional electrogenic neurons integrated within the postnatal visual cortical circuit.

  16. Cortical GABAergic Interneurons in Cross-Modal Plasticity following Early Blindness

    Sébastien Desgent


    Full Text Available Early loss of a given sensory input in mammals causes anatomical and functional modifications in the brain via a process called cross-modal plasticity. In the past four decades, several animal models have illuminated our understanding of the biological substrates involved in cross-modal plasticity. Progressively, studies are now starting to emphasise on cell-specific mechanisms that may be responsible for this intermodal sensory plasticity. Inhibitory interneurons expressing γ-aminobutyric acid (GABA play an important role in maintaining the appropriate dynamic range of cortical excitation, in critical periods of developmental plasticity, in receptive field refinement, and in treatment of sensory information reaching the cerebral cortex. The diverse interneuron population is very sensitive to sensory experience during development. GABAergic neurons are therefore well suited to act as a gate for mediating cross-modal plasticity. This paper attempts to highlight the links between early sensory deprivation, cortical GABAergic interneuron alterations, and cross-modal plasticity, discuss its implications, and further provide insights for future research in the field.

  17. Reduced GABAergic inhibition explains cortical hyperexcitability in the wobbler mouse model of ALS

    Nieto-Gonzalez, Jose Luis; Moser, Jakob; Lauritzen, Martin


    Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease of the central nervous system. Symptomatic and presymptomatic ALS patients demonstrate cortical hyperexcitability, which raises the possibility that alterations in inhibitory gamma-aminobutyric acid (GABA)ergic system could...

  18. Multiple distinct subtypes of GABAergic neurons in mouse visual cortex identified by triple immunostaining

    Yuri Gonchar


    Full Text Available The majority of cortical interneurons use GABA (gamma amino butyric acid as inhibitory neurotransmitter. GABAergic neurons are morphologically, connectionally, electrically and chemically heterogeneous. In rat cerebral cortex three distinct groups of GABAergic interneurons have been identifi ed by the expression of parvalbumin (PV, calretinin (CR and somatostatin (SOM. Recent studies in mouse cerebral cortex have revealed a different organization in which the CR and SOM populations are partially overlapping. Because CR and SOM neurons derive from different progenitors located in different embryonic structures, the coexpression of CR + SOM suggests that the chemical differentiation of interneurons is regulated postmitotically. Here, we have taken an important fi rst step towards understanding this process by triple immunostaining mouse visual cortex with a panel of antibodies, which has been used extensively for classifying developing interneurons. We have found at least 13 distinct groups of GABAergic neurons which include PV, CR, SOM, CCK (cholecystokinin, CR + SOM, CR + NPY (neuropeptide Y, CR + VIP (vasointestinal polypeptide, SOM + NPY, SOM + VIP, VIP + ChAT (choline acetyltransferase, CCK + NPY, CR + SOM + NPY and CR + SOM + VIP expressing cells. Triple immunostaining with PV, CR and SOM antibodies during postnatal development further showed that PV is never colocalized with CR and SOM. Importantly, expression of SOM and CR + SOM developed after the percentage of CR cells that do not express SOM has reached the mature level, suggesting that the chemical differentiation of SOM and CR + SOM neurons is a postnatal event, which may be controlled by transcriptional regulation.

  19. Layer-specific endocannabinoid-mediated long-term depression of GABAergic neurotransmission onto principal neurons in mouse visual cortex.

    Sun, Wenjuan; Wang, Laijian; Li, Shuo; Tie, Xiaoxiu; Jiang, Bin


    Visually induced endocannabinoid-mediated long-term depression of GABAergic neurotransmission (iLTD) mediates the maturation of GABAergic release in layer 2/3 of visual cortex. Here we examined whether the maturation of GABAergic transmission in other layers of visual cortex also requires endocannabinoids. The developmental plasticity of GABAergic neurotransmission onto the principal neurons in different layers of mouse visual cortex was examined in cortical slices by whole-cell recordings of inhibitory postsynaptic currents evoked by presynaptic inhibitory inputs. Theta burst stimulation of GABAergic inputs induced an endocannabinoid-mediated long-term depression of GABAergic neurotransmission onto pyramidal cells in layer 2/3 from postnatal day (P)10 to 30 and in layer 5 from P10 to 40, whereas that of GABAergic inputs did not induce iLTD onto star pyramidal neurons in layer 4 at any time postnatally, indicating that this plasticity is laminar-specific. The developmental loss of iLTD paralleled the maturation of GABAergic inhibition in both layer 2/3 and layer 5. Visual deprivation delayed the developmental loss of iLTD in layers 3 and 5 during a critical period, while 2 days of light exposure eliminated iLTD in both layers. Furthermore, the GABAergic synapses in layers 2/3 and 5 did not normally mature in the type 1 cannabinoid receptor knock-out mice, whereas those in layer 4 did not require endocannabinoid receptor for maturation. These results suggest that visually induced endocannabinoid-dependent iLTD mediates the maturation of GABAergic release in extragranular layer rather than in granular layer of mouse visual cortex.

  20. Dynamic GABAergic afferent modulation of AgRP neurons

    Garfield, Alastair S; Shah, Bhavik P; Burgess, Christian R; Li, Monica M; Li, Chia; Steger, Jennifer S; Madara, Joseph C; Campbell, John N; Kroeger, Daniel; Scammell, Thomas E; Tannous, Bakhos A; Myers, Martin G; Andermann, Mark L; Krashes, Michael J; Lowell, Bradford B


    Agouti-related peptide (AgRP) neurons of the arcuate nucleus of the hypothalamus (ARC) promote homeostatic feeding at times of caloric insufficiency, yet they are rapidly suppressed by food-related sensory cues prior to ingestion. Here we identify a highly selective inhibitory afferent to AgRP neurons that serves as a neural determinant of this rapid modulation. Specifically, GABAergic projections arising from the ventral compartment of the dorsomedial nucleus of the hypothalamus (vDMH) contribute to the pre-consummatory modulation of ARCAgRP neurons. In a manner reciprocal to ARCAgRP neurons, ARC-projecting leptin receptor (LepR)-expressing GABAergic DMH neurons exhibit rapid activation upon availability of food that additionally reflects the relative value of the food. Thus, DMHLepR neurons form part of the sensory network that relays real-time information about the nature and availability of food to dynamically modulate ARCAgRP neuron activity and feeding behavior. PMID:27643429

  1. Reelin secreted by GABAergic neurons regulates glutamate receptor homeostasis.

    Cecilia Gonzalez Campo

    Full Text Available BACKGROUND: Reelin is a large secreted protein of the extracellular matrix that has been proposed to participate to the etiology of schizophrenia. During development, reelin is crucial for the correct cytoarchitecture of laminated brain structures and is produced by a subset of neurons named Cajal-Retzius. After birth, most of these cells degenerate and reelin expression persists in postnatal and adult brain. The phenotype of neurons that bind secreted reelin and whether the continuous secretion of reelin is required for physiological functions at postnatal stages remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Combining immunocytochemical and pharmacological approaches, we first report that two distinct patterns of reelin expression are present in cultured hippocampal neurons. We show that in hippocampal cultures, reelin is secreted by GABAergic neurons displaying an intense reelin immunoreactivity (IR. We demonstrate that secreted reelin binds to receptors of the lipoprotein family on neurons with a punctate reelin IR. Secondly, using calcium imaging techniques, we examined the physiological consequences of reelin secretion blockade. Blocking protein secretion rapidly and reversibly changes the subunit composition of N-methyl-D-aspartate glutamate receptors (NMDARs to a predominance of NR2B-containing NMDARs. Addition of recombinant or endogenously secreted reelin rescues the effects of protein secretion blockade and reverts the fraction of NR2B-containing NMDARs to control levels. Therefore, the continuous secretion of reelin is necessary to control the subunit composition of NMDARs in hippocampal neurons. CONCLUSIONS/SIGNIFICANCE: Our data show that the heterogeneity of reelin immunoreactivity correlates with distinct functional populations: neurons synthesizing and secreting reelin and/or neurons binding reelin. Furthermore, we show that continuous reelin secretion is a strict requirement to maintain the composition of NMDARs. We propose

  2. Releasing the Cortical Brake by Non-Invasive Electromagnetic Stimulation? rTMS Induces LTD of GABAergic Neurotransmission.

    Lenz, Maximilian; Vlachos, Andreas


    Repetitive Transcranial Magnetic Stimulation (rTMS) is a non-invasive brain stimulation technique which modulates cortical excitability beyond the stimulation period. However, despite its clinical use rTMS-based therapies which prevent or reduce disabilities in a functionally significant and sustained manner are scarce. It remains unclear how rTMS-mediated changes in cortical excitability, which are not task- or input-specific, exert beneficial effects in some healthy subjects and patients. While experimental evidence exists that repetitive magnetic stimulation (rMS) is linked to the induction of long-term potentiation (LTP) of excitatory neurotransmission, less attention has been dedicated to rTMS-induced structural, functional and molecular adaptations at inhibitory synapses. In this review article we provide a concise overview on basic neuroscience research, which reveals an important role of local disinhibitory networks in promoting associative learning and memory. These studies suggest that a reduction in inhibitory neurotransmission facilitates the expression of associative plasticity in cortical networks under physiological conditions. Hence, it is interesting to speculate that rTMS may act by decreasing GABAergic neurotransmission onto cortical principal neurons. Indeed, evidence has been provided that rTMS is capable of modulating inhibitory networks. Consistent with this suggestion recent basic science work discloses that a 10 Hz rTMS protocol reduces GABAergic synaptic strength on principal neurons. These findings support a model in which rTMS-induced long-term depression (LTD) of GABAergic synaptic strength mediates changes in excitation/inhibition-balance of cortical networks, which may in turn facilitate (or restore) the ability of stimulated networks to express input- and task-specific associative synaptic plasticity.

  3. GABAergic actions on cholinergic laterodorsal tegmental neurons

    Kohlmeier, K A; Kristiansen, Uffe


    Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) play a critical role in regulation of behavioral state. Therefore, elucidation of mechanisms that control their activity is vital for understanding of how switching between wakefulness, sleep and anesthetic states is effectuated....... Therefore, we studied the actions of GABA agonists and antagonists on cholinergic LDT cells by performing patch clamp recordings in mouse brain slices. Under conditions where detection of Cl(-) -mediated events was optimized, GABA induced gabazine (GZ)-sensitive inward currents in the majority of LDT...

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

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


    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.

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

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


    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

  6. Late postnatal shifts of parvalbumin and nitric oxide synthase expression within the GABAergic and glutamatergic phenotypes of inferior colliculus neurons.

    Fujimoto, Hisataka; Konno, Kotaro; Watanabe, Masahiko; Jinno, Shozo


    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.

  7. 3D Clustering of GABAergic Neurons Enhances Inhibitory Actions on Excitatory Neurons in the Mouse Visual Cortex

    Teppei Ebina


    Full Text Available Neocortical neurons with similar functional properties assemble into spatially coherent circuits, but it remains unclear how inhibitory interneurons are organized. We applied in vivo two-photon functional Ca2+ imaging and whole-cell recording of synaptic currents to record visual responses of cortical neurons and analyzed their spatial arrangements. GABAergic interneurons were clustered in the 3D space of the mouse visual cortex, and excitatory neurons located within the clusters (insiders had a lower amplitude and sharper orientation tuning of visual responses than outsiders. Inhibitory synaptic currents recorded from the insiders were larger than those of the outsiders. Single, isolated interneurons did not show such a location-tuning/amplitude relationship. The two principal subtypes of interneurons, parvalbumin- and somatostatin-expressing neurons, also formed clusters with only slightly overlapping each other and exhibited a different location-tuning relationship. These findings suggest that GABAergic interneurons and their subgroups form clusters to make their inhibitory function more effective than isolated interneurons.

  8. Multiple embryonic origins of nitric oxide synthase-expressing GABAergic neurons of the neocortex

    Lorenza eMagno


    Full Text Available Cortical GABAergic interneurons in rodents originate in three subcortical regions: the medial ganglionic eminence (MGE, the lateral/caudal ganglionic eminence (LGE/CGE and the preoptic area (POA. Each of these neuroepithelial precursor domains contributes different interneuron subtypes to the cortex. nNOS-expressing neurons represent a heterogenous population of cortical interneurons. We examined the development of these cells in the mouse embryonic cortex and their abundance and distribution in adult animals. Using genetic lineage tracing in transgenic mice we find that nNOS type I cells originate only in the MGE whereas type II cells have a triple origin in the MGE, LGE/CGE and POA. The two populations are born at different times during development, occupy different layers in the adult cortex and have distinct neurochemical profiles. nNOS neurons are more numerous in the adult cortex than previously reported and constitute a significant proportion of the cortical interneuron population. Our data suggest that the heterogeneity of nNOS neurons in the cortex can be attributed to their multiple embryonic origins which likely impose distinct genetic specification programs.

  9. The origin of cortical neurons

    J.G. Parnavelas


    Full Text Available Neurons of the mammalian cerebral cortex comprise two broad classes: pyramidal neurons, which project to distant targets, and the inhibitory nonpyramidal cells, the cortical interneurons. Pyramidal neurons are generated in the germinal ventricular zone, which lines the lateral ventricles, and migrate along the processes of radial glial cells to their positions in the developing cortex in an `inside-out' sequence. The GABA-containing nonpyramidal cells originate for the most part in the ganglionic eminence, the primordium of the basal ganglia in the ventral telencephalon. These cells follow tangential migratory routes to enter the cortex and are in close association with the corticofugal axonal system. Once they enter the cortex, they move towards the ventricular zone, possibly to obtain positional information, before they migrate radially in the direction of the pial surface to take up their positions in the developing cortex. The mechanisms that guide interneurons throughout these long and complex migratory routes are currently under investigation.

  10. SAT1, a glutamine transporter, is preferentially expressed in GABAergic neurons

    Tom Tallak Solbu


    Full Text Available Subsets of GABAergic neurons are able to maintain high frequency discharge patterns, which requires efficient replenishment of the releasable pool of GABA. Although glutamine is considered a preferred precursor of GABA, the identity of transporters involved in glutamine uptake by GABAergic neurons remains elusive. Molecular analyses revealed that SAT1 (Slc38a1 features system A characteristics with a preferential affinity for glutamine, and that SAT1 mRNA expression is associated with GABAergic neurons. By generating specific antibodies against SAT1 we show that this glutamine carrier is particularly enriched in GABAergic neurons. Cellular SAT1 distribution resembles that of GAD67, an essential GABA synthesis enzyme, suggesting that SAT1 can be involved in translocating glutamine into GABAergic neurons to facilitate inhibitory neurotransmitter generation.

  11. Transcriptional control of GABAergic neuron development in the dorsal spinal cord

    Huang Jing; Wu Shengxi


    GABAergic neurons are the major inhibitory interneurons that powerfully control the function of spinal neuronalnet works. In recent years, tremendous progresses have been made in understanding the transcriptional control of GABAergic neuron development in the dorsal spinal cord. New experimental approaches provide a relatively high throughput way to study the molecular regulation of subgroup fate determination. Our understanding of the molecular mechanisms on GABAergic neuron development in the dorsal spinal cord is rapidly expanding. Recent studies have defined several transcription factors that play essential roles in GABAergic neuron development in the spinal dorsal horn. Here, we review results of very recent analyses of the mechanisms that specify the GABAergic neuron development in the dorsal spinal cord, especially the progresses in the homeodomain (HD) and basic-helix-loop-helix(bHLH) containing transcription factors.

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

    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

  13. Diversity among principal and GABAergic neurons of the anterior olfactory nucleus

    Rachel eKay


    Full Text Available Understanding the cellular components of neural circuits is an essential step in discerning regional function. The anterior olfactory nucleus (AON is reciprocally connected to both the ipsi- and contralateral olfactory bulb (OB and piriform cortex (PC, and, as a result, can broadly influence the central processing of odor information. While both the AON and PC are simple cortical structures, the regions differ in many ways including their general organization, internal wiring and synaptic connections with other brain areas. The present work used targeted whole-cell patch clamping to investigate the morphological and electrophysiological properties of the AON’s two main neuronal populations: excitatory projection neurons and inhibitory interneurons. Retrograde fluorescent tracers placed into either the OB or PC identified projection neurons. Two classes were observed with different physiological signatures and locations (superficial and deep pyramidal neurons, suggesting the AON contains independent efferent channels. Transgenic mice in which GABA-containing cells expressed green fluorescent protein were used to assess inhibitory neurons. These cells were further identified as containing one or more of seven molecular markers including three calcium-binding proteins (calbindin, calretinin, parvalbumin or four neuropeptides (somatostatin, vasoactive intestinal peptide, neuropeptide Y, cholecystokinin. The proportion of GABAergic cells containing these markers varied across subregions reinforcing notions that the AON has local functional subunits. At least five classes of inhibitory cells were observed: fast-spiking multipolar, regular-spiking multipolar, superficial neurogliaform, deep neurogliaform, and horizontal neurons. While some of these cell types are similar to those reported in the PC and other cortical regions, the AON also has unique populations. These studies provide the first examination of the cellular components of this simple

  14. Control of cortical neuronal migration by glutamate and GABA

    Heiko J Luhmann


    Full Text Available Neuronal migration in the cortex is controlled by the paracrine action of the classical neurotransmitters glutamate and GABA. Glutamate controls radial migration of pyramidal neurons by acting primarily on NMDA receptors and regulates tangential migration of inhibitory interneurons by activating non-NMDA and NMDA receptors. GABA, acting on ionotropic GABAA-rho and GABAA receptors, has a dichotomic action on radially migrating neurons by acting as a GO signal in lower layers and as a STOP signal in upper cortical plate (CP, respectively. Metabotropic GABAB receptors promote radial migration into the CP and tangential migration of interneurons. Besides GABA, the endogenous GABAergic agonist taurine is a relevant agonist controlling radial migration. To a smaller extent glycine receptor activation can also influence radial and tangential migration. Activation of glutamate and GABA receptors causes increases in intracellular Ca2+ transients, which promote neuronal migration by acting on the cytoskeleton. Pharmacological or genetic manipulation of glutamate or GABA receptors during early corticogenesis induce heterotopic cell clusters in upper layers and loss of cortical lamination, i.e. neuronal migration disorders which can be associated with neurological or neuropsychiatric diseases. The pivotal role of NMDA and ionotropic GABA receptors in cortical neuronal migration is of major clinical relevance, since a number of drugs acting on these receptors (e.g. anti-epileptics, anesthetics, alcohol may disturb the normal migration pattern when present during early corticogenesis.

  15. Spatio-temporal extension in site of origin for cortical calretinin neurons in primates

    Ana eHladnik


    Full Text Available The vast majority of cortical GABAergic neurons can be defined by parvalbumin, somatostatin or calretinin expression. In most mammalians parvalbumin and somatostatin interneurons have constant proportions, each representing 5-7% of the total neuron number. In contrast, there is a 3 fold increase in the proportion of calretinin interneurons, which do not exceed 4% in rodents and reach 12% in higher order areas of primate cerebral cortex. In rodents almost all parvalbumin and somatostatin interneurons originate from the medial part of the subpallial proliferative structure, the ganglionic eminence (GE, while almost all calretinin interneurons originate from its caudal part. The spatial pattern of cortical GABAergic neurons origin from the GE is preserved in the monkey and human brain. However, it could be expected that the evolution is changing developmental rules to enable considerable expansion of calretinin interneuron population. During the early fetal period in primates cortical GABAergic neurons are almost entirely generated in the subpallium, as in rodents. Already at that time the primate caudal ganglionic eminence (CGE shows a relative increase in size and production of calretinin interneurons. During the second trimester of gestation, that is the main neurogenetic stage in primates without clear correlates found in rodents, the pallial production of cortical GABAergic neurons together with the extended persistence of the GE is observed. We propose that the CGE could be the main source of calretinin interneurons for the posterior and lateral cortical regions, but not for the frontal cortex. The associative granular frontal cortex represents around one third of the cortical surface and contains almost half of cortical calretinin interneurons. The majority of calretinin interneurons destined for the frontal cortex could be generated in the pallium, especially in the newly evolved outer subventricular zone that becomes the main pool of

  16. Mesenchymal stem cells enhance GABAergic transmission in co-cultured hippocampal neurons.

    Mauri, Mario; Lentini, Daniela; Gravati, Marta; Foudah, Dana; Biella, Gerardo; Costa, Barbara; Toselli, Mauro; Parenti, Marco; Coco, Silvia


    Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent stem cells endowed with neurotrophic potential combined with immunological properties, making them a promising therapeutic tool for neurodegenerative disorders. However, the mechanisms through which MSCs promote the neurological recovery following injury or inflammation are still largely unknown, although cell replacement and paracrine mechanisms have been hypothesized. In order to find out what are the mechanisms of the trophic action of MSCs, as compared to glial cells, on CNS neurons, we set up a co-culture system where rat MSCs (or cortical astrocytes) were used as a feeding layer for hippocampal neurons without any direct contact between the two cell types. The analysis of hippocampal synaptogenesis, synaptic vesicle recycling and electrical activity show that MSCs were capable to support morphological and functional neuronal differentiation. The proliferation of hippocampal glial cells induced by the release of bioactive substance(s) from MSCs was necessary for neuronal survival. Furthermore, MSCs selectively increased hippocampal GABAergic pre-synapses. This effect was paralleled with a higher expression of the potassium/chloride KCC2 co-transporter and increased frequency and amplitude of mIPSCs and sIPSCs. The enhancement of GABA synapses was impaired by the treatment with K252a, a Trk/neurotrophin receptor blocker, and by TrkB receptor bodies hence suggesting the involvement of BDNF as a mediator of such effects. The results obtained here indicate that MSC-secreted factors induce glial-dependent neuronal survival and trigger an augmented GABAergic transmission in hippocampal cultures, highlighting a new effect by which MSCs could promote CNS repair. Our results suggest that MSCs may be useful in those neurological disorders characterized by an impairment of excitation versus inhibition balance.

  17. The neuronal identity bias behind neocortical GABAergic plasticity.

    Allene, Camille; Lourenço, Joana; Bacci, Alberto


    In the neocortex, different types of excitatory and inhibitory neurons connect to one another following a detailed blueprint, defining functionally-distinct subnetworks, whose activity and modulation underlie complex cognitive functions. We review the cell-autonomous plasticity of perisomatic inhibition onto principal excitatory neurons. We propose that the tendency of different cortical layers to exhibit depression or potentiation of perisomatic inhibition is dictated by the specific identities of principal neurons (PNs). These are mainly defined by their projection targets and by their preference to be innervated by specific perisomatic-targeting basket cell types. Therefore, principal neurons responsible for relaying information to subcortical nuclei are differentially inhibited and show specific forms of plasticity compared to other PNs that are specialized in more associative functions. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Wierda, Keimpe D B; Sørensen, Jakob Balslev


    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...

  19. Sodium salicylate suppresses GABAergic inhibitory activity in neurons of rodent dorsal raphe nucleus.

    Yan Jin

    Full Text Available Sodium salicylate (NaSal, a tinnitus inducing agent, can activate serotonergic (5-HTergic neurons in the dorsal raphe nucleus (DRN and can increase serotonin (5-HT level in the inferior colliculus and the auditory cortex in rodents. To explore the underlying neural mechanisms, we first examined effects of NaSal on neuronal intrinsic properties and the inhibitory synaptic transmissions in DRN slices of rats by using whole-cell patch-clamp technique. We found that NaSal hyperpolarized the resting membrane potential, decreased the input resistance, and suppressed spontaneous and current-evoked firing in GABAergic neurons, but not in 5-HTergic neurons. In addition, NaSal reduced GABAergic spontaneous and miniature inhibitory postsynaptic currents in 5-HTergic neurons. We next examined whether the observed depression of GABAergic activity would cause an increase in the excitability of 5-HTergic neurons using optogenetic technique in DRN slices of the transgenic mouse with channelrhodopsin-2 expressed in GABAergic neurons. When the GABAergic inhibition was enhanced by optical stimulation to GABAergic neurons in mouse DRN, NaSal significantly depolarized the resting membrane potential, increased the input resistance and increased current-evoked firing of 5-HTergic neurons. However, NaSal would fail to increase the excitability of 5-HTergic neurons when the GABAergic synaptic transmission was blocked by picrotoxin, a GABA receptor antagonist. Our results indicate that NaSal suppresses the GABAergic activities to raise the excitability of local 5-HTergic neural circuits in the DRN, which may contribute to the elevated 5-HT level by NaSal in the brain.


    E.S. Petrova


    Full Text Available Gamma-aminobutyric acid (GABA is a major inhibitory neurotransmitter in the central nervous system. Enzyme glutamate decarboxylase (GAD-67 is a marker of GABA-ergic neurons. The purpose of this study is to examine the distribution of GAD-67-immunopositive neurons in the striatum of rats under experimental conditions, reproducing brief focal cerebral ischemia. Endovascular occlusion of the left middle cerebral artery in rats was performed. Duration of circulatory disorders was 30 min, the time of reperfusion was 48 hours. With counting GAD-67-immunopositive neurons in the striatum was found that the number of GABA-ergic neurons in the striatum ipsilateral hemisphere is reduced by 40%. In the contralateral hemisphere, the distribution and structure of the neurons is not different from controls. It is shown that GABA-ergic neurons are less susceptible to damage, as compared to other neurons phenotypes.

  1. Ptf1a triggers GABAergic neuronal cell fates in the retina

    Parain Karine


    Full Text Available Abstract Background In recent years, considerable knowledge has been gained on the molecular mechanisms underlying retinal cell fate specification. However, hitherto studies focused primarily on the six major retinal cell classes (five types of neurons of one type of glial cell, and paid little attention to the specification of different neuronal subtypes within the same cell class. In particular, the molecular machinery governing the specification of the two most abundant neurotransmitter phenotypes in the retina, GABAergic and glutamatergic, is largely unknown. In the spinal cord and cerebellum, the transcription factor Ptf1a is essential for GABAergic neuron production. In the mouse retina, Ptf1a has been shown to be involved in horizontal and most amacrine neurons differentiation. Results In this study, we examined the distribution of neurotransmitter subtypes following Ptf1a gain and loss of function in the Xenopus retina. We found cell-autonomous dramatic switches between GABAergic and glutamatergic neuron production, concomitant with profound defects in the genesis of amacrine and horizontal cells, which are mainly GABAergic. Therefore, we investigated whether Ptf1a promotes the fate of these two cell types or acts directly as a GABAergic subtype determination factor. In ectodermal explant assays, Ptf1a was found to be a potent inducer of the GABAergic subtype. Moreover, clonal analysis in the retina revealed that Ptf1a overexpression leads to an increased ratio of GABAergic subtypes among the whole amacrine and horizontal cell population, highlighting its instructive capacity to promote this specific subtype of inhibitory neurons. Finally, we also found that within bipolar cells, which are typically glutamatergic interneurons, Ptf1a is able to trigger a GABAergic fate. Conclusion Altogether, our results reveal for the first time in the retina a major player in the GABAergic versus glutamatergic cell specification genetic pathway.

  2. Physiological and morphological characterization of GABAergic neurons in the medial amygdala.

    Bian, Xiling


    GABAergic neurons in the medial amygdala (MeA) have been indicated in information processing in reproductive behavior and fear/anxiety. However, basic knowledge of their physiological and morphological properties is still very limited, probably due to the technical challenge to selectively record the GABAergic neurons. In this study, I characterized properties of the MeA GABAergic neurons by performing whole-cell patch clamp recordings from brain slices of adult knock-in mice selectively expressing green fluorescence protein (GFP) in GABAergic neurons. The majority (73%) of GABAergic neurons exhibiting low threshold calcium spike were classified as type I neurons, with morphological properties of being bitufted or stellate, and dendrites either aspiny or covered by various shapes of spines. Axonal collaterals of some neurons were observed near somata as well as in other amygdaloid nuclei. Neurons incapable of generate low threshold calcium spikes were divided into two types. Type II neurons (11%) exhibited hyperpolarization-activated sag and higher input resistance (>400 MΩ). Most Type II neurons exhibited asymmetric dendritic trees extending towards the superficial layer covered with long neck dendritic spines. The axons of type II neurons formed large collaterals and projected to other amygdaloid nuclei. Type III neurons (16%) lack prominent hyperpolarization-activated sag and possessed lower input resistance (neurons were local interneurons with smooth multipolar dendritic trees. Since both MeA and nearby amygdaloid nuclei are involved in fear/anxiety processing, two types of MeA GABAergic projection neurons and a third type of interneurons that might participate in anxiety-related behavior were revealed by my present study.

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

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


    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

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

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


    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.

  5. Bayesian network classifiers for categorizing cortical GABAergic interneurons.

    Mihaljević, Bojan; Benavides-Piccione, Ruth; Bielza, Concha; DeFelipe, Javier; Larrañaga, Pedro


    An accepted classification of GABAergic interneurons of the cerebral cortex is a major goal in neuroscience. A recently proposed taxonomy based on patterns of axonal arborization promises to be a pragmatic method for achieving this goal. It involves characterizing interneurons according to five axonal arborization features, called F1-F5, and classifying them into a set of predefined types, most of which are established in the literature. Unfortunately, there is little consensus among expert neuroscientists regarding the morphological definitions of some of the proposed types. While supervised classifiers were able to categorize the interneurons in accordance with experts' assignments, their accuracy was limited because they were trained with disputed labels. Thus, here we automatically classify interneuron subsets with different label reliability thresholds (i.e., such that every cell's label is backed by at least a certain (threshold) number of experts). We quantify the cells with parameters of axonal and dendritic morphologies and, in order to predict the type, also with axonal features F1-F4 provided by the experts. Using Bayesian network classifiers, we accurately characterize and classify the interneurons and identify useful predictor variables. In particular, we discriminate among reliable examples of common basket, horse-tail, large basket, and Martinotti cells with up to 89.52% accuracy, and single out the number of branches at 180 μm from the soma, the convex hull 2D area, and the axonal features F1-F4 as especially useful predictors for distinguishing among these types. These results open up new possibilities for an objective and pragmatic classification of interneurons.

  6. Different correlation patterns of cholinergic and GABAergic interneurons with striatal projection neurons

    Avital eAdler


    Full Text Available The striatum is populated by a single projection neuron group, the medium spiny neurons (MSNs, and several groups of interneurons. Two of the electrophysiologically well-characterized striatal interneuron groups are the tonically active neurons (TANs, which are presumably cholinergic interneurons, and the fast spiking interneurons (FSIs, presumably parvalbumin (PV expressing GABAergic interneurons. To better understand striatal processing it is thus crucial to define the functional relationship between MSNs and these interneurons in the awake and behaving animal. We used multiple electrodes and standard physiological methods to simultaneously record MSN spiking activity and the activity of TANs or FSIs from monkeys engaged in a classical conditioning paradigm. All three cell populations were highly responsive to the behavioral task. However, they displayed different average response profiles and a different degree of response synchronization (signal correlation. TANs displayed the most transient and synchronized response, MSNs the most diverse and sustained response and FSIs were in between on both parameters. We did not find evidence for direct monosynaptic connectivity between the MSNs and either the TANs or the FSIs. However, while the cross correlation histograms of TAN to MSN pairs were flat, those of FSI to MSN displayed positive asymmetrical broad peaks. The FSI-MSN correlogram profile implies that the spikes of MSNs follow those of FSIs and both are driven by a common, most likely cortical, input. Thus, the two populations of striatal interneurons are probably driven by different afferents and play complementary functional roles in the physiology of the striatal microcircuit.

  7. Nicotine increases GABAergic input on rat dorsal raphe serotonergic neurons through alpha7 nicotinic acetylcholine receptor.

    Hernández-Vázquez, F; Chavarría, K; Garduño, J; Hernández-López, S; Mihailescu, S P


    The dorsal raphe nucleus (DRN) contains large populations of serotonergic (5-HT) neurons. This nucleus receives GABAergic inhibitory afferents from many brain areas and from DRN interneurons. Both GABAergic and 5-HT DRN neurons express functional nicotinic acetylcholine receptors (nAChRs). Previous studies have demonstrated that nicotine increases 5-HT release and 5-HT DRN neuron discharge rate by stimulating postsynaptic nAChRs and by increasing glutamate and norepinephrine release inside DRN. However, the influence of nicotine on the GABAergic input to 5-HT DRN neurons was poorly investigated. Therefore, the aim of this work was to determine the effect of nicotine on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of 5-HT DRN neurons and the subtype of nAChR(s) involved in this response. Experiments were performed in coronal slices obtained from young Wistar rats. GABAergic sIPSCs were recorded from post hoc-identified 5-HT DRN neurons with the whole cell voltage patch-clamp technique. Administration of nicotine (1 μM) increased sIPSC frequency in 72% of identified 5-HT DRN neurons. This effect was not reproduced by the α4β2 nAChR agonist RJR-2403 and was not influenced by TTX (1 μM). It was mimicked by the selective agonist for α7 nAChR, PNU-282987, and exacerbated by the positive allosteric modulator of the same receptor, PNU-120596. The nicotine-induced increase in sIPSC frequency was independent on voltage-gated calcium channels and dependent on Ca(2+)-induced Ca(2+) release (CICR). These results demonstrate that nicotine increases the GABAergic input to most 5-HT DRN neurons, by activating α7 nAChRs and producing CICR in DRN GABAergic terminals.

  8. Local and commissural IC neurons make axosomatic inputs on large GABAergic tectothalamic neurons.

    Ito, Tetsufumi; Oliver, Douglas L


    Large GABAergic (LG) neurons are a distinct type of neuron in the inferior colliculus (IC) identified by their dense vesicular glutamate transporter 2 (VGLUT2)-containing axosomatic synaptic terminals. Yet the sources of these terminals are unknown. Since IC glutamatergic neurons express VGLUT2, and IC neurons are known to have local collaterals, we tested the hypothesis that these excitatory, glutamatergic axosomatic inputs on LG neurons come from local axonal collaterals and commissural IC neurons. We injected a recombinant viral tracer into the IC which enabled Golgi-like green fluorescent protein (GFP) labeling in both dendrites and axons. In all cases, we found terminals positive for both GFP and VGLUT2 (GFP+/VGLUT2+) that made axosomatic contacts on LG neurons. One to six axosomatic contacts were made on a single LG cell body by a single axonal branch. The GFP-labeled neurons giving rise to the VGLUT2+ terminals on LG neurons were close by. The density of GFP+/VGLUT2+ terminals on the LG neurons was related to the number of nearby GFP-labeled cells. On the contralateral side, a smaller number of LG neurons received axosomatic contacts from GFP+/VGLUT2+ terminals. In cases with a single GFP-labeled glutamatergic neuron, the labeled axonal plexus was flat, oriented in parallel to the fibrodendritic laminae, and contacted 9-30 LG cell bodies within the plexus. Our data demonstrated that within the IC microcircuitry there is a convergence of inputs from local IC excitatory neurons on LG cell bodies. This suggests that LG neurons are heavily influenced by the activity of the nearby laminar glutamatergic neurons in the IC.

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

    Wierda, Keimpe D B; Sørensen, Jakob B


    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.

  10. Unique functional properties of somatostatin-expressing GABAergic neurons in mouse barrel cortex.

    Gentet, L.J.; Kremer, Y.; Taniguchi, H.; Huang, Z.J.; Staiger, J.F.; Petersen, C.C.H.


    Neocortical GABAergic neurons have diverse molecular, structural and electrophysiological features, but the functional correlates of this diversity are largely unknown. We found unique membrane potential dynamics of somatostatin-expressing (SOM) neurons in layer 2/3 of the primary somatosensory

  11. GABAergic inputs from direct and indirect striatal projection neurons onto cholinergic interneurons in the primate putamen.

    Gonzales, Kalynda Kari; Pare, Jean-Francois; Wichmann, Thomas; Smith, Yoland


    Striatal cholinergic interneurons (ChIs) are involved in reward-dependent learning and the regulation of attention. The activity of these neurons is modulated by intrinsic and extrinsic γ-aminobutyric acid (GABA)ergic and glutamatergic afferents, but the source and relative prevalence of these diverse regulatory inputs remain to be characterized. To address this issue, we performed a quantitative ultrastructural analysis of the GABAergic and glutamatergic innervation of ChIs in the postcommissural putamen of rhesus monkeys. Postembedding immunogold localization of GABA combined with peroxidase immunostaining for choline acetyltransferase showed that 60% of all synaptic inputs to ChIs originate from GABAergic terminals, whereas 21% are from putatively glutamatergic terminals that establish asymmetric synapses, and 19% from other (non-GABAergic) sources of symmetric synapses. Double pre-embedding immunoelectron microscopy using substance P and Met-/Leu-enkephalin antibodies to label GABAergic terminals from collaterals of "direct" and "indirect" striatal projection neurons, respectively, revealed that 47% of the indirect pathway terminals and 36% of the direct pathway terminals target ChIs. Together, substance P- and enkephalin-positive terminals represent 24% of all synapses onto ChIs in the monkey putamen. These findings show that ChIs receive prominent GABAergic inputs from multiple origins, including a significant contingent from axon collaterals of direct and indirect pathway projection neurons. Copyright © 2013 Wiley Periodicals, Inc.

  12. Development and maturation of embryonic cortical neurons grafted into the damaged adult motor cortex

    Nissrine Ballout


    Full Text Available Injury to the human central nervous system can lead to devastating consequences due to its poor ability to self-repair. Neural transplantation aimed at replacing lost neurons and restore functional circuitry has proven to be a promising therapeutical avenue. We previously reported in adult rodent animal models with cortical lesions that grafted fetal cortical neurons could effectively re-establish specific patterns of projections and synapses. The current study was designed to provide a detailed characterization of the spatio-temporal in vivo development of fetal cortical transplanted cells within the lesioned adult motor cortex and their corresponding axonal projections. We show here that as early as two weeks after grafting, cortical neuroblasts transplanted into damaged adult motor cortex developed appropriate projections to cortical and subcortical targets. Grafted cells initially exhibited characteristics of immature neurons, which then differentiated into mature neurons with appropriate cortical phenotypes where most were glutamatergic and few were GABAergic. All cortical subtypes identified with the specific markers CTIP2, Cux1, FOXP2 and Tbr1 were generated after grafting as evidenced with BrdU co-labeling.The set of data provided here is of interest as it sets biological standards for future studies aimed at replacing fetal cells with embryonic stem cells as a source of cortical neurons.

  13. Muscarinic M1 receptors regulate propofol modulation of GABAergic transmission in rat ventrolateral preoptic neurons.

    Zhang, Yu; Yu, Tian; Liu, Yang; Qian, Kun; Yu, Bu-Wei


    GABAergic neurons within the ventrolateral preoptic area (VLPO) play an important role in sleep-wakefulness regulation. Propofol, a widely used systemic anesthetic, has lately been reported to excite noradrenaline (NA)-inhibited type of VLPO neurons. Present study tested if acetylcholine system takes part in the propofol modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat VLPO neurons using a conventional whole-cell patch clamp technique. Propofol reversibly decreased mIPSC frequency without affecting the current amplitude, indicating that propofol acts presynaptically to decrease the probability of spontaneous GABA release. The propofol action on GABAergic mIPSC frequency was completely blocked by atropine, a nonselective muscarinic acetylcholine (mACh) receptor antagonist, and pirenzepine, a selective M1 receptor antagonist. These results suggest that propofol acts on M1 receptors on GABAergic nerve terminals projecting to VLPO neurons to inhibit spontaneous GABA release. The M1 receptor-mediated modulation of GABAergic transmission onto VLPO neurons may contribute to the regulation of loss of consciousness induced by propofol.

  14. Neuregulin 3 Mediates Cortical Plate Invasion and Laminar Allocation of GABAergic Interneurons

    Giorgia Bartolini


    Full Text Available Neural circuits in the cerebral cortex consist of excitatory pyramidal cells and inhibitory interneurons. These two main classes of cortical neurons follow largely different genetic programs, yet they assemble into highly specialized circuits during development following a very precise choreography. Previous studies have shown that signals produced by pyramidal cells influence the migration of cortical interneurons, but the molecular nature of these factors has remained elusive. Here, we identified Neuregulin 3 (Nrg3 as a chemoattractive factor expressed by developing pyramidal cells that guides the allocation of cortical interneurons in the developing cortical plate. Gain- and loss-of-function approaches reveal that Nrg3 modulates the migration of interneurons into the cortical plate in a process that is dependent on the tyrosine kinase receptor ErbB4. Perturbation of Nrg3 signaling in conditional mutants leads to abnormal lamination of cortical interneurons. Nrg3 is therefore a critical mediator in the assembly of cortical inhibitory circuits.

  15. Neuron-astrocyte interaction enhance GABAergic synaptic transmission in a manner dependent on key metabolic enzymes.

    Przemysław eKaczor


    Full Text Available GABA is the major inhibitory neurotransmitter in the adult brain and mechanisms of GABAergic inhibition have been intensely investigated in the past decades. Recent studies provided evidence for an important role of astrocytes in shaping GABAergic currents. One of the most obvious, but yet poorly understood, mechanisms of the cross-talk between GABAergic currents and astrocytes is metabolism including neurotransmitter homeostasis. In particular, how modulation of GABAergic currents by astrocytes depends on key enzymes involved in cellular metabolism remains largely unknown. To address this issue, we have considered two simple models of neuronal cultures: nominally astrocyte-free neuronal culture (NC and neuronal-astrocytic co-cultures (ANCC and miniature Inhibitory Postsynaptic Currents (mIPSCs were recorded in control conditions and in the presence of respective enzyme blockers. We report that enrichment of neuronal culture with astrocytes results in a marked increase in mIPSC frequency. This enhancement of GABAergic activity was accompanied by increased number of GAD65 and vGAT puncta, indicating that at least a part of the frequency enhancement was due to increased number of synaptic contacts. Inhibition of glutamine synthetase (with MSO strongly reduced mIPSC frequency in ANCC but had no effect in NC. Moreover, treatment of ANCC with inhibitor of glycogen phosphorylase (BAYU6751 or with selective inhibitor of astrocytic Krebs cycle,fluoroacetate, resulted in a marked reduction of mIPSC frequency in ANCC having no effect in NC. We conclude that GABAergic synaptic transmission strongly depends on neuron-astrocyte interaction in a manner dependent on key metabolic enzymes as well as on the Krebs cycle.

  16. Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans

    Mahoney, Timothy R.; Luo, Shuo; Round, Elaine K.; Brauner, Martin; Gottschalk, Alexander; Thomas, James H.; Nonet, Michael L.


    The Caenorhabditis elegans defecation motor program (DMP) is a highly coordinated rhythmic behavior that requires two GABAergic neurons that synapse onto the enteric muscles. One class of DMP mutants, called anterior body wall muscle contraction and expulsion defective (aex) mutants, exhibits similar defects to those caused by the loss of these two neurons. Here, we demonstrate that aex-2 encodes a G-protein–coupled receptor (GPCR) and aex-4 encodes an exocytic SNAP25 homologue. We found that aex-2 functions in the nervous system and activates a Gsα signaling pathway to regulate defecation. aex-4, on the other hand, functions in the intestinal epithelial cells. Furthermore, we show that aex-5, which encodes a pro-protein convertase, functions in the intestine to regulate the DMP and that its secretion from the intestine is impaired in aex-4 mutants. Activation of the Gsα GPCR pathway in GABAergic neurons can suppress the defecation defect of the intestinal mutants aex-4 and aex-5. Lastly, we demonstrate that activation of GABAergic neurons using the light-gated cation channel channelrhodopsin-2 is sufficient to suppress the behavioral defects of aex-2, aex-4, and aex-5. These results genetically place intestinal genes aex-4 and aex-5 upstream of GABAergic GPCR signaling. We propose a model whereby the intestinal genes aex-4 and aex-5 control the DMP by regulating the secretion of a signal, which activates the neuronal receptor aex-2. PMID:18852466

  17. Quantitative study of NPY-expressing GABAergic neurons and axons in rat spinal dorsal horn.

    Polgár, Erika; Sardella, Thomas C P; Watanabe, Masahiko; Todd, Andrew J


    Between 25-40% of neurons in laminae I-III are GABAergic, and some of these express neuropeptide Y (NPY). We previously reported that NPY-immunoreactive axons form numerous synapses on lamina III projection neurons that possess the neurokinin 1 receptor (NK1r). The aims of this study were to determine the proportion of neurons and GABAergic boutons in this region that contain NPY, and to look for evidence that they selectively innervate different neuronal populations. We found that 4-6% of neurons in laminae I-III were NPY-immunoreactive and based on the proportions of neurons that are GABAergic, we estimate that NPY is expressed by 18% of inhibitory interneurons in laminae I-II and 9% of those in lamina III. GABAergic boutons were identified by the presence of the vesicular GABA transporter (VGAT) and NPY was found in 13-15% of VGAT-immunoreactive boutons in laminae I-II, and 5% of those in lamina III. For both the lamina III NK1r-immunoreactive projection neurons and protein kinase Cγ (PKCγ)-immunoreactive interneurons in lamina II, we found that around one-third of the VGAT boutons that contacted them were NPY-immunoreactive. However, based on differences in the sizes of these boutons and the strength of their NPY-immunoreactivity, we conclude that these originate from different populations of interneurons. Only 6% of VGAT boutons presynaptic to large lamina I projection neurons that lacked NK1rs contained NPY. These results show that NPY-containing neurons make up a considerable proportion of the inhibitory interneurons in laminae I-III, and that their axons preferentially target certain classes of dorsal horn neuron.

  18. Rac-GTPases Regulate Microtubule Stability and Axon Growth of Cortical GABAergic Interneurons.

    Tivodar, Simona; Kalemaki, Katerina; Kounoupa, Zouzana; Vidaki, Marina; Theodorakis, Kostas; Denaxa, Myrto; Kessaris, Nicoletta; de Curtis, Ivan; Pachnis, Vassilis; Karagogeos, Domna


    Cortical interneurons are characterized by extraordinary functional and morphological diversity. Although tremendous progress has been made in uncovering molecular and cellular mechanisms implicated in interneuron generation and function, several questions still remain open. Rho-GTPases have been implicated as intracellular mediators of numerous developmental processes such as cytoskeleton organization, vesicle trafficking, transcription, cell cycle progression, and apoptosis. Specifically in cortical interneurons, we have recently shown a cell-autonomous and stage-specific requirement for Rac1 activity within proliferating interneuron precursors. Conditional ablation of Rac1 in the medial ganglionic eminence leads to a 50% reduction of GABAergic interneurons in the postnatal cortex. Here we examine the additional role of Rac3 by analyzing Rac1/Rac3 double-mutant mice. We show that in the absence of both Rac proteins, the embryonic migration of medial ganglionic eminence-derived interneurons is further impaired. Postnatally, double-mutant mice display a dramatic loss of cortical interneurons. In addition, Rac1/Rac3-deficient interneurons show gross cytoskeletal defects in vitro, with the length of their leading processes significantly reduced and a clear multipolar morphology. We propose that in the absence of Rac1/Rac3, cortical interneurons fail to migrate tangentially towards the pallium due to defects in actin and microtubule cytoskeletal dynamics.

  19. Organization and properties of GABAergic neurons in solitary tract nucleus (NTS).

    Bailey, Timothy W; Appleyard, Suzanne M; Jin, Young-Ho; Andresen, Michael C


    Cranial visceral afferents enter the brain at the solitary tract nucleus (NTS). GABAergic neurons are scattered throughout the NTS, but their relation to solitary tract (ST) afferent pathways is imprecisely known. We hypothesized that most GABAergic NTS neurons would be connected only indirectly to the ST. We identified GABAergic neurons in brain stem horizontal slices using transgenic mice in which enhanced green fluorescent protein (EGFP) expression was linked to glutamic acid decarboxylase expression (GAD(+)). Finely graded electrical shocks to ST recruit ST-synchronized synaptic events with all-or-none thresholds and individual waveforms did not change with greater suprathreshold intensities--evidence consistent with initiation by single afferent axons. Most (approximately 70%) GAD(+) neurons received ST-evoked excitatory postsynaptic currents (EPSCs) that had minimally variant latencies (jitter, SD of latency 200 micros including inhibitory postsynaptic currents (IPSCs), indicating indirect connections (polysynaptic). Shocks of suprathreshold intensity delivered adjacent (50-300 microm) to the ST failed to excite non-ST inputs to second-order neurons, suggesting a paucity of axons passing near to ST that connected to these neurons. Despite expectations, we found similar ST synaptic patterns in GAD(+) and unlabeled neurons. Generally, ST information that arrived indirectly had small amplitudes (EPSCs and IPSCs) and frequency-dependent failures that reached >50% for IPSCs to bursts of stimuli. This ST afferent pathway organization is strongly use-dependent--a property that may tune signal propagation within and beyond NTS.

  20. 5-HT7 receptor modulates GABAergic transmission in the rat dorsal raphe nucleus and controls cortical release of serotonin

    Magdalena eKusek; Joanna eSowa; Katarzyna eKamińska; Krystyna eGołembiowska; Krzysztof eTokarski; Grzegorz eHess


    The 5-HT7 receptor is one of the several serotonin (5-HT) receptor subtypes that are expressed in the dorsal raphe nucleus (DRN). Some earlier findings suggested that 5-HT7 receptors in the DRN were localized on GABAergic interneurons modulating the activity of 5-HT projection neurons. The aim of the present study was to find out how the 5-HT7 receptor modulates the GABAergic synaptic input to putative 5-HT DRN neurons, and whether blockade of the 5-HT7 receptor would affect the release of 5-...

  1. Serotonin modulation of cortical neurons and networks

    Pau eCelada


    Full Text Available The serotonergic pathways originating in the dorsal and median raphe nuclei (DR and MnR, respectively are critically involved in cortical function. Serotonin (5-HT, acting on postsynaptic and presynaptic receptors, is involved in cognition, mood, impulse control and motor functions by 1 modulating the activity of different neuronal types, and 2 varying the release of other neurotransmitters, such as glutamate, GABA, acetylcholine and dopamine. Also, 5-HT seems to play an important role in cortical development. Of all cortical regions, the frontal lobe is the area most enriched in serotonergic axons and 5-HT receptors. 5-HT and selective receptor agonists modulate the excitability of cortical neurons and their discharge rate through the activation of several receptor subtypes, of which the 5-HT1A, 5-HT1B, 5-HT2A and 5-HT3 subtypes play a major role. Little is known, however, on the role of other excitatory receptors moderately expressed in cortical areas, such as 5-HT2C, 5-HT4, 5-HT6 and 5-HT7. In vitro and in vivo studies suggest that 5-HT1A and 5-HT2A receptors are key players and exert opposite effects on the activity of pyramidal neurons in the medial prefrontal cortex (mPFC. The activation of 5-HT1A receptors in mPFC hyperpolarizes pyramidal neurons whereas that of 5-HT2A receptors results in neuronal depolarization, reduction of the afterhyperpolarization and increase of excitatory postsynaptic currents (EPSCs and of discharge rate. 5-HT can also stimulate excitatory (5-HT2A and 5-HT3 and inhibitory (5-HT1A receptors in GABA interneurons to modulate synaptic GABA inputs onto pyramidal neurons. Likewise, the pharmacological manipulation of various 5-HT receptors alters oscillatory activity in PFC, suggesting that 5-HT is also involved in the control of cortical network activity. A better understanding of the actions of 5-HT in PFC may help to develop treatments for mood and cognitive disorders associated with an abnormal function of the

  2. Optogenetic activation of septal GABAergic afferents entrains neuronal firing in the medial habenula

    Choi, Kyuhyun; Lee, Youngin; Lee, Changwoo; Hong, Seokheon; Lee, Soonje; Kang, Shin Jung; Shin, Ki Soon


    The medial habenula (MHb) plays an important role in nicotine-related behaviors such as nicotine aversion and withdrawal. The MHb receives GABAergic input from the medial septum/diagonal band of Broca (MS/DB), yet the synaptic mechanism that regulates MHb activity is unclear. GABA (γ -aminobutyric acid) is a major inhibitory neurotransmitter activating both GABAA receptors and GABAB receptors. Depending on intracellular chloride concentration, however, GABAA receptors also function in an excitatory manner. In the absence of various synaptic inputs, we found that MHb neurons displayed spontaneous tonic firing at a rate of about ~4.4 Hz. Optogenetic stimulation of MS/DB inputs to the MHb evoked GABAA receptor-mediated synaptic currents, which produced stimulus-locked neuronal firing. Subsequent delayed yet lasting activation of GABAB receptors attenuated the intrinsic tonic firing. Consequently, septal GABAergic input alone orchestrates both excitatory GABAA and inhibitory GABAB receptors, thereby entraining the firing of MHb neurons. PMID:27703268

  3. Early GABAergic circuitry in the cerebral cortex.

    Luhmann, Heiko J; Kirischuk, Sergei; Sinning, Anne; Kilb, Werner


    In the cerebral cortex GABAergic signaling plays an important role in regulating early developmental processes, for example, neurogenesis, migration and differentiation. Transient cell populations, namely Cajal-Retzius in the marginal zone and thalamic input receiving subplate neurons, are integrated as active elements in transitory GABAergic circuits. Although immature pyramidal neurons receive GABAergic synaptic inputs already at fetal stages, they are integrated into functional GABAergic circuits only several days later. In consequence, GABAergic synaptic transmission has only a minor influence on spontaneous network activity during early corticogenesis. Concurrent with the gradual developmental shift of GABA action from excitatory to inhibitory and the maturation of cortical synaptic connections, GABA becomes more important in synchronizing neuronal network activity.

  4. GABAergic RIP-Cre neurons in the arcuate nucleus selectively regulate energy expenditure.

    Kong, Dong; Tong, Qingchun; Ye, Chianping; Koda, Shuichi; Fuller, Patrick M; Krashes, Michael J; Vong, Linh; Ray, Russell S; Olson, David P; Lowell, Bradford B


    Neural regulation of energy expenditure is incompletely understood. By genetically disrupting GABAergic transmission in a cell-specific fashion, and by combining this with selective pharmacogenetic activation and optogenetic mapping techniques, we have uncovered an arcuate-based circuit that selectively drives energy expenditure. Specifically, mice lacking synaptic GABA release from RIP-Cre neurons have reduced energy expenditure, become obese and are extremely sensitive to high-fat diet-induced obesity, the latter due to defective diet-induced thermogenesis. Leptin's ability to stimulate thermogenesis, but not to reduce feeding, is markedly attenuated. Acute, selective activation of arcuate GABAergic RIP-Cre neurons, which monosynaptically innervate PVH neurons projecting to the NTS, rapidly stimulates brown fat and increases energy expenditure but does not affect feeding. Importantly, this response is dependent upon GABA release from RIP-Cre neurons. Thus, GABAergic RIP-Cre neurons in the arcuate selectively drive energy expenditure, contribute to leptin's stimulatory effect on thermogenesis, and protect against diet-induced obesity.

  5. Upregulation of barrel GABAergic neurons is associated with cross-modal plasticity in olfactory deficit.

    Hong Ni

    Full Text Available BACKGROUND: Loss of a sensory function is often followed by the hypersensitivity of other modalities in mammals, which secures them well-awareness to environmental changes. Cellular and molecular mechanisms underlying cross-modal sensory plasticity remain to be documented. METHODOLOGY/PRINCIPAL FINDINGS: Multidisciplinary approaches, such as electrophysiology, behavioral task and immunohistochemistry, were used to examine the involvement of specific types of neurons in cross-modal plasticity. We have established a mouse model that olfactory deficit leads to a whisking upregulation, and studied how GABAergic neurons are involved in this cross-modal plasticity. In the meantime of inducing whisker tactile hypersensitivity, the olfactory injury recruits more GABAergic neurons and their fine processes in the barrel cortex, as well as upregulates their capacity of encoding action potentials. The hyperpolarization driven by inhibitory inputs strengthens the encoding ability of their target cells. CONCLUSION/SIGNIFICANCE: The upregulation of GABAergic neurons and the functional enhancement of neuronal networks may play an important role in cross-modal sensory plasticity. This finding provides the clues for developing therapeutic approaches to help sensory recovery and substitution.

