Synthesizing Enzyme Glutamic Acid Decarboxylase Research Articles

Protective roles of hepatic gamma-aminobutyric acid signaling in acute ethanol exposure-induced liver injury.

Alcoholic liver disease (ALD) is a consequence of heavy and prolonged alcohol consumptions. We previously demonstrated a hepatic gamma-aminobutyric acid (GABA) signaling system that protects the liver from toxic injury. The present study was designed to investigate the role of the hepatic GABA signaling system in the process of acute ethanol exposure-induced liver injury. Our results showed that the expression of GABA synthesizing enzyme glutamic acid decarboxylase and type A GABA receptor (GABAA R) subunits was upregulated in ethanol-treated mice compared with saline-treated controls. Remarkably, pretreatment of mice with GABA (1.5mg kg-1 body weight, intraperitoneal injection [i.p.]) or with the GABAA R agonist muscimol (1.2mg kg-1 body weight, i.p.) protected the liver against ethanol toxicity and improved liver function, whereas pretreatment of mice with the GABAA R antagonist bicuculline (2.0mg kg-1 body weight, i.p.) worsened the liver function. Further analyses suggest that GABAA R-mediated signaling protects the liver from ethanol injury by, at least partially, inhibiting the IRE1α-ASK1-JNK pro-apoptotic pathway in hepatocytes in the process of ethanol-induced endoplasmic reticulum stress response.

GABAergic innervation of the ciliary ganglion in macaque monkeys – A light and electron microscopic study

AbstractThe vertebrate ciliary ganglion (CG) is a relay station in the parasympathetic pathway activating the iris sphincter and ciliary muscle to mediate pupillary constriction and lens accommodation, respectively. While the postganglionic motoneurons in the CG are cholinergic, as are their inputs, there is evidence from avian studies that GABA may also be involved. Here, we used light and electron microscopic methods to examine the GABAergic innervation of the CG in Macaca fascicularis monkeys. Immunohistochemistry for the gamma aminobutyric acid synthesizing enzyme glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) revealed that all CG neurons are contacted by ChAT‐positive terminals. A subpopulation of 17.5% of CG neurons was associated with terminal boutons expressing GAD‐immunoreactivity in addition. Double‐labeling for GAD and synaptophysin confirmed that these were synaptic terminals. Electron microscopic analysis in conjunction with GABA‐immunogold staining showed that (1) GAD‐positive terminals mainly target dendrites and spines in the perisomatic neuropil of CG neurons; (2) GABA is restricted to a specific terminal type, which displays intermediate features lying between classically excitatory and inhibitory endings; and (3) if a CG neuron is contacted by GABA‐positive terminals, virtually all perisomatic terminals supplying it show GABA immunoreactivity. The source of this GABAergic input and whether GABA contributes to a specific CG function remains to be investigated. Nevertheless, our data indicate that the innervation of the ciliary ganglion is more complex than previously thought, and that GABA may play a neuromodulatory role in the control of lens or pupil function. J. Comp. Neurol. 525:1–1, 2017. © 2016 Wiley Periodicals, Inc.

GABAergic innervation of the ciliary ganglion in macaque monkeys - A light and electron microscopic study.

The vertebrate ciliary ganglion (CG) is a relay station in the parasympathetic pathway activating the iris sphincter and ciliary muscle to mediate pupillary constriction and lens accommodation, respectively. While the postganglionic motoneurons in the CG are cholinergic, as are their inputs, there is evidence from avian studies that GABA may also be involved. Here, we used light and electron microscopic methods to examine the GABAergic innervation of the CG in Macaca fascicularis monkeys. Immunohistochemistry for the gamma aminobutyric acid synthesizing enzyme glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) revealed that all CG neurons are contacted by ChAT-positive terminals. A subpopulation of 17.5% of CG neurons was associated with terminal boutons expressing GAD-immunoreactivity in addition. Double-labeling for GAD and synaptophysin confirmed that these were synaptic terminals. Electron microscopic analysis in conjunction with GABA-immunogold staining showed that (1) GAD-positive terminals mainly target dendrites and spines in the perisomatic neuropil of CG neurons; (2) GABA is restricted to a specific terminal type, which displays intermediate features lying between classically excitatory and inhibitory endings; and (3) if a CG neuron is contacted by GABA-positive terminals, virtually all perisomatic terminals supplying it show GABA immunoreactivity. The source of this GABAergic input and whether GABA contributes to a specific CG function remains to be investigated. Nevertheless, our data indicate that the innervation of the ciliary ganglion is more complex than previously thought, and that GABA may play a neuromodulatory role in the control of lens or pupil function. J. Comp. Neurol. 525:1517-1531, 2017. © 2016 Wiley Periodicals, Inc.

