GAD67 mRNA Expression Research Articles

High-Dose Methamphetamine Acutely Activates the Striatonigral Pathway to Increase Striatal Glutamate and Mediate Long-Term Dopamine Toxicity

Methamphetamine (METH) has been shown to increase the extracellular concentrations of both dopamine (DA) and glutamate (GLU) in the striatum. Dopamine, glutamate, or their combined effects have been hypothesized to mediate striatal DA nerve terminal damage. Although it is known that METH releases DA via reverse transport, it is not known how METH increases the release of GLU. We hypothesized that METH increases GLU indirectly via activation of the basal ganglia output pathways. METH increased striatonigral GABAergic transmission, as evidenced by increased striatal GAD65 mRNA expression and extracellular GABA concentrations in substantia nigra pars reticulata (SNr). The METH-induced increase in nigral extracellular GABA concentrations was D1 receptor-dependent because intranigral perfusion of the D1 DA antagonist SCH23390 (10 microm) attenuated the METH-induced increase in GABA release in the SNr. Additionally, METH decreased extracellular GABA concentrations in the ventromedial thalamus (VM). Intranigral perfusion of the GABA-A receptor antagonist, bicuculline (10 microm), blocked the METH-induced decrease in extracellular GABA in the VM and the METH-induced increase in striatal GLU. Intranigral perfusion of either a DA D1 or GABA-A receptor antagonist during the systemic administrations of METH attenuated the striatal DA depletions when measured 1 week later. These results show that METH enhances D1-mediated striatonigral GABAergic transmission (1), which in turn activates GABA-A receptors in the SNr (2), leading to a decrease in GABAergic nigrothalamic activity (3), an increase in corticostriatal GLU release (4), and a consequent long-term depletion of striatal DA content (5).

Differential effects of prolonged high frequency stimulation and of excitotoxic lesion of the subthalamic nucleus on dopamine denervation‐induced cellular defects in the rat striatum and globus pallidus

This study examined the effects of prolonged (4 days) high frequency stimulation (HFS) of the subthalamic nucleus (STN), in comparison with those of STN lesion, on the dopamine denervation-mediated cellular changes in the basal ganglia in a Wistar rat model of Parkinson's disease. STN HFS counteracted the dopamine lesion-induced increase in GAD67 mRNA expression in the output structures of the basal ganglia, as shown previously after STN lesion, providing cellular support for the similar antiparkinsonian benefits produced by the two surgical procedures. The dopamine denervation-induced increase in GAD67 mRNA levels in the globus pallidus was partially antagonized after HFS and totally reversed after ibotenate-induced STN lesion. The overexpression of striatal enkephalin mRNA tended to be further increased by HFS but was antagonized by STN lesion. The decrease in striatal substance P mRNA levels was affected neither by STN HFS nor lesion. As STN HFS for two hours was previously found not to interfere with the effects of dopamine lesion in the globus pallidus and striatum, the present data provide strong evidence that the effects of STN surgery in these structures involve long-term adaptive processes and that the rearrangements mediated by HFS and lesion are, at least in part, different.