  6. High-Degree Neurons Feed Cortical Computations.

    Nicholas M Timme


    Full Text Available Recent work has shown that functional connectivity among cortical neurons is highly varied, with a small percentage of neurons having many more connections than others. Also, recent theoretical developments now make it possible to quantify how neurons modify information from the connections they receive. Therefore, it is now possible to investigate how information modification, or computation, depends on the number of connections a neuron receives (in-degree or sends out (out-degree. To do this, we recorded the simultaneous spiking activity of hundreds of neurons in cortico-hippocampal slice cultures using a high-density 512-electrode array. This preparation and recording method combination produced large numbers of neurons recorded at temporal and spatial resolutions that are not currently available in any in vivo recording system. We utilized transfer entropy (a well-established method for detecting linear and nonlinear interactions in time series and the partial information decomposition (a powerful, recently developed tool for dissecting multivariate information processing into distinct parts to quantify computation between neurons where information flows converged. We found that computations did not occur equally in all neurons throughout the networks. Surprisingly, neurons that computed large amounts of information tended to receive connections from high out-degree neurons. However, the in-degree of a neuron was not related to the amount of information it computed. To gain insight into these findings, we developed a simple feedforward network model. We found that a degree-modified Hebbian wiring rule best reproduced the pattern of computation and degree correlation results seen in the real data. Interestingly, this rule also maximized signal propagation in the presence of network-wide correlations, suggesting a mechanism by which cortex could deal with common random background input. These are the first results to show that the extent to

  7. Effect of mescaline on single cortical neurones.

    Bradshaw, C M; Roberts, M H; Szabadi, E


    The effects of mescaline upon single cortical neurones were studied, using the microiontophoretic technique. Mescaline elicited excitatory and depressant responses similar to those evoked by noradrenaline (NA) and 5-hydroxytryptamine (5-HI). The responses to NA and mescaline were usually in the same direction, the neurone being either excited by both drugs or depressed by both drugs. The correlation between the effects of mescaline and 5-HT, however, was less consistent. The beta-adrenoceptor blocking agent MJ-1999 and the 5-HT antagonist methysergide were both effective in antagonizing mescaline responses.


    GABAergic interneurons comprise the bulk of local inhibitory neuronal circuitry in cortex and hippocampus and a subpopulation of these interneurons contain the calcium binding protein, parvalbumin (PV). A previous report indicated that severe hypothyroidism reduced PV immunoreact...

  9. Nicotine modulates GABAergic transmission to dopaminergic neurons in substantia nigra pars compacta

    Cheng XIAO; Ke-chun YANG; Chun-yi ZHOU; Guo-zhang JIN; Jie WU; Jiang-hong YE


    Aim: Dopaminergic neurons in the substantia nigra pars compacta (SNc) play important roles in motor control and drug addiction. As the major afferent, GABAergic innervation controls the activity of SNc dopaminergic neurons. Although it is clear that nicotine modulates SNc dopaminergic neurons by activating subtypes of somatodendritic nicotinic acetylcholine receptors (nAChRs), the detailed mechanisms of this activation remain to be addressed.Methods: In the current study, we recorded GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIP-SCs) from dissociated SNc dopaminergic neurons that were obtained using an enzyme-free procedure. These neurons preserved some functional terminals after isolation, including those that release GABA.Results: We found that both extra- and intra-cellular calcium modulates sIPSCs in these neurons. Furthermore, both nicotine and endogenous acetylcholine enhance the frequency of sIPSCs. Moreover, endogenous acetylcholine tonically facilitates sIPSC frequency, primarily by activating the a4B2* nAChRs on the GABAergic terminals.Conclusion: Nicotine facilitates GABA release onto SNc dopaminergic neurons mainly via the activation of presynaptic a4B2* nAChRs.

  10. GABAergic Mechanisms in Schizophrenia

    de Jonge, Jeroen C; Vinkers, Christiaan H; Hulshoff Pol, Hilleke E


    Schizophrenia is a psychiatric disorder characterized by hallucinations, delusions, disorganized thinking, and impairments in cognitive functioning. Evidence from postmortem studies suggests that alterations in cortical γ-aminobutyric acid (GABAergic) neurons contribute to the clinical features...... of schizophrenia. In vivo measurement of brain GABA levels using magnetic resonance spectroscopy (MRS) offers the possibility to provide more insight into the relationship between problems in GABAergic neurotransmission and clinical symptoms of schizophrenia patients. This study reviews and links alterations...... in the GABA system in postmortem studies, animal models, and human studies in schizophrenia. Converging evidence implicates alterations in both presynaptic and postsynaptic components of GABAergic neurotransmission in schizophrenia, and GABA may thus play an important role in the pathophysiology...

  11. Depolarization by K+ and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: vesicular versus non-vesicular release of GABA

    Belhage, B; Hansen, Gert Helge; Schousboe, A


    Neurotransmitter release and changes in the concentration of intracellular free calcium ([Ca++]i) were studied in cultured GABAergic cerebral cortical neurons, from mice, upon depolarization with either an unphysiologically high potassium concentration (55 mM) or the physiological excitatory...... neurotransmitter glutamate (100 microM). Both depolarizing stimuli exerted prompt increases in the release of preloaded [3H]GABA as well as in [Ca++]i. However, the basic properties of transmitter release and the increase in [Ca++]i under a variety of conditions were different during stimulation with K...... in nature whereas that induced by the neurotransmitter glutamate is not....

  12. Development of GPCR modulation of GABAergic transmission in chicken nucleus laminaris neurons.

    Zheng-Quan Tang

    Full Text Available Neurons in the nucleus laminaris (NL of birds act as coincidence detectors and encode interaural time difference to localize the sound source in the azimuth plane. GABAergic transmission in a number of CNS nuclei including the NL is subject to a dual modulation by presynaptic GABA(B receptors (GABA(BRs and metabotropic glutamate receptors (mGluRs. Here, using in vitro whole-cell patch clamp recordings from acute brain slices of the chick, we characterized the following important but unknown properties pertaining to such a dual modulation: (1 emergence of functional GABA synapses in NL neurons; (2 the temporal onset of neuromodulation mediated by GABA(BRs and mGluRs; and (3 the physiological conditions under which GABA(BRs and mGluRs are activated by endogenous transmitters. We found that (1 GABA(AR-mediated synaptic responses were observed in about half of the neurons at embryonic day 11 (E11; (2 GABA(BR-mediated modulation of the GABAergic transmission was detectable at E11, whereas the modulation by mGluRs did not emerge until E15; and (3 endogenous activity of GABA(BRs was induced by both low- (5 or 10 Hz and high-frequency (200 Hz stimulation of the GABAergic pathway, whereas endogenous activity of mGluRs was induced by high- (200 Hz but not low-frequency (5 or 10 Hz stimulation of the glutamatergic pathway. Furthermore, the endogenous activity of mGluRs was mediated by group II but not group III members. Therefore, autoreceptor-mediated modulation of GABAergic transmission emerges at the same time when the GABA synapses become functional. Heteroreceptor-mediated modulation appears at a later time and is receptor type dependent in vitro.

  13. Hyperbaric oxygenation alleviates chronic constriction injury (CCI)-induced neuropathic pain and inhibits GABAergic neuron apoptosis in the spinal cord.

    Fu, Huiqun; Li, Fenghua; Thomas, Sebastian; Yang, Zhongjin


    Dysfunction of GABAergic inhibitory controls contributes to the development of neuropathic pain. We examined our hypotheses that (1) chronic constriction injury (CCI)-induced neuropathic pain is associated with increased spinal GABAergic neuron apoptosis, and (2) hyperbaric oxygen therapy (HBO) alleviates CCI-induced neuropathic pain by inhibiting GABAergic neuron apoptosis. Male rats were randomized into 3 groups: CCI, CCI+HBO and the control group (SHAM). Mechanical allodynia was tested daily following CCI procedure. HBO rats were treated at 2.4 atmospheres absolute (ATA) for 60min once per day. The rats were euthanized and the spinal cord harvested on day 8 and 14 post-CCI. Detection of GABAergic cells and apoptosis was performed. The percentages of double positive stained cells (NeuN/GABA), cleaved caspase-3 or Cytochrome C in total GABAergic cells or in total NeuN positive cells were calculated. HBO significantly alleviated mechanical allodynia. CCI-induced neuropathic pain was associated with significantly increased spinal apoptotic GABA-positive neurons. HBO considerably decreased these spinal apoptotic cells. Cytochrome-C-positive neurons and cleaved caspase-3-positive neurons were also significantly higher in CCI rats. HBO significantly decreased these positive cells. Caspase-3 mRNA was also significantly higher in CCI rats. HBO reduced mRNA expression of caspase-3. CCI-induced neuropathic pain was associated with increased apoptotic GABAergic neurons induced by activation of key proteins of mitochondrial apoptotic pathways in the dorsal horn of the spinal cord. HBO alleviated CCI-induced neuropathic pain and reduced GABAergic neuron apoptosis. The beneficial effect of HBO may be via its inhibitory role in CCI-induced GABAergic neuron apoptosis by suppressing mitochondrial apoptotic pathways in the spinal cord. Increased apoptotic GABAergic neurons induced by activation of key proteins of mitochondrial apoptotic pathways in the dorsal horn of the spinal

  14. Pyramidal cells make specific connections onto smooth (GABAergic neurons in mouse visual cortex.

    Rita Bopp


    Full Text Available One of the hallmarks of neocortical circuits is the predominance of recurrent excitation between pyramidal neurons, which is balanced by recurrent inhibition from smooth GABAergic neurons. It has been previously described that in layer 2/3 of primary visual cortex (V1 of cat and monkey, pyramidal cells filled with horseradish peroxidase connect approximately in proportion to the spiny (excitatory, 95% and 81%, respectively and smooth (GABAergic, 5% and 19%, respectively dendrites found in the neuropil. By contrast, a recent ultrastructural study of V1 in a single mouse found that smooth neurons formed 51% of the targets of the superficial layer pyramidal cells. This suggests that either the neuropil of this particular mouse V1 had a dramatically different composition to that of V1 in cat and monkey, or that smooth neurons were specifically targeted by the pyramidal cells in that mouse. We tested these hypotheses by examining similar cells filled with biocytin in a sample of five mice. We found that the average composition of the neuropil in V1 of these mice was similar to that described for cat and monkey V1, but that the superficial layer pyramidal cells do form proportionately more synapses with smooth dendrites than the equivalent neurons in cat or monkey. These distributions may underlie the distinct differences in functional architecture of V1 between rodent and higher mammals.

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

    Barber, Melissa; Pierani, Alessandra


    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.

  16. Electrical remodeling of preoptic GABAergic neurons involves the Kv1.5 subunit.

    Iustin V Tabarean

    Full Text Available The electrogenic machinery of an excitable cell can adapt in response to changes in input, genetic deficit or in pathological conditions, however the underlying molecular mechanisms are not understood. In cases of genetic deletion it is commonly observed that a channel subunit from the same family replaces the missing one. We have previously reported that Kv4.2-/- preoptic GABAergic neurons display identical firing characteristics to those of wild-type neurons despite having reduced A-type currents, and that, surprisingly, they present a robust upregulation of a delayed rectifier current, the nature of which is unknown. Here, using pharmacology, qPCR and Western blots we report that, although the wild-type neurons express several Kv subunits, the upregulated current is conducted by the Kv1.5 subunit exclusively. Thus, this study reveals the molecular nature of a novel mechanism of electrical remodeling in central neurons.

  17. Acetaminophen induces apoptosis in rat cortical neurons.

    Inmaculada Posadas

    Full Text Available BACKGROUND: Acetaminophen (AAP is widely prescribed for treatment of mild pain and fever in western countries. It is generally considered a safe drug and the most frequently reported adverse effect associated with acetaminophen is hepatotoxicity, which generally occurs after acute overdose. During AAP overdose, encephalopathy might develop and contribute to morbidity and mortality. Our hypothesis is that AAP causes direct neuronal toxicity contributing to the general AAP toxicity syndrome. METHODOLOGY/PRINCIPAL FINDINGS: We report that AAP causes direct toxicity on rat cortical neurons both in vitro and in vivo as measured by LDH release. We have found that AAP causes concentration-dependent neuronal death in vitro at concentrations (1 and 2 mM that are reached in human plasma during AAP overdose, and that are also reached in the cerebrospinal fluid of rats for 3 hours following i.p injection of AAP doses (250 and 500 mg/kg that are below those required to induce acute hepatic failure in rats. AAP also increases both neuronal cytochrome P450 isoform CYP2E1 enzymatic activity and protein levels as determined by Western blot, leading to neuronal death through mitochondrial-mediated mechanisms that involve cytochrome c release and caspase 3 activation. In addition, in vivo experiments show that i.p. AAP (250 and 500 mg/kg injection induces neuronal death in the rat cortex as measured by TUNEL, validating the in vitro data. CONCLUSIONS/SIGNIFICANCE: The data presented here establish, for the first time, a direct neurotoxic action by AAP both in vivo and in vitro in rats at doses below those required to produce hepatotoxicity and suggest that this neurotoxicity might be involved in the general toxic syndrome observed during patient APP overdose and, possibly, also when AAP doses in the upper dosing schedule are used, especially if other risk factors (moderate drinking, fasting, nutritional impairment are present.

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

    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.

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

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


    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.

  20. The response of GABAergic and cholinergic neurons to transient cerebral ischemia.

    Francis, A; Pulsinelli, W


    The vulnerability of striatal and hippocampal neurons to ischemia was studied by measuring the activity of neurotransmitter-related enzymes after transient forebrain ischemia in rats. Activities of glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) were measured 6 h to 8 days after 20, 30 or 40 min of forebrain ischemia, as markers for GABAergic and cholinergic neurons respectively. Transient forebrain ischemia resulted in depression of striatal GAD activity while striatal CAT and hippocampal GAD activities were unaffected. Striatal GAD activity progressively decreased during the first 24 h postischemia and remained depressed 5--8 days later, suggesting irreversible damage to this population of neurons. The stability of striatal CAT and hippocampal GAD activity indicates that these cells were resistant to the present ischemic conditions.

  1. [Local GABA-ergic modulation of serotonergic neuron activity in the nucleus raphe magnus].

    Iniushkin, A N; Merkulova, N A; Orlova, A O; Iniushkina, E M


    In voltage-clamp experimental on slices of the rat brainstem the effects of 5-HT and GABA on serotonergic neurons of nucleus raphe magnus were investigated. Local applications of 5-HT induced an increase in IPCSs frequency and amplitude in 45% of serotonergic cells. The effect suppressed by the blocker of fast sodium channels tetradotoxin. Antagonist of GABA receptor gabazine blocked IPSCs in neurons both sensitive and non-sensitive to 5-HT action. Applications of GABA induced a membrane current (I(GABA)), which was completely blocked by gabazine. The data suggest self-control of the activity of serotonergic neurons in nucleus raphe magnus by negative feedback loop via local GABAergic interneurons.

  2. Possible role of GABAergic depolarization in neocortical neurons in generating hyperexcitatory behaviors during emergence from sevoflurane anesthesia in the rat

    Byung‑Gun Lim


    Full Text Available Hyperexcitatory behaviors occurring after sevoflurane anesthesia are of serious clinical concern, but the underlying mechanism is unknown. These behaviors may result from the potentiation by sevoflurane of GABAergic depolarization/excitation in neocortical neurons, cells implicated in the genesis of consciousness and arousal. The current study sought to provide evidence for this hypothesis with rats, the neocortical neurons of which are known to respond to GABA (γ-aminobutyric acid with depolarization/excitation at early stages of development (i.e., until the second postnatal week and with hyperpolarization/inhibition during adulthood. Employing behavioral tests and electrophysiological recordings in neocortical slice preparations, we found: (1 sevoflurane produced PAHBs (post-anesthetic hyperexcitatory behaviors in postnatal day (P1–15 rats, whereas it failed to elicit PAHBs in P16 or older rats; (2 GABAergic PSPs (postsynaptic potentials were depolarizing/excitatory in the neocortical neurons of P5 and P10 rats, whereas mostly hyperpolarizing/inhibitory in the cells of adult rats; (3 at P14–15, <50% of rats had PAHBs and, in general, the cells of the animals with PAHBs exhibited strongly depolarizing GABAergic PSPs, whereas those without PAHBs showed hyperpolarizing or weakly depolarizing GABAergic PSPs; (4 bumetanide [inhibitor of the Cl− importer NKCC (Na+–K+–2Cl− cotransporter] treatment at P5 suppressed PAHBs and depolarizing GABAergic responses; and (5 sevoflurane at 1% (i.e., concentration <1 minimum alveolar concentration potentiated depolarizing GABAergic PSPs in the neurons of P5 and P10 rats and of P14–15 animals with PAHBs, evoking action potentials in ≥50% of these cells. On the basis of these results, we conclude that sevoflurane may produce PAHBs by potentiating GABAergic depolarization/excitation in neocortical neurons.

  3. Possible Role of GABAergic Depolarization in Neocortical Neurons in Generating Hyperexcitatory Behaviors during Emergence from Sevoflurane Anesthesia in the Rat

    Byung-Gun Lim


    Full Text Available Hyperexcitatory behaviors occurring after sevoflurane anesthesia are of serious clinical concern, but the underlying mechanism is unknown. These behaviors may result from the potentiation by sevoflurane of GABAergic depolarization/excitation in neocortical neurons, cells implicated in the genesis of consciousness and arousal. The current study sought to provide evidence for this hypothesis with rats, the neocortical neurons of which are known to respond to GABA (γ-aminobutyric acid with depolarization/excitation at early stages of development (i.e., until the second postnatal week and with hyperpolarization/inhibition during adulthood. Employing behavioral tests and electrophysiological recordings in neocortical slice preparations, we found: (1 sevoflurane produced PAHBs (post-anesthetic hyperexcitatory behaviors in postnatal day (P1–15 rats, whereas it failed to elicit PAHBs in P16 or older rats; (2 GABAergic PSPs (postsynaptic potentials were depolarizing/excitatory in the neocortical neurons of P5 and P10 rats, whereas mostly hyperpolarizing/inhibitory in the cells of adult rats; (3 at P14–15, <50 % of rats had PAHBs and, in general, the cells of the animals with PAHBs exhibited strongly depolarizing GABAergic PSPs, whereas those without PAHBs showed hyperpolarizing or weakly depolarizing GABAergic PSPs; (4 bumetanide [inhibitor of the Cl− importer NKCC (Na+ -K+−2Cl− cotransporter] treatment at P5 suppressed PAHBs and depolarizing GABAergic responses; and (5 sevoflurane at 1 % (i.e., concentration <1 minimum alveolar concentration potentiated depolarizing GABAergic PSPs in the neurons of P5 and P10 rats and of P14–15 animals with PAHBs, evoking action potentials in ≥50% of these cells. On the basis of these results, we conclude that sevoflurane may produce PAHBs by potentiating GABAergic depolarization/excitation in neocortical neurons.

  4. [The distribution of GABA-ergic neurons in rat neocortex in the postnatal period after the perinatal hypoxia].

    Khozhaĭ, L I; Otelin, V A


    The distribution of GABA-ergic neurons in different areas of the neocortex (frontal, sensorimotor, visual cortex) was studied in Wistar rats at different time periods of postnatal development after their exposure to perinatal hypoxia. To identify these neurons, the antibodies against GAD-67, the marker of GABA-ergic neurons, were used. It was found that the exposure to perinatal hypoxia caused a significant reduction in the number of GAD-67-expressing neurons in both upper and deep layers of the cortex in juvenile age (day 20 of postnatal period), that persisted until the prepubertal period (day 40). In experimental animals at postnatal day 40, the numbers of neurons that synthesized GAD-67, were two times lower in each of the layers of the neocortex than those in control animals. It is suggested that a drastic reduction in the number of GABA-ergic neurons in the neocortex could be a result of the damaging effects of acute perinatal hypoxia on the processes of progenitor cell migration from the subventricular zone, or on the synthesis of the factors controlling these migration processes as well as on GABA-ergic neuron maturation, leading to a delay of GAD-67 expression.

  5. GFR alpha-1 is expressed in parvalbumin GABAergic neurons in the hippocampus.

    Sarabi, A; Hoffer, B J; Olson, L; Morales, M


    Glial cell line derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons. GDNF binds with high affinity to GDNF-family receptor alpha-1 (GFR alpha-1). This receptor is expressed in different areas of the brain, including the hippocampus and dentate gyrus. By using in situ hybridization and immunohistochemistry, we found that 19% to 37% of glutamic acid decarboxylase (GAD) expressing neurons co-expressed GFR alpha-1 in the hippocampus. GFR alpha-1/GAD co-expression was found mainly in the stratum (s) pyramidale (29-37%) and s. oriens (20-25%). Further characterization of GFR alpha-1 expressing interneurons, based on their calcium-binding protein immunoreactivity, demonstrated that many parvalbumin (PV) immunoreactive neurons express GFR alpha-1 in the s. pyramidale of CA1 (72%), CA2 (70%) and CA3 (70%) subfields of the hippocampus. GFR alpha-1/PV double labeled neurons were also detected in the s. oriens of CA1 (52%), CA2 (27%) and CA3 (36%) subfields. The expression of GFR alpha-1 in principal neurons and in a specific sub-population of GABAergic neurons (PV-containing neurons) suggest that GDNF might modulate, in a selective manner, functions of the entire adult hippocampus.

  6. GABAergic neuron-specific loss of Ube3a causes Angelman syndrome-like EEG abnormalities and enhances seizure susceptibility

    Judson, Matthew C.; Wallace, Michael L.; Sidorov, Michael S.; Burette, Alain C.; Gu, Bin; van Woerden, Geeske M.; King, Ian F.; Han, Ji Eun; Zylka, Mark J.; Elgersma, Ype; Weinberg, Richard J.; Philpot, Benjamin D.


    SUMMARY Loss of maternal UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorder associated with severe epilepsy. We previously implicated GABAergic deficits onto layer (L) 2/3 pyramidal neurons in the pathogenesis of neocortical hyperexcitability, and perhaps epilepsy, in AS model mice. Here we investigate consequences of selective Ube3a loss from either GABAergic or glutamatergic neurons, focusing on the development of hyperexcitability within L2/3 neocortex and in broader circuit and behavioral contexts. We find that GABAergic Ube3a loss causes AS-like increases in neocortical EEG delta power, enhances seizure susceptibility, and leads to presynaptic accumulation of clathrin-coated vesicles (CCVs) – all without decreasing GABAergic inhibition onto L2/3 pyramidal neurons. Conversely, glutamatergic Ube3a loss fails to yield EEG abnormalities, seizures, or associated CCV phenotypes, despite impairing tonic inhibition onto L2/3 pyramidal neurons. These results substantiate GABAergic Ube3a loss as the principal cause of circuit hyperexcitability in AS mice, lending insight into ictogenic mechanisms in AS. PMID:27021170

  7. GABAergic Neuron-Specific Loss of Ube3a Causes Angelman Syndrome-Like EEG Abnormalities and Enhances Seizure Susceptibility.

    Judson, Matthew C; Wallace, Michael L; Sidorov, Michael S; Burette, Alain C; Gu, Bin; van Woerden, Geeske M; King, Ian F; Han, Ji Eun; Zylka, Mark J; Elgersma, Ype; Weinberg, Richard J; Philpot, Benjamin D


    Loss of maternal UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorder associated with severe epilepsy. We previously implicated GABAergic deficits onto layer (L) 2/3 pyramidal neurons in the pathogenesis of neocortical hyperexcitability, and perhaps epilepsy, in AS model mice. Here we investigate consequences of selective Ube3a loss from either GABAergic or glutamatergic neurons, focusing on the development of hyperexcitability within L2/3 neocortex and in broader circuit and behavioral contexts. We find that GABAergic Ube3a loss causes AS-like increases in neocortical EEG delta power, enhances seizure susceptibility, and leads to presynaptic accumulation of clathrin-coated vesicles (CCVs)-all without decreasing GABAergic inhibition onto L2/3 pyramidal neurons. Conversely, glutamatergic Ube3a loss fails to yield EEG abnormalities, seizures, or associated CCV phenotypes, despite impairing tonic inhibition onto L2/3 pyramidal neurons. These results substantiate GABAergic Ube3a loss as the principal cause of circuit hyperexcitability in AS mice, lending insight into ictogenic mechanisms in AS.

  8. Structural organization of long-range GABAergic projection system of the hippocampus

    Shozo Jinno


    Full Text Available GABA is a key mediator of neural activity in the mammalian central nervous system, and a diverse set of GABAergic neurons utilize GABA as transmitter. It has been widely accepted that GABAergic neurons typically serve as interneurons while glutamatergic principal cells send excitatory signals to remote areas. In general, glutamatergic projection neurons monosynaptically innervate both principal cells and local GABAergic interneurons in each target area, and these GABAergic cells play a vital role in modulation of the activity of principal cells. The formation and recall of sensory, motor and cognitive representations require coordinated fast communication among multiple areas of the cerebral cortex, which are thought to be mostly mediated by glutamatergic neurons. However, there is an increasing body of evidence showing that specific subpopulations of cortical GABAergic neurons send long-range axonal projections to subcortical and other cortical areas. In particular, a variety of GABAergic neurons in the hippocampus project to neighboring and remote areas. Using anatomical, molecular and electrophysiological approaches, several types of GABAergic projection neurons have been shown to exist in the hippocampus. The target areas of these cells are the subiculum and other retrohippocampal areas, the medial septum and the contralateral dentate gyrus. The long-range GABAergic projection system of the hippocampus may serve to coordinate precisely the multiple activity patterns of widespread cortical cell assemblies in different brain states and among multiple functionally related areas.

  9. Pentobarbital enhances GABAergic neurotransmission to cardiac parasympathetic neurons, which is prevented by expression of GABA(A) epsilon subunit.

    Irnaten, Mustapha; Walwyn, Wendy M; Wang, Jijiang; Venkatesan, Priya; Evans, Cory; Chang, Kyoung S K; Andresen, Michael C; Hales, Tim G; Mendelowitz, David


    Pentobarbital decreases the gain of the baroreceptor reflex on the order of 50%, and this blunting is caused nearly entirely by decreasing cardioinhibitory parasympathetic activity. The most likely site of action of pentobarbital is the gamma-aminobutyric acid type A (GABA(A)) receptor. The authors tested whether pentobarbital augments the inhibitory GABAergic neurotransmission to cardiac parasympathetic neurons, and whether expression of the GABA(A) epsilon subunit prevents this facilitation. The authors used a novel approach to study the effect of pentobarbital on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with a fluorescent tracer and were visually identified for patch clamp recording. The effects of pentobarbital on spontaneous GABAergic synaptic events were tested. An adenovirus was used to express the epsilon subunit of the GABA(A) receptor in cardiac parasympathetic neurons to examine whether this transfection alters pentobarbital-mediated changes in GABAergic neurotransmission. Pentobarbital increased the duration but not the frequency or amplitude of spontaneous GABAergic currents in cardiac parasympathetic neurons. Transfection of cardiac parasympathetic neurons with the epsilon subunit of the GABA(A) receptor prevented the pentobarbital-evoked facilitation of GABAergic currents. Pentobarbital, at clinically relevant concentrations, prolongs the duration of spontaneous inhibitory postsynaptic currents that impinge on cardiac parasympathetic neurons. This action would augment the inhibition of cardiac parasympathetic neurons, reduce parasympathetic cardioinhibitory activity, and increase heart rate. Expression of the GABA(A) receptor epsilon subunit in cardiac parasympathetic neurons renders the GABA receptors insensitive to pentobarbital.

  10. Stearic acid protects primary cultured cortical neurons against oxidative stress

    Ze-jian WANG; Cui-ling LIANG; Guang-mei LI; Cai-yi YU; Ming YIN


    Aim: To observe the effects of stearic acid against oxidative stress in primary cultured cortical neurons. Methods: Cortical neurons were exposed to glutamate,hydrogen peroxide (H202), or NaN3 insult in the presence or absence of stearic acid. Cell viability of cortical neurons was determined by MTT assay and LDH release. Endogenous antioxidant enzymes activity[superoxide dismutases (SOD),glutathione peroxidase (GSH-Px), and catalase (CAT)] and lipid peroxidation in cultured cortical neurons were evaluated using commercial kits. {3-[1(p-chloro-benzyl)-5-(isopropyl)-3-t-butylthiondol-2-yl]-2,2-dimethylpropanoic acid, Na}[MK886; 5 pmol/L; a noncompetitive inhibitor of proliferator-activated receptor(PPAR)α], bisphenol A diglycidyl ether (BADGE; 100 μmol/L; an antagonist of PPARγ), and cycloheximide (CHX; 30 μmol/L, an inhibitor of protein synthesis)were tested for their effects on the neuroprotection afforded by stearic acid.Western blotting was used to determine the PPARγ protein level in cortical neurons.Results: Stearic acid dose-dependently protected cortical neurons against glutamate or H202 injury and increased glutamate uptake in cultured neurons.This protection was concomitant to the inhibition of lipid peroxidation and to the promotion activity of Cu/Zn SOD and CAT in cultured cortical neurons. Its neuroprotective effects were completely blocked by BADGE and CHX. After incubation with H2O2 for 24 h, the expression of the PPARγ protein decreased significantly (P<0.05), and the inhibitory effect of H2O2 on the expression of PPARγ can be attenuated by stearic acid. Conclusion: Stearic acid can protect cortical neurons against oxidative stress by boosting the internal antioxidant enzymes.Its neuroprotective effect may be mainly mediated by the activation of PPARγ and new protein synthesis in cortical neurons.

  11. BDNF promotes differentiation and maturation of adult-born neurons through GABAergic transmission.

    Waterhouse, Emily G; An, Juan Ji; Orefice, Lauren L; Baydyuk, Maryna; Liao, Guey-Ying; Zheng, Kang; Lu, Bai; Xu, Baoji


    Brain-derived neurotrophic factor (BDNF) has been implicated in regulating adult neurogenesis in the subgranular zone (SGZ) of the dentate gyrus; however, the mechanism underlying this regulation remains unclear. In this study, we found that Bdnf mRNA localized to distal dendrites of dentate gyrus granule cells isolated from wild-type (WT) mice, but not from Bdnf(klox/klox) mice where the long 3' untranslated region (UTR) of Bdnf mRNA is truncated. KCl-induced membrane depolarization stimulated release of dendritic BDNF translated from long 3' UTR Bdnf mRNA in cultured hippocampal neurons, but not from short 3' UTR Bdnf mRNA. Bdnf(klox/klox) mice exhibited reduced expression of glutamic acid decarboxylase 65 (a GABA synthase), increased proliferation of progenitor cells, and impaired differentiation and maturation of newborn neurons in the SGZ. These deficits in adult neurogenesis were rescued with administration of phenobarbital, an enhancer of GABA(A) receptor activity. Furthermore, we observed similar neurogenesis deficits in mice where the receptor for BDNF, TrkB, was selectively abolished in parvalbumin (PV)-expressing GABAergic interneurons. Thus, our data suggest that locally synthesized BDNF in dendrites of granule cells promotes differentiation and maturation of progenitor cells in the SGZ by enhancing GABA release, at least in part, from PV-expressing GABAergic interneurons.

  12. Long-range GABAergic connections distributed throughout the neocortex and their possible function

    Nobuaki eTamamaki


    Full Text Available Features and functions of long range GABAergic projection neurons in the developing cerebral cortex have been reported previously, although until now their significance in the adult cerebral cortex has remained uncertain. The septo-hippocampal circuit is one exception – in this system, long range mature GABAergic projection neurons have been well analyzed and their contribution to the generation of theta-oscillatory behavior in the hippocampus has been documented. To have a clue to the function of the GABAergic projection neurons in the neocortex, we view the long range GABAergic projections those participating in the cortico-cortical, cortico-fugal, and afferent projections in the cerebral cortex. Then, we consider the possibility that the GABAergic projection neurons are involved in the generation, modification, and/or synchronization of oscillations in mature neocortical neuron activity. When markers that identify the GABAergic projection neurons are examined in anatomical and developmental studies, it is clear that neuronal NO synthetase (nNOS-immunoreactivity can readily identify GABAergic projection fibers (i.e. those longer than 1.5 mm. To elucidate the role of the GABAergic projection neurons in the neocortex, it will be necessary to clarify the network constructed by nNOS-positive GABAergic projection neurons and their postsynaptic targets. Thus, our long-range goals will be to label and manipulate (including deleting the GABAergic projection neurons using genetic tools driven by a nNOS promoter. We recognize that this may be a complex endeavor, as most excitatory neurons in the murine neocortex express nNOS transiently. Nevertheless, additional studies characterizing long range GABAergic projection neurons will have great value to the overall understanding of mature cortical function.

  13. Ginkgolides protects cultured cortical neurons against excitotoxic and oxidative insults

    ZHANGYu-Yang; YUQing-Hai; YOUSong; SHENGLi


    AIM: The neurotoxicity of glutamate is associated with neurological disorders including hypoxic-ischaemic brain injury. Studies using cultured cortical neurons have demonstrated that exposure to glutamate produced delayed degeneration of mature neurons. Oxygen free radicals generated during injury have been postulated to be a major cause of neuronal cell

  14. The changing roles of neurons in the cortical subplate

    Michael J Friedlander


    Full Text Available Neurons may serve different functions over the course of an organism’s life. Recent evidence suggests that cortical subplate neurons including those that reside in the white matter may perform longitudinal multi-tasking at different stages of development. These cells play a key role in early cortical development in coordinating thalamocortical reciprocal innervation. At later stages of development, they become integrated within the cortical microcircuitry. This type of longitudinal multi-tasking can enhance the capacity for information processing by populations of cells serving different functions over the lifespan. Subplate cells are initially derived when cells from the ventricular zone underlying the cortex migrate to the cortical preplate that is subsequently split by the differentiating neurons of the cortical plate with some neurons locating in the marginal zone and others settling below in the subplate (SP. While the cortical plate neurons form most of the cortical layers (layers 2-6, the marginal zone neurons form layer 1 and the SP neurons become interstitial cells of the white matter as well as forming a compact sublayer along the bottom of layer 6. After serving as transient innervation targets for thalamocortical axons, most of these cells die and layer 4 neurons become innervated by thalamic axons. However, 10-20% survives, remaining into adulthood along the bottom of layer 6 and as a scattered population of interstitial neurons in the white matter. Surviving subplate cells’ axons project throughout the overlying laminae, reaching layer 1 and issuing axon collaterals within white matter and in lower layer 6. This suggests that they participate in local synaptic networks, as well. Moreover, they receive excitatory and inhibitory synaptic inputs, potentially monitoring outputs from axon collaterals of cortical efferents, from cortical afferents and/or from each other. We explore our understanding of the functional connectivity of

  15. Tangentially migrating neurons assemble a primary cilium that promotes their reorientation to the cortical plate.

    Baudoin, Jean-Pierre; Viou, Lucie; Launay, Pierre-Serge; Luccardini, Camilla; Espeso Gil, Sergio; Kiyasova, Vera; Irinopoulou, Théano; Alvarez, Chantal; Rio, Jean-Paul; Boudier, Thomas; Lechaire, Jean-Pierre; Kessaris, Nicoletta; Spassky, Nathalie; Métin, Christine


    In migrating neurons, the centrosome nucleates and anchors a polarized network of microtubules that directs organelle movements. We report here that the mother centriole of neurons migrating tangentially from the medial ganglionic eminence (MGE) assembles a short primary cilium and exposes this cilium to the cell surface by docking to the plasma membrane in the leading process. Primary cilia are built by intraflagellar transport (IFT), which is also required for Sonic hedgehog (Shh) signal transduction in vertebrates. We show that Shh pathway perturbations influenced the leading process morphology and dynamics of MGE cells. Whereas Shh favored the exit of MGE cells away from their tangential migratory paths in the developing cortex, cyclopamine or invalidation of IFT genes maintained MGE cells in the tangential paths. Our findings show that signals transmitted through the primary cilium promote the escape of future GABAergic interneurons from their tangential routes to colonize the cortical plate.

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

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


    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.

  17. 5-HT7 receptor modulates GABAergic transmission in the rat dorsal raphe nucleus and controls cortical release of serotonin

    Magdalena eKusek


    Full Text Available The 5-HT7 receptor is one of the several serotonin (5-HT receptor subtypes that are expressed in the dorsal raphe nucleus (DRN. Some earlier findings suggested that 5-HT7 receptors in the DRN were localized on GABAergic interneurons modulating the activity of 5-HT projection neurons. The aim of the present study was to find out how the 5-HT7 receptor modulates the GABAergic synaptic input to putative 5-HT DRN neurons, and whether blockade of the 5-HT7 receptor would affect the release of 5-HT in the target structure. Male Wistar rats with microdialysis probes implanted in the prefrontal cortex (PFC received injections of the 5-HT7 receptor antagonist (2R-1-[(3-hydroxyphenylsulfonyl]-2-[2-(4-methyl-1 piperidinylethyl]pyrrolidine hydrochloride (SB 269970, which induced an increase in the levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA in the PFC. In another set of experiments whole-cell recordings from presumed projection neurons were carried out using DRN slices. SB 269970 application resulted in depolarization and in an increase in the firing frequency of the cells. In order to activate 5-HT7 receptors, 5-carboxamidotryptamine (5-CT was applied in the presence of N-[2-[4-(2-methoxyphenyl-1piperazinyl]ethyl]-N-2- pyridinylcyclohexanecarboxamide (WAY100635. Hyperpolarization of cells and a decrease in the firing frequency were observed after activation of the 5-HT7 receptor. Blockade of 5-HT7 receptors caused

  18. Relating normalization to neuronal populations across cortical areas.

    Ruff, Douglas A; Alberts, Joshua J; Cohen, Marlene R


    Normalization, which divisively scales neuronal responses to multiple stimuli, is thought to underlie many sensory, motor, and cognitive processes. In every study where it has been investigated, neurons measured in the same brain area under identical conditions exhibit a range of normalization, ranging from suppression by nonpreferred stimuli (strong normalization) to additive responses to combinations of stimuli (no normalization). Normalization has been hypothesized to arise from interactions between neuronal populations, either in the same or different brain areas, but current models of normalization are not mechanistic and focus on trial-averaged responses. To gain insight into the mechanisms underlying normalization, we examined interactions between neurons that exhibit different degrees of normalization. We recorded from multiple neurons in three cortical areas while rhesus monkeys viewed superimposed drifting gratings. We found that neurons showing strong normalization shared less trial-to-trial variability with other neurons in the same cortical area and more variability with neurons in other cortical areas than did units with weak normalization. Furthermore, the cortical organization of normalization was not random: neurons recorded on nearby electrodes tended to exhibit similar amounts of normalization. Together, our results suggest that normalization reflects a neuron's role in its local network and that modulatory factors like normalization share the topographic organization typical of sensory tuning properties.

  19. Reciprocal cholinergic and GABAergic modulation of the small ventrolateral pacemaker neurons of Drosophila's circadian clock neuron network.

    Lelito, Katherine R; Shafer, Orie T


    The relatively simple clock neuron network of Drosophila is a valuable model system for the neuronal basis of circadian timekeeping. Unfortunately, many key neuronal classes of this network are inaccessible to electrophysiological analysis. We have therefore adopted the use of genetically encoded sensors to address the physiology of the fly's circadian clock network. Using genetically encoded Ca(2+) and cAMP sensors, we have investigated the physiological responses of two specific classes of clock neuron, the large and small ventrolateral neurons (l- and s-LN(v)s), to two neurotransmitters implicated in their modulation: acetylcholine (ACh) and γ-aminobutyric acid (GABA). Live imaging of l-LN(v) cAMP and Ca(2+) dynamics in response to cholinergic agonist and GABA application were well aligned with published electrophysiological data, indicating that our sensors were capable of faithfully reporting acute physiological responses to these transmitters within single adult clock neuron soma. We extended these live imaging methods to s-LN(v)s, critical neuronal pacemakers whose physiological properties in the adult brain are largely unknown. Our s-LN(v) experiments revealed the predicted excitatory responses to bath-applied cholinergic agonists and the predicted inhibitory effects of GABA and established that the antagonism of ACh and GABA extends to their effects on cAMP signaling. These data support recently published but physiologically untested models of s-LN(v) modulation and lead to the prediction that cholinergic and GABAergic inputs to s-LN(v)s will have opposing effects on the phase and/or period of the molecular clock within these critical pacemaker neurons.

  20. Neurogliaform and Ivy cells: a major class of nNOS expressing GABAergic neurons

    Caren eArmstrong


    Full Text Available Neurogliaform and Ivy cells are members of an abundant class of neuronal nitric oxide synthase (nNOS expressing GABAergic interneurons found in diverse brain regions. These cells have a defining dense local axonal plexus, and display unique synaptic properties, including a biphasic postsynaptic response with both a slow GABAA component, and a GABAB component, following even a single action potential. The type of transmission displayed by these cells has been termed ‘volume transmission,’ distinct from both tonic and classical synaptic transmission. Electrical connections are also notable in that, unlike other classes, neurogliaform family cells will form gap junctions not only with other neurogliaform cells, but also with members of other GABAergic cell classes. In this review we focus on neurogliaform and Ivy cells throughout the hippocampal formation, where recent studies highlight their role in feedforward inhibition, uncover their ability to display a phenomenon called persistent firing, and reveal their modulation by opioids. The unique properties of this class of cells, their abundance, rich connectivity, and modulation by clinically relevant drugs make them an attractive target for future studies in vivo during different behavioral and pharmacological conditions.

  1. Fgf16 is required for specification of GABAergic neurons and oligodendrocytes in the zebrafish forebrain.

    Ayumi Miyake

    Full Text Available Fibroblast growth factor (Fgf signaling plays crucial roles in various developmental processes including those in the brain. We examined the role of Fgf16 in the formation of the zebrafish brain. The knockdown of fgf16 decreased cell proliferation in the forebrain and midbrain. fgf16 was also essential for development of the ventral telencephalon and diencephalon, whereas fgf16 was not required for dorsoventral patterning in the midbrain. fgf16 was additionally required for the specification and differentiation of γ-aminobutyric acid (GABAergic interneurons and oligodendrocytes, but not for those of glutamatergic neurons in the forebrain. Cross talk between Fgf and Hedgehog (Hh signaling was critical for the specification of GABAergic interneurons and oligodendrocytes. The expression of fgf16 in the forebrain was down-regulated by the inhibition of Hh and Fgf19 signaling, but not by that of Fgf3/Fgf8 signaling. The fgf16 morphant phenotype was similar to that of the fgf19 morphant and embryos blocked Hh signaling. The results of the present study indicate that Fgf16 signaling, which is regulated by the downstream pathways of Hh-Fgf19 in the forebrain, is involved in forebrain development.

  2. Differential sensitivity of GABAergic and glycinergic inputs to orexin-A in preganglionic cardiac vagal neurons of newborn rats

    Ji-jiang WANG; Yong-hua CHEN; Ke-yong LI; Feng-yan SUN


    Aim: To test the effect of orexin-A (hypocretin-1), a neuropeptide synthesized in the lateral hypothalamus and the perifornical area, on the glycinergic inputs and the GABAergic inputs of cardiac vagal neurons (CVN). Methods: The effects of orexin-A at three concentrations (20 nmol/L, 100 nmol/L, 500 nmol/L) on the glycinergic inputs and the GABAergic inputs were investigated by using retrograde fluorescent labeling of cardiac neurons (CVN) in the nucleus ambiguus (NA) and the voltage patch-clamp technique. Results: Orexin-A dose-dependently increased the frequency of both the glycinergic and the GABAergic spontaneous inhibitory postsynaptic currents (sIPSC). However, at a lower concentration (20 nmol/L) of orexin-A, although the frequency of the glycinergic sIPSC was significantly increased, the frequency of the GABAergic sIPSC was not significantly changed. Conclusion: The glycinergic inputs and the GABAergic inputs have different sensitivities to orexin-A, which suggests that the two kinds of inhibitory inputs might play different roles in the synaptic control of cardiac vagal functions.

  3. Further characterization of autoantibodies to GABAergic neurons in the central nervous system produced by a subset of children with autism

    Wills Sharifia


    Full Text Available Abstract Background Autism is a neurodevelopmental disorder characterized by impairments in social interaction and deficits in verbal and nonverbal communication, together with the presence of repetitive behaviors or a limited repertoire of activities and interests. The causes of autism are currently unclear. In a previous study, we determined that 21% of children with autism have plasma autoantibodies that are immunoreactive with a population of neurons in the cerebellum that appear to be Golgi cells, which are GABAergic interneurons. Methods We have extended this analysis by examining plasma immunoreactivity in the remainder of the brain. To determine cell specificity, double-labeling studies that included one of the calcium-binding proteins that are commonly colocalized in GABAergic neurons (calbindin, parvalbumin or calretinin were also carried out to determine which GABAergic neurons are immunoreactive. Coronal sections through the rostrocaudal extent of the macaque monkey brain were reacted with plasma from each of seven individuals with autism who had previously demonstrated positive Golgi cell staining, as well as six negative controls. In addition, brain sections from adult male mice were similarly examined. Results In each case, specific staining was observed for neurons that had the morphological appearance of interneurons. By double-labeling sections with plasma and with antibodies directed against γ-aminobutyric acid (GABA, we determined that all autoantibody-positive neurons were GABAergic. However, not all GABAergic neurons were autoantibody-positive. Calbindin was colabeled in several of the autoantibody-labeled cells, while parvalbumin colabeling was less frequently observed. Autoantibody-positive cells rarely expressed calretinin. Sections from the mouse brain processed similarly to the primate sections also demonstrated immunoreactivity to interneurons distributed throughout the neocortex and many subcortical regions. Some

  4. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs in rat hippocampal GABAergic interneurons.

    Son, Jong-Hyun; Winzer-Serhan, Ursula H


    Hippocampal inhibitory interneurons are a diverse population of cells widely scattered in the hippocampus, where they regulate hippocampal circuit activity. The hippocampus receives cholinergic projections from the basal forebrain, and functional studies have suggested the presence of different subtypes of nicotinic acetylcholine receptors (AChRs) on gamma-aminobutyric acid (GABA)ergic interneurons. Single-cell polymerase chain reaction analysis had confirmed that several nAChR subunit mRNAs are co-expressed with glutamate decarboxylase 67 (GAD67), the marker for GABAergic interneurons. In this anatomical study, we systematically investigated the co-expression of GAD67 with different nAChR subunits by using double in situ hybridization with a digoxigenin-labeled GAD67 probe and (35)S-labeled probes for nAChR subunits (alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4). The results revealed that most GAD67-positive interneurons expressed beta2, and 67 % also expressed alpha7 mRNA. In contrast, mRNA expression of other subunits was limited; only 13 % of GAD67-positive neurons co-expressed alpha4, and less than 10% expressed transcripts for alpha2, alpha3, alpha5, or beta4. Most GAD67/alpha2 co-expression was located in CA1/CA3 stratum oriens, and GAD67/alpha5 co-expression was predominantly detected in CA1/CA3 stratum radiatum/lacunosum moleculare and the dentate gyrus. Expression of alpha6 and beta3 mRNAs was rarely detected in the hippocampus, and mRNAs were not co-expressed with GAD67. These findings suggest that the majority of nicotinic responses in GABAergic interneurons should be mediated by a homomeric alpha7 or heteromeric alpha7*-containing nAChRs. Other possible combinations such as alpha2beta2*, alpha4beta2*, or alpha5beta2* heteromeric nAChRs could contribute to functional nicotinic response in subsets of GABAergic interneurons but overall would have a minor role.

  5. Characterization of GABAergic neurons in the mouse lateral septum: a double fluorescence in situ hybridization and immunohistochemical study using tyramide signal amplification.

    Changjiu Zhao

    Full Text Available Gamma-aminobutyric acid (GABA neurotransmission in the lateral septum (LS is implicated in modulating various behavioral processes, including emotional reactivity and maternal behavior. However, identifying the phenotype of GABAergic neurons in the CNS has been hampered by the longstanding inability to reliably detect somal immunoreactivity for GABA or glutamic acid decarboxylase (GAD, the enzyme that produces GABA. In this study, we designed unique probes for both GAD65 (GAD2 and GAD67 (GAD1, and used fluorescence in Situ hybridization (FISH with tyramide signal amplification (TSA to achieve unequivocal detection of cell bodies of GABAergic neurons by GAD mRNAs. We quantitatively characterized the expression and chemical phenotype of GABAergic neurons across each subdivision of LS and in cingulate cortex (Cg and medial preoptic area (MPOA in female mice. Across LS, almost all GAD65 mRNA-expressing neurons were found to contain GAD67 mRNA (approximately 95-98%, while a small proportion of GAD67 mRNA-containing neurons did not express GAD65 mRNA (5-14%. Using the neuronal marker NeuN, almost every neuron in LS (> 90% was also found to be GABA-positive. Interneuron markers using calcium-binding proteins showed that LS GABAergic neurons displayed immunoreactivity for calbindin (CB or calretinin (CR, but not parvalbumin (PV; almost all CB- or CR-immunoreactive neurons (98-100% were GABAergic. The proportion of GABAergic neurons immunoreactive for CB or CR varied depending on the subdivisions examined, with the highest percentage of colocalization in the caudal intermediate LS (LSI (approximately 58% for CB and 35% for CR. These findings suggest that the vast majority of GABAergic neurons within the LS have the potential for synthesizing GABA via the dual enzyme systems GAD65 and GAD67, and each subtype of GABAergic neurons identified by distinct calcium-binding proteins may exert unique roles in the physiological function and neuronal circuitry of

  6. Interactions between ethanol and the endocannabinoid system at GABAergic synapses on basolateral amygdala principal neurons.

    Talani, Giuseppe; Lovinger, David M


    The basolateral amygdala (BLA) plays crucial roles in stimulus value coding, as well as drug and alcohol dependence. Ethanol alters synaptic transmission in the BLA, while endocannabinoids (eCBs) produce presynaptic depression at BLA synapses. Recent studies suggest interactions between ethanol and eCBs that have important consequences for alcohol drinking behavior. To determine how ethanol and eCBs interact in the BLA, we examined the physiology and pharmacology of GABAergic synapses onto BLA pyramidal neurons in neurons from young rats. Application of ethanol at concentrations relevant to intoxication increased, in both young and adult animals, the frequency of spontaneous and miniature GABAergic inhibitory postsynaptic currents, indicating a presynaptic site of ethanol action. Ethanol did not potentiate sIPSCs during inhibition of adenylyl cyclase while still exerting its effect during inhibition of protein kinase A. Activation of type 1 cannabinoid receptors (CB1) in the BLA inhibited GABAergic transmission via an apparent presynaptic mechanism, and prevented ethanol potentiation. Surprisingly, ethanol potentiation was also prevented by CB1 antagonists/inverse agonists. Brief depolarization of BLA pyramidal neurons suppressed GABAergic transmission (depolarization-induced suppression of inhibition [DSI]), an effect previously shown to be mediated by postsynaptic eCB release and presynaptic CB1 activation. A CB1-mediated suppression of GABAergic transmission was also produced by combined afferent stimulation at 0.1 Hz (LFS), and postsynaptic loading with the eCB arachidonoyl ethanolamide (AEA). Both DSI and LFS-induced synaptic depression were prevented by ethanol. Our findings indicate antagonistic interactions between ethanol and eCB/CB1 modulation at GABAergic BLA synapses that may contribute to eCB roles in ethanol seeking and drinking.

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

    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


    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: PMID:28012274

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

    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


    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. Hypothalamic Vasopressinergic Projections Innervate Central Amygdala GABAergic Neurons: Implications for Anxiety and Stress Coping

    Hernández, Vito S.; Hernández, Oscar R.; Perez de la Mora, Miguel; Gómora, María J.; Fuxe, Kjell; Eiden, Lee E.; Zhang, Limei


    The arginine-vasopressin (AVP)-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs) are known for their role in hydro-electrolytic balance control via their projections to the neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula and other brain regions in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA). The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS), consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptor mRNAs were not detected, using the same method. Water-deprivation (WD) for 24 h, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze (EPM) test, and this effect was mimicked by bilateral microinfusion of AVP into the CeA. Anxious behavior induced by either WD or AVP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of CeA inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala. PMID:27932956

  10. Hypothalamic vasopressinergic projections innervate central amygdala GABAergic neurons: implications for anxiety and stress coping

    Vito Salvador Hernandez


    Full Text Available The arginine-vasopressin (AVP-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs are known for their role in hydro-electrolytic balance control via their projections to neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula, and other brain regions, in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA. The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS, consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN and supraoptic (SON nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptors mRNA were not detected, using the same method. Water-deprivation for 24 hrs, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze test, and this effect was mimicked by bilateral microinfusion of VP into the CeA. Anxious behavior induced by either water deprivation or VP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of central amygdala inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala.

  11. 5-HT7 receptor modulates GABAergic transmission in the rat dorsal raphe nucleus and controls cortical release of serotonin.

    Kusek, Magdalena; Sowa, Joanna; Kamińska, Katarzyna; Gołembiowska, Krystyna; Tokarski, Krzysztof; Hess, Grzegorz


    The 5-HT7 receptor is one of the several serotonin (5-HT) receptor subtypes that are expressed in the dorsal raphe nucleus (DRN). Some earlier findings suggested that 5-HT7 receptors in the DRN were localized on GABAergic interneurons modulating the activity of 5-HT projection neurons. The aim of the present study was to find out how the 5-HT7 receptor modulates the GABAergic synaptic input to putative 5-HT DRN neurons, and whether blockade of the 5-HT7 receptor would affect the release of 5-HT in the target structure. Male Wistar rats with microdialysis probes implanted in the prefrontal cortex (PFC) received injections of the 5-HT7 receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride (SB 269970), which induced an increase in the levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the PFC. In another set of experiments whole-cell recordings from presumed projection neurons were carried out using DRN slices. SB 269970 application resulted in depolarization and in an increase in the firing frequency of the cells. In order to activate 5-HT7 receptors, 5-carboxamidotryptamine (5-CT) was applied in the presence of N-[2-[4-(2-methoxyphenyl)-1piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY100635). Hyperpolarization of cells and a decrease in the firing frequency were observed after activation of the 5-HT7 receptor. Blockade of 5-HT7 receptors caused a decrease in the mean frequency of spontaneous inhibitory postsynaptic currents (sIPSCs), while its activation induced an increase. The mechanism of these effects appears to involve tonically-active 5-HT7 receptors modulating firing and/or GABA release from inhibitory interneurons which regulate the activity of DRN serotonergic projection neurons.

  12. The cholinergic agonist carbachol increases the frequency of spontaneous GABAergic synaptic currents in dorsal raphe serotonergic neurons in the mouse.

    Yang, C; Brown, R E


    Dorsal raphe nucleus (DRN) serotonin (5-HT) neurons play an important role in feeding, mood control and stress responses. One important feature of their activity across the sleep-wake cycle is their reduced firing during rapid-eye-movement (REM) sleep which stands in stark contrast to the wake/REM-on discharge pattern of brainstem cholinergic neurons. A prominent model of REM sleep control posits a reciprocal interaction between these cell groups. 5-HT inhibits cholinergic neurons, and activation of nicotinic receptors can excite DRN 5-HT neurons but the cholinergic effect on inhibitory inputs is incompletely understood. Here, in vitro, in DRN brain slices prepared from GAD67-GFP knock-in mice, a brief (3 min) bath application of carbachol (50 μM) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in GFP-negative, putative 5-HT neurons but did not affect miniature (tetrodotoxin-insensitive) IPSCs. Carbachol had no direct postsynaptic effect. Thus, carbachol likely increases the activity of local GABAergic neurons which synapse on 5-HT neurons. Removal of dorsal regions of the slice including the ventrolateral periaqueductal gray (vlPAG) region where GABAergic neurons projecting to the DRN have been identified, abolished the effect of carbachol on sIPSCs whereas the removal of ventral regions containing the oral region of the pontine reticular nucleus (PnO) did not. In addition, carbachol directly excited GFP-positive, GABAergic vlPAG neurons. Antagonism of both muscarinic and nicotinic receptors completely abolished the effects of carbachol. We suggest cholinergic neurons inhibit DRN 5-HT neurons when acetylcholine levels are lower i.e. during quiet wakefulness and the beginning of REM sleep periods, in part via excitation of muscarinic and nicotinic receptors located on local vlPAG and DRN GABAergic neurons. Higher firing rates or burst firing of cholinergic neurons associated with attentive wakefulness or phasic REM sleep periods

  13. A very large number of GABAergic neurons are activated in the tuberal hypothalamus during paradoxical (REM sleep hypersomnia.

    Emilie Sapin

    Full Text Available We recently discovered, using Fos immunostaining, that the tuberal and mammillary hypothalamus contain a massive population of neurons specifically activated during paradoxical sleep (PS hypersomnia. We further showed that some of the activated neurons of the tuberal hypothalamus express the melanin concentrating hormone (MCH neuropeptide and that icv injection of MCH induces a strong increase in PS quantity. However, the chemical nature of the majority of the neurons activated during PS had not been characterized. To determine whether these neurons are GABAergic, we combined in situ hybridization of GAD(67 mRNA with immunohistochemical detection of Fos in control, PS deprived and PS hypersomniac rats. We found that 74% of the very large population of Fos-labeled neurons located in the tuberal hypothalamus after PS hypersomnia were GAD-positive. We further demonstrated combining MCH immunohistochemistry and GAD(67in situ hybridization that 85% of the MCH neurons were also GAD-positive. Finally, based on the number of Fos-ir/GAD(+, Fos-ir/MCH(+, and GAD(+/MCH(+ double-labeled neurons counted from three sets of double-staining, we uncovered that around 80% of the large number of the Fos-ir/GAD(+ neurons located in the tuberal hypothalamus after PS hypersomnia do not contain MCH. Based on these and previous results, we propose that the non-MCH Fos/GABAergic neuronal population could be involved in PS induction and maintenance while the Fos/MCH/GABAergic neurons could be involved in the homeostatic regulation of PS. Further investigations will be needed to corroborate this original hypothesis.