Lower Glutamic Acid Decarboxylase 65-kDa Isoform Messenger RNA and Protein Levels in the Prefrontal Cortex in Schizoaffective Disorder but Not Schizophrenia

BackgroundAltered gamma-aminobutyric acid (GABA) signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in patients with schizophrenia and schizoaffective disorder. Levels of the GABA–synthesizing enzyme glutamic acid decarboxylase 67-kDa isoform (GAD67) in the PFC have been consistently reported to be lower in patients with these disorders, but the status of the second GABA–synthesizing enzyme, glutamic acid decarboxylase 65-kDa isoform (GAD65), remains unclear. MethodsGAD65 messenger RNA (mRNA) levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. In a subset of subject pairs, GAD65 relative protein levels were quantified by confocal immunofluorescence microscopy. ResultsMean GAD65 mRNA levels were 13.6% lower in subjects with schizoaffective disorder but did not differ in subjects with schizophrenia relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein levels within subjects with schizoaffective disorder were not attributable to factors commonly comorbid with the diagnosis. ConclusionsIn concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in patients with schizoaffective disorder relative to patients with schizophrenia, these findings may support an interpretation that GAD65 downregulation provides a homeostatic response complementary to GAD67 downregulation that serves to reduce inhibition in the face of lower PFC network activity.

GABAergic System in β-Cells: From Autoimmunity Target to Regeneration Tool

The γ-aminobutyric acid (GABA) is a product of decarboxylation of the amino acid glutamate mediated by the synthesizing enzyme glutamic acid decarboxylase (GAD) (1–3). Although GABA is a major inhibitory neurotransmitter of the brain, it is produced at high levels in pancreatic islets (4). β-Cells store GABA in synaptic-like microvesicles, and upon its secretion, GABA exerts many paracrine functions in pancreatic islets (4). While the total function of GABA in β-cells is incompletely understood (4), its synthesizing enzyme GAD is possibly one of the most significant pancreatic islet β-cell autoantigens (5). GAD is a primary target of autoantibodies, and anti-GAD antibodies are associated with the development of type 1 diabetes (T1D) (5). GABA activates three types of membrane receptors: GABAA and GABAC, which are ligand-gated Cl− channels, and GABAB, a ligand-gated Ca2+ or K+ channel (6). It has been demonstrated that β-cells mainly express the GABAB receptor (GABABR) and the GABAA receptor (GABAAR) and produce GABA through GAD (Fig. 1) (4); GABA colocalizes with insulin as shown by confocal microscopy (4,7). Activation of GABA receptors in islet β-cells increases insulin release (8), exerts protective and regenerative effects on islet β-cells (9), and reduces apoptosis in cultured islets (9). GABA has also been shown to increase DNA synthesis in the pancreatic cell line INS-1, and when injected in vivo it increased the number of Ki67+ islet β-cells (Fig. 1). Thus, GABA increases β-cell proliferation in vivo and in vitro, protects INS-1 cells from streptozotocin (STZ)-induced apoptosis, and prevents hyperglycemia in murine models of diabetes (9). FIG. 1. Regenerative and immunological abilities of the inhibitory neurotransmitter GABA. Extracellular glutamate, the precursor of …

Genetic variation in GAD1 is associated with cortical thickness in the parahippocampal gyrus