GABA Neurons in the Human Prefrontal Cortex

Back to table of contents Previous article Next article Images in NeuroscienceFull AccessGABA Neurons in the Human Prefrontal CortexTakanori Hashimoto, M.D., Ph.D., David W. Volk, M.D., Ph.D., and David A. Lewis, M.D., Takanori HashimotoSearch for more papers by this author, M.D., Ph.D., David W. VolkSearch for more papers by this author, M.D., Ph.D., and David A. LewisSearch for more papers by this author, M.D., Pittsburgh, Pa.Published Online:22 Dec 2014https://doi.org/10.1176/ajp.161.10.1764AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail About 25% of cortical neurons utilize the inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Subclasses of GABA neurons (left panel) differ in their morphological, biochemical, and functional characteristics, indicating that they likely play distinct roles in regulating cortical circuitry. Some GABA neurons (a) provide inhibitory synapses to the cell bodies of nearby excitatory pyramidal neurons (Py) and to their initial axon segments; these appear to be positioned to regulate the firing of pyramidal neurons. OtherGABA neurons (b) provide inhibitory synapses to the distal portions of pyramidal neuron dendrites, where they modulate neighboring excitatory inputs to pyramidal neurons. The synapses of a third group of GABA neurons (c) provide inhibitory inputs to other GABA neurons, resulting in a downstream “disinhibition” of pyramidal neurons. These three subclasses of GABA neurons express different calcium-binding proteins. The (a) type of GABA neurons express parvalbumin (PV), whereas the (b) and (c) types of GABA neurons express calretinin (CR). The cell type-specific expression of parvalbumin and calretinin make it possible to use the expression of the messenger RNA (mRNA) encoding each protein to study the involvement of different subclasses of GABA neurons in brain diseases. As illustrated in the center panels, contiguous sections of normal human brain tissue from Brodmann’s area 9 were processed for in situ hybridization to detect the messenger RNA for GAD67 (an enzyme that synthesizes GABA), parvalbumin, and calretinin. GAD67 mRNA is expressed by all GABA neurons that are principally found in cortical layers II-VI, but not in the subjacent white matter (WM). Parvalbumin and calretinin mRNA are expressed selectively in subpopulations of GABA neurons, and these have different distributions across the cortical layers. The right series of panels illustrates changes in mRNA expression for these proteins in the same cortical region in subjects with schizophrenia. GAD67 mRNA expression is decreased, suggesting altered prefrontal GABA-mediated neurotransmission in schizophrenia. Furthermore, the expression of parvalbumin mRNA is decreased, whereas the expression of calretinin mRNA is unchanged. These findings, as a group, suggest that prefrontal GABA neurons regulating pyramidal neuron firing are more prominently affected in schizophrenia than are other subpopulations of GABA neurons. Dysregulation of pyramidal neuron firing may contribute to cognitive dysfunction in schizophrenia.Address reprint requests to Dr. Tamminga, UT Southwestern Medical Center, Department of Psychiatry, 5323 Harry Hines Blvd., #NC5.914, Dallas, TX 75390-9070; [email protected] (e-mail). Figure. GABA Neurons in the Human Prefrontal Cortex FiguresReferencesCited byDetailsCited ByUnique gene alterations are induced in FACS-purified Fos-positive neurons activated during cue-induced relapse to heroin seeking21 November 2012 | Journal of Neurochemistry, Vol. 124, No. 1Selective epigenetic alteration of layer I GABAergic neurons isolated from prefrontal cortex of schizophrenia patients using laser-assisted microdissection30 January 2007 | Molecular Psychiatry, Vol. 12, No. 4NeuroReport, Vol. 17, No. 6The Journal of Comparative Neurology, Vol. 490, No. 3 Volume 161Issue 10 October 2004Pages 1764-1764 Metrics PDF download History Published online 22 December 2014 Published in print 1 October 2004

GABA neurons in the human prefrontal cortex.

Back to table of contents Previous article Next article Images in NeuroscienceFull AccessGABA Neurons in the Human Prefrontal CortexTakanori Hashimoto, M.D., Ph.D., David W. Volk, M.D., Ph.D., and David A. Lewis, M.D., Takanori HashimotoSearch for more papers by this author, M.D., Ph.D., David W. VolkSearch for more papers by this author, M.D., Ph.D., and David A. LewisSearch for more papers by this author, M.D., Pittsburgh, Pa.Published Online:22 Dec 2014https://doi.org/10.1176/ajp.161.10.1764AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail About 25% of cortical neurons utilize the inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Subclasses of GABA neurons (left panel) differ in their morphological, biochemical, and functional characteristics, indicating that they likely play distinct roles in regulating cortical circuitry. Some GABA neurons (a) provide inhibitory synapses to the cell bodies of nearby excitatory pyramidal neurons (Py) and to their initial axon segments; these appear to be positioned to regulate the firing of pyramidal neurons. OtherGABA neurons (b) provide inhibitory synapses to the distal portions of pyramidal neuron dendrites, where they modulate neighboring excitatory inputs to pyramidal neurons. The synapses of a third group of GABA neurons (c) provide inhibitory inputs to other GABA neurons, resulting in a downstream “disinhibition” of pyramidal neurons. These three subclasses of GABA neurons express different calcium-binding proteins. The (a) type of GABA neurons express parvalbumin (PV), whereas the (b) and (c) types of GABA neurons express calretinin (CR). The cell type-specific expression of parvalbumin and calretinin make it possible to use the expression of the messenger RNA (mRNA) encoding each protein to study the involvement of different subclasses of GABA neurons in brain diseases. As illustrated in the center panels, contiguous sections of normal human brain tissue from Brodmann’s area 9 were processed for in situ hybridization to detect the messenger RNA for GAD67 (an enzyme that synthesizes GABA), parvalbumin, and calretinin. GAD67 mRNA is expressed by all GABA neurons that are principally found in cortical layers II-VI, but not in the subjacent white matter (WM). Parvalbumin and calretinin mRNA are expressed selectively in subpopulations of GABA neurons, and these have different distributions across the cortical layers. The right series of panels illustrates changes in mRNA expression for these proteins in the same cortical region in subjects with schizophrenia. GAD67 mRNA expression is decreased, suggesting altered prefrontal GABA-mediated neurotransmission in schizophrenia. Furthermore, the expression of parvalbumin mRNA is decreased, whereas the expression of calretinin mRNA is unchanged. These findings, as a group, suggest that prefrontal GABA neurons regulating pyramidal neuron firing are more prominently affected in schizophrenia than are other subpopulations of GABA neurons. Dysregulation of pyramidal neuron firing may contribute to cognitive dysfunction in schizophrenia.Address reprint requests to Dr. Tamminga, UT Southwestern Medical Center, Department of Psychiatry, 5323 Harry Hines Blvd., #NC5.914, Dallas, TX 75390-9070; [email protected] (e-mail). Figure. GABA Neurons in the Human Prefrontal Cortex FiguresReferencesCited byDetailsCited ByUnique gene alterations are induced in FACS-purified Fos-positive neurons activated during cue-induced relapse to heroin seeking21 November 2012 | Journal of Neurochemistry, Vol. 124, No. 1Selective epigenetic alteration of layer I GABAergic neurons isolated from prefrontal cortex of schizophrenia patients using laser-assisted microdissection30 January 2007 | Molecular Psychiatry, Vol. 12, No. 4NeuroReport, Vol. 17, No. 6The Journal of Comparative Neurology, Vol. 490, No. 3 Volume 161Issue 10 October 2004Pages 1764-1764 Metrics PDF download History Published online 22 December 2014 Published in print 1 October 2004