  14. Genetic dissection of GABAergic neural circuits in mouse neocortex

    Hiroki eTaniguchi


    Full Text Available Diverse and flexible cortical functions rely on the ability of neural circuits to perform multiple types of neuronal computations. GABAergic inhibitory interneurons significantly contribute to this task by regulating the balance of activity, synaptic integration, spiking, synchrony, and oscillation in a neural ensemble. GABAergic interneruons display a high degree of cellular diversity in morphology, physiology, connectivity, and gene expression. A considerable number of subtypes of GABAergic interneurons diversify modes of cortical inhibition, enabling various types of information processing in the cortex. Thus, comprehensively understanding fate specification, circuit assembly and physiological function of GABAergic interneurons is a key to elucidate the principles of cortical wiring and function. Recent advances in genetically encoded molecular tools have made a breakthrough to systematically study cortical circuitry at the molecular, cellular, circuit, and whole animal levels. However, the biggest obstacle to fully applying the power of these to analysis of GABAergic circuits was that there were no efficient and reliable methods to express them in subtypes of GABAergic interneurons. Here, I first summarize cortical interneuron diversity and current understanding of mechanisms, by which distinct classes of GABAergic interneurons are generated. I then review recent development in genetically encoded molecular tools for neural circuit research, and genetic targeting of GABAergic interneuron subtypes, particulary focusing on our recent effort to develop and characterize Cre/CreER knockin lines. Finally, I highlight recent success in genetic targeting of chandelier cells (ChCs, the most unique and distinct GABAergic interneuron subtype, and discuss what kind of questions need to be addressed to understand development and function of cortical inhibitory circuits.

  15. Effects of Morphology Constraint on Electrophysiological Properties of Cortical Neurons

    Zhu, Geng; Du, Liping; Jin, Lei; Offenhäusser, Andreas


    There is growing interest in engineering nerve cells in vitro to control architecture and connectivity of cultured neuronal networks or to build neuronal networks with predictable computational function. Pattern technologies, such as micro-contact printing, have been developed to design ordered neuronal networks. However, electrophysiological characteristics of the single patterned neuron haven’t been reported. Here, micro-contact printing, using polyolefine polymer (POP) stamps with high resolution, was employed to grow cortical neurons in a designed structure. The results demonstrated that the morphology of patterned neurons was well constrained, and the number of dendrites was decreased to be about 2. Our electrophysiological results showed that alterations of dendritic morphology affected firing patterns of neurons and neural excitability. When stimulated by current, though both patterned and un-patterned neurons presented regular spiking, the dynamics and strength of the response were different. The un-patterned neurons exhibited a monotonically increasing firing frequency in response to injected current, while the patterned neurons first exhibited frequency increase and then a slow decrease. Our findings indicate that the decrease in dendritic complexity of cortical neurons will influence their electrophysiological characteristics and alter their information processing activity, which could be considered when designing neuronal circuitries.

  16. Hypothalamic Non-AgRP, Non-POMC GABAergic Neurons Are Required for Postweaning Feeding and NPY Hyperphagia.

    Kim, Eun Ran; Wu, Zhaofei; Sun, Hao; Xu, Yuanzhong; Mangieri, Leandra R; Xu, Yong; Tong, Qingchun


    The hypothalamus is critical for feeding and body weight regulation. Prevailing studies focus on hypothalamic neurons that are defined by selectively expressing transcription factors or neuropeptides including those expressing proopiomelanocortin (POMC) and agouti-related peptides (AgRP). The Cre expression driven by the pancreas-duodenum homeobox 1 promoter is abundant in several hypothalamic nuclei but not in AgRP or POMC neurons. Using this line, we generated mice with disruption of GABA release from a major subset of non-POMC, non-AgRP GABAergic neurons in the hypothalamus. These mice exhibited a reduction in postweaning feeding and growth, and disrupted hyperphagic responses to NPY. Disruption of GABA release severely diminished GABAergic input to the paraventricular hypothalamic nucleus (PVH). Furthermore, disruption of GABA-A receptor function in the PVH also reduced postweaning feeding and blunted NPY-induced hyperphagia. Given the limited knowledge on postweaning feeding, our results are significant in identifying GABA release from a major subset of less appreciated hypothalamic neurons as a key mediator for postweaning feeding and NPY hyperphagia, and the PVH as one major downstream site that contributes significantly to the GABA action. Significance statement: Prevalent studies on feeding in the hypothalamus focus on well characterized, selective groups neurons [e.g., proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons], and as a result, the role of the majority of other hypothalamic neurons is largely neglected. Here, we demonstrated an important role for GABAergic projections from non-POMC non-AgRP neurons to the paraventricular hypothalamic nucleus in promoting postweaning (mainly nocturnal) feeding and mediating NPY-induced hyperphagia. Thus, these results signify an importance to study those yet to be defined hypothalamic neurons in the regulation of energy balance and reveal a neural basis for postweaning (nocturnal) feeding and

  17. Direct Induction and Functional Maturation of Forebrain GABAergic Neurons from Human Pluripotent Stem Cells

    Alfred Xuyang Sun


    Full Text Available Gamma-aminobutyric acid (GABA-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here, we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs into GABAergic neurons (iGNs with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6–8 weeks. Furthermore, in vitro, iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs. Upon transplantation into immunodeficient mice, human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together, our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons.

  18. Direct Induction and Functional Maturation of Forebrain GABAergic Neurons from Human Pluripotent Stem Cells.

    Sun, Alfred Xuyang; Yuan, Qiang; Tan, Shawn; Xiao, Yixin; Wang, Danlei; Khoo, Audrey Tze Ting; Sani, Levena; Tran, Hoang-Dai; Kim, Paul; Chiew, Yong Seng; Lee, Kea Joo; Yen, Yi-Chun; Ng, Huck Hui; Lim, Bing; Je, Hyunsoo Shawn


    Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here, we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore, in vitro, iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice, human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together, our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons.

  19. Action of peripherally administered cholecystokinin on monoaminergic and GABAergic neurons in the rat brain.



    Full Text Available In an acute study, cholecystokinin octapeptide sulfate (CCK in doses of 1, 10 or 100 micrograms/kg body weight was injected intraperitoneally into rats just prior to the dark cycle. Rats were sacrificed two hours following the CCK injection. Norepinephrine levels were elevated in the dorsal amygdala of rats injected with 10 micrograms of CCK as well as in the septum of rats injected with 1 and 10 micrograms of CCK. The dopamine level in the septum of rats injected with 1 microgram of CCK as well as the gamma-aminobutyric acid (GABA level in the lateral hypothalamus of rats injected with 10 micrograms of CCK were also elevated. In a chronic study, CCK (1 microgram/kg body weight/h was subcutaneously infused into rats with Alzet osmotic minipump for seven consecutive days. The daily food consumption did not change during the 7 days of CCK infusion. The dopamine turnover in the striatum accelerated and the GABA level increased. On the contrary, dopamine metabolism in the substantia nigra and locus coeruleus decreased. Furthermore, the serotonin level in the substantia nigra decreased. Norepinephrine levels decreased in the nucleus paraventricularis, the locus coeruleus and the substantia nigra. The results suggest that peripherally administered CCK may act on the monoaminergic neurons and GABAergic neurons in the brain.

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

    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


    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.

  1. Restoration of Mecp2 expression in GABAergic neurons is sufficient to rescue multiple disease features in a mouse model of Rett syndrome.

    Ure, Kerstin; Lu, Hui; Wang, Wei; Ito-Ishida, Aya; Wu, Zhenyu; He, Ling-Jie; Sztainberg, Yehezkel; Chen, Wu; Tang, Jianrong; Zoghbi, Huda Y


    The postnatal neurodevelopmental disorder Rett syndrome, caused by mutations in MECP2, produces a diverse array of symptoms, including loss of language, motor, and social skills and the development of hand stereotypies, anxiety, tremor, ataxia, respiratory dysrhythmias, and seizures. Surprisingly, despite the diversity of these features, we have found that deleting Mecp2 only from GABAergic inhibitory neurons in mice replicates most of this phenotype. Here we show that genetically restoring Mecp2 expression only in GABAergic neurons of male Mecp2 null mice enhanced inhibitory signaling, extended lifespan, and rescued ataxia, apraxia, and social abnormalities but did not rescue tremor or anxiety. Female Mecp2(+/-) mice showed a less dramatic but still substantial rescue. These findings highlight the critical regulatory role of GABAergic neurons in certain behaviors and suggest that modulating the excitatory/inhibitory balance through GABAergic neurons could prove a viable therapeutic option in Rett syndrome.

  2. Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat

    Nair-Roberts, R.G.; Chatelain-Badie, S.D.; Benson, E.; White-Cooper, H; BOLAM, J. P.; Ungless, M.A.


    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...

  3. Parvalbumin-Expressing GABAergic Neurons in Mouse Barrel Cortex Contribute to Gating a Goal-Directed Sensorimotor Transformation

    Shankar Sachidhanandam


    Full Text Available Sensory processing in neocortex is primarily driven by glutamatergic excitation, which is counterbalanced by GABAergic inhibition, mediated by a diversity of largely local inhibitory interneurons. Here, we trained mice to lick a reward spout in response to whisker deflection, and we recorded from genetically defined GABAergic inhibitory neurons in layer 2/3 of the primary somatosensory barrel cortex. Parvalbumin-expressing (PV, vasoactive intestinal peptide-expressing (VIP, and somatostatin-expressing (SST neurons displayed distinct action potential firing dynamics during task performance. Whereas SST neurons fired at low rates, both PV and VIP neurons fired at high rates both spontaneously and in response to whisker stimulation. After an initial outcome-invariant early sensory response, PV neurons had lower firing rates in hit trials compared to miss trials. Optogenetic inhibition of PV neurons during this time period enhanced behavioral performance. Hence, PV neuron activity might contribute causally to gating the sensorimotor transformation of a whisker sensory stimulus into licking motor output.

  4. 5-HT1A/7 receptor agonist excites cardiac vagal neurons via inhibition of both GABAergic and glycinergic inputs

    Yong-hua CHEN; Li-li HOU; Ji-jiang WANG


    Aim: To study the synaptic mechanisms involved in the 5-hydroxytryptaminel AF/7 (5-HT1A/7) receptor-mediated reflex control of cardiac vagal preganglionic neurons (CVPN). Methods: CVPN were retrogradely labeled and identified in brain stem slices of newborn rats, and their synaptic activity was examined using whole-cell patch-clamp. Results: 8-Hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT), an agonist of 5-HT1A/7 receptors, had no effect on the glutamatergic inputs of CVPN. In contrast, it significantly decreased the frequency and the amplitude of both the GABAergic and the glycinergic spontaneous inhibitory postsynaptic currents (slPSC). 8-OH-DPAT also caused significant amplitude decrease of the GABAergic currents evoked by stimulation of the nucleus tractus solitarius. Both the fre-quency inhibition and the amplitude inhibition of the GABAergic and the glycinergic sIPSC by 8-OH-DPAT had dose-dependent tendencies and could be reversed by WAY-100635, an antagonist of 5-HT1A/7 receptors. In the pre-exist-ence of tetrodotoxin, 8-OH-DPAT had no effect on the GABAergic or the glycinergic miniature inhibitory postsynaptic currents, and had no effect on the GABAergic or the glycinergic currents evoked by exogenous GABA or glycine. Conclusion:The 5-HT1A/7 receptor agonist excites CVPN indirectly via the inhibition of both the GABAergic and glycinergic inputs. These findings have at least in part re-vealed the synaptic mechanisms involved in the 5-HT1A/7 receptor-mediated reflex control of cardiac vagal nerves in intact animals.

  5. Distribution and compartmental organization of GABAergic medium-sized spiny neurons in the mouse Nucleus Accumbens

    Giuseppe eGangarossa


    Full Text Available The nucleus accumbens (NAc is a critical brain region involved in many reward-related behaviors. The NAc comprises major compartments the core and the shell, which encompass several subterritories. GABAergic medium-sized spiny neurons (MSNs constitute the output neurons of the NAc core and shell. While the functional organization of the NAc core outputs resembles the one described for the dorsal striatum, a simple classification of the NAc shell neurons has been difficult to define due to the complexity of the compartmental segregation of cells. We used a variety of BAC transgenic mice expressing enhanced green fluorescence (EGFP or the Cre-recombinase (Cre under the control of the promoter of dopamine D1, D2, and D3 receptors and of adenosine A2a receptor to dissect the microanatomy of the NAc. Moreover, using various immunological markers we characterized in detail the distribution of MSNs in the mouse NAc. In addition, cell-type specific ERK phosphorylation in the NAc subterritories was analyzed following acute administration of SKF81297 (a D1R-like agonist, quinpirole (a D2R-like agonist, apomorphine (a non-selective DA receptor agonist, raclopride (a D2R-like antagonist, and psychostimulant drugs, including cocaine and d-amphetamine. Each drug generated a unique topography and cell-type specific activation of ERK in the NAc. Our results show the existence of marked differences in the receptor expression pattern and functional activation of MSNs within the shell subterritories. This study emphasizes the anatomical and functional heterogeneity of the NAc, which will have to be considered in its further study.

  6. Enhancement of cortical network activity in vitro and promotion of GABAergic neurogenesis by stimulation with an electromagnetic field with a 150 MHz carrier wave pulsed with an alternating 10 and 16 Hz modulation.

    Alexandra eGramowski-Voss


    Full Text Available In recent years, various stimuli were identified capable of enhancing neurogenesis, a process which is dysfunctional in the senescent brain and in neurodegenerative and certain neuropsychiatric diseases. Applications of electromagnetic fields to brain tissue have been shown to affect cellular properties and their importance for therapies in medicine is recognized.In this study, differentiating murine cortical networks on multiwell microelectrode arrays were repeatedly exposed to an extremely low electromagnetic field (ELEMF with alternating 10 and 16 Hz frequencies piggy-backed onto a 150 MHz carrier frequency. The ELEMF exposure stimulated the electrical network activity and intensified the structure of bursts. Further, the exposure with an electromagnetic field within the first 28 days of the differentiation the network activity induced also reorganization within the burst structure. This effect was already most pronounced at 14 days in vitro after 10 days of exposure. Overall, the development of cortical activity under these conditions was accelerated. These functional electrophysiological changes were accompanied by morphological ones. The percentage of neurons in the neuron glia co-culture was increased without affecting the total number of cells, indicating an enhancement of neurogenesis. The ELEMF exposure selectively promoted the proliferation of a particular population of neurons, evidenced by the increased proportion of GABAergic neurons. The results support the initial hypothesis that this kind of ELEMF stimulation is a treatment option for specific indications with promising potential for CNS applications, especially for degenerative diseases such as Alzheimer’s disease and other dementias.

  7. GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon

    Ducray, Angélique; Krebs, Sandra H:; Schaller, Benoft;


    of the GFLs affected soma size of GABA-ir neurons. In contrast, only NRTN treatment significantly increased 5-HT-ir cells densities at 10 ng/ml (1.3-fold), while an augmentation was seen for GDNF and PSPN at 100 ng/ml (2.4-fold and 1.7-fold, respectively). ARTN had no effect on 5-HT-ir cell densities....... Morphological analysis of 5-HT-ir neurons revealed a significant increase of soma size, number of primary neurites/neuron and neurite length/neuron after GDNF exposure, while PSPN only affected soma size, and NRTN and ARTN failed to exert any effect. In conclusion, we identified GFLs as effective neurotrophic...... factors for VM GABAergic and serotonergic neurons, demonstrating characteristic individual action profiles emphasizing their important and distinct roles during brain development....

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

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


    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.

  9. Characterization of GABAergic neurons in rapid-eye-movement sleep controlling regions of the brainstem reticular formation in GAD67-green fluorescent protein knock-in mice.

    Brown, Ritchie E; McKenna, James T; Winston, Stuart; Basheer, Radhika; Yanagawa, Yuchio; Thakkar, Mahesh M; McCarley, Robert W


    Recent experiments suggest that brainstem GABAergic neurons may control rapid-eye-movement (REM) sleep. However, understanding their pharmacology/physiology has been hindered by difficulty in identification. Here we report that mice expressing green fluorescent protein (GFP) under the control of the GAD67 promoter (GAD67-GFP knock-in mice) exhibit numerous GFP-positive neurons in the central gray and reticular formation, allowing on-line identification in vitro. Small (10-15 microm) or medium-sized (15-25 microm) GFP-positive perikarya surrounded larger serotonergic, noradrenergic, cholinergic and reticular neurons, and > 96% of neurons were double-labeled for GFP and GABA, confirming that GFP-positive neurons are GABAergic. Whole-cell recordings in brainstem regions important for promoting REM sleep [subcoeruleus (SubC) or pontine nucleus oralis (PnO) regions] revealed that GFP-positive neurons were spontaneously active at 3-12 Hz, fired tonically, and possessed a medium-sized depolarizing sag during hyperpolarizing steps. Many neurons also exhibited a small, low-threshold calcium spike. GFP-positive neurons were tested with pharmacological agents known to promote (carbachol) or inhibit (orexin A) REM sleep. SubC GFP-positive neurons were excited by the cholinergic agonist carbachol, whereas those in the PnO were either inhibited or excited. GFP-positive neurons in both areas were excited by orexins/hypocretins. These data are congruent with the hypothesis that carbachol-inhibited GABAergic PnO neurons project to, and inhibit, REM-on SubC reticular neurons during waking, whereas carbachol-excited SubC and PnO GABAergic neurons are involved in silencing locus coeruleus and dorsal raphe aminergic neurons during REM sleep. Orexinergic suppression of REM during waking is probably mediated in part via excitation of acetylcholine-inhibited GABAergic neurons.

  10. Cortical cell and neuron density estimates in one chimpanzee hemisphere.

    Collins, Christine E; Turner, Emily C; Sawyer, Eva Kille; Reed, Jamie L; Young, Nicole A; Flaherty, David K; Kaas, Jon H


    The density of cells and neurons in the neocortex of many mammals varies across cortical areas and regions. This variability is, perhaps, most pronounced in primates. Nonuniformity in the composition of cortex suggests regions of the cortex have different specializations. Specifically, regions with densely packed neurons contain smaller neurons that are activated by relatively few inputs, thereby preserving information, whereas regions that are less densely packed have larger neurons that have more integrative functions. Here we present the numbers of cells and neurons for 742 discrete locations across the neocortex in a chimpanzee. Using isotropic fractionation and flow fractionation methods for cell and neuron counts, we estimate that neocortex of one hemisphere contains 9.5 billion cells and 3.7 billion neurons. Primary visual cortex occupies 35 cm(2) of surface, 10% of the total, and contains 737 million densely packed neurons, 20% of the total neurons contained within the hemisphere. Other areas of high neuron packing include secondary visual areas, somatosensory cortex, and prefrontal granular cortex. Areas of low levels of neuron packing density include motor and premotor cortex. These values reflect those obtained from more limited samples of cortex in humans and other primates.

  11. Retinoic acid from the meninges regulates cortical neuron generation.

    Siegenthaler, Julie A; Ashique, Amir M; Zarbalis, Konstantinos; Patterson, Katelin P; Hecht, Jonathan H; Kane, Maureen A; Folias, Alexandra E; Choe, Youngshik; May, Scott R; Kume, Tsutomu; Napoli, Joseph L; Peterson, Andrew S; Pleasure, Samuel J


    Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.

  12. GABAergic inhibition modulates intensity sensitivity of temporally patterned pulse trains in the inferior collicular neurons in big brown bats.

    Luan, Rui-Hong; Wu, Fei-Jian; Jen, Philip H-S; Sun, Xin-De


    The echolocating big brown bats (Eptesicus fuscus) emit trains of frequency-modulated (FM) biosonar signals with duration, amplitude, repetition rate, and sweep structure changing systematically during interception of their prey. In the present study, the sound stimuli of temporally patterned pulse trains at three different pulse repetition rates (PRRs) were used to mimic the sounds received during search, approach, and terminal stages of echolocation. Electrophysiological method was adopted in recordings from the inferior colliculus (IC) of midbrain. By means of iontophoretic application of bicuculline, the effect of GABAergic inhibition on the intensity sensitivity of IC neurons responding to three different PRRs of 10, 30 and 90 pulses per second (pps) was examined. The rate-intensity functions (RIFs) were acquired. The dynamic range (DR) of RIFs was considered as a criterion of intensity sensitivity. Comparing the average DR of RIFs at different PRRs, we found that the intensity sensitivity of some neurons improved, but that of other neurons decayed when repetition rate of stimulus trains increased from 10 to 30 and 90 pps. During application of bicuculline, the number of impulses responding to the different pulse trains increased under all stimulating conditions, while the DR differences of RIFs at different PRRs were abolished. The results indicate that GABAergic inhibition was involved in modulating the intensity sensitivity of IC neurons responding to pulse trains at different PRRs. Before and during bicuculline application, the percentage of changes in responses was maximal in lower stimulus intensity near to the minimum threshold (MT), and decreased gradually with the increment of stimulus intensity. This observation suggests that GABAergic inhibition contributes more effectively to the intensity sensitivity of the IC neurons responding to pulse trains at lower sound level.

  13. Rich-Club Organization in Effective Connectivity among Cortical Neurons

    Shimono, Masanori; Ito, Shinya; Yeh, Fang-Chin; Timme, Nicholas; Myroshnychenko, Maxym; Lapish, Christopher C.; Tosi, Zachary; Hottowy, Pawel; Smith, Wesley C.; Masmanidis, Sotiris C.; Litke, Alan M.; Sporns, Olaf; Beggs, John M.


    The performance of complex networks, like the brain, depends on how effectively their elements communicate. Despite the importance of communication, it is virtually unknown how information is transferred in local cortical networks, consisting of hundreds of closely spaced neurons. To address this, it is important to record simultaneously from hundreds of neurons at a spacing that matches typical axonal connection distances, and at a temporal resolution that matches synaptic delays. We used a 512-electrode array (60 μm spacing) to record spontaneous activity at 20 kHz from up to 500 neurons simultaneously in slice cultures of mouse somatosensory cortex for 1 h at a time. We applied a previously validated version of transfer entropy to quantify information transfer. Similar to in vivo reports, we found an approximately lognormal distribution of firing rates. Pairwise information transfer strengths also were nearly lognormally distributed, similar to reports of synaptic strengths. Some neurons transferred and received much more information than others, which is consistent with previous predictions. Neurons with the highest outgoing and incoming information transfer were more strongly connected to each other than chance, thus forming a “rich club.” We found similar results in networks recorded in vivo from rodent cortex, suggesting the generality of these findings. A rich-club structure has been found previously in large-scale human brain networks and is thought to facilitate communication between cortical regions. The discovery of a small, but information-rich, subset of neurons within cortical regions suggests that this population will play a vital role in communication, learning, and memory. SIGNIFICANCE STATEMENT Many studies have focused on communication networks between cortical brain regions. In contrast, very few studies have examined communication networks within a cortical region. This is the first study to combine such a large number of neurons (several

  14. α-MSH exerts direct postsynaptic excitatory effects on NTS neurons and enhances GABAergic signaling in the NTS.

    Mimee, A; Kuksis, M; Ferguson, A V


    The central melanocortin system plays an essential role in the regulation of energy balance. While anorexigenic effects of α-melanocyte-stimulating hormone (α-MSH) acting in the nucleus of the solitary tract (NTS), a critical medullary autonomic control center, have been established, the cellular events underlying these effects are less well characterized. In this study, we used whole-cell patch-clamp electrophysiology to examine firstly whether α-MSH exerts direct postsynaptic effects on the membrane potential of rat NTS neurons in slice preparation, and secondly whether α-MSH influences GABAergic signaling in the NTS. In normal artificial cerebrospinal fluid, perfusion of α-MSH (500 nM) resulted in a depolarization in 39% of cells (n=16, mean 6.14±0.54 mV), and a hyperpolarization in 22% of cells (n=9, -6.79±1.02 mV). Studies using tetrodotoxin to block neuronal communication revealed α-MSH exerts direct depolarizing effects on some NTS neurons, and indirect inhibitory effects on others. A third subset of neurons is simultaneously directly depolarized and indirectly hyperpolarized by α-MSH, resulting in a net lack of effect on membrane potential. The inhibitory inputs influenced by α-MSH were identified as GABAergic, as α-MSH increased the frequency, but not amplitude, of inhibitory postsynaptic currents (IPSCs) in 50% of NTS neurons. α-MSH had no effect on the frequency or amplitude of miniature IPSCs. Furthermore, pharmacological blockade of GABAA and GABAB receptors, and physical removal of all synaptic inputs via cellular dissociation, abolished hyperpolarizations induced by α-MSH. We conclude α-MSH exerts direct, postsynaptic excitatory effects on a subset of NTS neurons. By exciting GABAergic NTS neurons and presynaptically enhancing GABAergic signaling, α-MSH also indirectly inhibits other NTS cells. These findings provide critical insight into the cellular events underlying medullary melanocortin anorexigenic effects, and expand the

  15. 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.

    Aldana, Blanca I; Zhang, Yu; Lihme, Maria Fog; Bak, Lasse K; Nielsen, Jørgen E; Holst, Bjørn; Hyttel, Poul; Freude, Kristine K; Waagepetersen, Helle S


    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

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  16. Perisomatic GABAergic synapses of basket cells effectively control principal neuron activity in amygdala networks

    Veres, Judit M; Nagy, Gergő A; Hájos, Norbert


    Efficient control of principal neuron firing by basket cells is critical for information processing in cortical microcircuits, however, the relative contribution of their perisomatic and dendritic synapses to spike inhibition is still unknown. Using in vitro electrophysiological paired recordings we reveal that in the mouse basal amygdala cholecystokinin- and parvalbumin-containing basket cells provide equally potent control of principal neuron spiking. We performed pharmacological manipulations, light and electron microscopic investigations to show that, although basket cells innervate the entire somato-denditic membrane surface of principal neurons, the spike controlling effect is achieved primarily via the minority of synapses targeting the perisomatic region. As the innervation patterns of individual basket cells on their different postsynaptic partners show high variability, the impact of inhibitory control accomplished by single basket cells is also variable. Our results show that both basket cell types can powerfully regulate the activity in amygdala networks predominantly via their perisomatic synapses. DOI: PMID:28060701

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

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


    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.

  18. Multiple facets of GABAergic neurons and synapses: multiple fates of GABA signalling in epilepsies.

    Cossart, Rosa; Bernard, Christophe; Ben-Ari, Yehezkel


    Because blocking GABAergic neurotransmission in control tissue generates seizures and because GABA boosters control epilepsy in many patients, studies on epilepsies have been dominated by the axiom that seizures are generated by a failure of GABA-mediated inhibition. However, GABAergic interneurons and synapses are heterogeneous and have many roles that go beyond the straightforward concept of "inhibition of the target". Operation of such a diversified system cannot be ascribed to a single mechanism. In epileptic tissue, GABAergic networks undergo complex rewiring at the anatomical, physiological and functional levels; GABAergic synapses are still operative but show unique features, including excitatory effects. Therefore, inhibition is not a uniform notion and the concept of "failure" of inhibition in epilepsies must be reassessed. Seizures are not generated in a normal circuit in which GABA-mediated inhibition is simply impaired, but in a profoundly rewired network in which several properties of GABA function are altered. This review is part of the TINS Interneuron Diversity series.

  19. The aspirin metabolite salicylate enhances neuronal excitation in rat hippocampal CA1 area through reducing GABAergic inhibition.

    Gong, Neng; Zhang, Min; Zhang, Xiao-Bing; Chen, Lin; Sun, Guang-Chun; Xu, Tian-Le


    Salicylate is the major metabolite and active component of aspirin (acetylsalicylic acid), which is widely used in clinical medicine for treating inflammation, pain syndromes and cardiovascular disorders. The well-known mechanism underlying salicylate's action mainly involves the inhibition of cyclooxygenase and subsequent decrease in prostaglandin production. Recent evidence suggests that salicylate also affects neuronal function through interaction with specific membrane channels/receptors. However, the effect of salicylate on synaptic and neural network function remains largely unknown. In this study, we investigated the effect of sodium salicylate on the synaptic transmission and neuronal excitation in the hippocampal CA1 area of rats, a key structure for many complex brain functions. With electrophysiological recordings in hippocampal slices, we found that sodium salicylate significantly enhanced neuronal excitation through reducing inhibitory GABAergic transmission without affecting the basal excitatory synaptic transmission. Salicylate significantly inhibited the amplitudes of both evoked and miniature inhibitory postsynaptic currents, and directly reduced gamma-aminobutyric acid type A (GABA(A)) receptor-mediated responses in cultured rat hippocampal neurons. Together, our results suggest that the widely used aspirin might impair hippocampal synaptic and neural network functions through its actions on GABAergic neurotransmission. Given the capability of aspirin to penetrate the blood-brain barrier, the present data imply that aspirin intake may cause network hyperactivity and be potentially harmful in susceptible subpopulations.

  20. Striatal cholinergic interneurons and D2 receptor-expressing GABAergic medium spiny neurons regulate tardive dyskinesia.

    Bordia, Tanuja; Zhang, Danhui; Perez, Xiomara A; Quik, Maryka


    Tardive dyskinesia (TD) is a drug-induced movement disorder that arises with antipsychotics. These drugs are the mainstay of treatment for schizophrenia and bipolar disorder, and are also prescribed for major depression, autism, attention deficit hyperactivity, obsessive compulsive and post-traumatic stress disorder. There is thus a need for therapies to reduce TD. The present studies and our previous work show that nicotine administration decreases haloperidol-induced vacuous chewing movements (VCMs) in rodent TD models, suggesting a role for the nicotinic cholinergic system. Extensive studies also show that D2 dopamine receptors are critical to TD. However, the precise involvement of striatal cholinergic interneurons and D2 medium spiny neurons (MSNs) in TD is uncertain. To elucidate their role, we used optogenetics with a focus on the striatum because of its close links to TD. Optical stimulation of striatal cholinergic interneurons using cholineacetyltransferase (ChAT)-Cre mice expressing channelrhodopsin2-eYFP decreased haloperidol-induced VCMs (~50%), with no effect in control-eYFP mice. Activation of striatal D2 MSNs using Adora2a-Cre mice expressing channelrhodopsin2-eYFP also diminished antipsychotic-induced VCMs, with no change in control-eYFP mice. In both ChAT-Cre and Adora2a-Cre mice, stimulation or mecamylamine alone similarly decreased VCMs with no further decline with combined treatment, suggesting nAChRs are involved. Striatal D2 MSN activation in haloperidol-treated Adora2a-Cre mice increased c-Fos(+) D2 MSNs and decreased c-Fos(+) non-D2 MSNs, suggesting a role for c-Fos. These studies provide the first evidence that optogenetic stimulation of striatal cholinergic interneurons and GABAergic MSNs modulates VCMs, and thus possibly TD. Moreover, they suggest nicotinic receptor drugs may reduce antipsychotic-induced TD. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons.

    de Salas-Quiroga, Adán; Díaz-Alonso, Javier; García-Rincón, Daniel; Remmers, Floortje; Vega, David; Gómez-Cañas, María; Lutz, Beat; Guzmán, Manuel; Galve-Roperh, Ismael


    The CB1 cannabinoid receptor, the main target of Δ(9)-tetrahydrocannabinol (THC), the most prominent psychoactive compound of marijuana, plays a crucial regulatory role in brain development as evidenced by the neurodevelopmental consequences of its manipulation in animal models. Likewise, recreational cannabis use during pregnancy affects brain structure and function of the progeny. However, the precise neurobiological substrates underlying the consequences of prenatal THC exposure remain unknown. As CB1 signaling is known to modulate long-range corticofugal connectivity, we analyzed the impact of THC exposure on cortical projection neuron development. THC administration to pregnant mice in a restricted time window interfered with subcerebral projection neuron generation, thereby altering corticospinal connectivity, and produced long-lasting alterations in the fine motor performance of the adult offspring. Consequences of THC exposure were reminiscent of those elicited by CB1 receptor genetic ablation, and CB1-null mice were resistant to THC-induced alterations. The identity of embryonic THC neuronal targets was determined by a Cre-mediated, lineage-specific, CB1 expression-rescue strategy in a CB1-null background. Early and selective CB1 reexpression in dorsal telencephalic glutamatergic neurons but not forebrain GABAergic neurons rescued the deficits in corticospinal motor neuron development of CB1-null mice and restored susceptibility to THC-induced motor alterations. In addition, THC administration induced an increase in seizure susceptibility that was mediated by its interference with CB1-dependent regulation of both glutamatergic and GABAergic neuron development. These findings demonstrate that prenatal exposure to THC has long-lasting deleterious consequences in the adult offspring solely mediated by its ability to disrupt the neurodevelopmental role of CB1 signaling.

  2. Morphology and ontogeny of rat perirhinal cortical neurons.

    Furtak, Sharon Christine; Moyer, James Russell; Brown, Thomas Huntington


    Golgi-impregnated neurons from rat perirhinal cortex (PR) were classified into one of 15 distinct morphological categories (N = 6,891). The frequency of neurons in each cell class was determined as a function of the layer of PR and the age of the animal, which ranged from postnatal day 0 (P0) to young adulthood (P45). The developmental appearance of Golgi-impregnated neurons conformed to the expected "inside-out" pattern of development, meaning that cells populated in deep before superficial layers of PR. The relative frequencies of different cell types changed during the first 2 weeks of postnatal development. The largest cells, which were pyramidal and spiny multipolar neurons, appeared earliest. Aspiny stellate neurons were the last to appear. The total number of Golgi-impregnated neurons peaked at P10-12, corresponding to the time of eye-opening. This early increase in the number of impregnated neurons parallels observations in other cortical areas. The relative frequency of the 15 cell types remained constant between P14 to P45. The proportion of pyramidal neurons in PR ( approximately 50%) was much smaller than is typical of neocortex ( approximately 70%). A correspondingly larger proportion of PR neurons were nonpyramidal cells that are less common in neocortex. The relative frequency distribution of cell types creates an overall impression of considerable morphological diversity, which is arguably related to the particular manner in which this periallocortical brain region processes and stores information.

  3. Coordinated scaling of cortical and cerebellar numbers of neurons

    Suzana Herculano-Houzel


    Full Text Available While larger brains possess concertedly larger cerebral cortices and cerebella, the relative size of the cerebral cortex increases with brain size, but relative cerebellar size does not. In the absence of data on numbers of neurons in these structures, this discrepancy has been used to dispute the hypothesis that the cerebral cortex and cerebellum function and have evolved in concert and to support a trend towards neocorticalization in evolution. However, the rationale for interpreting changes in absolute and relative size of the cerebral cortex and cerebellum relies on the assumption that they reflect absolute and relative numbers of neurons in these structures across all species – an assumption that our recent studies have shown to be flawed. Here I show for the first time that the numbers of neurons in the cerebral cortex and cerebellum are directly correlated across 19 mammalian species of 4 different orders, including humans, and increase concertedly in a similar fashion both within and across the orders Eulipotyphla (Insectivora, Rodentia, Scandentia and Primata, such that on average a ratio of 3.6 neurons in the cerebellum to every neuron in the cerebral cortex is maintained across species. This coordinated scaling of cortical and cerebellar numbers of neurons provides direct evidence in favor of concerted function, scaling and evolution of these brain structures, and suggests that the common notion that equates cognitive advancement with neocortical expansion should be revisited to consider in its stead the coordinated scaling of neocortex and cerebellum as a functional ensemble.

  4. Involvement of glutamatergic and GABAergic transmission in MK-801-increased gamma band oscillation power in rat cortical electroencephalograms.

    Hiyoshi, T; Kambe, D; Karasawa, J; Chaki, S


    Hypofunction of the N-methyl-D-aspartic acid receptor (NMDAr) has been considered to play a crucial role in the pathophysiology of schizophrenia. In rodent electroencephalogram (EEG) studies, non-competitive NMDAr antagonists have been reported to produce aberrant basal gamma band oscillation (GBO), as observed in schizophrenia. Aberrations in GBO power have attracted attention as a translational biomarker for the development of novel antipsychotic drugs. However, the neuronal mechanisms as well as the pharmacological significance of NMDAr antagonist-induced aberrant GBO power have not been fully investigated. In the present study, to address the above questions, we examined the pharmacological properties of MK-801 (0.1 mg/kg)-increased basal GBO power in rat cortical EEG. Riluzole (3-10 mg/kg), a glutamate release inhibitor, reduced the MK-801-increased basal GBO power. In contrast, L-838,417 (1-3 mg/kg), an α2/3/5 subunit-selective GABAA receptor-positive allosteric modulator, enhanced the GBO increase. Antipsychotics such as haloperidol (0.05-0.3 mg/kg) and clozapine (1-10 mg/kg) dose-dependently attenuated the MK-801-increased GBO power. Likewise, LY379268 (0.3-3 mg/kg), an metabotropic glutamate 2/3 receptor (mGlu2/3 receptor) agonist, reduced the GBO increase in a dose-dependent manner, which was antagonized by an mGlu2/3 receptor antagonist LY341495. These results suggest that an increase in cortical GBO power induced by NMDAr hypofunction can be attributed to the aberrant activities of both excitatory pyramidal neurons and inhibitory interneurons in local circuits. The aberrant cortical GBO power reflecting cortical network dysfunction observed in schizophrenia might be a useful biomarker for the discovery of novel antipsychotic drugs.

  5. Broadband Macroscopic Cortical Oscillations Emerge from Intrinsic Neuronal Response Failures

    Amir eGoldental


    Full Text Available Broadband spontaneous macroscopic neural oscillations are rhythmic cortical firing which was extensively examined during the last century, however, their possible origination is still controversial. In this work we show how macroscopic oscillations emerge in solely excitatory random networks and without topological constraints. We experimentally and theoretically show that these oscillations stem from the counterintuitive underlying mechanism - the intrinsic stochastic neuronal response failures. These neuronal response failures, which are characterized by short-term memory, lead to cooperation among neurons, resulting in sub- or several- Hertz macroscopic oscillations which coexist with high frequency gamma oscillations. A quantitative interplay between the statistical network properties and the emerging oscillations is supported by simulations of large networks based on single-neuron in-vitro experiments and a Langevin equation describing the network dynamics. Results call for the examination of these oscillations in the presence of inhibition and external drives.

  6. A single GABAergic neuron mediates feedback of odor-evoked signals in the mushroom body of larval Drosophila

    Liria Monica Masuda-Nakagawa


    Full Text Available Inhibition has a central role in defining the selectivity of the responses of higher order neurons to sensory stimuli. However, the circuit mechanisms of regulation of these responses by inhibitory neurons are still unclear. In Drosophila, the mushroom bodies (MBs are necessary for olfactory memory, and by implication for the selectivity of learned responses to specific odors. To understand the circuitry of inhibition in the calyx (the input dendritic region of the MBs, and its relationship with MB excitatory activity, we used the simple anatomy of the Drosophila larval olfactory system to identify any inhibitory inputs that could contribute to the selectivity of MB odor responses. We found that a single neuron accounts for all detectable GABA innervation in the calyx of the MBs, and that this neuron has presynaptic terminals in the calyx and postsynaptic branches in the MB lobes (output axonal area. We call this neuron the larval anterior paired lateral (APL neuron, because of its similarity to the previously described adult APL neuron. Reconstitution of GFP partners (GRASP suggests that the larval APL makes extensive contacts with the MB intrinsic neurons, Kenyon Cells (KCs, but few contacts with incoming projection neurons. Using calcium imaging of neuronal activity in live larvae, we show that the larval APL responds to odors, in a mannner that requires output from KCs. Our data suggest that the larval APL is the sole GABAergic neuron that innervates the MB input region and carries inhibitory feedback from the MB output region, consistent with a role in modulating the olfactory selectivity of MB neurons.

  7. Transcriptional Networks Controlled by NKX2-1 in the Development of Forebrain GABAergic Neurons.

    Sandberg, Magnus; Flandin, Pierre; Silberberg, Shanni; Su-Feher, Linda; Price, James D; Hu, Jia Sheng; Kim, Carol; Visel, Axel; Nord, Alex S; Rubenstein, John L R


    The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissive chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach provides a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.

  8. Cortical neuronal mechanisms of sleep homeostasis.

    Vyazovskiy, Vladyslav V


    The longer we are awake, the deeper is our subsequent sleep. On the other hand, the shorter and more fragmented is our sleep, the more difficult it is for us to maintain wakefulness and stable cognitive performance the next day. This relationship between wakefulness and subsequent sleep becomes especially apparent after sleep deprivation or during chronic sleep restriction, which is experienced by millions of people in our society, as well as in multiple neurological, respiratory and other chronic diseases. Invariably, poor sleep leads to fatigue, sleepiness, marked cognitive deficits and impaired mood. The crucial question is what happens to the brain after a period of being awake or asleep, and where in the brain and why do these changes occur. This review summarizes information about neurophysiological substrates of sleep homeostatic processes at the cellular and network levels. It is suggested that sensory, behavioral and cognitive deficits after sleep deprivation resulting from the imbalance between local and global neuronal interactions can be reversed only by physiological sleep.

  9. 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.

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P; Geller, Alfred I


    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

  10. 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

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P.; Geller, Alfred I.


    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

  11. Expression of COUP-TFII Nuclear Receptor in Restricted GABAergic Neuronal Populations in the Adult Rat Hippocampus

    Fuentealba, Pablo; Klausberger, Thomas; Karayannis, Theofanis; Suen, Wai Yee; Huck, Jojanneke; Tomioka, Ryohei; Rockland, Kathleen; Capogna, Marco; Studer, Michèle; Morales, Marisela; Somogyi, Peter


    The COUP-TFII nuclear receptor, also known as NR2F2, is expressed in the developing ventral telencephalon and modulates the tangential migration of a set of subpallial neuronal progenitors during forebrain development. Little information is available about its expression patterns in the adult brain. We have identified the cell populations expressing COUP-TFII and the contribution of some of them to network activity in vivo. Expression of COUP-TFII by hippocampal pyramidal and dentate granule cells, as well as neurons in the neocortex, formed a gradient increasing from undetectable in the dorsal to very strong in the ventral sectors. In the dorsal hippocampal CA1 area, COUP-TFII was restricted to GABAergic interneurons and expressed in several, largely nonoverlapping neuronal populations. Immunoreactivity was present in calretinin-, neuronal nitric oxide synthase-, and reelin-expressing cells, as well as in subsets of cholecystokinin- or calbindin-expressing or radiatum-retrohippocampally projecting GABAergic cells, but not in parvalbumin-and/or somatostatin-expressing interneurons. In vivo recording and juxtacellular labeling of COUP-TFII-expressing cells revealed neurogliaform cells, basket cells in stratum radiatum and tachykinin-expressing radiatum dentate innervating interneurons, identified by their axodendritic distributions. They showed cell type-selective phase-locked firing to the theta rhythm but no activation during sharp wave/ripple oscillations. These basket cells in stratum radiatum and neurogliaform cells fired at the peak of theta oscillations detected extracellularly in stratum pyramidale, unlike previously reported ivy cells, which fired at the trough. The characterization of COUP-TFII-expressing neurons suggests that this developmentally important transcription factor plays cell type-specific role(s)in the adult hippocampus. PMID:20130170

  12. Meis/UNC-62 isoform dependent regulation of CoupTF-II/UNC-55 and GABAergic motor neuron subtype differentiation.

    Campbell, Richard F; Walthall, Walter W


    Gene regulatory networks orchestrate the assembly of functionally related cells within a cellular network. Subtle differences often exist among functionally related cells within such networks. How differences are created among cells with similar functions has been difficult to determine due to the complexity of both the gene and the cellular networks. In Caenorhabditis elegans, the DD and VD motor neurons compose a cross-inhibitory, GABAergic network that coordinates dorsal and ventral muscle contractions during locomotion. The Pitx2 homologue, UNC-30, acts as a terminal selector gene to create similarities and the Coup-TFII homologue, UNC-55, is necessary for creating differences between the two motor neuron classes. What is the organizing gene regulatory network responsible for initiating the expression of UNC-55 and thus creating differences between the DD and VD motor neurons? We show that the unc-55 promoter has modules that contain Meis/UNC-62 binding sites. These sites can be subdivided into regions that are capable of activating or repressing UNC-55 expression in different motor neurons. Interestingly, different isoforms of UNC-62 are responsible for the activation and the stabilization of unc-55 transcription. Furthermore, specific isoforms of UNC-62 are required for proper synaptic patterning of the VD motor neurons. Isoform specific regulation of differentiating neurons is a relatively unexplored area of research and presents a mechanism for creating differences among functionally related cells within a network. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Sonic Hedgehog Promotes Neurite Outgrowth of Primary Cortical Neurons Through Up-Regulating BDNF Expression.

    He, Weiliang; Cui, Lili; Zhang, Cong; Zhang, Xiangjian; He, Junna; Xie, Yanzhao


    Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary cortical neurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary cortical neurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary cortical neurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary cortical neurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary cortical neurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary cortical neurons at least partially through modulating BDNF expression.

  14. Short-term memory in networks of dissociated cortical neurons.

    Dranias, Mark R; Ju, Han; Rajaram, Ezhilarasan; VanDongen, Antonius M J


    Short-term memory refers to the ability to store small amounts of stimulus-specific information for a short period of time. It is supported by both fading and hidden memory processes. Fading memory relies on recurrent activity patterns in a neuronal network, whereas hidden memory is encoded using synaptic mechanisms, such as facilitation, which persist even when neurons fall silent. We have used a novel computational and optogenetic approach to investigate whether these same memory processes hypothesized to support pattern recognition and short-term memory in vivo, exist in vitro. Electrophysiological activity was recorded from primary cultures of dissociated rat cortical neurons plated on multielectrode arrays. Cultures were transfected with ChannelRhodopsin-2 and optically stimulated using random dot stimuli. The pattern of neuronal activity resulting from this stimulation was analyzed using classification algorithms that enabled the identification of stimulus-specific memories. Fading memories for different stimuli, encoded in ongoing neural activity, persisted and could be distinguished from each other for as long as 1 s after stimulation was terminated. Hidden memories were detected by altered responses of neurons to additional stimulation, and this effect persisted longer than 1 s. Interestingly, network bursts seem to eliminate hidden memories. These results are similar to those that have been reported from similar experiments in vivo and demonstrate that mechanisms of information processing and short-term memory can be studied using cultured neuronal networks, thereby setting the stage for therapeutic applications using this platform.

  15. A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons

    Sui-Yi Xu


    Full Text Available The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm2 onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco’s Modified Eagle’s Medium (HG-DMEM to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five days in vitro (DIV; cell growth was still substantial at 14 DIV. Neurons were identified by β-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting of β-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.

  16. Locus coeruleus stimulation recruits a broad cortical neuronal network and increases cortical perfusion.

    Toussay, Xavier; Basu, Kaustuv; Lacoste, Baptiste; Hamel, Edith


    The locus coeruleus (LC), the main source of brain noradrenalin (NA), modulates cortical activity, cerebral blood flow (CBF), glucose metabolism, and blood-brain barrier permeability. However, the role of the LC-NA system in the regulation of cortical CBF has remained elusive. This rat study shows that similar proportions (∼20%) of cortical pyramidal cells and GABA interneurons are contacted by LC-NA afferents on their cell soma or proximal dendrites. LC stimulation induced ipsilateral activation (c-Fos upregulation) of pyramidal cells and of a larger proportion (>36%) of interneurons that colocalize parvalbumin, somatostatin, or nitric oxide synthase compared with pyramidal cells expressing cyclooxygenase-2 (22%, p interneurons (16%, p BK, -52%, p < 0.05), and inward-rectifier (Kir, -40%, p < 0.05) K+ channels primarily impaired the hyperemic response. The data demonstrate that LC stimulation recruits a broad network of cortical excitatory and inhibitory neurons resulting in increased cortical activity and that K+ fluxes and EET signaling mediate a large part of the hemodynamic response.

  17. Accelerated intoxication of GABAergic synapses by botulinum neurotoxin A disinhibits stem cell-derived neuron networks prior to network silencing

    Phillip H Beske


    Full Text Available Botulinum neurotoxins (BoNTs are extremely potent toxins that specifically cleave SNARE proteins in peripheral synapses, preventing neurotransmitter release. Neuronal responses to BoNT intoxication are traditionally studied by quantifying SNARE protein cleavage in vitro or monitoring physiological paralysis in vivo. Consequently, the dynamic effects of intoxication on synaptic behaviors are not well understood. We have reported that mouse embryonic stem cell-derived neurons (ESNs are highly sensitive to BoNT based on molecular readouts of intoxication. Here we study the time-dependent changes in synapse- and network-level behaviors following addition of BoNT/A to spontaneously active networks of glutamatergic and GABAergic ESNs. Whole-cell patch-clamp recordings indicated that BoNT/A rapidly blocked synaptic neurotransmission, confirming that ESNs replicate the functional pathophysiology responsible for clinical botulism. Quantitation of spontaneous neurotransmission in pharmacologically isolated synapses revealed accelerated silencing of GABAergic synapses compared to glutamatergic synapses, which was consistent with the selective accumulation of cleaved SNAP-25 at GAD1+ presynaptic terminals at early timepoints. Different latencies of intoxication resulted in complex network responses to BoNT/A addition, involving rapid disinhibition of stochastic firing followed by network silencing. Synaptic activity was found to be highly sensitive to SNAP-25 cleavage, reflecting the functional consequences of the localized cleavage of the small subpopulation of SNAP-25 that is engaged in neurotransmitter release in the nerve terminal. Collectively these findings illustrate that use of synaptic function assays in networked neurons cultures offers a novel and highly sensitive approach for mechanistic studies of toxin:neuron interactions and synaptic responses to BoNT.

  18. Dense neuron clustering explains connectivity statistics in cortical microcircuits.

    Vladimir V Klinshov

    Full Text Available Local cortical circuits appear highly non-random, but the underlying connectivity rule remains elusive. Here, we analyze experimental data observed in layer 5 of rat neocortex and suggest a model for connectivity from which emerge essential observed non-random features of both wiring and weighting. These features include lognormal distributions of synaptic connection strength, anatomical clustering, and strong correlations between clustering and connection strength. Our model predicts that cortical microcircuits contain large groups of densely connected neurons which we call clusters. We show that such a cluster contains about one fifth of all excitatory neurons of a circuit which are very densely connected with stronger than average synapses. We demonstrate that such clustering plays an important role in the network dynamics, namely, it creates bistable neural spiking in small cortical circuits. Furthermore, introducing local clustering in large-scale networks leads to the emergence of various patterns of persistent local activity in an ongoing network activity. Thus, our results may bridge a gap between anatomical structure and persistent activity observed during working memory and other cognitive processes.

  19. KV7 Channels Regulate Firing during Synaptic Integration in GABAergic Striatal Neurons

    M. Belén Pérez-Ramírez


    Full Text Available Striatal projection neurons (SPNs process motor and cognitive information. Their activity is affected by Parkinson’s disease, in which dopamine concentration is decreased and acetylcholine concentration is increased. Acetylcholine activates muscarinic receptors in SPNs. Its main source is the cholinergic interneuron that responds with a briefer latency than SPNs during a cortical command. Therefore, an important question is whether muscarinic G-protein coupled receptors and their signaling cascades are fast enough to intervene during synaptic responses to regulate synaptic integration and firing. One of the most known voltage dependent channels regulated by muscarinic receptors is the KV7/KCNQ channel. It is not known whether these channels regulate the integration of suprathreshold corticostriatal responses. Here, we study the impact of cholinergic muscarinic modulation on the synaptic response of SPNs by regulating KV7 channels. We found that KV7 channels regulate corticostriatal synaptic integration and that this modulation occurs in the dendritic/spines compartment. In contrast, it is negligible in the somatic compartment. This modulation occurs on sub- and suprathreshold responses and lasts during the whole duration of the responses, hundreds of milliseconds, greatly altering SPNs firing properties. This modulation affected the behavior of the striatal microcircuit.

  20. KV7 Channels Regulate Firing during Synaptic Integration in GABAergic Striatal Neurons

    Pérez-Ramírez, M. Belén; Laville, Antonio; Tapia, Dagoberto; Lara-González, Esther; Bargas, José; Galarraga, Elvira


    Striatal projection neurons (SPNs) process motor and cognitive information. Their activity is affected by Parkinson's disease, in which dopamine concentration is decreased and acetylcholine concentration is increased. Acetylcholine activates muscarinic receptors in SPNs. Its main source is the cholinergic interneuron that responds with a briefer latency than SPNs during a cortical command. Therefore, an important question is whether muscarinic G-protein coupled receptors and their signaling cascades are fast enough to intervene during synaptic responses to regulate synaptic integration and firing. One of the most known voltage dependent channels regulated by muscarinic receptors is the KV7/KCNQ channel. It is not known whether these channels regulate the integration of suprathreshold corticostriatal responses. Here, we study the impact of cholinergic muscarinic modulation on the synaptic response of SPNs by regulating KV7 channels. We found that KV7 channels regulate corticostriatal synaptic integration and that this modulation occurs in the dendritic/spines compartment. In contrast, it is negligible in the somatic compartment. This modulation occurs on sub- and suprathreshold responses and lasts during the whole duration of the responses, hundreds of milliseconds, greatly altering SPNs firing properties. This modulation affected the behavior of the striatal microcircuit. PMID:26113994

  1. Optogenetic activation of serotonergic terminals facilitates GABAergic inhibitory input to orexin/hypocretin neurons

    Srikanta Chowdhury; Akihiro Yamanaka


    Orexin/hypocretin neurons play a crucial role in the regulation of sleep/wakefulness, primarily in the maintenance of wakefulness. These neurons innervate wide areas of the brain and receive diverse synaptic inputs including those from serotonergic (5-HT) neurons in the raphe nucleus. Previously we showed that pharmacological application of 5-HT directly inhibited orexin neurons via 5-HT1A receptors. However, it was still unclear how 5-HT neurons regulated orexin neurons since 5-HT neurons co...

  2. Computational Study of Subdural Cortical Stimulation: Effects of Simulating Anisotropic Conductivity on Activation of Cortical Neurons.

    Hyeon Seo

    Full Text Available Subdural cortical stimulation (SuCS is an appealing method in the treatment of neurological disorders, and computational modeling studies of SuCS have been applied to determine the optimal design for electrotherapy. To achieve a better understanding of computational modeling on the stimulation effects of SuCS, the influence of anisotropic white matter conductivity on the activation of cortical neurons was investigated in a realistic head model. In this paper, we constructed pyramidal neuronal models (layers 3 and 5 that showed primary excitation of the corticospinal tract, and an anatomically realistic head model reflecting complex brain geometry. The anisotropic information was acquired from diffusion tensor magnetic resonance imaging (DT-MRI and then applied to the white matter at various ratios of anisotropic conductivity. First, we compared the isotropic and anisotropic models; compared to the isotropic model, the anisotropic model showed that neurons were activated in the deeper bank during cathodal stimulation and in the wider crown during anodal stimulation. Second, several popular anisotropic principles were adapted to investigate the effects of variations in anisotropic information. We observed that excitation thresholds varied with anisotropic principles, especially with anodal stimulation. Overall, incorporating anisotropic conductivity into the anatomically realistic head model is critical for accurate estimation of neuronal responses; however, caution should be used in the selection of anisotropic information.

  3. Human Temporal Cortical Single Neuron Activity during Language: A Review

    George A. Ojemann


    Full Text Available Findings from recordings of human temporal cortical single neuron activity during several measures of language, including object naming and word reading are reviewed and related to changes in activity in the same neurons during recent verbal memory and verbal associative learning measures, in studies conducted during awake neurosurgery for the treatment of epilepsy. The proportion of neurons changing activity with language tasks was similar in either hemisphere. Dominant hemisphere activity was characterized by relative inhibition, some of which occurred during overt speech, possibly to block perception of one’s own voice. However, the majority seems to represent a dynamic network becoming active with verbal memory encoding and especially verbal learning, but inhibited during performance of overlearned language tasks. Individual neurons are involved in different networks for different aspects of language, including naming or reading and naming in different languages. The majority of the changes in activity were tonic sustained shifts in firing. Patterned phasic activity for specific language items was very infrequently recorded. Human single neuron recordings provide a unique perspective on the biologic substrate for language, for these findings are in contrast to many of the findings from other techniques for investigating this.