Patients with schizophrenia show widespread cortical thickness reductions throughout the brain. Likewise, reduced expression of the γ-Aminobutyric acid (GABA) synthesizing enzyme glutamic acid decarboxylase (GAD1) and a single nucleotide polymorphism (SNP) rs3749034 in the corresponding gene have been associated with schizophrenia. We tested whether this SNP is associated with reduced cortical thickness, an intermediate phenotype for schizophrenia. Because of the well known interactions between the GABAergic and dopaminergic systems, we examined whether associations between GAD1 rs3749034 and cortical thickness are modulated by the catechol-O-methyltransferase (COMT) Val158Met genotype. Structural MRI and genotype data was obtained from 94 healthy subjects enrolled in the Mind Clinical Imaging Consortium study to examine the relations between GAD1 genotype and cortical thickness. Our data show a robust reduction of cortical thickness in the left parahippocampal gyrus (PHG) in G allele homozygotes of GAD1 rs3749034. When we stratified our analyses according to the COMT Val158Met genotype, cortical thickness reductions of G allele homozygotes were only found in the presence of the Val allele. Genetic risk variants of schizophrenia in the GABAergic system might interact with the dopaminergic system and impact brain structure and functioning. Our findings point to the importance of the GABAergic system in the pathogenesis of schizophrenia.

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Differential regulation of glutamic acid decarboxylase gene expression after extinction of a recent memory vs. intermediate memory

Extinction reduces fear to stimuli that were once associated with an aversive event by no longer coupling the stimulus with the aversive event. Extinction learning is supported by a network comprising the amygdala, hippocampus, and prefrontal cortex. Previous studies implicate a critical role of GABA in extinction learning, specifically the GAD65 isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD). However, a detailed analysis of changes in gene expression of GAD in the subregions comprising the extinction network has not been undertaken. Here, we report changes in gene expression of the GAD65 and GAD67 isoforms of GAD, as measured by relative quantitative real-time RT-PCR, in subregions of the amygdala, hippocampus, and prefrontal cortex 24-26 h after extinction of a recent (1-d) or intermediate (14-d) fear memory. Our results show that extinction of a recent memory induces a down-regulation of Gad65 gene expression in the hippocampus (CA1, dentate gyrus) and an up-regulation of Gad67 gene expression in the infralimbic cortex. Extinguishing an intermediate memory increased Gad65 gene expression in the central amygdala. These results indicate a differential regulation of Gad gene expression after extinction of a recent memory vs. intermediate memory.

Expression of parvalbumin and glutamic acid decarboxylase-67 after acute administration of MK-801. Implications for the NMDA hypofunction model of schizophrenia

A reduction of GABAergic markers in postmortem tissue is consistently found in schizophrenia. This is generally mediated by a decreased expression of the calcium-binding protein, parvalbumin (PV), and the 67-kDa isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD(67)). Similar reductions of PV or GAD(67) are observed after repeated exposure to N-methyl-D-aspartate (NMDA) receptor antagonists but less attention has been paid to what occurs after their acute administration. Here, we have used in situ hybridization to examine the expression of PV and GAD(67) mRNAs at 4h and 24h after an acute administration of MK-801 (1mg/kg). Four hours after MK-801, the expression of PV mRNA decreased only in dentate gyrus of the hippocampus. Twenty four hours after this treatment, a reduction of the levels of PV mRNA was found in the medial prefrontal, orbitofrontal and entorhinal cortices, hippocampus and the basolateral nucleus of the amygdala. In contrast, no changes in the expression of GAD(67) were observed in any of the brain regions examined. Interestingly, the reduction in PV mRNA expression is observed in discrete corticolimbic subregions that have been implicated in schizophrenia, which is coincident with changes observed in postmortem tissue of schizophrenia brain. These findings indicate that acute administration of a NMDA antagonist delineate a pattern of changes in GABAergic markers different from those observed in postmortem tissue in schizophrenia inasmuch as only deficits in parvalbumin (but not GAD(67)) were seen.

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

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

Chemical neuroanatomy of the dorsal raphe nucleus and adjacent structures of the mouse brain

Serotonin neurons play a major role in many normal and pathological brain functions. In the rat these neurons have a varying number of cotransmitters, including neuropeptides. Here we studied, with histochemical techniques, the relation between serotonin, some other small-molecule transmitters, and a number of neuropeptides in the dorsal raphe nucleus (DRN) and the adjacent ventral periaqueductal gray (vPAG) of mouse, an important question being to establish possible differences from rat. Even if similarly distributed, the serotonin neurons in mouse lacked the extensive coexpression of nitric oxide synthase and galanin seen in rat. Although partly overlapping in the vPAG, no evidence was obtained for the coexistence of serotonin with dopamine, substance P, cholecystokinin, enkephalin, somatostatin, neurotensin, dynorphin, thyrotropin-releasing hormone, or corticotropin-releasing hormone. However, some serotonin neurons expressed the gamma-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase (GAD). Work in other laboratories suggests that, as in rat, serotonin neurons in the mouse midline DRN express the vesicular glutamate transporter 3, presumably releasing glutamate. Our study also shows that many of the neuropeptides studied (substance P, galanin, neurotensin, dynorphin, and corticotropin-releasing factor) are present in nerve terminal networks of varying densities close to the serotonin neurons, and therefore may directly or indirectly influence these cells. The apparently low numbers of coexisting messengers in mouse serotonin neurons, compared to rat, indicate considerable species differences with regard to the chemical neuronatomy of the DRN. Thus, extrapolation of DRN physiology, and possibly pathology, from rat to mouse, and even human, should be made with caution.