Glutamic acid decarboxylase 67 mRNA regulation in two globus pallidus neuron populations by dopamine and the subthalamic nucleus.

The globus pallidus (GP) consists of two neuron populations, distinguished according to their immunoreactivity for parvalbumin (PV). The PV-immunoreactive (PV+) neurons project preferentially to "downstream" targets such as the subthalamic and entopeduncular nuclei, whereas neurons lacking PV (PV- neurons) project preferentially to the striatum, suggesting a role for PV- cells in feedback to striatal neurons. Although dopamine D2 antagonist administration induces immediate early gene expression preferentially in PV- GP neurons, little is known about long-term regulation of PV- versus PV+ GP neurons. Nigral 6-hydroxydopamine (6-OHDA) lesions or repeated D2-class antagonist injections have been shown to increase pallidal expression of glutamate decarboxylase (GAD(67) isoform) mRNA. This increase in GAD(67) is believed to be secondary to activation of excitatory subthalamopallidal projections. The current study examined the effects of subthalamic nucleus (STN) lesion on 6-OHDA- or repeated D2 antagonist-induced changes in GP GAD(67) mRNA expression in PV+ and PV- neurons. Five or 21 d after nigral 6-OHDA injections or after 3, 7, or 21 d of D2 antagonist administration, GAD(67) mRNA increased in both the PV- and PV+ GP neurons, but the magnitude of the increase was significantly greater in PV- neurons. By contrast, STN lesion resulted in declines in GAD(67) mRNA in both cell populations, with the decreases in PV+ neurons exceeding those in PV- neurons. Furthermore, STN lesion completely blocked 6-OHDA- or D2 antagonist-induced GAD(67) mRNA increases in PV+ cells but only partly offset the GAD(67) mRNA increase in PV- pallidal neurons. Thus, the PV+ and PV- neurons are influenced in qualitatively similar ways by dopamine and the STN, but these cell types exhibit contrasting degrees of regulation by the dopaminergic and STN perturbations. This pattern of results has implications for pallidal control of striatal versus downstream basal ganglia nuclei.

Unchanged pancreatic beta-cell phenotype, function and sensitivity to cytokine-induced apoptosis in GAD-65 autoantigen-suppressed INS-1 cells

