Glutamic Acid Decarboxylase mRNA Levels Research Articles

GABAergic dysfunction in mGlu7 receptor-deficient mice as reflected by decreased levels of glutamic acid decarboxylase 65 and 67 kDa and increased reelin proteins in the hippocampus

Glutamate is the main excitatory neurotransmitter in the brain, while γ-aminobutyric acid (GABA) is a primary inhibitory neuromodulator. Both amino acids act through ionotropic and metabotropic receptors that are widely distributed in the central nervous system. There are at least eight subtypes of metabotropic glutamate receptors (mGlu), which have been divided into three groups (mGlu I, II, and III). The mGlu7 receptor subtype, which belongs to the mGlu III group, seems to play a special role, as it is abundant in brain structures that are known to be responsible for antidepressant and/or anxiolytic activity of drugs. In GABAergic neurons, GABA is synthesised from glutamate by the pyridoxal phosphate (PLP)-dependent enzyme glutamic acid decarboxylase (GAD). It is expressed as two major isoforms, GAD65 and GAD67, responsible for the synthesis of the vesicular and cytoplasmic pool of neurotransmitter, respectively. Moreover, GABAergic neurons express a variety of proteins such as reelin, involved in synaptic transmission and plasticity. The aim of our study was to investigate the regulation of GABA synthesis and the level of modulatory receptor for GABA in mice lacking mGlu7 receptor for glutamate. The levels of GAD mRNA, GADs, and reelin proteins in the hippocampi of mGlu7−/− and mGlu7−/+ mice were measured using in situ hybridisation, immunohistochemistry, and Western blotting (WB). GAD mRNAs in the CA and DG regions of the hippocampus were measured separately. The levels of GAD65, GAD67, and reelin proteins were determined in the homogenates using WB, and the number of stained neurons was estimated using a stereological method of counting. GABA B receptor level was measured using a radioligand binding assay. Our results show that the mRNA and protein levels of both GADs were decreased in the hippocampi of animals lacking the mGlu7 receptor. Decreased levels of GAD67 mRNA were found in both the CA and DG regions, while the decrease in GAD65 mRNA was observed mainly in the CA region of the hippocampus. The protein levels of GAD65 was lowered in mGlu7−/− animals only, while GAD67 and GABA B receptor number were decreased in both mGlu7+/− and mGlu7−/− mice when measured in the whole hippocampus. In contrast, reelin was shown to be increased both in mGlu7−/+ and mGlu7−/− mice. The results suggest that mGlu7 receptor is involved in the regulation of GABAergic system activity at the level of GABA synthesised enzymes, specific proteins expressed by GABAergic neurons and metabotropic receptor for GABA.

Metabotropic glutamate mGluR5 receptor blockade opposes abnormal involuntary movements and the increases in glutamic acid decarboxylase mRNA levels induced by l-DOPA in striatal neurons of 6-hydroxydopamine-lesioned rats

The present study examined the effect of a subchronic systemic administration of the glutamate metabotropic mGluR5 receptor antagonist MPEP on l-DOPA-induced dyskinesias and striatal gene expression in adult rats with a unilateral 6-OHDA lesion of dopamine neurons. The daily systemic administration of l-DOPA for 2 weeks induced a gradual increase in limb dyskinesia and axial dystonia. The subchronic systemic co-administration of MPEP reduced the severity of limb dyskinesia and axial dystonia over the whole duration of l-DOPA treatment. Subchronic l-DOPA administration was paralleled by a significant increase in mRNA levels of the two isoforms of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD67 and GAD65) and preprodynorphin (PPD). Single cell analysis on emulsion radioautographs indicated that l-DOPA-induced increases in GAD67 occurred predominantly in preproenkephalin-unlabeled striatonigral and, to a lesser extent, in preproenkephalin-labeled striatopallidal neurons. MPEP completely reversed the effects of l-DOPA on GAD67 and reduced the increases in GAD65 and PPD mRNA levels in striatonigral neurons. MPEP also reversed the small l-DOPA-induced increase in GAD67 mRNA levels in striatopallidal neurons. Altogether, the findings support the idea that the relative efficacy of mGluR5 receptor antagonists to oppose l-DOPA-induced abnormal involuntary movements involves an ability to oppose increases in GAD gene expression and GABA-mediated signaling in striatonigral and striatopallidal neurons. The results also confirm the potential usefulness of antagonists of mGluR5 receptors as adjuncts in the treatment of l-DOPA-induced dyskinesia in patients with Parkinson's disease.