Immunocytochemical evidence that monkey rod bipolar cells use GABA

Certain bipolar cells in most species immunostain for GABA or its synthesizing enzyme glutamic acid decarboxylase. However, it is unknown whether they actually release GABA and, if so, from which cellular compartment and by what release mechanism. We investigated these questions in monkey retina where rod bipolar cells immunostain for GABA. We found that rod bipolar cells immunostain for one isoform of GAD (GAD65) in their somas, dendrites and axon terminals. Near the fovea, the somatic stain of rod bipolar cells is weaker than that of horizontal cells but, at the periphery, it is stronger. Staining for the vesicular GABA transporter in monkey rod bipolar cells is negative. However, staining for the GABA transporter GAT3 is positive in the soma and primary dendrites (but not in the axon terminals). Staining for GAT3 is also positive in horizontal cells. Double staining of rod bipolar cells and the alpha subunit of the GABAA receptor reveals scarce GABAA puncta that appose rod bipolar dendrites. We conclude that monkey rod bipolar cells use GABA and discuss the possibility that they tonically release GABA from their dendrites using a reverse action of GAT3.

Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture

The well-characterized human teratocarcinoma line Ntera2 (NT2) can be differentiated into mature neurons. We have significantly shortened the time-consuming process for generating postmitotic neurons to approximately 4 weeks by introducing a differentiation protocol for free-floating cell aggregates and a subsequent purification step. Here, we characterize the neurochemical phenotypes of the neurons derived from this cell aggregate method. During differentiation, the NT2 cells lose immunoreactivity for vimentin and nestin filaments, which are characteristic for the immature state of neuronal precursors. Instead, they acquire typical neuronal markers such as beta-tubulin type III, microtubule-associated protein 2, and phosphorylated tau, but no astrocyte markers such as glial fibrillary acidic protein. They grow neural processes that express punctate immunoreactivity for synapsin and synaptotagmin suggesting the formation of presynaptic structures. Despite their common clonal origin, neurons cultured for 2-4 weeks in vitro comprise a heterogeneous population expressing several neurotransmitter phenotypes. Approximately 40% of the neurons display glutamatergic markers. A minority of neurons is immunoreactive for serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase. We have found no evidence for a dopaminergic phenotype. Subgroups of NT2 neurons respond to the application of nitric oxide donors with the synthesis of cGMP. A major subset shows immunoreactivity to the cholinergic markers choline acetyl-transferase, vesicular acetylcholine transporter, and the non-phosphorylated form of neurofilament H, all indicative of motor neurons. The NT2 system may thus be well suited for research related to motor neuron diseases.

Gabaergic mechanisms of hypothalamic nuclei in the expression of conditioned fear

The amygdala, the dorsal periaqueductal gray (dPAG), and the medial hypothalamus have long been recognized to be a neural system responsible for the generation and elaboration of unconditioned fear in the brain. It is also well known that this neural substrate is under a tonic inhibitory control exerted by GABA mechanisms. However, whereas there is a growing body of evidence to suggest that the amygdala and dPAG are also able to integrate conditioned fear, it is still unclear, however, how the distinct hypothalamic nuclei participate in fear conditioning. In this work we aimed to examine the extent to which the gabaergic mechanisms of this brain region are involved in conditioned fear using the fear-potentiated startle (FPS). Muscimol, a GABA-A receptor agonist, and semicarbazide, an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD), were used as an enhancer and inhibitor of the GABA mechanisms, respectively. Muscimol and semicarbazide were injected into the anterior hypothalamus (AHN), the dorsomedial part of the ventromedial nucleus (VMHDM), the dorsomedial (DMH) or the dorsal premammillary (PMD) nuclei of male Wistar rats before test sessions of the fear conditioning paradigm. The injections into the DMH and PMD did not produce any significant effects on FPS. On the other hand, muscimol injections into the AHN and VMHDM caused significant reduction in FPS. These results indicate that injections of muscimol and semicarbazide into the DMH and PMD fail to change the FPS, whereas the enhancement of the GABA transmission in the AHN and VMHDM produces a reduction of the conditioned fear responses. On the other hand, the inhibition of this transmission led to an increase of this conditioned response in the AHN. Thus, whereas DMH and PMD are known to be part of the caudal-most region of the medial hypothalamic defensive system, which integrates unconditioned fear, systems mediating conditioned fear select the AHN and VMHDM nuclei that belong to the rostral-most portion of the hypothalamic defense area. Thus, distinct subsets of neurons in the hypothalamus could mediate different aspects of the defensive responses.

