Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

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Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

2015-01-01

The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

Optogenetically enhanced axon regeneration: motor versus sensory neuron-specific stimulation.

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Ward, Patricia J; Clanton, Scott L; English, Arthur W

2018-02-01

Brief neuronal activation in injured peripheral nerves is both necessary and sufficient to enhance motor axon regeneration, and this effect is specific to the activated motoneurons. It is less clear whether sensory neurons respond in a similar manner to neuronal activation following peripheral axotomy. Further, it is unknown to what extent enhancement of axon regeneration with increased neuronal activity relies on a reflexive interaction within the spinal circuitry. We used mouse genetics and optical tools to evaluate the precision and selectivity of system-specific neuronal activation to enhance axon regeneration in a mixed nerve. We evaluated sensory and motor axon regeneration in two different mouse models expressing the light-sensitive cation channel, channelrhodopsin (ChR2). We selectively activated either sensory or motor axons using light stimulation combined with transection and repair of the sciatic nerve. Regardless of genotype, the number of ChR2-positive neurons whose axons had regenerated successfully was greater following system-specific optical treatment, with no effect on the number of ChR2-negative neurons (whether motor or sensory neurons). We conclude that acute system-specific neuronal activation is sufficient to enhance both motor and sensory axon regeneration. This regeneration-enhancing effect is likely cell autonomous. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Functional Maturation of Human Stem Cell-Derived Neurons in Long-Term Cultures.

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Rebecca S Lam

Full Text Available Differentiated neurons can be rapidly acquired, within days, by inducing stem cells to express neurogenic transcription factors. We developed a protocol to maintain long-term cultures of human neurons, called iNGNs, which are obtained by inducing Neurogenin-1 and Neurogenin-2 expression in induced pluripotent stem cells. We followed the functional development of iNGNs over months and they showed many hallmark properties for neuronal maturation, including robust electrical and synaptic activity. Using iNGNs expressing a variant of channelrhodopsin-2, called CatCh, we could control iNGN activity with blue light stimulation. In combination with optogenetic tools, iNGNs offer opportunities for studies that require precise spatial and temporal resolution. iNGNs developed spontaneous network activity, and these networks had excitatory glutamatergic synapses, which we characterized with single-cell synaptic recordings. AMPA glutamatergic receptor activity was especially dominant in postsynaptic recordings, whereas NMDA glutamatergic receptor activity was absent from postsynaptic recordings but present in extrasynaptic recordings. Our results on long-term cultures of iNGNs could help in future studies elucidating mechanisms of human synaptogenesis and neurotransmission, along with the ability to scale-up the size of the cultures.

Activation of temperature-sensitive TRPV1-like receptors in ARC POMC neurons reduces food intake.

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Jae Hoon Jeong

2018-04-01

Full Text Available Proopiomelanocortin (POMC neurons in the arcuate nucleus of the hypothalamus (ARC respond to numerous hormonal and neural signals, resulting in changes in food intake. Here, we demonstrate that ARC POMC neurons express capsaicin-sensitive transient receptor potential vanilloid 1 receptor (TRPV1-like receptors. To show expression of TRPV1-like receptors in ARC POMC neurons, we use single-cell reverse transcription-polymerase chain reaction (RT-PCR, immunohistochemistry, electrophysiology, TRPV1 knock-out (KO, and TRPV1-Cre knock-in mice. A small elevation of temperature in the physiological range is enough to depolarize ARC POMC neurons. This depolarization is blocked by the TRPV1 receptor antagonist and by Trpv1 gene knockdown. Capsaicin-induced activation reduces food intake that is abolished by a melanocortin receptor antagonist. To selectively stimulate TRPV1-like receptor-expressing ARC POMC neurons in the ARC, we generate an adeno-associated virus serotype 5 (AAV5 carrying a Cre-dependent channelrhodopsin-2 (ChR2-enhanced yellow fluorescent protein (eYFP expression cassette under the control of the two neuronal POMC enhancers (nPEs. Optogenetic stimulation of TRPV1-like receptor-expressing POMC neurons decreases food intake. Hypothalamic temperature is rapidly elevated and reaches to approximately 39 °C during treadmill running. This elevation is associated with a reduction in food intake. Knockdown of the Trpv1 gene exclusively in ARC POMC neurons blocks the feeding inhibition produced by increased hypothalamic temperature. Taken together, our findings identify a melanocortinergic circuit that links acute elevations in hypothalamic temperature with acute reductions in food intake.

Optogenetic stimulation of locus ceruleus neurons augments inhibitory transmission to parasympathetic cardiac vagal neurons via activation of brainstem α1 and β1 receptors.

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Wang, Xin; Piñol, Ramón A; Byrne, Peter; Mendelowitz, David

2014-04-30

Locus ceruleus (LC) noradrenergic neurons are critical in generating alertness. In addition to inducing cortical arousal, the LC also orchestrates changes in accompanying autonomic system function that compliments increased attention, such as during stress, excitation, and/or exposure to averse or novel stimuli. Although the association between arousal and increased heart rate is well accepted, the neurobiological link between the LC and parasympathetic neurons that control heart rate has not been identified. In this study, we test directly whether activation of noradrenergic neurons in the LC influences brainstem parasympathetic cardiac vagal neurons (CVNs). CVNs were identified in transgenic mice that express channel-rhodopsin-2 (ChR2) in LC tyrosine hydroxylase neurons. Photoactivation evoked a rapid depolarization, increased firing, and excitatory inward currents in ChR2-expressing neurons in the LC. Photostimulation of LC neurons did not alter excitatory currents, but increased inhibitory neurotransmission to CVNs. Optogenetic activation of LC neurons increased the frequency of isolated glycinergic IPSCs by 27 ± 8% (p = 0.003, n = 26) and augmented GABAergic IPSCs in CVNs by 21 ± 5% (p = 0.001, n = 26). Inhibiting α1, but not α2, receptors blocked the evoked responses. Inhibiting β1 receptors prevented the increase in glycinergic, but not GABAergic, IPSCs in CVNs. This study demonstrates LC noradrenergic neurons inhibit the brainstem CVNs that generate parasympathetic activity to the heart. This inhibition of CVNs would increase heart rate and risks associated with tachycardia. The receptors activated within this pathway, α1 and/or β1 receptors, are targets for clinically prescribed antagonists that promote slower, cardioprotective heart rates during heightened vigilant states.

PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording.

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Susana Q Lima

Full Text Available Neural circuits are exquisitely organized, consisting of many different neuronal subpopulations. However, it is difficult to assess the functional roles of these subpopulations using conventional extracellular recording techniques because these techniques do not easily distinguish spikes from different neuronal populations. To overcome this limitation, we have developed PINP (Photostimulation-assisted Identification of Neuronal Populations, a method of tagging neuronal populations for identification during in vivo electrophysiological recording. The method is based on expressing the light-activated channel channelrhodopsin-2 (ChR2 to restricted neuronal subpopulations. ChR2-tagged neurons can be detected electrophysiologically in vivo since illumination of these neurons with a brief flash of blue light triggers a short latency reliable action potential. We demonstrate the feasibility of this technique by expressing ChR2 in distinct populations of cortical neurons using two different strategies. First, we labeled a subpopulation of cortical neurons-mainly fast-spiking interneurons-by using adeno-associated virus (AAV to deliver ChR2 in a transgenic mouse line in which the expression of Cre recombinase was driven by the parvalbumin promoter. Second, we labeled subpopulations of excitatory neurons in the rat auditory cortex with ChR2 based on projection target by using herpes simplex virus 1 (HSV1, which is efficiently taken up by axons and transported retrogradely; we find that this latter population responds to acoustic stimulation differently from unlabeled neurons. Tagging neurons is a novel application of ChR2, used in this case to monitor activity instead of manipulating it. PINP can be readily extended to other populations of genetically identifiable neurons, and will provide a useful method for probing the functional role of different neuronal populations in vivo.

Linking Neurons to Network Function and Behavior by Two-Photon Holographic Optogenetics and Volumetric Imaging.

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Dal Maschio, Marco; Donovan, Joseph C; Helmbrecht, Thomas O; Baier, Herwig

2017-05-17

We introduce a flexible method for high-resolution interrogation of circuit function, which combines simultaneous 3D two-photon stimulation of multiple targeted neurons, volumetric functional imaging, and quantitative behavioral tracking. This integrated approach was applied to dissect how an ensemble of premotor neurons in the larval zebrafish brain drives a basic motor program, the bending of the tail. We developed an iterative photostimulation strategy to identify minimal subsets of channelrhodopsin (ChR2)-expressing neurons that are sufficient to initiate tail movements. At the same time, the induced network activity was recorded by multiplane GCaMP6 imaging across the brain. From this dataset, we computationally identified activity patterns associated with distinct components of the elicited behavior and characterized the contributions of individual neurons. Using photoactivatable GFP (paGFP), we extended our protocol to visualize single functionally identified neurons and reconstruct their morphologies. Together, this toolkit enables linking behavior to circuit activity with unprecedented resolution. Copyright © 2017 Elsevier Inc. All rights reserved.

Proliferating neuronal progenitors in the postnatal hippocampus transiently express the proneural gene Ngn2.

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Ozen, Ilknur; Galichet, Christophe; Watts, Colin; Parras, Carlos; Guillemot, François; Raineteau, Olivier

2007-05-01

Little is known of the transcription factors expressed by adult neural progenitors produced in the hippocampal neurogenic niche. Here, we study the expression of the proneural basic helix-loop-helix (bHLH) transcription factor Neurogenin-2 (Ngn2) in the adult hippocampus. We have characterized the pattern of expression of Ngn2 in the adult hippocampus using immunostaining for Ngn2 protein and a Ngn2-green fluorescent protein (GFP) reporter mouse strain. A significant proportion of Ngn2-expressing cells were mitotically active. Ngn2-GFP expression was restricted to the subgranular zone and declined with age. Neuronal markers were used to determine the phenotype of Ngn2-expressing cells. The vast majority of Ngn2-GFP-positive cells expressed the immature neuronal markers, doublecortin (DCX) and polysialic acid-neural cell adhesion molecule (PSA-NCAM). Finally, the pattern of Ngn2 expression was studied following seizure induction. Our data show an increase in neurogenesis, detected in these animals by bromodeoxyuridine (BrdU) and DCX staining that was contemporaneous with a marked increase in Ngn2-GFP-expression. Taken together, our results show that Ngn2-GFP represents a specific marker for neurogenesis and its modulation in the adult hippocampus. Ngn2 transient expression in proliferating neuronal progenitors supports the idea that it plays a significant role in adult neurogenesis.

Parvalbumin-expressing interneurons can act solo while somatostatin-expressing interneurons act in chorus in most cases on cortical pyramidal cells.

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Safari, Mir-Shahram; Mirnajafi-Zadeh, Javad; Hioki, Hiroyuki; Tsumoto, Tadaharu

2017-10-06

Neural circuits in the cerebral cortex consist primarily of excitatory pyramidal (Pyr) cells and inhibitory interneurons. Interneurons are divided into several subtypes, in which the two major groups are those expressing parvalbumin (PV) or somatostatin (SOM). These subtypes of interneurons are reported to play distinct roles in tuning and/or gain of visual response of pyramidal cells in the visual cortex. It remains unclear whether there is any quantitative and functional difference between the PV → Pyr and SOM → Pyr connections. We compared unitary inhibitory postsynaptic currents (uIPSCs) evoked by electrophysiological activation of single presynaptic interneurons with population IPSCs evoked by photo-activation of a mass of interneurons in vivo and in vitro in transgenic mice in which PV or SOM neurons expressed channelrhodopsin-2, and found that at least about 14 PV neurons made strong connections with a postsynaptic Pyr cell while a much larger number of SOM neurons made weak connections. Activation or suppression of single PV neurons modified visual responses of postsynaptic Pyr cells in 6 of 7 pairs whereas that of single SOM neurons showed no significant modification in 8 of 11 pairs, suggesting that PV neurons can act solo whereas most of SOM neurons may act in chorus on Pyr cells.

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

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Brill Monika S

2011-04-01

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

Activation of D2 dopamine receptor-expressing neurons in the nucleus accumbens increases motivation

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Soares-Cunha, Carina; Coimbra, Barbara; David-Pereira, Ana; Borges, Sonia; Pinto, Luisa; Costa, Patricio; Sousa, Nuno; Rodrigues, Ana J.

2016-01-01

Striatal dopamine receptor D1-expressing neurons have been classically associated with positive reinforcement and reward, whereas D2 neurons are associated with negative reinforcement and aversion. Here we demonstrate that the pattern of activation of D1 and D2 neurons in the nucleus accumbens (NAc) predicts motivational drive, and that optogenetic activation of either neuronal population enhances motivation in mice. Using a different approach in rats, we further show that activating NAc D2 neurons increases cue-induced motivational drive in control animals and in a model that presents anhedonia and motivational deficits; conversely, optogenetic inhibition of D2 neurons decreases motivation. Our results suggest that the classic view of D1–D2 functional antagonism does not hold true for all dimensions of reward-related behaviours, and that D2 neurons may play a more prominent pro-motivation role than originally anticipated. PMID:27337658

Drug-driven AMPA receptor redistribution mimicked by selective dopamine neuron stimulation.

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Matthew T C Brown

2010-12-01

Full Text Available Addictive drugs have in common that they cause surges in dopamine (DA concentration in the mesolimbic reward system and elicit synaptic plasticity in DA neurons of the ventral tegmental area (VTA. Cocaine for example drives insertion of GluA2-lacking AMPA receptors (AMPARs at glutamatergic synapes in DA neurons. However it remains elusive which molecular target of cocaine drives such AMPAR redistribution and whether other addictive drugs (morphine and nicotine cause similar changes through their effects on the mesolimbic DA system.We used in vitro electrophysiological techniques in wild-type and transgenic mice to observe the modulation of excitatory inputs onto DA neurons by addictive drugs. To observe AMPAR redistribution, post-embedding immunohistochemistry for GluA2 AMPAR subunit was combined with electron microscopy. We also used a double-floxed AAV virus expressing channelrhodopsin together with a DAT Cre mouse line to selectively express ChR2 in VTA DA neurons. We find that in mice where the effect of cocaine on the dopamine transporter (DAT is specifically blocked, AMPAR redistribution was absent following administration of the drug. Furthermore, addictive drugs known to increase dopamine levels cause a similar AMPAR redistribution. Finally, activating DA VTA neurons optogenetically is sufficient to drive insertion of GluA2-lacking AMPARs, mimicking the changes observed after a single injection of morphine, nicotine or cocaine.We propose the mesolimbic dopamine system as a point of convergence at which addictive drugs can alter neural circuits. We also show that direct activation of DA neurons is sufficient to drive AMPAR redistribution, which may be a mechanism associated with early steps of non-substance related addictions.

Thy1.2 YFP-16 transgenic mouse labels a subset of large-diameter sensory neurons that lack TRPV1 expression.

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Thomas E Taylor-Clark

Full Text Available The Thy1.2 YFP-16 mouse expresses yellow fluorescent protein (YFP in specific subsets of peripheral and central neurons. The original characterization of this model suggested that YFP was expressed in all sensory neurons, and this model has been subsequently used to study sensory nerve structure and function. Here, we have characterized the expression of YFP in the sensory ganglia (DRG, trigeminal and vagal of the Thy1.2 YFP-16 mouse, using biochemical, functional and anatomical analyses. Despite previous reports, we found that YFP was only expressed in approximately half of DRG and trigeminal neurons and less than 10% of vagal neurons. YFP-expression was only found in medium and large-diameter neurons that expressed neurofilament but not TRPV1. YFP-expressing neurons failed to respond to selective agonists for TRPV1, P2X(2/3 and TRPM8 channels in Ca2+ imaging assays. Confocal analysis of glabrous skin, hairy skin of the back and ear and skeletal muscle indicated that YFP was expressed in some peripheral terminals with structures consistent with their presumed non-nociceptive nature. In summary, the Thy1.2 YFP-16 mouse expresses robust YFP expression in only a subset of sensory neurons. But this mouse model is not suitable for the study of nociceptive nerves or the function of such nerves in pain and neuropathies.

Neurons That Underlie Drosophila melanogaster Reproductive Behaviors: Detection of a Large Male-Bias in Gene Expression in fruitless-Expressing Neurons

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Nicole R. Newell

2016-08-01

Full Text Available Male and female reproductive behaviors in Drosophila melanogaster are vastly different, but neurons that express sex-specifically spliced fruitless transcripts (fru P1 underlie these behaviors in both sexes. How this set of neurons can generate such different behaviors between the two sexes is an unresolved question. A particular challenge is that fru P1-expressing neurons comprise only 2–5% of the adult nervous system, and so studies of adult head tissue or whole brain may not reveal crucial differences. Translating Ribosome Affinity Purification (TRAP identifies the actively translated pool of mRNAs from fru P1-expressing neurons, allowing a sensitive, cell-type-specific assay. We find four times more male-biased than female-biased genes in TRAP mRNAs from fru P1-expressing neurons. This suggests a potential mechanism to generate dimorphism in behavior. The male-biased genes may direct male behaviors by establishing cell fate in a similar context of gene expression observed in females. These results suggest a possible global mechanism for how distinct behaviors can arise from a shared set of neurons.

Serotonergic neurons signal reward and punishment on multiple timescales

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Cohen, Jeremiah Y; Amoroso, Mackenzie W; Uchida, Naoshige

2015-01-01

Serotonin's function in the brain is unclear. One challenge in testing the numerous hypotheses about serotonin's function has been observing the activity of identified serotonergic neurons in animals engaged in behavioral tasks. We recorded the activity of dorsal raphe neurons while mice experienced a task in which rewards and punishments varied across blocks of trials. We ‘tagged’ serotonergic neurons with the light-sensitive protein channelrhodopsin-2 and identified them based on their responses to light. We found three main features of serotonergic neuron activity: (1) a large fraction of serotonergic neurons modulated their tonic firing rates over the course of minutes during reward vs punishment blocks; (2) most were phasically excited by punishments; and (3) a subset was phasically excited by reward-predicting cues. By contrast, dopaminergic neurons did not show firing rate changes across blocks of trials. These results suggest that serotonergic neurons signal information about reward and punishment on multiple timescales. DOI: http://dx.doi.org/10.7554/eLife.06346.001 PMID:25714923

Bcl-2 over-expression fails to prevent age-related loss of calretinin positive neurons in the mouse dentate gyrus

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Han Mingbo

2006-08-01

Full Text Available Abstract Background Cognitive performance declines with increasing age. Possible cellular mechanisms underlying this age-related functional decline remain incompletely understood. Early studies attributed this functional decline to age-related neuronal loss. Subsequent studies using unbiased stereological techniques found little or no neuronal loss during aging. However, studies using specific cellular markers found age-related loss of specific neuronal types. To test whether there is age-related loss of specific neuronal populations in the hippocampus, and subsequently, whether over-expression of the B-cell lymphoma protein-2 (Bcl-2 in these neurons could delay possible age-related neuronal loss, we examined calretinin (CR positive neurons in the mouse dentate gyrus during aging. Result In normal mice, there was an age-related loss of CR positive cells in the dentate gyrus. At the same region, there was no significant decrease of total numbers of neurons, which suggested that age-related loss of CR positive cells was due to the decrease of CR expression in these cells instead of cell death. In the transgenic mouse line over-expressing Bcl-2 in neurons, there was an age-related loss of CR positive cells. Interestingly, there was also an age-related neuronal loss in this transgenic mouse line. Conclusion These data suggest an age-related loss of CR positive neurons but not total neuronal loss in normal mice and this age-related neuronal change is not prevented by Bcl-2 over-expression.

Neuronal and glial expression of inward rectifier potassium channel subunits Kir2.x in rat dorsal root ganglion and spinal cord.

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Murata, Yuzo; Yasaka, Toshiharu; Takano, Makoto; Ishihara, Keiko

2016-03-23

Inward rectifier K(+) channels of the Kir2.x subfamily play important roles in controlling the neuronal excitability. Although their cellular localization in the brain has been extensively studied, only a few studies have examined their expression in the spinal cord and peripheral nervous system. In this study, immunohistochemical analyses of Kir2.1, Kir2.2, and Kir2.3 expression were performed in rat dorsal root ganglion (DRG) and spinal cord using bright-field and confocal microscopy. In DRG, most ganglionic neurons expressed Kir2.1, Kir2.2 and Kir2.3, whereas satellite glial cells chiefly expressed Kir2.3. In the spinal cord, Kir2.1, Kir2.2 and Kir2.3 were all expressed highly in the gray matter of dorsal and ventral horns and moderately in the white matter also. Within the gray matter, the expression was especially high in the substantia gelatinosa (lamina II). Confocal images obtained using markers for neuronal cells, NeuN, and astrocytes, Sox9, showed expression of all three Kir2 subunits in both neuronal somata and astrocytes in lamina I-III of the dorsal horn and the lateral spinal nucleus of the dorsolateral funiculus. Immunoreactive signals other than those in neuronal and glial somata were abundant in lamina I and II, which probably located mainly in nerve fibers or nerve terminals. Colocalization of Kir2.1 and 2.3 and that of Kir2.2 and 2.3 were present in neuronal and glial somata. In the ventral horn, motor neurons and interneurons were also immunoreactive with the three Kir2 subunits. Our study suggests that Kir2 channels composed of Kir2.1-2.3 subunits are expressed in neuronal and glial cells in the DRG and spinal cord, contributing to sensory transduction and motor control. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Optogenetics of the Spinal Cord: Use of Channelrhodopsin Proteins for Interrogation of Spinal Cord Circuits.

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Rahman, Habibur; Nam, Youngpyo; Kim, Jae-Hong; Lee, Won-Ha; Suk, Kyoungho

2017-12-29

Spinal cord circuits play a key role in receiving and transmitting somatosensory information from the body and the brain. They also contribute to the timing and coordination of complex patterns of movement. Under disease conditions, such as spinal cord injury and neuropathic pain, spinal cord circuits receive pain signals from peripheral nerves, and are involved in pain development via neurotransmitters and inflammatory mediators released from neurons and glial cells. Despite the importance of spinal cord circuits in sensory and motor functions, many questions remain regarding the relationship between activation of specific cells and behavioral responses. Optogenetics offers the possibility of understanding the complex cellular activity and mechanisms of spinal cord circuits, as well as having therapeutic potential for addressing spinal cord-related disorders. In this review, we discuss recent findings in optogenetic research employing the channelrhodopsin protein to assess the function of specific neurons and glia in spinal cord circuits ex vivo and in vivo. We also explore the possibilities and challenges of employing optogenetics technology in future therapeutic strategies for the treatment of spinal disorders. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Channel properties of Nax expressed in neurons.

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Masahito Matsumoto

Full Text Available Nax is a sodium-concentration ([Na+]-sensitive Na channel with a gating threshold of ~150 mM for extracellular [Na+] ([Na+]o in vitro. We previously reported that Nax was preferentially expressed in the glial cells of sensory circumventricular organs including the subfornical organ, and was involved in [Na+] sensing for the control of salt-intake behavior. Although Nax was also suggested to be expressed in the neurons of some brain regions including the amygdala and cerebral cortex, the channel properties of Nax have not yet been adequately characterized in neurons. We herein verified that Nax was expressed in neurons in the lateral amygdala of mice using an antibody that was newly generated against mouse Nax. To investigate the channel properties of Nax expressed in neurons, we established an inducible cell line of Nax using the mouse neuroblastoma cell line, Neuro-2a, which is endogenously devoid of the expression of Nax. Functional analyses of this cell line revealed that the [Na+]-sensitivity of Nax in neuronal cells was similar to that expressed in glial cells. The cation selectivity sequence of the Nax channel in cations was revealed to be Na+ ≈ Li+ > Rb+ > Cs+ for the first time. Furthermore, we demonstrated that Nax bound to postsynaptic density protein 95 (PSD95 through its PSD95/Disc-large/ZO-1 (PDZ-binding motif at the C-terminus in neurons. The interaction between Nax and PSD95 may be involved in promoting the surface expression of Nax channels because the depletion of endogenous PSD95 resulted in a decrease in Nax at the plasma membrane. These results indicated, for the first time, that Nax functions as a [Na+]-sensitive Na channel in neurons as well as in glial cells.

Optogenetic activation of neocortical neurons in vivo with a sapphire-based micro-scale LED probe

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Niall eMcAlinden

2015-05-01

Full Text Available Optogenetics has proven to be a revolutionary technology in neuroscience and has advanced continuously over the past decade. However, optical stimulation technologies for in vivo need to be developed to match the advances in genetics and biochemistry that have driven this field. In particular, conventional approaches for in vivo optical illumination have a limitation on the achievable spatio-temporal resolution. Here we utilize a sapphire-based microscale gallium nitride light-emitting diode (µLED probe to activate neocortical neurons in vivo. The probes were designed to contain independently controllable multiple µLEDs, emitting at 450 nm wavelength with an irradiance of up to 2 W/mm2. Monte-Carlo stimulations predicted that optical stimulation using a µLED can modulate neural activity within a localized region. To validate this prediction, we tested this probe in the mouse neocortex that expressed channelrhodopsin-2 (ChR2 and compared the results with optical stimulation through a fiber at the cortical surface. We confirmed that both approaches reliably induced action potentials in cortical neurons and that the µLED probe evoked strong responses in deep neurons. Due to the possibility to integrate many optical stimulation sites onto a single shank, the µLED probe is thus a promising approach to control neurons locally in vivo.

Comparative functional expression of nAChR subtypes in rodent DRG neurons.

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Smith, Nathan J; Hone, Arik J; Memon, Tosifa; Bossi, Simon; Smith, Thomas E; McIntosh, J Michael; Olivera, Baldomero M; Teichert, Russell W

2013-01-01

We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly α3β4 and α6β4 nAChRs; (2) neurons that express predominantly α7 nAChRs; (3) neurons that express a combination of α3β4/α6β4 and α7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70-80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15-30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca(2) (+) transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.

[Knockdown of dopamine receptor D2 upregulates the expression of adiogenic genes in mouse primary mesencephalic neurons].

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Ding, Jiaqi; Chen, Xiaoli; Lin, Jiaji; Zhu, Junling; Li, Zhuyi

2018-01-01

Objective To study the effects of dopamine receptor D2 (DRD2) on the adipogenesis genes in mouse primary mesencephalic neurons. Methods The lentiviral vectors which expressed specific shRNA targeting DRD2 were constructed to decrease DRD2 expression in mouse primary mesencephalic neurons. High throughput sequencing (HTS) analysis was used to investigate gene expression changes between the DRD2 knock-down group and the negative control group. Real-time quantitative PCR (qRT-PCR) and Western blot analysis were applied to verify the differently expressed genes. Fatty acids were measured by fatty acid detection kit. Results DRD2 expression was effectively down-regulated in mouse primary mesencephalic neurons by lentiviral vectors. HTS revealed adipogenesis genes were significantly up-regulated after DRD2 down-regulation, mainly including delta(14)-sterol reductase, acetyl-coenzyme A synthetase, insulin-induced gene 1 protein and especially stearoyl-coenzyme A desaturase 1 (SCD1, 4-fold upregulated). The qRT-PCR and Western blot analysis verified that SCD1 was upregulated 2.6 folds and 2 folds respectively by lentiviral DRD2-shRNA vectors. Moreover, the SCD1-related free fatty acids were significantly more increased than the negative control group. Conclusion DRD2 in primary mesencephalic neurons had a significant regulative effect on the adipogenesis genes. The up-regulation of SCD1 can accelerate the conversion of saturated fatty acids to monounsaturated fatty acids and prevent the damage of lipid toxicity to cells.

Phospholipase D1 increases Bcl-2 expression during neuronal differentiation of rat neural stem cells.

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Park, Shin-Young; Ma, Weina; Yoon, Sung Nyo; Kang, Min Jeong; Han, Joong-Soo

2015-01-01

We studied the possible role of phospholipase D1 (PLD1) in the neuronal differentiation, including neurite formation of neural stem cells. PLD1 protein and PLD activity increased during neuronal differentiation. Bcl-2 also increased. Downregulation of PLD1 by transfection with PLD1 siRNA or a dominant-negative form of PLD1 (DN-PLD1) inhibited both neurite outgrowth and Bcl-2 expression. PLD activity was dramatically reduced by a PLCγ (phospholipase Cγ) inhibitor (U73122), a Ca(2+)chelator (BAPTA-AM), and a PKCα (protein kinase Cα) inhibitor (RO320432). Furthermore, treatment with arachidonic acid (AA) which is generated by the action of PLA2 (phospholipase A2) on phosphatidic acid (a PLD1 product), increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, indicating that PLA2 is involved in the differentiation process resulting from PLD1 activation. PGE2 (prostaglandin E2), a cyclooxygenase product of AA, also increased during neuronal differentiation. Moreover, treatment with PGE2 increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, and this effect was inhibited by a PKA inhibitor (Rp-cAMP). As expected, inhibition of p38 MAPK resulted in loss of CREB activity, and when CREB activity was blocked with CREB siRNA, Bcl-2 production also decreased. We also showed that the EP4 receptor was required for the PKA/p38MAPK/CREB/Bcl-2 pathway. Taken together, these observations indicate that PLD1 is activated by PLCγ/PKCα signaling and stimulate Bcl-2 expression through PLA2/Cox2/EP4/PKA/p38MAPK/CREB during neuronal differentiation of rat neural stem cells.

The glucagon-like peptide 2 receptor is expressed in enteric neurons and not in the epithelium of the intestine

DEFF Research Database (Denmark)

Pedersen, Jens; B. Pedersen, Nis; Brix, Sophie W.

2015-01-01

fibrillary acidic protein in these isolated tissue fractions was quantified with qRT-PCR. Expression of the Glp2r was confined to compartments containing enteric neurons and receptor expression was absent in the epithelium. Our findings provide evidence for the expression of the GLP-2R in intestinal...... compartments rich in enteric neurons and, importantly they exclude significant expression in the epithelium of rat jejunal mucosa....

Cerebellar nuclei neurons show only small excitatory responses to optogenetic olivary stimulation in transgenic mice: in vivo and in vitro studies

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Huo eLu

2016-03-01

Full Text Available To study the olivary input to the cerebellar nuclei (CN we used optogenetic stimulation in transgenic mice expressing channelrhodopsin-2 (ChR2 in olivary neurons. We obtained in vivo extracellular Purkinje cell (PC and CN recordings in anesthetized mice while stimulating the contralateral inferior olive (IO with a blue laser (single pulse, 10 - 50 ms duration. Peri-stimulus histograms were constructed to show the spike rate changes after optical stimulation. Among 29 CN neurons recorded, 15 showed a decrease in spike rate of variable strength and duration, and only 1 showed a transient spiking response. These results suggest that direct olivary input to CN neurons is usually overridden by stronger Purkinje cell inhibition triggered by climbing fiber responses. To further investigate the direct input from the climbing fiber collaterals we also conducted whole cell recordings in brain slices, where we used local stimulation with blue light. Due to the expression of ChR2 in Purkinje cell axons as well as the IO in our transgenic line, strong inhibitory responses could be readily triggered with optical stimulation (13 of 15 neurons. After blocking this inhibition with GABAzine, only in 5 of 13 CN neurons weak excitatory responses were revealed. Therefore our in vitro results support the in vivo findings that the excitatory input to CN neurons from climbing fiber collaterals in adult mice is masked by the inhibition under normal conditions.

Progranulin is expressed within motor neurons and promotes neuronal cell survival

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Kay Denis G

2009-10-01

Full Text Available Abstract Background Progranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U associated with the formation of ubiquitinated inclusions. Recent reports indicate that progranulin has neurotrophic effects, which, if confirmed would make progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival. Results In situ hybridisation and immunohistochemical techniques revealed that the progranulin gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human progranulin gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months. This is mediated at least in part through

A mammalian conserved element derived from SINE displays enhancer properties recapitulating Satb2 expression in early-born callosal projection neurons.

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Kensuke Tashiro

Full Text Available Short interspersed repetitive elements (SINEs are highly repeated sequences that account for a significant proportion of many eukaryotic genomes and are usually considered "junk DNA". However, we previously discovered that many AmnSINE1 loci are evolutionarily conserved across mammalian genomes, suggesting that they may have acquired significant functions involved in controlling mammalian-specific traits. Notably, we identified the AS021 SINE locus, located 390 kbp upstream of Satb2. Using transgenic mice, we showed that this SINE displays specific enhancer activity in the developing cerebral cortex. The transcription factor Satb2 is expressed by cortical neurons extending axons through the corpus callosum and is a determinant of callosal versus subcortical projection. Mouse mutants reveal a crucial function for Sabt2 in corpus callosum formation. In this study, we compared the enhancer activity of the AS021 locus with Satb2 expression during telencephalic development in the mouse. First, we showed that the AS021 enhancer is specifically activated in early-born Satb2(+ neurons. Second, we demonstrated that the activity of the AS021 enhancer recapitulates the expression of Satb2 at later embryonic and postnatal stages in deep-layer but not superficial-layer neurons, suggesting the possibility that the expression of Satb2 in these two subpopulations of cortical neurons is under genetically distinct transcriptional control. Third, we showed that the AS021 enhancer is activated in neurons projecting through the corpus callosum, as described for Satb2(+ neurons. Notably, AS021 drives specific expression in axons crossing through the ventral (TAG1(-/NPY(+ portion of the corpus callosum, confirming that it is active in a subpopulation of callosal neurons. These data suggest that exaptation of the AS021 SINE locus might be involved in enhancement of Satb2 expression, leading to the establishment of interhemispheric communication via the corpus callosum

Cerebellins are differentially expressed in selective subsets of neurons throughout the brain.

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Seigneur, Erica; Südhof, Thomas C

2017-10-15

Cerebellins are secreted hexameric proteins that form tripartite complexes with the presynaptic cell-adhesion molecules neurexins or 'deleted-in-colorectal-cancer', and the postsynaptic glutamate-receptor-related proteins GluD1 and GluD2. These tripartite complexes are thought to regulate synapses. However, cerebellins are expressed in multiple isoforms whose relative distributions and overall functions are not understood. Three of the four cerebellins, Cbln1, Cbln2, and Cbln4, autonomously assemble into homohexamers, whereas the Cbln3 requires Cbln1 for assembly and secretion. Here, we show that Cbln1, Cbln2, and Cbln4 are abundantly expressed in nearly all brain regions, but exhibit strikingly different expression patterns and developmental dynamics. Using newly generated knockin reporter mice for Cbln2 and Cbln4, we find that Cbln2 and Cbln4 are not universally expressed in all neurons, but only in specific subsets of neurons. For example, Cbln2 and Cbln4 are broadly expressed in largely non-overlapping subpopulations of excitatory cortical neurons, but only sparse expression was observed in excitatory hippocampal neurons of the CA1- or CA3-region. Similarly, Cbln2 and Cbln4 are selectively expressed, respectively, in inhibitory interneurons and excitatory mitral projection neurons of the main olfactory bulb; here, these two classes of neurons form dendrodendritic reciprocal synapses with each other. A few brain regions, such as the nucleus of the lateral olfactory tract, exhibit astoundingly high Cbln2 expression levels. Viewed together, our data show that cerebellins are abundantly expressed in relatively small subsets of neurons, suggesting specific roles restricted to subsets of synapses. © 2017 Wiley Periodicals, Inc.

Visualization of Oxytocin Release that Mediates Paired Pulse Facilitation in Hypothalamic Pathways to Brainstem Autonomic Neurons

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Piñol, Ramón A.; Jameson, Heather; Popratiloff, Anastas; Lee, Norman H.; Mendelowitz, David

2014-01-01

Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN) contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2) expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV). We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs), neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection. PMID:25379676

Optogenetic activation of leptin- and glucose-regulated GABAergic neurons in dorsomedial hypothalamus promotes food intake via inhibitory synaptic transmission to paraventricular nucleus of hypothalamus

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Zesemdorj Otgon-Uul

2016-08-01

Full Text Available Objective: The dorsomedial hypothalamus (DMH has been considered an orexigenic nucleus, since the DMH lesion reduced food intake and body weight and induced resistance to diet-induced obesity. The DMH expresses feeding regulatory neuropeptides and receptors including neuropeptide Y (NPY, cocaine- and amphetamine-regulated transcript (CART, cholecystokinin (CCK, leptin receptor, and melanocortin 3/4 receptors. However, the principal neurons generating the orexigenic function in the DMH remain to be defined. This study aimed to clarify the role of the DMH GABAergic neurons in feeding regulation by using optogenetics and electrophysiological techniques. Methods: We generated the mice expressing ChRFR-C167A, a bistable chimeric channelrhodopsin, selectively in GABAergic neurons of DMH via locally injected adeno-associated virus 2. Food intake after optogenetic activation of DMH GABAergic neurons was measured. Electrophysiological properties of DMH GABAergic neurons were measured using slice patch clamp. Results: Optogenetic activation of DMH GABAergic neurons promoted food intake. Leptin hyperpolarized and lowering glucose depolarized half of DMH GABAergic neurons, suggesting their orexigenic property. Optical activation of axonal terminals of DMH GABAergic neurons at the paraventricular nucleus of hypothalamus (PVN, where anorexigenic neurons are localized, increased inhibitory postsynaptic currents on PVN neurons and promoted food intake. Conclusion: DMH GABAergic neurons are regulated by metabolic signals leptin and glucose and, once activated, promote food intake via inhibitory synaptic transmission to PVN. Keywords: Dorsomedial hypothalamus, GABAergic neuron, Feeding, Leptin, Glucose, Optogenetics

IP3-dependent intracellular Ca2+ release is required for cAMP-induced c-fos expression in hippocampal neurons

International Nuclear Information System (INIS)

Zhang, Wenting; Tingare, Asmita; Ng, David Chi-Heng; Johnson, Hong W.; Schell, Michael J.; Lord, Rebecca L.; Chawla, Sangeeta

2012-01-01

Highlights: ► cAMP-induced c-fos expression in hippocampal neurons requires a submembraneous Ca 2+ pool. ► The submembraneous Ca 2+ pool derives from intracellular ER stores. ► Expression of IP 3 -metabolizing enzymes inhibits cAMP-induced c-fos expression. ► SRE-mediated and CRE-mediated gene expression is sensitive to IP 3 -metabolizing enzymes. ► Intracellular Ca 2+ release is required for cAMP-induced nuclear translocation of TORC1. -- Abstract: Ca 2+ and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca 2+ and cAMP signals, including some that are Ca 2+ -responsive, some that are cAMP-responsive and some that detect coincident Ca 2+ and cAMP signals. Because Ca 2+ and cAMP can influence each other’s amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca 2+ are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca 2+ buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP 3 levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements – the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP 3 metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by depletion of intracellular Ca 2+ stores. Our data indicate that Ca 2+ release from IP 3 -sensitive pools is required for cAMP-induced transcription in hippocampal neurons.

Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons

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Fabio eTescarollo

2014-08-01

Full Text Available The excitatory neurotransmitter glutamate has been reported to have a major impact on brain energy metabolism. Using primary cultures of rat hippocampal neurons, we observed that glutamate reduces glucose utilization in this cell type, suggesting alteration in mitochondrial oxidative metabolism. The aquaglyceroporin AQP9 and the monocarboxylate transporter MCT2, two transporters for oxidative energy substrates, appear to be present in mitochondria of these neurons. Moreover, they not only co-localize but they interact with each other as they were found to co-immunoprecipitate from hippocampal neuron homogenates. Exposure of cultured hippocampal neurons to glutamate 100 µM for 1 hour led to enhanced expression of both AQP9 and MCT2 at the protein level without any significant change at the mRNA level. In parallel, a similar increase in the protein expression of LDHA was evidenced without an effect on the mRNA level. These data suggest that glutamate exerts an influence on neuronal energy metabolism likely through a regulation of the expression of some key mitochondrial proteins.

