Progesterone regulation of GABAA receptor plasticity in adult rat supraoptic nucleus

Marked plasticity in GABAA receptor signalling occurs in adult oxytocin neurons of the supraoptic nucleus (SON) through the modulation of GABAA receptor alpha subunits during pregnancy. The present studies were undertaken to examine the potential mechanisms underlying this plasticity. In vivo microdialysis experiments in conscious rats revealed that no significant changes in extracellular GABA concentrations occurred within the SON over the last two days of pregnancy and the time of parturition itself. In situ hybridization studies examined the effects of gonadal steroid manipulation upon the GABAA receptor subunits expressed by SON neurons (alpha1, alpha2, beta2 and gamma2 subunits) and demonstrated that cellular levels of the alpha1 subunit were increased following 8 days oestrogen and progesterone treatment. Estrogen alone or allopregnanolone, the progesterone derivative, had no effect on alpha1 subunit mRNA expression in the SON. Immunocytochemical experiments demonstrated progesterone receptors in many neural populations but not within the SON of late pregnant rats. These studies indicate that alterations in endogenous GABA release within the SON are unlikely to be responsible for the GABAA receptor plasticity exhibited by oxytocin neurons in late pregnancy. Rather, data demonstrate that the fluctuating concentrations of progesterone during pregnancy act indirectly on SON neurons to modulate alpha1 subunit mRNA expression. Together, these experiments provide evidence for the ligand-independent induction of GABAA receptor plasticity in the adult brain by progesterone.     

Withdrawal from progesterone increases expression of alpha4, beta1, and delta GABA(A) receptor subunits in neurons in the periaqueductal gray matter in female Wistar rats

Premenstrual dysphoric disorder (PMDD) shows comorbidity with other psychiatric conditions such as panic disorder (PD). The symptoms of both conditions are exacerbated during the late luteal phase of the menstrual cycle, when progesterone levels fall sharply. The present study investigated the effect of withdrawal from progesterone (PWD) on expression of alpha4, beta1, and delta GABA(A) receptor subunits in neurons within the panic circuitry of the midbrain periaqueductal gray matter (PAG) in adult female Wistar rats. Immunostaining for alpha4, beta1, and delta GABA(A) receptor subunits was present in neurons throughout the PAG in vehicle-treated animals (VEH), in rats after 24 hours withdrawal from a progesterone dosing regime (PWD, 5 mg kg(-1) i.p. twice daily for 6 days), and in animals maintained on progesterone for 7 days (HP). Compared to HP and VEH animals, which did not differ significantly from each other, the number of immunostained neurons present in the PAG of PWD rats was significantly higher. The effect was most pronounced in the dorsolateral column of the PAG. The parallel changes in the three GABA(A) receptor subunits suggests that falling progesterone levels may be associated with expression of new receptors of the alpha4beta1delta subtype. This could lead to functional changes in GABAergic transmission within the PAG. We suggest that changes in GABA(A) receptor-mediated inhibitory tone in the PAG consequent to withdrawal from progesterone may contribute to the increased anxiety and susceptibility to panic seen during the late luteal phase of the menstrual cycle in PMDD and PD patients.     

Gonadal steroids regulate GABAA receptor subunit mRNA expression in NT2-N neurons

Changes in gonadal steroid hormone levels during the menstrual cycle affect seizure frequency in women with catamenial epilepsy. Since GABA(A) receptors (GABARs) contribute to the prevention and termination of seizures by reducing neuronal excitability, we hypothesized that fluctuating gonadal steroid levels might affect GABAR subunit expression, which could alter inhibitory tone leading to increased seizure activity. To address this question in a simplified environment in vitro, we examined the effects of gonadal steroids on NT2-N neuronal cells. We have previously shown that NT2-N cells express functional GABARs, and that the expression pattern of GABAR subunits is regulated by chronic benzodiazepine exposure and hypoxia. NT2-N neurons were exposed to progesterone (0.1 microM), beta-estradiol (3 nM), or vehicle (DMSO) for 2 days or 7 days prior to RNA harvesting. GABAR subunit mRNA levels were assessed by semiquantitative RT-PCR normalized to actin levels. Progesterone exposure for 7 days increased alpha2 and gamma3 and decreased alpha5 subunit mRNAs, while beta-estradiol caused significant increases in alpha3, beta3 and epsilon expression. Further analysis revealed differential regulation of alpha4, alpha5, epsilon and pi subunit expression. Plots of relative PCR density in progesterone-treated cells for alpha2 vs. alpha5, alpha5 vs. gamma3 and alpha2 vs. gamma3 showed correlation between samples, suggesting coordinate regulation. Both progesterone and estrogen nuclear receptor mRNAs were detected by RT-PCR, and 2 days but not 7 days estrogen exposure upregulated progesterone receptor mRNA. Gonadal steroid fluctuations regulate GABA(A) receptor subunit expression in NT2-N cells. Such changes, if observed in vivo, could affect seizure frequency.     

Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus

The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.     