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.

Middle‐Aged Female Rats Lack the Dynamic Changes in GAD67 mRNA Levels Observed in Young Females on the Day of a Luteinising Hormone Surge

The hypothalamic decapeptide gonadotrophin-releasing hormone (GnRH), modulates gonadotrophin synthesis and secretion and is essential for the preovulatory luteinising hormone (LH) surge. As females age, there is a gradual attenuation and eventual loss of the preovulatory LH surge and oestrous cyclicity. Data from previous studies have demonstrated evidence of compromised GnRH neuronal function at this time. The present study begins to explore the hypothesis that the age-related attenuation of the LH surge and decline in GnRH neuronal function are due, in part, to increased inhibitory influences on GnRH neurones. In situ hybridisation (ISH) was used to assess relative levels of mRNA for one isoform of glutamic acid decarboxylase (GAD), the rate-limiting enzyme for GABA synthesis. Ovariectomised young and middle-aged rats were injected with oestradiol benzoate and progesterone in a regimen for LH surge induction. Animals were killed at time points prior to, during the ascending phase, and during the peak and early descending phase of the LH surge. Dynamic changes in GAD(67) mRNA levels were observed in young but not middle-aged females in two regions known to be important for LH surge induction, the rostral proeptic area in the region of the organum vasculosum of the lamina terminalis (OVLT) and in the ventral periventricular preoptic area. Furthermore, GAD(67) mRNA levels were elevated in middle-aged relative to young females in the region of the OVLT at the time of LH surge induction and in the ventral periventricular preoptic area prior to surge induction. Age-related differences were not observed in other brain regions analysed. These data suggest that GABA synthesis may be elevated in middle-aged relative to young females in specific brain regions at critical times in conjunction with the LH surge, and that the lack of dynamic changes in GABA levels in these regions may contribute to the attenuated LH surge observed in middle-aged females.

Chronic exercise increases GAD gene expression in the caudal hypothalamus of spontaneously hypertensive rats

Previous studies have suggested that a γ-amino-butyric acid (GABA) deficit in the caudal hypothalamus (CH) of the spontaneously hypertensive rat (SHR) contributes to elevated levels of arterial pressure. The purpose of this study was to examine if SHR that underwent exercise training demonstrated a blunted development of hypertension and greater levels of glutamic acid decarboxylase (GAD) mRNA transcripts in the caudal hypothalamus. SHR were randomly paired and assigned to either a trained group (T; n=9) or a non-trained control group (NT; n=9). Trained animals were exercised for 10 weeks on a motorized treadmill while NT animals concurrently rested on a mock-treadmill. Following the 10-week training period, Northern blot analyses of mRNA for both the 65-kDa (GAD 65) and 67-kDa (GAD 67) isoforms of GAD were performed on tissue from caudal hypothalamic and cerebellar control brain regions. Exercise training simultaneously blunted the developmental rise in blood pressure in SHR (Δ59±9 mmHg in trained versus Δ77±9 mmHg in non-trained; P<0.03) and increased both GAD 65 (147±44%) and GAD 67 (162±77%) mRNA transcript levels in the CH ( P<0.05). In contrast, no difference was detected in GAD mRNA levels in the cerebellum between T and NT SHR. These findings are consistent with our previous functional studies and demonstrate that exercise can significantly and specifically upregulate GAD gene transcript levels in the caudal hypothalamus of hypertensive rats.