  4. Ketamine alters cortical integration of GABAergic interneurons and induces long-term sex-dependent impairments in transgenic Gad67-GFP mice

    Aligny, C; Roux, C; Dourmap, N; Ramdani, Y; Do-Rego, J-C; Jégou, S; Leroux, P; Leroux-Nicollet, I; Marret, S; Gonzalez, B J


    Ketamine, a non-competitive N-methyl-D-aspartate (NMDA) antagonist, widely used as an anesthetic in neonatal pediatrics, is also an illicit drug named Super K or KitKat consumed by teens and young adults. In the immature brain, despite several studies indicating that NMDA antagonists are neuroprotective against excitotoxic injuries, there is more and more evidence indicating that these molecules exert a deleterious effect by suppressing a trophic function of glutamate. In the present study, we show using Gad67-GFP mice that prenatal exposure to ketamine during a time-window in which GABAergic precursors are migrating results in (i) strong apoptotic death in the ganglionic eminences and along the migratory routes of GABAergic interneurons; (ii) long-term deficits in interneuron density, dendrite numbers and spine morphology; (iii) a sex-dependent deregulation of γ-aminobutyric acid (GABA) levels and GABA transporter expression; (iv) sex-dependent changes in the response to glutamate-induced calcium mobilization; and (v) the long-term sex-dependent behavioral impairment of locomotor activity. In conclusion, using a preclinical approach, the present study shows that ketamine exposure during cortical maturation durably affects the integration of GABAergic interneurons by reducing their survival and differentiation. The resulting molecular, morphological and functional modifications are associated with sex-specific behavioral deficits in adults. In light of the present data, it appears that in humans, ketamine could be deleterious for the development of the brain of preterm neonates and fetuses of addicted pregnant women. PMID:24991763

  5. Leading role of thalamic over cortical neurons during postinhibitory rebound excitation

    Grenier, F.; Timofeev, I.; Steriade, M.


    The postinhibitory rebound excitation is an intrinsic property of thalamic and cortical neurons that is implicated in a variety of normal and abnormal operations of neuronal networks, such as slow or fast brain rhythms during different states of vigilance as well as seizures. We used dual simultaneous intracellular recordings of thalamocortical neurons from the ventrolateral nucleus and neurons from the motor cortex, together with thalamic and cortical field potentials, to investigate the temporal relations between thalamic and cortical events during the rebound excitation that follows prolonged periods of stimulus-induced inhibition. Invariably, the rebound spike-bursts in thalamocortical cells occurred before the rebound depolarization in cortical neurons and preceded the peak of the depth-negative, rebound field potential in cortical areas. Also, the inhibitory-rebound sequences were more pronounced and prolonged in cortical neurons when elicited by thalamic stimuli, compared with cortical stimuli. The role of thalamocortical loops in the rebound excitation of cortical neurons was shown further by the absence of rebound activity in isolated cortical slabs. However, whereas thalamocortical neurons remained hyperpolarized after rebound excitation, because of the prolonged spike-bursts in inhibitory thalamic reticular neurons, the rebound depolarization in cortical neurons was prolonged, suggesting the role of intracortical excitatory circuits in this sustained activity. The role of intrathalamic events in triggering rebound cortical activity should be taken into consideration when analyzing information processes at the cortical level; at each step, corticothalamic volleys can set into action thalamic inhibitory neurons, leading to rebound spike-bursts that are transferred back to the cortex, thus modifying cortical activities. PMID:9811903

  6. Slow Bursting Neurons of Mouse Cortical Layer 6b Are Depolarized by Hypocretin/Orexin and Major Transmitters of Arousal

    Wenger Combremont, Anne-Laure; Bayer, Laurence; Dupré, Anouk; Mühlethaler, Michel; Serafin, Mauro


    Neurons firing spontaneously in bursts in the absence of synaptic transmission have been previously recorded in different layers of cortical brain slices. It has been suggested that such neurons could contribute to the generation of alternating UP and DOWN states, a pattern of activity seen during slow-wave sleep. Here, we show that in layer 6b (L6b), known from our previous studies to contain neurons highly responsive to the wake-promoting transmitter hypocretin/orexin (hcrt/orx), there is a set of neurons, endowed with distinct intrinsic properties, which displayed a strong propensity to fire spontaneously in rhythmic bursts. In response to small depolarizing steps, they responded with a delayed firing of action potentials which, upon higher depolarizing steps, invariably inactivated and were followed by a depolarized plateau potential and a depolarizing afterpotential. These cells also displayed a strong hyperpolarization-activated rectification compatible with the presence of an Ih current. Most L6b neurons with such properties were able to fire spontaneously in bursts. Their bursting activity was of intrinsic origin as it persisted not only in presence of blockers of ionotropic glutamatergic and GABAergic receptors but also in a condition of complete synaptic blockade. However, a small number of these neurons displayed a mix of intrinsic bursting and synaptically driven recurrent UP and DOWN states. Most of the bursting L6b neurons were depolarized and excited by hcrt/orx through a direct postsynaptic mechanism that led to tonic firing and eventually inactivation. Similarly, they were directly excited by noradrenaline, histamine, dopamine, and neurotensin. Finally, the intracellular injection of these cells with dye and their subsequent Neurolucida reconstruction indicated that they were spiny non-pyramidal neurons. These results lead us to suggest that the propensity for slow rhythmic bursting of this set of L6b neurons could be directly impeded by hcrt

  7. The effect of multivalent Sonic hedgehog on differentiation of human embryonic stem cells into dopaminergic and GABAergic neurons.

    Vazin, Tandis; Ashton, Randolph S; Conway, Anthony; Rode, Nikhil A; Lee, Susan M; Bravo, Verenice; Healy, Kevin E; Kane, Ravi S; Schaffer, David V


    Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions, where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh), whose posttranslational lipid modifications and assembly into multimers enhance its biological potency, potentially through receptor clustering. Investigations of Shh typically utilize recombinant, monomeric protein, and thus the impact of multivalency on ligand potency is unexplored. Among its many activities, Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies, compared to the monomeric Shh, increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%, respectively. Thus, multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients.

  8. Probabilistic identification of cerebellar cortical neurones across species.

    Gert Van Dijck

    Full Text Available Despite our fine-grain anatomical knowledge of the cerebellar cortex, electrophysiological studies of circuit information processing over the last fifty years have been hampered by the difficulty of reliably assigning signals to identified cell types. We approached this problem by assessing the spontaneous activity signatures of identified cerebellar cortical neurones. A range of statistics describing firing frequency and irregularity were then used, individually and in combination, to build Gaussian Process Classifiers (GPC leading to a probabilistic classification of each neurone type and the computation of equi-probable decision boundaries between cell classes. Firing frequency statistics were useful for separating Purkinje cells from granular layer units, whilst firing irregularity measures proved most useful for distinguishing cells within granular layer cell classes. Considered as single statistics, we achieved classification accuracies of 72.5% and 92.7% for granular layer and molecular layer units respectively. Combining statistics to form twin-variate GPC models substantially improved classification accuracies with the combination of mean spike frequency and log-interval entropy offering classification accuracies of 92.7% and 99.2% for our molecular and granular layer models, respectively. A cross-species comparison was performed, using data drawn from anaesthetised mice and decerebrate cats, where our models offered 80% and 100% classification accuracy. We then used our models to assess non-identified data from awake monkeys and rabbits in order to highlight subsets of neurones with the greatest degree of similarity to identified cell classes. In this way, our GPC-based approach for tentatively identifying neurones from their spontaneous activity signatures, in the absence of an established ground-truth, nonetheless affords the experimenter a statistically robust means of grouping cells with properties matching known cell classes. Our

  9. Axon guidance of rat cortical neurons by microcontact printed gradients.

    Fricke, Rita; Zentis, Peter D; Rajappa, Lionel T; Hofmann, Boris; Banzet, Marko; Offenhäusser, Andreas; Meffert, Simone H


    Substrate-bound gradients expressed in numerous spatio-temporal patterns play a crucial role during the development of complex neural circuits. A deeper understanding of the axon guidance mechanism is provided by studying the effect of a defined substrate-bound cue on a confined neural network. In this study, we constructed a discontinuous substrate-bound gradient to control neuronal cell position, the path of neurite growth, and axon directionality. A variety of gradient patterns, with slight changes in slope, width, and length were designed and fabricated by microcontact printing using laminin/poly-l-lysine (PLL) or PLL alone. The gradients were tested for neurite growth and their impact on axon guidance of embryonic rat cortical neurons. The neurite length was determined and the axon was evaluated by Tau-1 immunostaining. We found that the microgradients of laminin/PLL and PLL directed neurons' adhesion, differentially controlled the neurite growth, and guided up to 84% of the axons. The effect of the protein micropattern on axon guidance and neurite growth depended on the protein and geometric parameters used. Our approach proved to be very successful in guiding axons of single multipolar neurons with very high efficiency. It could thereby be useful to engineer defined neural networks for analyzing signal processing of functional circuits, as well as to unravel fundamental questions of the axon guidance mechanism.

  10. Order-based representation in random networks of cortical neurons.

    Goded Shahaf


    Full Text Available The wide range of time scales involved in neural excitability and synaptic transmission might lead to ongoing change in the temporal structure of responses to recurring stimulus presentations on a trial-to-trial basis. This is probably the most severe biophysical constraint on putative time-based primitives of stimulus representation in neuronal networks. Here we show that in spontaneously developing large-scale random networks of cortical neurons in vitro the order in which neurons are recruited following each stimulus is a naturally emerging representation primitive that is invariant to significant temporal changes in spike times. With a relatively small number of randomly sampled neurons, the information about stimulus position is fully retrievable from the recruitment order. The effective connectivity that makes order-based representation invariant to time warping is characterized by the existence of stations through which activity is required to pass in order to propagate further into the network. This study uncovers a simple invariant in a noisy biological network in vitro; its applicability under in vivo constraints remains to be seen.

  11. GABAergic neuron deficit as an idiopathic generalized epilepsy mechanism: the role of BRD2 haploinsufficiency in juvenile myoclonic epilepsy.

    Libor Velíšek

    Full Text Available Idiopathic generalized epilepsy (IGE syndromes represent about 30% of all epilepsies. They have strong, but elusive, genetic components and sex-specific seizure expression. Multiple linkage and population association studies have connected the bromodomain-containing gene BRD2 to forms of IGE. In mice, a null mutation at the homologous Brd2 locus results in embryonic lethality while heterozygous Brd2+/- mice are viable and overtly normal. However, using the flurothyl model, we now show, that compared to the Brd2+/+ littermates, Brd2+/- males have a decreased clonic, and females a decreased tonic-clonic, seizure threshold. Additionally, long-term EEG/video recordings captured spontaneous seizures in three out of five recorded Brd2+/- female mice. Anatomical analysis of specific regions of the brain further revealed significant differences in Brd2+/- vs +/+ mice. Specifically, there were decreases in the numbers of GABAergic (parvalbumin- or GAD67-immunopositive neurons along the basal ganglia pathway, i.e., in the neocortex and striatum of Brd2+/- mice, compared to Brd2+/+ mice. There were also fewer GABAergic neurons in the substantia nigra reticulata (SNR, yet there was a minor, possibly compensatory increase in the GABA producing enzyme GAD67 in these SNR cells. Further, GAD67 expression in the superior colliculus and ventral medial thalamic nucleus, the main SNR outputs, was significantly decreased in Brd2+/- mice, further supporting GABA downregulation. Our data show that the non-channel-encoding, developmentally critical Brd2 gene is associated with i sex-specific increases in seizure susceptibility, ii the development of spontaneous seizures, and iii seizure-related anatomical changes in the GABA system, supporting BRD2's involvement in human IGE.

  12. A possible role of the non-GAT1 GABA transporters in transfer of GABA from GABAergic to glutamatergic neurons in mouse cerebellar neuronal cultures.

    Suñol, C; Babot, Z; Cristòfol, R; Sonnewald, U; Waagepetersen, H S; Schousboe, A


    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

  13. Ibuprofen augments bilirubin toxicity in rat cortical neuronal culture.

    Berns, Monika; Toennessen, Margit; Koehne, Petra; Altmann, Rodica; Obladen, Michael


    Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin-albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic cortical neurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 microg/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p < 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p < 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 microM unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 microg/mL ibuprofen caused additional loss of cell viability and increased LDH release (p < 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-ala-asp-fluoromethyl ketone abolished ibuprofen- and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 microM reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism.

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

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


    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.

  15. Properties of bilateral spinocerebellar activation of cerebellar cortical neurons

    Pontus eGeborek


    Full Text Available We aimed to explore the cerebellar cortical inputs from two spinocerebellar pathways, the spinal border cell-component of the ventral spinocerebellar tract (SBC-VSCT and the dorsal spinocerebellar tract (DSCT, respectively, in the sublobule C1 of the cerebellar posterior lobe. The two pathways were activated by electrical stimulation of the contralateral lateral funiculus (coLF and the ipsilateral LF (iLF at lower thoracic levels. Most granule cells in sublobule C1 did not respond at all but part of the granule cell population displayed high-intensity responses to either coLF or iLF stimulation. As a rule, Golgi cells and Purkinje cell simple spikes responded to input from both LFs, although Golgi cells could be more selective. In addition, a small population of granule cells responded to input from both the coLF and the iLF. However, in these cases, similarities in the temporal topography and magnitude of the responses suggested that the same axons were stimulated from the two LFs, i.e. that the axons of individual spinocerebellar neurons could be present in both funiculi. This was also confirmed for a population of spinal neurons located within known locations of SBC-VSCT neurons and dorsal horn DSCT neurons. We conclude that bilateral spinocerebellar responses can occur in cerebellar granule cells, but the VSCT and DSCT systems that provide the input can also be organized bilaterally. The implications for the traditional functional separation of VSCT and DSCT systems and the issue whether granule cells primarily integrate functionally similar information or not are discussed.

  16. IL-6 mediated degeneration of forebrain GABAergic interneurons and cognitive impairment in aged mice through activation of neuronal NADPH oxidase.

    Laura L Dugan

    Full Text Available BACKGROUND: Multiple studies have shown that plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6 are elevated in patients with important and prevalent adverse health conditions, including atherosclerosis, diabetes, obesity, obstructive sleep apnea, hypertension, and frailty. Higher plasma levels of IL-6, in turn, increase the risk of many conditions associated with aging including age-related cognitive decline. However, the mechanisms underlying this association between IL-6 and cognitive vulnerability remain unclear. METHODS AND FINDINGS: We investigated the role of IL-6 in brain aging in young (4 mo and aged (24 mo wild-type C57BL6 and genetically-matched IL-6(-/- mice, and determined that IL-6 was necessary and sufficient for increased neuronal expression of the superoxide-producing immune enzyme, NADPH-oxidase, and this was mediated by non-canonical NFkappaB signaling. Furthermore, superoxide production by NADPH-oxidase was directly responsible for age-related loss of parvalbumin (PV-expressing GABAergic interneurons, neurons essential for normal information processing, encoding, and retrieval in hippocampus and cortex. Targeted deletion of IL-6 or elimination of superoxide by chronic treatment with a superoxide-dismutase mimetic prevented age-related loss of PV-interneurons and reversed age-related cognitive deficits on three standard tests of spatial learning and recall. CONCLUSIONS: Present results indicate that IL-6 mediates age-related loss of critical PV-expressing GABAergic interneurons through increased neuronal NADPH-oxidase-derived superoxide production, and that rescue of these interneurons preserves cognitive performance in aging mice, suggesting that elevated peripheral IL-6 levels may be directly and mechanistically linked to long-lasting cognitive deficits in even normal older individuals. Further, because PV-interneurons are also selectively affected by commonly used anesthetic agents and drugs, our findings

  17. Human temporal cortical single neuron activity during working memory maintenance.

    Zamora, Leona; Corina, David; Ojemann, George


    The Working Memory model of human memory, first introduced by Baddeley and Hitch (1974), has been one of the most influential psychological constructs in cognitive psychology and human neuroscience. However the neuronal correlates of core components of this model have yet to be fully elucidated. Here we present data from two studies where human temporal cortical single neuron activity was recorded during tasks differentially affecting the maintenance component of verbal working memory. In Study One we vary the presence or absence of distracting items for the entire period of memory storage. In Study Two we vary the duration of storage so that distractors filled all, or only one-third of the time the memory was stored. Extracellular single neuron recordings were obtained from 36 subjects undergoing awake temporal lobe resections for epilepsy, 25 in Study one, 11 in Study two. Recordings were obtained from a total of 166 lateral temporal cortex neurons during performance of one of these two tasks, 86 study one, 80 study two. Significant changes in activity with distractor manipulation were present in 74 of these neurons (45%), 38 Study one, 36 Study two. In 48 (65%) of those there was increased activity during the period when distracting items were absent, 26 Study One, 22 Study Two. The magnitude of this increase was greater for Study One, 47.6%, than Study Two, 8.1%, paralleling the reduction in memory errors in the absence of distracters, for Study One of 70.3%, Study Two 26.3% These findings establish that human lateral temporal cortex is part of the neural system for working memory, with activity during maintenance of that memory that parallels performance, suggesting it represents active rehearsal. In 31 of these neurons (65%) this activity was an extension of that during working memory encoding that differed significantly from the neural processes recorded during overt and silent language tasks without a recent memory component, 17 Study one, 14 Study two

  18. Neuronal Sirt3 protects against excitotoxic injury in mouse cortical neuron culture.

    Sun Hee Kim

    Full Text Available BACKGROUND: Sirtuins (Sirt, a family of nicotinamide adenine nucleotide (NAD dependent deacetylases, are implicated in energy metabolism and life span. Among the known Sirt isoforms (Sirt1-7, Sirt3 was identified as a stress responsive deacetylase recently shown to play a role in protecting cells under stress conditions. Here, we demonstrated the presence of Sirt3 in neurons, and characterized the role of Sirt3 in neuron survival under NMDA-induced excitotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: To induce excitotoxic injury, we exposed primary cultured mouse cortical neurons to NMDA (30 µM. NMDA induced a rapid decrease of cytoplasmic NAD (but not mitochondrial NAD in neurons through poly (ADP-ribose polymerase-1 (PARP-1 activation. Mitochondrial Sirt3 was increased following PARP-1 mediated NAD depletion, which was reversed by either inhibition of PARP-1 or exogenous NAD. We found that massive reactive oxygen species (ROS produced under this NAD depleted condition mediated the increase in mitochondrial Sirt3. By transfecting primary neurons with a Sirt3 overexpressing plasmid or Sirt3 siRNA, we showed that Sirt3 is required for neuroprotection against excitotoxicity. CONCLUSIONS: This study demonstrated for the first time that mitochondrial Sirt3 acts as a prosurvival factor playing an essential role to protect neurons under excitotoxic injury.

  19. Exogenous Reelin modifies the migratory behavior of neurons depending on cortical location.

    Britto, Joanne M; Tait, Karen J; Lee, Ean Phing; Gamble, Robin S; Hattori, Mitsuharu; Tan, Seong-Seng


    Malformations of cortical development can arise when projection neurons generated in the germinal zones fail to migrate properly into the cortical plate. This process is critically dependent on the Reelin glycoprotein, which when absent leads to an inversion of cortical layers and blurring of borders. Reelin has other functions including supporting neuron migration and maintaining their trajectories; however, the precise role on glial fiber-dependent or -independent migration of neurons remains controversial. In this study, we wish to test the hypothesis that migrating cortical neurons at different levels of the cortical wall have differential responses to Reelin. We exposed neurons migrating across the cortical wall to exogenous Reelin and monitored their migratory behavior using time-lapse imaging. Our results show that, in the germinal zones, exogenous Reelin retarded neuron migration and altered their trajectories. This behavior is in contrast to the response of neurons located in the intermediate zone (IZ), possibly because Reelin receptors are not expressed in this zone. In the reeler cortex, Reelin receptors are expressed in the IZ and exposure to exogenous Reelin was able to rescue the migratory defect. These studies demonstrate that migrating neurons have nonequivalent responses to Reelin depending on their location within the cortical wall. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail:

  20. Responses to Gamma-Aminobutyric Acid of Rat Visual Cortical Neurons in Tissue Slices


    Neurol. 234: 242-263. Peters, A. and Proskauer, c. C. (1980) Synaptic relationships between a multipolar stellate cell and a pyramidal neuron in rat...APR 1986 2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Responses to Gamma-Aminobutyric Acid of Rat Visual Cortical Neurons in...AIR FORCE MEDICAL CENTER Title of Thesis: Responses to Gamma-Aminobutyric Acid of Rat Visual Cortical Neurons in Tissue Slices Name of Candidate

  1. GABAergic cell type diversity in the basolateral amygdala.

    Capogna, Marco


    Here I review the diversity of GABAergic neurons in the rodent basolateral amygdala (BLA). In spite of the recent identification of the role played by certain neurons of BLA in learning and memory of fear, the diversity of GABAergic neurons has not been fully explored. I describe analogies and differences between GABAergic neurons in BLA and cerebral cortex. Emphasis is given to a comprehensive functional, neurochemical and anatomical classification of GABAergic neuron types. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Alterations in cortical thickness and neuronal density in the frontal cortex of Albert Einstein.

    Anderson, B; Harvey, T


    Neuronal density, neuron size, and the number of neurons under 1 mm2 of cerebral cortical surface area were measured in the right pre-frontal cortex of Albert Einstein and five elderly control subjects. Measurement of neuronal density used the optical dissector technique on celloidin-embedded cresyl violet-stained sections. The neurons counted provided a systematic random sample for the measurement of cell body cross-sectional area. Einstein's cortex did not differ from the control subjects in the number of neurons under 1 mm2 of cerebral cortex or in mean neuronal size. Because Einstein's cortex was thinner than the controls he had a greater neuronal density.

  3. Mechanisms of GABAergic Homeostatic Plasticity

    Peter Wenner


    Full Text Available Homeostatic plasticity ensures that appropriate levels of activity are maintained through compensatory adjustments in synaptic strength and cellular excitability. For instance, excitatory glutamatergic synapses are strengthened following activity blockade and weakened following increases in spiking activity. This form of plasticity has been described in a wide array of networks at several different stages of development, but most work and reviews have focussed on the excitatory inputs of excitatory neurons. Here we review homeostatic plasticity of GABAergic neurons and their synaptic connections. We propose a simplistic model for homeostatic plasticity of GABAergic components of the circuitry (GABAergic synapses onto excitatory neurons, excitatory connections onto GABAergic neurons, cellular excitability of GABAergic neurons: following chronic activity blockade there is a weakening of GABAergic inhibition, and following chronic increases in network activity there is a strengthening of GABAergic inhibition. Previous work on GABAergic homeostatic plasticity supports certain aspects of the model, but it is clear that the model cannot fully account for some results which do not appear to fit any simplistic rule. We consider potential reasons for these discrepancies.

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

    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


    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.

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

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


    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

  6. Entorhinal cortical innervation of parvalbumin-containing neurons (Basket and Chandelier cells) in the rat Ammon's horn.

    Kiss, J; Buzsaki, G; Morrow, J S; Glantz, S B; Leranth, C


    Physiological data suggest that in the CA1-CA3 hippocampal areas of rats, entorhinal cortical efferents directly influence the activity of interneurons, in addition to pyramidal cells. To verify this hypothesis, the following experiments were performed: 1) light microscopic double-immunostaining for parvalbumin and the anterograde tracer Phaseolus vulgaris-leucoagglutinin injected into the entorhinal cortex; 2) light and electron microscopic analysis of cleaved spectrin-immunostained (i.e., degenerating axons and boutons) hippocampal sections following entorhinal cortex lesion; and 3) an electron microscopic study of parvalbumin-immunostained hippocampal sections after entorhinal cortex lesion. The results demonstrate that in the stratum lacunosum-moleculare of the CA1 and CA3 regions, entorhinal cortical axons form asymmetric synaptic contacts on parvalbumin-containing dendritic shafts. In the stratum lacunosum-moleculare, parvalbumin-immunoreactive dendrites represent processes of GABAergic, inhibitory basket and chandelier cells; these interneurons innervate the perisomatic area and axon initial segments of pyramidal cells, respectively. A feed-forward activation of these neurons by the entorhinal input may explain the strong, short-latency inhibition of pyramidal cells.

  7. Cortical neuron loss in post-traumatic higher brain dysfunction using (123)I-iomazenil SPECT.

    Nakagawara, Jyoji; Kamiyama, Kenji; Takahashi, Masaaki; Nakamura, Hirohiko


    In patients with higher brain dysfunction (HBD) after mild traumatic brain injury (MTBI), diagnostic imaging of cortical neuron loss in the frontal lobes was studied using SPECT with (123)I-iomazenil (IMZ), as a radioligand for central benzodiazepine receptor (BZR). Statistical imaging analysis using three-dimensional stereotactic surface projections (3D-SSP) for (123)I-IMZ SPECT was performed in 17 patients. In all patients with HBD defined by neuropsychological tests, cortical neuron loss was indicated in the bilateral medial frontal lobes in 14 patients (83 %). A comparison between the group of 17 patients and the normal database demonstrated common areas of cortical neuron loss in the bilateral medial frontal lobes involving the medial frontal gyrus (MFG) and the anterior cingulate gyrus (ACG). In an assessment of cortical neuron loss in the frontal medial cortex using the stereotactic extraction estimation (SEE) method (level 3), significant cortical neuron loss was observed within bilateral MFG in 9 patients and unilateral MFG in 4, and bilateral ACG in 12 and unilateral ACG in 3. Fourteen patients showed significant cortical neuron loss in bilateral MFG or ACG. In patients with MTBI, HBD seemed to correlate with selective cortical neuron loss within the bilateral MFG or ACG where the responsible lesion could be. 3D-SSP and SEE level 3 analysis for (123)I-IMZ SPECT could be valuable for diagnostic imaging of HBD after MTBI.

  8. IgLON cell adhesion molecules are shed from the cell surface of cortical neurons to promote neuronal growth.

    Sanz, Ricardo; Ferraro, Gino B; Fournier, Alyson E


    Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature cortical neurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature cortical neurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature cortical neurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from cortical neurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature cortical neurons.

  9. IgLON Cell Adhesion Molecules Are Shed from the Cell Surface of Cortical Neurons to Promote Neuronal Growth*

    Sanz, Ricardo; Ferraro, Gino B.; Fournier, Alyson E.


    Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature cortical neurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature cortical neurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature cortical neurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from cortical neurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature cortical neurons. PMID:25538237

  10. Hippocampal GABAergic interneurons coexpressing alpha7-nicotinic receptors and connexin-36 are able to improve neuronal viability under oxygen-glucose deprivation.

    Voytenko, L P; Lushnikova, I V; Savotchenko, A V; Isaeva, E V; Skok, M V; Lykhmus, O Yu; Patseva, M A; Skibo, G G


    The hippocampal interneurons are very diverse by chemical profiles and rather inconsistent by sensitivity to CI. Some hippocampal GABAergic interneurons survive certain time after ischemia while ischemia-sensitive interneurons and pyramidal neurons are damaged. GABAergic signaling, nicotinic receptors expressing α7-subunit (α7nAChRs(+)) and connexin-36 (Cx36(+), electrotonic gapjunctions protein) contradictory modulate post-ischemic environment. We hypothesized that hippocampal ischemia-resistant GABAergic interneurons coexpressing glutamate decarboxylase-67 isoform (GAD67(+)), α7nAChRs(+), Cx36(+) are able to enhance neuronal viability. To check this hypothesis the histochemical and electrophysiological investigations have been performed using rat hippocampal organotypic culture in the condition of 30-min oxygen-glucose deprivation (OGD). Post-OGD reoxygenation (4h) revealed in CA1 pyramidal layer numerous damaged cells, decreased population spike amplitude and increased pair-pulse depression. In these conditions GAD67(+) interneurons displayed the OGD-resistance and significant increase of GABA synthesis/metabolism (GAD67-immunofluorescence, mitochondrial activity). The α7nAChRs(+) and Cx36(+) co-localizations were revealed in resistant GAD67(+) interneurons. Under OGD: GABAA-receptors (GABAARs) blockade increased cell damage and exacerbated the pair-pulse depression in CA1 pyramidal layer; α7nAChRs and Cx36-channels separate blockades sufficiently decreased cell damage while interneuronal GAD67-immunofluorescence and mitochondrial activity were similar to the control. Thus, hippocampal GABAergic interneurons co-expressing α7nAChRs and Cx36 remained resistant certain time after OGD and were able to modulate CA1 neuron survival through GABAARs, α7nAChRs and Cx36-channels activity. The enhancements of the neuronal viability together with GABA synthesis/metabolism normalization suggest cooperative neuroprotective mechanism that could be used for increase in

  11. C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment.

    Voss, Anne K; Britto, Joanne M; Dixon, Mathew P; Sheikh, Bilal N; Collin, Caitlin; Tan, Seong-Seng; Thomas, Tim


    Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Cortical neuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates cortical neuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a cortical neuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient cortical neurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in cortical neurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.

  12. Shaping inhibition: activity dependent structural plasticity of GABAergic synapses

    Carmen E Flores


    Full Text Available Inhibitory transmission through the neurotransmitter Ɣ-aminobutyric acid (GABA shapes network activity in the mammalian cerebral cortex by filtering synaptic incoming information and dictating the activity of principal cells. The incredibly diverse population of cortical neurons that use GABA as neurotransmitter shows an equally diverse range of mechanisms that regulate changes in the strength of GABAergic synaptic transmission and allow them to dynamically follow and command the activity of neuronal ensembles. Similarly to glutamatergic synaptic transmission, activity-dependent functional changes in inhibitory neurotransmission are accompanied by alterations in GABAergic synapse structure that range from morphological reorganization of postsynaptic density to de novo formation and elimination of inhibitory contacts. Here we review several aspects of structural plasticity of inhibitory synapses, including its induction by different forms of neuronal activity, behavioral and sensory experience and the molecular mechanisms and signaling pathways involved. We discuss the functional consequences of GABAergic synapse structural plasticity for information processing and memory formation in view of the heterogenous nature of the structural plasticity phenomena affecting inhibitory synapses impinging on somatic and dendritic compartments of cortical and hippocampal neurons.

  13. The Memory-Impairing Effects of Septal GABA Receptor Activation Involve GABAergic Septo-Hippocampal Projection Neurons

    Krebs-Kraft, Desiree L.; Wheeler, Marina G.; Parent, Marise B.


    Septal infusions of the [gamma]-aminobutyric acid (GABA)[subscript A] agonist muscimol impair memory, and the effect likely involves the hippocampus. GABA[subscript A] receptors are present on the perikarya of cholinergic and GABAergic septo-hippocampal (SH) projections. The current experiments determined whether GABAergic SH projections are…

  14. NADPH diaphorase-positive neurons in the lizard hippocampus: a distinct subpopulation of GABAergic interneurons.

    Dávila, J C; Megías, M; Andreu, M J; Real, M A; Guirado, S


    We analyzed the distribution and light-microscopic features of the NADPH diaphorase-containing structures in the lizard hippocampus, likely to correspond to nitric oxide synthase-containing cells and fibers, and thus likely to release nitric oxide. We also studied co-localization of NADPH diaphorase with the neurotransmitter GABA, the calcium-binding protein parvalbumin, and the neuropeptide somatostatin, in order to examine whether putative nitric oxide-synthesizing neurons represent a different subpopulation of GABA cells, on which the authors recently reported in lizards. We also studied co-localization of NADPH diaphorase with parvalbumin or somatostatin in mice to ascertain whether the characteristics of this population in reptiles parallel the situation in mammals. Most of the positive NADPH diaphorase neurons were stained in a Golgi-like manner and were in the plexiform layers of the lizard hippocampus with morphologies ranging from bipolar to multipolar. Co-localization with GABA was 100%, and NADPH diaphorase-positive neurons in the lizard hippocampus did not contain parvalbumin or somatostatin. The results indicate that putative nitric oxide-synthesizing neurons represent a distinct subpopulation of GABA interneurons in the lizard hippocampus. Two different types of fibers were described in the plexiform layers: one type bearing thick varicosities, and the other thinner ones. We discuss the possibility that at least part of the positive fibers arise from a hypothalamic aminergic nucleus contacting the third ventricle, the periventricular hypothalamic organ. Most radial glia were stained almost completely and formed typical end-feet both at the pia and around capillaries. The results of this study confirm that the capacity for synthesizing nitric oxide is linked to a determined set of neuronal markers depending on the specific brain region, and they provide new resemblances between hippocampal regions in different classes of vertebrates.

  15. Euchromatin histone methyltransferase 1 regulates cortical neuronal network development

    Bart Martens, Marijn; Frega, Monica; Classen, Jessica; Epping, Lisa; Bijvank, Elske; Benevento, Marco; van Bokhoven, Hans; Tiesinga, Paul; Schubert, Dirk; Nadif Kasri, Nael


    Heterozygous mutations or deletions in the human Euchromatin histone methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a neurodevelopmental disorder that is characterized by autistic-like features and severe intellectual disability (ID). Neurodevelopmental disorders including ID and autism may be related to deficits in activity-dependent wiring of brain circuits during development. Although Kleefstra syndrome has been associated with dendritic and synaptic defects in mice and Drosophila, little is known about the role of EHMT1 in the development of cortical neuronal networks. Here we used micro-electrode arrays and whole-cell patch-clamp recordings to investigate the impact of EHMT1 deficiency at the network and single cell level. We show that EHMT1 deficiency impaired neural network activity during the transition from uncorrelated background action potential firing to synchronized network bursting. Spontaneous bursting and excitatory synaptic currents were transiently reduced, whereas miniature excitatory postsynaptic currents were not affected. Finally, we show that loss of function of EHMT1 ultimately resulted in less regular network bursting patterns later in development. These data suggest that the developmental impairments observed in EHMT1-deficient networks may result in a temporal misalignment between activity-dependent developmental processes thereby contributing to the pathophysiology of Kleefstra syndrome. PMID:27767173

  16. Developmental changes of TrkB signaling in response to exogenous brain-derived neurotrophic factor in primary cortical neurons.

    Zhou, Xianju; Xiao, Hua; Wang, Hongbing


    Neocortical circuits are most sensitive to sensory experience during a critical period of early development. Previous studies implicate that brain-derived neurotrophic factor (BDNF) and GABAergic inhibition may control the timing of the critical period. By using an in vitro maturation model, we found that neurons at DIV (day in vitro) 7, around a period when functional synapses start to form and GABAergic inhibition emerges, displayed the most dynamic activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB by exogenous BDNF. The BDNF-stimulated transcriptional up-regulation of CREB target genes was also the highest in DIV 7 neurons. The basal level of ERK1/2 and CREB activity, as well as the expression of CREB target genes, increased along with maturation, and neurons at DIV 13 and 22 displayed less dynamic responses to BDNF. Furthermore, we found that the developmentally regulated GABAergic inhibition correlated with the decline of BDNF-mediated signaling during maturation. BDNF stimulation along with suppression of GABAergic inhibition enhanced the activation of ERK1/2-CREB signaling and gene transcription in mature neurons. Conversely, BDNF stimulation along with enhancement of GABAergic inhibition reduced the overall induction of intracellular signaling in younger neurons. We propose that the less dynamic molecular changes may play a certain role in the loss of plasticity during maturation.

  17. Protective effects of berberine against amyloid beta-induced toxicity in cultured rat cortical neurons

    Jing Wang; Yanjun Zhang; Shuai Du; Mixia Zhang


    Berberine, a major constituent of Coptidis rhizoma, exhibits neural protective effects. The present study analyzed the potential protective effect of berberine against amyloid G-induced cytotoxicity in rat cerebral cortical neurons. Alzheimer's disease cell models were treated with 0.5 and 2 μmol/Lberberine for 36 hours to inhibit amyloid G-induced toxicity. Methyl thiazolyl tetrazolium assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining results showed that berberine significantly increased cell viability and reduced cell apoptosis in primary cultured rat cortical neurons. In addition, western blot analysis revealed a protective effect of berberine against amyloid β-induced toxicity in cultured cortical neurons, which coincided with significantly decreased abnormal up-regulation of activated caspase-3. These results showed that berberine exhibited a protective effect against amyloid 13-induced cytotoxicity in cultured rat cortical neurons.

  18. Characterization of age-dependent and progressive cortical neuronal degeneration in presenilin conditional mutant mice.

    Mary Wines-Samuelson

    Full Text Available Presenilins are the major causative genes of familial Alzheimer's disease (AD. Our previous study has demonstrated essential roles of presenilins in memory and neuronal survival. Here, we explore further how loss of presenilins results in age-related, progressive neurodegeneration in the adult cerebral cortex, where the pathogenesis of AD occurs. To circumvent the requirement of presenilins for embryonic development, we used presenilin conditional double knockout (Psen cDKO mice, in which presenilin inactivation is restricted temporally and spatially to excitatory neurons of the postnatal forebrain beginning at 4 weeks of age. Increases in the number of degenerating (Fluoro-Jade B+, 7.6-fold and apoptotic (TUNEL+, 7.4-fold neurons, which represent approximately 0.1% of all cortical neurons, were first detected at 2 months of age when there is still no significant loss of cortical neurons and volume in Psen cDKO mice. By 4 months of age, significant loss of cortical neurons (approximately 9% and gliosis was found in Psen cDKO mice. The apoptotic cell death is associated with caspase activation, as shown by increased numbers of cells immunoreactive for active caspases 9 and 3 in the Psen cDKO cortex. The vulnerability of cortical neurons to loss of presenilins is region-specific with cortical neurons in the lateral cortex most susceptible. Compared to the neocortex, the increase in apoptotic cell death and the extent of neurodegeneration are less dramatic in the Psen cDKO hippocampus, possibly in part due to increased neurogenesis in the aging dentate gyrus. Neurodegeneration is also accompanied with mitochondrial defects, as indicated by reduced mitochondrial density and altered mitochondrial size distribution in aging Psen cortical neurons. Together, our findings show that loss of presenilins in cortical neurons causes apoptotic cell death occurring in a very small percentage of neurons, which accumulates over time and leads to substantial loss

  19. Distribution of neurons in functional areas of the mouse cerebral cortex reveals quantitatively different cortical zones.

    Herculano-Houzel, Suzana; Watson, Charles; Paxinos, George


    How are neurons distributed along the cortical surface and across functional areas? Here we use the isotropic fractionator (Herculano-Houzel and Lent, 2005) to analyze the distribution of neurons across the entire isocortex of the mouse, divided into 18 functional areas defined anatomically. We find that the number of neurons underneath a surface area (the N/A ratio) varies 4.5-fold across functional areas and neuronal density varies 3.2-fold. The face area of S1 contains the most neurons, followed by motor cortex and the primary visual cortex. Remarkably, while the distribution of neurons across functional areas does not accompany the distribution of surface area, it mirrors closely the distribution of cortical volumes-with the exception of the visual areas, which hold more neurons than expected for their volume. Across the non-visual cortex, the volume of individual functional areas is a shared linear function of their number of neurons, while in the visual areas, neuronal densities are much higher than in all other areas. In contrast, the 18 functional areas cluster into three different zones according to the relationship between the N/A ratio and cortical thickness and neuronal density: these three clusters can be called visual, sensory, and, possibly, associative. These findings are remarkably similar to those in the human cerebral cortex (Ribeiro et al., 2013) and suggest that, like the human cerebral cortex, the mouse cerebral cortex comprises two zones that differ in how neurons form the cortical volume, and three zones that differ in how neurons are distributed underneath the cortical surface, possibly in relation to local differences in connectivity through the white matter. Our results suggest that beyond the developmental divide into visual and non-visual cortex, functional areas initially share a common distribution of neurons along the parenchyma that become delimited into functional areas according to the pattern of connectivity established later.

  20. Slow cortical rhythms: from single-neuron electrophysiology to whole-brain imaging in vivo

    Olcese, U.; Faraguna, U.


    The slow cortical oscillation is the major brain rhythm occurring during sleep, and has been the object of thorough investigation for over thirty years. Despite all these efforts, the function and the neuronal mechanisms behind slow cortical rhythms remain only partially understood. In this review

  1. Slow cortical rhythms: from single-neuron electrophysiology to whole-brain imaging in vivo

    U. Olcese; U. Faraguna


    The slow cortical oscillation is the major brain rhythm occurring during sleep, and has been the object of thorough investigation for over thirty years. Despite all these efforts, the function and the neuronal mechanisms behind slow cortical rhythms remain only partially understood. In this review w

  2. GABA regulates the multidirectional tangential migration of GABAergic interneurons in living neonatal mice.

    Hiroyuki Inada

    Full Text Available Cortical GABAergic interneurons originate from ganglionic eminences and tangentially migrate into the cortical plate at early developmental stages. To elucidate the characteristics of this migration of GABAergic interneurons in living animals, we established an experimental design specialized for in vivo time-lapse imaging of the neocortex of neonate mice with two-photon laser-scanning microscopy. In vesicular GABA/glycine transporter (VGAT-Venus transgenic mice from birth (P0 through P3, we observed multidirectional tangential migration of genetically-defined GABAergic interneurons in the neocortical marginal zone. The properties of this migration, such as the motility rate (distance/hr, the direction moved, and the proportion of migrating neurons to stationary neurons, did not change through P0 to P3, although the density of GABAergic neurons at the marginal zone decreased with age. Thus, the characteristics of the tangential motility of individual GABAergic neurons remained constant in development. Pharmacological block of GABA(A receptors and of the Na⁺-K⁺-Cl⁻ cotransporters, and chelating intracellular Ca²⁺, all significantly reduced the motility rate in vivo. The motility rate and GABA content within the cortex of neonatal VGAT-Venus transgenic mice were significantly greater than those of GAD67-GFP knock-in mice, suggesting that extracellular GABA concentration could facilitate the multidirectional tangential migration. Indeed, diazepam applied to GAD67-GFP mice increased the motility rate substantially. In an in vitro neocortical slice preparation, we confirmed that GABA induced a NKCC sensitive depolarization of GABAergic interneurons in VGAT-Venus mice at P0-P3. Thus, activation of GABA(AR by ambient GABA depolarizes GABAergic interneurons, leading to an acceleration of their multidirectional motility in vivo.

  3. Presynaptic Glycine Receptors Increase GABAergic Neurotransmission in Rat Periaqueductal Gray Neurons

    Kwi-Hyung Choi


    Full Text Available The periaqueductal gray (PAG is involved in the central regulation of nociceptive transmission by affecting the descending inhibitory pathway. In the present study, we have addressed the functional role of presynaptic glycine receptors in spontaneous glutamatergic transmission. Spontaneous EPSCs (sEPSCs were recorded in mechanically dissociated rat PAG neurons using a conventional whole-cell patch recording technique under voltage-clamp conditions. The application of glycine (100 µM significantly increased the frequency of sEPSCs, without affecting the amplitude of sEPSCs. The glycine-induced increase in sEPSC frequency was blocked by 1 µM strychnine, a specific glycine receptor antagonist. The results suggest that glycine acts on presynaptic glycine receptors to increase the probability of glutamate release from excitatory nerve terminals. The glycine-induced increase in sEPSC frequency completely disappeared either in the presence of tetrodotoxin or Cd2+, voltage-gated Na+, or Ca2+ channel blockers, suggesting that the activation of presynaptic glycine receptors might depolarize excitatory nerve terminals. The present results suggest that presynaptic glycine receptors can regulate the excitability of PAG neurons by enhancing glutamatergic transmission and therefore play an important role in the regulation of various physiological functions mediated by the PAG.

  4. Rab, Arf, and Arl-Regulated Membrane Traffic in Cortical Neuron Migration.

    Tang, Bor Luen


    The migration of projection neurons from its birthplace in the subventricular zone to their final destination in the cortical plate is a complex process that requires a series of highly coordinated cellular events. Amongst the key factors involved in the processes are modulators of cytoskeletal dynamics, as well as cellular membrane traffic. Members of the small GTPases family responsible for the latter process, the Rabs and Arfs, have been recently implicated in cortical neuron migration. Rab5 and Rab11, which are key modulators of endocytosis and endocytic recycling respectively, ensure proper surface expression and distribution of N-cadherin, a key adhesion protein that tethers migrating neurons to the radial glia fiber tracts during pia-directed migration. Rab7, which is associated with lysosomal biogenesis and function, is important for the final step of terminal translocation when N-cadherin is downregulated by lysosomal degradation. Arf6 activity, which is known to be important in neuronal processes outgrowth, may negatively impact the multipolar-bipolar transition of cortical neurons undergoing radial migration, but the downstream effector of Arf6 in this regard is not yet known. In addition to the above, members of the Arl family which have been recently shown to be important in radial glia scaffold formation, would also be important for cortical neuron migration. In this short review, we discuss recent advances in our understanding of the importance of membrane traffic regulated by the Rab, Arf, and Arl family members in cortical neuron migration.

  5. Bilaminar co-culture of primary rat cortical neurons and glia.

    Shimizu, Saori; Abt, Anna; Meucci, Olimpia


    This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons. Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection. A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions(1). At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods(1-3). Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology(4), cellular and molecular biology(5-8), biochemistry(5), imaging and microscopy(4,6,7,9,10). The primary neurons extend axons and dendrites to form functional synapses(11), a process which is not observed in neuronal cell lines, although some

  6. Laminar segregation of GABAergic neurons in the avian nucleus isthmi pars magnocellularis: a retrograde tracer and comparative study.

    Faunes, Macarena; Fernández, Sara; Gutiérrez-Ibáñez, Cristián; Iwaniuk, Andrew N; Wylie, Douglas R; Mpodozis, Jorge; Karten, Harvey J; Marín, Gonzalo


    The isthmic complex is part of a visual midbrain circuit thought to be involved in stimulus selection and spatial attention. In birds, this circuit is composed of the nuclei isthmi pars magnocellularis (Imc), pars parvocellularis (Ipc), and pars semilunaris (SLu), all of them reciprocally connected to the ipsilateral optic tectum (TeO). The Imc conveys heterotopic inhibition to the TeO, Ipc, and SLu via widespread γ-aminobutyric acid (GABA)ergic axons that allow global competitive interactions among simultaneous sensory inputs. Anatomical studies in the chick have described a cytoarchitectonically uniform Imc nucleus containing two intermingled cell types: one projecting to the Ipc and SLu and the other to the TeO. Here we report that in passerine species, the Imc is segregated into an internal division displaying larger, sparsely distributed cells, and an external division displaying smaller, more densely packed cells. In vivo and in vitro injections of neural tracers in the TeO and the Ipc of the zebra finch demonstrated that neurons from the external and internal subdivisions project to the Ipc and the TeO, respectively, indicating that each Imc subdivision contains one of the two cell types hodologically defined in the chick. In an extensive survey across avian orders, we found that, in addition to passerines, only species of Piciformes and Rallidae exhibited a segregated Imc, whereas all other groups exhibited a uniform Imc. These results offer a comparative basis to investigate the functional role played by each Imc neural type in the competitive interactions mediated by this nucleus.

  7. Differential expression of K4-AP currents and Kv3.1 potassium channel transcripts in cortical neurons that develop distinct firing phenotypes.

    Massengill, J L; Smith, M A; Son, D I; O'Dowd, D K


    Maturation of electrical excitability during early postnatal development is critical to formation of functional neural circuitry in the mammalian neocortex. Little is known, however, about the changes in gene expression underlying the development of firing properties that characterize different classes of cortical neurons. Here we describe the development of cortical neurons with two distinct firing phenotypes, regular-spiking (RS) and fast-spiking (FS), that appear to emerge from a population of immature multiple-spiking (IMS) neurons during the first two postnatal weeks, both in vivo (within layer IV) and in vitro. We report the expression of a slowly inactivating, 4-AP-sensitive potassium current (K4-AP) at significantly higher density in FS compared with RS neurons. The same current is expressed at intermediate levels in IMS neurons. The kinetic, voltage-dependent, and pharmacological properties of the K4-AP current are similar to those observed by heterologous expression of Kv3.1 potassium channel mRNA. Single-cell RT-PCR analysis demonstrates that PCR products representing Kv3.1 transcripts are amplified more frequently from FS than RS neurons, with an intermediate frequency of Kv3.1 detection in neurons with immature firing properties. Taken together, these data suggest that the Kv3.1 gene encodes the K4-AP current and that expression of this gene is regulated in a cell-specific manner during development. Analysis of the effects of 4-AP on firing properties suggests that the K4-AP current is important for rapid action potential repolarization, fast after-hyperpolarization, brief refractory period, and high firing frequency characteristic of FS GABAergic interneurons.

  8. Three Types of Cortical Layer 5 Neurons That Differ in Brain-wide Connectivity and Function.

    Kim, Euiseok J; Juavinett, Ashley L; Kyubwa, Espoir M; Jacobs, Matthew W; Callaway, Edward M


    Cortical layer 5 (L5) pyramidal neurons integrate inputs from many sources and distribute outputs to cortical and subcortical structures. Previous studies demonstrate two L5 pyramid types: cortico-cortical (CC) and cortico-subcortical (CS). We characterize connectivity and function of these cell types in mouse primary visual cortex and reveal a new subtype. Unlike previously described L5 CC and CS neurons, this new subtype does not project to striatum [cortico-cortical, non-striatal (CC-NS)] and has distinct morphology, physiology, and visual responses. Monosynaptic rabies tracing reveals that CC neurons preferentially receive input from higher visual areas, while CS neurons receive more input from structures implicated in top-down modulation of brain states. CS neurons are also more direction-selective and prefer faster stimuli than CC neurons. These differences suggest distinct roles as specialized output channels, with CS neurons integrating information and generating responses more relevant to movement control and CC neurons being more important in visual perception.

  9. Neurodegenerative, with expression ATF-2 by p38 in cortical neurons.

    Hosseini, M; Ostad, N; Parivar, K; Ghahremani, M H


    DNA damage, as an important initiator of neuronal cell death, has been implicated in numerous neurodegenerative conditions. We previously delineated several pathways that control embryonic cortical neuronal cell death evoked by the DNA-damaging agent, camptothecin. The topisomerase-1 inhibitor, camptothecin, has been shown to induce cortical neuronal cell death in a reproducible and synchronistic manner. Primary embryonic neuronal cell culture cortical neurons were prepared. In the study, the survival % of neurons in camptothecin P38 group, after 6 hours (85%), 24 hours (64%) and 48 hours (50%), compared to camptothecin ATF-2 and P38 group after 4 hours (97 and 95%), have been significantly lower, and the expression % of neurons in camptothecin P38 group , after 6 hours (20%), 24 hours (40%) and 48 hours (55%), compared to camptothecin ATF-2 and P38 group after 4 hours (5 and 3%) have been significant lower (pATF-2 group after 24hours (30%), have been significant lower (pATF-2 in embryonic cortical neurons following DNA damage.

  10. Latency dependent development of related firing patterns of cultured cortical neurons

    le Feber, Jakob; van Pelt, Jaap; Rutten, Wim

    Networks of cortical neurons were grown over multi electrode arrays to enable simultaneous measurement of signals from multiple neurons. We described functional connectivity in these networks by relationships be¬tween individual electrodes, based on conditional firing probabilities. In this study we

  11. Hypoxic preconditioning differentially affects GABAergic and glutamatergic neuronal cells in the injured cerebellum of the neonatal rat.

    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

  12. Low level laser therapy reduces oxidative stress in cortical neurons in vitro

    Huang, Ying-Ying; Tedford, Clark E.; McCarthy, Thomas; Hamblin, Michael R.


    It is accepted that the mechanisms of low level laser therapy (LLLT) involves photons that are absorbed in the mitochondria of cells and lead to increase of mitochondrial metabolism resulting in more electron transport, increase of mitochondrial membrane potential, and more ATP production. Intracellular calcium changes are seen that correlate with mitochondrial stimulation. The situation with two other intermediates is more complex however: reactive oxygen species (ROS) and nitric oxide (NO). Evidence exists that low levels of ROS are produced by LLLT in normal cells that can be beneficial by (for instance) activating NF-kB. However high fluences of light can produce large amounts of ROS that can damage the cells. In oxidatively stressed cells the situation may be different. We exposed primary cultured cortical neurons to hydrogen peroxide (H2O2) or cobalt chloride (CoCl2) oxidative insults in the presence or absence of LLLT (810-nm laser at 0.3 or 3 J/cm2). Cell viability of cortical neurons was determined by lactate dehydrogenase assay. ROS in neurons was detected using an ROS probe, MitoRox with confocal microscopy. Results showed that LLLT dose-dependently reversed ROS production and protected cortical neurons against H2O2 or CoCl2 induced oxidative injury in cultured cortical neurons. Conclusion: LLLT can protect cortical neurons against oxidative stress by reversing the levels of ROS.

  13. MEC-17 deficiency leads to reduced α-tubulin acetylation and impaired migration of cortical neurons.

    Li, Lei; Wei, Dan; Wang, Qiong; Pan, Jing; Liu, Rong; Zhang, Xu; Bao, Lan


    Neuronal migration is a fundamental process during the development of the cerebral cortex and is regulated by cytoskeletal components. Microtubule dynamics can be modulated by posttranslational modifications to tubulin subunits. Acetylation of α-tubulin at lysine 40 is important in regulating microtubule properties, and this process is controlled by acetyltransferase and deacetylase. MEC-17 is a newly discovered α-tubulin acetyltransferase that has been found to play a major role in the acetylation of α-tubulin in different species in vivo. However, the physiological function of MEC-17 during neural development is largely unknown. Here, we report that MEC-17 is critical for the migration of cortical neurons in the rat. MEC-17 was strongly expressed in the cerebral cortex during development. MEC-17 deficiency caused migratory defects in the cortical projection neurons and interneurons, and perturbed the transition of projection neurons from the multipolar stage to the unipolar/bipolar stage in the intermediate zone of the cortex. Furthermore, knockdown of α-tubulin deacetylase HDAC6 or overexpression of tubulin(K40Q) to mimic acetylated α-tubulin could reduce the migratory and morphological defects caused by MEC-17 deficiency in cortical projection neurons. Thus, MEC-17, which regulates the acetylation of α-tubulin, appears to control the migration and morphological transition of cortical neurons. This finding reveals the importance of MEC-17 and α-tubulin acetylation in cortical development.

  14. Excitatory cortical neurons with multipolar shape establish neuronal polarity by forming a tangentially oriented axon in the intermediate zone.

    Hatanaka, Yumiko; Yamauchi, Kenta


    The formation of axon-dendrite polarity is crucial for neuron to make the proper information flow within the brain. Although the processes of neuronal polarity formation have been extensively studied using neurons in dissociated culture, the corresponding developmental processes in vivo are still unclear. Here, we illuminate the initial steps of morphological polarization of excitatory cortical neurons in situ, by sparsely labeling their neuroepithelial progenitors using in utero electroporation and then examining their neuronal progeny in brain sections and in slice cultures. Morphological analysis showed that an axon-like long tangential process formed in progeny cells in the intermediate zone (IZ). Time-lapse imaging analysis using slice culture revealed that progeny cells with multipolar shape, after alternately extending and retracting their short processes for several hours, suddenly elongated a long process tangentially. These cells then transformed into a bipolar shape, extending a pia-directed leading process, and migrated radially leaving the tangential process behind, which gave rise to an "L-shaped" axon. Our findings suggest that neuronal polarity in these cells is established de novo from a nonpolarized stage in vivo and indicate that excitatory cortical neurons with multipolar shape in the IZ initiate axon outgrowth before radial migration into the cortical plate.

  15. Sodium entry during action potentials of mammalian central neurons: incomplete inactivation and reduced metabolic efficiency in fast-spiking neurons

    Carter, Brett C.; Bean, Bruce P.


    We measured the time course of sodium entry during action potentials of mouse central neurons at 37 °C to examine how efficiently sodium entry is coupled to depolarization. In cortical pyramidal neurons, sodium entry was nearly completely confined to the rising phase of the spike: only ~25% more sodium enters than the theoretical minimum necessary for spike depolarization. However, in fast-spiking GABAergic neurons (cerebellar Purkinje cells and cortical interneurons), twice as much sodium en...

  16. Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity.

    Xu, Jin-Chong; Fan, Jing; Wang, Xueqing; Eacker, Stephen M; Kam, Tae-In; Chen, Li; Yin, Xiling; Zhu, Juehua; Chi, Zhikai; Jiang, Haisong; Chen, Rong; Dawson, Ted M; Dawson, Valina L


    Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human cortical neuron culture system from human embryonic stem cells or human inducible pluripotent stem cells that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid to FOXG1(+) neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human cortical neuronal cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate (NMDA) receptors, nitric oxide (NO), and poly(ADP-ribose) polymerase (PARP) (a cell death pathway called parthanatos that is distinct from apoptosis, necroptosis, and other forms of cell death). Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons.