Inflammatory mediator bradykinin increases population of sensory neurons expressing functional T-type Ca(2+) channels.

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Huang, Dongyang; Liang, Ce; Zhang, Fan; Men, Hongchao; Du, Xiaona; Gamper, Nikita; Zhang, Hailin

2016-04-29

T-type Ca(2+) channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca(2+) currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca(2+) channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca(2+) currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect CaV3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a 'reserve pool' of CaV3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Distinctive features of Phox2b-expressing neurons in the rat reticular formation dorsal to the trigeminal motor nucleus.

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Nagoya, Kouta; Nakamura, Shiro; Ikeda, Keiko; Onimaru, Hiroshi; Yoshida, Atsushi; Nakayama, Kiyomi; Mochizuki, Ayako; Kiyomoto, Masaaki; Sato, Fumihiko; Kawakami, Kiyoshi; Takahashi, Koji; Inoue, Tomio

2017-09-01

Phox2b encodes a paired-like homeodomain-containing transcription factor essential for development of the autonomic nervous system. Phox2b-expressing (Phox2b + ) neurons are present in the reticular formation dorsal to the trigeminal motor nucleus (RdV) as well as the nucleus of the solitary tract and parafacial respiratory group. However, the nature of Phox2b + RdV neurons is still unclear. We investigated the physiological and morphological properties of Phox2b + RdV neurons using postnatal day 2-7 transgenic rats expressing yellow fluorescent protein under the control of Phox2b. Almost all of Phox2b + RdV neurons were glutamatergic, whereas Phox2b-negative (Phox2b - ) RdV neurons consisted of a few glutamatergic, many GABAergic, and many glycinergic neurons. The majority (48/56) of Phox2b + neurons showed low-frequency firing (LF), while most of Phox2b - neurons (35/42) exhibited high-frequency firing (HF) in response to intracellularly injected currents. All, but one, Phox2b + neurons (55/56) did not fire spontaneously, whereas three-fourths of the Phox2b - neurons (31/42) were spontaneously active. K + channel and persistent Na + current blockers affected the firing of LF and HF neurons. The majority of Phox2b + (35/46) and half of the Phox2b - neurons (19/40) did not respond to stimulations of the mesencephalic trigeminal nucleus, the trigeminal tract, and the principal sensory trigeminal nucleus. Biocytin labeling revealed that about half of the Phox2b + (5/12) and Phox2b - RdV neurons (5/10) send their axons to the trigeminal motor nucleus. These results suggest that Phox2b + RdV neurons have distinct neurotransmitter phenotypes and firing properties from Phox2b - RdV neurons and might play important roles in feeding-related functions including suckling and possibly mastication. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons

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Chen Yong

2012-02-01

Full Text Available Abstract Background The purinergic P2X3 receptor (P2X3R expressed in the dorsal root ganglion (DRG sensory neuron and the P2X7 receptor (P2X7R expressed in the surrounding satellite glial cell (SGC are two major receptors participating in neuron-SGC communication in adult DRGs. Activation of P2X7Rs was found to tonically reduce the expression of P2X3Rs in DRGs, thus inhibiting the abnormal pain behaviors in adult rats. P2X receptors are also actively involved in sensory signaling in developing rodents. However, very little is known about the developmental change of P2X7Rs in DRGs and the interaction between P2X7Rs and P2X3Rs in those animals. We therefore examined the expression of P2X3Rs and P2X7Rs in postnatal rats and determined if P2X7R-P2X3R control exists in developing rats. Findings We immunostained DRGs of immature rats and found that P2X3Rs were expressed only in neurons and P2X7Rs were expressed only in SGCs. Western blot analyses indicated that P2X3R expression decreased while P2X7R expression increased with the age of rats. Electrophysiological studies showed that the number of DRG neurons responding to the stimulation of the P2XR agonist, α,β-meATP, was higher and the amplitudes of α,β-meATP-induced depolarizations were larger in immature DRG neurons. As a result, P2X3R-mediated flinching responses were much more pronounced in immature rats than those found in adult rats. When we reduced P2X7R expression with P2X7R-siRNA in postnatal and adult rats, P2X3R-mediated flinch responses were greatly enhanced in both rat populations. Conclusions These results show that the P2X7R expression increases as rats age. In addition, P2X7Rs in SGCs exert inhibitory control on the P2X3R expression and function in sensory neurons of immature rats, just as observed in adult rats. Regulation of P2X7R expression is likely an effective way to control P2X3R activity and manage pain relief in infants.

Activation of Brainstem Pro-opiomelanocortin Neurons Produces Opioidergic Analgesia, Bradycardia and Bradypnoea.

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Cerritelli, Serena; Hirschberg, Stefan; Hill, Rob; Balthasar, Nina; Pickering, Anthony E

2016-01-01

Opioids are widely used medicinally as analgesics and abused for hedonic effects, actions that are each complicated by substantial risks such as cardiorespiratory depression. These drugs mimic peptides such as β-endorphin, which has a key role in endogenous analgesia. The β-endorphin in the central nervous system originates from pro-opiomelanocortin (POMC) neurons in the arcuate nucleus and nucleus of the solitary tract (NTS). Relatively little is known about the NTSPOMC neurons but their position within the sensory nucleus of the vagus led us to test the hypothesis that they play a role in modulation of cardiorespiratory and nociceptive control. The NTSPOMC neurons were targeted using viral vectors in a POMC-Cre mouse line to express either opto-genetic (channelrhodopsin-2) or chemo-genetic (Pharmacologically Selective Actuator Modules). Opto-genetic activation of the NTSPOMC neurons in the working heart brainstem preparation (n = 21) evoked a reliable, titratable and time-locked respiratory inhibition (120% increase in inter-breath interval) with a bradycardia (125±26 beats per minute) and augmented respiratory sinus arrhythmia (58% increase). Chemo-genetic activation of NTSPOMC neurons in vivo was anti-nociceptive in the tail flick assay (latency increased by 126±65%, pneurons were found to project to key brainstem structures involved in cardiorespiratory control (nucleus ambiguus and ventral respiratory group) and endogenous analgesia (periaqueductal gray and midline raphe). Thus the NTSPOMC neurons may be capable of tuning behaviour by an opioidergic modulation of nociceptive, respiratory and cardiac control.

Combining microfluidics, optogenetics and calcium imaging to study neuronal communication in vitro.

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Renault, Renaud; Sukenik, Nirit; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis; Peyrin, Jean-Michel; Bottani, Samuel; Monceau, Pascal; Moses, Elisha; Vignes, Maéva

2015-01-01

In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.

Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons

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Hannah I. Bishop

2018-01-01

Full Text Available Voltage-gated K+ (Kv channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

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

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

2016-01-01

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

TRPA1 expression levels and excitability brake by KV channels influence cold sensitivity of TRPA1-expressing neurons.

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Memon, Tosifa; Chase, Kevin; Leavitt, Lee S; Olivera, Baldomero M; Teichert, Russell W

2017-06-14

The molecular sensor of innocuous (painless) cold sensation is well-established to be transient receptor potential cation channel, subfamily M, member 8 (TRPM8). However, the role of transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in noxious (painful) cold sensation has been controversial. We find that TRPA1 channels contribute to the noxious cold sensitivity of mouse somatosensory neurons, independent of TRPM8 channels, and that TRPA1-expressing neurons are largely non-overlapping with TRPM8-expressing neurons in mouse dorsal-root ganglia (DRG). However, relatively few TRPA1-expressing neurons (e.g., responsive to allyl isothiocyanate or AITC, a selective TRPA1 agonist) respond overtly to cold temperature in vitro, unlike TRPM8-expressing neurons, which almost all respond to cold. Using somatosensory neurons from TRPM8-/- mice and subtype-selective blockers of TRPM8 and TRPA1 channels, we demonstrate that responses to cold temperatures from TRPA1-expressing neurons are mediated by TRPA1 channels. We also identify two factors that affect the cold-sensitivity of TRPA1-expressing neurons: (1) cold-sensitive AITC-sensitive neurons express relatively more TRPA1 transcripts than cold-insensitive AITC-sensitive neurons and (2) voltage-gated potassium (K V ) channels attenuate the cold-sensitivity of some TRPA1-expressing neurons. The combination of these two factors, combined with the relatively weak agonist-like activity of cold temperature on TRPA1 channels, partially explains why few TRPA1-expressing neurons respond to cold. Blocking K V channels also reveals another subclass of noxious cold-sensitive DRG neurons that do not express TRPM8 or TRPA1 channels. Altogether, the results of this study provide novel insights into the cold-sensitivity of different subclasses of somatosensory neurons. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Visualization of oxytocin release that mediates paired pulse facilitation in hypothalamic pathways to brainstem autonomic neurons.

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Ramón A Piñol

Full Text Available Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2 expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV. We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs, neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection.

Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model.

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Jan Tønnesen

Full Text Available Intrastriatal grafts of stem cell-derived dopamine (DA neurons induce behavioral recovery in animal models of Parkinson's disease (PD, but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2 and halorhodopsin (NpHR, respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

The Relevance of AgRP Neuron-Derived GABA Inputs to POMC Neurons Differs for Spontaneous and Evoked Release.

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Rau, Andrew R; Hentges, Shane T

2017-08-02

Hypothalamic agouti-related peptide (AgRP) neurons potently stimulate food intake, whereas proopiomelanocortin (POMC) neurons inhibit feeding. Whether AgRP neurons exert their orexigenic actions, at least in part, by inhibiting anorexigenic POMC neurons remains unclear. Here, the connectivity between GABA-releasing AgRP neurons and POMC neurons was examined in brain slices from male and female mice. GABA-mediated spontaneous IPSCs (sIPSCs) in POMC neurons were unaffected by disturbing GABA release from AgRP neurons either by cell type-specific deletion of the vesicular GABA transporter or by expression of botulinum toxin in AgRP neurons to prevent vesicle-associated membrane protein 2-dependent vesicle fusion. Additionally, there was no difference in the ability of μ-opioid receptor (MOR) agonists to inhibit sIPSCs in POMC neurons when MORs were deleted from AgRP neurons, and activation of the inhibitory designer receptor hM4Di on AgRP neurons did not affect sIPSCs recorded from POMC neurons. These approaches collectively indicate that AgRP neurons do not significantly contribute to the strong spontaneous GABA input to POMC neurons. Despite these observations, optogenetic stimulation of AgRP neurons reliably produced evoked IPSCs in POMC neurons, leading to the inhibition of POMC neuron firing. Thus, AgRP neurons can potently affect POMC neuron function without contributing a significant source of spontaneous GABA input to POMC neurons. Together, these results indicate that the relevance of GABAergic inputs from AgRP to POMC neurons is state dependent and highlight the need to consider different types of transmitter release in circuit mapping and physiologic regulation. SIGNIFICANCE STATEMENT Agouti-related peptide (AgRP) neurons play an important role in driving food intake, while proopiomelanocortin (POMC) neurons inhibit feeding. Despite the importance of these two well characterized neuron types in maintaining metabolic homeostasis, communication between these

Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.

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David Ramonet

2011-04-01

Full Text Available Mutations in the leucine-rich repeat kinase 2 (LRRK2 gene cause late-onset, autosomal dominant familial Parkinson's disease (PD and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s through which familial mutations precipitate neuronal degeneration and PD.

Expression of the P2X2 receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

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Mizuno, Márcia Sanae; Crisma, Amanda Rabello; Borelli, Primavera; Castelucci, Patricia

2012-01-01

AIM: To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility. METHODS: The study examined the distribution of the P2X2 receptor (P2X2R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X2R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm2) and area profile (μm²) of P2X2R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area. RESULTS: In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X2R, nNOS, ChAT and CalR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X2R-IR was observed to co-localize 100% with that for nNOS, ChAT and CalR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm2) of P2X2R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CalR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the

Cre-expressing neurons in the cortical white matter of Ntsr1-Cre GN220 mice.

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Sundberg, Sofie C; Granseth, Björn

2018-03-23

Genetically modified mouse strains that express Cre-recombinase in specific neuronal sub-populations have become widely used tools for investigating neuronal function. The Ntsr1-Cre GN220 mouse expresses this enzyme in corticothalamic neurons in layer 6 of cerebral cortex. We observed that about 7% of Cre-expressing cells in the primary visual cortex are found within the white matter bordering layer 6. By using the immunohistochemical marker for layer 6 neurons, Forkhead box protein 2 (FoxP2), and fluorescently conjugated latex beads injected into the dorsal lateral geniculate nucleus, we show that about half of these cells are similar to and could belong to the layer 6 corticothalamic neuron population. The other half seems to be a distinct white matter (WM) neuron sub-population that we estimate to constitute 2-4% of the total cortical Cre-expressing population. Staining for the neuronal marker Neuronal nuclei (NeuN) revealed that about 15-40% of WM neurons are Cre-expressing. Thus, the potential contribution from WM neurons needs to be considered when interpreting the results from experiments using the Ntsr1-Cre GN220 mouse for investigating corticothalamic neuronal function. Copyright © 2018 Elsevier B.V. All rights reserved.

Optogenetic activation of dorsal raphe serotonin neurons enhances patience for future rewards.

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Miyazaki, Kayoko W; Miyazaki, Katsuhiko; Tanaka, Kenji F; Yamanaka, Akihiro; Takahashi, Aki; Tabuchi, Sawako; Doya, Kenji

2014-09-08

Serotonin is a neuromodulator that is involved extensively in behavioral, affective, and cognitive functions in the brain. Previous recording studies of the midbrain dorsal raphe nucleus (DRN) revealed that the activation of putative serotonin neurons correlates with the levels of behavioral arousal [1], rhythmic motor outputs [2], salient sensory stimuli [3-6], reward, and conditioned cues [5-8]. The classic theory on serotonin states that it opposes dopamine and inhibits behaviors when aversive events are predicted [9-14]. However, the therapeutic effects of serotonin signal-enhancing medications have been difficult to reconcile with this theory [15, 16]. In contrast, a more recent theory states that serotonin facilitates long-term optimal behaviors and suppresses impulsive behaviors [17-21]. To test these theories, we developed optogenetic mice that selectively express channelrhodopsin in serotonin neurons and tested how the activation of serotonergic neurons in the DRN affects animal behavior during a delayed reward task. The activation of serotonin neurons reduced the premature cessation of waiting for conditioned cues and food rewards. In reward omission trials, serotonin neuron stimulation prolonged the time animals spent waiting. This effect was observed specifically when the animal was engaged in deciding whether to keep waiting and was not due to motor inhibition. Control experiments showed that the prolonged waiting times observed with optogenetic stimulation were not due to behavioral inhibition or the reinforcing effects of serotonergic activation. These results show, for the first time, that the timed activation of serotonin neurons during waiting promotes animals' patience to wait for a delayed reward. Copyright © 2014 Elsevier Ltd. All rights reserved.

Homeostatic regulation of excitatory synapses on striatal medium spiny neurons expressing the D2 dopamine receptor.

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Thibault, Dominic; Giguère, Nicolas; Loustalot, Fabien; Bourque, Marie-Josée; Ducrot, Charles; El Mestikawy, Salah; Trudeau, Louis-Éric

2016-05-01

Striatal medium spiny neurons (MSNs) are contacted by glutamatergic axon terminals originating from cortex, thalamus and other regions. The striatum is also innervated by dopaminergic (DAergic) terminals, some of which release glutamate as a co-transmitter. Despite evidence for functional DA release at birth in the striatum, the role of DA in the establishment of striatal circuitry is unclear. In light of recent work suggesting activity-dependent homeostatic regulation of glutamatergic terminals on MSNs expressing the D2 DA receptor (D2-MSNs), we used primary co-cultures to test the hypothesis that stimulation of DA and glutamate receptors regulates the homeostasis of glutamatergic synapses on MSNs. Co-culture of D2-MSNs with mesencephalic DA neurons or with cortical neurons produced an increase in spines and functional glutamate synapses expressing VGLUT2 or VGLUT1, respectively. The density of VGLUT2-positive terminals was reduced by the conditional knockout of this gene from DA neurons. In the presence of both mesencephalic and cortical neurons, the density of synapses reached the same total, compatible with the possibility of a homeostatic mechanism capping excitatory synaptic density. Blockade of D2 receptors increased the density of cortical and mesencephalic glutamatergic terminals, without changing MSN spine density or mEPSC frequency. Combined blockade of AMPA and NMDA glutamate receptors increased the density of cortical terminals and decreased that of mesencephalic VGLUT2-positive terminals, with no net change in total excitatory terminal density or in mEPSC frequency. These results suggest that DA and glutamate signaling regulate excitatory inputs to striatal D2-MSNs at both the pre- and postsynaptic level, under the influence of a homeostatic mechanism controlling functional output of the circuit.

touché is required for touch evoked generator potentials within vertebrate sensory neurons

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Low, Sean E.; Ryan, Joel; Sprague, Shawn M.; Hirata, Hiromi; Cui, Wilson W.; Zhou, Weibin; Hume, Richard I.; Kuwada, John Y.; Saint-Amant, Louis

2010-01-01

The process by which light-touch in vertebrates is transformed into an electrical response in cutaneous mechanosensitive neurons is a largely unresolved question. To address this question we undertook a forward genetic screen in zebrafish (Danio rerio) to identify mutants exhibiting abnormal touch-evoked behaviors, despite the presence of sensory neurons and peripheral neurites. One family, subsequently named touché, was found to harbor a recessive mutation which produced offspring that were unresponsive to light-touch, but responded to a variety of other sensory stimuli. The optogenetic activation of motor behaviors by touché mutant sensory neurons expressing ChannelRhodopsin-2 suggested that the synaptic output of sensory neurons was intact, consistent with a defect in sensory neuron activation. To explore sensory neuron activation we developed an in vivo preparation permitting the precise placement of a combined electrical and tactile stimulating probe upon eGFP positive peripheral neurites. In wild type larva electrical and tactile stimulation of peripheral neurites produced action potentials detectable within the cell body. In a subset of these sensory neurons an underlying generator potential could be observed in response to subthreshold tactile stimuli. A closer examination revealed that the amplitude of the generator potential was proportional to the stimulus amplitude. When assayed touché mutant sensory neurons also responded to electrical stimulation of peripheral neurites similar to wild type larvae, however tactile stimulation of these neurites failed to uncover a subset of sensory neurons possessing generator potentials. These findings suggest that touché is required for generator potentials, and that generator potentials underlie responsiveness to light-touch in zebrafish. PMID:20631165

Dopamine receptor gene expression by enkephalin neurons in rat forebrain

International Nuclear Information System (INIS)

Le Moine, C.; Normand, E.; Guitteny, A.F.; Fouque, B.; Teoule, R.; Bloch, B.

1990-01-01

In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex. Hybridization with D2 and preproenkephalin A probes in adjacent sections, as well as combined hybridization with the two probes in the same sections, demonstrated that all detectable enkephalin neurons in the striatum contained the D2 receptor mRNA. Large neurons in caudate putamen, which were unlabeled with the preproenkephalin A probe and which may have been cholinergic, also expressed the D2 receptor gene. Haloperidol treatment (14 or 21 days) provoked an increase in mRNA content for D2 receptor and preproenkephalin A in the striatum. This suggests that the increase in D2 receptor number observed after haloperidol treatment is due to increased activity of the D2 gene. These results indicate that in the striatum, the enkephalin neurons are direct targets for dopamine liberated from mesostriatal neurons

Dopamine receptor gene expression by enkephalin neurons in rat forebrain

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Le Moine, C.; Normand, E.; Guitteny, A.F.; Fouque, B.; Teoule, R.; Bloch, B. (Universite de Bordeaux II (France))

1990-01-01

In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex. Hybridization with D2 and preproenkephalin A probes in adjacent sections, as well as combined hybridization with the two probes in the same sections, demonstrated that all detectable enkephalin neurons in the striatum contained the D2 receptor mRNA. Large neurons in caudate putamen, which were unlabeled with the preproenkephalin A probe and which may have been cholinergic, also expressed the D2 receptor gene. Haloperidol treatment (14 or 21 days) provoked an increase in mRNA content for D2 receptor and preproenkephalin A in the striatum. This suggests that the increase in D2 receptor number observed after haloperidol treatment is due to increased activity of the D2 gene. These results indicate that in the striatum, the enkephalin neurons are direct targets for dopamine liberated from mesostriatal neurons.

Neurochemical phenotype of cytoglobin‑expressing neurons in the rat hippocampus

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Hundahl, Christian Ansgar; Fahrenkrug, Jan; Hannibal, Jens

2014-01-01

in a subpopulation of brain neurons. Recently, it has been shown that stress upregulates Cygb expression in the brain and the majority of neuronal nitric oxide synthase (nNOS)-positive neurons, an enzyme that produces NO, co-express Cygb. However, there are more neurons expressing Cygb than nNOS, thus a large number...... of Cygb neurons remain uncharacterized by the neurochemical content. The aim of the present study was to provide an additional and more detailed neurochemical phenotype of Cygb-expressing neurons in the rat hippocampus. The rat hippocampus was chosen due to the abundance of Cygb, as well as this limbic...... structure being an important target in a number of neurodegenerative diseases. Using triple immunohistochemistry, it was demonstrated that nearly all the parvalbumin- and heme oxygenase 1-positive neurons co-express Cygb and to a large extent, these neuron populations are distinct from the population...

Neuronal MHC Class I Expression Is Regulated by Activity Driven Calcium Signaling.

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Dan Lv

Full Text Available MHC class I (MHC-I molecules are important components of the immune system. Recently MHC-I have been reported to also play important roles in brain development and synaptic plasticity. In this study, we examine the molecular mechanism(s underlying activity-dependent MHC-I expression using hippocampal neurons. Here we report that neuronal expression level of MHC-I is dynamically regulated during hippocampal development after birth in vivo. Kainic acid (KA treatment significantly increases the expression of MHC-I in cultured hippocampal neurons in vitro, suggesting that MHC-I expression is regulated by neuronal activity. In addition, KA stimulation decreased the expression of pre- and post-synaptic proteins. This down-regulation is prevented by addition of an MHC-I antibody to KA treated neurons. Further studies demonstrate that calcium-dependent protein kinase C (PKC is important in relaying KA simulation activation signals to up-regulated MHC-I expression. This signaling cascade relies on activation of the MAPK pathway, which leads to increased phosphorylation of CREB and NF-κB p65 while also enhancing the expression of IRF-1. Together, these results suggest that expression of MHC-I in hippocampal neurons is driven by Ca2+ regulated activation of the MAPK signaling transduction cascade.

Gene Expression and the Diversity of Identified Neurons

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Buck, L.; Stein, R.; Palazzolo, M.; Anderson, D. J.; Axel, R.

1983-01-01

Nervous systems consist of diverse populations of neurons that are anatomically and functionally distinct. The diversity of neurons and the precision with which they are interconnected suggest that specific genes or sets of genes are activated in some neurons but not expressed in others. Experimentally, this problem may be considered at two levels. First, what is the total number of genes expressed in the brain, and how are they distributed among the different populations of neurons? Second, ...

Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

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Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A.

2014-01-01

Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders

Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

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Park, Kyoung Ho [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Yeo, Sang Won, E-mail: swyeo@catholic.ac.kr [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Troy, Frederic A., E-mail: fatroy@ucdavis.edu [Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, CA 95616 (United States); Xiamen University, School of Medicine, Xiamen City (China)

2014-10-17

Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons

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Fomina Alla F

2008-09-01

Full Text Available Abstract Background Olfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment. Results We demonstrate that cultured olfactory sensory neurons express endogenous odorant receptors. Lentiviral vector-mediated gene transfer enables successful ectopic expression of odorant receptors. We show that the ectopically expressed mouse I7 is functional in the cultured olfactory sensory neurons. When two different odorant receptors are ectopically expressed simultaneously, both receptor proteins co-localized in the same olfactory sensory neurons up to 10 days in vitro. Conclusion This culture technique provided an efficient method to culture olfactory sensory neurons whose morphology, molecular characteristics and maturation progression resembled those observed in vivo. Using this system, regulation of odorant receptor expression and its ligand specificity can be studied in its intrinsic cellular environment.

Differential expression of alpha-synuclein in hippocampal neurons.

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Katsutoshi Taguchi

Full Text Available α-Synuclein is the major pathological component of synucleinopathies including Parkinson's disease and dementia with Lewy bodies. Recent studies have demonstrated that α-synuclein also plays important roles in the release of synaptic vesicles and synaptic membrane recycling in healthy neurons. However, the precise relationship between the pathogenicity and physiological functions of α-synuclein remains to be elucidated. To address this issue, we investigated the subcellular localization of α-synuclein in normal and pathological conditions using primary mouse hippocampal neuronal cultures. While some neurons expressed high levels of α-synuclein in presynaptic boutons and cell bodies, other neurons either did not or only very weakly expressed the protein. These α-synuclein-negative cells were identified as inhibitory neurons by immunostaining with specific antibodies against glutamic acid decarboxylase (GAD, parvalbumin, and somatostatin. In contrast, α-synuclein-positive synapses were colocalized with the excitatory synapse marker vesicular glutamate transporter-1. This expression profile of α-synuclein was conserved in the hippocampus in vivo. In addition, we found that while presynaptic α-synuclein colocalizes with synapsin, a marker of presynaptic vesicles, it is not essential for activity-dependent membrane recycling induced by high potassium treatment. Exogenous supply of preformed fibrils generated by recombinant α-synuclein was shown to promote the formation of Lewy body (LB -like intracellular aggregates involving endogenous α-synuclein. GAD-positive neurons did not form LB-like aggregates following treatment with preformed fibrils, however, exogenous expression of human α-synuclein allowed intracellular aggregate formation in these cells. These results suggest the presence of a different mechanism for regulation of the expression of α-synuclein between excitatory and inhibitory neurons. Furthermore, α-synuclein expression

Nociceptive DRG neurons express muscle lim protein upon axonal injury.

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Levin, Evgeny; Andreadaki, Anastasia; Gobrecht, Philipp; Bosse, Frank; Fischer, Dietmar

2017-04-04

Muscle lim protein (MLP) has long been regarded as a cytosolic and nuclear muscular protein. Here, we show that MLP is also expressed in a subpopulation of adult rat dorsal root ganglia (DRG) neurons in response to axonal injury, while the protein was not detectable in naïve cells. Detailed immunohistochemical analysis of L4/L5 DRG revealed ~3% of MLP-positive neurons 2 days after complete sciatic nerve crush and maximum ~10% after 4-14 days. Similarly, in mixed cultures from cervical, thoracic, lumbar and sacral DRG ~6% of neurons were MLP-positive after 2 days and maximal 17% after 3 days. In both, histological sections and cell cultures, the protein was detected in the cytosol and axons of small diameter cells, while the nucleus remained devoid. Moreover, the vast majority could not be assigned to any of the well characterized canonical DRG subpopulations at 7 days after nerve injury. However, further analysis in cell culture revealed that the largest population of MLP expressing cells originated from non-peptidergic IB4-positive nociceptive neurons, which lose their ability to bind the lectin upon axotomy. Thus, MLP is mostly expressed in a subset of axotomized nociceptive neurons and can be used as a novel marker for this population of cells.

Expanding the Repertoire of Optogenetically Targeted Cells with an Enhanced Gene Expression System

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Kenji F. Tanaka

2012-08-01

Full Text Available Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems.

Parallel and patterned optogenetic manipulation of neurons in the brain slice using a DMD-based projector.

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Sakai, Seiichiro; Ueno, Kenichi; Ishizuka, Toru; Yawo, Hiromu

2013-01-01

Optical manipulation technologies greatly advanced the understanding of the neuronal network and its dysfunctions. To achieve patterned and parallel optical switching, we developed a microscopic illumination system using a commercial DMD-based projector and a software program. The spatiotemporal patterning of the system was evaluated using acute slices of the hippocampus. The neural activity was optically manipulated, positively by the combination of channelrhodopsin-2 (ChR2) and blue light, and negatively by the combination of archaerhodopsin-T (ArchT) and green light. It is suggested that our projector-managing optical system (PMOS) would effectively facilitate the optogenetic analyses of neurons and their circuits. Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Submucosal neurons and enteric glial cells expressing the P2X7 receptor in rat experimental colitis.

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da Silva, Marcos Vinícius; Marosti, Aline Rosa; Mendes, Cristina Eusébio; Palombit, Kelly; Castelucci, Patricia

2017-06-01

The aim of this study was to evaluate the effect of ulcerative colitis on the submucosal neurons and glial cells of the submucosal ganglia of rats. 2,4,6-Trinitrobenzene sulfonic acid (TNBS; colitis group) was administered in the colon to induce ulcerative colitis, and distal colons were collected after 24h. The colitis rats were compared with those in the sham and control groups. Double labelling of the P2X7 receptor with calbindin (marker for intrinsic primary afferent neurons, IPANs, submucosal plexus), calretinin (marker for secretory and vasodilator neurons of the submucosal plexus), HuC/D and S100β was performed in the submucosal plexus. The density (neurons per area) of submucosal neurons positive for the P2X7 receptor, calbindin, calretinin and HuC/D decreased by 21%, 34%, 8.2% and 28%, respectively, in the treated group. In addition, the density of enteric glial cells in the submucosal plexus decreased by 33%. The profile areas of calbindin-immunoreactive neurons decreased by 25%. Histological analysis revealed increased lamina propria and decreased collagen in the colitis group. This study demonstrated that ulcerative colitis affected secretory and vasodilatory neurons, IPANs and enteric glia of the submucosal plexus expressing the P2X7 receptor. Copyright © 2017 Elsevier GmbH. All rights reserved.

Mean expression of the X chromosome is associated with neuronal density

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James Thomas Swingland

2012-11-01

Full Text Available Neurodegenerative diseases are characterised by neuronal loss. Neuronal loss causes a varying density of neurons across samples which confounds results from gene expression studies. Chromosome X is known to be specifically important in brain. We hypothesised the existence of a chromosomal signature of gene expression associated with the X-chromosome for neurological conditions not normally associated with that chromosome. The hypothesis was investigated using microarray datasets from studies on Parkinson's disease, Alzheimer's disease and Huntington's disease. Data were analysed using Chromowave, an analytical tool for detecting spatially extended expression changes across chromosomes. To examine associations with neuronal density, expressions from a set of neuron specific genes were extracted. The association between these genes and the expression patterns extracted by Chromowave was then analyzed. We observed an extended pattern of low expression of ChrX consistent in all the neurodegenerative disease brain datasets. There was a strong correlation between mean ChrX expression and the pattern extracted from the autosomal neuronal specific genes, but no correlation with mean autosomal expression. No chromosomal patterns associated with the neuron specific genes were found on other chromosomes. The chromosomal expression pattern was not present in datasets from blood cells. The ChrX:Autosome expression ratio was also higher in neuronal cells than in tissues with a mix of cell types.The results suggest that a loss of neurons manifests in gene expression experiments primarily as a reduction in mean expression of genes along ChrX. The most likely explanation for this finding relates to the documented general up-regulation of ChrX in brain tissue which, this work suggests, occurs primarily in neurons. The purpose and mechanisms behind this cell specific higher expression warrant further research, which may also help elucidate connectio

Characterization of claustral neurons by comparative gene expression profiling and dye-injection analyses

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Akiya eWatakabe

2014-05-01

Full Text Available The identity of the claustrum as a part of cerebral cortex, and in particular of the adjacent insular cortex, has been investigated by connectivity features and patterns of gene expression. In the present paper, we mapped the cortical and claustral expression of several cortical genes in rodent and macaque monkey brains (nurr1, latexin, cux2, and netrinG2 to further assess shared features between cortex and claustrum. In mice, these genes were densely expressed in the claustrum, but very sparsely in the cortex and not present in the striatum. To test whether the cortical vs. claustral cell types can be distinguished by co-expression of these genes, we performed a panel of double ISH in mouse and macaque brain. NetrinG2 and nurr1 genes were co-expressed across entire cortex and claustrum, but cux2 and nurr1 were co-expressed only in the insular cortex and claustrum. Latexin was expressed, in the macaque, only in the claustrum. The nurr1+ claustral neurons expressed VGluT1, a marker for cortical glutamatergic cells and send cortical projections. Taken together, our data suggest a partial commonality between claustral neurons and a subtype of cortical neurons in the monkey brain. Moreover, in the embryonic (E110 macaque brain, many nurr1+ neurons were scattered in the white matter between the claustrum and the insular cortex, possibly representing their migratory history. In a second set of experiments, we injected Lucifer Yellow intracellularly in mouse and rat slices to investigate whether dendrites of insular and claustral neurons can cross the border of the two brain regions. Dendrites of claustral neurons did not invade the overlying insular territory. In summary, gene expression profile of the claustrum is similar to that of the neocortex, in both rodent and macaque brains, but with modifications in density of expression and cellular co-localization of specific genes.

Layer 5 Callosal Parvalbumin-Expressing Neurons: A Distinct Functional Group of GABAergic Neurons.

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Zurita, Hector; Feyen, Paul L C; Apicella, Alfonso Junior

2018-01-01

Previous studies have shown that parvalbumin-expressing neurons (CC-Parv neurons) connect the two hemispheres of motor and sensory areas via the corpus callosum, and are a functional part of the cortical circuit. Here we test the hypothesis that layer 5 CC-Parv neurons possess anatomical and molecular mechanisms which dampen excitability and modulate the gating of interhemispheric inhibition. In order to investigate this hypothesis we use viral tracing to determine the anatomical and electrophysiological properties of layer 5 CC-Parv and parvalbumin-expressing (Parv) neurons of the mouse auditory cortex (AC). Here we show that layer 5 CC-Parv neurons had larger dendritic fields characterized by longer dendrites that branched farther from the soma, whereas layer 5 Parv neurons had smaller dendritic fields characterized by shorter dendrites that branched nearer to the soma. The layer 5 CC-Parv neurons are characterized by delayed action potential (AP) responses to threshold currents, lower firing rates, and lower instantaneous frequencies compared to the layer 5 Parv neurons. Kv1.1 containing K + channels are the main source of the AP repolarization of the layer 5 CC-Parv and have a major role in determining both the spike delayed response, firing rate and instantaneous frequency of these neurons.

Sonic hedgehog expressing and responding cells generate neuronal diversity in the medial amygdala

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Machold Robert P

2010-05-01

Full Text Available Abstract Background The mammalian amygdala is composed of two primary functional subdivisions, classified according to whether the major output projection of each nucleus is excitatory or inhibitory. The posterior dorsal and ventral subdivisions of the medial amygdala, which primarily contain inhibitory output neurons, modulate specific aspects of innate socio-sexual and aggressive behaviors. However, the development of the neuronal diversity of this complex and important structure remains to be fully elucidated. Results Using a combination of genetic fate-mapping and loss-of-function analyses, we examined the contribution and function of Sonic hedgehog (Shh-expressing and Shh-responsive (Nkx2-1+ and Gli1+ neurons in the medial amygdala. Specifically, we found that Shh- and Nkx2-1-lineage cells contribute differentially to the dorsal and ventral subdivisions of the postnatal medial amygdala. These Shh- and Nkx2-1-lineage neurons express overlapping and non-overlapping inhibitory neuronal markers, such as Calbindin, FoxP2, nNOS and Somatostatin, revealing diverse fate contributions in discrete medial amygdala nuclear subdivisions. Electrophysiological analysis of the Shh-derived neurons additionally reveals an important functional diversity within this lineage in the medial amygdala. Moreover, inducible Gli1CreER(T2 temporal fate mapping shows that early-generated progenitors that respond to Shh signaling also contribute to medial amygdala neuronal diversity. Lastly, analysis of Nkx2-1 mutant mice demonstrates a genetic requirement for Nkx2-1 in inhibitory neuronal specification in the medial amygdala distinct from the requirement for Nkx2-1 in cerebral cortical development. Conclusions Taken together, these data reveal a differential contribution of Shh-expressing and Shh-responding cells to medial amygdala neuronal diversity as well as the function of Nkx2-1 in the development of this important limbic system structure.

Nucleus Accumbens Dopamine D2-Receptor Expressing Neurons Control Behavioral Flexibility in a Place Discrimination Task in the IntelliCage

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Macpherson, Tom; Morita, Makiko; Wang, Yanyan; Sasaoka, Toshikuni; Sawa, Akira; Hikida, Takatoshi

2016-01-01

Considerable evidence has demonstrated a critical role for the nucleus accumbens (NAc) in the acquisition and flexibility of behavioral strategies. These processes are guided by the activity of two discrete neuron types, dopamine D1- or D2-receptor expressing medium spiny neurons (D1-/D2-MSNs). Here we used the IntelliCage, an automated…

Phrenic motor neuron adenosine 2A receptors elicit phrenic motor facilitation.

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Seven, Yasin B; Perim, Raphael R; Hobson, Orinda R; Simon, Alec K; Tadjalli, Arash; Mitchell, Gordon S

2018-04-15

Although adenosine 2A (A 2A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A 2A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A 2A receptor siRNA injections. A 2A receptor siRNA injections selectively knocked down A 2A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A 2A receptors relevant to A 2A receptor-induced phrenic motor facilitation. Upregulation of A 2A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury. Cervical spinal adenosine 2A (A 2A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A 2A receptors for pMF are unknown. This is an important question since the physiological outcome of A 2A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A 2A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A 2A receptor immunostaining significantly colocalized with NeuN-positive neurons (89 ± 2%). Intrapleural siRNA injections were used to selectively knock down A 2A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A 2A receptor knock-down was verified by a ∼45% decrease in A 2A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, p

Connections between EM2-containing terminals and GABA/μ-opioid receptor co-expressing neurons in the rat spinal trigeminal caudal nucleus

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Li, Meng-Ying; Wu, Zhen-Yu; Lu, Ya-Cheng; Yin, Jun-Bin; Wang, Jian; Zhang, Ting; Dong, Yu-Lin; Wang, Feng

2014-01-01

Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect via the μ-opioid receptor (MOR). To provide morphological evidence for the pain control effect of EM2, the synaptic connections between EM2-immunoreactive (IR) axonal terminals and γ-amino butyric acid (GABA)/MOR co-expressing neurons in lamina II of the spinal trigeminal caudal nucleus (Vc) were investigated in the rat. Dense EM2-, MOR- and GABA-IR fibers and terminals were mainly observed in lamina II of the Vc. Within lamina II, GABA- and MOR-neuronal cell bodies were also encountered. The results of immunofluorescent histochemical triple-staining showed that approximately 14.2 or 18.9% of GABA-IR or MOR-IR neurons also showed MOR- or GABA-immunopositive staining in lamina II; approximately 45.2 and 36.1% of the GABA-IR and MOR-IR neurons, respectively, expressed FOS protein in their nuclei induced by injecting formalin into the left lower lip of the mouth. Most of the GABA/MOR, GABA/FOS, and MOR/FOS double-labeled neurons made close contacts with EM2-IR fibers and terminals. Immuno-electron microscopy confirmed that the EM2-IR terminals formed synapses with GABA-IR or MOR-IR dendritic processes and neuronal cell bodies in lamina II of the Vc. These results suggest that EM2 might participate in pain transmission and modulation by binding to MOR-IR and GABAergic inhibitory interneuron in lamina II of the Vc to exert inhibitory effect on the excitatory interneuron in lamina II and projection neurons in laminae I and III. PMID:25386121

Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

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Chihiro Sugiyama

2015-01-01

Full Text Available In vitro estimating strategies for potential neurotoxicity are required to screen multiple substances. In a previous study, we showed that exposure to low-concentrations of some chemicals, such as organotin, decreased the expression of GluR2 protein, which is a subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA-type glutamate receptors, and led to neuronal vulnerability. This result suggested that GluR2 decreases as an index of neuronal cell sensitivity and vulnerability to various toxic insults. Accordingly, we developed a versatile method that is a large scale determination of GluR2 protein expression in the presence of environmental chemicals by means of AlphaLISA technology. Various analytical conditions were optimized, and then GluR2 protein amount was measured by the method using AlphaLISA. The GluR2 amounts were strongly correlated with that of measured by western blotting, which is currently used to determine GluR2 expression. An ideal standard curve could be written with the authentic GluR2 protein from 0 ng to 100 ng. Subsequently, twenty environmental chemicals were screened and nitenpyram was identified as a chemical which lead to decrease in GluR2 protein expression. This assay may provide a tool for detecting neurotoxic chemicals according to decreases in GluR2 protein expression.