Serotonergic regulation of neuropeptide and glutamic acid decarboxylase mRNA levels in the rat striatum and globus pallidus: studies with fluoxetine and DOI

The serotonergic regulation of neuropeptide and glutamic acid decarboxylase (GAD) mRNA levels in the rat basal ganglia was investigated by determining the effects of chronic treatment with the serotonin uptake blocker fluoxetine and the serotonin 5-HT 2 agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrobromide (DOI). Fluoxetine (10 mg/kg) induced a reduction of preproenkephalin and GAD65 mRNA levels in the caudate-putamen and nucleus accumbens core and shell after 5 days of treatment. In addition, GAD65 mRNA levels were reduced in the globus pallidus. These changes appeared to be transient as they were not found after 14 days of fluoxetine treatment. DOI (7 mg/kg), administered for 9 days, induced a decrease of preprodynorphin mRNA levels in the caudate-putamen and the nucleus accumbens core and shell. No regional differentiation in the effects of fluoxetine and DOI was observed. Based on the present results, we propose that an increased 5-HT tone may reduce enkephalin and GABA mRNA levels in striatal regions and in the globus pallidus. Our results further show that preproenkephalin mRNA is not affected by chronic 5-HT 2 receptor stimulation, indicating that the fluoxetine-induced decrease in preproenkephalin mRNA levels involves other 5-HT receptors than the 5-HT 2 receptor. Preprodynorphin mRNA levels, on the other hand, were found to be reduced after chronic 5-HT 2 receptor stimulation. This observation, together with our previous finding that the 5-HT 2 antagonist ritanserin tends to increase preprodynorphin mRNA levels, suggests a 5-HT 2-mediated tonic inhibition of preprodynorphin mRNA levels.

Modulation of basal ganglia neurotransmission by the classical antipsychotic fluphenazine is due in part to the blockade of dopamine D 1-receptors

Classical antipsychotics, such as fluphenazine, influence neurotransmission by blocking both dopamine D 1- and D 2-receptors which in turn results in widespread adaptive changes in the neurochemistry of the basal ganglia. The purpose of the present study was to determine the role of D 1-receptors in mediating some of these neurochemical events, including changes in D 1- and D 2-receptor binding, and the expression of preproenkephalin and glutamic acid decarboxylase mRNAs. For these experiments, rats were given a depot injection of fluphenazine decanoate or injected twice daily for 21 days with the D 1-receptor antagonist SCH-23390. An additional group received both fluphenazine and SCH-23390 and controls were given saline. Fluphenazine administration decreased D 2-receptor binding throughout the basal ganglia while SCH-23390 was without effect. In contrast to the uniform reduction in D 2-receptor binding, fluphenazine altered D 1-receptor binding in a region-dependent manner. Region-dependent changes were also observed in animals given SCH-23390 which increased binding in the entopeduncular nucleus and posterior caudate-putamen without affecting other brain regions. Both fluphenazine and SCH-23390 significantly enhanced preproenkephalin and glutamic acid decarboxylase (GAD) mRNA expression in the anterior striatum. Fluphenazine also increased GAD mRNA levels in the entopeduncular nucleus. Together, these results indicate that the attenuation of D 1-receptor-mediated neurotransmission modulates a number of clinically relevant neurochemical processes in the basal ganglia.

Castration decreases single cell levels of mRNA encoding glutamic acid decarboxylase in the diagonal band of broca and the sexually dimorphic nucleus of the preoptic area.