  17. Distribution of GABA-ergic Neurons in the Rhesus Monkey Substantia Nigra%猕猴黑质GABA能神经元的分布

    董大翠; 蔡秋云; 姚大卫; 陈活彝


    The higher evolutive rhesus monkeys were used as experimental materials. GABA-ergic neurons were observed under a light microscope. The GABA-ergic neurons in the pars reticulata of substantion nigra were numerous, dominant in the shape of polygonal or shuttle. The GABA neurons in the pars compacta of substantia nigra were less than in the pars reticulata, with the shape dominant in polygonal, round or shuttle.Many GABA-like positive fibres were observed at the inferior colliculus level of substantia nigra.%采用进化程度较高的灵长类动物猕猴作为实验材料,在光镜下观察黑质的GABA能神经元。在黑质网状部GABA能神经元数量多,以多角形和梭形为主,致密部的GABA能神经元较网状部略稀少,胞体形态以多角形、圆形和梭形为主;在下丘平面还可见到许多GABA样阳性神经纤维。

  18. Cell surface domain specific postsynaptic currents evoked by identified GABAergic neurones in rat hippocampus in vitro.

    Maccaferri, G; Roberts, J D; Szucs, P; Cottingham, C A; Somogyi, P


    1. Inhibitory postsynaptic currents (IPSCs) evoked in CA1 pyramidal cells (n = 46) by identified interneurones (n = 43) located in str. oriens were recorded in order to compare their functional properties and to determine the effect of synapse location on the apparent IPSC kinetics as recorded using somatic voltage clamp at -70 mV and nearly symmetrical [Cl-]. 2. Five types of visualised presynaptic interneurone, oriens-lacunosum moleculare (O-LMC), basket (BC), axo-axonic (AAC), bistratified (BiC) and oriens-bistratified (O-BiC) cells, were distinguished by immunocytochemistry and/or synapse location using light and electron microscopy. 3. Somatostatin immunoreactive O-LMCs, innervating the most distal dendritic shafts and spines, evoked the smallest amplitude (26 +/- 10 pA, s.e.m., n = 8) and slowest IPSCs (10-90 % rise time, 6.2 +/- 0.6 ms; decay, 20.8 +/- 1.7 ms, n = 8), with no paired-pulse modulation of the second IPSC (93 +/- 4 %) at 100 ms interspike interval. In contrast, parvalbumin-positive AACs evoked larger amplitude (308 +/- 103 pA, n = 7) and kinetically faster (rise time, 0.8 +/- 0.1 ms; decay 11.2 +/- 0.9 ms, n = 7) IPSCs showing paired-pulse depression (to 68 +/- 5 %, n = 6). Parvalbumin- or CCK-positive BCs (n = 9) terminating on soma/dendrites, BiCs (n = 4) and O-BiCs (n = 7) innervating dendrites evoked IPSCs with intermediate kinetic parameters. The properties of IPSCs and sensitivity to bicuculline indicated that they were mediated by GABAA receptors. 4. In three cases, kinetically complex, multiphasic IPSCs, evoked by an action potential in the recorded basket cells, suggested that coupled interneurones, possibly through electrotonic junctions, converged on the same postsynaptic neurone. 5. The population of O-BiCs (4 of 4 somatostatin positive) characterised in this study had horizontal dendrites restricted to str. oriens/alveus and innervated stratum radiatum and oriens. Other BiCs had radial dendrites as described earlier. The parameters

  19. Inhibitory synapse formation in a co-culture model incorporating GABAergic medium spiny neurons and HEK293 cells stably expressing GABAA receptors.

    Brown, Laura E; Fuchs, Celine; Nicholson, Martin W; Stephenson, F Anne; Thomson, Alex M; Jovanovic, Jasmina N


    Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed

  20. Genetic evidence for p75NTR-dependent tetraploidy in cortical projection neurons from adult mice.

    López-Sánchez, Noelia; Frade, José M


    A subpopulation of chick retinal projection neurons becomes tetraploid during development, an event prevented by blocking antibodies against p75 neurotrophin receptor (p75(NTR)). We have used an optimized flow cytometric assay, based on the analysis of unfixed brain cell nuclei, to study whether p75(NTR)-dependent neuronal tetraploidization takes place in the cerebral cortex, giving rise to projection neurons as well. We show that 3% of neurons in both murine neocortex and chick telencephalic derivatives are tetraploid, and that in the mouse ~85% of these neurons express the immediate early genes Erg-1 and c-Fos, indicating that they are functionally active. Tetraploid cortical neurons (65-80%) express CTIP2, a transcription factor specific for subcortical projection neurons in the mouse neocortex. During the period in which these neurons are born, p75(NTR) is detected in differentiating neurons undergoing DNA replication. Accordingly, p75(NTR)-deficient mice contain a reduced proportion of both NeuN and CTIP2-positive neocortical tetraploid neurons, thus providing genetic evidence for the participation of p75(NTR) in the induction of neuronal tetraploidy in the mouse neocortex. In the striatum tetraploidy is mainly associated with long-range projection neurons as well since ~80% of tetraploid neurons in this structure express calbindin, a marker of neostriatal-matrix spiny neurons, known to establish long-range projections to the substantia nigra and globus pallidus. In contrast, only 20% of tetraploid cortical neurons express calbindin, which is mainly expressed in layers II-III, where CTIP2 is absent. We conclude that tetraploidy mainly affects long-range projection neurons, being facilitated by p75(NTR) in the neocortex.

  1. Morphometric characteristics of Neuropeptide Y immunoreactive neurons of human cortical amygdaloid nucleus

    Mališ Miloš


    Full Text Available Introduction Cortical amygdaloid nucleus belongs to the corticomedial part of the amygdaloid complex. In this nucleus there are neurons that produce neuropetide Y. This peptide has important roles in sleeping, learning, memory, gastrointestinal regulation, anxiety, epilepsy, alcoholism and depression. Material and methods We investigated morphometric characteristics (numbers of primary dendrites, longer and shorter diameters of cell bodies and maximal radius of dendritic arborization of NPY immunoreactive neurons of human cortical amygdaloid nucleus on 6 male adult human brains, aged 46 to 77 years, by immunohistochemical avidin-biotin technique. Results Our investigation has shown that in this nucleus there is a moderate number of NPY immunoreactive neurons. 67% of found neurons were nonpyramidal, while 33% were pyramidal. Among the nonpyramidal neurons the dominant groups were multipolar neurons (41% - of which 25% were multipolar irregular, and 16% multipolar oval. Among the pyramidal neurons the dominant groups were the neurons with triangular shape of cell body (21%. All found NPY immunoreactive neurons (pyramidal and nonpyramidal altogether had intervals of values of numbers of primary dendrites 2 to 6, longer diameters of cell bodies 13 to 38 µm, shorter diameters of cell bodies 9 to 20 µm and maximal radius of dendritic arborization 50 to 340 µm. More than a half of investigated neurons (57% had 3 primary dendrites. Discussion and conclusion The other researchers did not find such percentage of pyramidal immunoreactive neurons in this amygdaloid nucleus. If we compare our results with the results of the ather researchers we can conclude that all pyramidal NPY immunoreactive neurons found in this human amygdaloid nucleus belong to the class I of neurons, and that all nonpyramidal NPY immunoreactive neurons belong to the class II of neurons described by other researchers. We suppose that all found pyramidal neurons were projectional.

  2. The Impact of CXCR4 Blockade on the Survival of Rat Brain Cortical Neurons

    Merino, José Joaquín; Garcimartín, Alba; López-Oliva, María Elvira; Benedí, Juana; González, María Pilar


    Background: Chemokine receptor type 4 (CXCR4) plays a role in neuronal survival/cell repair and also contributes to the progression of cancer and neurodegenerative diseases. Chemokine ligand 12 (CXCL12) binds to CXCR4. In this study, we have investigated whether CXCR4 blockade by AMD3100 (a CXCR4 antagonist, member of bicyclam family) may affect neuronal survival in the absence of insult. Thus, we have measured the mitochondrial membrane potential (MMP), Bax and Bcl-2 protein translocation, and cytochrome c release in AMD3100-treated brain cortical neurons at 7 DIV (days in vitro). Methods: For this aim, AMD3100 (200 nM) was added to cortical neurons for 24 h, and several biomarkers like cell viability, reactive oxygen species (ROS) generation, lactate dehydrogenase (LDH) release, caspase-3/9 activity, proteins Bax and Bcl-2 translocation, and cytochrome c release were analyzed by immunoblot. Results: CXCR4 blockade by AMD3100 (200 nM, 24 h) induces mitochondrial hyperpolarization and increases caspase-3/9 hyperpolarization without affecting LDH release as compared to untreated controls. AMD3100 also increases cytochrome c release and promotes Bax translocation to the mitochondria, whereas it raises cytosolic Bcl-2 levels in brain cortical neurons. Conclusion: CXCR4 blockade induces cellular death via intrinsic apoptosis in rat brain cortical neurons in absence of insult. PMID:27916896

  3. Sulfite triggers sustained calcium overload in cultured cortical neurons via a redox-dependent mechanism.

    Wang, Xiao; Cao, Hui; Guan, Xin-Lei; Long, Li-Hong; Hu, Zhuang-Li; Ni, Lan; Wang, Fang; Chen, Jian-Guo; Wu, Peng-Fei


    Sulfite is a compound commonly used as preservative in foods and pharmaceuticals. Many studies have examined the neurotoxicity of sulfite, but its effect on neuronal calcium homeostasis has not yet been reported. Here, we observed the effect of sulfite on the cytosolic free calcium concentration ([Ca(2+)]i) in cultured cortical neurons using Fura-2/AM based calcium imaging technique. Sulfite (250-1000μM) caused a sustained increase in [Ca(2+)]i in the neurons via a dose-dependent manner. In Ca(2+)-free solution, sulfite failed to increase [Ca(2+)]i. After the depletion of the intracellular calcium store, the effect of sulfite on the [Ca(2+)]i was largely abolished. Pharmacological inhibition of phospholipase C (PLC)-inositol 1,4,5-triphosphate (IP3) signaling pathway blocked sulfite-induced increase of [Ca(2+)]i. Interestingly, antioxidants such as trolox and dithiothreitol, abolished the increase of [Ca(2+)]i induced by sulfite. Exposure to sulfite triggered generation of sulfur- and oxygen-centered free radicals in neurons and increased oxidative stress both in the cultured cortical neurons and the prefrontal cortex of rats. Furthemore, sulfite decreased cell viability in cultured cortical neurons via a calcium-dependent manner. Thus, our current study suggests that the redox-dependent calcium overload triggered by sulfite in cortical neuronsmay be involved in its neurotoxicity. Copyright © 2016. Published by Elsevier Ireland Ltd.

  4. Synaptic Targets of Medial Septal Projections in the Hippocampus and Extrahippocampal Cortices of the Mouse.

    Unal, Gunes; Joshi, Abhilasha; Viney, Tim J; Kis, Viktor; Somogyi, Peter


    Temporal coordination of neuronal assemblies among cortical areas is essential for behavioral performance. GABAergic projections from the medial septum and diagonal band complex exclusively innervate GABAergic interneurons in the rat hippocampus, contributing to the coordination of neuronal activity, including the generation of theta oscillations. Much less is known about the synaptic target neurons outside the hippocampus. To reveal the contribution of synaptic circuits involving the medial septum of mice, we have identified postsynaptic cortical neurons in wild-type and parvalbumin-Cre knock-in mice. Anterograde axonal tracing from the septum revealed extensive innervation of the hippocampus as well as the subiculum, presubiculum, parasubiculum, the medial and lateral entorhinal cortices, and the retrosplenial cortex. In all examined cortical regions, many septal GABAergic boutons were in close apposition to somata or dendrites immunopositive for interneuron cell-type molecular markers, such as parvalbumin, calbindin, calretinin, N-terminal EF-hand calcium-binding protein 1, cholecystokinin, reelin, or a combination of these molecules. Electron microscopic observations revealed septal boutons forming axosomatic or axodendritic type II synapses. In the CA1 region of hippocampus, septal GABAergic projections exclusively targeted interneurons. In the retrosplenial cortex, 93% of identified postsynaptic targets belonged to interneurons and the rest to pyramidal cells. These results suggest that the GABAergic innervation from the medial septum and diagonal band complex contributes to temporal coordination of neuronal activity via several types of cortical GABAergic interneurons in both hippocampal and extrahippocampal cortices. Oscillatory septal neuronal firing at delta, theta, and gamma frequencies may phase interneuron activity.

  5. Oxytocin-induced antinociception in the spinal cord is mediated by a subpopulation of glutamatergic neurons in lamina I-II which amplify GABAergic inhibition

    Schlichter Rémy


    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.

  6. An interneuron progenitor maintains neurogenic potential in vivo and differentiates into GABAergic interneurons after transplantation in the postnatal rat brain.

    Wang, Qi; Hong, Peiwei; Gao, Hui; Chen, Yuntian; Yang, Qi; Jiang, Mei; Li, Hedong


    Dysfunction of cortical GABAergic interneurons are involved in numerous neurological disorders including epilepsy, schizophrenia and autism; and replenishment of these cells by transplantation strategy has proven to be a feasible and effective method to help revert the symptoms in several animal models. To develop methodology of generating transplantable GABAergic interneurons for therapy, we previously reported the isolation of a v-myc-induced GABAergic interneuron progenitor clone GE6 from embryonic ganglionic eminence (GE). These cells can proliferate and form functional inhibitory synapses in culture. Here, we tested their differentiation behavior in vivo by transplanting them into the postnatal rat forebrain. We found that GE6 cells migrate extensively in the neonatal forebrain and differentiate into both neurons and glia, but preferentially into neurons when compared with a sister progenitor clone CTX8. The neurogenic potential of GE6 cells is also maintained after transplantation into a non-permissive environment such as adult cortex or when treated with inflammatory cytokine in culture. The GE6-derived neurons were able to mature in vivo as GABAergic interneurons expressing GABAergic, not glutamatergic, presynaptic puncta. Finally, we propose that v-myc-induced human interneuron progenitor clones could be an alternative cell source of transplantable GABAergic interneurons for treating related neurological diseases in future clinic.

  7. Capsaicin protects cortical neurons against ischemia/reperfusion injury via down-regulating NMDA receptors.

    Huang, Ming; Cheng, Gen; Tan, Han; Qin, Rui; Zou, Yimin; Wang, Yun; Zhang, Ying


    Capsaicin, the ingredient responsible for the pungent taste of hot chili peppers, is widely used in the study and management of pain. Recently, its neuroprotective effect has been described in multiple studies. Herein, we investigated the underlying mechanisms for the neuroprotective effect of capsaicin. Direct injection of capsaicin (1 or 3nmol) into the peri-infarct area reduced the infarct volume and improved neurological behavioral scoring and motor coordination function in the middle cerebral artery occlusion (MCAO)/reperfusion model in rats. The time window of the protective effect of capsaicin was within 1h after reperfusion, when excitotoxicity is the main reason of cell death. In cultured cortical neurons, administration of capsaicin attenuated glutamate-induced excitotoxic injury. With respect to the mechanisms of the neuroprotective effect of capsaicin, reduced calcium influx after glutamate stimulation was observed following capsaicin pretreatment in cortical neurons. Trpv1 knock-out abolished the inhibitory effect of capsaicin on glutamate-induced calcium influx and subsequent neuronal death. Reduced expression of GluN1 and GluN2B, subunits of NMDA receptor, was examined after capsaicin treatment in cortical neurons. In summary, our studies reveal that the neuroprotective effect of capsaicin in cortical neurons is TRPV1-dependent and down-regulation of the expression and function of NMDA receptors contributes to the protection afforded by capsaicin. Copyright © 2017. Published by Elsevier Inc.

  8. Betaine attenuates memory impairment after water-immersion restraint stress and is regulated by the GABAergic neuronal system in the hippocampus.

    Kunisawa, Kazuo; Kido, Kiwamu; Nakashima, Natsuki; Matsukura, Takuya; Nabeshima, Toshitaka; Hiramatsu, Masayuki


    GABA mediated neuronal system regulates hippocampus-dependent memory and stress responses by controlling plasticity and neuronal excitability. Here, we demonstrate that betaine ameliorates water-immersion restraint stress (WIRS)-induced memory impairments. This improvement was inhibited by a betaine/GABA transporter-1 (GABA transporter-2: GAT2) inhibitor, NNC 05-2090. In this study, we investigated whether memory amelioration by betaine was mediated by the GABAergic neuronal system. Adult male mice were co-administered betaine and GABA receptor antagonists after WIRS. We also examined whether memory impairment after WIRS was attenuated by GABA receptor agonists. The memory functions were evaluated using a novel object recognition test 3-6 days after WIRS and/or the step-down type passive avoidance test at 7-8 days. The co-administration of the GABAA receptor antagonist bicuculline (1mg/kg) or the GABAB receptor antagonist phaclofen (10mg/kg) 1h after WIRS suppressed the memory-improving effects induced by betaine. Additionally, the administration of the GABAA receptor agonist muscimol (1mg/kg) or the GABAB receptor agonist baclofen (10mg/kg) 1h after WIRS attenuated memory impairments. These results were similar to the data observed with betaine. The treatment with betaine after WIRS significantly decreased the expression of GABA transaminase, and this effect was partially blocked by NNC 05-2090 in the hippocampus. WIRS caused a transient increase in hippocampal GABA levels and the changes after WIRS were not affected by betaine treatment in an in vivo microdialysis study. These results suggest that the beneficial effects of betaine may be mediated in part by changing the GABAergic neuronal system. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Pre-differentiation of human neural stem cells into GABAergic neurons prior to transplant results in greater repopulation of the damaged brain and accelerates functional recovery after transient ischemic stroke.

    Abeysinghe, Hima C S; Bokhari, Laita; Quigley, Anita; Choolani, Mahesh; Chan, Jerry; Dusting, Gregory J; Crook, Jeremy M; Kobayashi, Nao R; Roulston, Carli L


    Despite attempts to prevent brain injury during the hyperacute phase of stroke, most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study, we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ), and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain. Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival, cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy. Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype, showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days, suggesting an additional trophic role of these GABAergic cells. In contrast, undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar. Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery.

  10. Disruption of Transient Serotonin Accumulation by Non-Serotonin-Producing Neurons Impairs Cortical Map Development

    Xiaoning Chen


    Full Text Available Polymorphisms that alter serotonin transporter SERT expression and functionality increase the risks for autism and psychiatric traits. Here, we investigate how SERT controls serotonin signaling in developing CNS in mice. SERT is transiently expressed in specific sets of glutamatergic neurons and uptakes extrasynaptic serotonin during perinatal CNS development. We show that SERT expression in glutamatergic thalamocortical axons (TCAs dictates sensory map architecture. Knockout of SERT in TCAs causes lasting alterations in TCA patterning, spatial organizations of cortical neurons, and dendritic arborization in sensory cortex. Pharmacological reduction of serotonin synthesis during the first postnatal week rescues sensory maps in SERTGluΔ mice. Furthermore, knockdown of SERT expression in serotonin-producing neurons does not impair barrel maps. We propose that spatiotemporal SERT expression in non-serotonin-producing neurons represents a determinant in early life genetic programming of cortical circuits. Perturbing this SERT function could be involved in the origin of sensory and cognitive deficits associated with neurodevelopmental disorders.


    The insecticidal and neurotoxic effects of pyrethroids result from prolonged sodium channel inactivation, which causes alterations in neuronal firing and communication. Previously, we determined the relative potencies of 11 type I and type II pyrethroid insecticides using microel...

  12. Baicalein reverts L-valine-induced persistent sodium current up-modulation in primary cortical neurons.

    Caioli, Silvia; Candelotti, Elena; Pedersen, Jens Z; Saba, Luana; Antonini, Alessia; Incerpi, Sandra; Zona, Cristina


    L-valine is a branched-chain amino acid (BCAA) largely used as dietary integrator by athletes and involved in some inherited rare diseases such as maple syrup urine disease. This pathology is caused by an altered BCAA metabolism with the accumulation of toxic keto acids in tissues and body fluids with consequent severe neurological symptoms. In animal models of BCAA accumulation, increased oxidative stress levels and lipid peroxidation have been reported. The aim of this study was to analyze both whether high BCAA concentrations in neurons induce reactive oxygen species (ROS) production and whether, by performing electrophysiological recordings, the neuronal functional properties are modified. Our results demonstrate that in primary cortical cultures, a high dose of valine increases ROS production and provokes neuronal hyperexcitability because the action potential frequencies and the persistent sodium current amplitudes increase significantly compared to non-treated neurons. Since Baicalein, a flavone obtained from the Scutellaria root, has been shown to act as a strong antioxidant with neuroprotective effects, we evaluated its possible antioxidant activity in primary cortical neurons chronically exposed to L-valine. The preincubation of cortical neurons with Baicalein prevents the ROS production and is able to revert both the neuronal hyperexcitability and the increase of the persistent sodium current, indicating a direct correlation between the ROS production and the altered physiological parameters. In conclusion, our data show that the electrophysiological alterations of cortical neurons elicited by high valine concentration are due to the increase in ROS production, suggesting much caution in the intake of BCAA dietary integrators.

  13. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication

    Gefei Wang


    Full Text Available Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i. but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy.

  14. Procedure for recording the simultaneous activity of single neurons distributed across cortical areas during sensory discrimination

    Hernández, Adrián; Nácher, Verónica; Luna, Rogelio; Alvarez, Manuel; Zainos, Antonio; Cordero, Silvia; Camarillo, Liliana; Vázquez, Yuriria; Lemus, Luis; Romo, Ranulfo


    We report a procedure for recording the simultaneous activity of single neurons distributed across five cortical areas in behaving monkeys. The procedure consists of a commercially available microdrive adapted to a commercially available neural data collection system. The critical advantage of this procedure is that, in each cortical area, a configuration of seven microelectrodes spaced 250–500 μm can be inserted transdurally and each can be moved independently in the z axis. For each microelectrode, the data collection system can record the activity of up to five neurons together with the local field potential (LFP). With this procedure, we normally monitor the simultaneous activity of 70–100 neurons while trained monkeys discriminate the difference in frequency between two vibrotactile stimuli. Approximately 20–60 of these neurons have response properties previously reported in this task. The neuronal recordings show good signal-to-noise ratio, are remarkably stable along a 1-day session, and allow testing several protocols. Microelectrodes are removed from the brain after a 1-day recording session, but are reinserted again the next day by using the same or different x-y microelectrode array configurations. The fact that microelectrodes can be moved in the z axis during the recording session and that the x-y configuration can be changed from day to day maximizes the probability of studying simultaneous interactions, both local and across distant cortical areas, between neurons associated with the different components of this task. PMID:18946031

  15. Neuropeptide Y protects cerebral cortical neurons by regulating microglial immune function

    Qijun Li; Changzheng Dong; Wenling Li; Wei Bu; Jiang Wu; Wenqing Zhao


    Neuropeptide Y has been shown to inhibit the immunological activity of reactive microglia in the rat cerebral cortex, to reduce N-methyl-D-aspartate current (INMDA) in cortical neurons, and protect neurons. In this study, after primary cultured microglia from the cerebral cortex of rats were treated with lipopolysaccharide, interleukin-1β and tumor necrosis factor-α levels in the cell culture medium increased, and mRNA expression of these cytokines also increased. After primary cultured cortical neurons were incubated with the lipopolysaccharide-treated microg-lial conditioned medium, peak INMDA in neurons increased. These effects of lipopolysaccharide were suppressed by neuropeptide Y. After addition of the neuropeptide Y Y1 receptor antago-nist BIBP3226, the effects of neuropeptide Y completely disappeared. These results suggest that neuropeptide Y prevents excessive production of interleukin-1β and tumor necrosis factor-α by inhibiting microglial reactivity. This reduces INMDA in rat cortical neurons, preventing excitotoxic-ity, thereby protecting neurons.

  16. Coupling (reduced Graphene Oxide to Mammalian Primary Cortical Neurons In Vitro

    Antonina M. Monaco


    Full Text Available Neuronal nanoscale interfacing aims at identifying or designing nanostructured smart materials and validating their applications as novel biocompatible scaffolds with active properties for neuronal networks formation, nerve regeneration, and bidirectional biosignal coupling. Among several carbon-based nanomaterials, Graphene recently attracted great interest for biological applications, given its unique mechanical, optical, electronic properties, and its recent technological applications. Here we explore the use of Graphene Oxide (GO and reduced Graphene Oxide (rGO as biocompatible culture substrates for primary neuronal networks developing ex vivo. We quantitatively studied cytotoxicity and cellular viability as well as single-cell and network-level electrophysiological properties of neurons in vitro. Our results confirm previous reports, employing immortalized cell lines or pluripotent stem cells, and extend them to mammalian primary cortical neurons: GO and rGO are biocompatible substrates and do not alter neuronal excitable properties.

  17. Effects of sex and deletion of neuropeptide Y2 receptors from GABAergic neurons on affective and alcohol drinking behaviors in mice

    Nora M McCall


    Full Text Available A large literature has demonstrated that neuropeptide Y (NPY regulates many emotional and reward-related behaviors via its primary receptors, Y1R and Y2R. Classically, NPY actions at postsynaptic Y1R decrease anxiety, depression, and alcohol drinking, while its actions at presynaptic Y2R produce the opposite behavioral phenotypes. However, emerging evidence suggests that activation of Y2R can also produce anxiolysis in a brain region and neurotransmitter system-dependent fashion. Further, numerous human and rodent studies have reported that females display higher levels of anxiety, depression, and alcohol drinking. In this study, we evaluated sex differences and the role of Y2R on GABAergic transmission in these behaviors using a novel transgenic mouse that lacks Y2R specifically in VGAT-expressing neurons (VGAT-Y2R knockout. First, we confirmed our genetic manipulation by demonstrating that Y2R protein expression was decreased and that a Y2R agonist could not alter GABAergic transmission in the extended amygdala, a limbic brain region critically implicated in the regulation of anxiety and alcohol drinking behaviors, using immunofluorescence and slice electrophysiology. Then, we tested male and female VGAT-Y2R knockout mice on a series of behavioral assays for anxiety, depression, fear, anhedonia, and alcohol drinking. We found that females displayed greater basal anxiety, higher levels of ethanol consumption, and faster fear conditioning than males, and that knockout mice exhibited enhanced depressive-like behavior in the forced swim test. Together, these results confirm previous studies that demonstrate higher expression of negative affective and alcohol drinking behaviors in females than males, and they highlight the importance of Y2R function in GABAergic systems in the expression of depressive-like behavior.

  18. The Effects of Dopamine and Estrogen upon Cortical Parvalbumin Expression


    positive interneurons because studies indicate that the parvalbumin containing subclass of GABAergic neurons are contacted by mesocortical dopamine fibers...that both dopamine and estrogen enhance the maturation of cortical interneurons that express the calcium binding protein, parvalbumin , in the developing... parvalbumin expression in the deep cortical layers in the in vivo model. Dopamine D1 and D2 receptors are located on parvalbumin containing interneurons

  19. Parvalbumin disappears from GABAergic CA1 neurons of the gerbil hippocampus with seizure onset while its presence persists in the perforant path.

    Scotti, A L; Kalt, G; Bollag, O; Nitsch, C


    Mongolian gerbils are epilepsy prone animals, a trait observable at the behavioural level during the 2nd month of life. As a unique species difference, gerbils express the calcium-binding protein parvalbumin (PV) in the perforant path from the entorhinal cortex to the hippocampus. In this study, we determined the time of appearance of PV in the layer II neurons of the entorhinal cortex and the perforant path terminals in gerbils between post-natal days 30 and 50. Signs of low grade seizures were observed in few animals from P40 onward. PV stain in the entorhinal cortex and perforant path terminals was already detectable at P30, well before the onset of behavioural seizures and did not change with age. It is suggested that the presence of PV in this pathway may be related to the generation early in life of an epileptogenic focus in the limbic forebrain. Altered inhibitory hippocampal circuits have also been suggested as a cause of seizures in the gerbil. Therefore, we quantitated hippocampal GABA-immunoreactive neurons and the PV-immunoreactive subpopulation. A group of gerbils with a high density of stained pyramidal interneurons in CA1 and one lacking PV-stained perikarya could be distinguished at P40 and P50. The density of GABA-immunoreactive nerve cells however, remained the same in both groups and through the ages studied. Thus, perikaryal PV is lost from intact GABAergic nerve cells at the same time as behavioural seizures are observed. The loss of PV from GABAergic neurons may affect their functional properties and be instrumental for the maintainance of behavioural seizures.

  20. Simultaneous measurement of neuronal activity and cortical hemodynamics by unshielded magnetoencephalography and near-infrared spectroscopy

    Seki, Yusuke; Miyashita, Tsuyoshi; Kandori, Akihiko; Maki, Atsushi; Koizumi, Hideaki


    The correlation between neuronal activity and cortical hemodynamics, namely, neurovascular coupling (NVC), is important to shed light on the mechanism of a variety of brain functions or neuronal diseases. NVC can be studied by simultaneously measuring neuronal activity and cortical hemodynamics. Consequently, noninvasive measurements of the NVC have been widely studied using both electroencephalography (EEG) and functional magnetic resonance imaging (fMRI). However, electromagnetic interference between EEG and fMRI is still a major problem. On the other hand, near-infrared spectroscopy (NIRS) is another promising tool for detecting cortical hemodynamics because it can be combined with EEG or magnetoencephalography (MEG) without any electromagnetic interference. Accordingly, in the present study, a simultaneous measurement system-combining an unshielded MEG using a two-dimensional gradiometer based on a low-T superconducting quantum interference device (SQUID) and an NIRS using nonmagnetic thin probes-was developed. This combined system was used to simultaneously measure both an auditory-evoked magnetic field and blood flow change in the auditory cortex. It was experimentally demonstrated that the combined unshielded MEG/NIRS system can simultaneously measure neuronal activity and cortical hemodynamics.

  1. Network bursts in cortical neuronal cultures: 'noise - versus pacemaker'- driven neural network simulations

    Gritsun, T.; Stegenga, J.; le Feber, Jakob; Rutten, Wim


    In this paper we address the issue of spontaneous bursting activity in cortical neuronal cultures and explain what might cause this collective behavior using computer simulations of two different neural network models. While the common approach to acivate a passive network is done by introducing

  2. Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons.

    Hevner, R.F.; Daza, R.A.; Rubenstein, J.L.; Stunnenberg, H.G.; Olavarria, J.F.; Englund, C.


    Cortical projection neurons exhibit diverse morphological, physiological, and molecular phenotypes, but it is unknown how many distinct types exist. Many projection cell phenotypes are associated with laminar fate (radial position), but each layer may also contain multiple types of projection cells.

  3. Regulation of Cerebral Cortical Size and Neuron Number by Fibroblast Growth Factors: Implications for Autism

    Vaccarino, Flora M.; Grigorenko, Elena L.; Smith, Karen Muller; Stevens, Hanna E.


    Increased brain size is common in children with autism spectrum disorders. Here we propose that an increased number of cortical excitatory neurons may underlie the increased brain volume, minicolumn pathology and excessive network excitability, leading to sensory hyper-reactivity and seizures, which are often found in autism. We suggest that…

  4. Long-term exposure of mice to nucleoside analogues disrupts mitochondrial DNA maintenance in cortical neurons.

    Yulin Zhang

    Full Text Available Nucleoside analogue reverse transcriptase inhibitor (NRTI, an integral component of highly active antiretroviral therapy (HAART, was widely used to inhibit HIV replication. Long-term exposure to NRTIs can result in mitochondrial toxicity which manifests as lipoatrophy, lactic acidosis, cardiomyopathy and myopathy, as well as polyneuropathy. But the cerebral neurotoxicity of NRTIs is still not well known partly due to the restriction of blood-brain barrier (BBB and the complex microenvironment of the central nervous system (CNS. In this study, the Balb/c mice were administered 50 mg/kg stavudine (D4T, 100 mg/kg zidovudine (AZT, 50 mg/kg lamivudine (3TC or 50 mg/kg didanosine (DDI per day by intraperitoneal injection, five days per week for one or four months, and primary cortical neurons were cultured and exposed to 25 µM D4T, 50 µM AZT, 25 µM 3TC or 25 µM DDI for seven days. Then, single neuron was captured from mouse cerebral cortical tissues by laser capture microdissection. Mitochondrial DNA (mtDNA levels of the primary cultured cortical neurons, and captured neurons or glial cells, and the tissues of brains and livers and muscles were analyzed by relative quantitative real-time PCR. The data showed that mtDNA did not lose in both NRTIs exposed cultured neurons and one month NRTIs treated mouse brains. In four months NRTIs treated mice, brain mtDNA levels remained unchanged even if the mtDNA levels of liver (except for 3TC and muscle significantly decreased. However, mtDNA deletion was significantly higher in the captured neurons from mtDNA unchanged brains. These results suggest that long-term exposure to NRTIs can result in mtDNA deletion in mouse cortical neurons.

  5. Coconut oil attenuates the effects of amyloid-β on cortical neurons in vitro.

    Nafar, Firoozeh; Mearow, Karen M


    Dietary supplementation has been studied as an approach to ameliorating deficits associated with aging and neurodegeneration. We undertook this pilot study to investigate the effects of coconut oil supplementation directly on cortical neurons treated with amyloid-β (Aβ) peptide in vitro. Our results indicate that neuron survival in cultures co-treated with coconut oil and Aβ is rescued compared to cultures exposed only to Aβ. Coconut oil co-treatment also attenuates Aβ-induced mitochondrial alterations. The results of this pilot study provide a basis for further investigation of the effects of coconut oil, or its constituents, on neuronal survival focusing on mechanisms that may be involved.

  6. 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

    Hanotel, Julie; Bessodes, Nathalie; Thélie, Aurore


    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...

  7. Effects of the analgesic acetaminophen (Paracetamol) and its para-aminophenol metabolite on viability of mouse-cultured cortical neurons.

    Schultz, Stephen; DeSilva, Mauris; Gu, Ting Ting; Qiang, Mei; Whang, Kyumin


    Acetaminophen has been used as an analgesic for more than a hundred years, but its mechanism of action has remained elusive. Recently, it has been shown that acetaminophen produces analgesia by the activation of the brain endocannabinoid receptor CB1 through its para-aminophenol (p-aminophenol) metabolite. The objective of this study was to determine whether p-aminophenol could be toxic for in vitro developing mouse cortical neurons as a first step in establishing a link between acetaminophen use and neuronal apoptosis. We exposed developing mouse cortical neurons to various concentrations of drugs for 24 hr in vitro. Acetaminophen itself was not toxic to developing mouse cortical neurons at therapeutic concentrations of 10-250 μg/ml. However, concentrations of p-aminophenol from 1 to 100 μg/ml produced significant (p < 0.05) loss of mouse cortical neuron viability at 24 hr compared to the controls. The naturally occurring endocannabinoid anandamide also caused similar 24-hr loss of cell viability in developing mouse cortical neurons at concentrations from 1 to 100 μg/ml, which indicates the mechanism of cell death could be through the cannabinoid receptors. The results of our experiments have shown a detrimental effect of the acetaminophen metabolite p-aminophenol on in vitro developing cortical neuron viability which could act through CB1 receptors of the endocannabinoid system. These results could be especially important in recommending an analgesic for children or individuals with traumatic brain injury who have developing cortical neurons.

  8. Necdin regulates p53 acetylation via Sirtuin1 to modulate DNA damage response in cortical neurons.

    Hasegawa, Koichi; Yoshikawa, Kazuaki


    Sirtuin1 (Sirt1), a mammalian homolog of yeast Sir2, deacetylates the tumor suppressor protein p53 and attenuates p53-mediated cell death. Necdin, a p53-interacting protein expressed predominantly in postmitotic neurons, is a melanoma antigen family protein that promotes neuronal differentiation and survival. In mammals, the necdin gene (Ndn) is maternally imprinted, and mutant mice carrying mutated paternal Ndn show abnormalities of neuronal development. Here we report that necdin regulates the acetylation status of p53 via Sirt1 to suppress p53-dependent apoptosis in postmitotic neurons. Double-immunostaining analysis demonstrated that necdin colocalizes with Sirt1 in postmitotic neurons of mouse embryonic forebrain in vivo. Coimmunoprecipitation and in vitro binding analyses revealed that necdin interacts with both p53 and Sirt1 to potentiate Sirt1-mediated p53 deacetylation by facilitating their association. Primary cortical neurons prepared from paternal Ndn-deficient mice have high p53 acetylation levels and are sensitive to the DNA-damaging compounds camptothecin and hydrogen peroxide. Moreover, DNA transfection per se increases p53 acetylation and apoptosis in paternal Ndn-deficient neurons, whereas small interfering RNA-mediated p53 knockdown completely blocks these changes. However, Sirt1 knockdown increases both acetylated p53 level and apoptosis in wild-type neurons but fails to affect them in paternal Ndn-deficient neurons. In organotypic forebrain slice cultures treated with hydrogen peroxide, p53 is accumulated and colocalized with necdin and Sirt1 in cortical neurons. These results suggest that necdin downregulates p53 acetylation levels by forming a stable complex with p53 and Sirt1 to protect neurons from DNA damage-induced apoptosis.

  9. Salidroside protects cortical neurons against glutamate-induced cytotoxicity by inhibiting autophagy.

    Yin, Wei-Yong; Ye, Qiang; Huang, Huan-Jie; Xia, Nian-Ge; Chen, Yan-Yan; Zhang, Yi; Qu, Qiu-Min


    Recent evidence suggests that glutamate-induced cytotoxicity contributes to autophagic neuron death and is partially mediated by increased oxidative stress. Salidroside has been demonstrated to have neuroprotective effects in glutamate-induced neuronal damage. The precise mechanism of its regulatory role in neuronal autophagy is, however, poorly understood. This study aimed to probe the effects and mechanisms of salidroside in glutamate-induced autophagy activation in cultured rat cortical neurons. Cell viability assay, Western blotting, coimmunoprecipitation, and small interfering RNA were performed to analyze autophagy activities during glutamate-evoked oxidative injury. We found that salidroside protected neonatal neurons from glutamate-induced apoptotic cell death. Salidroside significantly attenuated the LC3-II/LC3-I ratio and expression of Beclin-1, but increased (SQSTM1)/p62 expression under glutamate exposure. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, decreased LC3-II/LC3-I ratio, attenuated glutamate-induced cell injury, and mimicked some of the protective effects of salidroside against glutamate-induced cell injury. Molecular analysis demonstrated that salidroside inhibited cortical neuron autophagy in response to glutamate exposure through p53 signaling by increasing the accumulation of cytoplasmic p53. Salidroside inhibited the glutamate-induced dissociation of the Bcl-2-Beclin-1 complex with minor affects on the PI3K/Akt/mTOR signaling pathways. These data demonstrate that the inhibition of autophagy could be responsible for the neuroprotective effects of salidroside on glutamate-induced neuronal injury.

  10. Towards a theory of cortical columns: From spiking neurons to interacting neural populations of finite size.

    Tilo Schwalger


    Full Text Available Neural population equations such as neural mass or field models are widely used to study brain activity on a large scale. However, the relation of these models to the properties of single neurons is unclear. Here we derive an equation for several interacting populations at the mesoscopic scale starting from a microscopic model of randomly connected generalized integrate-and-fire neuron models. Each population consists of 50-2000 neurons of the same type but different populations account for different neuron types. The stochastic population equations that we find reveal how spike-history effects in single-neuron dynamics such as refractoriness and adaptation interact with finite-size fluctuations on the population level. Efficient integration of the stochastic mesoscopic equations reproduces the statistical behavior of the population activities obtained from microscopic simulations of a full spiking neural network model. The theory describes nonlinear emergent dynamics such as finite-size-induced stochastic transitions in multistable networks and synchronization in balanced networks of excitatory and inhibitory neurons. The mesoscopic equations are employed to rapidly integrate a model of a cortical microcircuit consisting of eight neuron types, which allows us to predict spontaneous population activities as well as evoked responses to thalamic input. Our theory establishes a general framework for modeling finite-size neural population dynamics based on single cell and synapse parameters and offers an efficient approach to analyzing cortical circuits and computations.

  11. Thalamus-derived molecules promote survival and dendritic growth of developing cortical neurons.

    Sato, Haruka; Fukutani, Yuma; Yamamoto, Yuji; Tatara, Eiichi; Takemoto, Makoto; Shimamura, Kenji; Yamamoto, Nobuhiko


    The mammalian neocortex is composed of various types of neurons that reflect its laminar and area structures. It has been suggested that not only intrinsic but also afferent-derived extrinsic factors are involved in neuronal differentiation during development. However, the role and molecular mechanism of such extrinsic factors are almost unknown. Here, we attempted to identify molecules that are expressed in the thalamus and affect cortical cell development. First, thalamus-specific molecules were sought by comparing gene expression profiles of the developing rat thalamus and cortex using microarrays, and by constructing a thalamus-enriched subtraction cDNA library. A systematic screening by in situ hybridization showed that several genes encoding extracellular molecules were strongly expressed in sensory thalamic nuclei. Exogenous and endogenous protein localization further demonstrated that two extracellular molecules, Neuritin-1 (NRN1) and VGF, were transported to thalamic axon terminals. Application of NRN1 and VGF to dissociated cell culture promoted the dendritic growth. An organotypic slice culture experiment further showed that the number of primary dendrites in multipolar stellate neurons increased in response to NRN1 and VGF, whereas dendritic growth of pyramidal neurons was not promoted. These molecules also increased neuronal survival of multipolar neurons. Taken together, these results suggest that the thalamus-specific molecules NRN1 and VGF play an important role in the dendritic growth and survival of cortical neurons in a cell type-specific manner.

  12. MicroRNA targeting of CoREST controls polarization of migrating cortical neurons.

    Volvert, Marie-Laure; Prévot, Pierre-Paul; Close, Pierre; Laguesse, Sophie; Pirotte, Sophie; Hemphill, James; Rogister, Florence; Kruzy, Nathalie; Sacheli, Rosalie; Moonen, Gustave; Deiters, Alexander; Merkenschlager, Matthias; Chariot, Alain; Malgrange, Brigitte; Godin, Juliette D; Nguyen, Laurent


    The migration of cortical projection neurons is a multistep process characterized by dynamic cell shape remodeling. The molecular basis of these changes remains elusive, and the present work describes how microRNAs (miRNAs) control neuronal polarization during radial migration. We show that miR-22 and miR-124 are expressed in the cortical wall where they target components of the CoREST/REST transcriptional repressor complex, thereby regulating doublecortin transcription in migrating neurons. This molecular pathway underlies radial migration by promoting dynamic multipolar-bipolar cell conversion at early phases of migration, and later stabilization of cell polarity to support locomotion on radial glia fibers. Thus, our work emphasizes key roles of some miRNAs that control radial migration during cerebral corticogenesis.

  13. Huntingtin-Mediated Multipolar-Bipolar Transition of Newborn Cortical Neurons Is Critical for Their Postnatal Neuronal Morphology.

    Barnat, Monia; Le Friec, Julien; Benstaali, Caroline; Humbert, Sandrine


    In the developing cortex, projection neurons undergo multipolar-bipolar transition, radial-directed migration, and maturation. The contribution of these developmental steps to the structure of the adult cortex is not completely understood. Here, we report that huntingtin (HTT), the protein mutated in Huntington's disease, is enriched in polarizing projection neurons. The depletion of HTT in postmitotic projection neurons leads to the mislocalization of layer-specific neuronal populations in the mouse neocortex. HTT is required for the multipolar-bipolar transition of projection neurons and for the maintenance of their bipolar shape during their radial migration. HTT mediates these effects in vivo through the regulation of RAB11-dependent N-Cadherin trafficking. Importantly, HD pathological HTT alters RAB11-dependent neuronal migration. Finally, we show that the cortical defects resulting from the postmitotic loss of HTT specifically during embryonic development affect neuronal morphology at adulthood. Our data reveal a new HTT-RAB11-N-Cadherin pathway regulating multipolar-bipolar transition with direct implications for mature brain. VIDEO ABSTRACT.

  14. Low concentrations of alcohol inhibit BDNF-dependent GABAergic plasticity via L-type Ca2+ channel inhibition in developing CA3 hippocampal pyramidal neurons.

    Zucca, Stefano; Valenzuela, C Fernando


    Fetal alcohol spectrum disorder (FASD) is associated with learning and memory alterations that could be, in part, a consequence of hippocampal damage. The CA3 hippocampal subfield is one of the regions affected by ethanol (EtOH), including exposure during the third trimester-equivalent (i.e., neonatal period in rats). However, the mechanism of action of EtOH is poorly understood. In CA3 pyramidal neurons from neonatal rats, dendritic BDNF release causes long-term potentiation of the frequency of GABAA receptor-mediated spontaneous postsynaptic currents (LTP-GABAA) and this mechanism is thought to play a role in GABAergic synapse maturation. Here, we show that short- and long-term exposure of neonatal male rats to low EtOH concentrations abolishes LTP-GABAA by inhibiting L-type voltage-gated Ca2+ channels. These findings support the recommendation that even light drinking should be avoided during pregnancy.

  15. GABAergic cell types in the lizard hippocampus.

    Guirado, S; Dávila, J C


    The neurochemical classification of GABAergic cells in the lizard hippocampus resulted in a further division into four major, non-overlapping subtypes. Each GABAergic cell subtype displays specific targets on the principal hippocampal neurons. The synaptic targets of the GABA/neuropeptide subtype are the distal apical dendrites of principal neurons. Calretinin- and parvalbumin-containing GABAergic cells synapse on the cell body and proximal dendrites of principal cells. Calbindin is expressed in a distinct group of interneurons, the synapses of which are directed to the dendrites of principal neurons. Finally, another subtype displays NADPH-diaphorase activity, but its synaptic target has not been established.

  16. Cortical hypoexcitation defines neuronal responses in the immediate aftermath of traumatic brain injury.

    Victoria Philippa Anne Johnstone

    Full Text Available Traumatic brain injury (TBI from a blow to the head is often associated with complex patterns of brain abnormalities that accompany deficits in cognitive and motor function. Previously we reported that a long-term consequence of TBI, induced with a closed-head injury method modelling human car and sporting accidents, is neuronal hyper-excitation in the rat sensory barrel cortex that receives tactile input from the face whiskers. Hyper-excitation occurred only in supra-granular layers and was stronger to complex than simple stimuli. We now examine changes in the immediate aftermath of TBI induced with same injury method. At 24 hours post-trauma significant sensorimotor deficits were observed and characterisation of the cortical population neuronal responses at that time revealed a depth-dependent suppression of neuronal responses, with reduced responses from supragranular layers through to input layer IV, but not in infragranular layers. In addition, increased spontaneous firing rate was recorded in cortical layers IV and V. We postulate that this early post-injury suppression of cortical processing of sensory input accounts for immediate post-trauma sensory morbidity and sets into train events that resolve into long-term cortical hyper-excitability in upper sensory cortex layers that may account for long-term sensory hyper-sensitivity in humans with TBI.

  17. The release of glutamate from cortical neurons regulated by BDNF via the TrkB/Src/PLC-γ1 pathway.

    Zhang, Zitao; Fan, Jin; Ren, Yongxin; Zhou, Wei; Yin, Guoyong


    The brain-derived neurotrophic factor (BDNF) participates in the regulation of cortical neurons by influencing the release of glutamate. However, the specific mechanisms are unclear. Hence, we isolated and cultured the cortical neurons of Sprague Dawley rats. Specific inhibitors of TrkB, Src, PLC-γ1, Akt, and MEK1/2 (i.e., K252a, PP2, U73122, LY294002, and PD98059, respectively) were used to treat cortical neurons and to detect the glutamate release from cortical neurons stimulated with BDNF. BDNF significantly increased glutamate release, and simultaneously enhanced phosphorylation levels of TrkB, Src, PLC-γ, Akt, and Erk1/2. For BDNF-stimulated cortical neurons, K252a inhibited glutamate release and inhibited the phosphorylation levels of TrkB, Src, PLC-γ, Erk1/2, and Akt (P PLC-γ1 (P 0.05). U73122 inhibited the glutamate release from BDNF-stimulated cortical neurons, but had no influence on the phosphorylation levels of TrkB, Src, Erk1/2, or Akt (P > 0.05). LY294002 and PD98059 did not affect the BDNF-stimulated glutamate release and did not inhibit the phosphorylation levels of TrkB, Src, or PLC-γ1. In summary, BDNF stimulated the glutamate release from cortical neurons via the TrkB/Src/PLC-γ1 signaling pathway.

  18. Brain-derived neurotrophic factor stimulates energy metabolism in developing cortical neurons.

    Burkhalter, Julia; Fiumelli, Hubert; Allaman, Igor; Chatton, Jean-Yves; Martin, Jean-Luc


    Brain-derived neurotrophic factor (BDNF) promotes the biochemical and morphological differentiation of selective populations of neurons during development. In this study we examined the energy requirements associated with the effects of BDNF on neuronal differentiation. Because glucose is the preferred energy substrate in the brain, the effect of BDNF on glucose utilization was investigated in developing cortical neurons via biochemical and imaging studies. Results revealed that BDNF increases glucose utilization and the expression of the neuronal glucose transporter GLUT3. Stimulation of glucose utilization by BDNF was shown to result from the activation of Na+/K+-ATPase via an increase in Na+ influx that is mediated, at least in part, by the stimulation of Na+-dependent amino acid transport. The increased Na+-dependent amino acid uptake by BDNF is followed by an enhancement of overall protein synthesis associated with the differentiation of cortical neurons. Together, these data demonstrate the ability of BDNF to stimulate glucose utilization in response to an enhanced energy demand resulting from increases in amino acid uptake and protein synthesis associated with the promotion of neuronal differentiation by BDNF.

  19. Cortical regulation of striatal projection neurons and interneurons in a Parkinson's disease rat model

    Jia-jia Wu


    Full Text Available Striatal neurons can be either projection neurons or interneurons, with each type exhibiting distinct susceptibility to various types of brain damage. In this study, 6-hydroxydopamine was injected into the right medial forebrain bundle to induce dopamine depletion, and/or ibotenic acid was injected into the M1 cortex to induce motor cortex lesions. Immunohistochemistry and western blot assay showed that dopaminergic depletion results in significant loss of striatal projection neurons marked by dopamine- and cyclic adenosine monophosphate-regulated phosphoprotein, molecular weight 32 kDa, calbindin, and μ-opioid receptor, while cortical lesions reversed these pathological changes. After dopaminergic deletion, the number of neuropeptide Y-positive striatal interneurons markedly increased, which was also inhibited by cortical lesioning. No noticeable change in the number of parvalbumin-positive interneurons was found in 6-hydroxydopamine-treated rats. Striatal projection neurons and interneurons show different susceptibility to dopaminergic depletion. Further, cortical lesions inhibit striatal dysfunction and damage induced by 6-hydroxydopamine, which provides a new possibility for clinical treatment of Parkinson's disease.

  20. Dynamics of cortical neuronal ensembles transit from decision making to storage for later report.

    Ponce-Alvarez, Adrián; Nácher, Verónica; Luna, Rogelio; Riehle, Alexa; Romo, Ranulfo


    Decisions based on sensory evaluation during single trials may depend on the collective activity of neurons distributed across brain circuits. Previous studies have deepened our understanding of how the activity of individual neurons relates to the formation of a decision and its storage for later report. However, little is known about how decision-making and decision maintenance processes evolve in single trials. We addressed this problem by studying the activity of simultaneously recorded neurons from different somatosensory and frontal lobe cortices of monkeys performing a vibrotactile discrimination task. We used the hidden Markov model to describe the spatiotemporal pattern of activity in single trials as a sequence of firing rate states. We show that the animal's decision was reliably maintained in frontal lobe activity through a selective state sequence, initiated by an abrupt state transition, during which many neurons changed their activity in a concomitant way, and for which both latency and variability depended on task difficulty. Indeed, transitions were more delayed and more variable for difficult trials compared with easy trials. In contrast, state sequences in somatosensory cortices were weakly decision related, had less variable transitions, and were not affected by the difficulty of the task. In summary, our results suggest that the decision process and its subsequent maintenance are dynamically linked by a cascade of transient events in frontal lobe cortices.

  1. Various tolerances to arsenic trioxide between human cortical neurons and leukemic cells

    ZHOU Jin; MENG Ran; SUI Xinhua; LI Wenbin; YANG Baofeng


    Arsenic trioxide (As2O3) is very effective for treatment of acute promyelocytic leukaemia (APL) but little can pass through the blood-brain-barrier (BBB),which limits its use in the prevention and treatment of central nervous system leukaemia (CNSL). Before creating a non-invasive method to help As2O3 's access, the safe and effective therapeutic concentration of As2O3 in the CNS ought to be known. The changes of apoptosis biomarkers, [Ca2+]i and PKC activity of both leukaemia cells and human cortical neurons, were monitored before and after being treated with As2O3 in vitro with laser confocal microscopy and Western blot. NSE concentration, the neuron invasive biomarker, was monitored by enzyme immunoassay (NSE-EIA). This study revealed that cortical neuron was more tolerable to As2O3 compared to NB4. 1.0 μmol / L As2O3 showed little influence on cortical neuron but effectively promoted apoptosis and induced differentiation of NB4.

  2. Bioreactor Transient Exposure Activates Specific Neurotrophic Pathway in Cortical Neurons

    Zimmitti, V.; Benedetti, E.; Caracciolo, V.; Sebastiani, P.; Di Loreto, S.


    Altered gravity forces might influence neuroplasticity and can provoke changes in biochemical mechanisms. In this contest, neurotrophins have a pivotal role, particularly nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). A suspension of dissociated cortical cells from rat embryos was exposed to 24 h of microgravity before plating in normal adherent culture system. Expression and transductional signalling pathways of NGF and BDNF were assessed at the end of maturational process (8-10 days in vitro). Rotating wall vessel bioreactor (RWV) pre-exposition did not induce changes in NGF expression and its high affinity receptor TrkA. On the contrary both BDNF expression and its high affinity receptor TrkB were strongly up-regulated, inducing Erk-5, but not Erk-1/2 activation and, in turn, MEF2C over-expression and activation. According to our previous and present results, we postulate that relatively short microgravitational stimuli, applied to neural cells during the developmental stage, exert a long time activation of specific neurotrophic pathways.

  3. Minimum neuron density for synchronized bursts in a rat cortical culture on multi-electrode arrays.

    Ito, D; Tamate, H; Nagayama, M; Uchida, T; Kudoh, S N; Gohara, K


    To investigate the minimum neuron and neurite densities required for synchronized bursts, we cultured rat cortical neurons on planar multi-electrode arrays (MEAs) at five plating densities (2500, 1000, 500, 250, and 100 cells/mm(2)) using two culture media: Neuron Culture Medium and Dulbecco's Modified Eagle Medium supplemented with serum (DMEM/serum). Long-term recording of spontaneous electrical activity clarified that the cultures exhibiting synchronized bursts required an initial plating density of at least 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum. Immediately after electrical recording, immunocytochemistry of microtubule-associated protein 2 (MAP2) and Neurofilament 200 kD (NF200) was performed directly on MEAs to investigate the actual densities of neurons and neurites forming the networks. Immunofluorescence observation revealed that the construction of complicated neuronal networks required the same initial plating density as for synchronized bursts, and that overly sparse cultures showed significant decreases of neurons and neurites. We also found that the final densities of surviving neurons at 1 month decreased greatly compared with the initial plating densities and became saturated in denser cultures. In addition, the area of neurites and the number of nuclei were saturated in denser cultures. By comparing both the results of electrophysiological recording and immunocytochemical observation, we revealed that there is a minimum threshold of neuron densities that must be met for the exhibition of synchronized bursts. Interestingly, these minimum densities of MAP2-positive final neurons did not differ between the two culture media; the density was approximately 50 neurons/mm(2). This value was obtained in the cultures with the initial plating densities of 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum.

  4. FMRP regulates multipolar to bipolar transition affecting neuronal migration and cortical circuitry.

    La Fata, Giorgio; Gärtner, Annette; Domínguez-Iturza, Nuria; Dresselaers, Tom; Dawitz, Julia; Poorthuis, Rogier B; Averna, Michele; Himmelreich, Uwe; Meredith, Rhiannon M; Achsel, Tilmann; Dotti, Carlos G; Bagni, Claudia


    Deficiencies in fragile X mental retardation protein (FMRP) are the most common cause of inherited intellectual disability, fragile X syndrome (FXS), with symptoms manifesting during infancy and early childhood. Using a mouse model for FXS, we found that Fmrp regulates the positioning of neurons in the cortical plate during embryonic development, affecting their multipolar-to-bipolar transition (MBT). We identified N-cadherin, which is crucial for MBT, as an Fmrp-regulated target in embryonic brain. Furthermore, spontaneous network activity and high-resolution brain imaging revealed defects in the establishment of neuronal networks at very early developmental stages, further confirmed by an unbalanced excitatory and inhibitory network. Finally, reintroduction of Fmrp or N-cadherin in the embryo normalized early postnatal neuron activity. Our findings highlight the critical role of Fmrp in the developing cerebral cortex and might explain some of the clinical features observed in patients with FXS, such as alterations in synaptic communication and neuronal network connectivity.