The enzyme glutamic acid decarboxylase (GAD) represents a major autoantigen in the early immunopathogenesis of T-cell-mediated destruction of pancreatic beta-cells in type 1 diabetes mellitus. Transgenic suppression of GAD expression in beta-cells of the non obese diabetic (NOD) mouse completely prevents development of the disease (1). Suppression of GAD-expression may thus provide a tool to prevent recurrence of autoimmune beta-cell destruction in transplanted pancreatic beta-cells in type 1 diabetic patients. Recent studies have shown that apoptosis may contribute to pancreatic beta-cell death in autoimmune diabetes. The aim of this study was the functional and phenotypic characterization of insulin producing pancreatic beta-cells with suppressed autoantigenic GAD-65 mRNA expression. GAD-65 expression in the rat pancreatic beta-cell line INS-1 was suppressed by stable transfection of vectors, that allow the overexpression of antisense mRNA for the rat isoform GAD-65 constitutively under the control of the CMV promoter. By reverse transcription polymerase chain reaction significant suppression of GAD-65 mRNA expression by 90% can be demonstrated. In INS-1 cell lines that stably express antisense GAD-65 mRNA, glucose-dependent insulin secretion (insulin RIA), beta-cell specific gene expression (RT-PCR for insulin, BETA2, glucokinase, GLUT2, PDX-1, Pax-4) and cytokine (IL-1beta, TNFalpha, IFNgamma, Mix) induced-apoptosis was tested. No significant alterations of these parameters were observed upon suppression of GAD-65 expression as compared to control cells. In summary these results demonstrate, that forced reduction of expression of the potential autoantigen GAD does not alter phenotype and function of insulin producing cells. Protection from pancreatic beta-cell destruction after GAD-suppression does not seem to be due to altered sensitivity to cytokine induced apoptosis.

Brain Neurosteroids in Gender-Related Aggression Induced by Social Isolation

Genetic, environmental, or hormonal factors and their interactions have been implicated in the expression of gender-related aggressive behavior in humans. Several independent lines of evidence support the role of hormonal and environmental factors in the aggressive behavior of experimental animals. Social isolation (SI) for 2-4 weeks in male but not in female mice results in the expression of aggression to a same-sex intruder. Long-term treatment (3 weeks) with anabolic steroids during SI in female mice induces aggressive behavior toward a male intruder of a severity similar to that observed in socially isolated (SI) male mice. The induced aggression in male and female mice is associated with a decrease of brain allopreg-nanolone (Allo). In SI male mice, aggression can be prevented by treatment with L-methionine (MET), which has also been shown to decrease reelin and GAD67 mRNA expression and maintain normal brain Allo content. The histone deacetylase inhibitor valproic acid can reverse this process, suggesting that histone tail acetylation may reverse the action of MET. We conclude that during social isolation, aggression can be controlled either by preventing Allo downregulation (e.g., by treatment with MET) or by direct administration of Allo or of agents (e.g., fluoxetine) that upregulate brain Allo content in SI mice.

Normalization of glutamate decarboxylase gene expression in the entopeduncular nucleus of rats with a unilateral 6-hydroxydopamine lesion correlates with increased gabaergic input following intermittent but not continuous levodopa

The expression of mRNA encoding for the 67 kilodalton isoform of glutamate decarboxylase (GAD67) was examined by in situ hybridization histochemistry in the entopeduncular nucleus (EP) of adult rats with a 6-hydroxydopamine unilaterally lesion of dopamine neurons. Our results provide original evidence that continuous or intermittent levodopa administration is equally effective at reversing the lesion-induced increase in GAD67 mRNA expression in the EP when compared with vehicle controls. To characterize the GABAergic interactions that may mediate levodopa-induced alterations in the EP, double-labeling in situ hybridization was conducted with a combination of GAD67 radioactive and preproenkephalin or preprotachykinin digoxigenin-labeled complementary RNA probes in the striatum. Levels of GAD67 mRNA labeling were significantly increased by intermittent, but not continuous levodopa. Analysis at the cellular level in a dorsal sector of the striatum revealed that GAD67 mRNA levels increased predominantly in preproenkephalin-unlabeled neuronal profiles, presumably striatal/EP neurons (+99.3%). Saturation analyses of 3H-flunitrazepam binding to GABA A receptors in the EP showed that the increase in GAD67 mRNA in preproenkephalin-unlabeled neurons by intermittent levodopa paralleled a significant decrease in number of GABA A receptors (Bmax) in the EP ipsilateral to the lesion. Continuous levodopa failed to alter striatal GAD67 mRNA levels, or the number or affinity of GABA A receptors when compared with vehicle-treated controls. These results suggest the normalization of GAD gene expression in the EP by intermittent levodopa involves an increase in GABAergic inhibition by striatonigral/EP neurons of the direct pathway. Conversely, the effects of continuous levodopa on GAD mRNA levels in the EP do not appear to be mediated by GABA.