Using quantitative in situ hybridization histochemistry (ISHH), we determined the effect of castration on single cell levels of glutamic acid decarboxylase (GAD) mRNA in discrete hypothalamic regions of the male rat brain associated with the control of gonadotropin secretion. A 48-base oligodeoxynucleotide probe was used to detect with equal affinity the two isoforms of GAD message, GAD65 and GAD67. GAD message also was quantitated in a number of selected areas of the brain to contrast GAD gene expression amongst several populations of GABAergic neurons. Comparison of 11 brain regions demonstrated a 9.3-fold range in the quantity of single cell GAD mRNA with levels being highest in the amygdala and the diagonal band of Broca, moderate in the piriform cortex, caudate nucleus, substantia innominata, globus pallidus, cingulate cortex and medial septal nucleus, and lowest in the lateral septal nucleus and the medial preoptic nucleus (MPN). Castration markedly reduced single cell GAD mRNA levels in the DBB and the MPN, two discrete hypothalamic structures known to contain dendritic fields, cell bodies, and axons of GnRH neurons projecting to the median eminence. A striking finding was a dense core of steroid-sensitive GABAergic neurons within the MPN comprising the sexually dimorphic nucleus of the preoptic area (SDN-POA). Similar to the MPN as a whole, the amount of GAD mRNA expressed by cells in the SDN-POA of sham operated control rats was greater than in castrated animals. GAD mRNA levels were inversely related to serum LH titers, suggesting a role for these neurons in the mechanism controlling gonadal steroid negative feedback on LH secretion. This report provides the basis for future work to determine if GAD65, GAD67 or whether both isoforms are affected by gonadal steroid input.

Gene Expression for Glutamic Acid Decarboxylase Is Reduced Without Loss of Neurons in Prefrontal Cortex of Schizophrenics

Up-regulation of gamma-aminobutyric acidA (GABAA) receptors and decreased GABA uptake in the cerebral cortex of schizophrenics suggest altered GABAergic transmission, which could be caused by primary disturbance of GABA synapses or by decreased production of the transmitter. Decreased production could be due to a shutdown in GABA production or to loss of GABA neurons caused by cell death or their failure to migrate to the cortex during brain development. To discriminate between these possibilities, we quantified levels of messenger RNA (mRNA) for the 67-kd isoform of glutamic acid decarboxylase (GAD), the key enzyme in GABA synthesis, and the number and laminar distribution of GAD mRNA--expressing neurons in the dorsolateral prefrontal cortex (DLPFC) of schizophrenics and matched controls, using in situ hybridization-histochemistry, densitometry, and cell-counting methods. These data were compared with the total number of neurons, the number of small, round or ovoid neurons 8 to 15 microns in diameter, and overall frontal lobe volume. As a control, mRNA levels for type II calcium-calmodulin-dependent protein kinase (CamIIK) were quantified. Schizophrenics showed a pronounced decrease in GAD mRNA levels in neurons of layer I (40%) and layer II (48%) and an overall 30% decrease in layers III to VI. There were also strong overall reductions in GAD mRNA levels. The CamIIK mRNA levels showed no significant differences between samples. No differences were found in the total number of neurons nor in small, round or ovoid neurons, which should include a majority of the GABA cells. Prefrontal gray and white matter volume did not differ significantly between controls and schizophrenics. The prefrontal cortex of schizophrenics shows reduced expression for GAD in the absence of significant cell loss. This may be brought about by an activity-dependent down-regulation associated with the functional hypoactivity of the DLPFC. The lack of significant alterations in cell numbers in the DLPFC and frontal lobe volume in schizophrenics also implies that overall cortical neuronal migration had not been compromised in development. Previous reports of altered neuronal distribution in the subcortical white matter of schizophrenic brains in comparison with that of controls may indicate disturbances of migration or programmed cell death in the cortical subplate, leading to altered connection formation in the overlying cortex of schizophrenics and activity-dependent down-regulation of neurotransmitter-related gene expression.