  5. Poloxamer-188 and citicoline provide neuronal membrane integrity and protect membrane stability in cortical spreading depression.

    Yıldırım, Timur; Eylen, Alpaslan; Lule, Sevda; Erdener, Sefik Evren; Vural, Atay; Karatas, Hulya; Ozveren, Mehmet Faik; Dalkara, Turgay; Gursoy-Ozdemir, Yasemin


    Under pathological conditions such as brain trauma, subarachnoid hemorrhage and stroke, cortical spreading depression (CSD) or peri-infarct depolarizations contribute to brain damage in animal models of neurological disorders as well as in human neurological diseases. CSD causes transient megachannel opening on the neuronal membrane, which may compromise neuronal survival under pathological conditions. Poloxamer-188 (P-188) and citicoline are neuroprotectants with membrane sealing properties. The aim of this study is to investigate the effect of P-188 and citicoline on the neuronal megachannel opening induced by CSD in the mouse brain. We have monitored megachannel opening with propidium iodide, a membrane impermeable fluorescent dye and, demonstrate that P-188 and citicoline strikingly decreased CSD-induced neuronal PI influx in cortex and hippocampal dentate gyrus. Therefore, these agents may be providing neuroprotection by blocking megachannel opening, which may be related to their membrane sealing action and warrant further investigation for treatment of traumatic brain injury and ischemic stroke.

  6. Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.

    Li, Hailong; Aksenova, Marina; Bertrand, Sarah J; Mactutus, Charles F; Booze, Rosemarie


    Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

  7. Changes in long-range connectivity and neuronal reorganization in partial cortical deafferentation model of epileptogenesis.

    Kuśmierczak, M; Lajeunesse, F; Grand, L; Timofeev, I


    Severe brain injuries can trigger epileptogenesis, a latent period that eventually leads to epilepsy. Previous studies have demonstrated that changes in local connectivity between cortical neurons are a part of the epileptogenic processes. In the present study we aimed to investigate whether changes in long-range connectivity are also involved in epileptogenesis. We performed a large unilateral transection (undercut) of the white matter below the suprasylvian gyrus in cats. After about 2 months, we either injected retrograde tracer (cholera toxin, sub-unit B, CTB) or performed Golgi staining. We analyzed distribution of retrogradely labeled neurons, counted dendritic spines in the neocortex (Golgi staining), and analyzed dendritic orientation in control conditions and after the injury. We found a significant increase in the number of detected cells at the frontal parts of the injured hemisphere, which suggests that the process of axonal sprouting occurs in the deafferented area. The increase in the number of retrogradely stained neurons was accompanied with a significant decrease in neocortical spine density in the undercut area, a reduction in vertical and an increase in horizontal orientation of neuronal processes. The present study shows global morphological changes underlying epileptogenesis. An increased connectivity in the injured cortical regions accompanied with a decrease in spine density suggests that excitatory synapses might be formed on dendritic shafts, which probably contributes to the altered neuronal excitability that was described in previous studies on epileptogenesis.

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

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


    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.

  9. Calcium imaging of cortical neurons using Fura-2 AM.

    Barreto-Chang, Odmara L; Dolmetsch, Ricardo E


    Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

  10. The adaptation of spike backpropagation delays in cortical neurons

    Yossi eBuskila


    Full Text Available We measured the action potential backpropagation delays in apical dendrites of layer 5 pyramidal neurons of the somatosensory cortex under different stimulation regimes that exclude synaptic involvement. These delays showed robust features and did not correlate to either transient change in the stimulus strength or low frequency stimulation of suprathreshold membrane oscillations. However, our results indicate that backpropagation delays correlate with high frequency (>10 Hz stimulation of membrane oscillations, and that persistent suprathreshold sinusoidal stimulation injected directly into the soma results in an increase of the backpropagation delay, suggesting an intrinsic adaptation of the bAP, which does not involve any synaptic modifications. Moreover, the calcium chelator BAPTA eliminated the alterations in the backpropagation delays, strengthening the hypothesis that increased calcium concentration in the dendrites modulates dendritic excitability and can impact the backpropagation velocity. These results emphasize the impact of dendritic excitability on bAP velocity along the dendritic tree, which affects the precision of the bAP arrival at the synapse during specific stimulus regimes, and is capable of shifting the extent and polarity of synaptic strength during suprathreshold synaptic processes such as STDP.

  11. Crambescidin 816 induces calcium influx though glutamate receptors in primary cultures of cortical neurons

    Víctor Martín Vázquez


    In summary, our data suggest that the cytotoxic effect of 10 μM Cramb816 in cortical neurons may be related to an increase in the cytosolic calcium concentration elicited by the toxin, which is shown to be mediated by glutamate receptor activation. Further studies analyzing the effect of glutamate receptor blockers on the cytotoxic effect of Cramb816 are needed to confirm this hypothesis.

  12. GABA concentration and GABAergic neuron populations in limbic areas are differentially altered by brain serotonin deficiency in Tph2 knockout mice.

    Waider, Jonas; Proft, Florian; Langlhofer, Georg; Asan, Esther; Lesch, Klaus-Peter; Gutknecht, Lise


    While tryptophan hydroxylase-2 (Tph2) null mutant (Tph2(-/-)) mice are completely deficient in brain serotonin (5-HT) synthesis, the formation of serotonergic neurons and pathfinding of their projections are not impaired. However, 5-HT deficiency, during development and in the adult, might affect morphological and functional parameters of other neural systems. To assess the influence of 5-HT deficiency on γ-amino butyric acid (GABA) systems, we carried out measurements of GABA concentrations in limbic brain regions of adult male wildtype (wt), heterozygous (Tph2(+/-)) and Tph2(-/-) mice. In addition, unbiased stereological estimation of GABAergic interneuron numbers and density was performed in subregions of amygdala and hippocampus. Amygdala and prefrontal cortex displayed significantly increased and decreased GABA concentrations, respectively, exclusively in Tph2(+/-) mice while no changes were detected between Tph2(-/-) and wt mice. In contrast, in the hippocampus, increased GABA concentrations were found in Tph2(-/-) mice. While total cell density in the anterior basolateral amygdala did not differ between genotypes, the number and density of the GABAergic interneurons were significantly decreased in Tph2(-/-) mice, with the group of parvalbumin (PV)-immunoreactive (ir) interneurons contributing somewhat less to the decrease than that of non-PV-ir GABAergic interneurons. Major morphological changes were also absent in the dorsal hippocampus, and only a trend toward reduced density of PV-ir cells was observed in the CA3 region of Tph2(-/-) mice. Our findings are the first to document that life-long reduction or complete lack of brain 5-HT transmission causes differential changes of GABA systems in limbic regions which are key players in emotional learning and memory processes. The changes likely reflect a combination of developmental alterations and functional adaptations of emotion circuits to balance the lack of 5-HT, and may underlie altered emotional

  13. Antioxidant and Protective Mechanisms against Hypoxia and Hypoglycaemia in Cortical Neurons in Vitro

    José Joaquín Merino


    Full Text Available In the present work, we have studied whether cell death could be induced in cortical neurons from rats subjected to different period of O2 deprivation and low glucose (ODLG. This “in vitro” model is designed to emulate the penumbra area under ischemia. In these conditions, cortical neurons displayed loss of mitochondrial respiratory ability however, nor necrosis neither apoptosis occurred despite ROS production. The absence of cellular death could be a consequence of increased antioxidant responses such as superoxide dismutase-1 (SOD1 and GPX3. In addition, the levels of reduced glutathione were augmented and HIF-1/3α overexpressed. After long periods of ODLG (12–24 h cortical neurons showed cellular and mitochondrial membrane alterations and did not recuperate cellular viability during reperfusion. This could mean that therapies directed toward prevention of cellular and mitochondrial membrane imbalance or cell death through mechanisms other than necrosis or apoptosis, like authophagy, may be a way to prevent ODLG damage.

  14. Cyclooxygenase-2 contributes to VX-induced cell death in cultured cortical neurons.

    Tenn, Catherine C; Weiss, M Tracy; Beaup, Claire; Peinnequin, Andre; Wang, Yushan; Dorandeu, Frederic


    The link between cell death and increased cyclooxygenases-2 (COX-2) activity has not been clearly established. In this study, we examined whether COX-2 activation contributed to the mechanism of neurotoxicity produced by an organophosphorous nerve agent in cultured rat cortical neurons. Exposure of neuronal cells to the nerve agent, VX resulted in an increase in COX enzyme activity in the culture media. A concentration dependent increase in the activity levels of COX-2 enzyme was observed while there was little to no effect on COX-1. In addition, COX-2 mRNA and protein levels increased several hours post-VX exposure. Pre-treatment of the cortical cells with the COX-2 selective inhibitor, NS 398 resulted in a decrease in both the enzyme activity and prostaglandin (PGE(2) and PGF(2α)) release, as well as in a reduction in cell death. These findings indicate that the increase in COX-2 activity may contribute to the mechanism of VX-induced neurotoxicity in cultured rat cortical neuron.

  15. The presence of cortical neurons in striatal-cortical co-cultures alters the effects of dopamine and BDNF on Medium Spiny Neuron dendritic development

    Rachel D Penrod


    Full Text Available Medium spiny neurons (MSNs are the major striatal neuron and receive synaptic input from both glutamatergic and dopaminergic afferents. These synapses are made on MSN dendritic spines, which undergo density and morphology changes in association with numerous disease and experience-dependent states. Despite wide interest in the structure and function of mature MSNs, relatively little is known about MSN development. Furthermore, most in vitro studies of MSN development have been done in simple striatal cultures that lack any type of non-autologous synaptic input, leaving open the question of how MSN development is affected by a complex environment that includes other types of neurons, glia, and accompanying secreted and cell-associated cues. Here we characterize the development of MSNs in striatal-cortical co-culture, including quantitative morphological analysis of dendritic arborization and spine development, describing progressive changes in density and morphology of developing spines. Overall, MSN growth is much more robust in the striatal-cortical co-culture compared to striatal mono-culture. Inclusion of dopamine in the co-culture further enhances MSN dendritic arborization and spine density, but the effects of dopamine on dendritic branching are only significant at later times in development. In contrast, exogenous Brain Derived Neurotrophic Factor (BDNF has only a minimal effect on MSN development in the co-culture, but significantly enhances MSN dendritic arborization in striatal mono-culture. Importantly, inhibition of NMDA receptors in the co-culture significantly enhances the effect of exogenous BDNF, suggesting that the efficacy of BDNF depends on the cellular environment. Combined, these studies identify specific periods of MSN development that may be particularly sensitive to perturbation by external factors and demonstrate the importance of studying MSN development in a complex signaling environment.

  16. Local connections of excitatory neurons in motor-associated cortical areas of the rat

    Kaneko, Takeshi


    In spite of recent progress in brain sciences, the local circuit of the cerebral neocortex, including motor areas, still remains elusive. Morphological works on excitatory cortical circuitry from thalamocortical (TC) afferents to corticospinal neurons (CSNs) in motor-associated areas are reviewed here. First, TC axons of motor thalamic nuclei have been re-examined by the single-neuron labeling method. There are middle layer (ML)-targeting and layer (L) 1-preferring TC axon types in motor-associated areas, being analogous to core and matrix types, respectively, of Jones (1998) in sensory areas. However, the arborization of core-like motor TC axons spreads widely and disregards the columnar structure that is the basis of information processing in sensory areas, suggesting that motor areas adopt a different information-processing framework such as area-wide laminar organization. Second, L5 CSNs receive local excitatory inputs not only from L2/3 pyramidal neurons but also from ML spiny neurons, the latter directly processing cerebellar information of core-like TC neurons (TCNs). In contrast, basal ganglia information is targeted to apical dendrites of L2/3 and L5 pyramidal neurons through matrix TCNs. Third, L6 corticothalamic neurons (CTNs) are most densely innervated by ML spiny neurons located just above CTNs. Since CTNs receive only weak connections from L2/3 and L5 pyramidal neurons, the TC recurrent circuit composed of TCNs, ML spiny neurons and CTNs appears relatively independent of the results of processing in L2/3 and L5. It is proposed that two circuits sharing the same TC projection and ML neurons are embedded in the neocortex: one includes L2/3 and L5 neurons, processes afferent information in a feedforward way and sends the processed information to other cortical areas and subcortical regions; and the other circuit participates in a dynamical system of the TC recurrent circuit and may serve as the basis of autonomous activity of the neocortex. PMID

  17. Axonal shearing in mature cortical neurons induces attempted regeneration and the reestablishment of neurite polarity.

    Blizzard, Catherine A; King, Anna E; Haas, Matilda A; O'Toole, David A; Vickers, James C; Dickson, Tracey C


    While functional recovery after injury is limited, it has become evident that the mature central nervous system does retain some ability to regenerate. This study investigated the intrinsic capacity of relatively mature cortical neurons (21 days in vitro) to respond to axonal loss. Neurons, growing as clusters on poly-L-lysine, were completely sheared of axons through chemical and mechanical disruption and transferred to either an intact astrocyte monolayer or a substrate of poly-L-lysine. Injured neurons exhibited a regenerative sprouting response that was independent of neuronal cell division or neural progenitors, as demonstrated by negative bromodeoxyuridine (BrdU) and the neuronal precursor intermediate filament nestin, labeling. At 24 h after injury, neurons had extended appropriately polarized neurites, demonstrated by compartmentalized microtubule-associated proteins MAP2 and tau immunolabeling. Newly sprouting axons were tipped by growth cones; however, growth cones on the tips of sprouting axons (mean area, 26.32 +/- 2.20 microm) were significantly (pregenerating neurons exhibited distinct axonal dynamics, with a significant (pneuronal structural plasticity and defining the role of astrocyte reactivity in the response to trauma.

  18. Chronic ciguatoxin treatment induces synaptic scaling through voltage gated sodium channels in cortical neurons.

    Martín, Víctor; Vale, Carmen; Rubiolo, Juan A; Roel, Maria; Hirama, Masahiro; Yamashita, Shuji; Vieytes, Mercedes R; Botana, Luís M


    Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin

  19. Kinesin KIF4A transports integrin β1 in developing axons of cortical neurons.

    Heintz, Tristan G; Heller, Janosch P; Zhao, Rongrong; Caceres, Alfredo; Eva, Richard; Fawcett, James W


    CNS axons have poor regenerative ability compared to PNS axons, and mature axons regenerate less well than immature embryonic axons. The loss of regenerative ability with maturity is accompanied by the setting up of a selective transport filter in axons, restricting the types of molecule that are present. We confirm that integrins (represented by subunits β1 and α5) are present in early cortical axons in vitro but are excluded from mature axons. Ribosomal protein and L1 show selective axonal transport through association with kinesin kif4A; we have therefore examined the hypothesis that integrin transport might also be in association with kif4A. Kif4A is present in all processes of immature cortical neurons cultured at E18, then downregulated by 14days in vitro, coinciding with the exclusion of integrin from axons. Kif4a co-localises with β1 integrin in vesicles in neurons and non-neuronal cells, and the two molecules co-immunoprecipitate. Knockdown of KIF4A expression with shRNA reduced the level of integrin β1 in axons of developing neurons and reduced neurite elongation on laminin, an integrin-dependent substrate. Overexpression of kif4A triggered apoptosis in neuronal and non-neuronal cells. In mature neurons expression of kif4A-GFP at a modest level did not kill the cells, and the kif4A was detectable in their axons. However this was not accompanied by an increase in integrin β1 axonal transport, suggesting that kif4A is not the only integrin transporter, and that integrin exclusion from axons is controlled by factors other than the kif4A level. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Regulation of Extrasynaptic GABAA α4 Receptors by Ethanol-Induced Protein Kinase A, but Not Protein Kinase C Activation in Cultured Rat Cerebral Cortical Neurons.

    Carlson, Stephen L; Bohnsack, J Peyton; Patel, Vraj; Morrow, A Leslie


    Ethanol produces changes in GABAA receptor trafficking and function that contribute to ethanol dependence symptomatology. Extrasynaptic γ-aminobutyric acid A receptors (GABAA-R) mediate inhibitory tonic current and are of particular interest because they are potentiated by physiologically relevant doses of ethanol. Here, we isolate GABAA α4δ receptors by western blotting in subsynaptic fractions to investigate protein kinase A (PKA) and protein kinase C (PKC) modulation of ethanol-induced receptor trafficking, while extrasynaptic receptor function is determined by measurement of tonic inhibition and responses evoked by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or PKA/PKC modulators. Ethanol exposure (1 hour) did not alter GABAA α4 receptor abundance, but it increased tonic current amplitude, an effect that was prevented by inhibiting PKA, but not PKC. Direct activation of PKA, but not PKC, increased the abundance and tonic current of extrasynaptic α4δ receptors. In contrast, prolonged ethanol exposure (4 hours) reduced α4δ receptor abundance as well as tonic current, and this effect was also PKA dependent. Finally, PKC activation by ethanol or phorbol-12,13-dibutyrate (PdBu) had no effect on extrasynaptic α4δ subunit abundance or activity. We conclude that ethanol alters extrasynaptic α4δ receptor function and expression in cortical neurons in a PKA-dependent manner, but ethanol activation of PKC does not influence these receptors. These results could have clinical relevance for therapeutic strategies to restore normal GABAergic functioning for the treatment of alcohol use disorders.

  1. Visualization of cortical projection neurons with retrograde TET-off lentiviral vector.

    Akiya Watakabe

    Full Text Available We are interested in identifying and characterizing various projection neurons that constitute the neocortical circuit. For this purpose, we developed a novel lentiviral vector that carries the tetracycline transactivator (tTA and the transgene under the TET Responsive Element promoter (TRE on a single backbone. By pseudotyping such a vector with modified rabies G-protein, we were able to express palmitoylated-GFP (palGFP or turboFP635 (RFP in corticothalamic, corticocortical, and corticopontine neurons of mice. The high-level expression of the transgene achieved by the TET-Off system enabled us to observe characteristic elaboration of neuronal processes for each cell type. At higher magnification, we were able to observe fine structures such as boutons and spines as well. We also injected our retrograde TET-Off vector to the marmoset cortex and proved that it can be used to label the long-distance cortical connectivity of millimeter scale. In conclusion, our novel retrograde tracer provides an attractive option to investigate the morphologies of identified cortical projection neurons of various species.

  2. Selective loss of parvalbumin-positive GABAergic interneurons in the cerebral cortex of maternally stressed Gad1-heterozygous mouse offspring.

    Uchida, T; Furukawa, T; Iwata, S; Yanagawa, Y; Fukuda, A


    Exposure to maternal stress (MS) and mutations in GAD1, which encodes the γ-aminobutyric acid (GABA) synthesizing enzyme glutamate decarboxylase (GAD) 67, are both risk factors for psychiatric disorders. However, the relationship between these risk factors remains unclear. Interestingly, the critical period of MS for psychiatric disorders in offspring corresponds to the period of GABAergic neuron neurogenesis and migration in the fetal brain, that is, in the late stage of gestation. Indeed, decrement of parvalbumin (PV)-positive GABAergic interneurons in the medial prefrontal cortex (mPFC) and hippocampus (HIP) has often been observed in schizophrenia patients. In the present study, we used GAD67-green fluorescent protein (GFP) knock-in mice (that is, mice in which the Gad1 gene is heterozygously deleted; GAD67(+/GFP)) that underwent prenatal stress from embryonic day 15.0 to 17.5 and monitored PV-positive GABAergic neurons to address the interaction between Gad1 disruption and stress. Administration of 5-bromo-2-deoxyuridine revealed that neurogenesis of GFP-positive GABAergic neurons, but not cortical plate cells, was significantly diminished in fetal brains during MS. Differential expression of glucocorticoid receptors by different progenitor cell types may underlie this differential outcome. Postnatally, the density of PV-positive, but not PV-negative, GABAergic neurons was significantly decreased in the mPFC, HIP and somatosensory cortex but not in the motor cortex of GAD67(+/GFP) mice. By contrast, these findings were not observed in wild-type (GAD67(+/+)) offspring. These results suggest that prenatal stress, in addition to heterozygous deletion of Gad1, could specifically disturb the proliferation of neurons destined to be PV-positive GABAergic interneurons.

  3. Analytical characterization of spontaneous firing in networks of developing rat cultured cortical neurons

    Tateno, Takashi; Kawana, Akio; Jimbo, Yasuhiko


    We have used a multiunit electrode array in extracellular recording to investigate changes in the firing patterns in networks of developing rat cortical neurons. The spontaneous activity of continual asynchronous firing or the alternation of asynchronous spikes and synchronous bursts changed over time so that activity in the later stages consisted exclusively of synchronized bursts. The spontaneous coordinated activity in bursts produced a variability in interburst interval (IBI) sequences that is referred to as ``form.'' The stochastic and nonlinear dynamical analysis of IBI sequences revealed that these sequences reflected a largely random process and that the form for relatively immature neurons was largely oscillatory while the form for the more mature neurons was Poisson-like. The observed IBI sequences thus showed changes in form associated with both the intrinsic properties of the developing cells and the neural response to correlated synaptic inputs due to interaction between the developing neural circuits.

  4. Effect of cholecystokinin-8 on in vitro cultured rat cortical neurons against apoptosis

    Ying Liu; Jiangbao Zhou


    BACKGROUND: Cholecystokinin (CCK-8) can regulate the synthesis of NO, release of amino acid substance and suppress Ca2+ inflow. It is unknown about neuroprotection of CCK-8 on neuronal apoptosis and its relationship with nerve growth factor (NGF).OBJECTryE: To investigate the protective effect of CCK-8 on in vitro cultured rat cortical neurons against apoptosis induced by glutamate, and explore its effect on expression of NGF in the neurons during apoptosis.DESIGN: Randomized controlled experiment on the basis of cells.SETTING: Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University.MATERIALS: Eighty SD rats of 1-day old; DMEM/F12 culture medium (Biochrom Company, Germany);Fetal bovine serum (TBD Company, Tianjin); CCK-8 (Sigma Company, USA). Glutamate (Bioengineering Company, Shanghai); TUNEL kit and NGF- in situ hybridization kit (Boster Bioengineering Company,Wuhan); anti-NGF polyclonal antibody (Santa-Cluz Company); NGF immunocytochemistry kit (Zhongshan Company, Beijing).METHODS: The experiments were carried out in Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University from December 2004 to September 2005. Primary cultured cortical neurons from SD rats of 1-day oldwere incubated for 7 days. The cultured cells were divided randomly into 3 groups:experimental group, model group and control group. Neurons in experimental groups were added CCK-8 of 1 ×10-6, 1 ×10-7, 1 ×10-8 μ mol/L respectively, and then added 50 μmol/L glutamate solution a hour later. Neurons in model groups were treated with 50 μ mol/L glutamate solution. In the control group, cells were treated with normal medium. Apoptosis of cultured cortical neurons were observed by fluorescent microscope, the expression of NGF protein and mRNA were determined respectively by immunocytochemistry and in situ hybridization, and apoptosis of cortical neurons was detected with terminal deoxynucleotidyl transferase-mediated nick

  5. Immediate Effects of Repetitive Magnetic Stimulation on Single Cortical Pyramidal Neurons

    Banerjee, Jineta; Sorrell, Mary E.; Celnik, Pablo A.; Pelled, Galit


    Repetitive Transcranial Magnetic Stimulation (rTMS) has been successfully used as a non-invasive therapeutic intervention for several neurological disorders in the clinic as well as an investigative tool for basic neuroscience. rTMS has been shown to induce long-term changes in neuronal circuits in vivo. Such long-term effects of rTMS have been investigated using behavioral, imaging, electrophysiological, and molecular approaches, but there is limited understanding of the immediate effects of TMS on neurons. We investigated the immediate effects of high frequency (20 Hz) rTMS on the activity of cortical neurons in an effort to understand the underlying cellular mechanisms activated by rTMS. We used whole-cell patch-clamp recordings in acute rat brain slices and calcium imaging of cultured primary neurons to examine changes in neuronal activity and intracellular calcium respectively. Our results indicate that each TMS pulse caused an immediate and transient activation of voltage gated sodium channels (9.6 ± 1.8 nA at -45 mV, p value rTMS stimulation induced action potentials in a subpopulation of neurons, and significantly increased the steady state current of the neurons at near threshold voltages (at -45 mV: before TMS: I = 130 ± 17 pA, during TMS: I = 215 ± 23 pA, p value = 0.001). rTMS stimulation also led to a delayed increase in intracellular calcium (153.88 ± 61.94% increase from baseline). These results show that rTMS has an immediate and cumulative effect on neuronal activity and intracellular calcium levels, and suggest that rTMS may enhance neuronal responses when combined with an additional motor, sensory or cognitive stimulus. Thus, these results could be translated to optimize rTMS protocols for clinical as well as basic science applications. PMID:28114421

  6. Concentration-Dependent Dual Role of Thrombin In Protection of Cultured Rat Cortical Neurons

    García, Paul S.; Ciavatta, Vincent T.; Fidler, Jonathan A.; Woodbury, Anna; Levy, Jerrold H.; Tyor, William R.


    Background Thrombin’s role in the nervous system is not well understood. Under conditions of blood-brain barrier compromise (e.g., neurosurgery or stroke), thrombin can result in neuroapoptosis and the formation of glial scars. Despite this, preconditioning with thrombin has been found to be neuroprotective in models of cerebral ischemia and intracerebral hemorrhage. Methods We investigated the effects of physiologically relevant concentrations of thrombin on cortical neurons using two culture-based assays. We examined thrombin’s effect on neurites by quantitative analysis of fluorescently labeled neurons. To characterize thrombin’s effects on neuron survival, we spectrophotometrically measured changes in enzymatic activity. Using receptor agonists and thrombin inhibitors, we separately examined the role of thrombin and its receptor in neuroprotection. Results We found that low concentrations of thrombin (1 nM) enhances neurite growth and branching, neuron viability, and protects against excitotoxic damage. In contrast, higher concentrations of thrombin (100 nM) are potentially detrimental to neuronal health as evidenced by inhibition of neurite growth. Lower concentrations of thrombin resulted in equivalent neuroprotection as the antifibrinolytic, aprotinin, and the direct thrombin inhibitor, argatroban. Interestingly, exogenous application of the species-specific thrombin inhibitor, antithrombin III, was detrimental to neuronal health; suggesting that some endogenous thrombin is necessary for optimal neuron health in our culture system. Activation of the thrombin receptor, protease-activated receptor - 1 (PAR-1), via micromolar concentrations of the thrombin receptor agonist peptide, TRAP, did not adversely affect neuronal viability. Conclusions An optimal concentration of thrombin exists to enhance neuronal health. Neurotoxic effects of thrombin do not involve activation of PAR receptors and thus separate pharmacologic manipulation of thrombin’s receptor

  7. Homocysteine Aggravates Cortical Neural Cell Injury through Neuronal Autophagy Overactivation following Rat Cerebral Ischemia-Reperfusion

    Yaqian Zhao


    Full Text Available Elevated homocysteine (Hcy levels have been reported to be involved in neurotoxicity after ischemic stroke. However, the underlying mechanisms remain incompletely understood to date. In the current study, we hypothesized that neuronal autophagy activation may be involved in the toxic effect of Hcy on cortical neurons following cerebral ischemia. Brain cell injury was determined by hematoxylin-eosin (HE staining and TdT-mediated dUTP Nick-End Labeling (TUNEL staining. The level and localization of autophagy were detected by transmission electron microscopy, western blot and immunofluorescence double labeling. The oxidative DNA damage was revealed by immunofluorescence of 8-Hydroxy-2′-deoxyguanosine (8-OHdG. Hcy treatment aggravated neuronal cell death, significantly increased the formation of autophagosomes and the expression of LC3B and Beclin-1 in the brain cortex after middle cerebral artery occlusion-reperfusion (MCAO. Immunofluorescence analysis of LC3B and Beclin-1 distribution indicated that their expression occurred mainly in neurons (NeuN-positive and hardly in astrocytes (GFAP-positive. 8-OHdG expression was also increased in the ischemic cortex of Hcy-treated animals. Conversely, LC3B and Beclin-1 overexpression and autophagosome accumulation caused by Hcy were partially blocked by the autophagy inhibitor 3-methyladenine (3-MA. Hcy administration enhanced neuronal autophagy, which contributes to cell death following cerebral ischemia. The oxidative damage-mediated autophagy may be a molecular mechanism underlying neuronal cell toxicity of elevated Hcy level.

  8. Ect2, an ortholog of Drosophila Pebble, regulates formation of growth cones in primary cortical neurons

    Tsuji, Takahiro; Higashida, Chiharu; Aoki, Yoshihiko; Islam, Mohammad Saharul; Dohmoto, Mitsuko; Higashida, Haruhiro


    In collaboration with Marshall Nirenberg, we performed in vivo RNA interference (RNAi) genome-wide screening in Drosophila embryos. Pebble has been shown to be involved in Drosophila neuronal development. We have also reported that depletion of Ect2, a mammalian ortholog of Pebble, induces differentiation in NG108-15 neuronal cells. However, the precise role of Ect2 in neuronal development has yet to be studied. Here, we confirmed in PC12 pheochromocytoma cells that inhibition of Ect2 expression by RNAi stimulated neurite outgrowth, and in the mouse embryonic cortex that Ect2 was accumulated throughout the ventricular and subventricular zones with neuronal progenitor cells. Next, the effects of Ect2 depletion were studied in primary cultures of mouse embryonic cortical neurons: Loss of Ect2 did not affect the differentiation stages of neuritogenesis, the number of neurites, or axon length, while the numbers of growth cones and growth cone-like structures were increased. Taken together, our results suggest that Ect2 contributes to neuronal morphological differentiation through regulation of growth cone dynamics. PMID:22366651

  9. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

    Luigi Bozzo

    Full Text Available Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM. To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM or switched to different glucose concentrations (0.5 or 10 mM. None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  10. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves


    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  11. Clinacanthus nutans Protects Cortical Neurons Against Hypoxia-Induced Toxicity by Downregulating HDAC1/6.

    Tsai, Hsin-Da; Wu, Jui-Sheng; Kao, Mei-Han; Chen, Jin-Jer; Sun, Grace Y; Ong, Wei-Yi; Lin, Teng-Nan


    Many population-based epidemiological studies have unveiled an inverse correlation between intake of herbal plants and incidence of stroke. C. nutans is a traditional herbal medicine widely used for snake bite, viral infection and cancer in Asian countries. However, its role in protecting stroke damage remains to be studied. Despite of growing evidence to support epigenetic regulation in the pathogenesis and recovery of stroke, a clear understanding of the underlying molecular mechanisms is still lacking. In the present study, primary cortical neurons were subjected to in vitro oxygen-glucose deprivation (OGD)-reoxygenation and hypoxic neuronal death was used to investigate the interaction between C. nutans and histone deacetylases (HDACs). Using pharmacological agents (HDAC inhibitor/activator), loss-of-function (HDAC siRNA) and gain-of-function (HDAC plasmid) approaches, we demonstrated an early induction of HDAC1/2/3/8 and HDAC6 in neurons after OGD insult. C. nutans extract selectively inhibited HDAC1 and HDAC6 expression and attenuated neuronal death. Results of reporter analysis further revealed that C. nutans suppressed HDAC1 and HDAC6 transcription. Besides ameliorating neuronal death, C. nutans also protected astrocytes and endothelial cells from hypoxic-induced cell death. In summary, results support ability for C. nutans to suppress post-hypoxic HDACs activation and mitigate against OGD-induced neuronal death. This study further opens a new avenue for the use of herbal medicines to regulate epigenetic control of brain injury.

  12. Modulating motility of intracellular vesicles in cortical neurons with nanomagnetic forces on-chip.

    Kunze, Anja; Murray, Coleman Tylor; Godzich, Chanya; Lin, Jonathan; Owsley, Keegan; Tay, Andy; Di Carlo, Dino


    Vesicle transport is a major underlying mechanism of cell communication. Inhibiting vesicle transport in brain cells results in blockage of neuronal signals, even in intact neuronal networks. Modulating intracellular vesicle transport can have a huge impact on the development of new neurotherapeutic concepts, but only if we can specifically interfere with intracellular transport patterns. Here, we propose to modulate motion of intracellular lipid vesicles in rat cortical neurons based on exogenously bioconjugated and cell internalized superparamagnetic iron oxide nanoparticles (SPIONs) within microengineered magnetic gradients on-chip. Upon application of 6-126 pN on intracellular vesicles in neuronal cells, we explored how the magnetic force stimulus impacts the motion pattern of vesicles at various intracellular locations without modulating the entire cell morphology. Altering vesicle dynamics was quantified using, mean square displacement, a caging diameter and the total traveled distance. We observed a de-acceleration of intercellular vesicle motility, while applying nanomagnetic forces to cultured neurons with SPIONs, which can be explained by a decrease in motility due to opposing magnetic force direction. Ultimately, using nanomagnetic forces inside neurons may permit us to stop the mis-sorting of intracellular organelles, proteins and cell signals, which have been associated with cellular dysfunction. Furthermore, nanomagnetic force applications will allow us to wirelessly guide axons and dendrites by exogenously using permanent magnetic field gradients.

  13. Ect2, an ortholog of Drosophila Pebble, regulates formation of growth cones in primary cortical neurons.

    Tsuji, Takahiro; Higashida, Chiharu; Aoki, Yoshihiko; Islam, Mohammad Saharul; Dohmoto, Mitsuko; Higashida, Haruhiro


    In collaboration with Marshall Nirenberg, we performed in vivo RNA interference (RNAi) genome-wide screening in Drosophila embryos. Pebble has been shown to be involved in Drosophila neuronal development. We have also reported that depletion of Ect2, a mammalian ortholog of Pebble, induces differentiation in NG108-15 neuronal cells. However, the precise role of Ect2 in neuronal development has yet to be studied. Here, we confirmed in PC12 pheochromocytoma cells that inhibition of Ect2 expression by RNAi stimulated neurite outgrowth, and in the mouse embryonic cortex that Ect2 was accumulated throughout the ventricular and subventricular zones with neuronal progenitor cells. Next, the effects of Ect2 depletion were studied in primary cultures of mouse embryonic cortical neurons: Loss of Ect2 did not affect the differentiation stages of neuritogenesis, the number of neurites, or axon length, while the numbers of growth cones and growth cone-like structures were increased. Taken together, our results suggest that Ect2 contributes to neuronal morphological differentiation through regulation of growth cone dynamics.

  14. Effects of inorganic lead on the differentiation and growth of cortical neurons in culture.

    Kern, M; Audesirk, T; Audesirk, G


    Lead exposure has devastating effects on the developing nervous system, producing morphological, cognitive, and behavioral deficits. To elucidate some of the mechanisms of lead neurotoxicity, we have examined its effects on the differentiation of several types of cultured neurons. Previously, we reported the effects of inorganic lead on several parameters of growth and differentiation of E18 rat hippocampal neurons and two types of neuroblastoma cells cultured in medium with 2% fetal calf serum (FCS) (Audesirk et al., 1991). In the present study, we report the effects of concentrations of lead ranging from 1nM to 1 mM on the differentiation of hippocampal neurons cultured in medium containing 10% FCS. In addition, we investigated lead effects on neurons isolated from the motor cortex region of the E18 rat embryo. Cortical neurons were exposed to lead in concentrations ranging from 0.1 nM to 1 mM in medium with either 10% FCS or 2% FCS for 48 hr. The effects of lead tended to be multimodal. Neurite initiation, which is highly sensitive to neurotoxic compounds, was inhibited by lead at both high and low concentrations, with no effects at intermediate levels. Medium with 10% FCS enhanced certain growth parameters and tended to reduce the effects of lead. There was an overall consistency in the effects of lead on motor cortex and hippocampal neurons.

  15. Dopamine D1-histamine H3 receptor heteromers provide a selective link to MAPK signaling in GABAergic neurons of the direct striatal pathway.

    Moreno, Estefanía; Hoffmann, Hanne; Gonzalez-Sepúlveda, Marta; Navarro, Gemma; Casadó, Vicent; Cortés, Antoni; Mallol, Josefa; Vignes, Michel; McCormick, Peter J; Canela, Enric I; Lluís, Carme; Moratalla, Rosario; Ferré, Sergi; Ortiz, Jordi; Franco, Rafael


    Previously, using artificial cell systems, we identified receptor heteromers between the dopamine D(1) or D(2) receptors and the histamine H(3) receptor. In addition, we demonstrated two biochemical characteristics of the dopamine D(1) receptor-histamine H(3) receptor heteromer. We have now extended this work to show the dopamine D(1) receptor-histamine H(3) receptor heteromer exists in the brain and serves to provide a novel link between the MAPK pathway and the GABAergic neurons in the direct striatal efferent pathway. Using the biochemical characteristics identified previously, we found that the ability of H(3) receptor activation to stimulate p44 and p42 extracellular signal-regulated MAPK (ERK 1/2) phosphorylation was only observed in striatal slices of mice expressing D(1) receptors but not in D(1) receptor-deficient mice. On the other hand, the ability of both D(1) and H(3) receptor antagonists to block MAPK activation induced by either D(1) or H(3) receptor agonists was also found in striatal slices. Taken together, these data indicate the occurrence of D(1)-H(3) receptor complexes in the striatum and, more importantly, that H(3) receptor agonist-induced ERK 1/2 phosphorylation in striatal slices is mediated by D(1)-H(3) receptor heteromers. Moreover, H(3) receptor-mediated phospho-ERK 1/2 labeling co-distributed with D(1) receptor-containing but not with D(2) receptor-containing striatal neurons. These results indicate that D(1)-H(3) receptor heteromers work as processors integrating dopamine- and histamine-related signals involved in controlling the function of striatal neurons of the direct striatal pathway.

  16. Modulation of specific sensory cortical areas by segregated basal forebrain cholinergic neurons demonstrated by neuronal tracing and optogenetic stimulation in mice

    Irene eChaves-Coira


    Full Text Available Neocortical cholinergic activity plays a fundamental role in sensory processing and cognitive functions. Previous results have suggested a refined anatomical and functional topographical organization of basal forebrain (BF projections that may control cortical sensory processing in a specific manner. We have used retrograde anatomical procedures to demonstrate the existence of specific neuronal groups in the BF involved in the control of specific sensory cortices. Fluoro-gold and Fast Blue fluorescent retrograde tracers were deposited into the primary somatosensory (S1 and primary auditory (A1 cortices in mice. Our results revealed that the BF is a heterogeneous area in which neurons projecting to different cortical areas are segregated into different neuronal groups. Most of the neurons located in the horizontal limb of the diagonal band of Broca (HDB projected to the S1 cortex, indicating that this area is specialized in the sensory processing of tactile stimuli. However, the nucleus basalis magnocellularis (B nucleus shows a similar number of cells projecting to the S1 as to the A1 cortices. In addition, we analyzed the cholinergic effects on the S1 and A1 cortical sensory responses by optogenetic stimulation of the BF neurons in urethane-anesthetized transgenic mice. We used transgenic mice expressing the light-activated cation channel, channelrhodopsin-2, tagged with a fluorescent protein (ChR2-YFP under the control of the choline-acetyl transferase promoter (ChAT. Cortical evoked potentials were induced by whisker deflections or by auditory clicks. According to the anatomical results, optogenetic HDB stimulation induced more extensive facilitation of tactile evoked potentials in S1 than auditory evoked potentials in A1, while optogenetic stimulation of the B nucleus facilitated either tactile or auditory evoked potentials equally. Consequently, our results suggest that cholinergic projections to the cortex are organized into segregated

  17. Modulation of Specific Sensory Cortical Areas by Segregated Basal Forebrain Cholinergic Neurons Demonstrated by Neuronal Tracing and Optogenetic Stimulation in Mice

    Chaves-Coira, Irene; Barros-Zulaica, Natali; Rodrigo-Angulo, Margarita; Núñez, Ángel


    Neocortical cholinergic activity plays a fundamental role in sensory processing and cognitive functions. Previous results have suggested a refined anatomical and functional topographical organization of basal forebrain (BF) projections that may control cortical sensory processing in a specific manner. We have used retrograde anatomical procedures to demonstrate the existence of specific neuronal groups in the BF involved in the control of specific sensory cortices. Fluoro-Gold (FlGo) and Fast Blue (FB) fluorescent retrograde tracers were deposited into the primary somatosensory (S1) and primary auditory (A1) cortices in mice. Our results revealed that the BF is a heterogeneous area in which neurons projecting to different cortical areas are segregated into different neuronal groups. Most of the neurons located in the horizontal limb of the diagonal band of Broca (HDB) projected to the S1 cortex, indicating that this area is specialized in the sensory processing of tactile stimuli. However, the nucleus basalis magnocellularis (B) nucleus shows a similar number of cells projecting to the S1 as to the A1 cortices. In addition, we analyzed the cholinergic effects on the S1 and A1 cortical sensory responses by optogenetic stimulation of the BF neurons in urethane-anesthetized transgenic mice. We used transgenic mice expressing the light-activated cation channel, channelrhodopsin-2, tagged with a fluorescent protein (ChR2-YFP) under the control of the choline-acetyl transferase promoter (ChAT). Cortical evoked potentials were induced by whisker deflections or by auditory clicks. According to the anatomical results, optogenetic HDB stimulation induced more extensive facilitation of tactile evoked potentials in S1 than auditory evoked potentials in A1, while optogenetic stimulation of the B nucleus facilitated either tactile or auditory evoked potentials equally. Consequently, our results suggest that cholinergic projections to the cortex are organized into segregated

  18. Acetylcholine modulates transient outward potassium channel in acutely isolated cerebral cortical neurons of rats

    Lanwei Cui; Tao Sun; Lihui Qu; Yurong Li; Haixia Wen


    BACKGROUND:The neuronal transient outward potassium channel has been shown to be highly associated with acetylcholine.However,the influence of acetylcholine on the transient outward potassium current in cerebral cortical neurons remains poorly understood.OBJECTIVE:To investigate acetylcholine modulation on transient outward potassium current in rat parietal cortical neurons using the whole-cell patch-clamp technique.DESIGN,TIME AND SETTING:A neuroelectrophysiology study was performed at the Department of Physiology,Harbin Medical University between January 2005 and January 2006.MATERIALS:Wistar rats were provided by the Animal Research Center,the Second Hospital of Harbin Medical University;PC-IIC patch-clamp amplifier and IBBClamp data collection analysis system were provided by Huazhong University for Science and Technology,Wuhan,China;PP-83 microelectrode puller was purchased from Narrishage,Japan.METHODS:The parietal somatosensory cortical neurons were acutely dissociated,and the modulation of acetylcholine (0.1,1,10,100 μmol/L) on transient outward potassium channel was recorded using the whole-cell patch-clamp technique.MAIN OUTCOME MEASURES:Influence of acetylcholine on transient outward potassium current,potassium channel activation,and inactivation.RESULTS:The inhibitory effect of acetylcholine on transient outward potassium current was dose- and voltage-dependent (P<0.01).Acetylcholine was found to significantly affect the activation process of transient outward potassium current,i.e.,the activation curve of transient outward potassium current was left-shifted,while the inactivation curve was shifted to hyperpolarization.Acetylcholine significantly prolonged the time constant of recovery from inactivation of transient outward potassium current (P<0.01).CONCLUSION:These results suggest that acetylcholine inhibits transient outward potassium current by regulating activation and inactivation processes of the transient outward potassium channel.

  19. Cell Signaling Mechanisms by which Geniposide Regulates Insulin- Degrading Enzyme Expression in Primary Cortical Neurons.

    Zhang, Yonglan; Xia, Zhining; Liu, Jianhui; Yin, Fei


    An increasing number of studies have demonstrated that insulin-degrading enzyme (IDE) plays an essential role in both the degradation and its activity of β-amyloid (Aβ). Therefore, the regulation of IDE expression and/or modification of IDE-dependent actions are two emerging strategies for the treatment of Alzheimer's disease (AD). We previously observed that geniposide, a novel agonist of glucagon-like peptide 1 receptor (GLP-1R), could attenuate Aβ-induced neurotoxicity by regulating the expression of IDE in primary cortical neurons. However, the signal transduction mechanisms underlying this effect were not elucidated. The present study, therefore examined and explored the cell signaling transduction and molecular mechanisms by which geniposide induces the expression of IDE in primary cortical neurons. The current study revealed that LY294002 (an inhibitor for phosphatidyl inositol 3-kinase, PI3K), PP1 (inhibitor for c-Src), GW9662 (antagonist for peroxisome proliferator-activated receptor γ, PPARγ), H89 (an inhibitor for protein kinase A, PKA) and AG1478 (an antagonist for epidermal growth factor receptor, EGFR) prohibited the up-regulation of IDE induced by geniposide in primary cortical neurons. Further, geniposide also enhanced the phosphorylation of PPARγ and accelerated the release of phosphorylated FoxO1 (forkhead box O1) from nuclear fraction to the cytosol. Moreover, geniposide directly activated the activity of IDE promoter in PC12 cells, which confirmed the presence of the GLP-1 receptor. Taken together, our findings reveal for the first time the cell signaling transduction pathway of geniposide regulating the expression of IDE in neurons.

  20. A new role for TIMP-1 in modulating neurite outgrowth and morphology of cortical neurons.

    Adlane Ould-yahoui

    Full Text Available BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1 displays pleiotropic activities, both dependent and independent of its inhibitory activity on matrix metalloproteinases (MMPs. In the central nervous system (CNS, TIMP-1 is strongly upregulated in reactive astrocytes and cortical neurons following excitotoxic/inflammatory stimuli, but no information exists on its effects on growth and morphology of cortical neurons. PRINCIPAL FINDINGS: We found that 24 h incubation with recombinant TIMP-1 induced a 35% reduction in neurite length and significantly increased growth cones size and the number of F-actin rich microprocesses. TIMP-1 mediated reduction in neurite length affected both dendrites and axons after 48 h treatment. The effects on neurite length and morphology were not elicited by a mutated form of TIMP-1 inactive against MMP-1, -2 and -3, and still inhibitory for MMP-9, but were mimicked by a broad spectrum MMP inhibitor. MMP-9 was poorly expressed in developing cortical neurons, unlike MMP-2 which was present in growth cones and whose selective inhibition caused neurite length reductions similar to those induced by TIMP-1. Moreover, TIMP-1 mediated changes in cytoskeleton reorganisation were not accompanied by modifications in the expression levels of actin, betaIII-tubulin, or microtubule assembly regulatory protein MAP2c. Transfection-mediated overexpression of TIMP-1 dramatically reduced neuritic arbour extension in the absence of detectable levels of released extracellular TIMP-1. CONCLUSIONS: Altogether, TIMP-1 emerges as a modulator of neuronal outgrowth and morphology in a paracrine and autrocrine manner through the inhibition, at least in part, of MMP-2 and not MMP-9. These findings may help us understand the role of the MMP/TIMP system in post-lesion pre-scarring conditions.

  1. 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.

    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


    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.

  2. Schisandrin B protects rat cortical neurons against Abeta1-42-induced neurotoxicity.

    Wang, Bin; Wang, Xue-Mei


    In the present study, we investigated the neuroprotective effects of schisandrin B on amyloid-beta1-42-induced toxicity and its potential mechanisms in rat cortical neuron cells. Amyloid beta1-42 significantly reduced cell viability and increased apoptosis. Pretreatment with schisandrin B prior to amyloid-beta1-42 exposure significantly elevated cell viability and reduced apoptosis. The anti-apoptotic effect of schisandrin B in rat cortical neurons was mediated by up-regulation of the anti-apoptotic protein Bcl-2 and down-regulation of the pro-apoptotic protein Bax. Schisandrin B also reduced the release of mitochondrial cytochrome c into cytosol and decreased caspase-9 and caspase-3 activities. Furthermore, schisandrin B increased activities of anti-oxidant reduced glutathione and decreased production of oxidative glutathione. Taken together, these results suggest that schisandrin B protected primary cultures of rat cortical cells against amyloid-beta1-42-induced neurotoxicity through anti-apoptosis involved in a mitochondria-mediated pathway and anti-oxidant action. Schisandrin B may represent a potential treatment strategy for Alzheimer's disease.

  3. CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

    Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing


    A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p ACM (p ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

  4. Cortical excitatory neurons become protected from cell division during neurogenesis in an Rb family-dependent manner.

    Oshikawa, Mio; Okada, Kei; Nakajima, Kazunori; Ajioka, Itsuki


    Cell cycle dysregulation leads to abnormal proliferation and cell death in a context-specific manner. Cell cycle progression driven via the Rb pathway forces neurons to undergo S-phase, resulting in cell death associated with the progression of neuronal degeneration. Nevertheless, some Rb- and Rb family (Rb, p107 and p130)-deficient differentiating neurons can proliferate and form tumors. Here, we found in mouse that differentiating cerebral cortical excitatory neurons underwent S-phase progression but not cell division after acute Rb family inactivation in differentiating neurons. However, the differentiating neurons underwent cell division and proliferated when Rb family members were inactivated in cortical progenitors. Differentiating neurons generated from Rb(-/-); p107(-/-); p130(-/-) (Rb-TKO) progenitors, but not acutely inactivated Rb-TKO differentiating neurons, activated the DNA double-strand break (DSB) repair pathway without increasing trimethylation at lysine 20 of histone H4 (H4K20), which has a role in protection against DNA damage. The activation of the DSB repair pathway was essential for the cell division of Rb-TKO differentiating neurons. These results suggest that newly born cortical neurons from progenitors become epigenetically protected from DNA damage and cell division in an Rb family-dependent manner.

  5. Near infrared radiation rescues mitochondrial dysfunction in cortical neurons after oxygen-glucose deprivation.

    Yu, Zhanyang; Liu, Ning; Zhao, Jianhua; Li, Yadan; McCarthy, Thomas J; Tedford, Clark E; Lo, Eng H; Wang, Xiaoying


    Near infrared radiation (NIR) is known to penetrate and affect biological systems in multiple ways. Recently, a series of experimental studies suggested that low intensity NIR may protect neuronal cells against a wide range of insults that mimic diseases such as stroke, brain trauma and neurodegeneration. However, the potential molecular mechanisms of neuroprotection with NIR remain poorly defined. In this study, we tested the hypothesis that low intensity NIR may attenuate hypoxia/ischemia-induced mitochondrial dysfunction in neurons. Primary cortical mouse neuronal cultures were subjected to 4 h oxygen-glucose deprivation followed by reoxygenation for 2 h, neurons were then treated with a 2 min exposure to 810-nm NIR. Mitochondrial function markers including MTT reduction and mitochondria membrane potential were measured at 2 h after treatment. Neurotoxicity was quantified 20 h later. Our results showed that 4 h oxygen-glucose deprivation plus 20 h reoxygenation caused 33.8 ± 3.4 % of neuron death, while NIR exposure significantly reduced neuronal death to 23.6 ± 2.9 %. MTT reduction rate was reduced to 75.9 ± 2.7 % by oxygen-glucose deprivation compared to normoxic controls, but NIR exposure significantly rescued MTT reduction to 87.6 ± 4.5 %. Furthermore, after oxygen-glucose deprivation, mitochondria membrane potential was reduced to 48.9 ± 4.39 % of normoxic control, while NIR exposure significantly ameliorated this reduction to 89.6 ± 13.9 % of normoxic control. Finally, NIR significantly rescued OGD-induced ATP production decline at 20 min after NIR. These findings suggest that low intensity NIR can protect neurons against oxygen-glucose deprivation by rescuing mitochondrial function and restoring neuronal energetics.

  6. Silencing gamma-aminobutyric acid A receptor alpha 1 subunit expression and outward potassium current in developing cortical neurons

    Tao Bo; Jiang Li; Jian Li; Xingfang Li; Kaihui Xing


    We used RNA interference (RNAi) to disrupt synthesis of the cortical neuronal γ-aminobutyric acid A receptor (GABAAR) α1 in rats during development, and measured outward K+ currents during neuronal electrical activity using whole-cell patch-clamp techniques. Three pairs of small interfering RNA (siRNA) for GABAAR α1 subunit were designed using OligoEngine RNAi software. This siRNA was found to effectively inhibited GABAAR α1 mRNA expression in cortical neuronal culture in vitro, but did not significantly affect neuronal survival. Outward K+ currents were decreased, indicating that GABAAR α1 subunits in developing neurons participate in neuronal function by regulating outward K+ current.

  7. Cortical regulation of dopamine depletion-induced dendritic spine loss in striatal medium spiny neurons.

    Neely, M D; Schmidt, D E; Deutch, A Y


    The proximate cause of Parkinson's disease is striatal dopamine depletion. Although no overt toxicity to striatal neurons has been reported in Parkinson's disease, one of the consequences of striatal dopamine loss is a decrease in the number of dendritic spines on striatal medium spiny neurons (MSNs). Dendrites of these neurons receive cortical glutamatergic inputs onto the dendritic spine head and dopaminergic inputs from the substantia nigra onto the spine neck. This synaptic arrangement suggests that dopamine gates corticostriatal glutamatergic drive onto spines. Using triple organotypic slice cultures composed of ventral mesencephalon, striatum, and cortex of the neonatal rat, we examined the role of the cortex in dopamine depletion-induced dendritic spine loss in MSNs. The striatal dopamine innervation was lesioned by treatment of the cultures with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) or by removing the mesencephalon. Both MPP+ and mesencephalic ablation decreased MSN dendritic spine density. Analysis of spine morphology revealed that thin spines were preferentially lost after dopamine depletion. Removal of the cortex completely prevented dopamine depletion-induced spine loss. These data indicate that the dendritic remodeling of MSNs seen in parkinsonism occurs secondary to increases in corticostriatal glutamatergic drive, and suggest that modulation of cortical activity may be a useful therapeutic strategy in Parkinson's disease.

  8. Interplay between kinesin-1 and cortical dynein during axonal outgrowth and microtubule organization in Drosophila neurons.

    del Castillo, Urko; Winding, Michael; Lu, Wen; Gelfand, Vladimir I


    In this study, we investigated how microtubule motors organize microtubules in Drosophila neurons. We showed that, during the initial stages of axon outgrowth, microtubules display mixed polarity and minus-end-out microtubules push the tip of the axon, consistent with kinesin-1 driving outgrowth by sliding antiparallel microtubules. At later stages, the microtubule orientation in the axon switches from mixed to uniform polarity with plus-end-out. Dynein knockdown prevents this rearrangement and results in microtubules of mixed orientation in axons and accumulation of microtubule minus-ends at axon tips. Microtubule reorganization requires recruitment of dynein to the actin cortex, as actin depolymerization phenocopies dynein depletion, and direct recruitment of dynein to the membrane bypasses the actin requirement. Our results show that cortical dynein slides 'minus-end-out' microtubules from the axon, generating uniform microtubule arrays. We speculate that differences in microtubule orientation between axons and dendrites could be dictated by differential activity of cortical dynein.

  9. Dynamic changes in proprotein convertase 2 activity in cortical neurons after ischemia/reperfusion and oxygen-glucose deprivation

    Shuqin Zhan; An Zhou; Chelsea Piper; Tao Yang


    In this study, a rat model of transient focal cerebral ischemia was established by performing 100 minutes of middle cerebral artery occlusion, and an in vitro model of experimental oxygen-glucose deprivation using cultured rat cortical neurons was established. Proprotein convertase 2 activity gradually decreased in the ischemic cortex with increasing duration of reperfusion. In cultured rat cortical neurons, the number of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling-positive neurons significantly increased and proprotein convertase 2 activity also decreased gradually with increasing duration of oxygen-glucose deprivation. These experimental findings indicate that proprotein convertase 2 activity decreases in ischemic rat cortex after reperfusion, as well as in cultured rat cortical neurons after oxygen-glucose deprivation. These changes in enzyme activity may play an important pathological role in brain injury.

  10. Intermediate Progenitor Cohorts Differentially Generate Cortical Layers and Require Tbr2 for Timely Acquisition of Neuronal Subtype Identity.

    Mihalas, Anca B; Elsen, Gina E; Bedogni, Francesco; Daza, Ray A M; Ramos-Laguna, Kevyn A; Arnold, Sebastian J; Hevner, Robert F


    Intermediate progenitors (IPs) amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

  11. Intermediate Progenitor Cohorts Differentially Generate Cortical Layers and Require Tbr2 for Timely Acquisition of Neuronal Subtype Identity

    Anca B. Mihalas


    Full Text Available Intermediate progenitors (IPs amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

  12. An in silico agent-based model demonstrates Reelin function in directing lamination of neurons during cortical development.