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

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

Time-course of SKF-81297-induced increase in glutamic acid decarboxylase 65 and 67 mRNA levels in striatonigral neurons and decrease in GABA A receptor α1 subunit mRNA levels in the substantia nigra, pars reticulata, in adult rats with a unilateral 6-hydroxydopamine lesion

Striatal projection neurons use GABA as their neurotransmitter and express the rate-limiting synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter vGAT. The chronic systemic administration of an agonist of dopamine D1/D5-preferring receptors is known to alter GAD mRNA levels in striatonigral neurons in intact and dopamine-depleted rats. In the present study, the effects of a single or subchronic systemic administration of the dopamine D1/D5-preferring receptor agonist SKF-81297 on GAD65, GAD67, PPD and vGAT mRNA levels in the striatum and GABA A receptor α1 subunit mRNA levels in the substantia nigra, pars reticulata, were measured in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion. After a single injection of SKF-81297, striatal GAD65 mRNA levels were significantly increased at 3 but not 72 h. In contrast, striatal GAD67 mRNA levels were increased and nigral α1 mRNA levels were decreased at 72 but not 3 h. Single cell analysis on double-labeled sections indicated that increased GAD or vGAT mRNA levels after acute SKF-81297 occurred in striatonigral neurons identified by their lack of preproenkephalin expression. Subchronic SKF-81297 induced significant increases in striatal GAD67, GAD65, preprodynorphin and vGAT mRNA levels and decreases in nigral α1 mRNA levels. In the striatum contralateral to the 6-OHDA lesion, subchronic but not acute SKF-81297 induced a significant increase in GAD65 mRNA levels. The other mRNA levels were not significantly altered. Finally, striatal GAD67 mRNA levels were negatively correlated with nigral α1 mRNA levels in the dopamine-depleted but not dopamine-intact side. The results suggest that different signaling pathways are involved in the modulation by dopamine D1/D5 receptors of GAD65 and GAD67 mRNA levels in striatonigral neurons. They also suggest that the down-regulation of nigral GABA A receptors is linked to the increase in striatal GAD67 mRNA levels in the dopamine-depleted striatum.

Diabetes affects the expression of GABA and potassium chloride cotransporter in the spinal cord: A study in streptozotocin diabetic rats

Painful diabetic neuropathy is associated to hyperexcitability and spontaneous hyperactivity of spinal cord neurons. The underlying pathophysiological mechanisms are not clear. Increases in excitatory neurotransmission at the spinal cord, involving glutamate and SP, seem to account for the abnormal neuronal activity, but inhibitory influences were never evaluated. This study aims to analyse the expression of GABA, its synthesizing enzyme glutamic acid decarboxylase (GAD) and the potassium chloride cotransporter (KCC2), in the spinal dorsal horn of streptozotocin (STZ)-induced diabetic rats. Four weeks after saline or STZ (60mg/kg) injection, animals were sacrificed and the spinal segments L2-L3 were removed and immunoreacted for GABA, GAD and KCC2, or processed for western blotting for KCC2. Densitometric quantification was performed in the superficial dorsal horn (laminae I, II and III) of immunoreacted sections and in the immunoblots. STZ rats presented a significant increase of GABA expression in laminae II and III when compared with control animals, while no differences were detected in GAD expression. A significant decrease in KCC2 expression was detected by immunohistochemistry in laminae I and II, which was confirmed by immunoblotting. Increased GABA levels, along with decrease in KCC2 expression, may underlie the abnormal neuronal activity detected in the spinal cord of diabetic rats. Reduction in KCC2 expression was shown to lead to increases in intracellular chloride concentration and, in such condition, GABA binding to GABA(A) receptor induces membrane depolarization, provoking neuronal excitation rather than inhibition. Based on these findings, we propose that a loss of GABA-mediated inhibitory tone at the spinal cord may result in neuronal hyperexcitability and spontaneous hyperactivity during diabetes.