Dopaminergic and glutamatergic blocking drugs differentially regulate glutamic acid decarboxylase mRNA in mouse brain

Dopaminergic and glutamatergic inputs play an important role in regulating the activity of GABAergic neurons in basal ganglia. To understand more fully the biochemical interactions between these neurotransmitter systems, the effects of blocking dopamine and glutamate ( N- methyl- d-aspartate ) (NMDA) receptors on the expression of glutamic acid decarboxylase (GAD) mRNA were examined. Persistent blockade of dopamine receptors was achieved by daily injections of EEDQ, a relatively non-selective irreversible D 1 and D 2 dopamine receptor antagonist, or FNM, a relatively selective irreversible D 2 dopamine receptor antagonist. Persistent blockade of NMDA receptors was achieved by continuously infusing dizocilpine (MK-801), a non-competitive NMDA receptor antagonist. The levels of GAD mRNA in mouse brain were measured by in situ hybridization histochemistry following treatment with these agents. Repeated administration of EEDQ increased the levels of GAD mRNA in corpus striatum and frontal and parietal cortex; the first significant effects were seen after 4 days of treatment. Treatment with FNM elicited effects similar to those produced by EEDQ, except FNM also significantly increased GAD mRNA in nucleus accumbens. Neither EEDQ nor FNM produced significant effects on GAD mRNA in olfactory tubercle or septum. Infusion of MK-801 produced a rapid and marked decrease in the levels of GAD mRNA in corpus striatum, nucleus accumbens, olfactory tubercle, septum and frontal and parietal cortex; significant changes were seen as early as 2 days of treatment. No significant effects were seen in globus pallidus. Cellular analysis of emulsion autoradiograms from corpus striatum revealed that MK-801 reduced the amount of GAD mRNA in individual cells as well as the proportion of cells expressing high levels of GAD mRNA. These results suggest that dopamine, though its interaction with D 2 dopamine receptors, exerts an inhibitory effect on the expression of GAD mRNA, and that glutamate, though its interaction with NMDA receptors, exerts a stimulatory effect on GAD mRNA expression. They show further that the regulation of gene expression by dopamine receptors or NMDA receptors is different in different regions of the brain.

Activity-dependent changes in GAD and preprotachykinin mRNAs in visual cortex of adult monkeys.

Tachykinin-immunoreactive neurons are a subgroup of the GABA neuronal population in layer IVC of monkey primary visual cortex. Following brief periods of monocular deprivation in adult monkeys, immunoreactivity for both GABA and tachykinins is dramatically reduced in layer IV cells that lie within the deprived ocular dominance columns of this cortical area. The present study shows that these activity-dependent changes are associated with changes in mRNA levels but over different time courses. Radioactive antisense riboprobes derived from monkey-specific cDNAs were used to localize glutamic acid decarboxylase (GAD) and beta-preprotachykinin (beta PPT) mRNAs by in situ hybridization histochemistry. GAD and beta PPT mRNAs decreased in deprived ocular dominance columns of adult monkeys when neural activity was abolished in one eye by intraocular injections of tetrodotoxin (TTX). beta PPT mRNA levels fell within 5 d of deprivation and thus appeared to parallel the fall in immunodetectable tachykinin levels. By contrast, reduced GAD mRNA levels were detectable only after 15 d of deprivation and long after the fall in immunoreactive GAD and GABA levels has maximized. These results suggest that tachykinin gene expression is regulated by transcriptional mechanisms as part of the first response to reduced neural activity whereas the initial downregulation of immunoreactive GAD and GABA depends on posttranscriptional mechanisms. Following a more prolonged period of deprivation, a secondary mechanism for GAD regulation appears to be engaged at the level of gene transcription or possibly by changes in mRNA stability.