Characterization of the neurochemical content of neuronal populations of the lamina terminalis activated by acute hydromineral challenge

The lamina terminalis (LT) contains three main regions, namely the subfornical organ (SFO), the median preoptic nucleus (MnPO) and the vascular organ of the LT (OVLT). Although LT is recognized of paramount importance in the regulation of hydromineral homeostasis, identity of the neurocircuits interconnecting the SFO and OVLT to the MnPO is not known. Furthermore, the phenotype of neuronal populations activated during acute hydromineral challenge is not yet determined. By using the high cellular resolution of the in situ hybridization histochemistry (ISHH), we investigated whether a furosemide-induced fluid and electrolyte depletion might modify both putative GABAergic and glutamatergic systems within the LT. We show that acute furosemide treatment (4 h) significantly reduced the expression of GAD67 mRNA, the active holoenzyme predictive of GABA synthesis, within the SFO. A strong tendency toward a reduction of GAD67 signal was also observed in the OVLT and MnPO. The hydromineral challenge did not alter the expression of GAD65 and type 2 vesicular glutamate transporter (vGlut2) mRNA in all the structures of the LT. Furosemide treatment was associated with a reduction in the population of GAD67-containing neurons in the periphery of the SFO and dorsal part of the MnPO. Contrastingly, GAD65-containing cells were shown to be increased in the OVLT and no change was observed for the vGlut2-containing neurons in the whole LT. By combining ISHH with immunohistochemistry (Fos immunoreactivity), we report that furosemide-induced water and sodium depletion did essentially recruit a glutamatergic network throughout the LT, although GABAergic neurons were specifically activated in the ring of the SFO and in the OVLT. The MnPO, the region of the LT that is considered as being an integrative area for sensory inputs arising from the SFO and OVLT, showed exclusive activation of excitatory neuronal populations. Taken together these results suggest that acute water and Na + depletion diminish the efficacy of the GABAergic system and mainly activates excitatory neuronal pathways in the regions of the LT.

Characterization of the neurochemical content of neuronal populations of the lamina terminalis activated by acute hydromineral challenge

The lamina terminalis (LT) contains three main regions, namely the subfornical organ (SFO), the median preoptic nucleus (MnPO) and the vascular organ of the LT (OVLT). Although LT is recognized of paramount importance in the regulation of hydromineral homeostasis, identity of the neurocircuits interconnecting the SFO and OVLT to the MnPO is not known. Furthermore, the phenotype of neuronal populations activated during acute hydromineral challenge is not yet determined. By using the high cellular resolution of the in situ hybridization histochemistry (ISHH), we investigated whether a furosemide-induced fluid and electrolyte depletion might modify both putative GABAergic and glutamatergic systems within the LT. We show that acute furosemide treatment (4 h) significantly reduced the expression of GAD67 mRNA, the active holoenzyme predictive of GABA synthesis, within the SFO. A strong tendency toward a reduction of GAD67 signal was also observed in the OVLT and MnPO. The hydromineral challenge did not alter the expression of GAD65 and type 2 vesicular glutamate transporter (vGlut2) mRNA in all the structures of the LT. Furosemide treatment was associated with a reduction in the population of GAD67-containing neurons in the periphery of the SFO and dorsal part of the MnPO. Contrastingly, GAD65-containing cells were shown to be increased in the OVLT and no change was observed for the vGlut2-containing neurons in the whole LT. By combining ISHH with immunohistochemistry (Fos immunoreactivity), we report that furosemide-induced water and sodium depletion did essentially recruit a glutamatergic network throughout the LT, although GABAergic neurons were specifically activated in the ring of the SFO and in the OVLT. The MnPO, the region of the LT that is considered as being an integrative area for sensory inputs arising from the SFO and OVLT, showed exclusive activation of excitatory neuronal populations. Taken together these results suggest that acute water and Na+ depletion diminish the efficacy of the GABAergic system and mainly activates excitatory neuronal pathways in the regions of the LT.

Differential effects of antipsychotics on haloperidol-induced vacuous chewing movements and subcortical gene expression in the rat

The behavioral and neurochemical effects of switching from typical to atypical medications have not been evaluated in the rodent models of tardive dyskinesia. Thus, we treated rats with haloperidol–decanoate for 12 weeks, and assessed the effects of additional treatment with olanzapine, haloperidol, clozapine, or vehicle on vacuous chewing movements and expression of transcripts for dopamine receptors, tyrosine hydroxylase, δ-opioid receptor, prodynorphin, preproenkephalin, glutamic acid decarboxylase-65 (glutamic acid decarboxylase (GAD)-65) and GAD-67 and N-methyl-D-aspartate (NMDA) receptor subunits in the striatum and its efferent pathways. Haloperidol–decanoate induced vacuous chewing movements extinguished following an additional 4 weeks of treatment with vehicle, olanzapine or haloperidol, but not clozapine. Post-treatment, vacuous chewing movements in the clozapine group were significantly higher than the vehicle, olanzapine and haloperidol groups. GAD-67 mRNA expression in the globus pallidus was decreased following additional treatment with olanzapine or haloperidol, but not clozapine. Changes in expression of other transcripts were not detected. These findings demonstrate important differences in the effects of typical and atypical antipsychotics on chronic vacuous chewing movements.