ASIC3, an acid-sensing ion channel, is expressed in metaboreceptive sensory neurons

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Fierro Leonardo

2005-11-01

Full Text Available Abstract Background ASIC3, the most sensitive of the acid-sensing ion channels, depolarizes certain rat sensory neurons when lactic acid appears in the extracellular medium. Two functions have been proposed for it: 1 ASIC3 might trigger ischemic pain in heart and muscle; 2 it might contribute to some forms of touch mechanosensation. Here, we used immunocytochemistry, retrograde labelling, and electrophysiology to ask whether the distribution of ASIC3 in rat sensory neurons is consistent with either of these hypotheses. Results Less than half (40% of dorsal root ganglion sensory neurons react with anti-ASIC3, and the population is heterogeneous. They vary widely in cell diameter and express different growth factor receptors: 68% express TrkA, the receptor for nerve growth factor, and 25% express TrkC, the NT3 growth factor receptor. Consistent with a role in muscle nociception, small ( Conclusion Our data indicates that: 1 ASIC3 is expressed in a restricted population of nociceptors and probably in some non-nociceptors; 2 co-expression of ASIC3 and CGRP, and the absence of P2X3, are distinguishing properties of a class of sensory neurons, some of which innervate blood vessels. We suggest that these latter afferents may be muscle metaboreceptors, neurons that sense the metabolic state of muscle and can trigger pain when there is insufficient oxygen.

Fluctuating Estrogen and Progesterone Receptor Expression in Brainstem Norepinephrine Neurons through the Rat Estrous Cycle

NARCIS (Netherlands)

Haywood, S.A.; Simonian, S.X.; Beek, van der E.M.; Bicknell, R.J.; Herbison, A.E.

1999-01-01

Norepinephrine (NE) neurons within the nucleus tractus solitarii (NTS; A2 neurons) and ventrolateral medulla (A1 neurons) represent gonadal steroid-dependent components of several neural networks regulating reproduction. Previous studies have shown that both A1 and A2 neurons express estrogen

PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

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Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

2018-02-01

To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

Behavioral analysis of Drosophila transformants expressing human taste receptor genes in the gustatory receptor neurons.

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Adachi, Ryota; Sasaki, Yuko; Morita, Hiromi; Komai, Michio; Shirakawa, Hitoshi; Goto, Tomoko; Furuyama, Akira; Isono, Kunio

2012-06-01

Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.

Fragile X Proteins FMRP and FXR2P Control Synaptic GluA1 Expression and Neuronal Maturation via Distinct Mechanisms

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Weixiang Guo

2015-06-01

Full Text Available Fragile X mental retardation protein (FMRP and its autosomal paralog FXR2P are selective neuronal RNA-binding proteins, and mice that lack either protein exhibit cognitive deficits. Although double-mutant mice display more severe learning deficits than single mutants, the molecular mechanism behind this remains unknown. In the present study, we discovered that FXR2P (also known as FXR2 is important for neuronal dendritic development. FMRP and FXR2P additively promote the maturation of new neurons by regulating a common target, the AMPA receptor GluA1, but they do so via distinct mechanisms: FXR2P binds and stabilizes GluA1 mRNA and enhances subsequent protein expression, whereas FMRP promotes GluA1 membrane delivery. Our findings unveil important roles for FXR2P and GluA1 in neuronal development, uncover a regulatory mechanism of GluA1, and reveal a functional convergence between fragile X proteins in neuronal development.

Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

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Guntur, Ananya R.; Gu, Pengyu; Takle, Kendra; Chen, Jingyi; Xiang, Yang; Yang, Chung-Hui

2015-01-01

The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. We found that ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response—a function of CC cells—when they encounter strong UV, an aversive stimulus for young larvae. PMID:26443856

Maximal COX-2 and ppRb expression in neurons occurs during early Braak stages prior to the maximal activation of astrocytes and microglia in Alzheimer's disease

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Arendt Thomas

2005-11-01

Full Text Available Abstract Neuronal expression of cyclooxygenase-2 (COX-2 and cell cycle proteins is suggested to contribute to neurodegeneration during Alzheimer's disease (AD. The stimulus that induces COX-2 and cell cycle protein expression in AD is still elusive. Activated glia cells are shown to secrete substances that can induce expression of COX-2 and cell cycle proteins in vitro. Using post mortem brain tissue we have investigated whether activation of microglia and astrocytes in AD brain can be correlated with the expression of COX-2 and phosphorylated retinoblastoma protein (ppRb. The highest levels of neuronal COX-2 and ppRb immunoreactivity are observed in the first stages of AD pathology (Braak 0–II, Braak A. No significant difference in COX-2 or ppRb neuronal immunoreactivity is observed between Braak stage 0 and later Braak stages for neurofibrillary changes or amyloid plaques. The mean number of COX-2 or ppRb immunoreactive neurons is significantly decreased in Braak stage C compared to Braak stage A for amyloid deposits. Immunoreactivity for glial markers KP1, CR3/43 and GFAP appears in the later Braak stages and is significantly increased in Braak stage V-VI compared to Braak stage 0 for neurofibrillary changes. In addition, a significant negative correlation is observed between the presence of KP1, CR3/43 and GFAP immunoreactivity and the presence of neuronal immunoreactivity for COX-2 and ppRb. These data show that maximal COX-2 and ppRb immunoreactivity in neurons occurs during early Braak stages prior to the maximal activation of astrocytes and microglia. In contrast to in vitro studies, post mortem data do not support a causal relation between the activation of microglia and astrocytes and the expression of neuronal COX-2 and ppRb in the pathological cascade of AD.

Optically-Induced Neuronal Activity Is Sufficient to Promote Functional Motor Axon Regeneration In Vivo.

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Patricia J Ward

Full Text Available Peripheral nerve injuries are common, and functional recovery is very poor. Beyond surgical repair of the nerve, there are currently no treatment options for these patients. In experimental models of nerve injury, interventions (such as exercise and electrical stimulation that increase neuronal activity of the injured neurons effectively enhance axon regeneration. Here, we utilized optogenetics to determine whether increased activity alone is sufficient to promote motor axon regeneration. In thy-1-ChR2/YFP transgenic mice in which a subset of motoneurons express the light-sensitive cation channel, channelrhodopsin (ChR2, we activated axons in the sciatic nerve using blue light immediately prior to transection and surgical repair of the sciatic nerve. At four weeks post-injury, direct muscle EMG responses evoked with both optical and electrical stimuli as well as the ratio of these optical/electrical evoked EMG responses were significantly greater in mice that received optical treatment. Thus, significantly more ChR2+ axons successfully re-innervated the gastrocnemius muscle in mice that received optical treatment. Sections of the gastrocnemius muscles were reacted with antibodies to Synaptic Vesicle Protein 2 (SV2 to quantify the number of re-occupied motor endplates. The number of SV2+ endplates was greater in mice that received optical treatment. The number of retrogradely-labeled motoneurons following intramuscular injection of cholera toxin subunit B (conjugated to Alexa Fluor 555 was greater in mice that received optical treatment. Thus, the acute (1 hour, one-time optical treatment resulted in robust, long-lasting effects compared to untreated animals as well as untreated axons (ChR2-. We conclude that neuronal activation is sufficient to promote motor axon regeneration, and this regenerative effect is specific to the activated neurons.

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

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

2012-01-01

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

Tetramethylpyrazine suppresses transient oxygen-glucose deprivation-induced connexin32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathway in cultured hippocampal neurons.

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Gong, Gu; Yuan, Libang; Cai, Lin; Ran, Maorong; Zhang, Yulan; Gong, Huaqu; Dai, Xuemei; Wu, Wei; Dong, Hailong

2014-01-01

Tetramethylpyrazine (TMP) has been widely used in China as a drug for the treatment of various diseases. Recent studies have suggested that TMP has a protective effect on ischemic neuronal damage. However, the exact mechanism is still unclear. This study aims to investigate the mechanism of TMP mediated ischemic hippocampal neurons injury induced by oxygen-glucose deprivation (OGD). The effect of TMP on hippocampal neurons viability was detected by MTT assay, LDH release assay and apoptosis rate was measured by flow cytometry. TMP significantly suppressed neuron apoptosis in a concentration-dependent manner. TMP could significantly reduce the elevated levels of connexin32 (Cx32) induced by OGD. Knockdown of Cx32 by siRNA attenuated OGD injury. Moreover, our study showed that viability was increased in siRNA-Cx32-treated-neurons, and neuron apoptosis was suppressed by activating Bcl-2 expression and inhibiting Bax expression. Over expression of Cx32 could decrease neurons viability and increase LDH release. Furthermore, OGD increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the neuron injury and Cx32 up-regulation. Taken together, TMP can reverse the OGD-induced Cx32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathways.

Tetramethylpyrazine suppresses transient oxygen-glucose deprivation-induced connexin32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathway in cultured hippocampal neurons.

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Gu Gong

Full Text Available Tetramethylpyrazine (TMP has been widely used in China as a drug for the treatment of various diseases. Recent studies have suggested that TMP has a protective effect on ischemic neuronal damage. However, the exact mechanism is still unclear. This study aims to investigate the mechanism of TMP mediated ischemic hippocampal neurons injury induced by oxygen-glucose deprivation (OGD. The effect of TMP on hippocampal neurons viability was detected by MTT assay, LDH release assay and apoptosis rate was measured by flow cytometry. TMP significantly suppressed neuron apoptosis in a concentration-dependent manner. TMP could significantly reduce the elevated levels of connexin32 (Cx32 induced by OGD. Knockdown of Cx32 by siRNA attenuated OGD injury. Moreover, our study showed that viability was increased in siRNA-Cx32-treated-neurons, and neuron apoptosis was suppressed by activating Bcl-2 expression and inhibiting Bax expression. Over expression of Cx32 could decrease neurons viability and increase LDH release. Furthermore, OGD increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the neuron injury and Cx32 up-regulation. Taken together, TMP can reverse the OGD-induced Cx32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathways.

Exclusive neuronal expression of SUCLA2 in the human brain

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Dobolyi, Arpád; Ostergaard, Elsebet; Bagó, Attila G

2015-01-01

associated with SUCLA2 mutations, the precise localization of SUCLA2 protein has never been investigated. Here, we show that immunoreactivity of A-SUCL-β in surgical human cortical tissue samples was present exclusively in neurons, identified by their morphology and visualized by double labeling...... was absent in glial cells, identified by antibodies directed against the glial markers GFAP and S100. Furthermore, in situ hybridization histochemistry demonstrated that SUCLA2 mRNA was present in Nissl-labeled neurons but not glial cells labeled with S100. Immunoreactivity of the GTP-forming β subunit (G......-SUCL-β) encoded by SUCLG2, or in situ hybridization histochemistry for SUCLG2 mRNA could not be demonstrated in either neurons or astrocytes. Western blotting of post mortem brain samples revealed minor G-SUCL-β immunoreactivity that was, however, not upregulated in samples obtained from diabetic versus non...

Distinct Laterality in Forelimb-Movement Representations of Rat Primary and Secondary Motor Cortical Neurons with Intratelencephalic and Pyramidal Tract Projections.

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Soma, Shogo; Saiki, Akiko; Yoshida, Junichi; Ríos, Alain; Kawabata, Masanori; Sakai, Yutaka; Isomura, Yoshikazu

2017-11-08

Two distinct motor areas, the primary and secondary motor cortices (M1 and M2), play crucial roles in voluntary movement in rodents. The aim of this study was to characterize the laterality in motor cortical representations of right and left forelimb movements. To achieve this goal, we developed a novel behavioral task, the Right-Left Pedal task, in which a head-restrained male rat manipulates a right or left pedal with the corresponding forelimb. This task enabled us to monitor independent movements of both forelimbs with high spatiotemporal resolution. We observed phasic movement-related neuronal activity (Go-type) and tonic hold-related activity (Hold-type) in isolated unilateral movements. In both M1 and M2, Go-type neurons exhibited bias toward contralateral preference, whereas Hold-type neurons exhibited no bias. The contralateral bias was weaker in M2 than M1. Moreover, we differentiated between intratelencephalic (IT) and pyramidal tract (PT) neurons using optogenetically evoked spike collision in rats expressing channelrhodopsin-2. Even in identified PT and IT neurons, Hold-type neurons exhibited no lateral bias. Go-type PT neurons exhibited bias toward contralateral preference, whereas IT neurons exhibited no bias. Our findings suggest a different laterality of movement representations of M1 and M2, in each of which IT neurons are involved in cooperation of bilateral movements, whereas PT neurons control contralateral movements. SIGNIFICANCE STATEMENT In rodents, the primary and secondary motor cortices (M1 and M2) are involved in voluntary movements via distinct projection neurons: intratelencephalic (IT) neurons and pyramidal tract (PT) neurons. However, it remains unclear whether the two motor cortices (M1 vs M2) and the two classes of projection neurons (IT vs PT) have different laterality of movement representations. We optogenetically identified these neurons and analyzed their functional activity using a novel behavioral task to monitor movements

P2X receptors, sensory neurons and pain.

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Bele, Tanja; Fabbretti, Elsa

2015-01-01

Pain represents a very large social and clinical problem since the current treatment provides insufficient pain relief. Plasticity of pain receptors together with sensitisation of sensory neurons, and the role of soluble mediators released from non-neuronal cells render difficult to understand the spatial and temporal scale of pain development, neuronal responses and disease progression. In pathological conditions, ATP is one of the most powerful mediators that activates P2X receptors that behave as sensitive ATP-detectors, such as neuronal P2X3 receptor subtypes and P2X4 and P2X7 receptors expressed on non-neuronal cells. Dissecting the molecular mechanisms occurring in sensory neurons and in accessory cells allows to design appropriate tissue- and cell- targeted approaches to treat chronic pain.

Polarized axonal surface expression of neuronal KCNQ potassium channels is regulated by calmodulin interaction with KCNQ2 subunit.

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John P Cavaretta

Full Text Available KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4 membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal

The multifaceted effects of agmatine on functional recovery after spinal cord injury through Modulations of BMP-2/4/7 expressions in neurons and glial cells.

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Yu Mi Park

Full Text Available Presently, few treatments for spinal cord injury (SCI are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm, a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm(2 weight for 1 min at thoracic vertebra (Th 9 segment. Mice that received an intraperitoneal (i.p. injection of Agm (100 mg/kg/day within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following

The Multifaceted Effects of Agmatine on Functional Recovery after Spinal Cord Injury through Modulations of BMP-2/4/7 Expressions in Neurons and Glial Cells

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Park, Yu Mi; Lee, Won Taek; Bokara, Kiran Kumar; Seo, Su Kyoung; Park, Seung Hwa; Kim, Jae Hwan; Yenari, Midori A.; Park, Kyung Ah; Lee, Jong Eun

2013-01-01

Presently, few treatments for spinal cord injury (SCI) are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm), a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm2 weight for 1 min at thoracic vertebra (Th) 9 segment. Mice that received an intraperitoneal (i.p.) injection of Agm (100 mg/kg/day) within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs) are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST) demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following SCI. PMID

Expression of Sirtuins in the Retinal Neurons of Mice, Rats, and Humans

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Hongdou Luo

2017-11-01

Full Text Available Sirtuins are a class of histone deacetylases (HDACs that have been shown to regulate a range of pathophysiological processes such as cellular aging, inflammation, metabolism, and cell proliferation. There are seven mammalian Sirtuins (SIRT1-7 that play important roles in stress response, aging, and neurodegenerative diseases. However, the location and function of Sirtuins in neurons are not well defined. This study assessed the retinal expression of Sirtuins in mice, rats, and humans and measured the expression of Sirtuins in aged and injured retinas. Expression of all 7 Sirtuins was confirmed by Western blot and Real-Time PCR analysis in all three species. SIRT1 is highly expressed in mouse, rat, and human retinas, whereas SIRT2-7 expression was relatively lower in human retinas. Immunofluorescence was also used to examine the expression and localization of Sirtuins in rat retinal neurons. Importantly, we demonstrate a marked reduction of SIRT1 expression in aged retinal neurons as well as retinas injured by acute ischemia-reperfusion. On the other hand, none of the other Sirtuins exhibit any significant age-related changes in expression except for SIRT5, which was significantly higher in the retinas of adults compared to both young and aged rats. Our work presents the first composite analysis of Sirtuins in the retinal neurons of mice, rats, and humans, and suggests that increasing the expression and activity of SIRT1 may be beneficial for the treatment of glaucoma and other age-related eye dysfunction.

Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.

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Sulk, Mathias; Seeliger, Stephan; Aubert, Jerome; Schwab, Verena D; Cevikbas, Ferda; Rivier, Michel; Nowak, Pawel; Voegel, Johannes J; Buddenkotte, Jörg; Steinhoff, Martin

2012-04-01

Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea.

Differential Gene Expression in Gonadotropin-Releasing Hormone Neurons of Male and Metestrous Female Mice.

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Vastagh, Csaba; Rodolosse, Annie; Solymosi, Norbert; Farkas, Imre; Auer, Herbert; Sárvári, Miklós; Liposits, Zsolt

2015-01-01

Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the regulation of the hypothalamic-pituitary gonadal axis in a sex-specific manner. We hypothesized that the differences seen in reproductive functions of males and females are associated with a sexually dimorphic gene expression profile of GnRH neurons. We compared the transcriptome of GnRH neurons obtained from intact metestrous female and male GnRH-green fluorescent protein transgenic mice. About 1,500 individual GnRH neurons from each sex were sampled with laser capture microdissection followed by whole-transcriptome amplification for gene expression profiling. Under stringent selection criteria (fold change >1.6, adjusted p value 0.01), Affymetrix Mouse Genome 430 PM array analysis identified 543 differentially expressed genes. Sexual dimorphism was most apparent in gene clusters associated with synaptic communication, signal transduction, cell adhesion, vesicular transport and cell metabolism. To validate microarray results, 57 genes were selected, and 91% of their differential expression was confirmed by real-time PCR. Similarly, 88% of microarray results were confirmed with PCR from independent samples obtained by patch pipette harvesting and pooling of 30 GnRH neurons from each sex. We found significant differences in the expression of genes involved in vesicle priming and docking (Syt1, Cplx1), GABAergic (Gabra3, Gabrb3, Gabrg2) and glutamatergic (Gria1, Grin1, Slc17a6) neurotransmission, peptide signaling (Sstr3, Npr2, Cxcr4) and the regulation of intracellular ion homeostasis (Cacna1, Cacnb1, Cacng5, Kcnq2, Kcnc1). The striking sexual dimorphism of the GnRH neuron transcriptome we report here contributes to a better understanding of the differences in cellular mechanisms of GnRH neurons in the two sexes. © 2015 S. Karger AG, Basel.

Enhancement of high glucose-induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2 /Akt/NF-κB pathway.

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Onphachanh, Xaykham; Lee, Hyun Jik; Lim, Jae Ryong; Jung, Young Hyun; Kim, Jun Sung; Chae, Chang Woo; Lee, Sei-Jung; Gabr, Amr Ahmed; Han, Ho Jae

2017-09-01

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM-associated neuronal cell death. Previous investigators reported on a genome-wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose-induced neuronal cell death and the effect of melatonin against high glucose-induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN-induced putative kinase 1 (PINK1) and LC-3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker™ fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V-positive cells. In addition, high glucose-stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N-acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT 2 receptor-specific inhibitor 4-P-PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin-regulated mitochondrial ROS production, cleaved caspase-3 and caspase-9 expressions, and the number of annexin V-positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT 2 /Akt/NF-κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions. © 2017 The Authors. Journal of Pineal Research

All-Optical Electrophysiology for Disease Modeling and Pharmacological Characterization of Neurons.

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Werley, Christopher A; Brookings, Ted; Upadhyay, Hansini; Williams, Luis A; McManus, Owen B; Dempsey, Graham T

2017-09-11

A key challenge for establishing a phenotypic screen for neuronal excitability is measurement of membrane potential changes with high throughput and accuracy. Most approaches for probing excitability rely on low-throughput, invasive methods or lack cell-specific information. These limitations stimulated the development of novel strategies for characterizing the electrical properties of cultured neurons. Among these was the development of optogenetic technologies (Optopatch) that allow for stimulation and recording of membrane voltage signals from cultured neurons with single-cell sensitivity and millisecond temporal resolution. Neuronal activity is elicited using blue light activation of the channelrhodopsin variant 'CheRiff'. Action potentials and synaptic signals are measured with 'QuasAr', a rapid and sensitive voltage-indicating protein with near-infrared fluorescence that scales proportionately with transmembrane potential. This integrated technology of optical stimulation and recording of electrical signals enables investigation of neuronal electrical function with unprecedented scale and precision. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Long-term exposure to endogenous levels of tributyltin decreases GluR2 expression and increases neuronal vulnerability to glutamate

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Nakatsu, Yusuke; Kotake, Yaichiro; Takishita, Tomoko; Ohta, Shigeru

2009-01-01

Tributyltin (TBT), an endocrine-disrupting chemical, has been used commercially as a heat stabilizer, agricultural pesticide and component of antifouling paints. In this study, we investigated the effect of long-term exposure to endogenous levels of TBT on neuronal glutamate receptors. Cultured rat cortical neurons were exposed to 1-50 nM TBT for 9 days (from day 2 to day 10 in vitro). The number of neurons was reduced by long-term exposure to 50 nM TBT, but not to 1-20 nM TBT. Long-term exposure to 20 nM TBT decreased the mRNA expression of glutamate receptors NR1, NR2A, GluR1 and GluR2, and increased that of NR2B, GluR3 and GluR4. GluR2 protein was also reduced by long-term exposure to TBT. Because AMPA receptor lacking GluR2 exhibits Ca 2+ permeability, we investigated whether Ca 2+ influx or glutamate toxicity was affected. Indeed, glutamate-induced Ca 2+ influx was increased in TBT-treated neurons. Consistent with this, neurons became more susceptible to glutamate toxicity as a result of long-term exposure to TBT and this susceptibility was abolished by an antagonist of GluR2-lacking AMPA receptor. Thus, it is suggested that long-term exposure to endogenous levels of TBT induces a decrease of GluR2 protein, causing neurons become more susceptible to glutamate toxicity.

Long-term exposure to endogenous levels of tributyltin decreases GluR2 expression and increases neuronal vulnerability to glutamate.

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Nakatsu, Yusuke; Kotake, Yaichiro; Takishita, Tomoko; Ohta, Shigeru

2009-10-15

Tributyltin (TBT), an endocrine-disrupting chemical, has been used commercially as a heat stabilizer, agricultural pesticide and component of antifouling paints. In this study, we investigated the effect of long-term exposure to endogenous levels of TBT on neuronal glutamate receptors. Cultured rat cortical neurons were exposed to 1-50 nM TBT for 9 days (from day 2 to day 10 in vitro). The number of neurons was reduced by long-term exposure to 50 nM TBT, but not to 1-20 nM TBT. Long-term exposure to 20 nM TBT decreased the mRNA expression of glutamate receptors NR1, NR2A, GluR1 and GluR2, and increased that of NR2B, GluR3 and GluR4. GluR2 protein was also reduced by long-term exposure to TBT. Because AMPA receptor lacking GluR2 exhibits Ca2+ permeability, we investigated whether Ca2+ influx or glutamate toxicity was affected. Indeed, glutamate-induced Ca2+ influx was increased in TBT-treated neurons. Consistent with this, neurons became more susceptible to glutamate toxicity as a result of long-term exposure to TBT and this susceptibility was abolished by an antagonist of GluR2-lacking AMPA receptor. Thus, it is suggested that long-term exposure to endogenous levels of TBT induces a decrease of GluR2 protein, causing neurons become more susceptible to glutamate toxicity.

Transcriptional regulation of gene expression clusters in motor neurons following spinal cord injury

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Westerdahl Ann-Charlotte

2010-06-01

Full Text Available Abstract Background Spinal cord injury leads to neurological dysfunctions affecting the motor, sensory as well as the autonomic systems. Increased excitability of motor neurons has been implicated in injury-induced spasticity, where the reappearance of self-sustained plateau potentials in the absence of modulatory inputs from the brain correlates with the development of spasticity. Results Here we examine the dynamic transcriptional response of motor neurons to spinal cord injury as it evolves over time to unravel common gene expression patterns and their underlying regulatory mechanisms. For this we use a rat-tail-model with complete spinal cord transection causing injury-induced spasticity, where gene expression profiles are obtained from labeled motor neurons extracted with laser microdissection 0, 2, 7, 21 and 60 days post injury. Consensus clustering identifies 12 gene clusters with distinct time expression profiles. Analysis of these gene clusters identifies early immunological/inflammatory and late developmental responses as well as a regulation of genes relating to neuron excitability that support the development of motor neuron hyper-excitability and the reappearance of plateau potentials in the late phase of the injury response. Transcription factor motif analysis identifies differentially expressed transcription factors involved in the regulation of each gene cluster, shaping the expression of the identified biological processes and their associated genes underlying the changes in motor neuron excitability. Conclusions This analysis provides important clues to the underlying mechanisms of transcriptional regulation responsible for the increased excitability observed in motor neurons in the late chronic phase of spinal cord injury suggesting alternative targets for treatment of spinal cord injury. Several transcription factors were identified as potential regulators of gene clusters containing elements related to motor neuron hyper

Transcriptional regulation of gene expression clusters in motor neurons following spinal cord injury.

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Ryge, Jesper; Winther, Ole; Wienecke, Jacob; Sandelin, Albin; Westerdahl, Ann-Charlotte; Hultborn, Hans; Kiehn, Ole

2010-06-09

Spinal cord injury leads to neurological dysfunctions affecting the motor, sensory as well as the autonomic systems. Increased excitability of motor neurons has been implicated in injury-induced spasticity, where the reappearance of self-sustained plateau potentials in the absence of modulatory inputs from the brain correlates with the development of spasticity. Here we examine the dynamic transcriptional response of motor neurons to spinal cord injury as it evolves over time to unravel common gene expression patterns and their underlying regulatory mechanisms. For this we use a rat-tail-model with complete spinal cord transection causing injury-induced spasticity, where gene expression profiles are obtained from labeled motor neurons extracted with laser microdissection 0, 2, 7, 21 and 60 days post injury. Consensus clustering identifies 12 gene clusters with distinct time expression profiles. Analysis of these gene clusters identifies early immunological/inflammatory and late developmental responses as well as a regulation of genes relating to neuron excitability that support the development of motor neuron hyper-excitability and the reappearance of plateau potentials in the late phase of the injury response. Transcription factor motif analysis identifies differentially expressed transcription factors involved in the regulation of each gene cluster, shaping the expression of the identified biological processes and their associated genes underlying the changes in motor neuron excitability. This analysis provides important clues to the underlying mechanisms of transcriptional regulation responsible for the increased excitability observed in motor neurons in the late chronic phase of spinal cord injury suggesting alternative targets for treatment of spinal cord injury. Several transcription factors were identified as potential regulators of gene clusters containing elements related to motor neuron hyper-excitability, the manipulation of which potentially could be

Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons.

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Vernon, Claire G; Swanson, Geoffrey T

2017-03-22

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2 -/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth. SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and

Expression of the ghrelin receptor gene in neurons of the medulla oblongata of the rat.

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Bron, Romke; Yin, Lei; Russo, Domenico; Furness, John B

2013-08-15

There is ambiguity concerning the distribution of neurons that express the ghrelin receptor (GHSR) in the medulla oblongata. In the current study we used a sensitive nonradioactive method to investigate GHSR mRNA distribution by in situ hybridization. Strong expression of the GHSR gene was confirmed in neurons of the facial nucleus (FacN, 7), the dorsal vagal complex (DVC), and the semicompact (but not compact) nucleus ambiguus (AmbSC and AmbC). In addition, expression of GHSR was found in other regions, where it had not been described before. GHSR-positive neurons were observed in the gustatory rostral nucleus tractus solitarius and in areas involved in vestibulo-ocular processing (such as the medial vestibular nucleus and the nucleus abducens). GHSR expression was also noted in ventral areas associated with cardiorespiratory control, including the gigantocellular reticular nucleus, the lateral paragigantocellular nucleus, the rostral and caudal ventrolateral medulla, the (pre)-Bötzinger complex, and the rostral and caudal ventrolateral respiratory group. However, GHSR-positive neurons in ventrolateral areas did not express markers for cardiovascular presympathetic vasomotor neurons, respiratory propriobulbar rhythmogenic neurons, or sensory interneurons. GHSR-positive cells were intermingled with catecholamine neurons in the dorsal vagal complex but these populations did not overlap. Thus, the ghrelin receptor occurs in the medulla oblongata in 1) second-order sensory neurons processing gustatory, vestibulo-ocular, and visceral sensation; 2) cholinergic somatomotor neurons of the FacN and autonomic preganglionic neurons of the DMNX and AmbSC; 3) cardiovascular neurons in the DVC, Gi, and LPGi; 4) neurons of as yet unknown function in the ventrolateral medulla. Copyright © 2013 Wiley Periodicals, Inc., A Wiley Company.

Ellagic acid protects against neuron damage in ischemic stroke through regulating the ratio of Bcl-2/Bax expression.

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Liu, Qing-Shan; Deng, Ran; Li, Shuran; Li, Xu; Li, Keqin; Kebaituli, Gulibanumu; Li, Xueli; Liu, Rui

2017-08-01

An oxygen-glucose deprivation and reoxygenation model in primary cultured rat cortical neurons was developed for this study to investigate the effects of ellagic acid (EA), a low-molecular-weight polyphenol, on neuron cells and their function, and to evaluate whether EA can be safely utilized by humans as a functional food or therapeutic agent. Administration of EA significantly decreased the volume of cerebrum infarction and the neurological deficit scores of the rats; EA treatment also increased the number of Bcl-2-positive cells and the ratio of Bcl-2-positive to Bax-positive neurons in the semidarkness zone near the brain ischemic focus in the photothrombotic cerebral ischemia model. Treatment of EA resulted in increased neuron viability, cell nuclear integrity, and the ratio of Bcl-2/Bax expression in the primary cultured neuron model; EA treatment also lead to a decrease in the number of apoptotic cells. Our results therefore suggest a specific mechanism for the beneficial effects of EA, providing new insights into how it provides neuroprotection. To the best of our knowledge, these results represent new insights on the mechanisms of the brain cell protective activity of EA. Thus, EA may be used in functional foods or medicines to help treat nerve dysfunction, neurodegenerative disease, and aging.

PPARγ transcriptionally regulates the expression of insulin-degrading enzyme in primary neurons

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Du, Jing; Zhang, Lang; Liu, Shubo; Zhang, Chi; Huang, Xiuqing; Li, Jian; Zhao, Nanming; Wang, Zhao

2009-01-01

Insulin-degrading enzyme (IDE) is a protease that has been demonstrated to play a key role in degrading both Aβ and insulin and deficient in IDE function is associated with Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2) pathology. However, little is known about the cellular and molecular regulation of IDE expression. Here we show IDE levels are markedly decreased in DM2 patients and positively correlated with the peroxisome proliferator-activated receptor γ (PPARγ) levels. Further studies show that PPARγ plays an important role in regulating IDE expression in rat primary neurons through binding to a functional peroxisome proliferator-response element (PPRE) in IDE promoter and promoting IDE gene transcription. Finally, we demonstrate that PPARγ participates in the insulin-induced IDE expression in neurons. These results suggest that PPARγ transcriptionally induces IDE expression which provides a novel mechanism for the use of PPARγ agonists in both DM2 and AD therapies.

A possible new mechanism for the control of miRNA expression in neurons.

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Kinjo, Erika Reime; Higa, Guilherme Shigueto Vilar; de Sousa, Erica; Casado, Otávio Augusto Nocera; Damico, Marcio Vinicius; Britto, Luiz Roberto G; Kihara, Alexandre Hiroaki

2013-10-01

The control of gene expression by miRNAs has been widely investigated in different species and cell types. Following a probabilistic rather than a deterministic regimen, the action of these short nucleotide sequences on specific genes depends on intracellular concentration, which in turn reflects the balance between biosynthesis and degradation. Recent studies have described the involvement of XRN2, an exoribonuclease, in miRNA degradation and PAPD4, an atypical poly(A) polymerase, in miRNA stability. Herein, we examined the expression of XRN2 and PAPD4 in developing and adult rat hippocampi. Combining bioinformatics and real-time PCR, we demonstrated that XRN2 and PAPD4 expression is regulated by the uncorrelated action of transcription factors, resulting in distinct gene expression profiles during development. Analyses of nuclei position and nestin labeling revealed that both proteins progressively accumulated during neuronal differentiation, and that they are weakly expressed in immature neurons and absent in glial and endothelial cells. Despite the differences in subcellular localization, both genes were concurrently identified within identical neuronal subpopulations, including specific inhibitory interneurons. Thus, we cope with a singular circumstance in biology: an almost complete intersected expression of functional-opposed genes, reinforcing that their antagonistically driven actions on miRNAs "make sense" if simultaneously present at the same cells. Considering that the transcriptome in the nervous system is finely tuned to physiological processes, it was remarkable that miRNA stability-related genes were concurrently identified in neurons that play essential roles in cognitive functions such as memory and learning. In summary, this study reveals a possible new mechanism for the control of miRNA expression. © 2013 Elsevier Inc. All rights reserved.

FTO is expressed in neurones throughout the brain and its expression is unaltered by fasting.

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McTaggart, James S; Lee, Sheena; Iberl, Michaela; Church, Chris; Cox, Roger D; Ashcroft, Frances M

2011-01-01

Single-nucleotide polymorphisms in the first intron of the ubiquitously expressed FTO gene are associated with obesity. Although the physiological functions of FTO remain unclear, food intake is often altered when Fto expression levels are manipulated. Furthermore, deletion of FTO from neurones alone has a similar effect on food intake to deletion of FTO in all tissues. These results indicate that FTO expression in the brain is particularly important. Considerable focus has been placed on the dynamic regulation of Fto mRNA expression in the hypothalamus after short-term (16-48 hour) fasting, but results have been controversial. There are no studies that quantify FTO protein levels across the brain, and assess its alteration following short-term fasting. Using immunohistochemistry, we found that FTO protein is widely expressed in mouse brain, and present in the majority of neurones. Using quantitative Western blotting and RT-qPCR we show that FTO protein and mRNA levels in the hypothalamus, cerebellum and rostral brain are relatively uniform, and levels in the brain are higher than in skeletal muscles of the lower limbs. Fasting for 18 hours does not alter the expression pattern, or levels, of FTO protein and mRNA. We further show that the majority of POMC neurones, which are critically involved in food intake regulation, also express FTO, but that the percentage of FTO-positive POMC neurones is not altered by fasting. In summary, we find no evidence that Fto/FTO expression is regulated by short-term (18-hour) fasting. Thus, it is unlikely that the hunger and increased post-fasting food intake caused by such food deprivation is driven by alterations in Fto/FTO expression. The widespread expression of FTO in neurones also suggests that physiological studies of this protein should not be limited to the hypothalamus.

FTO is expressed in neurones throughout the brain and its expression is unaltered by fasting.

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James S McTaggart

Full Text Available Single-nucleotide polymorphisms in the first intron of the ubiquitously expressed FTO gene are associated with obesity. Although the physiological functions of FTO remain unclear, food intake is often altered when Fto expression levels are manipulated. Furthermore, deletion of FTO from neurones alone has a similar effect on food intake to deletion of FTO in all tissues. These results indicate that FTO expression in the brain is particularly important. Considerable focus has been placed on the dynamic regulation of Fto mRNA expression in the hypothalamus after short-term (16-48 hour fasting, but results have been controversial. There are no studies that quantify FTO protein levels across the brain, and assess its alteration following short-term fasting. Using immunohistochemistry, we found that FTO protein is widely expressed in mouse brain, and present in the majority of neurones. Using quantitative Western blotting and RT-qPCR we show that FTO protein and mRNA levels in the hypothalamus, cerebellum and rostral brain are relatively uniform, and levels in the brain are higher than in skeletal muscles of the lower limbs. Fasting for 18 hours does not alter the expression pattern, or levels, of FTO protein and mRNA. We further show that the majority of POMC neurones, which are critically involved in food intake regulation, also express FTO, but that the percentage of FTO-positive POMC neurones is not altered by fasting. In summary, we find no evidence that Fto/FTO expression is regulated by short-term (18-hour fasting. Thus, it is unlikely that the hunger and increased post-fasting food intake caused by such food deprivation is driven by alterations in Fto/FTO expression. The widespread expression of FTO in neurones also suggests that physiological studies of this protein should not be limited to the hypothalamus.

BDNF regulates the expression and distribution of vesicular glutamate transporters in cultured hippocampal neurons.

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Carlos V Melo

Full Text Available BDNF is a pro-survival protein involved in neuronal development and synaptic plasticity. BDNF strengthens excitatory synapses and contributes to LTP, presynaptically, through enhancement of glutamate release, and postsynaptically, via phosphorylation of neurotransmitter receptors, modulation of receptor traffic and activation of the translation machinery. We examined whether BDNF upregulated vesicular glutamate receptor (VGLUT 1 and 2 expression, which would partly account for the increased glutamate release in LTP. Cultured rat hippocampal neurons were incubated with 100 ng/ml BDNF, for different periods of time, and VGLUT gene and protein expression were assessed by real-time PCR and immunoblotting, respectively. At DIV7, exogenous application of BDNF rapidly increased VGLUT2 mRNA and protein levels, in a dose-dependent manner. VGLUT1 expression also increased but only transiently. However, at DIV14, BDNF stably increased VGLUT1 expression, whilst VGLUT2 levels remained low. Transcription inhibition with actinomycin-D or α-amanitine, and translation inhibition with emetine or anisomycin, fully blocked BDNF-induced VGLUT upregulation. Fluorescence microscopy imaging showed that BDNF stimulation upregulates the number, integrated density and intensity of VGLUT1 and VGLUT2 puncta in neurites of cultured hippocampal neurons (DIV7, indicating that the neurotrophin also affects the subcellular distribution of the transporter in developing neurons. Increased VGLUT1 somatic signals were also found 3 h after stimulation with BDNF, further suggesting an increased de novo transcription and translation. BDNF regulation of VGLUT expression was specifically mediated by BDNF, as no effect was found upon application of IGF-1 or bFGF, which activate other receptor tyrosine kinases. Moreover, inhibition of TrkB receptors with K252a and PLCγ signaling with U-73122 precluded BDNF-induced VGLUT upregulation. Hippocampal neurons express both isoforms during

The retrotrapezoid nucleus neurons expressing Atoh1 and Phox2b are essential for the respiratory response to CO₂

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Ruffault, Pierre Louis; D’Autréaux, Fabien; Hayes, John A.