Loss of glutamate decarboxylase mRNA-containing neurons in the rat dentate gyrus following pilocarpine-induced seizures

In situ hybridization methods were used to determine if glutamic acid decarboxylase (GAD) mRNA-containing neurons within the hilus of the dentate gyrus are vulnerable to seizure-induced damage in a model of chronic seizures. Sprague-Dawley rats were injected intraperitoneally with pilocarpine, and the hippocampal formation was studied histologically at 1, 2, 4, and 8 week intervals after pilocarpine-induced seizures. In situ hybridization histochemistry, using a digoxigenin-labeled GAD cRNA probe, demonstrated a substantial decrease in the number of GAD mRNA-containing neurons in the hilus of the dentate gyrus in the pilocarpine-treated rats as compared to controls at all time intervals. Additional neuronanatomical studies, including cresyl violet staining, neuronal degeneration methods, and histochemical localization of glial fibrillary acidic protein, suggested that the decrease in the number of GAD mRNA-containing neurons was related to neuronal loss rather than to a decrease in GAD mRNA levels. The loss of GAD mRNA-containing neurons in the hilus contrasted with the relative preservation of labeled putative basket cells along the inner margin of the granule cell layer. Quantitative analyses of labeled neurons in three regions of the dentate gyrus in the 1 and 2 week groups showed statistically significant decreases in the mean number of GAD mRNA-containing neurons in the hilus of both groups of experimental animals. No significant differences were found in the molecular layer or the granule cell layer, which included labeled neurons along the lower margin of the granule cell layer. The results indicate that, in this model, a subpopulation of GAD mRNA-containing neurons within the dentate gyrus is selectively vulnerable to seizure-induced damage. Such differential vulnerability appears to be another indication of the heterogeneity of GABA neurons.

Abundant Gs? mRNA in basket cells of the dentate gyrus in adult rat hippocampus

In situ hybridization histochemistry (ISHH) has been used to study the differential distribution and relative abundance of mRNAs encoding a stimulatory alpha subunit of the G-protein (Gs alpha) and glutamic acid decarboxylase (GAD) in the dorsal hippocampus in adult rat brain. The present quantitative study shows that GABAergic neurons containing high levels of GAD mRNA, express considerably more Gs alpha message than excitatory principal neurons, the granule cells of the dentate gyrus and the pyramidal cells of CA1 subfield. A subpopulation of basket cells of the dentate gyrus exhibited a uniquely high level of Gs alpha mRNA, in addition to GAD. These findings may indicate a specific functional role for Gs alpha in these GABAergic neurons in the hippocampus.

6-Hydroxydopamine lesions of the nigrostriatal pathway alter the expression of glutamate decarboxylase messenger RNA in rat globus pallidus projection neurons

In situ hybridization was used to study the effect of 6-hydroxydopamine-induced damage to the midbrain dopaminergic neurons on the level of glutamate decarboxylase mRNA in globus pallidus neurons in the rat. Some animals received an injection of Fluoro-gold in the entopeduncular nucleus or the substantia nigra prior to the 6-hydroxydopamine lesion in order to identify glutamic acid decarboxylase mRNA levels in pallidal neurons that project to one of these targets. Analysis was carried out on a sample of all pallidal neurons as well as neurons that were identified as projection neurons in control and lesioned groups. The loss of the dopamine-containing neurons in the substantia nigra resulted in significant increases in the percentage of globus pallidus neurons that expressed glutamate decarboxylase mRNA and in the amount of glutamate decarboxylase mRNA per globus pallidus neuron. These increases were noted in a sample of all pallidal neurons, as well as pallidal neurons that were identified as projecting to either the entopeduncular nucleus or the substantia nigra. In control animals, glutamate decarboxylase mRNA was clearly identified in globus pallidus neurons projecting to the entopeduncular nucleus, indicating that this recently reported projection is at least partially GABAergic. The results of this study indicate that substantia nigra dopaminergic neurons regulate globus pallidus neurons in the rat, and that removal of the dopaminergic input to the corpus striatum results in a significant increase in the amount of glutamate decarboxylase mRNA in pallidal neurons. The decreased firing rate of pallidal neurons that is seen following the loss of dopamine input appears to be accompanied by an increase in the level of glutamate decarboxylase mRNA in these neurons.

Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus.