Disease-specific alterations in glutamatergic neurotransmission on inhibitory interneurons in the prefrontal cortex in schizophrenia

Glutamatergic modulation of inhibitory interneurons plays a crucial role in shaping the flow of information in the cerebral cortex. In a cohort of postmortem human brains from schizophrenia ( n = 20), bipolar disorder ( n = 20) and normal control ( n = 20) subjects, we colocalized the mRNA for the N-methyl- d-aspartate (NMDA) receptor NR2A subunit, labeled with [ 35S], and the mRNA for the γ-aminobutyric acid (GABA) synthesizing enzyme glutamic acid decarboxylase (GAD) 67, labeled with digoxigenin. We found that the density of GAD 67+ neurons in layers 2–5 of the prefrontal cortex was decreased by 27–36% in both schizophrenia and bipolar disorder. In addition, the density of the GAD 67+/NR2A+ neurons was decreased by 57% and 49% in layers 3 and 4, respectively, in schizophrenia, but it was unchanged in bipolar disorder. These findings raise the possibility that glutamatergic innervation of inhibitory interneurons via the NMDA receptor in the prefrontal cortex may be selectively altered in schizophrenia.

Differential alterations of kainate receptor subunits in inhibitory interneurons in the anterior cingulate cortex in schizophrenia and bipolar disorder

The aim of this study was to examine whether glutamatergic inputs onto GABA interneurons via the kainate receptor in the anterior cingulate cortex may be altered in schizophrenia and bipolar disorder. Hence, in a cohort of 60 post-mortem human brains from schizophrenia, bipolar disorder, and normal control subjects, we simultaneously labeled the mRNA for the GluR5 or GluR6 subunit of the kainate receptor with [ 35S] and the mRNA for the 67 kD isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) 67 with digoxigenin using an immunoperoxidase method. The density of the GAD 67 mRNA-containing neurons that co-expressed GluR5 mRNA was decreased by 43% and 40% in layer 2 of the anterior cingulate cortex in schizophrenia and bipolar disorder, respectively. In contrast, the density of the GAD 67 mRNA-containing cells that expressed GluR6 mRNA was unaltered in either condition. Furthermore, the amount of GluR5 or GluR6 mRNA in the GAD 67 mRNA-expressing cells that contained a detectable level of these transcripts was also unchanged. Finally, the density of cells that did not contain GAD 67 mRNA, which presumably included all pyramidal neurons, but expressed the mRNA for the GluR5 or GluR6 subunit was not altered. Thus, glutamatergic modulation of inhibitory interneurons, but not pyramidal neurons, via kainate receptors containing the GluR5 subunit appears to be selectively altered in the anterior cingulate cortex in schizophrenia and bipolar disorder.

Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro

The vulnerability of brain cells to neurologic insults varies greatly, depending on their neuronal subpopulation. However, cells surviving pathological insults such as ischemia or brain trauma may undergo structural changes, e.g., altered process growth, that could compromise brain function. In this study, we examined the effect of glutamate excitotoxicity on dendrite growth from surviving cortical GABAergic neurons in vitro. Glutamate exposure did not affect GABAergic neuron viability, however, it significantly reduced dendrite growth from GABAergic neurons. This effect was blocked by the AMPA receptor antagonists NBQX and CFM-2, and mimicked by AMPA, but not NMDA. Glutamate excitotoxicity also caused an NMDA receptor-mediated decrease in the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) immunoreactivity from GABAergic neurons, measured using immunocytochemical and Western blot techniques. GAD is necessary for GABA synthesis; however, reduction of GABA by 3-mercaptopropionic acid (3-MPA), which inhibits GABA synthesis, did not alter dendrite growth. These results suggest that GABAergic cortical neurons are relatively resistant to excitotoxic-induced cell death, but they can display morphological and biochemical alterations which may impair their function.