2015-01-01

. Photostimulation of these neurons entrains the respiratory rhythm. Conversely, abrogating expression of Atoh1 or Phox2b or glutamatergic transmission in these cells curtails the phrenic nerve response to low pH in embryonic preparations and abolishes the respiratory chemoreflex in behaving animals. Thus, the RTN...

Selective expression of KCNS3 potassium channel α-subunit in parvalbumin-containing GABA neurons in the human prefrontal cortex.

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Danko Georgiev

Full Text Available The cognitive deficits of schizophrenia appear to be associated with altered cortical GABA neurotransmission in the subsets of inhibitory neurons that express either parvalbumin (PV or somatostatin (SST. Identification of molecular mechanisms that operate selectively in these neurons is essential for developing targeted therapeutic strategies that do not influence other cell types. Consequently, we sought to identify, in the human cortex, gene products that are expressed selectively by PV and/or SST neurons, and that might contribute to their distinctive functional properties. Based on previously reported expression patterns in the cortex of mice and humans, we selected four genes: KCNS3, LHX6, KCNAB1, and PPP1R2, encoding K(+ channel Kv9.3 modulatory α-subunit, LIM homeobox protein 6, K(+ channel Kvβ1 subunit, and protein phosphatase 1 regulatory subunit 2, respectively, and examined their colocalization with PV or SST mRNAs in the human prefrontal cortex using dual-label in situ hybridization with (35S- and digoxigenin-labeled antisense riboprobes. KCNS3 mRNA was detected in almost all PV neurons, but not in SST neurons, and PV mRNA was detected in >90% of KCNS3 mRNA-expressing neurons. LHX6 mRNA was detected in almost all PV and >90% of SST neurons, while among all LHX6 mRNA-expressing neurons 50% expressed PV mRNA and >44% expressed SST mRNA. KCNAB1 and PPP1R2 mRNAs were detected in much larger populations of cortical neurons than PV or SST neurons. These findings indicate that KCNS3 is a selective marker of PV neurons, whereas LHX6 is expressed by both PV and SST neurons. KCNS3 and LHX6 might be useful for characterizing cell-type specific molecular alterations of cortical GABA neurotransmission and for the development of novel treatments targeting PV and/or SST neurons in schizophrenia.

TRPA1 is functionally expressed primarily by IB4-binding, non-peptidergic mouse and rat sensory neurons.

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Marie E Barabas

Full Text Available Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J DRG neurons. Approximately 80% of all small-diameter (<27 µm neurons from lumbar 1-6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79% or cinnamaldehyde (84% were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81% cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66% of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2-4% responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter responded to AITC (6% or cinnamaldehyde (4%, confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is

TRPA1 Is Functionally Expressed Primarily by IB4-Binding, Non-Peptidergic Mouse and Rat Sensory Neurons

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Stucky, Cheryl L.

2012-01-01

Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (neurons from lumbar 1–6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2–4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally expressed

Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

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Bron Dominique

2008-04-01

Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

Visualization of odor-induced neuronal activity by immediate early gene expression

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Bepari Asim K

2012-11-01

Full Text Available Abstract Background Sensitive detection of sensory-evoked neuronal activation is a key to mechanistic understanding of brain functions. Since immediate early genes (IEGs are readily induced in the brain by environmental changes, tracing IEG expression provides a convenient tool to identify brain activity. In this study we used in situ hybridization to detect odor-evoked induction of ten IEGs in the mouse olfactory system. We then analyzed IEG induction in the cyclic nucleotide-gated channel subunit A2 (Cnga2-null mice to visualize residual neuronal activity following odorant exposure since CNGA2 is a key component of the olfactory signal transduction pathway in the main olfactory system. Results We observed rapid induction of as many as ten IEGs in the mouse olfactory bulb (OB after olfactory stimulation by a non-biological odorant amyl acetate. A robust increase in expression of several IEGs like c-fos and Egr1 was evident in the glomerular layer, the mitral/tufted cell layer and the granule cell layer. Additionally, the neuronal IEG Npas4 showed steep induction from a very low basal expression level predominantly in the granule cell layer. In Cnga2-null mice, which are usually anosmic and sexually unresponsive, glomerular activation was insignificant in response to either ambient odorants or female stimuli. However, a subtle induction of c-fos took place in the OB of a few Cnga2-mutants which exhibited sexual arousal. Interestingly, very strong glomerular activation was observed in the OB of Cnga2-null male mice after stimulation with either the neutral odor amyl acetate or the predator odor 2, 3, 5-trimethyl-3-thiazoline (TMT. Conclusions This study shows for the first time that in vivo olfactory stimulation can robustly induce the neuronal IEG Npas4 in the mouse OB and confirms the odor-evoked induction of a number of IEGs. As shown in previous studies, our results indicate that a CNGA2-independent signaling pathway(s may activate the

In vivo transgenic expression of collybistin in neurons of the rat cerebral cortex.

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Fekete, Christopher D; Goz, Roman U; Dinallo, Sean; Miralles, Celia P; Chiou, Tzu-Ting; Bear, John; Fiondella, Christopher G; LoTurco, Joseph J; De Blas, Angel L

2017-04-01

Collybistin (CB) is a guanine nucleotide exchange factor selectively localized to γ-aminobutyric acid (GABA)ergic and glycinergic postsynapses. Active CB interacts with gephyrin, inducing the submembranous clustering and the postsynaptic accumulation of gephyrin, which is a scaffold protein that recruits GABA A receptors (GABA A Rs) at the postsynapse. CB is expressed with or without a src homology 3 (SH3) domain. We have previously reported the effects on GABAergic synapses of the acute overexpression of CB SH3- or CB SH3+ in cultured hippocampal (HP) neurons. In the present communication, we are studying the effects on GABAergic synapses after chronic in vivo transgenic expression of CB2 SH3- or CB2 SH3+ in neurons of the adult rat cerebral cortex. The embryonic precursors of these cortical neurons were in utero electroporated with CB SH3- or CB SH3+ DNAs, migrated to the appropriate cortical layer, and became integrated in cortical circuits. The results show that: 1) the strength of inhibitory synapses in vivo can be enhanced by increasing the expression of CB in neurons; and 2) there are significant differences in the results between in vivo and in culture studies. J. Comp. Neurol. 525:1291-1311, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The neuronal Ca(2+) -binding protein 2 (NECAB2) interacts with the adenosine A(2A) receptor and modulates the cell surface expression and function of the receptor.

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Canela, Laia; Luján, Rafael; Lluís, Carme; Burgueño, Javier; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Ciruela, Francisco

2007-09-01

Heptaspanning membrane also known as G protein-coupled receptors (GPCR) do interact with a variety of intracellular proteins whose function is regulate receptor traffic and/or signaling. Using a yeast two-hybrid screen, NECAB2, a neuronal calcium binding protein, was identified as a binding partner for the adenosine A(2A) receptor (A(2A)R) interacting with its C-terminal domain. Co-localization, co-immunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and NECAB2 in both transfected HEK-293 cells and also in rat striatum. Immunoelectron microscopy detection of NECAB2 and A(2A)R in the rat striatopallidal structures indicated that both proteins are co-distributed in the same glutamatergic nerve terminals. The interaction of NECAB2 with A(2A)R modulated the cell surface expression, the ligand-dependent internalization and the receptor-mediated activation of the MAPK pathway. Overall, these results show that A(2A)R interacts with NECAB2 in striatal neurones co-expressing the two proteins and that the interaction is relevant for A(2A)R function.

Distinct populations of GABAergic neurons in mouse rhombomere 1 express but do not require the homeodomain transcription factor PITX2.

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Waite, Mindy R; Skaggs, Kaia; Kaviany, Parisa; Skidmore, Jennifer M; Causeret, Frédéric; Martin, James F; Martin, Donna M

2012-01-01

Hindbrain rhombomere 1 (r1) is located caudal to the isthmus, a critical organizer region, and rostral to rhombomere 2 in the developing mouse brain. Dorsal r1 gives rise to the cerebellum, locus coeruleus, and several brainstem nuclei, whereas cells from ventral r1 contribute to the trochlear and trigeminal nuclei as well as serotonergic and GABAergic neurons of the dorsal raphe. Recent studies have identified several molecular events controlling dorsal r1 development. In contrast, very little is known about ventral r1 gene expression and the genetic mechanisms regulating its formation. Neurons with distinct neurotransmitter phenotypes have been identified in ventral r1 including GABAergic, serotonergic, and cholinergic neurons. Here we show that PITX2 marks a distinct population of GABAergic neurons in mouse embryonic ventral r1. This population appears to retain its GABAergic identity even in the absence of PITX2. We provide a comprehensive map of markers that places these PITX2-positive GABAergic neurons in a region of r1 that intersects and is potentially in communication with the dorsal raphe. Copyright © 2011 Elsevier Inc. All rights reserved.

Short-term memory in networks of dissociated cortical neurons.

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

2013-01-30

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.

Different requirements for GFRα2-signaling in three populations of cutaneous sensory neurons.

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Kupari, Jussi; Airaksinen, Matti S

2014-01-01

Many primary sensory neurons in mouse dorsal root ganglia (DRG) express one or several GFRα's, the ligand-binding receptors of the GDNF family, and their common signaling receptor Ret. GFRα2, the principal receptor for neurturin, is expressed in most of the small nonpeptidergic DRG neurons, but also in some large DRG neurons that start to express Ret earlier. Previously, GFRα2 has been shown to be crucial for the soma size of small nonpeptidergic nociceptors and for their target innervation of glabrous epidermis. However, little is known about this receptor in other Ret-expressing DRG neuron populations. Here we have investigated two populations of Ret-positive low-threshold mechanoreceptors that innervate different types of hair follicles on mouse back skin: the small C-LTMRs and the large Aβ-LTMRs. Using GFRα2-KO mice and immunohistochemistry we found that, similar to the nonpeptidergic nociceptors, GFRα2 controls the cell size but not the survival of both C-LTMRs and Aβ-LTMRs. In contrast to the nonpeptidergic neurons, GFRα2 is not required for the target innervation of C-LTMRs and Aβ-LTMRs in the back skin. These results suggest that different factors drive target innervation in these three populations of neurons. In addition, the observation that the large Ret-positive DRG neurons lack GFRα2 immunoreactivity in mature animals suggests that these neurons switch their GFRα signaling pathways during postnatal development.

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules.

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Grison, Alice; Zucchelli, Silvia; Urzì, Alice; Zamparo, Ilaria; Lazarevic, Dejan; Pascarella, Giovanni; Roncaglia, Paola; Giorgetti, Alejandro; Garcia-Esparcia, Paula; Vlachouli, Christina; Simone, Roberto; Persichetti, Francesca; Forrest, Alistair R R; Hayashizaki, Yoshihide; Carloni, Paolo; Ferrer, Isidro; Lodovichi, Claudia; Plessy, Charles; Carninci, Piero; Gustincich, Stefano

2014-08-27

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson's disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown. By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca2+ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains. Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

Postresuscitative Changes of Brain-Derived Neurotrophic Factor (BDNF Protein Expression: Association With Neuronal Death

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M. Sh. Avrushchenko

2017-01-01

Full Text Available Aim of the study: to evaluate expression level of BDNF and its association with the postresuscitative neuronal death in highly hypoxia-sensitive brain regions.Materials and methods. Cardiac arrest in adult albino male rats was evoked by intrathoracic clamping of supracardiac bundle of vessels for 10 min. Pyramidal neurons of the hippocampus and Purkinje cells of the cerebellum were analyzed at various time points after resuscitation (days 1, 4, 7, 14. Shame-operated rats served as controls. The expression of BDNF protein was immunohistochemically determined. The BDNF expression level was determined by evalution on the base of the average optical density. The number of neurons with different BDNF expression levels and the total number of neurons per 1 mm of the layer length were computed. Image analysis systems (Intel personal computer, Olympus BX-41 microscope, ImageScopeM, ImageJ 1,48v and MS Excel 2007 software packages were used in the study. Data statistical processing was performed with the aid of Statistica 7.0 program and Kolmogorov-Smirnov λ-test, Mann-Whitney U-test and Student's t-test.Results. The dynamics of postresuscitative shifts of BDNF immunoreactivity in neuronal populations of hippocampal pyramidal cells and cerebellar Purkinje cells was established. It was shown that the level of BDNF expression within the two neuronal populations decreased, that was accompanied by neuronal death. In the Purkinje cell population the neuronal death occurred by the 4th day after resuscitation, while in the hippocampus, it occurs only by the 7th day. Notably, only BDNF-negative neurons or neurons with low level of BDNF expression died in both neuronal populations.Conclusion. The results of the study indicate the existence of an interrelation between the shifts in BDNF expression and the postresuscitative neuronal death. It was shown that only the cells with none or poor BDNF expression underwent death in highly hypoxia-sensitive neuronal

Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury

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Zhang, Ming Dong; Tortoriello, Giuseppe; Hsueh, Brian

2014-01-01

, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1....../2 neurons are much more abundant in DRGs than the Ca2+-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn....... In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca2+-binding proteins in pain-related DRG neurons...

Liraglutide Modulates Appetite and Body Weight Via GLP-1R-Expressing Glutamatergic Neurons.

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Adams, Jessica M; Pei, Hongjuan; Sandoval, Darleen A; Seeley, Randy J; Chang, Rui B; Liberles, Stephen D; Olson, David P

2018-05-18

Glucagon-like peptide-1 receptor (GLP-1R) agonists are FDA-approved weight loss drugs. Despite their widespread use, the sites of action through which GLP-1R agonists (GLP1RAs) impact appetite and body weight are still not fully understood. Here, we determined whether GLP-1Rs in either GABAergic or glutamatergic neurons are necessary for the acute and chronic effects of the GLP1RA liraglutide on food intake, visceral illness, body weight and neural network activation. We found that mice lacking GLP-1Rs in vGAT -expressing GABAergic neurons responded identically to controls in all parameters measured, whereas deletion of GLP-1Rs in vGlut2 -expressing glutamatergic neurons eliminated liraglutide-induced weight loss and visceral illness and severely attenuated its effects on feeding. Concomitantly, deletion of GLP-1Rs from glutamatergic neurons completely abolished the neural network activation observed after liraglutide administration. We conclude that liraglutide activates a dispersed but discrete neural network to mediate its physiological effects, and that these effects require GLP-1R expression on glutamatergic but not GABAergic neurons. © 2018 by the American Diabetes Association.

Expression of Nrf2 in neurodegenerative diseases.

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Ramsey, Chenere P; Glass, Charles A; Montgomery, Marshall B; Lindl, Kathryn A; Ritson, Gillian P; Chia, Luis A; Hamilton, Ronald L; Chu, Charleen T; Jordan-Sciutto, Kelly L

2007-01-01

In response to oxidative stress, the nuclear factor E2-related factor 2 (Nrf2) transcription factor translocates from the cytoplasm into the nucleus and transactivates expression of genes with antioxidant activity. Despite this cellular mechanism, oxidative damage is abundant in Alzheimer and Parkinson disease (AD and PD). To investigate mechanisms by which Nrf2 activity may be aberrant or insufficient in neurodegenerative conditions, we assessed Nrf2 localization in affected brain regions of AD, Lewy body variant of AD (LBVAD), and PD. By immunohistochemistry, Nrf2 is expressed in both the nucleus and the cytoplasm of neurons in normal hippocampi with predominant expression in the nucleus. In AD and LBVAD, Nrf2 was predominantly cytoplasmic in hippocampal neurons and was not a major component of beta amyloid plaques or neurofibrillary tangles. By immunoblotting, we observed a significant decrease in nuclear Nrf2 levels in AD cases. In contrast, Nrf2 was strongly nuclear in PD nigral neurons but cytoplasmic in substantia nigra of normal, AD, and LBVAD cases. These findings suggest that Nrf2-mediated transcription is not induced in neurons in AD despite the presence of oxidative stress. In PD, nuclear localization of Nrf2 is strongly induced, but this response may be insufficient to protect neurons from degeneration.

Effects of lipopolysaccharide-induced inflammation on expression of growth-associated genes by corticospinal neurons

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Lieberman AR

2006-01-01

Full Text Available Abstract Background Inflammation around cell bodies of primary sensory neurons and retinal ganglion cells enhances expression of neuronal growth-associated genes and stimulates axonal regeneration. We have asked if inflammation would have similar effects on corticospinal neurons, which normally show little response to spinal cord injury. Lipopolysaccharide (LPS was applied onto the pial surface of the motor cortex of adult rats with or without concomitant injury of the corticospinal tract at C4. Inflammation around corticospinal tract cell bodies in the motor cortex was assessed by immunohistochemistry for OX42 (a microglia and macrophage marker. Expression of growth-associated genes c-jun, ATF3, SCG10 and GAP-43 was investigated by immunohistochemistry or in situ hybridisation. Results Application of LPS induced a gradient of inflammation through the full depth of the motor cortex and promoted c-Jun and SCG10 expression for up to 2 weeks, and GAP-43 upregulation for 3 days by many corticospinal neurons, but had very limited effects on neuronal ATF3 expression. However, many glial cells in the subcortical white matter upregulated ATF3. LPS did not promote sprouting of anterogradely labelled corticospinal axons, which did not grow into or beyond a cervical lesion site. Conclusion Inflammation produced by topical application of LPS promoted increased expression of some growth-associated genes in the cell bodies of corticospinal neurons, but was insufficient to promote regeneration of the corticospinal tract.

Divergent modulation of neuronal differentiation by caspase-2 and -9.

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Giuseppa Pistritto

Full Text Available Human Ntera2/cl.D1 (NT2 cells treated with retinoic acid (RA differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2 or -9 (si-Casp9 was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM, microtubule associated protein-2 (MAP2 and tyrosine hydroxylase (TH mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ∼100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells.

Neuron-NG2 Cell Synapses: Novel Functions for Regulating NG2 Cell Proliferation and Differentiation

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Qian-Kun Yang

2013-01-01

Full Text Available NG2 cells are a population of CNS cells that are distinct from neurons, mature oligodendrocytes, astrocytes, and microglia. These cells can be identified by their NG2 proteoglycan expression. NG2 cells have a highly branched morphology, with abundant processes radiating from the cell body, and express a complex set of voltage-gated channels, AMPA/kainate, and GABA receptors. Neurons notably form classical and nonclassical synapses with NG2 cells, which have varied characteristics and functions. Neuron-NG2 cell synapses could fine-tune NG2 cell activities, including the NG2 cell cycle, differentiation, migration, and myelination, and may be a novel potential therapeutic target for NG2 cell-related diseases, such as hypoxia-ischemia injury and periventricular leukomalacia. Furthermore, neuron-NG2 cell synapses may be correlated with the plasticity of CNS in adulthood with the synaptic contacts passing onto their progenies during proliferation, and synaptic contacts decrease rapidly upon NG2 cell differentiation. In this review, we highlight the characteristics of classical and nonclassical neuron-NG2 cell synapses, the potential functions, and the fate of synaptic contacts during proliferation and differentiation, with the emphasis on the regulation of the NG2 cell cycle by neuron-NG2 cell synapses and their potential underlying mechanisms.

GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster.

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Cheung, Samantha K; Scott, Kristin

2017-01-01

Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation.

Near-infrared (NIR) optogenetics using up-conversion system

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Hososhima, Shoko; Yuasa, Hideya; Ishizuka, Toru; Yawo, Hiromu

2015-03-01

Non-invasive remote control technologies designed to manipulate neural functions for a comprehensive and quantitative understanding of the neuronal network in the brain as well as for the therapy of neurological disorders have long been awaited. Recently, it has become possible to optically manipulate the neuronal activity using biological photo-reactive molecules such as channelrhodopsin-2 (ChR2). However, ChR2 and its relatives are mostly reactive to visible light which does not effectively penetrate through biological tissues. In contrast, near-infrared (NIR) light penetrates deep into the tissues because biological systems are almost transparent to light within this so-called `imaging window'. Here we used lanthanide nanoparticles (LNPs), which are composed of rare-earth elements, as luminous bodies to activate channelrhodopsins (ChRs) since they absorb low-energy NIR light to emit high-energy visible light (up-conversion). Neuron-glioma-hybrid ND-7/23 cells were cultured with LNP(NaYF4:Sc/Yb/Er) particles (peak emission, 543 nm) and transfected to express C1V1 (peak absorbance, 539 nm), a chimera of ChR1 and VChR1. The photocurrents were generated in response to NIR laser light (976 nm) to a level comparable to that evoked by a filtered Hg lamp (530-550 nm). NIR light pulses also evoked action potentials in the cultured neurons that expressed C1V1. It is suggested that the green luminescent light emitted from LNPs effectively activated C1V1 to generate the photocurrent. With the optimization of LNPs, acceptor photo-reactive biomolecules and optics, this system could be applied to non-invasively actuate neurons deep in the brain.

Intervention effects of ganoderma lucidum spores on epileptiform discharge hippocampal neurons and expression of neurotrophin-4 and N-cadherin.

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Shu-Qiu Wang

Full Text Available Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS, a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg(2+ free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE. Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i control, ii model (incubated with Mg(2+ free medium for 3 hours, iii GLS group I (incubated with Mg(2+ free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours and iv GLS group II (neurons incubated with Mg(2+ free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours. Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression.

Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells.

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Nakano, Rei; Edamura, Kazuya; Sugiya, Hiroshi; Narita, Takanori; Okabayashi, Ken; Moritomo, Tadaaki; Teshima, Kenji; Asano, Kazushi; Nakayama, Tomohiro

2013-10-01

To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Bone marrow from 6 adult dogs. BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx. Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons. Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

Transgenic labeling of higher order neuronal circuits linked to phospholipase C-β2-expressing taste bud cells in medaka fish.

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Ieki, Takashi; Okada, Shinji; Aihara, Yoshiko; Ohmoto, Makoto; Abe, Keiko; Yasuoka, Akihito; Misaka, Takumi

2013-06-01

The sense of taste plays a pivotal role in the food-selecting behaviors of vertebrates. We have shown that the fish ortholog of the phospholipase C gene (plc-β2) is expressed in a subpopulation of taste bud cells that transmit taste stimuli to the central nervous system to evoke favorable and aversive behaviors. We generated transgenic medaka expressing wheat germ agglutinin (WGA) under the control of a regulatory region of the medaka plc-β2 gene to analyze the neuronal circuit connected to these sensory cells. Immunohistochemical analysis of the transgenic fish 12 days post fertilization revealed that the WGA protein was transferred to cranial sensory ganglia and several nuclei in the hindbrain. WGA signals were also detected in the secondary gustatory nucleus in the hindbrain of 3-month-old transgenic fish. WGA signals were observed in several diencephalic and telencephalic regions in 9-month-old transgenic fish. The age-dependent increase in the labeled brain regions strongly suggests that labeling occurred at taste bud cells and progressively extended to cranial nerves and neurons in the central nervous system. These data are the first to demonstrate the tracing of higher order gustatory neuronal circuitry that is associated with a specific subpopulation of taste bud cells. These results provide insight into the basic neuronal architecture of gustatory information processing that is common among vertebrates. Copyright © 2012 Wiley Periodicals, Inc.

Cyclooxygenase 2 and neuronal nitric oxide synthase expression in the renal cortex are not interdependent in states of salt deficiency

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Castrop, H; Kammerl, M; Mann, Birgitte

2000-01-01

Neuronal nitric oxide synthase (nNOS) and cyclooxygenase-2 (COX-2) expression in the kidney are localized to the cortical thick ascending limb of the loop of Henle (cTALH), including the macula region, and increase after salt restriction. Because of the similar localization and regulation of n...... excretion. These findings suggest that under these conditions the control of nNOS and COX-2 gene expression in the macula densa regions of the kidney cortex are not dependent on each other....

cAMP/PKA signaling pathway contributes to neuronal apoptosis via regulating IDE expression in a mixed model of type 2 diabetes and Alzheimer's disease.

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Li, Huajie; Yang, Song; Wu, Jian; Ji, Lei; Zhu, Linfeng; Cao, Liping; Huang, Jinzhong; Jiang, Qingqing; Wei, Jiang; Liu, Meng; Mao, Keshi; Wei, Ning; Xie, Wei; Yang, Zhilong

2018-02-01

Type 2 diabetes (T2D) may play a relevant role in the development of Alzheimer's disease (AD), however, the underlying mechanism was not clear yet. We developed an animal model presenting both AD and T2D, morris water maze (MWM) test and recognition task were performed to trace the cognitive function. Fasting plasma glucose (FPG) and oral glucose tolerance test (OGTT) were determined to trace the metabolism evolution. TUNEL assay and apoptosis-related protein levels were analyzed for the detection of neuronal apoptosis. Cyclic adenosine monophosphate (cAMP) agonist bucladesine or protein kinase (PKA) inhibitor H-89 were used to determine the effects of cAMP/PKA signaling pathway on IDE expression and neuronal apoptosis. The results showed that T2D contributes to the AD progress by accelerating and worsening spatial memory and recognition dysfunctions. Metabolic parameters and glucose tolerance were significantly changed in the presence of the AD and T2D. The significantly induced neuronal apoptosis and increased pro-apoptotic proteins in mice with AD and T2D were also observed. We showed the decreased expression level of IDE and the activating of cAMP/PKA signaling pathway in AD and T2D mice. Further studies indicated that cAMP agonist decreased the expression level of IDE and induced the neuronal apoptosis in mice with AD and T2D; whereas PKA inhibitor H-89 treatment showed the completely opposite results. Our study indicated that, in the T2D and AD mice, cAMP/PKA signaling pathway and IDE may participate in the contribute role of T2D in accelerating the pathological process of AD via causing the accumulation of Aβ and neuronal apoptosis. © 2017 Wiley Periodicals, Inc.

Molecular Characterization of Native and Recom­binant Ionotrophic Glutamate Receptors Expressed in Neurons and Heterologous Systems

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Drasbek, Kim Ryun

2005-01-01

trafficking mediating the continuous replacement of synaptic receptors and is important for receptor tetramerization in the endoplasmatic reticulum. Given the many important properties of the GluR2 subunit, it was of great interest to investigate and compare synaptic properties in neuronal populations...... in synaptic currents of receptors from these neuronal preparations, miniature excitatory postsynaptic currents (mEPSCs) were recorded followed by single cell RT-PCR of the same neuron. Unfortunately, no population of GluR2 lacking neurons was detected by single cell RT-PCR, but a higher detection frequency...... expressing AMPARs with or without the GluR2 subunits. Earlier findings suggested that neurons cultured from spinal cord were devoid of GluR2 and expressed high amounts of GluR4. In contrast, GluR2 was detected in almost all cells from cortical cultures (Dai et al., 2001). To investigate differences...

Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation

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Berglund, Ken; Clissold, Kara; Li, Haofang E.; Wen, Lei; Park, Sung Young; Gleixner, Jan; Klein, Marguerita E.; Lu, Dongye; Barter, Joseph W.; Rossi, Mark A.; Augustine, George J.; Yin, Henry H.; Hochgeschwender, Ute

2016-01-01

Luminopsins are fusion proteins of luciferase and opsin that allow interrogation of neuronal circuits at different temporal and spatial resolutions by choosing either extrinsic physical or intrinsic biological light for its activation. Building on previous development of fusions of wild-type Gaussia luciferase with channelrhodopsin, here we expanded the utility of luminopsins by fusing bright Gaussia luciferase variants with either channelrhodopsin to excite neurons (luminescent opsin, LMO) or a proton pump to inhibit neurons (inhibitory LMO, iLMO). These improved LMOs could reliably activate or silence neurons in vitro and in vivo. Expression of the improved LMO in hippocampal circuits not only enabled mapping of synaptic activation of CA1 neurons with fine spatiotemporal resolution but also could drive rhythmic circuit excitation over a large spatiotemporal scale. Furthermore, virus-mediated expression of either LMO or iLMO in the substantia nigra in vivo produced not only the expected bidirectional control of single unit activity but also opposing effects on circling behavior in response to systemic injection of a luciferase substrate. Thus, although preserving the ability to be activated by external light sources, LMOs expand the use of optogenetics by making the same opsins accessible to noninvasive, chemogenetic control, thereby allowing the same probe to manipulate neuronal activity over a range of spatial and temporal scales. PMID:26733686

Activity of Tachykinin1-Expressing Pet1 Raphe Neurons Modulates the Respiratory Chemoreflex.

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Hennessy, Morgan L; Corcoran, Andrea E; Brust, Rachael D; Chang, YoonJeung; Nattie, Eugene E; Dymecki, Susan M

2017-02-15

Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized neuronal subtypes within the serotonergic neuronal system, borne out in functional studies, for the modulation of distinct facets of homeostasis. Such functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to coexpress other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the Tachykinin-1 ( Tac1 ) gene. Here, we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the serotonergic transcription factor gene Pet1 , referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic [clozapine -N- oxide (CNO)-hM4Di] perturbation of Tac1-Pet1 neuron activity blunted the ventilatory response of the respiratory CO 2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO 2 Tac1-Pet1 axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor regions. These findings demonstrate that the activity of a Pet1 neuron subtype with the potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, perhaps via motor outputs that engage muscles of respiration and maintain airway patency. These Tac1-Pet1 neurons may act downstream of Egr2-Pet1 serotonergic neurons, which were previously established in respiratory chemoreception, but do not innervate respiratory motor nuclei. SIGNIFICANCE STATEMENT Serotonin (5-HT) neurons modulate physiological processes and behaviors as diverse as body temperature, respiration, aggression, and mood. Using

TASK-2 Channels Contribute to pH Sensitivity of Retrotrapezoid Nucleus Chemoreceptor Neurons

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Wang, Sheng; Benamer, Najate; Zanella, Sébastien; Kumar, Natasha N.; Shi, Yingtang; Bévengut, Michelle; Penton, David; Guyenet, Patrice G.; Lesage, Florian

2013-01-01

Phox2b-expressing glutamatergic neurons of the retrotrapezoid nucleus (RTN) display properties expected of central respiratory chemoreceptors; they are directly activated by CO2/H+ via an unidentified pH-sensitive background K+ channel and, in turn, facilitate brainstem networks that control breathing. Here, we used a knock-out mouse model to examine whether TASK-2 (K2P5), an alkaline-activated background K+ channel, contributes to RTN neuronal pH sensitivity. We made patch-clamp recordings in brainstem slices from RTN neurons that were identified by expression of GFP (directed by the Phox2b promoter) or β-galactosidase (from the gene trap used for TASK-2 knock-out). Whereas nearly all RTN cells from control mice were pH sensitive (95%, n = 58 of 61), only 56% of GFP-expressing RTN neurons from TASK-2−/− mice (n = 49 of 88) could be classified as pH sensitive (>30% reduction in firing rate from pH 7.0 to pH 7.8); the remaining cells were pH insensitive (44%). Moreover, none of the recorded RTN neurons from TASK-2−/− mice selected based on β-galactosidase activity (a subpopulation of GFP-expressing neurons) were pH sensitive. The alkaline-activated background K+ currents were reduced in amplitude in RTN neurons from TASK-2−/− mice that retained some pH sensitivity but were absent from pH-insensitive cells. Finally, using a working heart–brainstem preparation, we found diminished inhibition of phrenic burst amplitude by alkalization in TASK-2−/− mice, with apneic threshold shifted to higher pH levels. In conclusion, alkaline-activated TASK-2 channels contribute to pH sensitivity in RTN neurons, with effects on respiration in situ that are particularly prominent near apneic threshold. PMID:24107938

Ablation of neurons expressing melanin-concentrating hormone (MCH) in adult mice improves glucose tolerance independent of MCH signaling.

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Whiddon, Benjamin B; Palmiter, Richard D

2013-01-30

Melanin-concentrating hormone (MCH)-expressing neurons have been ascribed many roles based on studies of MCH-deficient mice. However, MCH neurons express other neurotransmitters, including GABA, nesfatin, and cocaine-amphetamine-regulated transcript. The importance of these other signaling molecules made by MCH neurons remains incompletely characterized. To determine the roles of MCH neurons in vivo, we targeted expression of the human diphtheria toxin receptor (DTR) to the gene for MCH (Pmch). Within 2 weeks of diphtheria toxin injection, heterozygous Pmch(DTR/+) mice lost 98% of their MCH neurons. These mice became lean but ate normally and were hyperactive, especially during a fast. They also responded abnormally to psychostimulants. For these phenotypes, ablation of MCH neurons recapitulated knock-out of MCH, so MCH appears to be the critical neuromodulator released by these neurons. In contrast, MCH-neuron-ablated mice showed improved glucose tolerance when compared with MCH-deficient mutant mice and wild-type mice. We conclude that MCH neurons regulate glucose tolerance through signaling molecules other than MCH.

Pathophysiological role of prostaglandin E2-induced up-regulation of the EP2 receptor in motor neuron-like NSC-34 cells and lumbar motor neurons in ALS model mice.

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Kosuge, Yasuhiro; Miyagishi, Hiroko; Yoneoka, Yuki; Yoneda, Keiko; Nango, Hiroshi; Ishige, Kumiko; Ito, Yoshihisa

2017-07-04

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective degeneration of motor neurons. The primary triggers for motor neuronal death are still unknown, but inflammation is considered to be an important factor contributing to the pathophysiology of ALS both clinically and in ALS models. Prostaglandin E2 (PGE2) and its corresponding four E-prostanoid receptors play a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. It has also been shown that PGE2-EP2 signaling in glial cells (astrocytes or microglia) promotes motor neuronal death in G93A mice. The present study was designed to investigate the levels of expression of EP receptors in the spinal motor neurons of ALS model mice and to examine whether PGE2 alters the expression of EP receptors in differentiated NSC-34 cells, a motor neuron-like cell line. Immunohistochemical staining demonstrated that EP2 and EP3 immunoreactivity was localized in NeuN-positive large cells showing the typical morphology of motor neurons in mice. Semi-quantitative analysis showed that the immunoreactivity of EP2 in motor neurons was significantly increased in the early symptomatic stage in ALS model mice. In contrast, the level of EP3 expression remained constant, irrespective of age. In differentiated NSC-34 cells, bath application of PGE2 resulted in a concentration-dependent decrease of MTT reduction. Although PGE2 had no effect on cell survival at concentrations of less than 10 μM, pretreatment with 10 μM PGE2 significantly up-regulated EP2 and concomitantly potentiated cell death induced by 30 μM PGE2. These results suggest that PGE2 is an important effector for induction of the EP2 subtype in differentiated NSC-34 cells, and that not only EP2 up-regulation in glial cells but also EP2 up-regulation in motor neurons plays a pivotal role in the vulnerability of motor neurons in ALS model mice. Copyright © 2017 Elsevier Ltd. All rights

Marked change in microRNA expression during neuronal differentiation of human teratocarcinoma NTera2D1 and mouse embryonal carcinoma P19 cells

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Hohjoh, Hirohiko; Fukushima, Tatsunobu

2007-01-01

MicroRNAs (miRNAs) are small noncoding RNAs, with a length of 19-23 nucleotides, which appear to be involved in the regulation of gene expression by inhibiting the translation of messenger RNAs carrying partially or nearly complementary sequences to the miRNAs in their 3' untranslated regions. Expression analysis of miRNAs is necessary to understand their complex role in the regulation of gene expression during the development, differentiation and proliferation of cells. Here we report on the expression profile analysis of miRNAs in human teratocarcinoma NTere2D1, mouse embryonic carcinoma P19, mouse neuroblastoma Neuro2a and rat pheochromocytoma PC12D cells, which can be induced into differentiated cells with long neuritic processes, i.e., after cell differentiation, such that the resultant cells look similar to neuronal cells. The data presented here indicate marked changes in the expression of miRNAs, as well as genes related to neuronal development, occurred in the differentiation of NTera2D1 and P19 cells. Significant changes in miRNA expression were not observed in Neuro2a and PC12D cells, although they showed apparent morphologic change between undifferentiated and differentiated cells. Of the miRNAs investigated, the expression of miRNAs belonging to the miR-302 cluster, which is known to be specifically expressed in embryonic stem cells, and of miR-124a specific to the brain, appeared to be markedly changed. The miR-302 cluster was potently expressed in undifferentiated NTera2D1 and P19 cells, but hardly in differentiated cells, such that miR-124a showed an opposite expression pattern to the miR-302 cluster. Based on these observations, it is suggested that the miR-302 cluster and miR-124a may be useful molecular indicators in the assessment of degree of undifferentiation and/or differentiation in the course of neuronal differentiation

Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

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Ogren, John I; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L; Rothschild, Kenneth J

2015-05-15

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

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Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

2016-08-01

Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus.

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Jae Hoon Jeong

Full Text Available The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC, plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat.

Hierarchical clustering of gene expression patterns in the Eomes + lineage of excitatory neurons during early neocortical development

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Cameron David A

2012-08-01

Full Text Available Abstract Background Cortical neurons display dynamic patterns of gene expression during the coincident processes of differentiation and migration through the developing cerebrum. To identify genes selectively expressed by the Eomes + (Tbr2 lineage of excitatory cortical neurons, GFP-expressing cells from Tg(Eomes::eGFP Gsat embryos were isolated to > 99% purity and profiled. Results We report the identification, validation and spatial grouping of genes selectively expressed within the Eomes + cortical excitatory neuron lineage during early cortical development. In these neurons 475 genes were expressed ≥ 3-fold, and 534 genes ≤ 3-fold, compared to the reference population of neuronal precursors. Of the up-regulated genes, 328 were represented at the Genepaint in situ hybridization database and 317 (97% were validated as having spatial expression patterns consistent with the lineage of differentiating excitatory neurons. A novel approach for quantifying in situ hybridization patterns (QISP across the cerebral wall was developed that allowed the hierarchical clustering of genes into putative co-regulated groups. Forty four candidate genes were identified that show spatial expression with Intermediate Precursor Cells, 49 candidate genes show spatial expression with Multipolar Neurons, while the remaining 224 genes achieved peak expression in the developing cortical plate. Conclusions This analysis of differentiating excitatory neurons revealed the expression patterns of 37 transcription factors, many chemotropic signaling molecules (including the Semaphorin, Netrin and Slit signaling pathways, and unexpected evidence for non-canonical neurotransmitter signaling and changes in mechanisms of glucose metabolism. Over half of the 317 identified genes are associated with neuronal disease making these findings a valuable resource for studies of neurological development and disease.

Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

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Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

2016-02-01

Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. Copyright © 2015 Federation of European Biochemical Societies

Gene expression profiling of two distinct neuronal populations in the rodent spinal cord.