Ovarian steroids modulate learning, memory, and epileptic seizure activity, functions that are mediated in part by the hippocampus. Normal function depends on precise interactions between the inhibitory gamma-aminobutyric acid (GABA)ergic and excitatory glutamatergic neurons of the hippocampus. To determine whether estradiol and progesterone interact with GABAergic neurons, the levels of mRNA for glutamic acid decarboxylase (GAD), the rate-limiting enzyme for GABA synthesis, were measured by in situ hybridization histochemistry with 35S-labeled riboprobes complimentary to the feline GAD cDNA. The levels of mRNA for GAD were analyzed in selected region of the dorsal hippocampus and medial basal hypothalamus in ovariectomized, ovariectomized estradiol-treated, and ovariectomized estradiol- and progesterone-treated rats. In estradiol-treated rats, GAD mRNA levels increased in GABAergic neurons associated with the CA1 pyramidal cell layer, but not in the stratum oriens of CA1 or any other region of the hippocampus. Estradiol plus progesterone treatment reversed the estradiol-induced increase in GAD mRNA in CA1 and induced a small decrease in the hilus. No effect of estradiol or progesterone was observed in the dorsomedial, ventromedial, or arcuate nuclei of the hypothalamus. Estradiol or progesterone may alter cognitive performance and seizure activity by increasing or decreasing, respectively, the activity of GABAergic neurons in the hippocampus.

Differential effects of chronic ethanol administration on GABA A receptor α1 and α6 subunit mRNA levels in rat cerebellum

Chronic ethanol exposure alters muscimol, pentobarbital, and benzodiazepine agonist and inverse agonist effects on the function of GABA A receptor-gated Cl − channels in the central nervous system (CNS). We have recently shown that prolonged ethanol inhalation reduces the expression of GABA A receptor α1 and α2 subunit mRNAs in the rat cerebral cortex, with no effect on the level of α3 subunit transcripts, glutamic acid decarboxylase mRNA levels, or poly(A) + RNA levels. In the present study, rats were administered alcohol by liquid diet for 2 weeks using a pair-fed design. GABA A receptor α subunit mRNA levels were quantified by Northern analysis using specific cRNA probes. GABA A receptor α1 subunit mRNA levels were reduced in the cerebral cortex to the same extent as previously reported following prolonged ethanol inhalation. In the cerebellum, chronic ethanol ingestion reduced the levels of GABA A receptor α1 subunit mRNAs (4.8 and 4.4 kb) by 20–30% and increased the levels of GABA A receptor a6 subunit mRNA (2.7 kb) by 45%. GABA A receptor α2 and α3 subunit mRNAs were not detected in the cerebellum. Glutamic acid decarboxylase mRNA levels as well as poly(A) + RNA levels were not significantly altered following chronic ethanol exposure by liquid diet. Acute ethanol administration had no effect on GABA A receptor a6 subunit mRNA levels. However, acute administration of both Ro15-4513 and its vehicle control altered GABA A receptor α6 subunit mRNA levels in the cerebellum. Since GABA A receptor α6 subunits contain recognition sites for Ro15-4513, an inverse agonist, and an ethanol antagonist, the elevation in the expression of these subunits following chronic ethanol ingestion may account for increased sensitivity to inverse agonists after chronic ethanol administration and possibly contribute to the withdrawal syndrome. These data also suggest that chronic ethanol exposure regulates GABA A receptor gene expression by differential effects on the synthesis of specific subunits of GABA A receptors in the CNS.

Role of Dopaminergic D2 Receptors in the Regulation of Glutamic Acid Decarboxylase Messenger RNA in the Striatum of the Rat.