Norepinephrine–gamma-aminobutyric acid (GABA) interaction in limbic stress circuits: effects of reboxetine on GABAergic neurons

Background Reboxetine is a selective norepinephrine (NE) reuptake inhibitor that exerts significant antidepressant action. The current study assessed norepinephrine–γ-aminobutyric acid (GABA)-ergic mechanisms in reboxetine action, examining glutamic acid decarboxylase (GAD) mRNA expression in limbic neurocircuits following reboxetine within the context of chronic stress. Methods Male rats received 25 mg/kg reboxetine/day, p.o. Reboxetine and vehicle animals were exposed to 1 week of variable stress exposure or handling. Behavioral responses to stress (open field) were tested on day 7, and animals were killed on day 8 to assess neuroendocrine stress responses and limbic GAD65/67 mRNA regulation (in situ hybridization). Results Reboxetine significantly decreased behavioral reactivity in the open field. Reboxetine administration did not affect expression of GAD65/67 mRNA in handled rats; however, administration to stressed animals reduced GAD67 (but not GAD65) mRNA in the medial amygdaloid nucleus, posteromedial bed nucleus of the stria terminalis, and dentate gyrus. In contrast, GAD65 mRNA expression was increased by reboxetine in the lateral septum of stressed animals. Conclusions Norepinephrine pathways appear to modulate synthesis of GABA in central limbic stress circuits. Decreases in GABA synthetic capacity suggest reduced activation of stress-excitatory pathways and enhanced activation of stress-inhibitory circuits, and is consistent with a role for GABA in the antidepressant efficacy of NE reuptake inhibitors.

Experimental parkinsonism is associated with increased pallidal GAD gene expression and is reversed by site-directed antisense gene therapy.

The levels of mRNA encoding the two isoforms of glutamic acid decarboxylase (GAD(65) and GAD(67)) were measured throughout the pallidal complex in normal and acutely (i.e., 1 month duration) and chronically (i.e., 5 years duration) parkinsonian 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) -treated monkeys as well as in monkeys exposed to MPTP but asymptomatic for parkinsonism. GAD(65) mRNA labeling was modestly increased in the mid/caudal internal globus pallidus (GPi) but not in the external globus pallidus (GPe) in parkinsonian monkeys, compared with normal and asymptomatic monkeys. GAD(67) mRNA expression was highly increased in the mid/caudal GPi, and modestly increased in the GPe in parkinsonian monkeys compared with normal and asymptomatic animals. Infusion of GAD(67) antisense oligodeoxynucleotides bilaterally into the GPi resulted in a transient reversal of akinesia and bradykinesia that was not produced by infusion of missense oligodeoxynucleotides. These data emphasize the role of GAD enzyme (particularly GAD(67)) and GABA in the GPi for the expression of parkinsonian motor signs and suggest that selective manipulation of GABAergic neurotransmission in the GPi may have therapeutic potential for treating parkinsonism.

Baro-Activated Neurons With Pulse-Modulated Activity in the Rat Caudal Ventrolateral Medulla Express GAD67 mRNA

GABAergic neurons in the caudal ventrolateral medulla (CVLM) are believed to mediate the sympathetic baroreceptor reflex by inhibiting presympathetic neurons in the rostral ventrolateral medulla (RVLM). Accordingly, some CVLM neurons are activated by increased arterial pressure (AP; baro-activated), have activity strongly modulated by the AP pulse (pulse-modulated), and can be antidromically activated from the RVLM. This study examined whether baro-activated, pulse-modulated CVLM neurons are indeed GABAergic and examined their structures. We recorded extracellularly from 19 baro-activated, pulse-modulated CVLM neurons in chloralose-anesthetized rats. Most of these cells (13/19) were silenced by decreasing AP with nitroprusside, but some (6/19) remained active at low AP levels. They were also excited by phenyl biguanide (17/17) but inhibited by noxious tail pinch (8/11). Twelve baro-activated cells were filled with biotinamide and examined for expression of GAD67 mRNA. Because adjacent vagal motor neurons are also activated by increased AP, we examined choline acetyltransferase (ChAT) immunoreactivity. Most baro-activated cells (9/12) expressed high levels of GAD67 mRNA, the rest (3/12) displayed lower levels of GAD67 mRNA, but none showed ChAT immunoreactivity. In contrast, adjacent baro-inhibited CVLM cells had no GAD67 mRNA (n = 5) but were instead tyrosine hydroxylase immunoreactive (n = 7). Reconstruction of baro-activated CVLM neurons revealed axons that projected dorsomedially and rostrally with several axon collaterals. These data demonstrate the existence of GABAergic CVLM neurons with the physiological characteristics expected of interneurons that mediate the sympathetic baroreceptor reflex. In addition, baro-activated GABAergic CVLM neurons appear to integrate several types of inputs and provide inhibition to multiple targets.