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Jesper Ryge

Full Text Available BACKGROUND: In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. METHODOLOGY/PRINCIPAL FINDINGS: We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50-250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. CONCLUSIONS/SIGNIFICANCE: We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional

PERIPHERAL SENSORY NEURONS EXPRESSING MELANOPSIN RESPOND TO LIGHT

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Anna Matynia

2016-08-01

Full Text Available The ability of light to cause pain is paradoxical. The retina detects light but is devoid of nociceptors while the trigeminal sensory ganglia (TG contain nociceptors but not photoreceptors. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs are thought to mediate light-induced pain but recent evidence raises the possibility of an alternative light responsive pathway independent of the retina and optic nerve. Here, we show that melanopsin is expressed in both human and mouse TG neurons. In mice, they represent 3% of small TG neurons that are preferentially localized in the ophthalmic branch of the trigeminal nerve and are likely nociceptive C fibers and high-threshold mechanoreceptor Aδ fibers based on a strong size-function association. These isolated neurons respond to blue light stimuli with a delayed onset and sustained firing, similar to the melanopsin-dependent intrinsic photosensitivity observed in ipRGCs. Mice with severe bilateral optic nerve crush exhibit no light-induced responses including behavioral light aversion until treated with nitroglycerin, an inducer of migraine in people and migraine-like symptoms in mice. With nitroglycerin, these same mice with optic nerve crush exhibit significant light aversion. Furthermore, this retained light aversion remains dependent on melanopsin-expressing neurons. Our results demonstrate a novel light-responsive neural function independent of the optic nerve that may originate in the peripheral nervous system to provide the first direct mechanism for an alternative light detection pathway that influences motivated behavior.

Drosophila pheromone-sensing neurons expressing the ppk25 ion channel subunit stimulate male courtship and female receptivity.

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Vijayan, Vinoy; Thistle, Rob; Liu, Tong; Starostina, Elena; Pikielny, Claudio W

2014-03-01

As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25 is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating

Chronic zinc exposure decreases the surface expression of NR2A-containing NMDA receptors in cultured hippocampal neurons.

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Jia Zhu

Full Text Available Zinc distributes widely in the central nervous system, especially in the hippocampus, amygdala and cortex. The dynamic balance of zinc is critical for neuronal functions. Zinc modulates the activity of N-methyl-D-aspartate receptors (NMDARs through the direct inhibition and various intracellular signaling pathways. Abnormal NMDAR activities have been implicated in the aetiology of many brain diseases. Sustained zinc accumulation in the extracellular fluid is known to link to pathological conditions. However, the mechanism linking this chronic zinc exposure and NMDAR dysfunction is poorly understood.We reported that chronic zinc exposure reduced the numbers of NR1 and NR2A clusters in cultured hippocampal pyramidal neurons. Whole-cell and synaptic NR2A-mediated currents also decreased. By contrast, zinc did not affect NR2B, suggesting that chronic zinc exposure specifically influences NR2A-containg NMDARs. Surface biotinylation indicated that zinc exposure attenuated the membrane expression of NR1 and NR2A, which might arise from to the dissociation of the NR2A-PSD-95-Src complex.Chronic zinc exposure perturbs the interaction of NR2A to PSD-95 and causes the disorder of NMDARs in hippocampal neurons, suggesting a novel action of zinc distinct from its acute effects on NMDAR activity.

Endogenous α-crystallin inhibits expression of caspase-3 induced by hypoxia in retinal neurons.

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Ying, Xi; Peng, Yanli; Zhang, Jiaping; Wang, Xingli; Wu, Nan; Zeng, Yuxiao; Wang, Yi

2014-08-28

To investigate the expression of endogenous, hypoxic stress-induced α-crystallin and caspase-3 in rat retinal neurons in vitro. Retinal neurons were cultured from Long-Evans rats. The expression of endogenous α-crystallin was analyzed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, hypoxic exposure was performed in cultured cells, and the expression of endogenous α-crystallin and caspase-3 was assayed by Western blotting. Positive α-crystallin staining was observed in cultured retinal neurons, and expression of endogenous α-crystallin mRNA peaked 3-5d after inoculation (Pendogenous, hypoxic stress-induced α-crystallin expression increased gradually, peaking 6h after hypoxia. The expression was more abundant compared to the control (Pendogenous α-crystallin in retinal neurons, especially over-expression induced by hypoxic stress, results in the down regulation of caspase-3. The data suggest that endogenous α-crystallin may act as an endogenous neuroprotective factor in retinal neurons. Copyright © 2014 Elsevier Inc. All rights reserved.

Loss of translation elongation factor (eEF1A2) expression in vivo differentiates between Wallerian degeneration and dying-back neuronal pathology.

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Murray, Lyndsay M; Thomson, Derek; Conklin, Annalijn; Wishart, Thomas M; Gillingwater, Thomas H

2008-12-01

Wallerian degeneration and dying-back pathology are two well-known cellular pathways capable of regulating the breakdown and loss of axonal and synaptic compartments of neurons in vivo. However, the underlying mechanisms and molecular triggers of these pathways remain elusive. Here, we show that loss of translation elongation factor eEF1A2 expression in lower motor neurons and skeletal muscle fibres in homozygous Wasted mice triggered a dying-back neuropathy. Synaptic loss at the neuromuscular junction occurred in advance of axonal pathology and by a mechanism morphologically distinct from Wallerian degeneration. Dying-back pathology in Wasted mice was accompanied by reduced expression levels of the zinc finger protein ZPR1, as found in other dying-back neuropathies such as spinal muscular atrophy. Surprisingly, experimental nerve lesion revealed that Wallerian degeneration was significantly delayed in homozygous Wasted mice; morphological assessment revealed that approximately 80% of neuromuscular junctions in deep lumbrical muscles at 24 h and approximately 50% at 48 h had retained motor nerve terminals following tibial nerve lesion. This was in contrast to wild-type and heterozygous Wasted mice where < 5% of neuromuscular junctions had retained motor nerve terminals at 24 h post-lesion. These data show that eEF1A2 expression is required to prevent the initiation of dying-back pathology at the neuromuscular junction in vivo. In contrast, loss of eEF1A2 expression significantly inhibited the initiation and progression of Wallerian degeneration in vivo. We conclude that loss of eEF1A2 expression distinguishes mechanisms underlying dying-back pathology from those responsible for Wallerian degeneration in vivo and suggest that eEF1A2-dependent cascades may provide novel molecular targets to manipulate neurodegenerative pathways in lower motor neurons.

Sox2-Mediated Conversion of NG2 Glia into Induced Neurons in the Injured Adult Cerebral Cortex

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Christophe Heinrich

2014-12-01

Full Text Available The adult cerebral cortex lacks the capacity to replace degenerated neurons following traumatic injury. Conversion of nonneuronal cells into induced neurons has been proposed as an innovative strategy toward brain repair. Here, we show that retrovirus-mediated expression of the transcription factors Sox2 and Ascl1, but strikingly also Sox2 alone, can induce the conversion of genetically fate-mapped NG2 glia into induced doublecortin (DCX+ neurons in the adult mouse cerebral cortex following stab wound injury in vivo. In contrast, lentiviral expression of Sox2 in the unlesioned cortex failed to convert oligodendroglial and astroglial cells into DCX+ cells. Neurons induced following injury mature morphologically and some acquire NeuN while losing DCX. Patch-clamp recording of slices containing Sox2- and/or Ascl1-transduced cells revealed that a substantial fraction of these cells receive synaptic inputs from neurons neighboring the injury site. Thus, NG2 glia represent a potential target for reprogramming strategies toward cortical repair.

Preferential activation of HIF-2α adaptive signalling in neuronal-like cells in response to acute hypoxia.

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Miguel A S Martín-Aragón Baudel

Full Text Available Stroke causes severe neuronal damage as disrupted cerebral blood flow starves neurons of oxygen and glucose. The hypoxia inducible factors (HIF-1α and HIF-2α orchestrate oxygen homeostasis and regulate specific aspects of hypoxic adaptation. Here we show the importance of HIF-2α dependant signalling in neuronal adaptation to hypoxic insult. PC12 and NT2 cells were differentiated into neuronal-like cells using NGF and retinoic acid, and exposed to acute hypoxia (1% O2. Gene and protein expression was analysed by qPCR and immunoblotting and the neuronal-like phenotype was examined. PC12 and NT2 differentiation promoted neurite extension and expression of neuronal markers, NSE and KCC2. Induction of HIF-1α mRNA or protein was not detected in hypoxic neuronal-like cells, however marked induction of HIF-2α mRNA and protein expression was observed. Induction of HIF-1α target genes was also not detected in response to acute hypoxia, however significant induction of HIF-2α transcriptional targets was clearly evident. Furthermore, hypoxic insult dramatically reduced both neurite number and length, and attenuated expression of neuronal markers, NSE and KCC2. This correlated with an increase in expression of the neural progenitor and stem cell-like markers, CD44 and vimentin, suggesting HIF-2α molecular mechanisms could potentially promote regression of neuronal-like cells to a stem-like state and trigger neuronal recovery following ischaemic insult. Our findings suggest the HIF-2α pathway predominates over HIF-1α signalling in neuronal-like cells following acute hypoxia.

Pharmacological Characterisation of Nicotinic Acetylcholine Receptors Expressed in Human iPSC-Derived Neurons.

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Anna Chatzidaki

Full Text Available Neurons derived from human induced pluripotent stem cells (iPSCs represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels. Previous studies on iPSC-derived neuronal cells have reported the functional characterisation of a variety of receptors and ion channels, including glutamate receptors, γ-aminobutyric acid (GABA receptors and several voltage-gated ion channels. In the present study we have examined the expression and functional properties of nicotinic acetylcholine receptors (nAChRs in human iPSC-derived neurons. Gene expression analysis indicated the presence of transcripts encoding several nAChR subunits, with highest levels detected for α3-α7, β1, β2 and β4 subunits (encoded by CHRNA3-CHRNA7, CHRNB1, CHRNB2 and CHRNB4 genes. In addition, similarly high transcript levels were detected for the truncated dupα7 subunit transcript, encoded by the partially duplicated gene CHRFAM7A, which has been associated with psychiatric disorders such as schizophrenia. The functional properties of these nAChRs have been examined by calcium fluorescence and by patch-clamp recordings. The data obtained suggest that the majority of functional nAChRs expressed in these cells have pharmacological properties typical of α7 receptors. Large responses were induced by a selective α7 agonist (compound B, in the presence of the α7-selective positive allosteric modulator (PAM PNU-120596, which were blocked by the α7-selective antagonist methyllycaconitine (MLA. In addition, a small proportion of the neurons express nAChRs with properties typical of heteromeric (non-α7 containing nAChR subtypes. These cells therefore represent a great tool to advance our understanding of the properties of native human nAChRs, α7 in particular.

Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates

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Djenoune, Lydia; Khabou, Hanen; Joubert, Fanny; Quan, Feng B.; Nunes Figueiredo, Sophie; Bodineau, Laurence; Del Bene, Filippo; Burcklé, Céline; Tostivint, Hervé; Wyart, Claire

2014-01-01

Over 90 years ago, Kolmer and Agduhr identified spinal cerebrospinal fluid-contacting neurons (CSF-cNs) based on their morphology and location within the spinal cord. In more than 200 vertebrate species, they observed ciliated neurons around the central canal that extended a brush of microvilli into the cerebrospinal fluid (CSF). Although their morphology is suggestive of a primitive sensory cell, their function within the vertebrate spinal cord remains unknown. The identification of specific molecular markers for these neurons in vertebrates would benefit the investigation of their physiological roles. PKD2L1, a transient receptor potential channel that could play a role as a sensory receptor, has been found in cells contacting the central canal in mouse. In this study, we demonstrate that PKD2L1 is a specific marker for CSF-cNs in the spinal cord of mouse (Mus musculus), macaque (Macaca fascicularis) and zebrafish (Danio rerio). In these species, the somata of spinal PKD2L1+ CSF-cNs were located below or within the ependymal layer and extended an apical bulbous extension into the central canal. We found GABAergic PKD2L1-expressing CSF-cNs in all three species. We took advantage of the zebrafish embryo for its transparency and rapid development to identify the progenitor domains from which pkd2l1+ CSF-cNs originate. pkd2l1+ CSF-cNs were all GABAergic and organized in two rows—one ventral and one dorsal to the central canal. Their location and marker expression is consistent with previously described Kolmer–Agduhr cells. Accordingly, pkd2l1+ CSF-cNs were derived from the progenitor domains p3 and pMN defined by the expression of nkx2.2a and olig2 transcription factors, respectively. Altogether our results suggest that a system of CSF-cNs expressing the PKD2L1 channel is conserved in the spinal cord across bony vertebrate species. PMID:24834029

Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates

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Lydia eDjenoune

2014-05-01

Full Text Available Over ninety years ago, Kolmer and Agduhr identified spinal cerebrospinal fluid-contacting neurons (CSF-cNs based on their morphology and location within the spinal cord. In more than two hundred vertebrate species, they observed ciliated neurons around the central canal that extended a brush of microvilli into the cerebrospinal fluid (CSF. Although their morphology is suggestive of a primitive sensory cell, their function within the vertebrate spinal cord remains unknown. The identification of specific molecular markers for these neurons in vertebrates would benefit the investigation of their physiological roles. PKD2L1, a transient receptor potential channel that could play a role as a sensory receptor, has been found in cells contacting the central canal in mouse. In this study, we demonstrate that PKD2L1 is a specific marker for CSF-cNs in the spinal cord of mouse (Mus musculus, macaque (Macaca fascicularis and zebrafish (Danio rerio. In these species, the somata of spinal PKD2L1+ CSF-cNs were located below or within the ependymal layer and extended an apical bulbous extension into the central canal. We found GABAergic PKD2L1-expressing CSF-cNs in all three species. We took advantage of the zebrafish embryo for its transparency and rapid development to identify the progenitor domains from which pkd2l1+ CSF-cNs originate. pkd2l1+ CSF-cNs were all GABAergic and organized in two rows—one ventral and one dorsal to the central canal. Their location and marker expression is consistent with previously described Kolmer-Agduhr cells. Accordingly, pkd2l1+ CSF-cNs were derived from the progenitor domains p3 and pMN defined by the expression of nkx2.2a and olig2 transcription factors, respectively. Altogether our results suggest that a system of CSF-cNs expressing the PKD2L1 channel is conserved in the spinal cord across bony vertebrate species.

Functional characterisation of filamentous actin probe expression in neuronal cells.

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Shrujna Patel

Full Text Available Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.

Expression of diverse neuropeptide cotransmitters by identified motor neurons in Aplysia

International Nuclear Information System (INIS)

Church, P.J.; Lloyd, P.E.

1991-01-01

Neuropeptide synthesis was determined for individual identified ventral-cluster neurons in the buccal ganglia of Aplysia. Each of these cells was shown to be a motor neuron that innervates buccal muscles that generate biting and swallowing movements during feeding. Individual neurons were identified by a battery of physiological criteria and stained with intracellular injection of a vital dye, and the ganglia were incubated in 35S-methionine. Peptide synthesis was determined by measuring labeled peptides in extracts from individually dissected neuronal cell bodies analyzed by HPLC. Previously characterized peptides found to be synthesized included buccalin, FMRFamide, myomodulin, and the 2 small cardioactive peptides (SCPs). Each of these neuropeptides has been shown to modulate buccal muscle responses to motor neuron stimulation. Two other peptides were found to be synthesized in individual motor neurons. One peptide, which was consistently observed in neurons that also synthesized myomodulin, is likely to be the recently sequenced myomodulin B. The other peptide was observed in a subset of the neurons that synthesize FMRFamide. While identified motor neurons consistently synthesized the same peptide(s), neurons that innervate the same muscle often express different peptides. Neurons that synthesized the SCPs also contained SCP-like activity, as determined by snail heart bioassay. Our results indicate that every identified motor neuron synthesizes a subset of these methionine-containing peptides, and that several neurons consistently synthesize peptides that are likely to be processed from multiple precursors

Cre-expressing neurons in visual cortex of Ntsr1-Cre GN220 mice are corticothalamic and are depolarized by acetylcholine.

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Sundberg, Sofie Charlotte; Lindström, Sarah Helen; Sanchez, Gonzalo Manuel; Granseth, Björn

2018-01-01

The Ntsr1-Cre GN220 mouse expresses Cre-recombinase in corticothalamic (CT) neurons in neocortical layer 6. It is not known if the other major types of pyramidal neurons in this layer also express this enzyme. By electrophysiological recordings in slices and histological analysis of the uptake of retrogradely transported beads we show that Cre-positive neurons are CT and not corticocortical or corticoclaustral types. Furthermore, we show that Ntsr1-Cre-positive cells are immuno-positive for the nuclear transcription factor Forkhead box protein P2 (FoxP2). We conclude that Cre-expression is limited to a specific type of pyramidal neuron: CT. However, it appears as not all CT neurons are Cre-expressing; there are indications that the penetrance of the gene is about 90%. We demonstrate the utility of assigning a specific identity to individual neurons by determining that the CT neurons are potently modulated by acetylcholine acting on both nicotinic and muscarinic acetylcholine receptors. These results corroborate the suggested function of these neurons in regulating the gain of thalamocortical transfer of sensory information depending on attentional demand and state of arousal. © 2017 Wiley Periodicals, Inc.

Bach2 is involved in neuronal differentiation of N1E-115 neuroblastoma cells

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Shim, Ki Shuk; Rosner, Margit; Freilinger, Angelika; Lubec, Gert; Hengstschlaeger, Markus

2006-01-01

Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could represent a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status

Transgenic expression of B-50/GAP-43 in mature olfactory neurons triggers downregulation of native B-50/GAP-43 expression in immature olfactory neurons

NARCIS (Netherlands)

Holtmaat, Anthony J D G; Huizinga, C T; Margolis, F L; Gispen, Willem Hendrik; Verhaagen, J

1999-01-01

The adult mammalian olfactory neuroepithelium is an unusual neural tissue, since it maintains its capacity to form new neurons throughout life. Newly formed neurons differentiate in the basal layers of the olfactory neuroepithelium and express B-50/GAP-43, a protein implicated in neurite outgrowth.

Neuronal markers are expressed in human gliomas and NSE knockdown sensitizes glioblastoma cells to radiotherapy and temozolomide

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Yan, Tao; Skaftnesmo, Kai Ove; Leiss, Lina; Sleire, Linda; Wang, Jian; Li, Xingang; Enger, Per Øyvind

2011-01-01

Expression of neuronal elements has been identified in various glial tumors, and glioblastomas (GBMs) with neuronal differentiation patterns have reportedly been associated with longer survival. However, the neuronal class III β-tubulin has been linked to increasing malignancy in astrocytomas. Thus, the significance of neuronal markers in gliomas is not established. The expressions of class III β-tubulin, neurofilament protein (NFP), microtubule-associated protein 2 (MAP2) and neuron-specific enolase (NSE) were investigated in five GBM cell lines and two GBM biopsies with immunocytochemistry and Western blot. Moreover, the expression levels were quantified by real-time qPCR under different culture conditions. Following NSE siRNA treatment we used Electric cell-substrate impedance sensing (ECIS) to monitor cell growth and migration and MTS assays to study viability after irradiation and temozolomide treatment. Finally, we quantitated NSE expression in a series of human glioma biopsies with immunohistochemistry using a morphometry software, and collected survival data for the corresponding patients. The biopsies were then grouped according to expression in two halves which were compared by survival analysis. Immunocytochemistry and Western blotting showed that all markers except NFP were expressed both in GBM cell lines and biopsies. Notably, qPCR demonstrated that NSE was upregulated in cellular stress conditions, such as serum-starvation and hypoxia, while we found no uniform pattern for the other markers. NSE knockdown reduced the migration of glioma cells, sensitized them to hypoxia, radio- and chemotherapy. Furthermore, we found that GBM patients in the group with the highest NSE expression lived significantly shorter than patients in the low-expression group. Neuronal markers are aberrantly expressed in human GBMs, and NSE is consistently upregulated in different cellular stress conditions. Knockdown of NSE reduces the migration of GBM cells and sensitizes

Modulators of cytoskeletal reorganization in CA1 hippocampal neurons show increased expression in patients at mid-stage Alzheimer's disease.

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Patricia F Kao

2010-10-01

Full Text Available During the progression of Alzheimer's disease (AD, hippocampal neurons undergo cytoskeletal reorganization, resulting in degenerative as well as regenerative changes. As neurofibrillary tangles form and dystrophic neurites appear, sprouting neuronal processes with growth cones emerge. Actin and tubulin are indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that actin capping protein beta2 subunit, Capzb2, binds tubulin and, in the presence of tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons resulted in short, dystrophic neurites, seen in neurodegenerative diseases including AD. Here we demonstrate the statistically significant increase in the Capzb2 expression in the postmortem hippocampi in persons at mid-stage, Braak and Braak stage (BB III-IV, non-familial AD in comparison to controls. The dynamics of Capzb2 expression in progressive AD stages cannot be attributed to reactive astrocytosis. Moreover, the increased expression of Capzb2 mRNA in CA1 pyramidal neurons in AD BB III-IV is accompanied by an increased mRNA expression of brain derived neurotrophic factor (BDNF receptor tyrosine kinase B (TrkB, mediator of synaptic plasticity in hippocampal neurons. Thus, the up-regulation of Capzb2 and TrkB may reflect cytoskeletal reorganization and/or regenerative response occurring in hippocampal CA1 neurons at a specific stage of AD progression.

Differential Expression of Dopamine D5 Receptors across Neuronal Subtypes in Macaque Frontal Eye Field

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Adrienne Mueller

2018-02-01

Full Text Available Dopamine signaling in the prefrontal cortex (PFC is important for cognitive functions, yet very little is known about the expression of the D5 class of dopamine receptors (D5Rs in this region. To address this, we co-stained for D5Rs, pyramidal neurons (neurogranin+, putative long-range projection pyramidal neurons (SMI-32+, and several classes of inhibitory interneuron (parvalbumin+, calbindin+, calretinin+, somatostatin+ within the frontal eye field (FEF: an area within the PFC involved in the control of visual spatial attention. We then quantified the co-expression of D5Rs with markers of different cell types across different layers of the FEF. We show that: (1 D5Rs are more prevalent on pyramidal neurons than on inhibitory interneurons. (2 D5Rs are disproportionately expressed on putative long-range projecting pyramidal neurons. The disproportionately high expression of D5Rs on long-range projecting pyramidals, compared to interneurons, was particularly pronounced in layers II–III. Together these results indicate that the engagement of D5R-dependent mechanisms in the FEF varies depending on cell type and cortical layer, and suggests that non-locally projecting neurons contribute disproportionately to functions involving the D5R subtype.

Assessing sensory versus optogenetic network activation by combining (o)fMRI with optical Ca2+ recordings.

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Schmid, Florian; Wachsmuth, Lydia; Schwalm, Miriam; Prouvot, Pierre-Hugues; Jubal, Eduardo Rosales; Fois, Consuelo; Pramanik, Gautam; Zimmer, Claus; Faber, Cornelius; Stroh, Albrecht

2016-11-01

Encoding of sensory inputs in the cortex is characterized by sparse neuronal network activation. Optogenetic stimulation has previously been combined with fMRI (ofMRI) to probe functional networks. However, for a quantitative optogenetic probing of sensory-driven sparse network activation, the level of similarity between sensory and optogenetic network activation needs to be explored. Here, we complement ofMRI with optic fiber-based population Ca 2+ recordings for a region-specific readout of neuronal spiking activity in rat brain. Comparing Ca 2+ responses to the blood oxygenation level-dependent signal upon sensory stimulation with increasing frequencies showed adaptation of Ca 2+ transients contrasted by an increase of blood oxygenation level-dependent responses, indicating that the optical recordings convey complementary information on neuronal network activity to the corresponding hemodynamic response. To study the similarity of optogenetic and sensory activation, we quantified the density of cells expressing channelrhodopsin-2 and modeled light propagation in the tissue. We estimated the effectively illuminated volume and numbers of optogenetically stimulated neurons, being indicative of sparse activation. At the functional level, upon either sensory or optogenetic stimulation we detected single-peak short-latency primary Ca 2+ responses with similar amplitudes and found that blood oxygenation level-dependent responses showed similar time courses. These data suggest that ofMRI can serve as a representative model for functional brain mapping. © The Author(s) 2015.

Assessing sensory versus optogenetic network activation by combining (o)fMRI with optical Ca2+ recordings

Science.gov (United States)

Schmid, Florian; Wachsmuth, Lydia; Schwalm, Miriam; Prouvot, Pierre-Hugues; Jubal, Eduardo Rosales; Fois, Consuelo; Pramanik, Gautam; Zimmer, Claus; Stroh, Albrecht

2015-01-01

Encoding of sensory inputs in the cortex is characterized by sparse neuronal network activation. Optogenetic stimulation has previously been combined with fMRI (ofMRI) to probe functional networks. However, for a quantitative optogenetic probing of sensory-driven sparse network activation, the level of similarity between sensory and optogenetic network activation needs to be explored. Here, we complement ofMRI with optic fiber-based population Ca2+ recordings for a region-specific readout of neuronal spiking activity in rat brain. Comparing Ca2+ responses to the blood oxygenation level-dependent signal upon sensory stimulation with increasing frequencies showed adaptation of Ca2+ transients contrasted by an increase of blood oxygenation level-dependent responses, indicating that the optical recordings convey complementary information on neuronal network activity to the corresponding hemodynamic response. To study the similarity of optogenetic and sensory activation, we quantified the density of cells expressing channelrhodopsin-2 and modeled light propagation in the tissue. We estimated the effectively illuminated volume and numbers of optogenetically stimulated neurons, being indicative of sparse activation. At the functional level, upon either sensory or optogenetic stimulation we detected single-peak short-latency primary Ca2+ responses with similar amplitudes and found that blood oxygenation level-dependent responses showed similar time courses. These data suggest that ofMRI can serve as a representative model for functional brain mapping. PMID:26661247

EGL-13/SoxD specifies distinct O2 and CO2 sensory neuron fates in Caenorhabditis elegans.

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Jakob Gramstrup Petersen

2013-05-01

Full Text Available Animals harbor specialized neuronal systems that are used for sensing and coordinating responses to changes in oxygen (O2 and carbon dioxide (CO2. In Caenorhabditis elegans, the O2/CO2 sensory system comprises functionally and morphologically distinct sensory neurons that mediate rapid behavioral responses to exquisite changes in O2 or CO2 levels via different sensory receptors. How the diversification of the O2- and CO2-sensing neurons is established is poorly understood. We show here that the molecular identity of both the BAG (O2/CO2-sensing and the URX (O2-sensing neurons is controlled by the phylogenetically conserved SoxD transcription factor homolog EGL-13. egl-13 mutant animals fail to fully express the distinct terminal gene batteries of the BAG and URX neurons and, as such, are unable to mount behavioral responses to changes in O2 and CO2. We found that the expression of egl-13 is regulated in the BAG and URX neurons by two conserved transcription factors-ETS-5(Ets factor in the BAG neurons and AHR-1(bHLH factor in the URX neurons. In addition, we found that EGL-13 acts in partially parallel pathways with both ETS-5 and AHR-1 to direct BAG and URX neuronal fate respectively. Finally, we found that EGL-13 is sufficient to induce O2- and CO2-sensing cell fates in some cellular contexts. Thus, the same core regulatory factor, egl-13, is required and sufficient to specify the distinct fates of O2- and CO2-sensing neurons in C. elegans. These findings extend our understanding of mechanisms of neuronal diversification and the regulation of molecular factors that may be conserved in higher organisms.

Sexually dimorphic distribution of Prokr2 neurons revealed by the Prokr2-Cre mouse model.

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Mohsen, Zaid; Sim, Hosung; Garcia-Galiano, David; Han, Xingfa; Bellefontaine, Nicole; Saunders, Thomas L; Elias, Carol F

2017-12-01

Prokineticin receptor 2 (PROKR2) is predominantly expressed in the mammalian central nervous system. Loss-of-function mutations of PROKR2 in humans are associated with Kallmann syndrome due to the disruption of gonadotropin releasing hormone neuronal migration and deficient olfactory bulb morphogenesis. PROKR2 has been also implicated in the neuroendocrine control of GnRH neurons post-migration and other physiological systems. However, the brain circuitry and mechanisms associated with these actions have been difficult to investigate mainly due to the widespread distribution of Prokr2-expressing cells, and the lack of animal models and molecular tools. Here, we describe the generation, validation and characterization of a new mouse model that expresses Cre recombinase driven by the Prokr2 promoter, using CRISPR-Cas9 technology. Cre expression was visualized using reporter genes, tdTomato and GFP, in males and females. Expression of Cre-induced reporter genes was found in brain sites previously described to express Prokr2, e.g., the paraventricular and the suprachiasmatic nuclei, and the area postrema. The Prokr2-Cre mouse model was further validated by colocalization of Cre-induced GFP and Prokr2 mRNA. No disruption of Prokr2 expression, GnRH neuronal migration or fertility was observed. Comparative analysis of Prokr2-Cre expression in male and female brains revealed a sexually dimorphic distribution confirmed by in situ hybridization. In females, higher Cre activity was found in the medial preoptic area, ventromedial nucleus of the hypothalamus, arcuate nucleus, medial amygdala and lateral parabrachial nucleus. In males, Cre was higher in the amygdalo-hippocampal area. The sexually dimorphic pattern of Prokr2 expression indicates differential roles in reproductive function and, potentially, in other physiological systems.

Different populations of prostaglandin EP3 receptor-expressing preoptic neurons project to two fever-mediating sympathoexcitatory brain regions.

Science.gov (United States)

Nakamura, Y; Nakamura, K; Morrison, S F

2009-06-30

The central mechanism of fever induction is triggered by an action of prostaglandin E(2) (PGE(2)) on neurons in the preoptic area (POA) through the EP3 subtype of prostaglandin E receptor. EP3 receptor (EP3R)-expressing POA neurons project directly to the dorsomedial hypothalamus (DMH) and to the rostral raphe pallidus nucleus (rRPa), key sites for the control of thermoregulatory effectors. Based on physiological findings, we hypothesize that the febrile responses in brown adipose tissue (BAT) and those in cutaneous vasoconstrictors are controlled independently by separate neuronal pathways: PGE(2) pyrogenic signaling is transmitted from EP3R-expressing POA neurons via a projection to the DMH to activate BAT thermogenesis and via another projection to the rRPa to increase cutaneous vasoconstriction. In this case, DMH-projecting and rRPa-projecting neurons would constitute segregated populations within the EP3R-expressing neuronal group in the POA. Here, we sought direct anatomical evidence to test this hypothesis with a double-tracing experiment in which two types of the retrograde tracer, cholera toxin b-subunit (CTb), conjugated with different fluorophores were injected into the DMH and the rRPa of rats and the resulting retrogradely labeled populations of EP3R-immunoreactive neurons in the POA were identified with confocal microscopy. We found substantial numbers of EP3R-immunoreactive neurons in both the DMH-projecting and the rRPa-projecting populations. However, very few EP3R-immunoreactive POA neurons were labeled with both the CTb from the DMH and that from the rRPa, although a substantial number of neurons that were not immunoreactive for EP3R were double-labeled with both CTbs. The paucity of the EP3R-expressing neurons that send collaterals to both the DMH and the rRPa suggests that pyrogenic signals are sent independently to these caudal brain regions from the POA and that such pyrogenic outputs from the POA reflect different control mechanisms for BAT

NT2 derived neuronal and astrocytic network signalling.

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Eric J Hill

Full Text Available A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.

The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

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Helley, M P; Abate, W; Jackson, S K; Bennett, J H; Thompson, S W N

2015-12-03

The recent discovery that mammalian nociceptors express Toll-like receptors (TLRs) has raised the possibility that these cells directly detect and respond to pathogens with implications for either direct nociceptor activation or sensitization. A range of neuronal TLRs have been identified, however a detailed description regarding the distribution of expression of these receptors within sub-populations of sensory neurons is lacking. There is also some debate as to the composition of the TLR4 receptor complex on sensory neurons. Here we use a range of techniques to quantify the expression of TLR4, TLR7 and some associated molecules within neurochemically-identified sub-populations of trigeminal (TG) and dorsal root (DRG) ganglion sensory neurons. We also detail the pattern of expression and co-expression of two isoforms of lysophosphatidylcholine acyltransferase (LPCAT), a phospholipid remodeling enzyme previously shown to be involved in the lipopolysaccharide-dependent TLR4 response in monocytes, within sensory ganglia. Immunohistochemistry shows that both TLR4 and TLR7 preferentially co-localize with transient receptor potential vallinoid 1 (TRPV1) and purinergic receptor P2X ligand-gated ion channel 3 (P2X3), markers of nociceptor populations, within both TG and DRG. A gene expression profile shows that TG sensory neurons express a range of TLR-associated molecules. LPCAT1 is expressed by a proportion of both nociceptors and non-nociceptive neurons. LPCAT2 immunostaining is absent from neuronal profiles within both TG and DRG and is confined to non-neuronal cell types under naïve conditions. Together, our results show that nociceptors express the molecular machinery required to directly respond to pathogenic challenge independently from the innate immune system. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Levetiracetam differentially alters CD95 expression of neuronal cells and the mitochondrial membrane potential of immune and neuronal cells in vitro

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Susannah K Rogers

2014-02-01

Full Text Available Epilepsy is a neurological seizure disorder that affects over 100 million people worldwide. Levetiracetam, either alone, as monotherapy, or as adjunctive treatment, is widely used to control certain types of seizures. Despite its increasing popularity as a relatively safe and effective anti-convulsive treatment option, its mechanism(s of action are poorly understood. Studies have suggested neuronal, glial, and immune mechanisms of action. Understanding the precise mechanisms of action of Levetiracetam would be extremely beneficial in helping to understand the processes involved in seizure generation and epilepsy. Moreover, a full understanding of these mechanisms would help to create more efficacious treatments while minimizing side effects. The current study examined the effects of Levetiracetam on the mitochondrial membrane potential of neuronal and non-neuronal cells, in vitro, in order to determine if Levetiracetam influences metabolic processes in these cell types. In addition, this study sought to address possible immune-mediated mechanisms by determining if Levetiracetam alters the expression of immune receptor-ligand pairs. The results show that Levetiracetam induces expression of CD95 and CD178 on NGF-treated C17.2 neuronal cells. The results also show that Levetiracetam increases mitochondrial membrane potential on C17.2 neuronal cells in the presence of nerve growth factor. In contrast, Levetiracetam decreases the mitochondrial membrane potential of splenocytes and this effect was dependent on intact invariant chain, thus implicating immune cell interactions. These results suggest that both neuronal and non-neuronal anti-epileptic activities of Levetiracetam involve control over energy metabolism, more specifically, mΔΨ. Future studies are needed to further investigate this potential mechanism of action.

The Wnt Signaling Pathway Is Differentially Expressed during the Bovine Herpesvirus 1 Latency-Reactivation Cycle: Evidence That Two Protein Kinases Associated with Neuronal Survival, Akt3 and BMPR2, Are Expressed at Higher Levels during Latency.

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Workman, Aspen; Zhu, Liqian; Keel, Brittney N; Smith, Timothy P L; Jones, Clinton

2018-04-01

Sensory neurons in trigeminal ganglia (TG) of calves latently infected with bovine herpesvirus 1 (BoHV-1) abundantly express latency-related (LR) gene products, including a protein (ORF2) and two micro-RNAs. Recent studies in mouse neuroblastoma cells (Neuro-2A) demonstrated ORF2 interacts with β-catenin and a β-catenin coactivator, high-mobility group AT-hook 1 (HMGA1) protein, which correlates with increased β-catenin-dependent transcription and cell survival. β-Catenin and HMGA1 are readily detected in a subset of latently infected TG neurons but not TG neurons from uninfected calves or reactivation from latency. Consequently, we hypothesized that the Wnt/β-catenin signaling pathway is differentially expressed during the latency and reactivation cycle and an active Wnt pathway promotes latency. RNA-sequencing studies revealed that 102 genes associated with the Wnt/β-catenin signaling pathway were differentially expressed in TG during the latency-reactivation cycle in calves. Wnt agonists were generally expressed at higher levels during latency, but these levels decreased during dexamethasone-induced reactivation. The Wnt agonist bone morphogenetic protein receptor 2 (BMPR2) was intriguing because it encodes a serine/threonine receptor kinase that promotes neuronal differentiation and inhibits cell death. Another differentially expressed gene encodes a protein kinase (Akt3), which is significant because Akt activity enhances cell survival and is linked to herpes simplex virus 1 latency and neuronal survival. Additional studies demonstrated ORF2 increased Akt3 steady-state protein levels and interacted with Akt3 in transfected Neuro-2A cells, which correlated with Akt3 activation. Conversely, expression of Wnt antagonists increased during reactivation from latency. Collectively, these studies suggest Wnt signaling cooperates with LR gene products, in particular ORF2, to promote latency. IMPORTANCE Lifelong BoHV-1 latency primarily occurs in sensory neurons

Functional and neurochemical characterization of angiotensin type 1A receptor-expressing neurons in the nucleus of the solitary tract of the mouse.

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Carter, D A; Choong, Y-T; Connelly, A A; Bassi, J K; Hunter, N O; Thongsepee, N; Llewellyn-Smith, I J; Fong, A Y; McDougall, S J; Allen, A M

2017-10-01

Angiotensin II acts via two main receptors within the central nervous system, with the type 1A receptor (AT 1A R) most widely expressed in adult neurons. Activation of the AT 1 R in the nucleus of the solitary tract (NTS), the principal nucleus receiving central synapses of viscerosensory afferents, modulates cardiovascular reflexes. Expression of the AT 1 R occurs in high density within the NTS of most mammals, including humans, but the fundamental electrophysiological and neurochemical characteristics of the AT 1A R-expressing NTS neurons are not known. To address this, we have used a transgenic mouse, in which the AT 1A R promoter drives expression of green fluorescent protein (GFP). Approximately one-third of AT 1A R-expressing neurons express the catecholamine-synthetic enzyme tyrosine hydroxylase (TH), and a subpopulation of these stained for the transcription factor paired-like homeobox 2b (Phox2b). A third group, comprising approximately two-thirds of the AT 1A R-expressing NTS neurons, showed Phox2b immunoreactivity alone. A fourth group in the ventral subnucleus expressed neither TH nor Phox2b. In whole cell recordings from slices in vitro, AT 1A R-GFP neurons exhibited voltage-activated potassium currents, including the transient outward current and the M-type potassium current. In two different mouse strains, both AT 1A R-GFP neurons and TH-GFP neurons showed similar AT 1A R-mediated depolarizing responses to superfusion with angiotensin II. These data provide a comprehensive description of AT 1A R-expressing neurons in the NTS and increase our understanding of the complex actions of this neuropeptide in the modulation of viscerosensory processing. Copyright © 2017 the American Physiological Society.

Expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 in cocultures of dissociated DRG neurons and skeletal muscle cells in administration of NGF or NT-3

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Weiwei Zhang

2012-07-01

Full Text Available Both neurotrophins (NTs and target skeletal muscle (SKM cells are essential for the maintenance of the function of neurons and nerve-muscle communication. However, much less is known about the association of target SKM cells with distinct NTs on the expression of mRNAs for preprotachykinin (PPT, calcitonin-gene related peptide (CGRP, neurofilament 200 (NF-200, and microtubule associated protein 2 (MAP-2 in dorsal root ganglion (DRG sensory neurons. In the present study, a neuromuscular coculture model of dissociated dorsal root ganglion (DRG neurons and SKM cells was established. The morphology of DRG neurons and SKM cells in coculture was observed with an inverted phase contrast microscope. The effects of nerve growth factor (NGF or neurotrophin-3 (NT-3 on the expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 was analyzed by real time-PCR assay. The morphology of DRG neuronal cell bodies and SKM cells in neuromuscular coculture at different conditions was similar. The neurons presented evidence of dense neurite outgrowth in the presence of distinct NTs in neuromuscular cocultures. NGF and NT-3 increased mRNA levels of PPT, CGRP, and NF-200, but not MAP-2, in neuromuscular cocultures. These results offer new clues towards a better understanding of the association of target SKM cells with distinct NTs on the expression of mRNAs for PPT, CGRP, NF-200 and MAP-2, and implicate the association of target SKM cells and NTs with DRG sensory neuronal phenotypes.

Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons

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Jensen, Pia; Ducray, A D; Widmer, H R

2015-01-01

shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10days......, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells....... to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which...

NDRG2 promoted secreted miR-375 in microvesicles shed from M1 microglia, which induced neuron damage.

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Tang, Li-li; Wu, Yuan-bo; Fang, Chuan-qin; Qu, Ping; Gao, Zong-liang

2016-01-15

Microglia microvesicles (MVs) has shown to have significant biological functions under normal conditions. A diversity of miRNAs is involved in neuronal development, survival, function, and plasticity, but the exact functional role of NDRG2 and secreted miR-375 in MVs in neuron damage is poorly understood. We investigated the effect of NDRG2 and secreted miR-375 in MVs shed from M1 microglia on neuron damage. Expression of Nos2, Arg-1, miR-375, syntaxin-1A, NDRG2 and Pdk 1 were evaluated using RT-PCR or western blotting. Cell viability of N2A neuron was quantified by a MTT assay. Microglia can be polarized into different functional phenotypes. Expression of NDRG2 and Nos2 were significantly increased by LPS treatment on N9 cells, whereas treatment with IL-4 dramatically suppressed the expression of NDRG2 and remarkably elevated expression of Arg-1. Besides, MVs shed from LPS-treated N9 microglia significantly inhibited cell viability of N2A neurons and expression of syntaxin-1A, and NDRG2 interference reversed the up-regulated miR-375 in LPS-treated N9 microglia and MVs shed from LPS-treated N9 cells. Furthermore, NDRG2 could modulate miR-375 expression in N9 microglia and MVs. And miR-375 inhibitor remarkably elevated Pdk1 expression in N2A neurons. Finally, miR-375 inhibitor could reverse suppression effect of NDRG2 overexpression on cell viability of N2A neurons and expression of syntaxin-1A. Our results demonstrated that NDRG2 promoted secreted miR-375 in microvesicles shed from M1 microglia, which induced neuron damage. The suppression of NDRG2 and secreted miR-375 in MVs shed from M1 microglia may be potential targets for alleviation of neuron damage. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection.

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Chung, Chee Yeun; Seo, Hyemyung; Sonntag, Kai Christian; Brooks, Andrew; Lin, Ling; Isacson, Ole

2005-07-01

Molecular differences between dopamine (DA) neurons may explain why the mesostriatal DA neurons in the A9 region preferentially degenerate in Parkinson's disease (PD) and toxic models, whereas the adjacent A10 region mesolimbic and mesocortical DA neurons are relatively spared. To characterize innate physiological differences between A9 and A10 DA neurons, we determined gene expression profiles in these neurons in the adult mouse by laser capture microdissection, microarray analysis and real-time PCR. We found 42 genes relatively elevated in A9 DA neurons, whereas 61 genes were elevated in A10 DA neurons [> 2-fold; false discovery rate (FDR) neurotoxic or protective biochemical pathways. Three A9-elevated molecules [G-protein coupled inwardly rectifying K channel 2 (GIRK2), adenine nucleotide translocator 2 (ANT-2) and the growth factor IGF-1] and three A10-elevated peptides (GRP, CGRP and PACAP) were further examined in both alpha-synuclein overexpressing PC12 (PC12-alphaSyn) cells and rat primary ventral mesencephalic (VM) cultures exposed to MPP+ neurotoxicity. GIRK2-positive DA neurons were more vulnerable to MPP+ toxicity and overexpression of GIRK2 increased the vulnerability of PC12-alphaSyn cells to the toxin. Blocking of ANT decreased vulnerability to MPP+ in both cell culture systems. Exposing cells to IGF-1, GRP and PACAP decreased vulnerability of both cell types to MPP+, whereas CGRP protected PC12-alphaSyn cells but not primary VM DA neurons. These results indicate that certain differentially expressed molecules in A9 and A10 DA neurons may play key roles in their relative vulnerability to toxins and PD.

Expression of peroxisome proliferator-activated receptor-gamma in key neuronal subsets regulating glucose metabolism and energy homeostasis.

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Sarruf, David A; Yu, Fang; Nguyen, Hong T; Williams, Diana L; Printz, Richard L; Niswender, Kevin D; Schwartz, Michael W

2009-02-01

In addition to increasing insulin sensitivity and adipogenesis, peroxisome proliferator-activated receptor (PPAR)-gamma agonists cause weight gain and hyperphagia. Given the central role of the brain in the control of energy homeostasis, we sought to determine whether PPARgamma is expressed in key brain areas involved in metabolic regulation. Using immunohistochemistry, PPARgamma distribution and its colocalization with neuron-specific protein markers were investigated in rat and mouse brain sections spanning the hypothalamus, the ventral tegmental area, and the nucleus tractus solitarius. In several brain areas, nuclear PPARgamma immunoreactivity was detected in cells that costained for neuronal nuclei, a neuronal marker. In the hypothalamus, PPARgamma immunoreactivity was observed in a majority of neurons in the arcuate (including both agouti related protein and alpha-MSH containing cells) and ventromedial hypothalamic nuclei and was also present in the hypothalamic paraventricular nucleus, the lateral hypothalamic area, and tyrosine hydroxylase-containing neurons in the ventral tegmental area but was not expressed in the nucleus tractus solitarius. To validate and extend these histochemical findings, we generated mice with neuron-specific PPARgamma deletion using nestin cre-LoxP technology. Compared with littermate controls, neuron-specific PPARgamma knockout mice exhibited dramatic reductions of both hypothalamic PPARgamma mRNA levels and PPARgamma immunoreactivity but showed no differences in food intake or body weight over a 4-wk study period. We conclude that: 1) PPARgamma mRNA and protein are expressed in the hypothalamus, 2) neurons are the predominant source of PPARgamma in the central nervous system, although it is likely expressed by nonneuronal cell types as well, and 3) arcuate nucleus neurons that control energy homeostasis and glucose metabolism are among those in which PPARgamma is expressed.

Gene Expression in Accumbens GABA Neurons from Inbred Rats with Different Drug-Taking Behavior

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Sharp, B.M.; Chen, H.; Gong, S.; Wu, X.; Liu, Z.; Hiler, K.; Taylor, W.L.; Matta, S.G.

2011-01-01

Inbred Lewis and Fisher 344 rat strains differ greatly in drug self-administration; Lewis rats operantly self-administer drugs of abuse including nicotine, whereas Fisher self-administer poorly. As shown herein, operant food self-administration is similar. Based on their pivotal role in drug reward, we hypothesized that differences in basal gene expression in GABAergic neurons projecting from nucleus accumbens (NAcc) to ventral pallidum (VP) play a role in vulnerability to drug taking behavior. The transcriptomes of NAcc shell-VP GABAergic neurons from these two strains were analyzed in adolescents, using a multidisciplinary approach that combined stereotaxic ionotophoretic brain microinjections, laser-capture microdissection (LCM) and microarray measurement of transcripts. LCM enriched the gene transcripts detected in GABA neurons compared to the residual NAcc tissue: a ratio of neuron/residual > 1 and false discovery rate (FDR) 3 yielded 3,514. Strain-dependent differences in gene expression within GABA neurons were identified; 322 vs. 60 transcripts showed 1.5-fold vs. 2-fold differences in expression (FDR<5%). Classification by gene ontology showed these 322 transcripts were widely distributed, without categorical enrichment. This is most consistent with a global change in GABA neuron function. Literature-mining by Chilibot found 38 genes related to synaptic plasticity, signaling and gene transcription, all of which determine drug-abuse; 33 genes have no known association with addiction or nicotine. In Lewis rats, upregulation of Mint-1, Cask, CamkIIδ, Ncam1, Vsnl1, Hpcal1 and Car8 indicates these transcripts likely contribute to altered signaling and synaptic function in NAcc GABA projection neurons to VP. PMID:21745336

Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons

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Tomoko eSoga

2016-03-01

Full Text Available Kisspeptin, a newly discovered neuropeptide regulates gonadotropin-releasing hormone (GnRH. Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by kiss1 gene is a 145-amino acid- protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein coupled receptor 54 (GPR54 has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP labelled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP–GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1nM treatment for 36h on GnRH migration. Furthermore to determine kisspeptin-induced molecular pathways related with apoptosis, and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser captured EGFP–GnRH neurons by real time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd 26 in EGFP–GnRH neurons was up-regulated by the exposure to kisspeptin. These studies suggest that ankrd26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin-GPR54 signaling, which could be a potential pathway to suppress cell migration.

Dysregulation of RNA Mediated Gene Expression in Motor Neuron Diseases.

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Gonçalves, Inês do Carmo G; Rehorst, Wiebke A; Kye, Min Jeong

2016-01-01

Recent findings indicate an important role for RNA-mediated gene expression in motor neuron diseases, including ALS (amyotrophic lateral sclerosis) and SMA (spinal muscular atrophy). ALS, also known as Lou Gehrig's disease, is an adult-onset progressive neurodegenerative disorder, whereby SMA or "children's Lou Gehrig's disease" is considered a pediatric neurodevelopmental disorder. Despite the difference in genetic causes, both ALS and SMA share common phenotypes; dysfunction/loss of motor neurons that eventually leads to muscle weakness and atrophy. With advanced techniques in molecular genetics and cell biology, current data suggest that these two distinct motor neuron diseases share more than phenotypes; ALS and SMA have similar cellular pathological mechanisms including mitochondrial dysfunction, oxidative stress and dysregulation in RNA-mediated gene expression. Here, we will discuss the current findings on these two diseases with specific focus on RNA-mediated gene regulation including miRNA expression, pre-mRNA processing and RNA binding proteins.

The type III neurofilament peripherin is expressed in the tuberomammillary neurons of the mouse

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Julien Jean-Pierre

2008-02-01

Full Text Available Abstract Background Peripherin, a type III neuronal intermediate filament, is widely expressed in neurons of the peripheral nervous system and in selected central nervous system hindbrain areas with projections towards peripheral structures, such as cranial nerves and spinal cord neurons. Peripherin appears to play a role in neurite elongation during development and axonal regeneration, but its exact function is not known. We noticed high peripherin expression in the posterior hypothalamus of mice, and decided to investigate further the exact location of expression and function of peripherin in the mouse posterior hypothalamus. Results In situ hybridization indicated expression of peripherin in neurons with a distribution reminiscent of the histaminergic neurons, with little signal in any other part of the forebrain. Immunocytochemical staining for histidine decarboxylase and peripherin revealed extensive colocalization, showing that peripherin is produced by histaminergic neurons in all parts of the tuberomammillary nucleus. We next used histamine immunostaining in peripherin knockout, overexpressing and wild type mice to study if altered peripherin expression affects these neurons, but could not detect any visible difference in the appearance of these neurons or their axons. Peripherin knockout mice and heterozygotic littermates were used for measurement of locomotor activity, feeding, drinking, and energy expenditure. Both genotypes displayed diurnal rhythms with all the parameters higher during the dark period. The respiratory quotient, an indicator of the type of substrate being utilized, also exhibited a significant diurnal rhythm in both genotypes. The diurnal patterns and the average values of all the recorded parameters for 24 h, daytime and night time were not significantly different between the genotypes, however. Conclusion In conclusion, we have shown that peripherin is expressed in the tuberomammillary neurons of the mouse

Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord.

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Furness, John B; Cho, Hyun-Jung; Hunne, Billie; Hirayama, Haruko; Callaghan, Brid P; Lomax, Alan E; Brock, James A

2012-06-01

Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with

Positive regulation of raphe serotonin neurons by serotonin 2B receptors.

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Belmer, Arnauld; Quentin, Emily; Diaz, Silvina L; Guiard, Bruno P; Fernandez, Sebastian P; Doly, Stéphane; Banas, Sophie M; Pitychoutis, Pothitos M; Moutkine, Imane; Muzerelle, Aude; Tchenio, Anna; Roumier, Anne; Mameli, Manuel; Maroteaux, Luc

2018-06-01

Serotonin is a neurotransmitter involved in many psychiatric diseases. In humans, a lack of 5-HT 2B receptors is associated with serotonin-dependent phenotypes, including impulsivity and suicidality. A lack of 5-HT 2B receptors in mice eliminates the effects of molecules that directly target serotonergic neurons including amphetamine derivative serotonin releasers, and selective serotonin reuptake inhibitor antidepressants. In this work, we tested the hypothesis that 5-HT 2B receptors directly and positively regulate raphe serotonin neuron activity. By ex vivo electrophysiological recordings, we report that stimulation by the 5-HT 2B receptor agonist, BW723C86, increased the firing frequency of serotonin Pet1-positive neurons. Viral overexpression of 5-HT 2B receptors in these neurons increased their excitability. Furthermore, in vivo 5-HT 2B -receptor stimulation by BW723C86 counteracted 5-HT 1A autoreceptor-dependent reduction in firing rate and hypothermic response in wild-type mice. By a conditional genetic ablation that eliminates 5-HT 2B receptor expression specifically and exclusively from Pet1-positive serotonin neurons (Htr2b 5-HTKO mice), we demonstrated that behavioral and sensitizing effects of MDMA (3,4-methylenedioxy-methamphetamine), as well as acute behavioral and chronic neurogenic effects of the antidepressant fluoxetine, require 5-HT 2B receptor expression in serotonergic neurons. In Htr2b 5-HTKO mice, dorsal raphe serotonin neurons displayed a lower firing frequency compared to control Htr2b lox/lox mice as assessed by in vivo extracellular recordings and a stronger hypothermic effect of 5-HT 1A -autoreceptor stimulation was observed. The increase in head-twitch response to DOI (2,5-dimethoxy-4-iodoamphetamine) further confirmed the lower serotonergic tone resulting from the absence of 5-HT 2B receptors in serotonin neurons. Together, these observations indicate that the 5-HT 2B receptor acts as a direct positive modulator of serotonin Pet1

Transcription factors Foxa1 and Foxa2 are required for adult dopamine neurons maintenance

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Andrii eDomanskyi

2014-09-01

Full Text Available The proteins Foxa1 and Foxa2 belong to the forkhead family of transcription factors and are involved in the development of several tissues, including liver, pancreas, lung, prostate, and the neural system. Both Foxa1 and Foxa2 are also crucial for the specification and differentiation of dopamine (DA neurons during embryonic development, while about 30% of mice with an embryonic deletion of a single allele of the Foxa2 gene exhibit an age-related asymmetric loss of DA neurons and develop locomotor symptoms resembling Parkinson’s disease (PD. Notably, both Foxa1 and Foxa2 factors continue to be expressed in the adult dopamine system. To directly assess their functions selectively in adult DA neurons, we induced genetic deletions of Foxa1/2 transcription factors in mice using a tamoxifen inducible tissue-specific CreERT2 recombinase expressed under control of the dopamine transporter (DAT promoter (DATCreERT2. The conditional DA neurons-specific ablation of both genes, but not of Foxa2 alone, in early adulthood, caused a decline of striatal dopamine and its metabolites, along with locomotor deficits. At early pre-symptomatic stages, we observed a decline in aldehyde dehydrogenase family 1, subfamily A1 (Aldh1a1 protein expression in DA neurons. Further analyses revealed a decline of aromatic amino acid decarboxylase (AADC and a complete loss of DAT expression in these neurons. These molecular changes ultimately led to a reduction of DA neuron numbers in the substantia nigra pars compacta (SNpc of aged cFoxa1/2-/- mice, resembling the progressive course of PD in humans. Altogether, in this study, we address the molecular, cellular and functional role of both Foxa1 and Foxa2 factors in the maintenance of the adult dopamine system which may help to find better approaches for PD treatment.

ChR2 transgenic animals in peripheral sensory system: Sensing light as various sensations.

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Ji, Zhi-Gang; Wang, Hongxia

2016-04-01

Since the introduction of Channelrhodopsin-2 (ChR2) to neuroscience, optogenetics technology was developed, making it possible to activate specific neurons or circuits with spatial and temporal precision. Various ChR2 transgenic animal models have been generated and are playing important roles in revealing the mechanisms of neural activities, mapping neural circuits, controlling the behaviors of animals as well as exploring new strategy for treating the neurological diseases in both central and peripheral nervous system. An animal including humans senses environments through Aristotle's five senses (sight, hearing, smell, taste and touch). Usually, each sense is associated with a kind of sensory organ (eyes, ears, nose, tongue and skin). Is it possible that one could hear light, smell light, taste light and touch light? When ChR2 is targeted to different peripheral sensory neurons by viral vectors or generating ChR2 transgenic animals, the animals can sense the light as various sensations such as hearing, touch, pain, smell and taste. In this review, we focus on ChR2 transgenic animals in the peripheral nervous system. Firstly the working principle of ChR2 as an optogenetic actuator is simply described. Then the current transgenic animal lines where ChR2 was expressed in peripheral sensory neurons are presented and the findings obtained by these animal models are reviewed. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of activated ACM on expression of signal transducers in cerebral cortical neurons of rats.

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Wang, Xiaojing; Li, Zhengli; Zhu, Changgeng; Li, Zhongyu

2007-06-01

To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic factor (CNTF) and astrocytic conditioned medium (ACM) was collected to treat neurons for 4, 8 and 12 h. By using Western blot, the expression of calmodulin dependent protein kinase II (CaMK II), inducible nitric oxide synthase (iNOS) and adenylate cyclase (AC) was detected in neurons. The results showed that the expression of CaMK II, iNOS and AC was increased significantly in the neurons treated with ACM from 4 h to 12 h (PACM and such signal pathways as NOS-NO-cGMP, Ca2+/CaM-CaMK II and AC-cAMP-PKA might take part in the signal transduction of epileptogenesis.

Streptozotocin alters glucose transport, connexin expression and endoplasmic reticulum functions in neurons and astrocytes.

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Biswas, Joyshree; Gupta, Sonam; Verma, Dinesh Kumar; Singh, Sarika

2017-07-25

The study was undertaken to explore the cell-specific streptozotocin (STZ)-induced mechanistic alterations. STZ-induced rodent model is a well-established experimental model of Alzheimer's disease (AD) and in our previous studies we have established it as an in vitro screening model of AD by employing N2A neuronal cells. Therefore, STZ was selected in the present study to understand the STZ-induced cell-specific alterations by utilizing neuronal N2A and astrocytes C6 cells. Both neuronal and astrocyte cells were treated with STZ at 10, 50, 100 and 1000μM concentrations for 48h. STZ exposure caused significant decline in cellular viability and augmented cytotoxicity of cells involving astrocytes activation. STZ treatment also disrupted the energy metabolism by altered glucose uptake and its transport in both cells as reflected with decreased expression of glucose transporters (GLUT) 1/3. The consequent decrease in ATP level and decreased mitochondrial membrane potential was also observed in both the cells. STZ caused increased intracellular calcium which could cause the initiation of endoplasmic reticulum (ER) stress. Significant upregulation of ER stress-related markers were observed in both cells after STZ treatment. The cellular communication of astrocytes and neurons was altered as reflected by increased expression of connexin 43 along with DNA fragmentation. STZ-induced apoptotic death was evaluated by elevated expression of caspase-3 and PI/Hoechst staining of cells. In conclusion, study showed that STZ exert alike biochemical alterations, ER stress and cellular apoptosis in both neuronal and astrocyte cells. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Morphine regulates Argonaute 2 and TH expression and activity but not miR-133b in midbrain dopaminergic neurons.

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García-Pérez, Daniel; López-Bellido, Roger; Hidalgo, Juana M; Rodríguez, Raquel E; Laorden, Maria Luisa; Núñez, Cristina; Milanés, Maria Victoria

2015-01-01

Epigenetic changes such as microRNAs (miRs)/Ago2-induced gene silencing represent complex molecular signature that regulate cellular plasticity. Recent studies showed involvement of miRs and Ago2 in drug addiction. In this study, we show that changes in gene expression induced by morphine and morphine withdrawal occur with concomitant epigenetic modifications in the mesolimbic dopaminergic (DA) pathway [ventral tegmental area (VTA)/nucleus accumbens (NAc) shell], which is critically involved in drug-induced dependence. We found that acute or chronic morphine administration as well as morphine withdrawal did not modify miR-133b messenger RNA (mRNA) expression in the VTA, whereas Ago2 protein levels were decreased and increased in morphine-dependent rats and after morphine withdrawal, respectively. These changes were paralleled with enhanced and decreased NAc tyrosine hydroxylase (TH) protein (an early DA marker) in morphine-dependent rats and after withdrawal, respectively. We also observed changes in TH mRNA expression in the VTA that could be related to Ago2-induced translational repression of TH mRNA during morphine withdrawal. However, the VTA number of TH-positive neurons suffered no alterations after the different treatment. Acute morphine administration produced a marked increase in TH activity and DA turnover in the NAc (shell). In contrast, precipitated morphine withdrawal decreased TH activation and did not change DA turnover. These findings provide new information into the possible correlation between Ago2/miRs complex regulation and DA neurons plasticity during opiate addiction. © 2013 Society for the Study of Addiction.

High level over-expression of different NCX isoforms in HEK293 cell lines and primary neuronal cultures is protective following oxygen glucose deprivation.

Science.gov (United States)

Cross, Jane L; Boulos, Sherif; Shepherd, Kate L; Craig, Amanda J; Lee, Sharon; Bakker, Anthony J; Knuckey, Neville W; Meloni, Bruno P

2012-07-01

In this study we have assessed sodium-calcium exchanger (NCX) protein over-expression on cell viability in primary rat cortical neuronal and HEK293 cell cultures when subjected to oxygen-glucose deprivation (OGD). In cortical neuronal cultures, NCX2 and NCX3 over-expression was achieved using adenoviral vectors, and following OGD increased neuronal survival from ≈20% for control vector treated cultures to ≈80% for both NCX isoforms. In addition, we demonstrated that NCX2 and NCX3 over-expression in cortical neuronal cultures enables neurons to maintain intracellular calcium at significantly lower levels than control vector treated cultures when exposed to high (9mM) extracellular calcium challenge. Further assessment of NCX activity during OGD was performed using HEK293 cell lines generated to over-express NCX1, NCX2 or NCX3 isoforms. While it was shown that NCX isoform expression differed considerably in the different HEK293 cell lines, high levels of NCX over-expression was associated with increased resistance to OGD. Taken together, our findings show that high levels of NCX over-expression increases neuronal and HEK293 cell survival following OGD, improves calcium management in neuronal cultures and provides additional support for NCX as a therapeutic target to reduce ischemic brain injury. Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

APLP2 regulates neuronal stem cell differentiation during cortical development.

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Shariati, S Ali M; Lau, Pierre; Hassan, Bassem A; Müller, Ulrike; Dotti, Carlos G; De Strooper, Bart; Gärtner, Annette

2013-03-01

Expression of amyloid precursor protein (APP) and its two paralogues, APLP1 and APLP2 during brain development coincides with key cellular events such as neuronal differentiation and migration. However, genetic knockout and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. To address this issue, we analysed in depth the role of APLP2 during neurogenesis by silencing APLP2 in vivo in an APP/APLP1 double knockout mouse background. We find that under these conditions cortical progenitors remain in their undifferentiated state much longer, displaying a higher number of mitotic cells. In addition, we show that neuron-specific APLP2 downregulation does not impact the speed or position of migrating excitatory cortical neurons. In summary, our data reveal that APLP2 is specifically required for proper cell cycle exit of neuronal progenitors, and thus has a distinct role in priming cortical progenitors for neuronal differentiation.

Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila.

Science.gov (United States)

Tharadra, Sana Khalid; Medina, Adriana; Ray, Anandasankar

2013-01-01

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.

Axial level-specific regulation of neuronal development: lessons from PITX2.

Science.gov (United States)

Waite, Mindy R; Martin, Donna M

2015-02-01

Transcriptional regulation of gene expression is vital for proper control of proliferation, migration, differentiation, and survival of developing neurons. Pitx2 encodes a homeodomain transcription factor that is highly expressed in the developing and adult mammalian brain. In humans, mutations in PITX2 result in Rieger syndrome, characterized by defects in the development of the eyes, umbilicus, and teeth and variable abnormalities in the brain, including hydrocephalus and cerebellar hypoplasia. Alternative splicing of Pitx2 in the mouse results in three isoforms, Pitx2a, Pitx2b, and Pitx2c, each of which is expressed symmetrically along the left-right axis of the brain throughout development. Here, we review recent evidence for axial and brain region-specific requirements for Pitx2 during neuronal migration and differentiation, highlighting known isoform contributions. © 2014 Wiley Periodicals, Inc.

Neuronal Fibers and Neurotransmitter Receptor Expression in the Human Endolymphatic Sac

DEFF Research Database (Denmark)

Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas

2017-01-01

in intracranial pressure homeostasis. The anatomical location towards the sigmoid sinus would suggest a possible endo- and/or paracrine signaling. However, neuronal connections may also apply, but it remains very scarcely explored in the human ES. STUDY DESIGN: DNA micro-arrays and immunohistochemistry were used...... of genes specific for neuronal signaling was determined and results for selected key molecules verified by immunohistochemistry. Transmission electron microscopy was used for ultrastructural analysis. RESULTS: For the transmission electron microscopy analysis, a direct innervation of the ES was observed...... with unmyelinated fibers imbedded in the ES epithelial lining. The microarrays confirmed, that several molecules involved in neuronal signaling were found expressed significantly in the ES DNA profile, such as the Cholecystokinin peptide and related receptors, Dopamine receptors 2 and 5, vesicular monoamine...

Differential regulation of amyloid-β-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease

International Nuclear Information System (INIS)

Higgins, G.A.; Lewis, D.A.; Bahmanyar, S.; Goldgaber, D.; Gajdusek, D.C.; Young, W.G.; Morrison, J.H.; Wilson, M.C.

1988-01-01

The authors have mapped the neuroanatomical distribution of amyloid-β-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-β-protein mRNA appears to be expressed in all hippocampal neurons, but at different levels of abundance. In the central nervous system of monkey and normal aged human, image analysis shows that neurons of the dentate gyrus and cornu Ammonis fields contain a 2.5-times-greater hybridization signal than is present in neurons of the subiculum and entorhinal cortex. In contrast, in the Alzheimer disease hippocampal formation, the levels of amyloid-β-protein mRNA in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-β-protein gene expression may be altered in Alzheimer disease

Serotonin 2C receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis.

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Berglund, Eric D; Liu, Chen; Sohn, Jong-Woo; Liu, Tiemin; Kim, Mi Hwa; Lee, Charlotte E; Vianna, Claudia R; Williams, Kevin W; Xu, Yong; Elmquist, Joel K

2013-12-01

Energy and glucose homeostasis are regulated by central serotonin 2C receptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2C receptor-expressing neurons that mediate the effects of serotonin and serotonin 2C receptor agonists on energy and glucose homeostasis are unknown. Here, we show that mice lacking serotonin 2C receptors (Htr2c) specifically in pro-opiomelanocortin (POMC) neurons had normal body weight but developed glucoregulatory defects including hyperinsulinemia, hyperglucagonemia, hyperglycemia, and insulin resistance. Moreover, these mice did not show anorectic responses to serotonergic agents that suppress appetite and developed hyperphagia and obesity when they were fed a high-fat/high-sugar diet. A requirement of serotonin 2C receptors in POMC neurons for the maintenance of normal energy and glucose homeostasis was further demonstrated when Htr2c loss was induced in POMC neurons in adult mice using a tamoxifen-inducible POMC-cre system. These data demonstrate that serotonin 2C receptor-expressing POMC neurons are required to control energy and glucose homeostasis and implicate POMC neurons as the target for the effect of serotonin 2C receptor agonists on weight-loss induction and improved glycemic control.

Metformin Protects Neurons against Oxygen-Glucose Deprivation/Reoxygenation -Induced Injury by Down-Regulating MAD2B.

Science.gov (United States)

Meng, Xianfang; Chu, Guangpin; Yang, Zhihua; Qiu, Ping; Hu, Yue; Chen, Xiaohe; Peng, Wenpeng; Ye, Chen; He, Fang-Fang; Zhang, Chun

2016-01-01

Metformin, the common medication for type II diabetes, has protective effects on cerebral ischemia. However, the molecular mechanisms are far from clear. Mitotic arrest deficient 2-like protein 2 (MAD2B), an inhibitor of the anaphase-promoting complex (APC), is widely expressed in hippocampal and cortical neurons and plays an important role in mediating high glucose-induced neurotoxicity. The present study investigated whether metformin modifies the expression of MAD2B and to exert its neuroprotective effects in primary cultured cortical neurons during oxygen-glucose deprivation/reoxygenation (OGD/R), a widely used in vitro model of ischemia/reperfusion. Primary cortical neurons were cultured, deprived of oxygen-glucose for 1 h, and then recovered with oxygen-glucose for 12 h and 24 h. Cell viability was measured by detecting the levels of lactate dehydrogenase (LDH) in culture medium. The levels of MAD2B, cyclin B and p-histone 3 were measured by Western blot. Cell viability of neurons was reduced under oxygen-glucose deprivation/reoxygenation (OGD/R). The expression of MAD2B was increased under OGD/R. The levels of cyclin B1, which is a substrate of APC, were also increased. Moreover, OGD/R up-regulated the phosphorylation levels of histone 3, which is the induction of aberrant re-entry of post-mitotic neurons. However, pretreatment of neurons with metformin alleviated OGD/R-induced injury. Metformin further decreased the expression of MAD2B, cyclin B1 and phosphorylation levels of histone 3. Metformin exerts its neuroprotective effect through regulating the expression of MAD2B in neurons under OGD/R. © 2016 The Author(s) Published by S. Karger AG, Basel.

AgRP Neurons Control Systemic Insulin Sensitivity via Myostatin Expression in Brown Adipose Tissue.

Science.gov (United States)

Steculorum, Sophie M; Ruud, Johan; Karakasilioti, Ismene; Backes, Heiko; Engström Ruud, Linda; Timper, Katharina; Hess, Martin E; Tsaousidou, Eva; Mauer, Jan; Vogt, Merly C; Paeger, Lars; Bremser, Stephan; Klein, Andreas C; Morgan, Donald A; Frommolt, Peter; Brinkkötter, Paul T; Hammerschmidt, Philipp; Benzing, Thomas; Rahmouni, Kamal; Wunderlich, F Thomas; Kloppenburg, Peter; Brüning, Jens C

2016-03-24

Activation of Agouti-related peptide (AgRP) neurons potently promotes feeding, and chronically altering their activity also affects peripheral glucose homeostasis. We demonstrate that acute activation of AgRP neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue (BAT). AgRP neuron activation acutely reprograms gene expression in BAT toward a myogenic signature, including increased expression of myostatin. Interference with myostatin activity improves insulin sensitivity that was impaired by AgRP neurons activation. Optogenetic circuitry mapping reveals that feeding and insulin sensitivity are controlled by both distinct and overlapping projections. Stimulation of AgRP → LHA projections impairs insulin sensitivity and promotes feeding while activation of AgRP → anterior bed nucleus of the stria terminalis (aBNST)vl projections, distinct from AgRP → aBNSTdm projections controlling feeding, mediate the effect of AgRP neuron activation on BAT-myostatin expression and insulin sensitivity. Collectively, our results suggest that AgRP neurons in mice induce not only eating, but also insulin resistance by stimulating expression of muscle-related genes in BAT, revealing a mechanism by which these neurons rapidly coordinate hunger states with glucose homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Anti-Tribbles Pseudokinase 2 (TRIB2)-Immunization Modulates Hypocretin/Orexin Neuronal Functions.

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Tanaka, Susumu; Honda, Yoshiko; Honda, Makoto; Yamada, Hisao; Honda, Kazuki; Kodama, Tohru

2017-01-01

Recent findings showed that 16%-26% of narcolepsy patients were positive for anti-tribbles pseudokinase 2 (TRIB2) antibody, and the intracerebroventricular administration of immunoglobulin-G purified from anti-TRIB2 positive narcolepsy patients caused hypocretin/orexin neuron loss. We investigated the pathophysiological role of TRIB2 antibody using TRIB2-immunized rats and hypocretin/ataxin-3 transgenic (ataxin-3) mice. Plasma, cerebrospinal fluid (CSF), and hypothalamic tissues from TRIB2-immunized rats were collected. Anti-TRIB2 titers, hypocretin contents, mRNA expressions, the cell count of hypocretin neurons, and immunoreactivity of anti-TRIB2 antibodies on hypocretin neurons were investigated. The plasma from ataxin-3 mice was also used to determine the anti-TRIB2 antibody titer changes following the loss of hypocretin neurons. TRIB2 antibody titers increased in the plasma and CSF of TRIB2-immunized rats. The hypothalamic tissue immunostained with the sera from TRIB2-immunized rats revealed positive signals in the cytoplasm of hypcretin neurons. While no changes were found regarding hypothalamic hypocretin contents or cell counts, but there were significant decreases of the hypocretin mRNA level and release into the CSF. The plasma from over 26-week-old ataxin-3 mice, at the advanced stage of hypocretin cell destruction, showed positive reactions against TRIB2 antigen, and positive plasma also reacted with murine hypothalamic hypocretin neurons. Our results suggest that the general activation of the immune system modulates the functions of hypocretin neurons. The absence of a change in hypocretin cell populations suggested that factors other than anti-TRIB2 antibody play a part in the loss of hypocretin neurons in narcolepsy. The increased anti-TRIB2 antibody after the destruction of hypocretin neurons suggest that anti-TRIB2 antibody in narcolepsy patients is the consequence rather than the inciting cause of hypocretin cell destruction. © Sleep Research

GDE2 regulates subtype-specific motor neuron generation through inhibition of Notch signaling.

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Sabharwal, Priyanka; Lee, Changhee; Park, Sungjin; Rao, Meenakshi; Sockanathan, Shanthini

2011-09-22

The specification of spinal interneuron and motor neuron identities initiates within progenitor cells, while motor neuron subtype diversification is regulated by hierarchical transcriptional programs implemented postmitotically. Here we find that mice lacking GDE2, a six-transmembrane protein that triggers motor neuron generation, exhibit selective losses of distinct motor neuron subtypes, specifically in defined subsets of limb-innervating motor pools that correlate with the loss of force-generating alpha motor neurons. Mechanistically, GDE2 is expressed by postmitotic motor neurons but utilizes extracellular glycerophosphodiester phosphodiesterase activity to induce motor neuron generation by inhibiting Notch signaling in neighboring motor neuron progenitors. Thus, neuronal GDE2 controls motor neuron subtype diversity through a non-cell-autonomous feedback mechanism that directly regulates progenitor cell differentiation, implying that subtype specification initiates within motor neuron progenitor populations prior to their differentiation into postmitotic motor neurons. Copyright © 2011 Elsevier Inc. All rights reserved.

Shp2 in Forebrain Neurons Regulates Synaptic Plasticity, Locomotion, and Memory Formation in Mice

Science.gov (United States)

Kusakari, Shinya; Saitow, Fumihito; Ago, Yukio; Shibasaki, Koji; Sato-Hashimoto, Miho; Matsuzaki, Yasunori; Kotani, Takenori; Murata, Yoji; Hirai, Hirokazu; Matsuda, Toshio; Suzuki, Hidenori

2015-01-01

Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K+-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation. PMID:25713104

Beware of your Cre-Ation: lacZ expression impairs neuronal integrity and hippocampus-dependent memory.

Science.gov (United States)

Reichel, J M; Bedenk, B T; Gassen, N C; Hafner, K; Bura, S A; Almeida-Correa, S; Genewsky, A; Dedic, N; Giesert, F; Agarwal, A; Nave, K-A; Rein, T; Czisch, M; Deussing, J M; Wotjak, C T

2016-10-01

Expression of the lacZ-sequence is a widely used reporter-tool to assess the transgenic and/or transfection efficacy of a target gene in mice. Once activated, lacZ is permanently expressed. However, protein accumulation is one of the hallmarks of neurodegenerative diseases. Furthermore, the protein product of the bacterial lacZ gene is ß-galactosidase, an analog to the mammalian senescence-associated ß-galactosidase, a molecular marker for aging. Therefore we studied the behavioral, structural and molecular consequences of lacZ expression in distinct neuronal sub-populations. lacZ expression in cortical glutamatergic neurons resulted in severe impairments in hippocampus-dependent memory accompanied by marked structural alterations throughout the CNS. In contrast, GFP expression or the expression of the ChR2/YFP fusion product in the same cell populations did not result in either cognitive or structural deficits. GABAergic lacZ expression caused significantly decreased hyper-arousal and mild cognitive deficits. Attenuated structural and behavioral consequences of lacZ expression could also be induced in adulthood, and lacZ transfection in neuronal cell cultures significantly decreased their viability. Our findings provide a strong caveat against the use of lacZ reporter mice for phenotyping studies and point to a particular sensitivity of the hippocampus formation to detrimental consequences of lacZ expression. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Novel MeCP2 isoform-specific antibody reveals the endogenous MeCP2E1 expression in murine brain, primary neurons and astrocytes.

Directory of Open Access Journals (Sweden)

Robby M Zachariah

Full Text Available Rett Syndrome (RTT is a severe neurological disorder in young females, and is caused by mutations in the X-linked MECP2 gene. MECP2/Mecp2 gene encodes for two protein isoforms; MeCP2E1 and MeCP2E2 that are identical except for the N-terminus region of the protein. In brain, MECP2E1 transcripts are 10X higher, and MeCP2E1 is suggested to be the relevant isoform for RTT. However, due to the unavailability of MeCP2 isoform-specific antibodies, the endogenous expression pattern of MeCP2E1 is unknown. To gain insight into the expression of MeCP2E1 in brain, we have developed an anti-MeCP2E1 antibody and validated its specificity in cells exogenously expressing individual MeCP2 isoforms. This antibody does not show any cross-reactivity with MeCP2E2 and detects endogenous MeCP2E1 in mice brain, with no signal in Mecp2(tm1.1Bird y/- null mice. Additionally, we show the endogenous MeCP2E1 expression throughout different brain regions in adult mice, and demonstrate its highest expression in the brain cortex. Our results also indicate that MeCP2E1 is highly expressed in primary neurons, as compared to primary astrocytes. This is the first report of the endogenous MeCP2E1 expression at the protein levels, providing novel avenues for understanding different aspects of MeCP2 function.

Reduced neuronal size and mTOR pathway activity in the Mecp2 A140V Rett syndrome mouse model [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Sampathkumar Rangasamy

2016-09-01

Full Text Available Rett syndrome (RTT is a neurodevelopmental disorder caused by mutation in the X-linked MECP2 gene, encoding methyl-CpG-binding protein 2. We have created a mouse model (Mecp2 A140V “knock-in” mutant expressing the recurrent human MECP2 A140V mutation linked to an X-linked mental retardation/Rett syndrome phenotype. Morphological analyses focused on quantifying soma and nucleus size were performed on primary hippocampus and cerebellum granule neuron (CGN cultures from mutant (Mecp2A140V/y and wild type (Mecp2+/y male mice. Cultured hippocampus and cerebellar granule neurons from mutant animals were significantly smaller than neurons from wild type animals. We also examined soma size in hippocampus neurons from individual female transgenic mice that express both a mutant  (maternal allele and a wild type Mecp2 gene linked to an eGFP transgene (paternal allele. In cultures from such doubly heterozygous female mice, the size of neurons expressing the mutant (A140V allele also showed a significant reduction compared to neurons expressing wild type MeCP2, supporting a cell-autonomous role for MeCP2 in neuronal development. IGF-1 (insulin growth factor-1 treatment of neuronal cells from Mecp2 mutant mice rescued the soma size phenotype. We also found that Mecp2  mutation leads to down-regulation of the mTOR signaling pathway, known to be involved in neuronal size regulation. Our results suggest that i reduced neuronal size is an important in vitro cellular phenotype of Mecp2 mutation in mice, and ii MeCP2 might play a critical role in the maintenance of neuronal structure by modulation of the mTOR pathway. The definition of a quantifiable cellular phenotype supports using neuronal size as a biomarker in the development of a high-throughput, in vitro assay to screen for compounds that rescue small neuronal phenotype (“phenotypic assay”.

Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain.

Science.gov (United States)

Lindefors, N; Brene, S; Herrera-Marschitz, M; Persson, H

1989-01-01

In situ hybridization histochemistry and RNA blots were used to study the expression of glutamic acid decarboxylase (GAD) mRNA in rats with or without a unilateral lesion of midbrain dopamine neurons. Two populations of GAD mRNA positive neurons were found in the intact caudate-putamen, substantia nigra and fronto-parietal cortex. In caudate-putamen, only one out of ten of the GAD mRNA positive neurons expressed high levels, while in substantia nigra every second of the positive neurons expressed high levels of GAD mRNA. Relatively few, but intensively labelled neurons were found in the intact fronto-parietal cerebral cortex. In addition, one out of six of the GAD mRNA positive neurons in the fronto-parietal cortex showed a low labeling. On the ipsilateral side, the forebrain dopamine deafferentation induced an increase in the number of neurons expressing high levels of GAD mRNA in caudate-putamen, and a decrease in fronto-parietal cortex. A smaller decrease was also seen in substantia nigra. However, the total number of GAD mRNA positive neurons were not significantly changed in any of these brain regions. The changes in the levels of GAD mRNA after the dopamine lesion were confirmed by RNA blot analysis. Hence, midbrain dopamine neurons appear to control neuronal expression of GAD mRNA by a tonic down-regulation in a fraction of GAD mRNA positive neurons in caudate-putamen, and a tonic up-regulation in a fraction of GAD mRNA positive neurons in fronto-parietal cortex and substantia nigra.

Aberrant neuronal activity-induced signaling and gene expression in a mouse model of RASopathy.

Directory of Open Access Journals (Sweden)

Franziska Altmüller

2017-03-01

Full Text Available Noonan syndrome (NS is characterized by reduced growth, craniofacial abnormalities, congenital heart defects, and variable cognitive deficits. NS belongs to the RASopathies, genetic conditions linked to mutations in components and regulators of the Ras signaling pathway. Approximately 50% of NS cases are caused by mutations in PTPN11. However, the molecular mechanisms underlying cognitive impairments in NS patients are still poorly understood. Here, we report the generation and characterization of a new conditional mouse strain that expresses the overactive Ptpn11D61Y allele only in the forebrain. Unlike mice with a global expression of this mutation, this strain is viable and without severe systemic phenotype, but shows lower exploratory activity and reduced memory specificity, which is in line with a causal role of disturbed neuronal Ptpn11 signaling in the development of NS-linked cognitive deficits. To explore the underlying mechanisms we investigated the neuronal activity-regulated Ras signaling in brains and neuronal cultures derived from this model. We observed an altered surface expression and trafficking of synaptic glutamate receptors, which are crucial for hippocampal neuronal plasticity. Furthermore, we show that the neuronal activity-induced ERK signaling, as well as the consecutive regulation of gene expression are strongly perturbed. Microarray-based hippocampal gene expression profiling revealed profound differences in the basal state and upon stimulation of neuronal activity. The neuronal activity-dependent gene regulation was strongly attenuated in Ptpn11D61Y neurons. In silico analysis of functional networks revealed changes in the cellular signaling beyond the dysregulation of Ras/MAPK signaling that is nearly exclusively discussed in the context of NS at present. Importantly, changes in PI3K/AKT/mTOR and JAK/STAT signaling were experimentally confirmed. In summary, this study uncovers aberrant neuronal activity

Oxytocin-receptor-expressing neurons in the parabrachial nucleus regulate fluid intake.

Science.gov (United States)

Ryan, Philip J; Ross, Silvano I; Campos, Carlos A; Derkach, Victor A; Palmiter, Richard D

2017-12-01

Brain regions that regulate fluid satiation are not well characterized, yet are essential for understanding fluid homeostasis. We found that oxytocin-receptor-expressing neurons in the parabrachial nucleus of mice (Oxtr PBN neurons) are key regulators of fluid satiation. Chemogenetic activation of Oxtr PBN neurons robustly suppressed noncaloric fluid intake, but did not decrease food intake after fasting or salt intake following salt depletion; inactivation increased saline intake after dehydration and hypertonic saline injection. Under physiological conditions, Oxtr PBN neurons were activated by fluid satiation and hypertonic saline injection. Oxtr PBN neurons were directly innervated by oxytocin neurons in the paraventricular hypothalamus (Oxt PVH  neurons), which mildly attenuated fluid intake. Activation of neurons in the nucleus of the solitary tract substantially suppressed fluid intake and activated Oxtr PBN neurons. Our results suggest that Oxtr PBN neurons act as a key node in the fluid satiation neurocircuitry, which acts to decrease water and/or saline intake to prevent or attenuate hypervolemia and hypernatremia.

AP4M1 is abnormally expressed in oxygen-glucose deprived hippocampal neurons.

Science.gov (United States)

Zhang, J; Cheng, X Y; Sheng, G Y

2014-03-20

AP4M1 mutations have been suggested to be associated with autosomal recessive cerebral palsy syndrome. But the pathogenic mechanism remains uncertain. The purpose of this study is to investigate whether and how AP4M1 expression is changed in injured neurons. Primary cultured hippocampal neurons were prepared for this experiment. They were subjected to oxygen-glucose deprivation (OGD) leading to apoptosis, mimicking brain ischemia. Neuron-specific enolase (NSE) was labeled immunofluorescently to confirm that the purity of neuron was higher than 90%. Real-time PCR and western blotting were performed to measure the gene expression. AP4M1 was labeled with MAP2 or Tau-1 to observe the distribution. We found that the AP4M1 protein levels immediately after the procedure were similar between the OGD group and the sham group. However, down-regulation was observed 12h after the reperfusion, and became more notable at 24h. The real-time PCR showed similar results, except that the down-regulation of mRNA was able to be detected immediately after the OGD. Immunofluorescent labeling revealed AP4M1 distributed in the dendrites of normal neurons, but it redistributed to the axons after the OGD procedure. In conclusion, AP4M1 is not only down-regulated at both the mRNA and protein levels, but also redistributed from dendrites to axons in oxygen-glucose deprived hippocampal neurons. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Lactate promotes plasticity gene expression by potentiating NMDA signaling in neurons

KAUST Repository

Yang, Jiangyan; Ruchti, Evelyne; Petit, Jean Marie; Jourdain, Pascal; Grenningloh, Gabriele; Allaman, Igor; Magistretti, Pierre J.

2014-01-01

L-lactate is a product of aerobic glycolysis that can be used by neurons as an energy substrate. Here we report that in neurons L-lactate stimulates the expression of synaptic plasticity-related genes such as Arc, c-Fos, and Zif268 through a mechanism involving NMDA receptor activity and its downstream signaling cascade Erk1/2. L-lactate potentiates NMDA receptor-mediated currents and the ensuing increase in intracellular calcium. In parallel to this, L-lactate increases intracellular levels of NADH, thereby modulating the redox state of neurons. NADH mimics all of the effects of L-lactate on NMDA signaling, pointing to NADH increase as a primary mediator of L-lactate effects. The induction of plasticity genes is observed both in mouse primary neurons in culture and in vivo in the mouse sensory-motor cortex. These results provide insights for the understanding of the molecular mechanisms underlying the critical role of astrocyte-derived L-lactate in long-term memory and long-term potentiation in vivo. This set of data reveals a previously unidentified action of L-lactate as a signaling molecule for neuronal plasticity.

Lactate promotes plasticity gene expression by potentiating NMDA signaling in neurons

KAUST Repository

Yang, Jiangyan

2014-07-28

L-lactate is a product of aerobic glycolysis that can be used by neurons as an energy substrate. Here we report that in neurons L-lactate stimulates the expression of synaptic plasticity-related genes such as Arc, c-Fos, and Zif268 through a mechanism involving NMDA receptor activity and its downstream signaling cascade Erk1/2. L-lactate potentiates NMDA receptor-mediated currents and the ensuing increase in intracellular calcium. In parallel to this, L-lactate increases intracellular levels of NADH, thereby modulating the redox state of neurons. NADH mimics all of the effects of L-lactate on NMDA signaling, pointing to NADH increase as a primary mediator of L-lactate effects. The induction of plasticity genes is observed both in mouse primary neurons in culture and in vivo in the mouse sensory-motor cortex. These results provide insights for the understanding of the molecular mechanisms underlying the critical role of astrocyte-derived L-lactate in long-term memory and long-term potentiation in vivo. This set of data reveals a previously unidentified action of L-lactate as a signaling molecule for neuronal plasticity.

The histone demethylase Kdm6b regulates a mature gene expression program in differentiating cerebellar granule neurons.

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Wijayatunge, Ranjula; Liu, Fang; Shpargel, Karl B; Wayne, Nicole J; Chan, Urann; Boua, Jane-Valeriane; Magnuson, Terry; West, Anne E

2018-03-01

The histone H3 lysine 27 (H3K27) demethylase Kdm6b (Jmjd3) can promote cellular differentiation, however its physiological functions in neurons remain to be fully determined. We studied the expression and function of Kdm6b in differentiating granule neurons of the developing postnatal mouse cerebellum. At postnatal day 7, Kdm6b is expressed throughout the layers of the developing cerebellar cortex, but its expression is upregulated in newborn cerebellar granule neurons (CGNs). Atoh1-Cre mediated conditional knockout of Kdm6b in CGN precursors either alone or in combination with Kdm6a did not disturb the gross morphological development of the cerebellum. Furthermore, RNAi-mediated knockdown of Kdm6b in cultured CGN precursors did not alter the induced expression of early neuronal marker genes upon cell cycle exit. By contrast, knockdown of Kdm6b significantly impaired the induction of a mature neuronal gene expression program, which includes gene products required for functional synapse maturation. Loss of Kdm6b also impaired the ability of Brain-Derived Neurotrophic Factor (BDNF) to induce expression of Grin2c and Tiam1 in maturing CGNs. Taken together, these data reveal a previously unknown role for Kdm6b in the postmitotic stages of CGN maturation and suggest that Kdm6b may work, at least in part, by a transcriptional mechanism that promotes gene sensitivity to regulation by BDNF. Copyright © 2017 Elsevier Inc. All rights reserved.

Cocoa Enriched Diets Enhance Expression of Phosphatases and Decrease Expression of Inflammatory Molecules in Trigeminal Ganglion Neurons

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Cady, Ryan J.; Durham, Paul L.

2010-01-01

Activation of trigeminal nerves and release of neuropeptides that promote inflammation are implicated in the underlying pathology of migraine and temporomandibular joint (TMJ) disorders. The overall response of trigeminal nerves to peripheral inflammatory stimuli involves a balance between enzymes that promote inflammation, kinases, and those that restore homeostasis, phosphatases. The goal of this study was to determine the effects of a cocoa-enriched diet on the expression of key inflammatory proteins in trigeminal ganglion neurons under basal and inflammatory conditions. Rats were fed a control diet or an isocaloric diet enriched in cocoa for 14 days prior to an injection of noxious stimuli to cause acute or chronic excitation of trigeminal neurons. In animals fed a cocoa-enriched diet, basal levels of the mitogen-activated kinase (MAP) phosphatases MKP-1 and MKP-3 were elevated in neurons. Importantly, the stimulatory effects of acute or chronic peripheral inflammation on neuronal expression of the MAPK p38 and extracellular signal-regulated kinases (ERK) were significantly repressed in response to cocoa. Similarly, dietary cocoa significantly suppressed basal neuronal expression of calcitonin gene-related peptide (CGRP) as well as stimulated levels of the inducible form of nitric oxide synthase (iNOS), proteins implicated in the underlying pathology of migraine and TMJ disorders. To our knowledge, this is first evidence that a dietary supplement can cause upregulation of MKP, and that cocoa can prevent inflammatory responses in trigeminal ganglion neurons. Furthermore, our data provide evidence that cocoa contains biologically active compounds that would be beneficial in the treatment of migraine and TMJ disorders. PMID:20138852

Acute Cocaine Induces Fast Activation of D1 Receptor and Progressive Deactivation of D2 Receptor Strial Neurons: In Vivo Optical Microprobe [Ca2+]i Imaging

International Nuclear Information System (INIS)

Du, C.; Luo, Z.; Volkow, N.D.; Heintz, N.; Pan, Y.; Du, C.

2011-01-01

Cocaine induces fast dopamine increases in brain striatal regions, which are recognized to underlie its rewarding effects. Both dopamine D1 and D2 receptors are involved in cocaine's reward but the dynamic downstream consequences of cocaine effects in striatum are not fully understood. Here we used transgenic mice expressing EGFP under the control of either the D1 receptor (D1R) or the D2 receptor (D2R) gene and microprobe optical imaging to assess the dynamic changes in intracellular calcium ([Ca 2+ ] i ) responses (used as marker of neuronal activation) to acute cocaine in vivo separately for D1R- versus D2R-expressing neurons in striatum. Acute cocaine (8 mg/kg, i.p.) rapidly increased [Ca 2+ ] i in D1R-expressing neurons (10.6 ± 3.2%) in striatum within 8.3 ± 2.3 min after cocaine administration after which the increases plateaued; these fast [Ca 2+ ] i increases were blocked by pretreatment with a D1R antagonist (SCH23390). In contrast, cocaine induced progressive decreases in [Ca 2+ ] i in D2R-expressing neurons (10.4 ± 5.8%) continuously throughout the 30 min that followed cocaine administration; these slower [Ca 2+ ] i decreases were blocked by pretreatment with a D2R antagonist (raclopride). Since activation of striatal D1R-expressing neurons (direct-pathway) enhances cocaine reward, whereas activation of D2R expressing neurons suppresses it (indirect-pathway) (Lobo et al., 2010), this suggests that cocaine's rewarding effects entail both its fast stimulation ofD1R (resulting in abrupt activation of direct-pathway neurons) and a slower stimulation of D2R (resulting in longer-lasting deactivation of indirect-pathway neurons). We also provide direct in vivo evidence of D2R and D1R interactions in the striatal responses to acute cocaine administration.

Reduced Slc6a15 in Nucleus Accumbens D2-Neurons Underlies Stress Susceptibility.

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Chandra, Ramesh; Francis, T Chase; Nam, Hyungwoo; Riggs, Lace M; Engeln, Michel; Rudzinskas, Sarah; Konkalmatt, Prasad; Russo, Scott J; Turecki, Gustavo; Iniguez, Sergio D; Lobo, Mary Kay

2017-07-05

Previous research demonstrates that Slc6a15, a neutral amino acid transporter, is associated with depression susceptibility. However, no study examined Slc6a15 in the ventral striatum [nucleus accumbens (NAc)] in depression. Given our previous characterization of Slc6a15 as a striatal dopamine receptor 2 (D2)-neuron-enriched gene, we examined the role of Slc6a15 in NAc D2-neurons in mediating susceptibility to stress in male mice. First, we showed that Slc6a15 mRNA was reduced in NAc of mice susceptible to chronic social defeat stress (CSDS), a paradigm that produces behavioral and molecular adaptations that resemble clinical depression. Consistent with our preclinical data, we observed Slc6a15 mRNA reduction in NAc of individuals with major depressive disorder (MDD). The Slc6a15 reduction in NAc occurred selectively in D2-neurons. Next, we used Cre-inducible viruses combined with D2-Cre mice to reduce or overexpress Slc6a15 in NAc D2-neurons. Slc6a15 reduction in D2-neurons caused enhanced susceptibility to a subthreshold social defeat stress (SSDS) as observed by reduced social interaction, while a reduction in social interaction following CSDS was not observed when Slc6a15 expression in D2-neurons was restored. Finally, since both D2-medium spiny neurons (MSNs) and D2-expressing choline acetyltransferase (ChAT) interneurons express Slc6a15, we examined Slc6a15 protein in these interneurons after CSDS. Slc6a15 protein was unaltered in ChAT interneurons. Consistent with this, reducing Slc5a15 selectively in NAc D2-MSNs, using A2A-Cre mice that express Cre selectively in D2-MSNs, caused enhanced susceptibility to SSDS. Collectively, our data demonstrate that reduced Slc6a15 in NAc occurs in MDD individuals and that Slc6a15 reduction in NAc D2-neurons underlies stress susceptibility. SIGNIFICANCE STATEMENT Our study demonstrates a role for reduced Slc6a15, a neutral amino acid transporter, in nucleus accumbens (NAc) in depression and stress susceptibility. The

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability

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Speca, David J.; Ogata, Genki; Mandikian, Danielle; Bishop, Hannah I.; Wiler, Steve W.; Eum, Kenneth; Wenzel, H. Jürgen; Doisy, Emily T.; Matt, Lucas; Campi, Katharine L.; Golub, Mari S.; Nerbonne, Jeanne M.; Hell, Johannes W.; Trainor, Brian C.; Sack, Jon T.; Schwartzkroin, Philip A.; Trimmer, James S.

2014-01-01

The Kv2.1 delayed rectifier potassium channel exhibits high-level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity-dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1−/−) mice lacking this channel. Kv2.1−/− mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1−/− mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1−/− mice appears unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1−/− animals. Field recordings from hippocampal slices of Kv2.1−/− mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1−/− mice, long-term potentiation at the Schaffer collateral – CA1 synapse is decreased. Kv2.1−/− mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1−/− mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1−/− mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function. PMID:24494598

Spinal afferent neurons projecting to the rat lung and pleura express acid sensitive channels

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Kummer Wolfgang

2006-07-01

Full Text Available Abstract Background The acid sensitive ion channels TRPV1 (transient receptor potential vanilloid receptor-1 and ASIC3 (acid sensing ion channel-3 respond to tissue acidification in the range that occurs during painful conditions such as inflammation and ischemia. Here, we investigated to which extent they are expressed by rat dorsal root ganglion neurons projecting to lung and pleura, respectively. Methods The tracer DiI was either injected into the left lung or applied to the costal pleura. Retrogradely labelled dorsal root ganglion neurons were subjected to triple-labelling immunohistochemistry using antisera against TRPV1, ASIC3 and neurofilament 68 (marker for myelinated neurons, and their soma diameter was measured. Results Whereas 22% of pulmonary spinal afferents contained neither channel-immunoreactivity, at least one is expressed by 97% of pleural afferents. TRPV1+/ASIC3- neurons with probably slow conduction velocity (small soma, neurofilament 68-negative were significantly more frequent among pleural (35% than pulmonary afferents (20%. TRPV1+/ASIC3+ neurons amounted to 14 and 10% respectively. TRPV1-/ASIC3+ neurons made up between 44% (lung and 48% (pleura of neurons, and half of them presumably conducted in the A-fibre range (larger soma, neurofilament 68-positive. Conclusion Rat pleural and pulmonary spinal afferents express at least two different acid-sensitive channels that make them suitable to monitor tissue acidification. Patterns of co-expression and structural markers define neuronal subgroups that can be inferred to subserve different functions and may initiate specific reflex responses. The higher prevalence of TRPV1+/ASIC3- neurons among pleural afferents probably reflects the high sensitivity of the parietal pleura to painful stimuli.

Decreased adrenoceptor stimulation in heart failure rats reduces NGF expression by cardiac parasympathetic neurons.

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Hasan, Wohaib; Smith, Peter G

2014-04-01

Postganglionic cardiac parasympathetic and sympathetic nerves are physically proximate in atrial cardiac tissue allowing reciprocal inhibition of neurotransmitter release, depending on demands from central cardiovascular centers or reflex pathways. Parasympathetic cardiac ganglion (CG) neurons synthesize and release the sympathetic neurotrophin nerve growth factor (NGF), which may serve to maintain these close connections. In this study we investigated whether NGF synthesis by CG neurons is altered in heart failure, and whether norepinephrine from sympathetic neurons promotes NGF synthesis. NGF and proNGF immunoreactivity in CG neurons in heart failure rats following chronic coronary artery ligation was investigated. NGF immunoreactivity was decreased significantly in heart failure rats compared to sham-operated animals, whereas proNGF expression was unchanged. Changes in neurochemistry of CG neurons included attenuated expression of the cholinergic marker vesicular acetylcholine transporter, and increased expression of the neuropeptide vasoactive intestinal polypeptide. To further investigate norepinephrine's role in promoting NGF synthesis, we cultured CG neurons treated with adrenergic receptor (AR) agonists. An 82% increase in NGF mRNA levels was detected after 1h of isoproterenol (β-AR agonist) treatment, which increased an additional 22% at 24h. Antagonist treatment blocked isoproterenol-induced increases in NGF transcripts. In contrast, the α-AR agonist phenylephrine did not alter NGF mRNA expression. These results are consistent with β-AR mediated maintenance of NGF synthesis in CG neurons. In heart failure, a decrease in NGF synthesis by CG neurons may potentially contribute to reduced connections with adjacent sympathetic nerves. Copyright © 2013 Elsevier B.V. All rights reserved.

Correlating Anatomy and Function with Gene Expression in Individual Neurons by Combining in Vivo Labeling, Patch Clamp, and Single Cell RNA-seq

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Carsten K. Pfeffer

2017-11-01

Full Text Available The classification of neurons into distinct types is an ongoing effort aimed at revealing and understanding the diversity of the components of the nervous system. Recently available methods allow us to determine the gene expression pattern of individual neurons in the mammalian cerebral cortex to generate powerful categorization schemes. For a thorough understanding of neuronal diversity such genetic categorization schemes need to be combined with traditional classification parameters like position, axonal projection or response properties to sensory stimulation. Here we describe a method to link the gene expression of individual neurons with their position, axonal projection, or sensory response properties. Neurons are labeled in vivo based on their anatomical or functional properties and, using patch clamp pipettes, their RNA individually harvested in vitro for RNAseq. We validate the methodology using multiple established molecularly and anatomically distinct cell populations and explore molecular differences between uncharacterized neurons in mouse visual cortex. Gene expression patterns between L5 neurons projecting to frontal or contralateral cortex are distinct while L2 neurons differing in position, projection, or function are molecularly similar. With this method we can determine the genetic expression pattern of functionally and anatomically identified individual neurons.

In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

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Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

2010-07-01

This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

Lung inflammation induces IL-1β expression in hypoglossal neurons in rat brainstem

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Jafri, Anjum; Belkadi, Abdelmadjid; Zaidi, Syed I. A.; Getsy, Paulina; Wilson, Christopher G.; Martin, Richard J.

2013-01-01

Perinatal inflammation is associated with respiratory morbidity. Immune modulation of brainstem respiratory control centers may provide a link for this pathobiology. We exposed 11-day old rats to intratracheal lipopolysaccharide (LPS, 0.5 µg/g) to test the hypothesis that intrapulmonary inflammation increases expression of the proinflammatory cytokine IL-1β within respiratory-related brainstem regions. Intratracheal LPS resulted in a 32% increase in IL-1β protein expression in the medulla oblongata. In situ hybridization showed increased intensity of IL-1β mRNA but no change in neuronal numbers. Co-localization experiments showed that hypoglossal neurons express IL-1β mRNA and immunostaining showed a 43% increase in IL-1β protein-expressing cells after LPS exposure. LPS treatment also significantly increased microglial cell numbers though they did not express IL-1β mRNA. LPS-induced brainstem expression of neuronal IL-1β mRNA and protein may have implications for our understanding of the vulnerability of neonatal respiratory control in response to a peripheral pro-inflammatory stimulus. PMID:23648475

Macroglia-derived thrombospondin 2 regulates alterations of presynaptic proteins of retinal neurons following elevated hydrostatic pressure.

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Wang, Shuchao; Hu, Tu; Wang, Zhen; Li, Na; Zhou, Lihong; Liao, Lvshuang; Wang, Mi; Liao, Libin; Wang, Hui; Zeng, Leping; Fan, Chunling; Zhou, Hongkang; Xiong, Kun; Huang, Jufang; Chen, Dan

2017-01-01

Many studies on retinal injury and repair following elevated intraocular pressure suggest that the survival ratio of retinal neurons has been improved by various measures. However, the visual function recovery is far lower than expected. The homeostasis of retinal synapses in the visual signal pathway is the key structural basis for the delivery of visual signals. Our previous studies found that complicated changes in the synaptic structure between retinal neurons occurred much earlier than obvious degeneration of retinal ganglion cells in rat retinae. The lack of consideration of these earlier retinal synaptic changes in the rescue strategy may be partly responsible for the limited visual function recovery with the types of protective methods for retinal neurons used following elevated intraocular pressure. Thus, research on the modulatory mechanisms of the synaptic changes after elevated intraocular pressure injury may give new light to visual function rescue. In this study, we found that thrombospondin 2, an important regulator of synaptogenesis in central nervous system development, was distributed in retinal macroglia cells, and its receptor α2δ-1 was in retinal neurons. Cell cultures including mixed retinal macroglia cells/neuron cultures and retinal neuron cultures were exposed to elevated hydrostatic pressure for 2 h. The expression levels of glial fibrillary acidic protein (the marker of activated macroglia cells), thrombospondin 2, α2δ-1 and presynaptic proteins were increased following elevated hydrostatic pressure in mixed cultures, but the expression levels of postsynaptic proteins were not changed. SiRNA targeting thrombospondin 2 could decrease the upregulation of presynaptic proteins induced by the elevated hydrostatic pressure. However, in retinal neuron cultures, elevated hydrostatic pressure did not affect the expression of presynaptic or postsynaptic proteins. Rather, the retinal neuron cultures with added recombinant thrombospondin 2

Amygdala EphB2 Signaling Regulates Glutamatergic Neuron Maturation and Innate Fear.

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

2016-09-28

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

KCC2a expression in a human fetal lens epithelial cell line.

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Lauf, Peter K; Di Fulvio, Mauricio; Srivastava, Vinita; Sharma, Neelima; Adragna, Norma C

2012-01-01

The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin. Copyright © 2012 S. Karger AG, Basel.

Parallel expression of synaptophysin and evoked neurotransmitter release during development of cultured neurons

DEFF Research Database (Denmark)

Ehrhart-Bornstein, M; Treiman, M; Hansen, Gert Helge

1991-01-01

Primary cultures of GABAergic cerebral cortex neurons and glutamatergic cerebellar granule cells were used to study the expression of synaptophysin, a synaptic vesicle marker protein, along with the ability of each cell type to release neurotransmitter upon stimulation. The synaptophysin expression...... by quantitative immunoblotting and light microscope immunocytochemistry, respectively. In both cell types, a close parallelism was found between the temporal pattern of development in synaptophysin expression and neurotransmitter release. This temporal pattern differed between the two types of neurons....... The cerebral cortex neurons showed a biphasic time course of increase in synaptophysin content, paralleled by a biphasic pattern of development in their ability to release [3H]GABA in response to depolarization by glutamate or elevated K+ concentrations. In contrast, a monophasic, approximately linear increase...

Transcriptional Profiling of Cholinergic Neurons From Basal Forebrain Identifies Changes in Expression of Genes Between Sleep and Wake.

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Nikonova, Elena V; Gilliland, Jason DA; Tanis, Keith Q; Podtelezhnikov, Alexei A; Rigby, Alison M; Galante, Raymond J; Finney, Eva M; Stone, David J; Renger, John J; Pack, Allan I; Winrow, Christopher J

2017-06-01

To assess differences in gene expression in cholinergic basal forebrain cells between sleeping and sleep-deprived mice sacrificed at the same time of day. Tg(ChAT-eGFP)86Gsat mice expressing enhanced green fluorescent protein (eGFP) under control of the choline acetyltransferase (Chat) promoter were utilized to guide laser capture of cholinergic cells in basal forebrain. Messenger RNA expression levels in these cells were profiled using microarrays. Gene expression in eGFP(+) neurons was compared (1) to that in eGFP(-) neurons and to adjacent white matter, (2) between 7:00 am (lights on) and 7:00 pm (lights off), (3) between sleep-deprived and sleeping animals at 0, 3, 6, and 9 hours from lights on. There was a marked enrichment of ChAT and other markers of cholinergic neurons in eGFP(+) cells. Comparison of gene expression in these eGFP(+) neurons between 7:00 am and 7:00 pm revealed expected differences in the expression of clock genes (Arntl2, Per1, Per2, Dbp, Nr1d1) as well as mGluR3. Comparison of expression between spontaneous sleep and sleep-deprived groups sacrificed at the same time of day revealed a number of transcripts (n = 55) that had higher expression in sleep deprivation compared to sleep. Genes upregulated in sleep deprivation predominantly were from the protein folding pathway (25 transcripts, including chaperones). Among 42 transcripts upregulated in sleep was the cold-inducible RNA-binding protein. Cholinergic cell signatures were characterized. Whether the identified genes are changing as a consequence of differences in behavioral state or as part of the molecular regulatory mechanism remains to be determined. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

Efficient induction of dopaminergic neuron differentiation from induced pluripotent stem cells reveals impaired mitophagy in PARK2 neurons.

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Suzuki, Sadafumi; Akamatsu, Wado; Kisa, Fumihiko; Sone, Takefumi; Ishikawa, Kei-Ichi; Kuzumaki, Naoko; Katayama, Hiroyuki; Miyawaki, Atsushi; Hattori, Nobutaka; Okano, Hideyuki

2017-01-29

Patient-specific induced pluripotent stem cells (iPSCs) show promise for use as tools for in vitro modeling of Parkinson's disease. We sought to improve the efficiency of dopaminergic (DA) neuron induction from iPSCs by the using surface markers expressed in DA progenitors to increase the significance of the phenotypic analysis. By sorting for a CD184 high /CD44 - fraction during neural differentiation, we obtained a population of cells that were enriched in DA neuron precursor cells and achieved higher differentiation efficiencies than those obtained through the same protocol without sorting. This high efficiency method of DA neuronal induction enabled reliable detection of reactive oxygen species (ROS) accumulation and vulnerable phenotypes in PARK2 iPSCs-derived DA neurons. We additionally established a quantitative system using the mt-mKeima reporter system to monitor mitophagy in which mitochondria fuse with lysosomes and, by combining this system with the method of DA neuronal induction described above, determined that mitophagy is impaired in PARK2 neurons. These findings suggest that the efficiency of DA neuron induction is important for the precise detection of cellular phenotypes in modeling Parkinson's disease. Copyright © 2016. Published by Elsevier Inc.

A regulatory code for neuron-specific odor receptor expression.

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Anandasankar Ray

2008-05-01

Full Text Available Olfactory receptor neurons (ORNs must select-from a large repertoire-which odor receptors to express. In Drosophila, most ORNs express one of 60 Or genes, and most Or genes are expressed in a single ORN class in a process that produces a stereotyped receptor-to-neuron map. The construction of this map poses a problem of receptor gene regulation that is remarkable in its dimension and about which little is known. By using a phylogenetic approach and the genome sequences of 12 Drosophila species, we systematically identified regulatory elements that are evolutionarily conserved and specific for individual Or genes of the maxillary palp. Genetic analysis of these elements supports a model in which each receptor gene contains a zip code, consisting of elements that act positively to promote expression in a subset of ORN classes, and elements that restrict expression to a single ORN class. We identified a transcription factor, Scalloped, that mediates repression. Some elements are used in other chemosensory organs, and some are conserved upstream of axon-guidance genes. Surprisingly, the odor response spectra and organization of maxillary palp ORNs have been extremely well-conserved for tens of millions of years, even though the amino acid sequences of the receptors are not highly conserved. These results, taken together, define the logic by which individual ORNs in the maxillary palp select which odor receptors to express.

Differentiation of Spermatogonia Stem Cells into Functional Mature Neurons Characterized with Differential Gene Expression.

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Bojnordi, Maryam Nazm; Azizi, Hossein; Skutella, Thomas; Movahedin, Mansoureh; Pourabdolhossein, Fereshteh; Shojaei, Amir; Hamidabadi, Hatef Ghasemi

2017-09-01

Transplantation of embryonic stem cells (ESCs) is a promising therapeutic approach for the treatment of neurodegenerative diseases. However, ESCs are not usable clinically due to immunological and ethical limitations. The identification of an alternative safe cell source opens novel options via autologous transplantation in neuro-regeneration circumventing these problems. Here, we examined the neurogenic capacity of embryonic stem-like cells (ES-like cells) derived from the testis using neural growth factor inducers and utilized them to generate functional mature neurons. The neuronal differentiation of ES-like cells is induced in three stages. Stage 1 is related to embryoid body (EB) formation. To induce neuroprogenitor cells, EBs were cultured in the presence of retinoic acid, N 2 supplement and fibroblast growth factor followed by culturing in a neurobasal medium containing B 27 , N 2 supplements for additional 10 days, to allow the maturation and development of neuronal progenitor cells. The neurogenic differentiation was confirmed by immunostaining for markers of mature neurons. The differentiated neurons were positive for Tuj1 and Tau1. Real-time PCR dates indicated the expression of Nestin and Neuro D (neuroprogenitor markers) in induced cells at the second stage of the differentiation protocol. The differentiated mature neurons exhibited the specific neuron markers Map2 and β-tubulin. The functional maturity of neurons was confirmed by an electrophysiological analysis of passive and active neural membrane properties. These findings indicated a differentiation capacity of ES-like cells derived from the testis to functionally mature neurons, which proposes them as a novel cell source for neuroregenerative medicine.

Laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury.

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Boone, Deborah R; Sell, Stacy L; Hellmich, Helen Lee

2013-04-10

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function. Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al., has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types. We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 °C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving

Comparison of gene expression profile in embryonic mesencephalon and neuronal primary cultures.

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Dario Greco

Full Text Available In the mammalian central nervous system (CNS an important contingent of dopaminergic neurons are localized in the substantia nigra and in the ventral tegmental area of the ventral midbrain. They constitute an anatomically and functionally heterogeneous group of cells involved in a variety of regulatory mechanisms, from locomotion to emotional/motivational behavior. Midbrain dopaminergic neuron (mDA primary cultures represent a useful tool to study molecular mechanisms involved in their development and maintenance. Considerable information has been gathered on the mDA neurons development and maturation in vivo, as well as on the molecular features of mDA primary cultures. Here we investigated in detail the gene expression differences between the tissue of origin and ventral midbrain primary cultures enriched in mDA neurons, using microarray technique. We integrated the results based on different re-annotations of the microarray probes. By using knowledge-based gene network techniques and promoter sequence analysis, we also uncovered mechanisms that might regulate the expression of CNS genes involved in the definition of the identity of specific cell types in the ventral midbrain. We integrate bioinformatics and functional genomics, together with developmental neurobiology. Moreover, we propose guidelines for the computational analysis of microarray gene expression data. Our findings help to clarify some molecular aspects of the development and differentiation of DA neurons within the midbrain.

DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain

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Katherine E. Horn

2013-01-01

Full Text Available The transmembrane protein deleted in colorectal cancer (DCC and its ligand, netrin-1, regulate synaptogenesis during development, but their function in the mature central nervous system is unknown. Given that DCC promotes cell-cell adhesion, is expressed by neurons, and activates proteins that signal at synapses, we hypothesized that DCC expression by neurons regulates synaptic function and plasticity in the adult brain. We report that DCC is enriched in dendritic spines of pyramidal neurons in wild-type mice, and we demonstrate that selective deletion of DCC from neurons in the adult forebrain results in the loss of long-term potentiation (LTP, intact long-term depression, shorter dendritic spines, and impaired spatial and recognition memory. LTP induction requires Src activation of NMDA receptor (NMDAR function. DCC deletion severely reduced Src activation. We demonstrate that enhancing NMDAR function or activating Src rescues LTP in the absence of DCC. We conclude that DCC activation of Src is required for NMDAR-dependent LTP and certain forms of learning and memory.

Reduced sensory synaptic excitation impairs motor neuron function via Kv2.1 in spinal muscular atrophy.

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Fletcher, Emily V; Simon, Christian M; Pagiazitis, John G; Chalif, Joshua I; Vukojicic, Aleksandra; Drobac, Estelle; Wang, Xiaojian; Mentis, George Z

2017-07-01

Behavioral deficits in neurodegenerative diseases are often attributed to the selective dysfunction of vulnerable neurons via cell-autonomous mechanisms. Although vulnerable neurons are embedded in neuronal circuits, the contributions of their synaptic partners to disease process are largely unknown. Here we show that, in a mouse model of spinal muscular atrophy (SMA), a reduction in proprioceptive synaptic drive leads to motor neuron dysfunction and motor behavior impairments. In SMA mice or after the blockade of proprioceptive synaptic transmission, we observed a decrease in the motor neuron firing that could be explained by the reduction in the expression of the potassium channel Kv2.1 at the surface of motor neurons. Chronically increasing neuronal activity pharmacologically in vivo led to a normalization of Kv2.1 expression and an improvement in motor function. Our results demonstrate a key role of excitatory synaptic drive in shaping the function of motor neurons during development and the contribution of its disruption to a neurodegenerative disease.

Cultured subventricular zone progenitor cells transduced with neurogenin-2 become mature glutamatergic neurons and integrate into the dentate gyrus.

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Xia Chen

Full Text Available We have previously shown that transplantation of immature DCX+/NeuN+/Prox1+ neurons (found in the neonatal DG, but not undifferentiated neuronal progenitor cells (NPCs from ventral subventricular zone (SVZ, results in neuronal maturation in vivo within the dentate niche. Here we investigated whether we could enhance the integration of SVZ NPCs by forced expression of the proneural gene Neurogenin 2 (NEUROG2. NPCs cultured from neonatal GFP-transgenic rat SVZ for 7 days in a non-differentiating medium were transduced with a retrovirus encoding NEUROG2 and DsRed or the DsRed reporter gene alone (control. By 3 days post-transduction, the NEUROG2-transduced cells maintained in culture contained mostly immature neurons (91% DCX+; 76% NeuN+, whereas the control virus-transduced cells remained largely undifferentiated (30% DCX+; <1% NeuN+. At 6 weeks following transplantation into the DG of adult male rats, there were no neurons among the transplanted cells treated with the control virus but the majority of the NEUROG2-transduced DsRed+ SVZ cells became mature neurons (92% NeuN+; DCX-negative. Although the NEUROG2-transduced SVZ cells did not express the dentate granule neuron marker Prox1, most of the NEUROG2-transduced SVZ cells (78% expressed the glutamatergic marker Tbr1, suggesting the acquisition of a glutamatergic phenotype. Moreover, some neurons extended dendrites into the molecular layer, grew axons containing Ankyrin G+ axonal initial segments, and projected into the CA3 region, thus resembling mature DG granule neurons. A proportion of NEUROG2 transduced cells also expressed c-Fos and P-CREB, two markers of neuronal activation. We conclude that NEUROG2-transduction is sufficient to promote neuronal maturation and integration of transplanted NPCs from SVZ into the DG.

Glut2-dependent glucose-sensing controls thermoregulation by enhancing the leptin sensitivity of NPY and POMC neurons.