Levels of messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot and in situ hybridization analyses in the striatum of the rat, after chronic injections of two neuroleptics, sulpiride and haloperidol. The Northern blot analysis showed that the chronic injection of sulpiride at high doses (80 mg/kg, twice a day, 14 days) increased striatal GAD and PPE mRNA levels by 120% and 78% respectively, when compared to vehicle-injected rats. Haloperidol injections at relatively low doses (1 mg/kg, once a day, 14 days) produced parallel increases in GAD (40%) and PPE (52%) mRNA levels. After in situ hybridization densitometric measurements were performed on autoradiograms from rats treated with sulpiride, haloperidol or vehicle. The distribution of GAD and PPE mRNA signals in control rats was homogeneous along the rostrocaudal extension of the striatum. A similar increase was found along this axis after sulpiride (20%) and haloperidol (30%) treatments. The cellular observation of hybridization signals showed that grain density for GAD mRNA was increased in a majority of striatal cells after both treatments. By contrast, the PPE mRNA hybridization signal only increased in a subpopulation of neurons. The effects of such treatments were also analysed by measuring GAD activity in the striatum and in its output structures, the globus pallidus and the substantia nigra. After the administration of sulpiride, GAD activity was not modified in the striatum but increased in the globus pallidus (by 17%). After haloperidol treatment, GAD activity was increased in the globus pallidus (20%) and the substantia nigra (17%). It is concluded that the interruption of dopaminergic transmission, more precisely the D2 receptor blockade, promotes in striatopallidal neurons an increase in GAD mRNA accompanied by an increase in GAD activity and PPE mRNA. A possible regulation of GAD mRNA and GAD activity in striatonigral neurons is also discussed.

Expression of GAD mRNA in GABA interneurons of the rat medial frontal cortex

The distribution of glutamic acid decarboxylase (GAD) mRNA containing cells was studied in the rat medial frontal cortex (MFC). The neurons labelled by the 35S-labelled cDNA probe were distributed uniformly throughout all the layers and represented 16% of the total neuronal population. It was possible to distinguish two cell populations expressing high and low levels of GAD mRNA corresponding to 63% and 27% of labelled cells, respectively. Concerning the laminar distribution of these two populations of GAD mRNA containing neurons, no marked difference was observed between the various areas of the MFC.

Transplants of fetal substantia nigra regulate glutamic acid decarboxylase gene expression in host striatal neurons

Lesions of the dopaminergic innervation to the striatum result in increased activity of glutamic acid decarboxylase (GAD) and increased GAD mRNA in striatal GABAergic neurons. Here we show that solid transplants of dopamine-containing fetal mesencephalic tissue placed adjacent to the striatum can completely reverse the elevation of GAD mRNA in the striatum of adult rats with complete lesions of the nigrostriatal dopamine projections. The ability of the fetal transplants to re-establish control over gene expression in host target neurons indicates that there is a significant transneuronal influence of the transplanted neurons. Furthermore, striatal GAD mRNA levels appear to be a good marker of the functional impact of dopamine-producing transplants.

Parallel Decrease of Glutamic Acid Decarboxylase and Preproenkephalin mRNA in the Rat Striatum Following Chronic Treatment with a Dopaminergic D1 Antagonist and D2 Agonist

The levels of mRNA encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot analysis, in the dorsal and the ventral part of the striatum, following long-term treatments with drugs acting selectively on D1 or D2 dopaminergic receptors. Chronic injection of the selective D1 antagonist SCH 23390 elicited a significant decrease in level of both GAD and PPE mRNA (-30%) in the dorsal striatum, whereas no significant change was observed in the ventral striatum. Chronic administration of both SCH 23390 and RU 24926, a D2 agonist, decreased the GAD and PPE mRNA levels in the dorsal (-38 and -57%, respectively) as well as in the ventral (-70 and -60%, respectively) striatum. In the ventral striatum the marked reduction of GAD mRNA levels was paralleled by a significant decrease of Vmax values of GAD enzymatic activity (-41%). These results suggest that the decrease in content of both GAD and PPE mRNA, promoted by the chronic blockade of D1 receptors, is mainly due to the action of dopamine acting on unaffected D2 receptors. Indeed, this decrease is further amplified when the D2 agonist and the D1 antagonist are administered together. Our results substantiate further the molecular mechanisms by which dopamine acts on different populations of GABAergic and enkephalinergic neurons in the two striatal regions examined.