Modulation of glucocorticoid-induced GAD expression in pancreatic beta-cells by transcriptional activation of the GAD67 promoter and its possible effect on the development of diabetes.

GAD is a pancreatic beta-cell autoantigen in humans and nonobese diabetic (NOD) mice. Modulation of GAD expression in pancreatic beta-cells has been suggested to be associated with the development of autoimmune diabetes. Hormonal changes through environmental stimuli are considered to influence the expression of the disease. We determined whether steroid hormones would modulate the expression of GAD in pancreatic beta-cells. We treated NOD mouse beta-cells (MIN6N8a cells) with various steroids, including testosterone, estradiol, progesterone, and cortisol, and examined the expression of GAD67 mRNA. We found that only cortisol enhanced the expression of GAD67, whereas the other steroid hormones had no effect. When we treated MIN6N8a cells with a synthetic glucocorticoid, dexamethasone, we found that GAD67 mRNA expression was stimulated in a dose- and time-dependent manner. Cells treated with 100 nmol/l dexamethasone for 6 h showed a 10-fold increase in the expression of GAD67 mRNA and an increase in GAD67 protein. The upregulation of GAD67 expression in beta-cells by dexamethasone was found to be due to the transcriptional activation of the GAD67 promoter. We then examined whether dexamethasone would influence the development of diabetes in NOD mice. Injection of dexamethasone into neonatal NOD mice resulted in a significant increase in the expression of GAD67 mRNA in pancreatic beta-cells and the development of insulitis and diabetes. We conclude that glucocorticoid hormones can modulate GAD expression by the transcriptional activation of the GAD promoter and may influence the development of autoimmune diabetes in NOD mice.

Recovery of song preferences after excitotoxic HVC lesion in female canaries.

The courtship solicitation display (CSD) of the female canary is a model to study estrogen dependent auditory preferences for male songs. The forebrain auditory-vocal nucleus, HVC, is part of the circuit that determines such preferences. To further develop this model we show that bilateral excitotoxic lesions of the medial part of HVC involving between 18-60% of the bilateral nucleus are behaviorally effective while complete unilateral lesions are not. Further, we show that animals recover their song preferences over a period of several months after the lesion. This functional recovery does not involve anatomical recovery of the HVC. Even 9 months after the lesion, the HVC size of these females was similar to that of females sacrificed 2 days after the lesion and thus was 40 +/- 8% smaller compared to normal females. Further, ipsilaterally, the lesion procedure transiently disturbed the neurochemistry, such as GAD-mRNA expression, in the part of HVC that did not undergo cell death. These results suggest that the integrity of the lateral part of at least one HVC is required to perform CSD in response to relevant auditory stimuli.

Effects of intralaminar thalamic nuclei lesion on glutamic acid decarboxylase (GAD65 and GAD67) and cytochrome oxidase subunit I mRNA expression in the basal ganglia of the rat.

This study investigated the influence of thalamic inputs on neuronal metabolic activity in the rat basal ganglia. By means of in situ hybridization histochemistry, we examined the consequences of ibotenate-induced unilateral lesion of intralaminar thalamic nuclei on mRNA expression of cytochrome oxidase subunit-I (CoI) in the striatum and the subthalamic nucleus (STN) and of the two isoforms of glutamate decarboxylase (GAD65 and GAD67) in the striatum, globus pallidus (GP), entopeduncular nucleus (EP) and substantia nigra pars reticulata (SNr). In the striatum, GAD67 mRNA expression decreased selectively in the rostral part of the structure at 5 and 12 days postlesion (approximately -30%), whereas, GAD65 mRNA levels was downregulated only in the caudal striatum at 12 days (-29%). In both the striatum and STN, CoI mRNA expression decreased ipsilaterally at 5 and bilaterally at 12 days. In GP, GAD67 and GAD65 mRNA expression decreased ipsilaterally at 5 (-20% and -26%) and 12 days (-23% and -36%). In EP, selective bilateral decreases in GAD67 mRNA expression were found at 5 and 12 days (-50% and -40%). Conversely, in SNr, only GAD65 mRNA expression was reduced bilaterally at both time points. These data show that the thalamus exerts a widespread excitatory influence on the basal ganglia network that cannot be accounted for solely by its known direct connections. Given the recent data showing that intralaminar thalamic nuclei are a major nondopaminergic site of neurodegeneration in Parkinson's disease, these results may have a critical bearing on understanding the cellular basis of basal ganglia dysfunction in parkinsonism.