    James R Caffrey

    Full Text Available The characteristic six-layered appearance of the neocortex arises from the correct positioning of pyramidal neurons during development and alterations in this process can cause intellectual disabilities and developmental delay. Malformations in cortical development arise when neurons either fail to migrate properly from the germinal zones or fail to cease migration in the correct laminar position within the cortical plate. The Reelin signalling pathway is vital for correct neuronal positioning as loss of Reelin leads to a partially inverted cortex. The precise biological function of Reelin remains controversial and debate surrounds its role as a chemoattractant or stop signal for migrating neurons. To investigate this further we developed an in silico agent-based model of cortical layer formation. Using this model we tested four biologically plausible hypotheses for neuron motility and four biologically plausible hypotheses for the loss of neuron motility (conversion from migration. A matrix of 16 combinations of motility and conversion rules was applied against the known structure of mouse cortical layers in the wild-type cortex, the Reelin-null mutant, the Dab1-null mutant and a conditional Dab1 mutant. Using this approach, many combinations of motility and conversion mechanisms can be rejected. For example, the model does not support Reelin acting as a repelling or as a stopping signal. In contrast, the study lends very strong support to the notion that the glycoprotein Reelin acts as a chemoattractant for neurons. Furthermore, the most viable proposition for the conversion mechanism is one in which conversion is affected by a motile neuron sensing in the near vicinity neurons that have already converted. Therefore, this model helps elucidate the function of Reelin during neuronal migration and cortical development.

  13. Trans-anethole protects cortical neuronal cells against oxygen-glucose deprivation/reoxygenation.

    Ryu, Sangwoo; Seol, Geun Hee; Park, Hyeon; Choi, In-Young


    Trans-anethole has been studied on pharmacological properties such as anti-inflammation, anti-oxidative stress, antifungal and anticancer. However, to date, the anti-ischemic effects of trans-anethole have not been assessed. Therefore, we investigated the neuroprotection of trans-anethole against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cortical neuronal cell injury, an in vitro model of ischemia. The abilities of trans-anethole to block excitotoxicity, oxidative stress and mitochondrial dysfunction were evaluated in OGD/R-induced neurons. Trans-anethole significantly ameliorated OGD/R-induced neuronal cell injury by attenuating the intracellular calcium overload via the activation of NMDA receptors. Trans-anethole also inhibited OGD/R-induced reactive oxygen species overproduction, which may be derived from the scavenging activity in peroxyl radicals, assessed in an oxygen radical absorbance capacity assay. Furthermore, trans-anethole was shown to attenuate the depolarization of mitochondrial transmembrane. These results indicated that the neuroprotective effect of trans-anethole on OGD/R-induced neuronal injury might be due to its ability to inhibit excitotoxicity, oxidative stress and mitochondrial dysfunction. Considering these multiple pathways causing ischemic neuronal damage, the multi-functional effect of trans-anethole suggested that it may be effective in treating ischemic stroke.

  14. Opto-current-clamp actuation of cortical neurons using a strategically designed channelrhodopsin.

    Lei Wen

    Full Text Available BACKGROUND: Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1, has several advantages over channelrhodopsin-2 (ChR2 in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. METHODOLOGY/PRINCIPAL FINDINGS: The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp. The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5-10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5-10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. CONCLUSIONS/SIGNIFICANCE: The opto-current-clamp study suggests that the depolarization of a small number of neurons

  15. GABAergic ventrolateral pre‑optic nucleus neurons are involved in the mediation of the anesthetic hypnosis induced by propofol.

    Yuan, Jie; Luo, Zhuxin; Zhang, Yu; Zhang, Yi; Wang, Yuan; Cao, Song; Fu, Bao; Yang, Hao; Zhang, Lin; Zhou, Wenjing; Yu, Tian


    Intravenous anesthetics have been used clinically to induce unconsciousness for seventeen decades, however the mechanism of anesthetic‑induced unconsciousness remains to be fully elucidated. It has previously been demonstrated that anesthetics exert sedative effects by acting on endoge-nous sleep‑arousal circuits. However, few studies focus on the ventrolateral pre‑optic (VLPO) to locus coeruleus (LC) sleep‑arousal pathway. The present study aimed to investigate if VLPO is involved in unconsciousness induced by propofol. The present study additionally investigated if the inhibitory effect of propofol on LC neurons was mediated by activating VLPO neurons. Microinjection, target lesion and extracellular single‑unit recordings were used to study the role of the VLPO‑LC pathway in propofol anesthesia. The results demonstrated that GABAA agonist (THIP) or GABAA antagonist (gabazine) microinjections into VLPO altered the time of loss of righting reflex and the time of recovery of righting reflex. Furthermore, propofol suppressed the spontaneous firing activity of LC noradrenergic neurons. There was no significant difference observed in firing activity between VLPO sham lesion and VLPO lesion rats. The findings indicate that VLPO neurons are important in propofol‑induced unconsciousness, however are unlikely to contribute to the inhibitory effect of propofol on LC spontaneous firing activity.

  16. Identification and functional evidence of GABAergic neurons in parts of the brain of adult zebrafish (Danio rerio).

    Kim, Yong-Jung; Nam, Ryoung-Hee; Yoo, Young Mi; Lee, Chang-Joong


    The distribution of GABA-containing neurons was studied in the brain of the adult zebrafish by Nissl staining and immunohistochemistry. GABA immunoreactivity (GABA-IR) was demonstrated in parts of the brain such as olfactory bulb (OB), telencephalon, tectum stratum, and in the hypothalamus. GABA-IR appeared in the area where Nissl-stained cell bodies were abundant. The internal cellular layer of the OB was most densely stained by Nissl staining, and also showed a high level of GABA-IR. The telencephalon and the hypothalamus revealed a similar pattern to the OB in terms of Nissl staining and GABA-IR. However, the distribution and shape of stained cells of the tectum stratum were distinct from those in other regions: Nissl-stained neurons were ubiquitously present throughout all cellular layers including the stratum griseum centrale, the stratum album centrale (SAC), and the stratum periventriculare (SP). However, GABA-IR was weakly expressed in a limited number of neurons only in the SAC and SP. Whether GABA serves as an inhibitory neurotransmitter was also tested in the isolated telencephalon preparation by using extracellular field potential recordings. The synaptic activity recorded in the posterior dorsal telencephalon in response to the electrical stimulation of the anterior dorsal telencephalon was increased in the presence of the GABAA receptor antagonist, BMI, suggesting an inhibitory role for GABA-immunoreactive neurons in the adult brain of the zebrafish.

  17. Effect of Mimosa pudica (Linn.) extract on anxiety behaviour and GABAergic regulation of 5-HT neuronal activity in the mouse.

    Ayissi Mbomo, Rigobert; Gartside, Sasha; Ngo Bum, Elizabeth; Njikam, Njifutie; Okello, Ed; McQuade, Richard


    Mimosa pudica (Linn.) (M. pudica L.) is a plant used in some countries to treat anxiety and depression. In the present study we investigated the effects of an aqueous extract of M. pudica L. on mouse anxiety-like behaviour using the elevated T maze, and on regulation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neuronal activity using an in-vitro mouse brain slice preparation. Acute treatment with M. pudica L. extract had an anxiolytic effect on behaviour in the elevated T maze, specifically on inhibitory avoidance behaviour. Acute application of the extract alone had no effect on the activity of DRN 5-HT neurones. However, when co-applied with the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the extract enhanced the inhibitory effect of the THIP on DRN 5-HT neurones. These observed effects of M. pudica L. on both behaviour and GABA modulation of 5-HT neuronal activity are similar to the effects of diazepam, the established anxiolytic and positive modulator of the GABA(A) receptor. This study suggests that the aqueous extract of M. pudica L. contains a positive modulator of GABA(A) receptor function and provides impetus for further investigation of the neuropharmacologically active constituents of the extract.

  18. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting [Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China); Zheng, Ruimao, E-mail: [Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China); Zhu, Shigong, E-mail: [Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China)


    Highlights: • 14,15-EET inhibits OGD-induced apoptosis in cortical neurons. • Mitochondrial biogenesis of cortical neurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of cortical neurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

  19. CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons.

    Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing


    Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p ACM produced a significant increase in BKα1 and BKβ3 expression (p  0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.

  20. Estimation of the effective orientation of the SHG source in primary cortical neurons

    Psilodimitrakopoulos, Sotiris; Petegnief, Valérie; Soria, Guadalupe; Amat-Roldan, Ivan; Artigas, David; Planas, Anna M; Loza-Álvarez, Pablo


    In this paper we provide, for the first time to our knowledge, the effective orientation of the SHG source in cultured cortical neuronal processes in vitro. This is done by the use of the polarization sensitive second harmonic generation (PSHG) imaging microscopy technique. By performing a pixel-level resolution analysis we found that the SHG dipole source has a distribution of angles centered at θe =33.96°, with a bandwidth of ∆θe = 12.85°. This orientation can be related with the molecular...

  1. CNTF inhibits high voltage activated Ca2+ currents in fetal mouse cortical neurones

    Holm, Ninna R; Christophersen, Palle; Hounsgaard, Jørn;


    Neurotrophic factors yield neuroprotection by mechanisms that may be related to their effects as inhibitors of apoptosis as well as their effects on ion channels. The effect of ciliary neurotrophic factor (CNTF) on high-threshold voltage-activated Ca channels in cultured fetal mouse brain cortical...... neurones was investigated. Addition of CNTF into serum-free growth medium resulted in delayed reduction of the Ca2+ currents. The currents decreased to 50% after 4 h and stabilized at this level during incubation with CNTF for 48 h. Following removal of CNTF the inhibition was completely reversed after 18...

  2. Neuroprotective effects of L-carnitine against oxygenglucose deprivation in rat primary cortical neurons

    Yu Jin Kim


    Full Text Available &lt;b&gt;Purpose:&lt;/b&gt; Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD. &lt;b&gt;Methods:&lt;/b&gt; Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO and propidium iodide (PI were counted, and lactate dehydrogenase (LDH activity and reactive oxygen species (ROS levels were measured. The 3-(4,5-dimethylthiazol-2-yl-2,5- diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 μM, 10 μM, and 100 μM on OGD-induced neurotoxicity. &lt;B&gt;Results:&lt;/b&gt; Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 μM and 100 μM of L-carnitine compared with the untreated OGD group (P&lt;0.05. The application of L-carnitine at 100 μM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P&lt;0.05. &lt;B&gt;Conclusion:&lt;/b&gt; L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.



    Objective To research the effect of melatonin against glutamate excitotoxicity. Methods The model of glutamate-induced excitotoxic damage was built up in rat cerebral cortical cell culture. The effect of mela- tonin against excitotoxic injury was observed by determining the leakage rate of lactate dehydrogenase(LDH) from neurons. Results The leakage rate of LDH wasn't decreased markedly when cultures were exposed to melatonin be- fore, during or 6 h after glutamate treatment. The leakage rate of LDH was decreased significantly when melatonin was administered 0 h, 2 h or 4 h after the cultures were exposed to glutamate. The inhibitory function of melatonin on LDH leakage was most effective at 2 h and 4 h. Conclusion Melatonin has protective effects on neurons damaged by glutamate in a certain time limit.

  4. Golli Myelin Basic Proteins Modulate Voltage-Operated Ca(++) Influx and Development in Cortical and Hippocampal Neurons.

    Vt, Cheli; DA, Santiago González; V, Spreuer; V, Handley; At, Campagnoni; Pm, Paez


    The golli proteins, products of the myelin basic protein gene, are widely expressed in oligodendrocyte progenitor cells and neurons during the postnatal development of the brain. While golli appears to be important for oligodendrocyte migration and differentiation, its function in neuronal development is completely unknown. We have found that golli proteins function as new and novel modulators of voltage-operated Ca(++) channels (VOCCs) in neurons. In vitro, golli knock-out (KO) neurons exhibit decreased Ca(++) influx after plasma membrane depolarization and a substantial maturational delay. Increased expression of golli proteins enhances L-type Ca(++) entry and processes outgrowth in cortical neurons, and pharmacological activation of L-type Ca(++) channels stimulates maturation and prevents cell death in golli-KO neurons. In situ, Ca(++) influx mediated by L-type VOCCs was significantly decreased in cortical and hippocampal neurons of the golli-KO brain. These Ca(++) alterations affect cortical and hippocampal development and the proliferation and survival of neural progenitor cells during the postnatal development of the golli-KO brain. The CA1/3 sections and the dentate gyrus of the hippocampus were reduced in the golli-KO mice as well as the density of dendrites in the somatosensory cortex. Furthermore, the golli-KO mice display abnormal behavior including deficits in episodic memory and reduced anxiety. Because of the expression of the golli proteins within neurons in learning and memory centers of the brain, this work has profound implication in neurodegenerative diseases and neurological disorders.

  5. Imaging separation of neuronal from vascular effects of cocaine on rat cortical brain in vivo

    Yuan, Z.; Du, C.; Yuan, Z.; Luo, Z.; Volkow, N.D.; Pan, Y.; Du, C.


    MRI techniques to study brain function assume coupling between neuronal activity, metabolism and flow. However, recent evidence of physiological uncoupling between neuronal and cerebrovascular events highlights the need for methods to simultaneously measure these three properties. We report a multimodality optical approach that integrates dual-wavelength laser speckle imaging (measures changes in blood flow, blood volume and hemoglobin oxygenation), digital-frequency-ramping optical coherence tomography (images quantitative 3D vascular network) and Rhod2 fluorescence (images intracellular calcium for measure of neuronal activity) at high spatiotemporal resolutions (30 {micro}m, 10 Hz) and over a large field of view (3 x 5 mm{sup 2}). We apply it to assess cocaine's effects in rat cortical brain and show an immediate decrease 3.5 {+-} 0.9 min, phase (1) in the oxygen content of hemoglobin and the cerebral blood flow followed by an overshoot 7.1 {+-} 0.2 min, phase (2) lasting over 20 min whereas Ca{sup 2+} increased immediately (peaked at t = 4.1 {+-} 0.4 min) and remained elevated. This enabled us to identify a delay (2.9 {+-} 0.5 min) between peak neuronal and vascular responses in phase 2. The ability of this multimodality optical approach for simultaneous imaging at high spatiotemporal resolutions permits us to distinguish the vascular versus cellular changes of the brain, thus complimenting other neuroimaging modalities for brain functional studies (e. g., PET, fMRI).

  6. Molecular pathways underlying projection neuron production and migration during cerebral cortical development

    Chiaki eOhtaka-Maruyama


    Full Text Available Glutamatergic neurons of the mammalian cerebral cortex originate from the radial glia (RG progenitors in the ventricular zone (VZ. During corticogenesis, neuroblasts migrate toward the pial surface using two different migration modes. One is multipolar (MP migration with random directional movement, and the other is locomotion, which is a unidirectional movement guided by the RG fiber. After reaching their final destination, the neurons finalize their migration by terminal translocation, which is followed by maturation via dendrite extension to initiate synaptogenesis and thereby complete neural circuit formation. This switching of migration modes during cortical development is unique in mammals, which suggests that the RG-guided locomotion mode may contribute to the evolution of the mammalian neocortical 6-layer structure. Many factors have been reported to be involved in the regulation of this radial neuronal migration process. In general, the radial migration can be largely divided into four steps; (1 maintenance and departure from the VZ of neural progenitor cells, (2 MP migration and transition to bipolar cells, (3 RG-guided locomotion, and (4 terminal translocation and dendrite maturation. Among these, many different gene mutations or knockdown effects have resulted in failure of the MP to bipolar transition (step 2, suggesting that it is a critical step, particularly in radial migration. Moreover, this transition occurs at the subplate layer. In this review, we summarize recent advances in our understanding of the molecular mechanisms underlying each of these steps. Finally, we discuss the evolutionary aspects of neuronal migration in corticogenesis.

  7. Distinct regulation of activity-dependent transcription of immediate early genes in cultured rat cortical neurons.

    Fukuchi, Mamoru; Sanabe, Tomofumi; Watanabe, Toshifumi; Kubota, Takane; Tabuchi, Akiko; Tsuda, Masaaki


    The activity-regulated expression of immediate early genes (IEGs) contributes to long-lasting neuronal functions underlying long-term memory. However, their response properties following neuronal activity are unique and remain poorly understood. To address this knowledge gap, here we further investigated the response properties of two representative IEGs, c-fos and brain-derived neurotrophic factor (Bdnf). Treatment of cultured cortical cells with KCl produces a depolarization process that results in the increase of intracellular calcium concentration in a KCl concentration-dependent manner. Consistent with this increase, c-fos expression was induced in a KCl concentration-dependent manner. In contrast, however, Bdnf expression was optimally activated by both 25 and 50 mM concentration of KCl. Similar results were observed when the cells were treated with okadaic acid, which inhibits protein phosphatases and elicits the hyper-phosphorylation of signaling molecules. Thus, Bdnf expression is strictly regulated by a neuronal activity threshold in an all or nothing manner, whereas c-fos expression is activated in a neuronal activity-dependent manner. Our findings also suggest that these differential responses might be due to the presence or absence of a TATA box. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Modeling the Formation Process of Grouping Stimuli Sets through Cortical Columns and Microcircuits to Feature Neurons

    Frank Klefenz


    Full Text Available A computational model of a self-structuring neuronal net is presented in which repetitively applied pattern sets induce the formation of cortical columns and microcircuits which decode distinct patterns after a learning phase. In a case study, it is demonstrated how specific neurons in a feature classifier layer become orientation selective if they receive bar patterns of different slopes from an input layer. The input layer is mapped and intertwined by self-evolving neuronal microcircuits to the feature classifier layer. In this topical overview, several models are discussed which indicate that the net formation converges in its functionality to a mathematical transform which maps the input pattern space to a feature representing output space. The self-learning of the mathematical transform is discussed and its implications are interpreted. Model assumptions are deduced which serve as a guide to apply model derived repetitive stimuli pattern sets to in vitro cultures of neuron ensembles to condition them to learn and execute a mathematical transform.

  9. A novel role for PTEN in the inhibition of neurite outgrowth by Myelin-associated glycoprotein in cortical neurons

    Perdigoto, Ana Luisa; Chaudhry, Nagarathnamma; Barnes, Gregory N.; Filbin, Marie T.; Carter, Bruce D.


    Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including Myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects cortical neurons. Here, we demonstrate that cortical neurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAG’s inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or over expression of activated AKT in cortical neurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis. PMID:20869442

  10. Accelerated Intoxication of GABAergic Synapses by Botulinum Neurotoxin A Disinhibits Stem Cell-Derived Neuron Networks Prior to Network Silencing


    controls (Figure 2A). To con- firm that the reduction of APs was caused by the loss of synap- tic drive rather than decreased intrinsic excitability, passive...indicates a P < 0.05; ** indicates a P < 0.01; *** indicates a P < 0.001. significantly increased IBI and APs per burst within 10min, con- firming network...A may undergo retrograde transport and be released from motor neurons to intoxicate central synapses (Restani et al., 2012a,b; Marchand-Pauvert et al

  11. Basal forebrain neurons suppress amygdala kindling via cortical but not hippocampal cholinergic projections in rats.

    Ferencz, I; Leanza, G; Nanobashvili, A; Kokaia, M; Lindvall, O


    Intraventricular administration of the immunotoxin 192 IgG-saporin in rats has been shown to cause a selective loss of cholinergic afferents to the hippocampus and cortical areas, and to facilitate seizure development in hippocampal kindling. Here we demonstrate that this lesion also accelerates seizure progression when kindling is induced by electrical stimulations in the amygdala. However, whereas intraventricular 192 IgG-saporin facilitated the development of the initial stages of hippocampal kindling, the same lesion promoted the late stages of amygdala kindling. To explore the role of various parts of the basal forebrain cholinergic system in amygdala kindling, selective lesions of the cholinergic projections to either hippocampus or cortex were produced by intraparenchymal injections of 192 IgG-saporin into medial septum/vertical limb of the diagonal band or nucleus basalis, respectively. Cholinergic denervation of the cortical regions caused acceleration of amygdala kindling closely resembling that observed after the more widespread lesion induced by intraventricular 192 IgG-saporin. In contrast, removal of the cholinergic input to the hippocampus had no effect on the development of amygdala kindling. These data indicate that basal forebrain cholinergic neurons suppress kindling elicited from amygdala, and that this dampening effect is mediated via cortical but not hippocampal projections.

  12. Spatiotemporal memory is an intrinsic property of networks of dissociated cortical neurons.

    Ju, Han; Dranias, Mark R; Banumurthy, Gokulakrishna; VanDongen, Antonius M J


    The ability to process complex spatiotemporal information is a fundamental process underlying the behavior of all higher organisms. However, how the brain processes information in the temporal domain remains incompletely understood. We have explored the spatiotemporal information-processing capability of networks formed from dissociated rat E18 cortical neurons growing in culture. By combining optogenetics with microelectrode array recording, we show that these randomly organized cortical microcircuits are able to process complex spatiotemporal information, allowing the identification of a large number of temporal sequences and classification of musical styles. These experiments uncovered spatiotemporal memory processes lasting several seconds. Neural network simulations indicated that both short-term synaptic plasticity and recurrent connections are required for the emergence of this capability. Interestingly, NMDA receptor function is not a requisite for these short-term spatiotemporal memory processes. Indeed, blocking the NMDA receptor with the antagonist APV significantly improved the temporal processing ability of the networks, by reducing spontaneously occurring network bursts. These highly synchronized events have disastrous effects on spatiotemporal information processing, by transiently erasing short-term memory. These results show that the ability to process and integrate complex spatiotemporal information is an intrinsic property of generic cortical networks that does not require specifically designed circuits. Copyright © 2015 the authors 0270-6474/15/354040-12$15.00/0.

  13. Effects of the muscarinic antagonists pirenzepine and gallamine on spontaneous and evoked responses of rat cerebral cortical neurones.

    Swanson, T. H.; Phillis, J. W.


    1. The muscarinic receptor antagonists gallamine and pirenzepine were iontophoretically applied to rat cerebral cortical cholinoceptive neurones, including corticospinal neurones, to assess their effects on spontaneous firing, and firing induced by: stimulation of the nucleus basalis magnocellularis (NBM); contralateral hindpaw stimulation; application of acetylcholine (ACh); and application of glutamate. 2. Both compounds potently inhibited firing induced by ACh iontophoresis, whilst neither compound consistently altered firing induced by application of glutamate. 3. Gallamine was very effective and pirenzepine less effective, at inhibiting both spontaneous firing and the delayed firing induced by NBM stimulation. The short-latency excitations elicited by NBM stimulation were enhanced by these muscarinic antagonists. 4. Gallamine and pirenzepine enhanced cortical cholinoceptive cell firing induced by contralateral hindpaw stimulation. 5. It is concluded that gallamine depresses spontaneous activity more than pirenzepine, and that both compounds can affect the cortical cell firing evoked by stimulation of the NBM and of thalamo-cortical afferent fibres. PMID:3401638

  14. Endogenous polyamines regulate cortical neuronal excitability by blocking voltage-gated Na+ channels.

    Fleidervish, Ilya A; Libman, Lior; Katz, Efrat; Gutnick, Michael J


    Because the excitable properties of neurons in the neocortex depend on the characteristics of voltage-gated Na(+) channels, factors which regulate those characteristics can fundamentally modify the dynamics of cortical circuits. Here, we report on a novel neuromodulatory mechanism that links the availability of Na(+) channels to metabolism of polyamines (PAs) in the cerebral cortex. Using single channel and whole-cell recordings, we found that products of PA metabolism, the ubiquitous aliphatic polycations spermine and spermidine, are endogenous blockers of Na(+) channels in layer 5 pyramidal cells. Because the blockade is activity-dependent, it is particularly effective against Na(+) channels which fail to inactivate rapidly and thus underlie the persistent Na(+) current. At the level of the local cortical circuit, pharmacological depletion of PAs led to increased spontaneous spiking and periods of hypersynchronous discharge. Our data suggest that changes in PA levels, whether associated with normal brain states or pathological conditions, profoundly modify Na(+) channel availability and thereby shape the integrative behavior of single neurons and neocortical circuits.

  15. Dysregulated Expression of Neuregulin-1 by Cortical Pyramidal Neurons Disrupts Synaptic Plasticity

    Amit Agarwal


    Full Text Available Neuregulin-1 (NRG1 gene variants are associated with increased genetic risk for schizophrenia. It is unclear whether risk haplotypes cause elevated or decreased expression of NRG1 in the brains of schizophrenia patients, given that both findings have been reported from autopsy studies. To study NRG1 functions in vivo, we generated mouse mutants with reduced and elevated NRG1 levels and analyzed the impact on cortical functions. Loss of NRG1 from cortical projection neurons resulted in increased inhibitory neurotransmission, reduced synaptic plasticity, and hypoactivity. Neuronal overexpression of cysteine-rich domain (CRD-NRG1, the major brain isoform, caused unbalanced excitatory-inhibitory neurotransmission, reduced synaptic plasticity, abnormal spine growth, altered steady-state levels of synaptic plasticity-related proteins, and impaired sensorimotor gating. We conclude that an “optimal” level of NRG1 signaling balances excitatory and inhibitory neurotransmission in the cortex. Our data provide a potential pathomechanism for impaired synaptic plasticity and suggest that human NRG1 risk haplotypes exert a gain-of-function effect.

  16. Neurites regrowth of cortical neurons by GSK3beta inhibition independently of Nogo receptor 1.

    Seira, Oscar; Gavín, Rosalina; Gil, Vanessa; Llorens, Franc; Rangel, Alejandra; Soriano, Eduardo; del Río, José Antonio


    Lesioned axons do not regenerate in the adult mammalian CNS, owing to the over-expression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3beta (GSK3beta) and extracellular-related kinase (ERK) 1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3beta and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: (i) cerebellar granule cells and (ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3beta inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Finally, these regenerative effects were corroborated in the lesioned entorhino-hippocampal pathway in NgR1-/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections.

  17. Distinct Physiological Effects of Dopamine D4 Receptors on Prefrontal Cortical Pyramidal Neurons and Fast-Spiking Interneurons.

    Zhong, Ping; Yan, Zhen


    Dopamine D4 receptor (D4R), which is strongly linked to neuropsychiatric disorders, such as attention-deficit hyperactivity disorder and schizophrenia, is highly expressed in pyramidal neurons and GABAergic interneurons in prefrontal cortex (PFC). In this study, we examined the impact of D4R on the excitability of these 2 neuronal populations. We found that D4R activation decreased the frequency of spontaneous action potentials (sAPs) in PFC pyramidal neurons, whereas it induced a transient increase followed by a decrease of sAP frequency in PFC parvalbumin-positive (PV+) interneurons. D4R activation also induced distinct effects in both types of PFC neurons on spontaneous excitatory and inhibitory postsynaptic currents, which drive the generation of sAP. Moreover, dopamine substantially decreased sAP frequency in PFC pyramidal neurons, but markedly increased sAP frequency in PV+ interneurons, and both effects were partially mediated by D4R activation. In the phencyclidine model of schizophrenia, the decreasing effect of D4R on sAP frequency in both types of PFC neurons was attenuated, whereas the increasing effect of D4R on sAP in PV+ interneurons was intact. These results suggest that D4R activation elicits distinct effects on synaptically driven excitability in PFC projection neurons versus fast-spiking interneurons, which are differentially altered in neuropsychiatric disorder-related conditions.

  18. Self-organized two-state membrane potential transitions in a network of realistically modeled cortical neurons.

    Kang, Siu; Kitano, Katsunori; Fukai, Tomoki


    Recent studies have revealed that in vivo cortical neurons show spontaneous transitions between two subthreshold levels of the membrane potentials, 'up' and 'down' states. The neural mechanism of generating those spontaneous states transitions, however, remains unclear. Recent electrophysiological studies have suggested that those state transitions may occur through activation of a hyperpolarization-activated cation current (H-current), possibly by inhibitory synaptic inputs. Here, we demonstrate that two-state membrane potential fluctuations similar to those exhibited by in vivo neurons can be generated through a spike-timing-dependent self-organizing process in a network of inhibitory neurons and excitatory neurons expressing the H-current.

  19. Neuronal activity and TrkB ligands influence Kv3.1b and Kv3.2 expression in developing cortical interneurons.

    Grabert, J; Wahle, P


    Among the GABAergic neocortical interneurons, fast-spiking (FS) basket and chandelier cells are essential mediators for feed-forward inhibition, network synchrony and oscillations. The FS properties are in part mediated by the voltage-gated potassium channels Kv3.1b/3.2 which allow the fast repolarization of the membrane necessary for firing non-adapting action potentials at high frequencies. It has been recently reported that the FS phenotype fails to mature in BDNF knockout mice suggesting a role for neurotrophins. We now describe the role of neuronal activity and neurotrophins for Kv3.1b/3.2 expression using organotypic cultures of rat visual cortex as model system. Chronic activity deprivation from 2 days in vitro (DIV) prevented the postnatal developmental increase of Kv3.2, but not Kv3.1b mRNA expression. However, chronic activity deprivation failed to alter Kv3.1b and marginally delayed Kv3.2 protein expression. Activity deprivation by glutamate receptor blockade from 10 to 20 DIV reduced both mRNAs, whereas deprivation with tetrodotoxin (TTX) reduced both mRNAs and the Kv3.2 protein. Thalamic and cortical afferents in cocultures failed to alter the expression. BDNF and NT4 supplemented from 2 DIV onwards increased the expression of Kv3.1b, but not Kv3.2 mRNA in young cultures. Only NT4 increased the expression of both mRNAs later in development. Kv3 protein levels were not changed by exogenous tropomyosin-related kinase B (TrkB) ligands, but the levels decreased upon inhibiting the MAPK signaling suggesting a role for endogenous factors and in particular MEK2 signaling for translation. The results show that Kv3.1b/3.2 expression is differentially controlled by neuronal activity and neurotrophic factors.

  20. Female Mice Lacking Estrogen Receptor-α in Hypothalamic Proopiomelanocortin (POMC) Neurons Display Enhanced Estrogenic Response on Cortical Bone Mass.

    Farman, H H; Windahl, S H; Westberg, L; Isaksson, H; Egecioglu, E; Schele, E; Ryberg, H; Jansson, J O; Tuukkanen, J; Koskela, A; Xie, S K; Hahner, L; Zehr, J; Clegg, D J; Lagerquist, M K; Ohlsson, C


    Estrogens are important regulators of bone mass and their effects are mainly mediated via estrogen receptor (ER)α. Central ERα exerts an inhibitory role on bone mass. ERα is highly expressed in the arcuate (ARC) and the ventromedial (VMN) nuclei in the hypothalamus. To test whether ERα in proopiomelanocortin (POMC) neurons, located in ARC, is involved in the regulation of bone mass, we used mice lacking ERα expression specifically in POMC neurons (POMC-ERα(-/-)). Female POMC-ERα(-/-) and control mice were ovariectomized (OVX) and treated with vehicle or estradiol (0.5 μg/d) for 6 weeks. As expected, estradiol treatment increased the cortical bone thickness in femur, the cortical bone mechanical strength in tibia and the trabecular bone volume fraction in both femur and vertebrae in OVX control mice. Importantly, the estrogenic responses were substantially increased in OVX POMC-ERα(-/-) mice compared with the estrogenic responses in OVX control mice for cortical bone thickness (+126 ± 34%, P mass, ERα was silenced using an adeno-associated viral vector. Silencing of ERα in hypothalamic VMN resulted in unchanged bone mass. In conclusion, mice lacking ERα in POMC neurons display enhanced estrogenic response on cortical bone mass and mechanical strength. We propose that the balance between inhibitory effects of central ERα activity in hypothalamic POMC neurons in ARC and stimulatory peripheral ERα-mediated effects in bone determines cortical bone mass in female mice.

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

    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


    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.

  2. Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations.

    Härtig, W; Derouiche, A; Welt, K; Brauer, K; Grosche, J; Mäder, M; Reichenbach, A; Brückner, G


    Perineuronal nets (PNs) are known as chondroitin sulphate-rich, lattice-like coatings of the extracellular matrix. In the cortex of mammalian species investigated so far, they were mainly found around GABAergic neurons, but to a lesser degree also around pyramidal cells. Previous investigations in the rat revealed similar distribution patterns of fast-firing neurons expressing both the Kv3.1b subunit of voltage-gated potassium channels and the calcium-binding protein parvalbumin. In the present study, triple fluorescence labelling was applied for the simultaneous demonstration of PNs with the N-acetylgalactosamine-specific Wisteria floribunda agglutinin (WFA), parvalbumin-immunoreactivity (ir) with a monoclonal antibody and of Kv3.1b-ir with several rabbit antibodies. Subsets of non-pyramidal neurons - enwrapped by PNs and expressing parvalbumin and Kv3.1b - were detected in the rat and monkey neocortex and hippocampus. In the rat, faintly stained PNs were additionally found around several layer II/III and V pyramidal cells immunonegative for Kv3.1b, but contacted by Kv3.1b-containing boutons. In the monkey, more intensely labelled PNs frequently occurred around pyramidal cells which themselves appeared to be Kv3. 1b-immunopositive. We also observed minor Kv3.1b-ir and parvalbumin-ir cortical cell populations which were devoid of PNs; occasionally, nets were detected around neurons lacking both immunoreactivities. By confocal laser scanning microscopy, Kv3.1b-ir and WFA-binding sites were found adjoining at the soma and proximal dendritic surface, while lectin-binding sites usually extended on more distal dendritic segments and the axon initial segments which failed to express detectable Kv3.1b-ir. This spatial relationship of both markers was also confirmed by combined WFA-gold labelling and Kv3.1b-immunoperoxidase staining at the electron microscopic level. The data are used for a critical examination of current hypotheses concerning the functional role of PNs

  3. The dynamic brain: from spiking neurons to neural masses and cortical fields.

    Gustavo Deco


    Full Text Available The cortex is a complex system, characterized by its dynamics and architecture, which underlie many functions such as action, perception, learning, language, and cognition. Its structural architecture has been studied for more than a hundred years; however, its dynamics have been addressed much less thoroughly. In this paper, we review and integrate, in a unifying framework, a variety of computational approaches that have been used to characterize the dynamics of the cortex, as evidenced at different levels of measurement. Computational models at different space-time scales help us understand the fundamental mechanisms that underpin neural processes and relate these processes to neuroscience data. Modeling at the single neuron level is necessary because this is the level at which information is exchanged between the computing elements of the brain; the neurons. Mesoscopic models tell us how neural elements interact to yield emergent behavior at the level of microcolumns and cortical columns. Macroscopic models can inform us about whole brain dynamics and interactions between large-scale neural systems such as cortical regions, the thalamus, and brain stem. Each level of description relates uniquely to neuroscience data, from single-unit recordings, through local field potentials to functional magnetic resonance imaging (fMRI, electroencephalogram (EEG, and magnetoencephalogram (MEG. Models of the cortex can establish which types of large-scale neuronal networks can perform computations and characterize their emergent properties. Mean-field and related formulations of dynamics also play an essential and complementary role as forward models that can be inverted given empirical data. This makes dynamic models critical in integrating theory and experiments. We argue that elaborating principled and informed models is a prerequisite for grounding empirical neuroscience in a cogent theoretical framework, commensurate with the achievements in the

  4. Tissue-type plasminogen activator induces synaptic vesicle endocytosis in cerebral cortical neurons.

    Yepes, M; Wu, F; Torre, E; Cuellar-Giraldo, D; Jia, D; Cheng, L


    The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral cortical neurons tPA plays a central role coupling SVs exocytosis and endocytosis.

  5. Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells.

    Qi, Yuchen; Zhang, Xin-Jun; Renier, Nicolas; Wu, Zhuhao; Atkin, Talia; Sun, Ziyi; Ozair, M Zeeshan; Tchieu, Jason; Zimmer, Bastian; Fattahi, Faranak; Ganat, Yosif; Azevedo, Ricardo; Zeltner, Nadja; Brivanlou, Ali H; Karayiorgou, Maria; Gogos, Joseph; Tomishima, Mark; Tessier-Lavigne, Marc; Shi, Song-Hai; Studer, Lorenz


    Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

  6. Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

    Lingping Kong; Lingzhi Wu; Jie Zhang; Yaping Liao; Huaqiao Wang


    BACKGROUND:Numerous current studies have suggested that human telomerase reverse transcriptase (hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses;however,the mechanism of action remains unknown.OBJECTIVE:To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 (Aβ25-35).DESIGN,TIME AND SETTING:The randomized,controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research,Zhongshan School of Medicine,Sun Yat-sen University,China,from September 2005 to June 2008.MATERIALS:AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University,China.Human cortical neurons were derived from 12-20 week old aborted fetuses,obtained from the Guangzhou Maternal and Child Health Hospital,China.Mouse anti-Cdk5 and mouse anti-p16 monoclonal antibodies (Lab Vision,USA),and mouse anti-hTERT monoclonal antibody (Epitomics,USA),were used in this study.METHODS:(1) Recombinant adenovirus vectors,encoding hTERT (Ad-hTERT) and green fluorescent protein (Ad-GFP),were constructed using the AdEasy-1 Expression System.Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days.Likewise,human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days.Human embryonic cortical neurons in the control group were cultured as normal.(2) Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 μmol/L Aβ25-35 for 24 hours.Normal human embryonic cortical neurons treated with 10 μmol/L Aβ25-35 for 24 hours served as a model group.Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35.MAIN OUTCOME MEASURES:Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay

  7. Curcumin protects microglia and primary rat cortical neurons against HIV-1 gp120-mediated inflammation and apoptosis.

    Luyan Guo

    Full Text Available Curcumin is a molecule found in turmeric root that has anti-inflammatory, antioxidant, and anti-tumor properties and has been widely used as both an herbal drug and a food additive to treat or prevent neurodegenerative diseases. To explore whether curcumin is able to ameliorate HIV-1-associated neurotoxicity, we treated a murine microglial cell line (N9 and primary rat cortical neurons with curcumin in the presence or absence of neurotoxic HIV-1 gp120 (V3 loop protein. We found that HIV-1 gp120 profoundly induced N9 cells to produce reactive oxygen species (ROS, tumor necrosis factor-α (TNF-α and monocyte chemoattractant protein-1 (MCP-1. HIV-1 gp120 also induced apoptosis of primary rat cortical neurons. Curcumin exerted a powerful inhibitory effect against HIV-1 gp120-induced neuronal damage, reducing the production of ROS, TNF-α and MCP-1 by N9 cells and inhibiting apoptosis of primary rat cortical neurons. Curcumin may exert its biological activities through inhibition of the delayed rectification and transient outward potassium (K(+ current, as curcumin effectively reduced HIV-1 gp120-mediated elevation of the delayed rectification and transient outward K(+ channel current in neurons. We conclude that HIV-1 gp120 increases ROS, TNF-α and MCP-1 production in microglia, and induces cortical neuron apoptosis by affecting the delayed rectification and transient outward K(+ channel current. Curcumin reduces production of ROS and inflammatory mediators in HIV-1-gp120-stimulated microglia, and protects cortical neurons against HIV-1-mediated apoptosis, most likely through inhibition of HIV-1 gp120-induced elevation of the delayed rectification and transient outward K(+ current.

  8. Graded defragmentation of cortical neuronal firing during recovery of consciousness in rats.

    Vizuete, J A; Pillay, S; Ropella, K M; Hudetz, A G


    State-dependent neuronal firing patterns reflect changes in ongoing information processing and cortical function. A disruption of neuronal coordination has been suggested as the neural correlate of anesthesia. Here, we studied the temporal correlation patterns of ongoing spike activity, during a stepwise reduction of the volatile anesthetic desflurane, in the cerebral cortex of freely moving rats. We hypothesized that the recovery of consciousness from general anesthesia is accompanied by specific changes in the spatiotemporal pattern and correlation of neuronal activity. Sixty-four contact microelectrode arrays were chronically implanted in the primary visual cortex (contacts spanning 1.4-mm depth and 1.4-mm width) for recording of extracellular unit activity at four steady-state levels of anesthesia (8-2% desflurane) and wakefulness. Recovery of consciousness was defined as the regaining of the righting reflex (near 4%). High-intensity firing (HI) periods were segmented using a threshold (200-ms) representing the minimum in the neurons' bimodal interspike interval histogram under anesthesia. We found that the HI periods were highly fragmented in deep anesthesia and gradually transformed to a near-continuous firing pattern at wakefulness. As the anesthetic was withdrawn, HI periods became longer and increasingly correlated among the units both locally and across remote recording sites. Paradoxically, in 4 of 8 animals, HI correlation was also high at the deepest level of anesthesia (8%) when local field potentials (LFP) were burst-suppressed. We conclude that recovery from desflurane anesthesia is accompanied by a graded defragmentation of neuronal activity in the cerebral cortex. Hypersynchrony during deep anesthesia is an exception that occurs only with LFP burst suppression. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  9. Synaptic responsiveness of cortical and thalamic neurones during various phases of slow sleep oscillation in cat.

    Timofeev, I; Contreras, D; Steriade, M


    1. The fluctuations during various phases of the slow sleep oscillation (< 1 Hz) in synaptic responsiveness of motor cortical (Cx), thalamic reticular (RE) and thalamocortical (TC) neurones were investigated intracellularly in cats under ketamine-xylazine anaesthesia. Orthodromic responses to stimuli applied to brachium conjunctivum (BC) axons and corticothalamic pathways were studied. The phases of slow oscillation consist of a long-hyperpolarized, followed by a sharp depth-negative EEG deflection and a series of faster waves that are associated with the depolarization of Cx and RE neurones, while TC cells display a sequence of IPSPs within the spindle frequency. 2. BC-evoked bisynaptic excitatory postsynaptic potentials (EPSPs) in Cx and RE neurones were drastically reduced in amplitude during the long-lasting hyperpolarization and the early part of the depolarizing phase. By contrast, the BC-evoked monosynaptic EPSPs of TC cells were not diminished during the depth-positive EEG wave, but the hyperpolarization during this phase of the slow oscillation prevented TC neurones transferring prethalamic signals to the cortex. 3. At variance with the diminished bisynaptic EPSPs evoked in response to BC stimuli during the long-lasting hyperpolarization, Cx-evoked monosynaptic EPSPs in Cx cells increased linearly with hyperpolarization during this phase of the slow oscillation. Similarly, the amplitudes of Cx-evoked EPSPs in RE and TC cells were not diminished during the long-lasting hyperpolarization. 4. The diminished responsiveness of Cx and RE neurones to prethalamic volleys during the long-lasting hyperpolarization is attributed to gating processes at the level of TC cells that, because of their hyperpolarization, do not transfer prethalamic information to further relays. PMID:8814620

  10. Use of cortical neuronal networks for in vitro material biocompatibility testing.

    Charkhkar, Hamid; Frewin, Christopher; Nezafati, Maysam; Knaack, Gretchen L; Peixoto, Nathalia; Saddow, Stephen E; Pancrazio, Joseph J


    Neural interfaces aim to restore neurological function lost during disease or injury. Novel implantable neural interfaces increasingly capitalize on novel materials to achieve microscale coupling with the nervous system. Like any biomedical device, neural interfaces should consist of materials that exhibit biocompatibility in accordance with the international standard ISO10993-5, which describes in vitro testing involving fibroblasts where cytotoxicity serves as the main endpoint. In the present study, we examine the utility of living neuronal networks as functional assays for in vitro material biocompatibility, particularly for materials that comprise implantable neural interfaces. Embryonic mouse cortical tissue was cultured to form functional networks where spontaneous action potentials, or spikes, can be monitored non-invasively using a substrate-integrated microelectrode array. Taking advantage of such a platform, we exposed established positive and negative control materials to the neuronal networks in a consistent method with ISO 10993-5 guidance. Exposure to the negative controls, gold and polyethylene, did not significantly change the neuronal activity whereas the positive controls, copper and polyvinyl chloride (PVC), resulted in reduction of network spike rate. We also compared the functional assay with an established cytotoxicity measure using L929 fibroblast cells. Our findings indicate that neuronal networks exhibit enhanced sensitivity to positive control materials. In addition, we assessed functional neurotoxicity of tungsten, a common microelectrode material, and two conducting polymer formulations that have been used to modify microelectrode properties for in vivo recording and stimulation. These data suggest that cultured neuronal networks are a useful platform for evaluating the functional toxicity of materials intended for implantation in the nervous system.

  11. Phase dependent sign changes of GABAergic synaptic input explored in-silicio and in-vitro.

    Stiefel, Klaus M; Wespatat, Valérie; Gutkin, Boris; Tennigkeit, Frank; Singer, Wolf


    Inhibitory interactions play a crucial role in the synchronization of neuronal activity. Here we investigate the effect of GABAergic PSPs on spike timing in cortical neurons that exhibit an oscillatory modulation of their membrane potential. To this end we combined numerical simulations with in-vitro patch-clamp recordings from layer II/III pyramidal cells of the rat visual cortex. Special emphasis was placed on exploring how the reversal potential of the GABAergic synaptic currents (EGABA) and the phase relations of the PSPs relative to the oscillation cycles affect the timing of spikes riding on the depolarizing peaks of the oscillations. The simulations predicted: (1) With EGABA more negative than the oscillation minima PSPs are hyperpolarizing at all phases and thus delay or prevent spikes. (2) With EGABA being more positive than the oscillation maxima PSPs are depolarizing in a phase-independent way and lead to a phase advance of spikes. (3) In the intermediate case where EGABA lies within oscillation maxima and minima PSPs are either hyper- or depolarizing depending on their phase relations to the V(m) oscillations and can therefore either delay or advance spikes. Experiments conducted in this most interesting last configuration with biphasic PSPs agreed with the model predictions. Additional theoretical investigations revealed the effect of these PSP induced shifts in spike timing on synchronization in neuronal circuits. The results suggest that GABAergic mechanisms can assume highly specific timing functions in oscillatory networks.

  12. Aluminum—induced apoptosis in cultured cortical neurons and its effects on SAPK/JNK signal transduction pathway

    FuHJ; DongSZ


    Aluminum (Al) exposure and apoptotic cell death have been implicated in several neurodegenerative diseases.the mechanisms by which Al interacts with the nervous system are only partly understood.In this study,we used cultured cortical neurons to investigate the ability of Al to induce the apoptosis of neurons and to explore the role of SAPK/JNK signal transduction pathway on the apoptosis induced by Al.It was found that Al-induced degeneration of cortical neurons involved the DNA fragmentation characteristic of apoptosis.The rate of apoptosis increased significantly,which was measured by TdT-mediated dUTKP nick end labeling.Westerm blot analysis showed that SAPK/JNK activities of cortical neurons varied when the dose and exposure time of AlCl3 were different.Our study demonstrates that Al can induce the apoptosis of cortical neurons and SAPK/JNK signal transduction pathway may play a great role in the apoptosis.

  13. Potential protection of green tea polyphenols against 1800 MHz electromagnetic radiation-induced injury on rat cortical neurons.

    Liu, Mei-Li; Wen, Jian-Qiang; Fan, Yu-Bo


    Radiofrequency electromagnetic fields (EMF) are harmful to public health, but the certain anti-irradiation mechanism is not clear yet. The present study was performed to investigate the possible protective effects of green tea polyphenols against electromagnetic radiation-induced injury in the cultured rat cortical neurons. In this study, green tea polyphenols were used in the cultured cortical neurons exposed to 1800 MHz EMFs by the mobile phone. We found that the mobile phone irradiation for 24 h induced marked neuronal cell death in the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) and TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and protective effects of green tea polyphenols on the injured cortical neurons were demonstrated by testing the content of Bcl-2 Assaciated X protein (Bax) in the immunoprecipitation assay and Western blot assay. In our study results, the mobile phone irradiation-induced increases in the content of active Bax were inhibited significantly by green tea polyphenols, while the contents of total Bax had no marked changes after the treatment of green tea polyphenols. Our results suggested a neuroprotective effect of green tea polyphenols against the mobile phone irradiation-induced injury on the cultured rat cortical neurons.

  14. Neuroprotection with metformin and thymoquinone against ethanol-induced apoptotic neurodegeneration in prenatal rat cortical neurons

    Ullah Ikram


    Full Text Available Abstract Background Exposure to ethanol during early development triggers severe neuronal death by activating multiple stress pathways and causes neurological disorders, such as fetal alcohol effects or fetal alcohol syndrome. This study investigated the effect of ethanol on intracellular events that predispose developing neurons for apoptosis via calcium-mediated signaling. Although the underlying molecular mechanisms of ethanol neurotoxicity are not completely determined, mitochondrial dysfunction, altered calcium homeostasis and apoptosis-related proteins have been implicated in ethanol neurotoxicity. The present study was designed to evaluate the neuroprotective mechanisms of metformin (Met and thymoquinone (TQ during ethanol toxicity in rat prenatal cortical neurons at gestational day (GD 17.5. Results We found that Met and TQ, separately and synergistically, increased cell viability after ethanol (100 mM exposure for 12 hours and attenuated the elevation of cytosolic free calcium [Ca2+]c. Furthermore, Met and TQ maintained normal physiological mitochondrial transmembrane potential (ΔψM, which is typically lowered by ethanol exposure. Increased cytosolic free [Ca2+]c and lowered mitochondrial transmembrane potential after ethanol exposure significantly decreased the expression of a key anti-apoptotic protein (Bcl-2, increased expression of Bax, and stimulated the release of cytochrome-c from mitochondria. Met and TQ treatment inhibited the apoptotic cascade by increasing Bcl-2 expression. These compounds also repressed the activation of caspase-9 and caspase-3 and reduced the cleavage of PARP-1. Morphological conformation of cell death was assessed by TUNEL, Fluoro-Jade-B, and PI staining. These staining methods demonstrated more cell death after ethanol treatment, while Met, TQ or Met plus TQ prevented ethanol-induced apoptotic cell death. Conclusion These findings suggested that Met and TQ are strong protective agents against ethanol

  15. Effect of Anatomically Realistic Full-Head Model on Activation of Cortical Neurons in Subdural Cortical Stimulation—A Computational Study

    Seo, Hyeon; Kim, Donghyeon; Jun, Sung Chan


    Electrical brain stimulation (EBS) is an emerging therapy for the treatment of neurological disorders, and computational modeling studies of EBS have been used to determine the optimal parameters for highly cost-effective electrotherapy. Recent notable growth in computing capability has enabled researchers to consider an anatomically realistic head model that represents the full head and complex geometry of the brain rather than the previous simplified partial head model (extruded slab) that represents only the precentral gyrus. In this work, subdural cortical stimulation (SuCS) was found to offer a better understanding of the differential activation of cortical neurons in the anatomically realistic full-head model than in the simplified partial-head models. We observed that layer 3 pyramidal neurons had comparable stimulation thresholds in both head models, while layer 5 pyramidal neurons showed a notable discrepancy between the models; in particular, layer 5 pyramidal neurons demonstrated asymmetry in the thresholds and action potential initiation sites in the anatomically realistic full-head model. Overall, the anatomically realistic full-head model may offer a better understanding of layer 5 pyramidal neuronal responses. Accordingly, the effects of using the realistic full-head model in SuCS are compelling in computational modeling studies, even though this modeling requires substantially more effort.

  16. Ion channel density and threshold dynamics of repetitive firing in a cortical neuron model.

    Arhem, Peter; Blomberg, Clas


    Modifying the density and distribution of ion channels in a neuron (by natural up- and down-regulation, by pharmacological intervention or by spontaneous mutations) changes its activity pattern. In the present investigation, we analyze how the impulse patterns are regulated by the density of voltage-gated channels in a model neuron, based on voltage clamp measurements of hippocampal interneurons. At least three distinct oscillatory patterns, associated with three distinct regions in the Na-K channel density plane, were found. A stability analysis showed that the different regions are characterized by saddle-node, double-orbit, and Hopf bifurcation threshold dynamics, respectively. Single strongly graded action potentials occur in an area outside the oscillatory regions, but less graded action potentials occur together with repetitive firing over a considerable range of channel densities. The presently found relationship between channel densities and oscillatory behavior may be relevance for understanding principal spiking patterns of cortical neurons (regular firing and fast spiking). It may also be of relevance for understanding the action of pharmacological compounds on brain oscillatory activity.

  17. Bidirectional Regulation of Innate and Learned Behaviors That Rely on Frequency Discrimination by Cortical Inhibitory Neurons.

    Mark Aizenberg


    Full Text Available The ability to discriminate tones of different frequencies is fundamentally important for everyday hearing. While neurons in the primary auditory cortex (AC respond differentially to tones of different frequencies, whether and how AC regulates auditory behaviors that rely on frequency discrimination remains poorly understood. Here, we find that the level of activity of inhibitory neurons in AC controls frequency specificity in innate and learned auditory behaviors that rely on frequency discrimination. Photoactivation of parvalbumin-positive interneurons (PVs improved the ability of the mouse to detect a shift in tone frequency, whereas photosuppression of PVs impaired the performance. Furthermore, photosuppression of PVs during discriminative auditory fear conditioning increased generalization of conditioned response across tone frequencies, whereas PV photoactivation preserved normal specificity of learning. The observed changes in behavioral performance were correlated with bidirectional changes in the magnitude of tone-evoked responses, consistent with predictions of a model of a coupled excitatory-inhibitory cortical network. Direct photoactivation of excitatory neurons, which did not change tone-evoked response magnitude, did not affect behavioral performance in either task. Our results identify a new function for inhibition in the auditory cortex, demonstrating that it can improve or impair acuity of innate and learned auditory behaviors that rely on frequency discrimination.

  18. Lycopene Prevents Amyloid [Beta]-Induced Mitochondrial Oxidative Stress and Dysfunctions in Cultured Rat Cortical Neurons.

    Qu, Mingyue; Jiang, Zheng; Liao, Yuanxiang; Song, Zhenyao; Nan, Xinzhong


    Brains affected by Alzheimer's disease (AD) show a large spectrum of mitochondrial alterations at both morphological and genetic level. The causal link between β-amyloid (Aβ) and mitochondrial dysfunction has been established in cellular models of AD. We observed previously that lycopene, a member of the carotenoid family of phytochemicals, could counteract neuronal apoptosis and cell damage induced by Aβ and other neurotoxic substances, and that this neuroprotective action somehow involved the mitochondria. The present study aims to investigate the effects of lycopene on mitochondria in cultured rat cortical neurons exposed to Aβ. It was found that lycopene attenuated Aβ-induced oxidative stress, as evidenced by the decreased intracellular reactive oxygen species generation and mitochondria-derived superoxide production. Additionally, lycopene ameliorated Aβ-induced mitochondrial morphological alteration, opening of the mitochondrial permeability transition pores and the consequent cytochrome c release. Lycopene also improved mitochondrial complex activities and restored ATP levels in Aβ-treated neuron. Furthermore, lycopene prevented mitochondrial DNA damages and improved the protein level of mitochondrial transcription factor A in mitochondria. Those results indicate that lycopene protects mitochondria against Aβ-induced damages, at least in part by inhibiting mitochondrial oxidative stress and improving mitochondrial function. These beneficial effects of lycopene may account for its protection against Aβ-induced neurotoxicity.

  19. Cultured Cortical Neurons Can Perform Blind Source Separation According to the Free-Energy Principle.

    Takuya Isomura


    Full Text Available Blind source separation is the computation underlying the cocktail party effect--a partygoer can distinguish a particular talker's voice from the ambient noise. Early studies indicated that the brain might use blind source separation as a signal processing strategy for sensory perception and numerous mathematical models have been proposed; however, it remains unclear how the neural networks extract particular sources from a complex mixture of inputs. We discovered that neurons in cultures of dissociated rat cortical cells could learn to represent particular sources while filtering out other signals. Specifically, the distinct classes of neurons in the culture learned to respond to the distinct sources after repeating training stimulation. Moreover, the neural network structures changed to reduce free energy, as predicted by the free-energy principle, a candidate unified theory of learning and memory, and by Jaynes' principle of maximum entropy. This implicit learning can only be explained by some form of Hebbian plasticity. These results are the first in vitro (as opposed to in silico demonstration of neural networks performing blind source separation, and the first formal demonstration of neuronal self-organization under the free energy principle.

  20. Green Tea Polyphenols Attenuated Glutamate Excitotoxicity via Antioxidative and Antiapoptotic Pathway in the Primary Cultured Cortical Neurons.