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Mounien, Lourdes; Marty, Nell; Tarussio, David; Metref, Salima; Genoux, David; Preitner, Frédéric; Foretz, Marc; Thorens, Bernard

2010-06-01

The physiological contribution of glucose in thermoregulation is not completely established nor whether this control may involve a regulation of the melanocortin pathway. Here, we assessed thermoregulation and leptin sensitivity of hypothalamic arcuate neurons in mice with inactivation of glucose transporter type 2 (Glut2)-dependent glucose sensing. Mice with inactivation of Glut2-dependent glucose sensors are cold intolerant and show increased susceptibility to food deprivation-induced torpor and abnormal hypothermic response to intracerebroventricular administration of 2-deoxy-d-glucose compared to control mice. This is associated with a defect in regulated expression of brown adipose tissue uncoupling protein I and iodothyronine deiodinase II and with a decreased leptin sensitivity of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons, as observed during the unfed-to-refed transition or following i.p. leptin injection. Sites of central Glut-2 expression were identified by a genetic tagging approach and revealed that glucose-sensitive neurons were present in the lateral hypothalamus, the dorsal vagal complex, and the basal medulla but not in the arcuate nucleus. NPY and POMC neurons were, however, connected to nerve terminals from Glut2-expressing neurons. Thus, our data suggest that glucose controls thermoregulation and the leptin sensitivity of NPY and POMC neurons through activation of Glut2-dependent glucose-sensing neurons located outside of the arcuate nucleus.

Activation of Six1 Expression in Vertebrate Sensory Neurons.

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Shigeru Sato

Full Text Available SIX1 homeodomain protein is one of the essential key regulators of sensory organ development. Six1-deficient mice lack the olfactory epithelium, vomeronasal organs, cochlea, vestibule and vestibuloacoustic ganglion, and also show poor neural differentiation in the distal part of the cranial ganglia. Simultaneous loss of both Six1 and Six4 leads to additional abnormalities such as small trigeminal ganglion and abnormal dorsal root ganglia (DRG. The aim of this study was to understand the molecular mechanism that controls Six1 expression in sensory organs, particularly in the trigeminal ganglion and DRG. To this end, we focused on the sensory ganglia-specific Six1 enhancer (Six1-8 conserved between chick and mouse. In vivo reporter assays using both animals identified an important core region comprising binding consensus sequences for several transcription factors including nuclear hormone receptors, TCF/LEF, SMAD, POU homeodomain and basic-helix-loop-helix proteins. The results provided information on upstream factors and signals potentially relevant to Six1 regulation in sensory neurons. We also report the establishment of a new transgenic mouse line (mSix1-8-NLSCre that expresses Cre recombinase under the control of mouse Six1-8. Cre-mediated recombination was detected specifically in ISL1/2-positive sensory neurons of Six1-positive cranial sensory ganglia and DRG. The unique features of the mSix1-8-NLSCre line are the absence of Cre-mediated recombination in SOX10-positive glial cells and central nervous system and ability to induce recombination in a subset of neurons derived from the olfactory placode/epithelium. This mouse model can be potentially used to advance research on sensory development.

Heart failure-induced changes of voltage-gated Ca2+ channels and cell excitability in rat cardiac postganglionic neurons.

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Tu, Huiyin; Liu, Jinxu; Zhang, Dongze; Zheng, Hong; Patel, Kaushik P; Cornish, Kurtis G; Wang, Wei-Zhong; Muelleman, Robert L; Li, Yu-Long

2014-01-15

Chronic heart failure (CHF) is characterized by decreased cardiac parasympathetic and increased cardiac sympathetic nerve activity. This autonomic imbalance increases the risk of arrhythmias and sudden death in patients with CHF. We hypothesized that the molecular and cellular alterations of cardiac postganglionic parasympathetic (CPP) neurons located in the intracardiac ganglia and sympathetic (CPS) neurons located in the stellate ganglia (SG) possibly link to the cardiac autonomic imbalance in CHF. Rat CHF was induced by left coronary artery ligation. Single-cell real-time PCR and immunofluorescent data showed that L (Ca(v)1.2 and Ca(v)1.3), P/Q (Ca(v)2.1), N (Ca(v)2.2), and R (Ca(v)2.3) types of Ca2+ channels were expressed in CPP and CPS neurons, but CHF decreased the mRNA and protein expression of only the N-type Ca2+ channels in CPP neurons, and it did not affect mRNA and protein expression of all Ca2+ channel subtypes in the CPS neurons. Patch-clamp recording confirmed that CHF reduced N-type Ca2+ currents and cell excitability in the CPP neurons and enhanced N-type Ca2+ currents and cell excitability in the CPS neurons. N-type Ca2+ channel blocker (1 μM ω-conotoxin GVIA) lowered Ca2+ currents and cell excitability in the CPP and CPS neurons from sham-operated and CHF rats. These results suggest that CHF reduces the N-type Ca2+ channel currents and cell excitability in the CPP neurons and enhances the N-type Ca2+ currents and cell excitability in the CPS neurons, which may contribute to the cardiac autonomic imbalance in CHF.

Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

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Rehn, Matthias; Bader, Sandra; Bell, Anna; Diener, Martin

2013-09-01

We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped

Canonical TGF-β Signaling Negatively Regulates Neuronal Morphogenesis through TGIF/Smad Complex-Mediated CRMP2 Suppression.

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Nakashima, Hideyuki; Tsujimura, Keita; Irie, Koichiro; Ishizu, Masataka; Pan, Miao; Kameda, Tomonori; Nakashima, Kinichi

2018-05-16

Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-β (TGF-β) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-β family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-β family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-β receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-β signaling is involved in the pathogenesis of neurodevelopmental diseases. SIGNIFICANCE STATEMENT Canonical transforming growth factor-β (TGF-β) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-β signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-β signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our

Exposure to an open-field arena increases c-Fos expression in a subpopulation of neurons in the dorsal raphe nucleus, including neurons projecting to the basolateral amygdaloid complex

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Hale, M.W.; Hay-Schmidt, A.; Mikkelsen, J.D.

2008-01-01

Serotonergic systems in the dorsal raphe nucleus are thought to play an important role in the regulation of anxiety states. To investigate responses of neurons in the dorsal raphe nucleus to a mild anxiety-related stimulus, we exposed rats to an open-field, under low-light or high-light conditions....... Treatment effects on c-Fos expression in serotonergic and non-serotonergic cells in the midbrain raphe nuclei were determined 2 h following open-field exposure or home cage control (CO) conditions. Rats tested under both light conditions responded with increases in c-Fos expression in serotonergic neurons...... within subdivisions of the midbrain raphe nuclei compared with CO rats. However, the total numbers of serotonergic neurons involved were small suggesting that exposure to the open-field may affect a subpopulation of serotonergic neurons. To determine if exposure to the open-field activates a subset...

Neuronal type-specific gene expression profiling and laser-capture microdissection.

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Pietersen, Charmaine Y; Lim, Maribel P; Macey, Laurel; Woo, Tsung-Ung W; Sonntag, Kai C

2011-01-01

The human brain is an exceptionally heterogeneous structure. In order to gain insight into the neurobiological basis of neural circuit disturbances in various neurologic or psychiatric diseases, it is often important to define the molecular cascades that are associated with these disturbances in a neuronal type-specific manner. This can be achieved by the use of laser microdissection, in combination with molecular techniques such as gene expression profiling. To identify neurons in human postmortem brain tissue, one can use the inherent properties of the neuron, such as pigmentation and morphology or its structural composition through immunohistochemistry (IHC). Here, we describe the isolation of homogeneous neuronal cells and high-quality RNA from human postmortem brain material using a combination of rapid IHC, Nissl staining, or simple morphology with Laser-Capture Microdissection (LCM) or Laser Microdissection (LMD).

Morphine Preconditioning Downregulates MicroRNA-134 Expression Against Oxygen-Glucose Deprivation Injuries in Cultured Neurons of Mice.

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Meng, Fanjun; Li, Yan; Chi, Wenying; Li, Junfa

2016-07-01

Brain protection by narcotics such as morphine is clinically relevant due to the extensive use of narcotics in the perioperative period. Morphine preconditioning induces neuroprotection in neurons, but it remains uncertain whether microRNA-134 (miR-134) is involved in morphine preconditioning against oxygen-glucose deprivation-induced injuries in primary cortical neurons of mice. The present study examined this issue. After cortical neurons of mice were cultured in vitro for 6 days, the neurons were transfected by respective virus vector, such as lentiviral vector (LV)-miR-control-GFP, LV-pre-miR-134-GFP, LV-pre-miR-134-inhibitor-GFP for 24 hours; after being normally cultured for 3 days again, morphine preconditioning was performed by incubating the transfected primary neurons with morphine (3 μM) for 1 hour, and then neuronal cells were exposed to oxygen-glucose deprivation (OGD) for 1 hour and oxygen-glucose recovery for 12 hours. The neuronal cells survival rate and the amount of apoptotic neurons were determined by MTT assay or TUNEL staining at designated time; and the expression levels of miR-134 were detected using real-time reverse transcription polymerase chain reaction at the same time. The neuronal cell survival rate was significantly higher, and the amount of apoptotic neurons was significantly decreased in neurons preconditioned with morphine before OGD than that of OGD alone. The neuroprotection induced by morphine preconditioning was partially blocked by upregulating miR-134 expression, and was enhanced by downregulating miR-134 expression. The expression of miR-134 was significantly decreased in morphine-preconditioned neurons alone without transfection. By downregulating miR-134 expression, morphine preconditioning protects primary cortical neurons of mice against injuries induced by OGD.

DL-2-amino-3-phosphonopropionic acid protects primary neurons from oxygen-glucose deprivation induced injury.

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Cui, Di; Xu, Jun; Xu, Quanyi; Zuo, Guokun

2017-02-21

Cerebral infarction is a type of ischemic stroke and is one of the main causes of irreversible brain damage. Although multiple neuroprotective agents have been investigated recently, the potential of DL-2-amino-3-phosphonopropionic acid (DL-AP3) in treating oxygen-glucose deprivation (OGD)-induced neuronal injury, has not been clarified yet. This study was aimed to explore the role of DL-AP3 in primary neuronal cell cultures. Primary neurons were divided into four groups: (1) a control group that was not treated; (2) DL-AP3 group treated with 10 μM of DL-AP3; (3) OGD group, in which neurons were cultured under OGD conditions; and (4) OGD + DL-AP3 group, in which OGD model was first established and then the cells were treated with 10 μM of DL-AP3. Neuronal viability and apoptosis were measured using Cell Counting Kit-8 and flow cytometry. Expressions of phospho-Akt1 (p-Akt1) and cytochrome c were detected using Western blot. The results showed that DL-AP3 did not affect neuronal viability and apoptosis in DL-AP3 group, nor it changed p-Akt1 and cytochrome c expression (p > 0.05). In OGD + DL-AP3 group, DL-AP3 significantly attenuated the inhibitory effects of OGD on neuronal viability (p neurons from OGD-induced injury by affecting the viability and apoptosis of neurons, and by regulating the expressions of p-Akt1 and cytochrome c.

Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity

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Parras, Carlos M.; Schuurmans, Carol; Scardigli, Raffaella; Kim, Jaesang; Anderson, David J.; Guillemot, François

2002-01-01

The neural bHLH genes Mash1 and Ngn2 are expressed in complementary populations of neural progenitors in the central and peripheral nervous systems. Here, we have systematically compared the activities of the two genes during neural development by generating replacement mutations in mice in which the coding sequences of Mash1 and Ngn2 were swapped. Using this approach, we demonstrate that Mash1 has the capacity to respecify the identity of neuronal populations normally derived from Ngn2-expressing progenitors in the dorsal telencephalon and ventral spinal cord. In contrast, misexpression of Ngn2 in Mash1-expressing progenitors does not result in any overt change in neuronal phenotype. Taken together, these results demonstrate that Mash1 and Ngn2 have divergent functions in specification of neuronal subtype identity, with Mash1 having the characteristics of an instructive determinant whereas Ngn2 functions as a permissive factor that must act in combination with other factors to specify neuronal phenotypes. Moreover, the ectopic expression of Ngn2 can rescue the neurogenesis defects of Mash1 null mutants in the ventral telencephalon and sympathetic ganglia but not in the ventral spinal cord and the locus coeruleus, indicating that Mash1 contribution to the specification of neuronal fates varies greatly in different lineages, presumably depending on the presence of other determinants of neuronal identity. PMID:11825874

Connective tissue growth factor (CTGF/CCN2 is negatively regulated during neuron-glioblastoma interaction.

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Luciana F Romão

Full Text Available Connective-tissue growth factor (CTGF/CCN2 is a matricellular-secreted protein involved in complex processes such as wound healing, angiogenesis, fibrosis and metastasis, in the regulation of cell proliferation, migration and extracellular matrix remodeling. Glioblastoma (GBM is the major malignant primary brain tumor and its adaptation to the central nervous system microenvironment requires the production and remodeling of the extracellular matrix. Previously, we published an in vitro approach to test if neurons can influence the expression of the GBM extracellular matrix. We demonstrated that neurons remodeled glioma cell laminin. The present study shows that neurons are also able to modulate CTGF expression in GBM. CTGF immnoreactivity and mRNA levels in GBM cells are dramatically decreased when these cells are co-cultured with neonatal neurons. As proof of particular neuron effects, neonatal neurons co-cultured onto GBM cells also inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the transcription level. This inhibition seems to be contact-mediated, since conditioned media from embryonic or neonatal neurons do not affect CTGF expression in GBM cells. Furthermore, the inhibition of CTGF expression in GBM/neuronal co-cultures seems to affect the two main signaling pathways related to CTGF. We observed inhibition of TGFβ luciferase reporter assay; however phopho-SMAD2 levels did not change in these co-cultures. In addition levels of phospho-p44/42 MAPK were decreased in co-cultured GBM cells. Finally, in transwell migration assay, CTGF siRNA transfected GBM cells or GBM cells co-cultured with neurons showed a decrease in the migration rate compared to controls. Previous data regarding laminin and these results demonstrating that CTGF is down-regulated in GBM cells co-cultured with neonatal neurons points out an interesting view in the understanding of the tumor and cerebral microenvironment

Proliferative hypothalamic neurospheres express NPY, AGRP, POMC, CART and Orexin-A and differentiate to functional neurons.

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Lígia Sousa-Ferreira

Full Text Available Some pathological conditions with feeding pattern alterations, including obesity and Huntington disease (HD are associated with hypothalamic dysfunction and neuronal cell death. Additionally, the hypothalamus is a neurogenic region with the constitutive capacity to generate new cells of neuronal lineage, in adult rodents. The aim of the present work was to evaluate the expression of feeding-related neuropeptides in hypothalamic progenitor cells and their capacity to differentiate to functional neurons which have been described to be affected by hypothalamic dysfunction. Our study shows that hypothalamic progenitor cells from rat embryos grow as floating neurospheres and express the feeding-related neuropeptides Neuropeptide Y (NPY, Agouti-related Protein (AGRP, Pro-OpioMelanocortin (POMC, Cocaine-and-Amphetamine Responsive Transcript (CART and Orexin-A/Hypocretin-1. Moreover the relative mRNA expression of NPY and POMC increases during the expansion of hypothalamic neurospheres in proliferative conditions.Mature neurons were obtained from the differentiation of hypothalamic progenitor cells including NPY, AGRP, POMC, CART and Orexin-A positive neurons. Furthermore the relative mRNA expression of NPY, CART and Orexin-A increases after the differentiation of hypothalamic neurospheres. Similarly to the adult hypothalamic neurons the neurospheres-derived neurons express the glutamate transporter EAAT3. The orexigenic and anorexigenic phenotype of these neurons was identified by functional response to ghrelin and leptin hormones, respectively. This work demonstrates the presence of appetite-related neuropeptides in hypothalamic progenitor cells and neurons obtained from the differentiation of hypothalamic neurospheres, including the neuronal phenotypes that have been described by others as being affected by hypothalamic neurodegeneration. These in vitro models can be used to study hypothalamic progenitor cells aiming a therapeutic intervention to

Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation.

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Zheng, Xinde; Boyer, Leah; Jin, Mingji; Mertens, Jerome; Kim, Yongsung; Ma, Li; Ma, Li; Hamm, Michael; Gage, Fred H; Hunter, Tony

2016-06-10

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size.

The cell-specific pattern of cholecystokinin peptides in endocrine cells versus neurons is governed by the expression of prohormone convertases 1/3, 2, and 5/6

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Bundgaard, J.R.; Hannibal, J.; Zhu, X.

2008-01-01

Most peptide hormone genes are, in addition to endocrine cells, also expressed in neurons. The peptide hormone cholecystokinin (CCK) is expressed in different molecular forms in cerebral neurons and intestinal endocrine cells. To understand this difference, we examined the roles of the neuroendoc...

Distribution of the P2X2 receptor and chemical coding in ileal enteric neurons of obese male mice (ob/ob)

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Mizuno, Márcia Sanae; Crisma, Amanda Rabello; Borelli, Primavera; Schäfer, Bárbara Tavares; Silveira, Mariana Póvoa; Castelucci, Patricia

2014-01-01

AIM: To investigate the colocalization, density and profile of neuronal areas of enteric neurons in the ileum of male obese mice. METHODS: The small intestinal samples of male mice in an obese group (OG) (C57BL/6J ob/ob) and a control group (CG) (+/+) were used. The tissues were analyzed using a double immunostaining technique for immunoreactivity (ir) of the P2X2 receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT) and calretinin (Calr). Also, we investigated the density and profile of neuronal areas of the NOS-, ChAT- and Calr-ir neurons in the myenteric plexus. Myenteric neurons were labeled using an NADH-diaphorase histochemical staining method. RESULTS: The analysis demonstrated that the P2X2 receptor was expressed in the cytoplasm and in the nuclear and cytoplasmic membranes only in the CG. Neuronal density values (neuron/cm2) decreased 31% (CG: 6579 ± 837; OG: 4556 ± 407) and 16.5% (CG: 7796 ± 528; OG: 6513 ± 610) in the NOS-ir and calretinin-ir neurons in the OG, respectively (P < 0.05). Density of ChAT-ir (CG: 6200 ± 310; OG: 8125 ± 749) neurons significantly increased 31% in the OG (P < 0.05). Neuron size studies demonstrated that NOS, ChAT, and Calr-ir neurons did not differ significantly between the CG and OG groups. The examination of NADH-diaphorase-positive myenteric neurons revealed an overall similarity between the OG and CG. CONCLUSION: Obesity may exert its effects by promoting a decrease in P2X2 receptor expression and modifications in the density of the NOS-ir, ChAT-ir and CalR-ir myenteric neurons. PMID:25320527

Serotonin 2A receptor regulation of striatal neuropeptide gene expression is selective for tachykinin, but not enkephalin neurons following dopamine depletion.

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Basura, G J; Walker, P D

2001-08-15

Serotonin (5-HT) 2A receptor-mediated regulation of striatal preprotachykinin (PPT) and preproenkephalin (PPE) mRNAs was studied in adult rodents that had been subjected to near-total dopamine (DA) depletion as neonates. Two months following bilateral 6-hydroxydopamine (6-OHDA) lesion, PPT mRNA levels decreased 59-73% across dorsal subregions of the rostral and caudal striatum while PPE transcripts increased 61-94%. Four hours after a single injection of the serotonin 2A/2C receptor agonist, (+/-)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 1 mg/kg), PPT mRNA expression was significantly increased in DA-depleted rats across all dorsal subregions of the rostral and caudal striatum as compared to 6-OHDA-treated animals alone. In the intact rat, DOI did not influence PPT mRNA levels in the rostral striatum, but did raise expression in the caudal striatum where 5-HT2A receptors are prominent. DOI did not regulate PPE mRNA levels in any striatal sub-region of the intact or DA-depleted rat. Prior administration of the 5-HT2A/2C receptor antagonist, ritanserin (1 mg/kg) or the 5-HT2A receptor antagonist, ketanserin (1 mg/kg) completely blocked the DOI-induced increases in striatal PPT mRNA in both lesioned and intact animals. The ability of ketanserin to produce identical results as ritanserin suggests that 5-HT2A receptor-mediated regulation is selectively strengthened within tachykinin neurons of the rostral striatum which are suppressed by DA depletion. The selectivity suggests that 5-HT2A receptor upregulation following DA depletion is capable of regulating tachykinin biosynthesis without influencing enkephalin expression in striatal output neurons.

Co-release of glutamate and GABA from single vesicles in GABAergic neurons exogenously expressing VGLUT3

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Johannes eZimmermann

2015-09-01

Full Text Available The identity of the vesicle neurotransmitter transporter expressed by a neuron largely corresponds with the primary neurotransmitter that cell releases. However, the vesicular glutamate transporter subtype 3 (VGLUT3 is mainly expressed in non-glutamatergic neurons, including cholinergic, serotonergic, or GABAergic neurons. Though a functional role for glutamate release from these non-glutamatergic neurons has been demonstrated, the interplay between VGLUT3 and the neuron’s characteristic neurotransmitter transporter, particularly in the case of GABAergic neurons, at the synaptic and vesicular level is less clear. In this study, we explore how exogenous expression of VGLUT3 in striatal GABAergic neurons affects the packaging and release of glutamate and GABA in synaptic vesicles. We found that VGLUT3 expression in isolated, autaptic GABAergic neurons leads to action potential evoked release of glutamate. Under these conditions, glutamate and GABA could be packaged together in single vesicles release either spontaneously or asynchronously. However, the presence of glutamate in GABAergic vesicles did not affect uptake of GABA itself, suggesting a lack of synergy in vesicle filling for these transmitters. Finally, we found postsynaptic detection of glutamate released from GABAergic terminals difficult when bona fide glutamatergic synapses were present, suggesting that co-released glutamate cannot induce postsynaptic glutamate receptor clustering.

The influence of TSA and VPA on the in vitro differentiation of bone marrow mesenchymal stem cells into neuronal lineage cells: Gene expression studies.

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Fila-Danilow, Anna; Borkowska, Paulina; Paul-Samojedny, Monika; Kowalczyk, Malgorzata; Kowalski, Jan

2017-03-27

Epigenetic mechanisms regulate the transcription of genes, which can affect the differentiation of MSCs. The aim of the current work is to determine how the histone deacetylase inhibitors TSA and VPA affect the expression of neuronal lineage genes in a culture of rat MSCs (rMSCs). We analyzed the expression of early neuron marker gene (Tubb3), mature neuron markers genes (Vacht, Th, Htr2a) and the oligodendrocyte progenitor marker gene (GalC). Moreover, changes in the gene expression after three different periods of exposure to TSA and VPA were investigated for the first time. After six days of exposition to TSA and VPA, the expression of Tubb3 and GalC decreased, while the expression of Th increased. The highest increase of VAChT expression was observed after three days of TSA and VPA treatment. A decrease in Htr2a gene expression was observed after TSA treatment and an increase was observed after VPA treatment. We also observed that TSA and VPA inhibited cell proliferation and the formation of neurospheres in the rMSCs culture. The central findings of our study are that TSA and VPA affect the expression of neuronal lineage genes in an rMSCs culture. After exposure to TSA or VPA, the expression of early neuronal gene decreases but equally the expression of mature neuron genes increases. After TSA and VPA treatment ER of the oligodendrocyte progenitor marker decreased. TSA and VPA inhibit cell proliferation and the formation of neurospheres in rMSCs culture.

Anatomical and molecular consequences of Unilateral Naris Closure on two populations of olfactory sensory neurons expressing defined odorant receptors.

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Molinas, Adrien; Aoudé, Imad; Soubeyre, Vanessa; Tazir, Bassim; Cadiou, Hervé; Grosmaitre, Xavier

2016-07-28

Mammalian olfactory sensory neurons (OSNs), the primary elements of the olfactory system, are located in the olfactory epithelium lining the nasal cavity. Exposed to the environment, their lifespan is short. Consequently, OSNs are regularly regenerated and several reports show that activity strongly modulates their development and regeneration: the peripheral olfactory system can adjust to the amount of stimulus through compensatory mechanisms. Unilateral naris occlusion (UNO) was frequently used to investigate this mechanism at the entire epithelium level. However, there is little data regarding the effects of UNO at the cellular level, especially on individual neuronal populations expressing a defined odorant receptor. Here, using UNO during the first three postnatal weeks, we analyzed the anatomical and molecular consequences of sensory deprivation in OSNs populations expressing the MOR23 and M71 receptors. The density of MOR23-expressing neurons is decreased in the closed side while UNO does not affect the density of M71-expressing neurons. Using Real Time qPCR on isolated neurons, we observed that UNO modulates the transcript levels for transduction pathway proteins (odorant receptors, CNGA2, PDE1c). The transcripts modulated by UNO will differ between populations depending on the receptor expressed. These results suggest that sensory deprivation will have different effects on different OSNs' populations. As a consequence, early experience will shape the functional properties of OSNs differently depending on the type of odorant receptor they express. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Fingolimod phosphate attenuates oligomeric amyloid β-induced neurotoxicity via increased brain-derived neurotrophic factor expression in neurons.

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Yukiko Doi

Full Text Available The neurodegenerative processes that underlie Alzheimer's disease are mediated, in part, by soluble oligomeric amyloid β, a neurotoxic protein that inhibits hippocampal long-term potentiation, disrupts synaptic plasticity, and induces the production of reactive oxygen species. Here we show that the sphingosine-1-phosphate (S1P receptor (S1PR agonist fingolimod phosphate (FTY720-P-a new oral drug for multiple sclerosis-protects neurons against oligomeric amyloid β-induced neurotoxicity. We confirmed that primary mouse cortical neurons express all of the S1P receptor subtypes and FTY720-P directly affects the neurons. Treatment with FTY720-P enhanced the expression of brain-derived neurotrophic factor (BDNF in neurons. Moreover, blocking BDNF-TrkB signaling with a BDNF scavenger, TrkB inhibitor, or ERK1/2 inhibitor almost completely ablated these neuroprotective effects. These results suggested that the neuroprotective effects of FTY720-P are mediated by upregulated neuronal BDNF levels. Therefore, FTY720-P may be a promising therapeutic agent for neurodegenerative diseases, such as Alzheimer's disease.

Circadian Activators Are Expressed Days before They Initiate Clock Function in Late Pacemaker Neurons from Drosophila.

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Liu, Tianxin; Mahesh, Guruswamy; Houl, Jerry H; Hardin, Paul E

2015-06-03

Circadian pacemaker neurons in the Drosophila brain control daily rhythms in locomotor activity. These pacemaker neurons can be subdivided into early or late groups depending on whether rhythms in period (per) and timeless (tim) expression are initiated at the first instar (L1) larval stage or during metamorphosis, respectively. Because CLOCK-CYCLE (CLK-CYC) heterodimers initiate circadian oscillator function by activating per and tim transcription, a Clk-GFP transgene was used to mark when late pacemaker neurons begin to develop. We were surprised to see that CLK-GFP was already expressed in four of five clusters of late pacemaker neurons during the third instar (L3) larval stage. CLK-GFP is only detected in postmitotic neurons from L3 larvae, suggesting that these four late pacemaker neuron clusters are formed before the L3 larval stage. A GFP-cyc transgene was used to show that CYC, like CLK, is also expressed exclusively in pacemaker neurons from L3 larval brains, demonstrating that CLK-CYC is not sufficient to activate per and tim in late pacemaker neurons at the L3 larval stage. These results suggest that most late pacemaker neurons develop days before novel factors activate circadian oscillator function during metamorphosis. Copyright © 2015 the authors 0270-6474/15/358662-10$15.00/0.

Astrocyte-neuron crosstalk regulates the expression and subcellular localization of carbohydrate metabolism enzymes.

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Mamczur, Piotr; Borsuk, Borys; Paszko, Jadwiga; Sas, Zuzanna; Mozrzymas, Jerzy; Wiśniewski, Jacek R; Gizak, Agnieszka; Rakus, Dariusz

2015-02-01

Astrocytes releasing glucose- and/or glycogen-derived lactate and glutamine play a crucial role in shaping neuronal function and plasticity. Little is known, however, how metabolic functions of astrocytes, e.g., their ability to degrade glucosyl units, are affected by the presence of neurons. To address this issue we carried out experiments which demonstrated that co-culturing of rat hippocampal astrocytes with neurons significantly elevates the level of mRNA and protein for crucial enzymes of glycolysis (phosphofructokinase, aldolase, and pyruvate kinase), glycogen metabolism (glycogen synthase and glycogen phosphorylase), and glutamine synthetase in astrocytes. Simultaneously, the decrease of the capability of neurons to metabolize glucose and glutamine is observed. We provide evidence that neurons alter the expression of astrocytic enzymes by secretion of as yet unknown molecule(s) into the extracellular fluid. Moreover, our data demonstrate that almost all studied enzymes may localize in astrocytic nuclei and this localization is affected by the co-culturing with neurons which also reduces proliferative activity of astrocytes. Our results provide the first experimental evidence that the astrocyte-neuron crosstalk substantially affects the expression of basal metabolic enzymes in the both types of cells and influences their subcellular localization in astrocytes. © 2014 Wiley Periodicals, Inc.

Hindbrain Catecholamine Neurons Activate Orexin Neurons During Systemic Glucoprivation in Male Rats.

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Li, Ai-Jun; Wang, Qing; Elsarelli, Megan M; Brown, R Lane; Ritter, Sue

2015-08-01

Hindbrain catecholamine neurons are required for elicitation of feeding responses to glucose deficit, but the forebrain circuitry required for these responses is incompletely understood. Here we examined interactions of catecholamine and orexin neurons in eliciting glucoprivic feeding. Orexin neurons, located in the perifornical lateral hypothalamus (PeFLH), are heavily innervated by hindbrain catecholamine neurons, stimulate food intake, and increase arousal and behavioral activation. Orexin neurons may therefore contribute importantly to appetitive responses, such as food seeking, during glucoprivation. Retrograde tracing results showed that nearly all innervation of the PeFLH from the hindbrain originated from catecholamine neurons and some raphe nuclei. Results also suggested that many catecholamine neurons project collaterally to the PeFLH and paraventricular hypothalamic nucleus. Systemic administration of the antiglycolytic agent, 2-deoxy-D-glucose, increased food intake and c-Fos expression in orexin neurons. Both responses were eliminated by a lesion of catecholamine neurons innervating orexin neurons using the retrogradely transported immunotoxin, anti-dopamine-β-hydroxylase saporin, which is specifically internalized by dopamine-β-hydroxylase-expressing catecholamine neurons. Using designer receptors exclusively activated by designer drugs in transgenic rats expressing Cre recombinase under the control of tyrosine hydroxylase promoter, catecholamine neurons in cell groups A1 and C1 of the ventrolateral medulla were activated selectively by peripheral injection of clozapine-N-oxide. Clozapine-N-oxide injection increased food intake and c-Fos expression in PeFLH orexin neurons as well as in paraventricular hypothalamic nucleus neurons. In summary, catecholamine neurons are required for the activation of orexin neurons during glucoprivation. Activation of orexin neurons may contribute to appetitive responses required for glucoprivic feeding.

Arrays of microLEDs and astrocytes: biological amplifiers to optogenetically modulate neuronal networks reducing light requirement.

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Rolando Berlinguer-Palmini

Full Text Available In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2 and by means of a matrix of individually addressable super-bright microLEDs (μLEDs with an excitation peak at 470 nm. We combined Ca2+ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the μLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing μLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the μLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and μLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.

Arrays of microLEDs and astrocytes: biological amplifiers to optogenetically modulate neuronal networks reducing light requirement.

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Berlinguer-Palmini, Rolando; Narducci, Roberto; Merhan, Kamyar; Dilaghi, Arianna; Moroni, Flavio; Masi, Alessio; Scartabelli, Tania; Landucci, Elisa; Sili, Maria; Schettini, Antonio; McGovern, Brian; Maskaant, Pleun; Degenaar, Patrick; Mannaioni, Guido

2014-01-01

In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (μLEDs) with an excitation peak at 470 nm. We combined Ca2+ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the μLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing μLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the μLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and μLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.

The GluN2B subunit represents a major functional determinant of NMDA receptors in human induced pluripotent stem cell-derived cortical neurons

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Ioana Neagoe

2018-04-01

Full Text Available Abnormal signaling pathways mediated by N-methyl-d-aspartate receptors (NMDARs have been implicated in the pathogenesis of various CNS disorders and have been long considered as promising points of therapeutic intervention. However, few efforts have been previously described concerning evaluation of therapeutic modulators of NMDARs and their downstream pathways in human neurons with endogenous expression of NMDARs. In the present study, we assessed expression, functionality, and subunit composition of endogenous NMDARs in human induced pluripotent stem cell (hiPSC-derived cortical neurons (iCell Neurons and iCell GlutaNeurons. We initially confirmed the expected pharmacological response of iCell Neurons and iCell GlutaNeurons to NMDA by patch-clamp recordings. Subsequent pharmacological interrogation using GluN2 subunit-selective antagonists revealed the predominance of GluN2B in both iCell Neurons and iCell GlutaNeurons. This observation was also supported by qRT-PCR and Western blot analyses of GluN2 subunit expression as well as pharmacological experiments using positive allosteric modulators with distinct GluN2 subunit selectivity. We conclude that iCell Neurons and iCell GlutaNeurons express functional GluN2B-containing NMDARs and could serve as a valuable system for development and validation of GluN2B-modulating pharmaceutical agents. Keywords: Human induced pluripotent stem cell-derived neurons, iCell Neurons, iCell GlutaNeurons, NMDA receptors, GluN2B, Positive allosteric modulators

Optogenetic release of norepinephrine from cardiac sympathetic neurons alters mechanical and electrical function.

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Wengrowski, Anastasia M; Wang, Xin; Tapa, Srinivas; Posnack, Nikki Gillum; Mendelowitz, David; Kay, Matthew W

2015-02-01

Release of norepinephrine (NE) from sympathetic neurons enhances heart rate (HR) and developed force through activation of β-adrenergic receptors, and this sympathoexcitation is a key risk for the generation of cardiac arrhythmias. Studies of β-adrenergic modulation of cardiac function typically involve the administration of exogenous β-adrenergic receptor agonists to directly elicit global β-adrenergic receptor activation by bypassing the involvement of sympathetic nerve terminals. In this work, we use a novel method to activate sympathetic fibres within the myocardium of Langendorff-perfused hearts while measuring changes in electrical and mechanical function. The light-activated optogenetic protein channelrhodopsin-2 (ChR2) was expressed in murine catecholaminergic sympathetic neurons. Sympathetic fibres were then photoactivated to examine changes in contractile force, HR, and cardiac electrical activity. Incidence of arrhythmia was measured with and without exposure to photoactivation of sympathetic fibres, and hearts were optically mapped to detect changes in action potential durations and conduction velocities. Results demonstrate facilitation of both developed force and HR after photostimulated release of NE, with increases in contractile force and HR of 34.5 ± 5.5 and 25.0 ± 9.3%, respectively. Photostimulation of sympathetic fibres also made hearts more susceptible to arrhythmia, with greater incidence and severity. In addition, optically mapped action potentials displayed a small but significant shortening of the plateau phase (-5.5 ± 1.0 ms) after photostimulation. This study characterizes a powerful and clinically relevant new model for studies of cardiac arrhythmias generated by increasing the activity of sympathetic nerve terminals and the resulting activation of myocyte β-adrenergic receptors. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

Sustained expression of a neuron-specific isoform of the Taf1 gene in development stages and aging in mice

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Jambaldorj, Jamiyansuren; Makino, Satoshi; Munkhbat, Batmunkh; Tamiya, Gen

2012-01-01

Highlights: ► We identified the mouse homologue of neuron-specific TAF1 (N-Taf1). ► Taf1 mRNA was expressed in most tissues and cell lines. ► N-Taf1 mRNA was expressed in the brain and Neuroblastoma N2a cell lines. ► Taf1 and N-Taf1 showed different expression profile in development stage and aging. -- Abstract: TATA-box binding protein associated factor 1 (TAF1) protein is the largest and the essential component of the TFIID complex in the pathway of RNA polymerase II–mediated gene transcription, and it regulates transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1), which we reported previously, may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. In the present study, we cloned the full-length cDNA that encodes the mouse homologue of N-TAF1 (N-Taf1) protein. By carrying out of real time RT-PCR, we investigated the expression analysis of the N-Taf1 mRNA in mouse tissues and cell lines. As well as the human N-TAF1, the N-Taf1 showed limited expression in the brain and neuroblastoma, whereas Taf1 expressed elsewhere. Furthermore, in mouse embryo head or mouse brain, mRNA expression of TAF1 changes dramatically during development but N-Taf1 showed sustained expression. Our result suggests that the N-Taf1 gene has an important role in non-dividing neuronal cell rather than in cell division and proliferation during neurogenesis.

Sustained expression of a neuron-specific isoform of the Taf1 gene in development stages and aging in mice

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Jambaldorj, Jamiyansuren [Department of Pharmacology, Institute of Health Biosciences, Graduate School, The University of Tokushima, Tokushima 770-8503 (Japan); Advanced Molecular Epidemiology Research Institute, Yamagata University Faculty of Medicine, Yamagata 990-9585 (Japan); Central Scientific Research Laboratory, Institute of Medical Sciences, Ulaanbaatar (Mongolia); Makino, Satoshi, E-mail: smakino@genetix-h.com [Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu 520-2192 (Japan); Munkhbat, Batmunkh [Central Scientific Research Laboratory, Institute of Medical Sciences, Ulaanbaatar (Mongolia); Tamiya, Gen [Advanced Molecular Epidemiology Research Institute, Yamagata University Faculty of Medicine, Yamagata 990-9585 (Japan)

2012-08-24

Highlights: Black-Right-Pointing-Pointer We identified the mouse homologue of neuron-specific TAF1 (N-Taf1). Black-Right-Pointing-Pointer Taf1 mRNA was expressed in most tissues and cell lines. Black-Right-Pointing-Pointer N-Taf1 mRNA was expressed in the brain and Neuroblastoma N2a cell lines. Black-Right-Pointing-Pointer Taf1 and N-Taf1 showed different expression profile in development stage and aging. -- Abstract: TATA-box binding protein associated factor 1 (TAF1) protein is the largest and the essential component of the TFIID complex in the pathway of RNA polymerase II-mediated gene transcription, and it regulates transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1), which we reported previously, may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. In the present study, we cloned the full-length cDNA that encodes the mouse homologue of N-TAF1 (N-Taf1) protein. By carrying out of real time RT-PCR, we investigated the expression analysis of the N-Taf1 mRNA in mouse tissues and cell lines. As well as the human N-TAF1, the N-Taf1 showed limited expression in the brain and neuroblastoma, whereas Taf1 expressed elsewhere. Furthermore, in mouse embryo head or mouse brain, mRNA expression of TAF1 changes dramatically during development but N-Taf1 showed sustained expression. Our result suggests that the N-Taf1 gene has an important role in non-dividing neuronal cell rather than in cell division and proliferation during neurogenesis.

Characterization of Na+ and Ca2+ channels in zebrafish dorsal root ganglion neurons.

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Yu-Jin Won

Full Text Available BACKGROUND: Dorsal root ganglia (DRG somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available. METHODOLOGY/PRINCIPAL FINDINGS: We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na(+ currents (rapidly- and slowly-inactivating were discovered. Rapidly-inactivating I(Na were preferentially expressed in relatively large neurons, while slowly-inactivating I(Na was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I(Na components. Voltage-gated Ca(2+ currents (I(Ca were primarily (87% comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca(V2.2 (N-type Ca(2+ channels. A few DRG neurons (8% displayed a miniscule low-voltage-activated component. I(Ca in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability. CONCLUSIONS/SIGNIFICANCE: Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and

Metabotropic glutamate receptor 2 and corticotrophin-releasing factor receptor-1 gene expression is differently regulated by BDNF in rat primary cortical neurons