Glutamate NMDA receptor subunit R1 and GAD mRNA expression in human temporal lobe epilepsy.

1. Molecular mechanisms underlying increased hippocampal excitability in human temporal lobe epilepsy (TLE) are largely unknown. A disturbance of the imbalance between excitatory and inhibitory neurotransmission pathways in the epileptic hippocampus may contribute substantially to a decreased seizure threshold. 2. We have extended the investigation whether TLE is associated with changes in the expression of GAD67 and NMDAR1 by assessing the relative amounts of the mRNAs in human hippocampal samples by means of semiquantitative RT-PCR. The samples included 16 hippocampal slices obtained at surgery from intractable TLE (HS, n = 14; non-HS, n = 2) and 3 postmortem control hippocampi. 3. The ratio for the GAD/NMDAR1 transcripts was significantly higher in TLE cases when compared to the nonepileptic samples. Such findings are mainly a consequence of the increased amounts of GAD mRNA detected in the epileptic hippocampus. Compared with nonepileptic samples, and without correction for neuron losses, the amounts of NMDAR1 mRNA in HS are slightly reduced, and in the non-HS samples they are significantly increased, which is consistent with an increase of NMDAR1 in the hippocampal remaining neurons, as previously reported. 4. Our results also contribute to the indication of GAD67 mRNA upregulation in human TLE. A possible functional implication for the increased GAD mRNA levels could be a mechanism to reduce neuronal hyperexcitability, synchronization, and/or the spread of seizure.

Single-cell RT-PCR detects shifts in mRNA expression profiles of basal forebrain neurons during aging

The medial septum and nucleus of the diagonal band (MS/nDB) contain cholinergic and GABAergic neuronal populations that have been identified based on immunohistochemical staining and/or electrophysiological properties. We explored the molecular diversity of MS/nDB neurons using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) to assess gene expression profiles during aging in individual neurons acutely isolated from young (2–4 months) and aged (26–27 months) F344 rats. Neuronal gene expression profiles were characterized by detection of mRNAs for choline acetyltransferase (ChAT, cholinergic) and glutamate decarboxylase (GAD67, GABAergic), as well as mRNAs for calcium binding proteins (CaBPs) calbindin-D28k, calretinin and parvalbumin. Four major neuronal populations were identified: ChAT-positive (ChAT+) cells, GAD-positive (GAD+) cells, ChAT+/GAD+ cells and ChAT negative/GAD negative (ChAT−/GAD−) cells. With age, the percentage of cells expressing ChAT mRNA decreased from 53% in young to 40%, and the expression of GAD67 mRNA was reduced from 56 to 35% of the cells tested. The percentage of cells with detectable levels of both ChAT and GAD67 mRNA was reduced from 24% in young to 9% in aged. Concomitantly, the percentage of ChAT−/GAD− cells increased from 15 to 34% with age. Of the CaBPs, calretinin expression was observed most frequently in this study, and its detection decreased from 33 to 22% of the cells with age. Observations concerning the CaBPs were confirmed using in situ hybridization. These results suggest a shift in the mRNA expression profiles of MS/nDB neuronal populations during aging and exemplify the molecular diversity of cholinergic and GABAergic cells.

Potential Role of Endogenous Brain-Derived Neurotrophic Factor in Long-Term Neuronal Reorganization of the Superior Colliculus after Bilateral Visual Deprivation

The role of the brain-derived neurotrophic factor (BDNF), the BDNF receptor (TrkB), and the glutamic acid decarboxylase (GAD67) after neonatal, bilateral nerve deafferentiation during postnatal development was investigated in the rat superior colliculus (SC). BDNF and GAD67 mRNA expression were significantly increased in optic (Op) and intermediate gray (InG) layers at 5, 8, 15, and 21 days after birth, but not in adult animals. However, TrkB mRNA expression was not modified at any time tested. At 15 days, where changes in BDNF and GAD67 mRNAs were more evident, an upregulation of the NMDAR1A mRNA glutamate receptor in the Op and InG, a modification in the pattern of synaptic zinc in the superficial layers of SC, and an increased synaptophysin immunoreactivity in the Op was found. This indicates the existence of a synergic mechanism between BDNF and NMDA to determine refinement of connections after the loss of visual input in SC.