    Cong, Lin; Cao, Chang; Cheng, Yong; Qin, Xiao-Yan


    Green tea polyphenols are a natural product which has antioxidative and antiapoptotic effects. It has been shown that glutamate excitotoxicity induced oxidative stress is linked to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In this study we explored the neuroprotective effect of green teen polyphenols against glutamate excitotoxicity in the primary cultured cortical neurons. We found that green tea polyphenols protected against glutamate induced neurotoxicity in the cortical neurons as measured by MTT and TUNEL assays. Green tea polyphenols were then showed to inhibit the glutamate induced ROS release and SOD activity reduction in the neurons. Furthermore, our results demonstrated that green tea polyphenols restored the dysfunction of mitochondrial pro- or antiapoptotic proteins Bax, Bcl-2, and caspase-3 caused by glutamate. Interestingly, the neuroprotective effect of green tea polyphenols was abrogated when the neurons were incubated with siBcl-2. Taken together, these results demonstrated that green tea polyphenols protected against glutamate excitotoxicity through antioxidative and antiapoptotic pathways.

  1. Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery.

    Tornero, Daniel; Wattananit, Somsak; Grønning Madsen, Marita; Koch, Philipp; Wood, James; Tatarishvili, Jemal; Mine, Yutaka; Ge, Ruimin; Monni, Emanuela; Devaraju, Karthikeyan; Hevner, Robert F; Brüstle, Oliver; Lindvall, Olle; Kokaia, Zaal


    Stem cell-based approaches to restore function after stroke through replacement of dead neurons require the generation of specific neuronal subtypes. Loss of neurons in the cerebral cortex is a major cause of stroke-induced neurological deficits in adult humans. Reprogramming of adult human somatic cells to induced pluripotent stem cells is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells can be converted to functional cortical neurons that survive and give rise to behavioural recovery after transplantation in the stroke-injured cerebral cortex is not known. We have generated progenitors in vitro, expressing specific cortical markers and giving rise to functional neurons, from long-term self-renewing neuroepithelial-like stem cells, produced from adult human fibroblast-derived induced pluripotent stem cells. At 2 months after transplantation into the stroke-damaged rat cortex, the cortically fated cells showed less proliferation and more efficient conversion to mature neurons with morphological and immunohistochemical characteristics of a cortical phenotype and higher axonal projection density as compared with non-fated cells. Pyramidal morphology and localization of the cells expressing the cortex-specific marker TBR1 in a certain layered pattern provided further evidence supporting the cortical phenotype of the fated, grafted cells, and electrophysiological recordings demonstrated their functionality. Both fated and non-fated cell-transplanted groups showed bilateral recovery of the impaired function in the stepping test compared with vehicle-injected animals. The behavioural improvement at this early time point was most likely not due to neuronal replacement and reconstruction of circuitry. At 5 months after stroke in immunocompromised rats, there was no tumour formation and the grafted cells exhibited electrophysiological properties of mature neurons with evidence of integration in host circuitry. Our

  2. Cell-attached recordings of responses evoked by photorelease of GABA in the immature cortical neurons

    Marat eMinlebaev


    Full Text Available We present a novel non-invasive technique to measure the polarity of GABAergic responses based on cell-attached recordings of currents activated by laser-uncaging of GABA. For these recordings, a patch pipette was filled with a solution containing RuBi-GABA, and GABA was released from this complex by a laser beam conducted to the tip of the patch pipette via an optic fiber. In cell-attached recordings from neocortical and hippocampal neurons in postnatal days P2-5 rat brain slices in vitro, we found that laser-uncaging of GABA activates integral cell-attached currents mediated by tens of GABA(A channels. The initial response was inwardly directed, indicating a depolarizing response to GABA. The direction of the initial response was dependent on the pipette potential and analysis of its slope-voltage relationships revealed a depolarizing driving force of +11 mV for the currents through GABA channels. Initial depolarizing responses to GABA uncaging were inverted to hyperpolarizing in the presence of the NKCC1 blocker bumetanide. Current-voltage relationships of the currents evoked by Rubi-GABA uncaging using voltage-ramps at the peak of responses not only revealed a bumetanide-sensitive depolarizing reversal potential of the GABA(A receptor mediated responses, but also showed a strong voltage-dependent hysteresis. Upon desensitization of the uncaged-GABA response, current-voltage relationships of the currents through single GABA(A channels revealed depolarizing responses with the driving force values similar to those obtained for the initial response. Thus, cell-attached recordings of the responses evoked by local intrapipette GABA uncaging are suitable to assess the polarity of the GABA(A-Rs mediated signals in small cell compartments.

  3. Transcranial Direct Current Stimulation modulates cortical neuronal activity in Alzheimer’s Disease

    Sara eMarceglia


    Full Text Available Quantitative electroencephalography (qEEG showed that Alzheimer’s disease (AD is characterized by increased theta power, decreased alpha and beta power, and decreased coherence in the alpha and theta band in posterior regions. These abnormalities are thought to be associated with functional disconnections among cortical areas, death of cortical neurons, axonal pathology, and cholinergic deficits. Since transcranial Direct Current Stimulation (tDCS over the temporo-parietal area is thought to have beneficial effects in patients with AD, in this study we aimed to investigate whether tDCS benefits are related to tDCS-induced changes in cortical activity, as represented by qEEG.A weak anodal current (1.5 mA, 15 min was delivered bilaterally over the temporal-parietal lobe to 7 subjects with probable AD (Mini-Mental State Examination, MMSE score >20. EEG (21 electrodes, 10-20 international system was recorded for 5 minutes with eyes closed before (baseline, t0 and 30 minutes after anodal and cathodal tDCS ended (t1. At the same time points, patients performed a Word Recognition Task (WRT to assess working memory functions. The spectral power and the inter- and intra-hemispheric EEG coherence in different frequency bands (e.g., low frequencies, including delta and theta; high frequencies, including alpha and beta were calculated for each subject at t0 and t1. tDCS-induced changes in EEG neurophysiological markers were correlated with the performance of patients at the WRT.At baseline, qEEG features in AD patients confirmed that the decreased high frequency power was correlated with lower MMSE. After anodal tDCS, we observed an increase in the high-frequency power in the temporo-parietal area and an increase in the temporo-parieto-occipital coherence that correlated with the improvement at the WRT. In addition, cathodal tDCS produced a non-specific effect of decreased theta power all over the scalp that was not correlated with the clinical

  4. Transcranial Direct Current Stimulation Modulates Cortical Neuronal Activity in Alzheimer's Disease.

    Marceglia, Sara; Mrakic-Sposta, Simona; Rosa, Manuela; Ferrucci, Roberta; Mameli, Francesca; Vergari, Maurizio; Arlotti, Mattia; Ruggiero, Fabiana; Scarpini, Elio; Galimberti, Daniela; Barbieri, Sergio; Priori, Alberto


    Quantitative electroencephalography (qEEG) showed that Alzheimer's disease (AD) is characterized by increased theta power, decreased alpha and beta power, and decreased coherence in the alpha and theta band in posterior regions. These abnormalities are thought to be associated with functional disconnections among cortical areas, death of cortical neurons, axonal pathology, and cholinergic deficits. Since transcranial Direct Current Stimulation (tDCS) over the temporo-parietal area is thought to have beneficial effects in patients with AD, in this study we aimed to investigate whether tDCS benefits are related to tDCS-induced changes in cortical activity, as represented by qEEG. A weak anodal current (1.5 mA, 15 min) was delivered bilaterally over the temporal-parietal lobe to seven subjects with probable AD (Mini-Mental State Examination, MMSE score >20). EEG (21 electrodes, 10-20 international system) was recorded for 5 min with eyes closed before (baseline, t0) and 30 min after anodal and cathodal tDCS ended (t1). At the same time points, patients performed a Word Recognition Task (WRT) to assess working memory functions. The spectral power and the inter- and intra-hemispheric EEG coherence in different frequency bands (e.g., low frequencies, including delta and theta; high frequencies, including alpha and beta) were calculated for each subject at t0 and t1. tDCS-induced changes in EEG neurophysiological markers were correlated with the performance of patients at the WRT. At baseline, qEEG features in AD patients confirmed that the decreased high frequency power was correlated with lower MMSE. After anodal tDCS, we observed an increase in the high-frequency power in the temporo-parietal area and an increase in the temporo-parieto-occipital coherence that correlated with the improvement at the WRT. In addition, cathodal tDCS produced a non-specific effect of decreased theta power all over the scalp that was not correlated with the clinical observation at the WRT

  5. Genetics and Function of Neocortical GABAergic Interneurons in Neurodevelopmental Disorders

    E. Rossignol


    Full Text Available A dysfunction of cortical and limbic GABAergic circuits has been postulated to contribute to multiple neurodevelopmental disorders in humans, including schizophrenia, autism, and epilepsy. In the current paper, I summarize the characteristics that underlie the great diversity of cortical GABAergic interneurons and explore how the multiple roles of these cells in developing and mature circuits might contribute to the aforementioned disorders. Furthermore, I review the tightly controlled genetic cascades that determine the fate of cortical interneurons and summarize how the dysfunction of genes important for the generation, specification, maturation, and function of cortical interneurons might contribute to these disorders.

  6. Adenosine A1 receptor-mediated transactivation of the EGF receptor produces a neuroprotective effect on cortical neurons in vitro

    Ke-qiang XIE; Li-min ZHANG; Yan CAO; Jun ZHU; Lin-yin FENG


    Aim:To understand the mechanism of the transactivation of the epidermal growth factor receptor (EGFR) mediated by the adenosine A1 receptor (A1R).Methods:Primary cultured rat cortical neurons subjected to oxygen-glucose deprivation (OGD) and HEK293/A1R cells were treated with the A1R-specific agonist N6-cyclopentyladenosine (CPA).Phospho-EGFR,Akt,and ERK1/2 were observed by Western blot.An interaction between EGFR and AIR was detected using immunoprecipitation and immunocytochemistry.Results:The A1R agonist CPA causes protein kinase B (Akt) activation and protects primary cortical neurons from oxygen-glucose deprivation (OGD) insult.A1R and EGFR co-localize in the membranes of neurons and form an immunocomplex.A1R stimulation induces significant EGFR phosphorylation via a P13K and Src kinase signaling pathway;this stimulation provides a neuroprotective effect in cortical neurons.CPA leads to sustained phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) in cortical neurons,but only to transient phosphorylation in HEK 293/A1R cells.The response to the AtR agonist is mediated primarily through EGFR trans-activation that is dependent on pertussis toxin (PTX)-sensitive G1 protein and metalloproteases in HEK 293/A1R.Conclusion:A1R-mediated EGFR transactivation confers a neuroprotective effect in primary cortical neurons.P13 kinase and Src kinase play pivotal roles in this response.

  7. Bcl11a (Ctip1) Controls Migration of Cortical Projection Neurons through Regulation of Sema3c.

    Wiegreffe, Christoph; Simon, Ruth; Peschkes, Katharina; Kling, Carolin; Strehle, Michael; Cheng, Jin; Srivatsa, Swathi; Liu, Pentao; Jenkins, Nancy A; Copeland, Neal G; Tarabykin, Victor; Britsch, Stefan


    During neocortical development, neurons undergo polarization, oriented migration, and layer-type-specific differentiation. The transcriptional programs underlying these processes are not completely understood. Here, we show that the transcription factor Bcl11a regulates polarity and migration of upper layer neurons. Bcl11a-deficient late-born neurons fail to correctly switch from multipolar to bipolar morphology, resulting in impaired radial migration. We show that the expression of Sema3c is increased in migrating Bcl11a-deficient neurons and that Bcl11a is a direct negative regulator of Sema3c transcription. In vivo gain-of-function and rescue experiments demonstrate that Sema3c is a major downstream effector of Bcl11a required for the cell polarity switch and for the migration of upper layer neurons. Our data uncover a novel Bcl11a/Sema3c-dependent regulatory pathway used by migrating cortical neurons.

  8. Neuron-specific stimulus masking reveals interference in spike timing at the cortical level.

    Larson, Eric; Maddox, Ross K; Perrone, Ben P; Sen, Kamal; Billimoria, Cyrus P


    The auditory system is capable of robust recognition of sounds in the presence of competing maskers (e.g., other voices or background music). This capability arises despite the fact that masking stimuli can disrupt neural responses at the cortical level. Since the origins of such interference effects remain unknown, in this study, we work to identify and quantify neural interference effects that originate due to masking occurring within and outside receptive fields of neurons. We record from single and multi-unit auditory sites from field L, the auditory cortex homologue in zebra finches. We use a novel method called spike timing-based stimulus filtering that uses the measured response of each neuron to create an individualized stimulus set. In contrast to previous adaptive experimental approaches, which have typically focused on the average firing rate, this method uses the complete pattern of neural responses, including spike timing information, in the calculation of the receptive field. When we generate and present novel stimuli for each neuron that mask the regions within the receptive field, we find that the time-varying information in the neural responses is disrupted, degrading neural discrimination performance and decreasing spike timing reliability and sparseness. We also find that, while removing stimulus energy from frequency regions outside the receptive field does not significantly affect neural responses for many sites, adding a masker in these frequency regions can nonetheless have a significant impact on neural responses and discriminability without a significant change in the average firing rate. These findings suggest that maskers can interfere with neural responses by disrupting stimulus timing information with power either within or outside the receptive fields of neurons.

  9. Buffer capacity of rat cortical tissue as well as of cultured neurons and astrocytes.

    Katsura, K; Mellergård, P; Theander, S; Ouyang, Y B; Siesjö, B K


    The primary objective of this work was to assess the intrinsic nonbicarbonate buffer capacity (beta i) of cultured neurons and astrocytes and to compare the beta i values obtained to those of neocortical tissue. A second objective was to determine the pH dependence of beta i. Titration of homogenates of whole-brain cortical tissue and cultured neurons with NaOH and HCl gave beta i values of 25-30 mmol.l-1 x pH-1. The buffer capacity was essentially constant in the pH range of 6-7. Astrocytes showed a higher buffer capacity and a clear relationship between beta i and pH. However, beta i decreased when pH was reduced from 7 to 6. The beta i values derived from microspectrofluorometric studies on neurons and astrocytes were surprisingly variable, ranging from 10 to 50 mmol.l-1 x pH-1. The ammonia "step method" suggested that beta i increased dramatically when pH was lowered from 7 to 6 but the propionic "step method" failed to reveal such a pH dependence. Some techniques obviously give erroneous values for beta i, presumably because changes in buffer base concentration (due to transmembrane fluxes of H+, HCO3-, NH4+ or anions of weak acids) violate the principles upon which the calculations are based. From the results obtained by direct titration and with the propionate technique, we tentatively conclude that beta i in neurons and astrocytes are approximately 20 and 30 mmol.l-1 x pH-1, respectively. We further suggest that the term "intrinsic buffer capacity", as commonly used, is redefined.

  10. Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture.

    López, E; Arce, C; Oset-Gasque, M J; Cañadas, S; González, M P


    Cadmium is a toxic agent that it is also an environmental contaminant. Cadmium exposure may be implicated in some humans disorders related to hyperactivity and increased aggressiveness. This study presents data indicating that cadmium induces cellular death in cortical neurons in culture. This death could be mediated by an apoptotic and a necrotic mechanism. The apoptotic death may be mediated by oxidative stress with reactive oxygen species (ROS) formation which could be induced by mitochondrial membrane dysfunction since this cation produces: (a) depletion of mitochondrial membrane potential and (b) diminution of ATP levels with ATP release. Necrotic death could be mediated by lipid peroxidation induced by cadmium through an indirect mechanism (ROS formation). On the other hand, 40% of the cells survive cadmium action. This survival seems to be mediated by the ability of these cells to activate antioxidant defense systems, since cadmium reduced the intracellular glutathione levels and induced catalase and SOD activation in these cells.

  11. Neuronal dysfunction and disconnection of cortical hubs in non-demented subjects with elevated amyloid burden

    Drzezga, Alexander; Van Dijk, Koene R. A.; Sreenivasan, Aishwarya; Talukdar, Tanveer; Sullivan, Caroline; Schultz, Aaron P.; Sepulcre, Jorge; Putcha, Deepti; Greve, Doug; Johnson, Keith A.; Sperling, Reisa A.


    Disruption of functional connectivity between brain regions may represent an early functional consequence of β-amyloid pathology prior to clinical Alzheimer's disease. We aimed to investigate if non-demented older individuals with increased amyloid burden demonstrate disruptions of functional whole-brain connectivity in cortical hubs (brain regions typically highly connected to multiple other brain areas) and if these disruptions are associated with neuronal dysfunction as measured with fluorodeoxyglucose-positron emission tomography. In healthy subjects without cognitive symptoms and patients with mild cognitive impairment, we used positron emission tomography to assess amyloid burden and cerebral glucose metabolism, structural magnetic resonance imaging to quantify atrophy and novel resting state functional magnetic resonance imaging processing methods to calculate whole-brain connectivity. Significant disruptions of whole-brain connectivity were found in amyloid-positive patients with mild cognitive impairment in typical cortical hubs (posterior cingulate cortex/precuneus), strongly overlapping with regional hypometabolism. Subtle connectivity disruptions and hypometabolism were already present in amyloid-positive asymptomatic subjects. Voxel-based morphometry measures indicate that these findings were not solely a consequence of regional atrophy. Whole-brain connectivity values and metabolism showed a positive correlation with each other and a negative correlation with amyloid burden. These results indicate that disruption of functional connectivity and hypometabolism may represent early functional consequences of emerging molecular Alzheimer's disease pathology, evolving prior to clinical onset of dementia. The spatial overlap between hypometabolism and disruption of connectivity in cortical hubs points to a particular susceptibility of these regions to early Alzheimer's-type neurodegeneration and may reflect a link between synaptic dysfunction and functional

  12. Neurotoxicity of Ecstasy metabolites in rat cortical neurons, and influence of hyperthermia.

    Capela, João Paulo; Meisel, Andreas; Abreu, Artur Reis; Branco, Paula Sério; Ferreira, Luísa Maria; Lobo, Ana Maria; Remião, Fernando; Bastos, Maria Lurdes; Carvalho, Félix


    3,4-Methylenedioxymethamphetamine (MDMA or "Ecstasy") is a widely abused, psychoactive recreational drug. There is growing evidence that the MDMA neurotoxic profile may be highly dependent on both its hepatic metabolism and body temperature. Metabolism of MDMA involves N-demethylation to 3,4-methylenedioxyamphetamine (MDA), which is also a drug of abuse. MDMA and MDA are O-demethylenated to N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and alpha-methyldopamine (alpha-MeDA), respectively, both of which are catechols that can undergo oxidation to the corresponding ortho-quinones. In the presence of glutathione (GSH), ortho-quinones may be conjugated with GSH to form glutathionyl adducts. In this study, we evaluated the neurotoxicity of MDMA and three of its metabolites obtained by synthesis, N-Me-alpha-MeDA, alpha-MeDA, and 5-(GSH)-alpha-MeDA [5-(glutathion-S-yl)-alpha-methyldopamine] in rat cortical neuronal serum-free cultures under normal (36.5 degrees C) and hyperthermic (40 degrees C) conditions. Cell viability was assessed, and the mechanism of cell death was also evaluated. Our study shows that these metabolites are more neurotoxic [5-(GSH)-alpha-MeDA being the most toxic] than the parent compound MDMA. The neurotoxicity of MDMA metabolites was partially prevented by the antioxidants N-acetylcystein and also, in a minor extent, by alpha-phenyl-N-tert-butyl nitrone. All the tested compounds induced apoptotic cell death in cortical neurons, and their neurotoxic effect was potentiated under hyperthermic conditions. These data suggest that MDMA metabolites, especially under hyperthermic conditions, contribute to MDMA-induced neurotoxicity.

  13. Restoration of Progranulin Expression Rescues Cortical Neuron Generation in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia

    Susanna Raitano


    Full Text Available To understand how haploinsufficiency of progranulin (PGRN causes frontotemporal dementia (FTD, we created induced pluripotent stem cells (iPSCs from patients carrying the GRNIVS1+5G > C mutation (FTD-iPSCs. FTD-iPSCs were fated to cortical neurons, the cells most affected in FTD. Although generation of neuroprogenitors was unaffected, their further differentiation into CTIP2-, FOXP2-, or TBR1-TUJ1 double-positive cortical neurons, but not motorneurons, was significantly decreased in FTD-neural progeny. Zinc finger nuclease-mediated introduction of GRN cDNA into the AAVS1 locus corrected defects in cortical neurogenesis, demonstrating that PGRN haploinsufficiency causes inefficient cortical neuron generation. RNA sequencing analysis confirmed reversal of the altered gene expression profile following genetic correction. We identified the Wnt signaling pathway as one of the top defective pathways in FTD-iPSC-derived neurons, which was reversed following genetic correction. Differentiation of FTD-iPSCs in the presence of a WNT inhibitor mitigated defective corticogenesis. Therefore, we demonstrate that PGRN haploinsufficiency hampers corticogenesis in vitro.

  14. Evaluation of the Neuroactivity of ToxCast Compounds Using Multi-well Microelectrode Array Recordings in Primary Cortical Neurons

    Evaluation of the Neuroactivity of ToxCast Compounds Using Multi-well Microelectrode Array Recordings in Primary Cortical Neurons P Valdivia1, M Martin2, WR LeFew3, D Hall3, J Ross1, K Houck2 and TJ Shafer3 1Axion Biosystems, Atlanta GA and 2NCCT, 3ISTD, NHEERL, ORD, US EPA, RT...

  15. Expression of exogenous LIN28 contributes to proliferation and survival of mouse primary cortical neurons in vitro.

    Bhuiyan, M I H; Lee, J-H; Kim, S Y; Cho, K-O


    LIN28, an RNA-binding protein, is known to be involved in the regulation of many cellular processes, such as embryonic stem cell proliferation, cell fate succession, developmental timing, and oncogenesis. In this study, we investigated the effect of constitutively expressing exogenous LIN28 on neuronal cell proliferation and viability in vitro. Plasmids containing LIN28-green fluorescent protein (GFP) or GFP were introduced into the embryonic mouse brains at E14.5 by in utero electroporation. Two days after electroporation, embryonic cortices were harvested and cultured. It was found that transfected cells stably overexpressed LIN28 in vitro. Viability curve from live cell imaging showed that the number of GFP-expressing cells decreased over time in line with naive primary cortical neurons. In contrast, the number of LIN28-GFP-overexpressing neurons initially increased and remained high at later time-points in culture than GFP-expressing cells. Double immunofluorescence showed that at an early time in culture, the number of Ki-67/GFP double-positive cells was higher in the LIN28-GFP group than that of controls. Moreover, there were significantly lower numbers of condensed nuclei/GFP- and cleaved caspase-3/GFP-positive cells in the LIN28-GFP groups compared to control GFP. Furthermore, it was confirmed that the LIN28-GFP-expressing cells at days in vitro (DIV)13 were neuronal nuclei (NeuN)-positive mature neurons. Finally, the expression of insulin-like growth factor 2 (IGF-2) was induced in LIN28-expressing primary cortical neurons, which was not detected in controls. Taken together, our results indicate that the expression of exogenous LIN28 can promote the proliferation of neural progenitor cells and exert prosurvival effect on primary cortical neurons by inhibiting caspase-dependent apoptosis, possibly via upregulation of IGF-2. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  16. Ultrastructural changes of rat cortical neurons following ligustrazine intervention for cerebral ischemia/reperfusion injury

    Hui Zhang; Jianfeng Dong; Qiuzhen Zhao; Wen Song; Aihua Bo


    low-dose group and ligustrazine high-dose group received ligustrazine injections, 50 mg/kg and 100 mg/kg, respectively. Samples were collected at the same time as the model group.MAIN OUTCOME MEASURES: Alterations of the neuronal ultrastructure and main organelles were ob-served by electron microscopy.RESULTS: Forty Wistar rats were included in the final analysis. Plentiful ribosome and rough endoplasmic reticulum existed in the cytoplasm of cortical neurons in the normal group. Edema existed in the nucleus and cytoplasm of neurons in the model group. The cell membrane was damaged, resulting in the external erup-tion of certain cellular organelles. In the low-dose ligustrazine group, neuronal swelling was decreased in the cytoplasm, whereas cellular organelles were relatively increased. However, the mitochondria remained swollen. The double layer structure disappeared in parts of the mitochondrial membrane. The caryotheca was still broken, and neuronal damage was significantly decreased in the high-dose ligustrazine group. In ad-dition, cytoplasmic swelling was reduced andmost part of caryotheca was complete. Fragmentation of the cellular membrane was not detected. Mitochondrial cristae and the lysosome could also be detected. The number of rough endoplasmic reticulum and free ribosomes was increased, and the structure of great part of caryotheca was clear. In addition, the number of nuclear pore was increased. However, the nuclear hetero-chromatin was relatively reduced.CONCLUSION: In the rat, the protective effects of ligustrazine were significant on neuronal membrane structures and main organelles after cerebral ischemia/reperfusion. There was a dose-dependent effect be-tween neuronal changes and Ligustrazine.

  17. GABAergic circuit dysfunctions in neurodevelopmental disorders

    Bidisha eChattopadhyaya


    Full Text Available GABAergic interneurons control neuronal excitability, integration, and plasticity. Further, they regulate the generation of temporal synchrony and oscillatory behavior among networks of pyramidal neurons. Such oscillations within and across neural systems are believed to serve various complex functions, such as perception, movement initiation, and memory. Alterations in the development of GABAergic circuits have been implicated in various brain diseases with neurodevelopmental origin. Here, we highlight recent studies suggesting a role for alterations of GABA transmission in the pathophysiology of two neurodevelopmental diseases, schizophrenia and autism. We further discuss how manipulations of GABA signaling may be used for novel therapeutic interventions.

  18. Stromal derived factor-1 exerts differential regulation on distinct cortical cell populations in vitro

    Zeef Leo


    Full Text Available Abstract Background Stromal derived factor (SDF-1, an alpha chemokine, is a widely known chemoattractant in the immune system. A growing body of evidence now suggests multiple regulatory roles for SDF-1 in the developing nervous system. Results To investigate the role of SDF-1 signaling in the growth and differentiation of cortical cells, we performed numerous in vitro experiments, including gene chip and quantitative RT-PCR analysis. Using SDF-1 medium and AMD3100, a receptor antagonist, we demonstrate that the chemokine signaling regulates key events during early cortical development. First, SDF-1 signaling maintains cortical progenitors in proliferation, possibly through a mechanism involving connexin 43 mediated intercellular coupling. Second, SDF-1 signaling upregulates the differentiation of cortical GABAergic neurons, independent of sonic signaling pathway. Third, SDF-1 enables the elongation and branching of axons of cortical glutamatergic neurons. Finally, cortical cultures derived from CXCR4-/- mutants show a close parallel to AMD3100 treatment with reduced cell proliferation and differentiation of GABAergic neurons. Conclusion Results from this study show that SDF-1 regulates distinct cortical cell populations in vitro.

  19. IL-10 Protects Neurites in Oxygen-Glucose-Deprived Cortical Neurons through the PI3K/Akt Pathway.

    Longzai Lin

    Full Text Available IL-10, as a cytokine, has an anti-inflammatory cascade following various injuries, but it remains blurred whether IL-10 protects neurites of cortical neurons after oxygen-glucose deprivation injury. Here, we reported that IL-10, in a concentration-dependent manner, reduced neuronal apoptosis and increased neuronal survival in oxygen-glucose-deprived primary cortical neurons, producing an optimal protective effect at 20ng/ml. After staining NF-H and GAP-43, we found that IL-10 significantly protected neurites in terms of axon length and dendrite number by confocal microscopy. Furthermore, it induced the phosphorylation of AKT, suppressed the activation of caspase-3, and up-regulated the protein expression of GAP-43. In contrast, LY294002, a specific inhibitor of PI3K/AKT, reduced the level of AKT phosphorylation and GAP-43 expression, increased active caspase-3 expression and thus significantly weakened IL-10-mediated protective effect in the OGD-induced injury model. IL-10NA, the IL-10 neutralizing antibody, reduced the level of p-PI3K phosphorylation and increased the expression of active caspase-3. These findings suggest that IL-10 provides neuroprotective effects by protecting neurites through PI3K/AKT signaling pathway in oxygen-glucose-deprived primary cortical neurons.

  20. The Fas/Fas ligand death receptor pathway contributes to phenylalanine-induced apoptosis in cortical neurons.

    Xiaodong Huang

    Full Text Available Phenylketonuria (PKU, an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe. A recent study showed that the mitochondria-mediated (intrinsic apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic apoptotic pathway and endoplasmic reticulum (ER stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h, suggesting involvement of the Fas receptor (FasR-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.

  1. Regulation of action potential waveforms by axonal GABAA receptors in cortical pyramidal neurons.

    Yang Xia

    Full Text Available GABAA receptors distributed in somatodendritic compartments play critical roles in regulating neuronal activities, including spike timing and firing pattern; however, the properties and functions of GABAA receptors at the axon are still poorly understood. By recording from the cut end (bleb of the main axon trunk of layer -5 pyramidal neurons in prefrontal cortical slices, we found that currents evoked by GABA iontophoresis could be blocked by picrotoxin, indicating the expression of GABAA receptors in axons. Stationary noise analysis revealed that single-channel properties of axonal GABAA receptors were similar to those of somatic receptors. Perforated patch recording with gramicidin revealed that the reversal potential of the GABA response was more negative than the resting membrane potential at the axon trunk, suggesting that GABA may hyperpolarize the axonal membrane potential. Further experiments demonstrated that the activation of axonal GABAA receptors regulated the amplitude and duration of action potentials (APs and decreased the AP-induced Ca2+ transients at the axon. Together, our results indicate that the waveform of axonal APs and the downstream Ca2+ signals are modulated by axonal GABAA receptors.

  2. Herpes Simplex Virus-Type1 (HSV-1) Impairs DNA Repair in Cortical Neurons.

    De Chiara, Giovanna; Racaniello, Mauro; Mollinari, Cristiana; Marcocci, Maria Elena; Aversa, Giorgia; Cardinale, Alessio; Giovanetti, Anna; Garaci, Enrico; Palamara, Anna Teresa; Merlo, Daniela


    Several findings suggest that Herpes simplex virus-1 (HSV-1) infection plays a role in the neurodegenerative processes that characterize Alzheimer's disease (AD), but the underlying mechanisms have yet to be fully elucidated. Here we show that HSV-1 productive infection in cortical neurons causes the accumulation of DNA lesions that include both single (SSBs) and double strand breaks (DSBs), which are reported to be implicated in the neuronal loss observed in neurodegenerative diseases. We demonstrate that HSV-1 downregulates the expression level of Ku80, one of the main components of non-homologous end joining (NHEJ), a major pathway for the repair of DSBs. We also provide data suggesting that HSV-1 drives Ku80 for proteasomal degradation and impairs NHEJ activity, leading to DSB accumulation. Since HSV-1 usually causes life-long recurrent infections, it is possible to speculate that cumulating damages, including those occurring on DNA, may contribute to virus induced neurotoxicity and neurodegeneration, further suggesting HSV-1 as a risk factor for neurodegenerative conditions.

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

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


    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.

  4. Herpes Simplex Virus-Type1 (HSV-1) Impairs DNA Repair in Cortical Neurons

    De Chiara, Giovanna; Racaniello, Mauro; Mollinari, Cristiana; Marcocci, Maria Elena; Aversa, Giorgia; Cardinale, Alessio; Giovanetti, Anna; Garaci, Enrico; Palamara, Anna Teresa; Merlo, Daniela


    Several findings suggest that Herpes simplex virus-1 (HSV-1) infection plays a role in the neurodegenerative processes that characterize Alzheimer’s disease (AD), but the underlying mechanisms have yet to be fully elucidated. Here we show that HSV-1 productive infection in cortical neurons causes the accumulation of DNA lesions that include both single (SSBs) and double strand breaks (DSBs), which are reported to be implicated in the neuronal loss observed in neurodegenerative diseases. We demonstrate that HSV-1 downregulates the expression level of Ku80, one of the main components of non-homologous end joining (NHEJ), a major pathway for the repair of DSBs. We also provide data suggesting that HSV-1 drives Ku80 for proteasomal degradation and impairs NHEJ activity, leading to DSB accumulation. Since HSV-1 usually causes life-long recurrent infections, it is possible to speculate that cumulating damages, including those occurring on DNA, may contribute to virus induced neurotoxicity and neurodegeneration, further suggesting HSV-1 as a risk factor for neurodegenerative conditions. PMID:27803664

  5. Discontinuous Galerkin finite element method for solving population density functions of cortical pyramidal and thalamic neuronal populations.

    Huang, Chih-Hsu; Lin, Chou-Ching K; Ju, Ming-Shaung


    Compared with the Monte Carlo method, the population density method is efficient for modeling collective dynamics of neuronal populations in human brain. In this method, a population density function describes the probabilistic distribution of states of all neurons in the population and it is governed by a hyperbolic partial differential equation. In the past, the problem was mainly solved by using the finite difference method. In a previous study, a continuous Galerkin finite element method was found better than the finite difference method for solving the hyperbolic partial differential equation; however, the population density function often has discontinuity and both methods suffer from a numerical stability problem. The goal of this study is to improve the numerical stability of the solution using discontinuous Galerkin finite element method. To test the performance of the new approach, interaction of a population of cortical pyramidal neurons and a population of thalamic neurons was simulated. The numerical results showed good agreement between results of discontinuous Galerkin finite element and Monte Carlo methods. The convergence and accuracy of the solutions are excellent. The numerical stability problem could be resolved using the discontinuous Galerkin finite element method which has total-variation-diminishing property. The efficient approach will be employed to simulate the electroencephalogram or dynamics of thalamocortical network which involves three populations, namely, thalamic reticular neurons, thalamocortical neurons and cortical pyramidal neurons.

  6. Cortical overexpression of neuronal calcium sensor-1 induces functional plasticity in spinal cord following unilateral pyramidal tract injury in rat.

    Ping K Yip

    Full Text Available Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult cortical neurons forming the corticospinal tract (CST normally have low levels of the neuronal calcium sensor-1 (NCS1 protein. In primary cultured adult cortical neurons, the lentivector-induced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CST-denervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system.

  7. Ephrin-B1 controls the columnar distribution of cortical pyramidal neurons by restricting their tangential migration.

    Dimidschstein, Jordane; Passante, Lara; Dufour, Audrey; van den Ameele, Jelle; Tiberi, Luca; Hrechdakian, Tatyana; Adams, Ralf; Klein, Rüdiger; Lie, Dieter Chichung; Jossin, Yves; Vanderhaeghen, Pierre


    Neurons of the cerebral cortex are organized in layers and columns. Unlike laminar patterning, the mechanisms underlying columnar organization remain largely unexplored. Here, we show that ephrin-B1 plays a key role in this process through the control of nonradial steps of migration of pyramidal neurons. In vivo gain of function of ephrin-B1 resulted in a reduction of tangential motility of pyramidal neurons, leading to abnormal neuronal clustering. Conversely, following genetic disruption of ephrin-B1, cortical neurons displayed a wider lateral dispersion, resulting in enlarged ontogenic columns. Dynamic analyses revealed that ephrin-B1 controls the lateral spread of pyramidal neurons by limiting neurite extension and tangential migration during the multipolar phase. Furthermore, we identified P-Rex1, a guanine-exchange factor for Rac3, as a downstream ephrin-B1 effector required to control migration during the multipolar phase. Our results demonstrate that ephrin-B1 inhibits nonradial migration of pyramidal neurons, thereby controlling the pattern of cortical columns.

  8. Dendritic Na(+) spikes enable cortical input to drive action potential output from hippocampal CA2 pyramidal neurons.

    Sun, Qian; Srinivas, Kalyan V; Sotayo, Alaba; Siegelbaum, Steven A


    Synaptic inputs from different brain areas are often targeted to distinct regions of neuronal dendritic arbors. Inputs to proximal dendrites usually produce large somatic EPSPs that efficiently trigger action potential (AP) output, whereas inputs to distal dendrites are greatly attenuated and may largely modulate AP output. In contrast to most other cortical and hippocampal neurons, hippocampal CA2 pyramidal neurons show unusually strong excitation by their distal dendritic inputs from entorhinal cortex (EC). In this study, we demonstrate that the ability of these EC inputs to drive CA2 AP output requires the firing of local dendritic Na(+) spikes. Furthermore, we find that CA2 dendritic geometry contributes to the efficient coupling of dendritic Na(+) spikes to AP output. These results provide a striking example of how dendritic spikes enable direct cortical inputs to overcome unfavorable distal synaptic locale to trigger axonal AP output and thereby enable efficient cortico-hippocampal information flow.

  9. A possible role of the non-GAT1 GABA transporters in transfer of GABA from GABAergic to glutamatergic neurons in mouse cerebellar neuronal cultures

    Suñol, C; Babot, Z; Cristòfol, R


    . 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 GAT...... 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 m...... 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 GAT2 or GAT3 (HUGO nomenclature) could play a role....

  10. The neocortex of cetartiodactyls. II. Neuronal morphology of the visual and motor cortices in the giraffe (Giraffa camelopardalis).

    Jacobs, Bob; Harland, Tessa; Kennedy, Deborah; Schall, Matthew; Wicinski, Bridget; Butti, Camilla; Hof, Patrick R; Sherwood, Chet C; Manger, Paul R


    The present quantitative study extends our investigation of cetartiodactyls by exploring the neuronal morphology in the giraffe (Giraffa camelopardalis) neocortex. Here, we investigate giraffe primary visual and motor cortices from perfusion-fixed brains of three subadults stained with a modified rapid Golgi technique. Neurons (n = 244) were quantified on a computer-assisted microscopy system. Qualitatively, the giraffe neocortex contained an array of complex spiny neurons that included both "typical" pyramidal neuron morphology and "atypical" spiny neurons in terms of morphology and/or orientation. In general, the neocortex exhibited a vertical columnar organization of apical dendrites. Although there was no significant quantitative difference in dendritic complexity for pyramidal neurons between primary visual (n = 78) and motor cortices (n = 65), there was a significant difference in dendritic spine density (motor cortex > visual cortex). The morphology of aspiny neurons in giraffes appeared to be similar to that of other eutherian mammals. For cross-species comparison of neuron morphology, giraffe pyramidal neurons were compared to those quantified with the same methodology in African elephants and some cetaceans (e.g., bottlenose dolphin, minke whale, humpback whale). Across species, the giraffe (and cetaceans) exhibited less widely bifurcating apical dendrites compared to elephants. Quantitative dendritic measures revealed that the elephant and humpback whale had more extensive dendrites than giraffes, whereas the minke whale and bottlenose dolphin had less extensive dendritic arbors. Spine measures were highest in the giraffe, perhaps due to the high quality, perfusion fixation. The neuronal morphology in giraffe neocortex is thus generally consistent with what is known about other cetartiodactyls.

  11. Oxygen flux reduces Cux1 positive neurons and cortical growth in a gestational rodent model of growth restriction.

    Fletcher, Elaine; Wade, Jean; Georgala, Petrina A; Gillespie, Trudi L; Price, David J; Pilley, Elizabeth; Becher, Julie-Clare


    The mammalian cerebral cortex forms in an inside-out manner, establishing deep cortical layers before superficial layers and is regulated by transcription factors which influence cell differentiation. Preterm birth interrupts the trajectory of normal neurodevelopment and adverse perinatal exposures have been implicated in cortical injury. We hypothesise that growth restriction (GR) and fluctuating hyperoxia (ΔO2) impair cortical laminar development. Sprague-Dawley rats received 18% (non-restricted, NR) or 9% (growth restricted, GR) protein diet from E15-P7. Litters were reared in air or fluctuating hyperoxia (circa 10kPa) from P0 to P7. Cortical laminae were stained and measured. Neuronal subtypes were quantified using immunofluorescence for subtype-specific transcription factors (Satb2, Cux1, Ctip2, Tbr1). ΔO2 did not affect brain weight at P7 but reduced cortical thickness in both NR (pdevelopment in a rodent model with preferential disadvantage to superficial neurons. Copyright © 2016 Elsevier GmbH. All rights reserved.

  12. Power-law inter-spike interval distributions infer a conditional maximization of entropy in cortical neurons.

    Yasuhiro Tsubo

    Full Text Available The brain is considered to use a relatively small amount of energy for its efficient information processing. Under a severe restriction on the energy consumption, the maximization of mutual information (MMI, which is adequate for designing artificial processing machines, may not suit for the brain. The MMI attempts to send information as accurate as possible and this usually requires a sufficient energy supply for establishing clearly discretized communication bands. Here, we derive an alternative hypothesis for neural code from the neuronal activities recorded juxtacellularly in the sensorimotor cortex of behaving rats. Our hypothesis states that in vivo cortical neurons maximize the entropy of neuronal firing under two constraints, one limiting the energy consumption (as assumed previously and one restricting the uncertainty in output spike sequences at given firing rate. Thus, the conditional maximization of firing-rate entropy (CMFE solves a tradeoff between the energy cost and noise in neuronal response. In short, the CMFE sends a rich variety of information through broader communication bands (i.e., widely distributed firing rates at the cost of accuracy. We demonstrate that the CMFE is reflected in the long-tailed, typically power law, distributions of inter-spike intervals obtained for the majority of recorded neurons. In other words, the power-law tails are more consistent with the CMFE rather than the MMI. Thus, we propose the mathematical principle by which cortical neurons may represent information about synaptic input into their output spike trains.

  13. Effect of HDAC inhibitors on neuroprotection and neurite outgrowth in primary rat cortical neurons following ischemic insult.

    Hasan, Mohammad Rakibul; Kim, Ji-Hye; Kim, Youn Jung; Kwon, Kyoung Ja; Shin, Chan Young; Kim, Hahn Young; Han, Seol-Heui; Choi, Dong-Hee; Lee, Jongmin


    Histone deacetylase inhibitors (HDACi)-valproic acid (VPA) and trichostatin A (TSA) promote neurogenesis, neurite outgrowth, synaptic plasticity and neuroprotection. In this study, we investigated whether VPA and TSA promote post-ischemic neuroprotection and neuronal restoration in rat primary cortical neurons. On 6 days in vitro (DIV), cortical neurons were exposed to oxygen-glucose deprivation for 90 min. Cells were returned to normoxic conditions and cultured for 1, 3, or 7 days with or without VPA and TSA. Control cells were cultured in normoxic conditions only. On 7, 9, and 13 DIV, cells were measured neurite outgrowth using the Axiovision program and stained with Tunel staining kit. Microtubule associated protein-2 immunostaining and tunel staining showed significant recovery of neurite outgrowth and post-ischemic neuronal death by VPA or TSA treatment. We also determined levels of acetylated histone H3, PSD95, GAP 43 and synaptophysin. Significant increases in all three synaptic markers and acetylated histone H3 were observed relative to non-treated cells. Post-ischemic HDACi treatment also significantly raised levels of brain derived neurotrophic factor (BDNF) expression and secreted BDNF. Enhanced BDNF expression by HDACi treatment might have been involved in the post-ischemic neuroprotection and neuronal restorative effects. Our findings suggest that both VPA and TSA treatment during reoxygenation after ischemia may help post-ischemic neuroprotection and neuronal regeneration via increased BDNF expression and activation.

  14. Ethanol-induced disruption of Golgi apparatus morphology, primary neurite number and cellular orientation in developing cortical neurons.

    Powrozek, Teresa A; Olson, Eric C


    Prenatal ethanol exposure disrupts cortical neurite initiation and outgrowth, but prior studies have reported both ethanol-dependent growth promotion and inhibition. To resolve this ambiguity and better approximate in vivo conditions, we quantitatively analyzed neuronal morphology using a new, whole hemisphere explant model. In this model, Layer 6 (L6) cortical neurons migrate, laminate and extend neurites in an organotypic fashion. To selectively label L6 neurons, we performed ex utero electroporation of a GFP expression construct at embryonic day 13 and allowed the explants to develop for 2 days in vitro. Explants were exposed to (400 mg/dL) ethanol for either 4 or 24 h prior to fixation. Complete 3-D reconstructions were made of >80 GFP-positive neurons in each experimental condition. Acute responses to ethanol exposure included compaction of the Golgi apparatus accompanied by elaboration of supernumerary primary apical neurites, as well as a modest (∼15%) increase in higher order apical neurite length. With longer exposure time, ethanol exposure leads to a consistent, significant disorientation of the cell (cell body, primary apical neurite, and Golgi) with respect to the pial surface. The effects on cellular orientation were accompanied by decreased expression of cytoskeletal elements, microtubule-associated protein 2 and F-actin. These findings indicate that upon exposure to ethanol, developing L6 neurons manifest disruptions in Golgi apparatus and cytoskeletal elements which may in turn trigger selective and significant perturbations to primary neurite formation and neuronal polarity.

  15. Faithful SGCE imprinting in iPSC-derived cortical neurons: an endogenous cellular model of myoclonus-dystonia

    Grütz, Karen; Seibler, Philip; Weissbach, Anne; Lohmann, Katja; Carlisle, Francesca A.; Blake, Derek J.; Westenberger, Ana; Klein, Christine; Grünewald, Anne


    In neuropathology research, induced pluripotent stem cell (iPSC)-derived neurons are considered a tool closely resembling the patient brain. Albeit in respect to epigenetics, this concept has been challenged. We generated iPSC-derived cortical neurons from myoclonus-dystonia patients with mutations (W100G and R102X) in the maternally imprinted ε-sarcoglycan (SGCE) gene and analysed properties such as imprinting, mRNA and protein expression. Comparison of the promoter during reprogramming and differentiation showed tissue-independent differential methylation. DNA sequencing with methylation-specific primers and cDNA analysis in patient neurons indicated selective expression of the mutated paternal SGCE allele. While fibroblasts only expressed the ubiquitous mRNA isoform, brain-specific SGCE mRNA and ε-sarcoglycan protein were detected in iPSC-derived control neurons. However, neuronal protein levels were reduced in both mutants. Our phenotypic characterization highlights the suitability of iPSC-derived cortical neurons with SGCE mutations for myoclonus-dystonia research and, in more general terms, prompts the use of iPSC-derived cellular models to study epigenetic mechanisms impacting on health and disease. PMID:28155872

  16. Characterizing HSF1 Binding and Post-Translational Modifications of hsp70 Promoter in Cultured Cortical Neurons: Implications in the Heat-Shock Response.

    Andrea V Gómez

    Full Text Available Causes of lower induction of Hsp70 in neurons during heat shock are still a matter of debate. To further inquire into the mechanisms regulating Hsp70 expression in neurons, we studied the activity of Heat Shock Factor 1 (HSF1 and histone posttranslational modifications (PTMs at the hsp70 promoter in rat cortical neurons. Heat shock induced a transient and efficient translocation of HSF1 to neuronal nuclei. However, no binding of HSF1 at the hsp70 promoter was detected while it bound to the hsp25 promoter in cortical neurons during heat shock. Histone PTMs analysis showed that the hsp70 promoter harbors lower levels of histone H3 and H4 acetylation in cortical neurons compared to PC12 cells under basal conditions. Transcriptomic profiling data analysis showed a predominant usage of cryptic transcriptional start sites at hsp70 gene in the rat cerebral cortex, compared with the whole brain. These data support a weaker activation of hsp70 canonical promoter. Heat shock increased H3Ac at the hsp70 promoter in PC12 cells, which correlated with increased Hsp70 expression while no modifications occurred at the hsp70 promoter in cortical neurons. Increased histone H3 acetylation by Trichostatin A led to hsp70 mRNA and protein induction in cortical neurons. In conclusion, we found that two independent mechanisms maintain a lower induction of Hsp70 in cortical neurons. First, HSF1 fails to bind specifically to the hsp70 promoter in cortical neurons during heat shock and, second, the hsp70 promoter is less accessible in neurons compared to non-neuronal cells due to histone deacetylases repression.

  17. Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

    Ascoli, Giorgio A; Alonso-Nanclares, Lidia; Anderson, Stewart A; Barrionuevo, German; Benavides-Piccione, Ruth; Burkhalter, Andreas; Buzsáki, György; Cauli, Bruno; Defelipe, Javier; Fairén, Alfonso; Feldmeyer, Dirk; Fishell, Gord; Fregnac, Yves; Freund, Tamas F; Gardner, Daniel; Gardner, Esther P; Goldberg, Jesse H; Helmstaedter, Moritz; Hestrin, Shaul; Karube, Fuyuki; Kisvárday, Zoltán F; Lambolez, Bertrand; Lewis, David A; Marin, Oscar; Markram, Henry; Muñoz, Alberto; Packer, Adam; Petersen, Carl C H; Rockland, Kathleen S; Rossier, Jean; Rudy, Bernardo; Somogyi, Peter; Staiger, Jochen F; Tamas, Gabor; Thomson, Alex M; Toledo-Rodriguez, Maria; Wang, Yun; West, David C; Yuste, Rafael


    Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.

  18. Analysis of BH3-only proteins upregulated in response to oxygen/glucose deprivation in cortical neurons identifies Bmf but not Noxa as potential mediator of neuronal injury.

    Pfeiffer, S; Anilkumar, U; Chen, G; Ramírez-Peinado, S; Galindo-Moreno, J; Muñoz-Pinedo, C; Prehn, J H M


    Stress signaling in response to oxygen/glucose deprivation (OGD) and ischemic injury activates a group of pro-apoptotic genes, the Bcl-2 homology domain 3 (BH3)-only proteins, which are capable of activating the mitochondrial apoptosis pathway. Targeted studies previously identified the BH3-only proteins Puma, Bim and Bid to have a role in ischemic/hypoxic neuronal injury. We here investigated the transcriptional activation of pro-apoptotic BH3-only proteins after OGD-induced injury in murine neocortical neurons. We observed a potent and early upregulation of noxa at mRNA and protein level, and a significant increase in Bmf protein levels during OGD in neocortical neurons and in the ipsilateral cortex of mice subjected to transient middle cerebral artery occlusion (tMCAO). Surprisingly, gene deficiency in noxa reduced neither OGD- nor glutamate-induced neuronal injury in cortical neurons and failed to influence infarct size or neurological deficits after tMCAO. In contrast, bmf deficiency induced significant protection against OGD- or glutamate-induced injury in cultured neurons, and bmf-deficient mice showed reduced neurological deficits after tMCAO in vivo. Collectively, our data not only point to a role of Bmf as a BH3-only protein contributing to excitotoxic and ischemic neuronal injury but also demonstrate that the early and potent induction of noxa does not influence ischemic neuronal injury.

  19. Impairment of GABA transporter GAT-1 terminates cortical recurrent network activity via enhanced phasic inhibition

    Daniel Simon Razik


    Full Text Available In the central nervous system, GABA transporters (GATs very efficiently clear synaptically released GABA from the extracellular space, and thus exert a tight control on GABAergic inhibition. In neocortex, GABAergic inhibition is heavily recruited during recurrent phases of spontaneous action potential activity which alternate with neuronally quiet periods. Therefore, such activity should be quite sensitive to minute alterations of GAT function. Here, we explored the effects of a gradual impairment of GAT-1 and GAT-2/3 on spontaneous recurrent network activity – termed network bursts and silent periods – in organotypic slice cultures of rat neocortex. The GAT-1 specific antagonist NO-711 depressed activity already at nanomolar concentrations (IC50 for depression of spontaneous multiunit firing rate of 42 nM, reaching a level of 80% at 500-1000 nM. By contrast, the GAT-2/3 preferring antagonist SNAP-5114 had weaker and less consistent effects. Several lines of evidence pointed towards an enhancement of phasic GABAergic inhibition as the dominant activity-depressing mechanism: network bursts were drastically shortened, phasic GABAergic currents decayed slower, and neuronal excitability during ongoing activity was diminished. In silent periods, NO-711 had little effect on neuronal excitability or membrane resistance, quite in contrast to the effects of muscimol, a GABA mimetic which activates GABAA receptors tonically. Our results suggest that an enhancement of phasic GABAergic inhibition efficiently curtails cortical recurrent activity and may mediate antiepileptic effects of therapeutically relevant concentrations of GAT-1 antagonists.

  20. Protective effects of N-methyl-D-aspartate receptor antagonism on VX-induced neuronal cell death in cultured rat cortical neurons.

    Wang, Yushan; Weiss, M Tracy; Yin, Junfei; Tenn, Catherine C; Nelson, Peggy D; Mikler, John R


    Exposure of the central nervous system to organophosphorus (OP) nerve agents induces seizures and neuronal cell death. Here we report that the OP nerve agent, VX, induces apoptotic-like cell death in cultured rat cortical neurons. The VX effects on neurons were concentration-dependent, with an IC(50) of approximately 30 microM. Blockade of N-methyl-D-aspartate receptors (NMDAR) with 50 microM. D-2-amino-5-phosphonovalerate (APV) diminished 30 microM VX-induced total cell death, as assessed by alamarBlue assay and Hoechst staining. In contrast, neither antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) nor metabotropic glutamate receptors (mGluRs) had any effect on VX-induced neurotoxicity. VX-induced neuronal cell death could not be solely attributed to acetylcholinesterase (AChE) inhibition, since neither the reversible pharmacological cholinesterase inhibitor, physostigmine, nor the muscarinic receptor antagonist, atropine, affected VX-induced cell death. Importantly, APV was found to be therapeutically effective against VX-induced cell death up to 2 h post VX exposure. These results suggest that NMDARs, but not AMPARs or mGluRs, play important roles in VX-induced cell death in cultured rat cortical neurons. Based on their therapeutic effects, NMDAR antagonists may be beneficial in the treatment of VX-induced neurotoxicities.

  1. Network-state modulation of power-law frequency-scaling in visual cortical neurons.

    El Boustani, Sami; Marre, Olivier; Béhuret, Sébastien; Baudot, Pierre; Yger, Pierre; Bal, Thierry; Destexhe, Alain; Frégnac, Yves


    Various types of neural-based signals, such as EEG, local field potentials and intracellular synaptic potentials, integrate multiple sources of activity distributed across large assemblies. They have in common a power-law frequency-scaling structure at high frequencies, but it is still unclear whether this scaling property is dominated by intrinsic neuronal properties or by network activity. The latter case is particularly interesting because if frequency-scaling reflects the network state it could be used to characterize the functional impact of the connectivity. In intracellularly recorded neurons of cat primary visual cortex in vivo, the power spectral density of V(m) activity displays a power-law structure at high frequencies with a fractional scaling exponent. We show that this exponent is not constant, but depends on the visual statistics used to drive the network. To investigate the determinants of this frequency-scaling, we considered a generic recurrent model of cortex receiving a retinotopically organized external input. Similarly to the in vivo case, our in computo simulations show that the scaling exponent reflects the correlation level imposed in the input. This systematic dependence was also replicated at the single cell level, by controlling independently, in a parametric way, the strength and the temporal decay of the pairwise correlation between presynaptic inputs. This last model was implemented in vitro by imposing the correlation control in artificial presynaptic spike trains through dynamic-clamp techniques. These in vitro manipulations induced a modulation of the scaling exponent, similar to that observed in vivo and predicted in computo. We conclude that the frequency-scaling exponent of the V(m) reflects stimulus-driven correlations in the cortical network activity. Therefore, we propose that the scaling exponent could be used to read-out the "effective" connectivity responsible for the dynamical signature of the population signals measured

  2. Network-state modulation of power-law frequency-scaling in visual cortical neurons.

    Sami El Boustani


    Full Text Available Various types of neural-based signals, such as EEG, local field potentials and intracellular synaptic potentials, integrate multiple sources of activity distributed across large assemblies. They have in common a power-law frequency-scaling structure at high frequencies, but it is still unclear whether this scaling property is dominated by intrinsic neuronal properties or by network activity. The latter case is particularly interesting because if frequency-scaling reflects the network state it could be used to characterize the functional impact of the connectivity. In intracellularly recorded neurons of cat primary visual cortex in vivo, the power spectral density of V(m activity displays a power-law structure at high frequencies with a fractional scaling exponent. We show that this exponent is not constant, but depends on the visual statistics used to drive the network. To investigate the determinants of this frequency-scaling, we considered a generic recurrent model of cortex receiving a retinotopically organized external input. Similarly to the in vivo case, our in computo simulations show that the scaling exponent reflects the correlation level imposed in the input. This systematic dependence was also replicated at the single cell level, by controlling independently, in a parametric way, the strength and the temporal decay of the pairwise correlation between presynaptic inputs. This last model was implemented in vitro by imposing the correlation control in artificial presynaptic spike trains through dynamic-clamp techniques. These in vitro manipulations induced a modulation of the scaling exponent, similar to that observed in vivo and predicted in computo. We conclude that the frequency-scaling exponent of the V(m reflects stimulus-driven correlations in the cortical network activity. Therefore, we propose that the scaling exponent could be used to read-out the "effective" connectivity responsible for the dynamical signature of the population

  3. Neuronal and microglial regulators of cortical wiring: usual and novel guideposts

    Paola eSquarzoni


    Full Text Available Neocortex functioning relies on the formation of complex networks that begins to be assembled duri