Differential expression of GABA(A) receptor subunits in the distinct nuclei of the rat amygdala

Detailed knowledge of the anatomical distribution of different GABA(A) receptor subunits is crucial for understanding the physiological actions of GABA in individual brain areas and for developing drugs acting through the individual GABA receptor subtypes. Since the amygdala is a key brain structure in the processing of emotional information with distinct functions in each nucleus, GABA(A) receptors in the amygdala are an important target of treatment for emotional disorders. In this study, we analyzed by quantitative RT-PCR the expression levels of all GABA(A) receptor subunits in distinct nuclei of the amygdala, the central (Ce) and the lateral/basolateral (LA/BLA) amygdala. We found the strongest expression of the gamma(2) subunit mRNA in both the Ce and LA/BLA, modest expressions of alpha(1), alpha(2) and alpha(3) mRNAs in the LA/BLA and alpha(2) and gamma(1) mRNAs in the Ce, and weak expressions of alpha(6), rho(2) and rho(3) mRNAs in both regions. We further revealed the significantly different expressions of alpha(1), alpha(3), alpha(5), gamma(1), gamma(2), delta, epsilon and theta subunit mRNAs in the Ce and LA/BLA. Differences in the expression levels of GABA(A) receptor subunits suggest different sensitivity to a variety of drugs including benzodiazepines and anesthetics in amygdala nuclei with distinct functions.     

Downregulation of the alpha5 subunit of the GABA(A) receptor in the pilocarpine model of temporal lobe epilepsy

Specific subunits of gamma-aminobutyric acid (GABA)A receptors may be regulated differentially in animal models of temporal lobe epilepsy during the chronic stage. Although several subunits may be upregulated, other subunits may be downregulated in the hippocampal formation. The alpha5 subunit is of particular interest because of its relatively selective localization in the hippocampus and its potential role in tonic inhibition. In normal rats, immunolabeling of the alpha5 subunit was high in the dendritic layers of CA1 and CA2 and moderate in these regions of CA3. In chronic pilocarpine-treated rats displaying recurrent seizures, alpha5 subunit-labeling was substantially decreased in CA1 and nearly absent in CA2. Only slight decreases in immunolabeling were evident in CA3. In situ hybridization studies demonstrated that the alpha5 subunit mRNA was also strongly decreased in stratum pyramidale of CA1 and CA2. Thus, the alterations in localization of the alpha5 subunit peptide and its mRNA were highly correlated. The large decreases in labeling of the alpha5 subunit did not appear to be related to loss of pyramidal neurons in CA1 or CA2 since these neurons were generally preserved in pilocarpine-treated animals. No comparable decreases in labeling of the alpha2 subunit of the GABA(A) receptor were detected. These findings indicate that the alpha5 subunit of the GABA(A) receptor is capable of substantial and prolonged downregulation in remaining pyramidal neurons in a model of temporal lobe epilepsy. The results raise the possibility that presumptive extrasynaptic GABA(A) receptor subunits, such as the alpha5 subunit, may be regulated differently than synaptically located subunits, such as the alpha2 subunit, within the same brain regions in some pathological conditions.     

GABA(A) receptor changes in delta subunit-deficient mice: altered expression of alpha4 and gamma2 subunits in the forebrain

The delta subunit is a novel subunit of the pentameric gamma-aminobutyric acid (GABA)(A) receptor that conveys special pharmacological and functional properties to recombinant receptors and may be particularly important in mediating tonic inhibition. Mice that lack the delta subunit have been produced by gene-targeting technology, and these mice were studied with immunohistochemical and immunoblot methods to determine whether changes in GABA(A) receptors were limited to deletion of the delta subunit or whether alterations in other GABA(A) receptor subunits were also present in the delta subunit knockout (delta-/-) mice. Immunohistochemical studies of wild-type mice confirmed the restricted distribution of the delta subunit in the forebrain. Regions with moderate to high levels of delta subunit expression included thalamic relay nuclei, caudate-putamen, molecular layer of the dentate gyrus, and outer layers of the cerebral cortex. Virtually no delta subunit labeling was evident in adjacent regions, such as the thalamic reticular nucleus, hypothalamus, and globus pallidus. Comparisons of the expression of other subunits in delta-/- and wild-type mice demonstrated substantial changes in the alpha4 and gamma2 subunits of the GABA(A) receptor in the delta-/- mice. gamma2 Subunit expression was increased, whereas alpha4 subunit expression was decreased in delta-/- mice. Importantly, alterations of both the alpha4 and the gamma2 subunits were confined primarily to brain regions that normally expressed the delta subunit. This suggests that the additional subunit changes are directly linked to loss of the delta subunit and could reflect local changes in subunit composition and function of GABA(A) receptors in delta-/- mice.     

Identification of residues within GABA(A) receptor alpha subunits that mediate specific assembly with receptor beta subunits.

GABA(A) receptors can be constructed from a range of differing subunit isoforms: alpha, beta, gamma, delta, and epsilon. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of alpha and beta subunits. The expression of a gamma subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within alpha subunit isoforms are important in the assembly of receptors comprised of alphabeta and alphabetagamma subunits. Deletion of these residues from the alpha1 or alpha6 subunits results in retention of either alpha subunit isoform in the endoplasmic reticulum on coexpression with the beta3, or beta3 and gamma2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the alpha1 and beta3 subunits, but were without affect on the production of alpha/gamma complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional alpha1beta3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of alpha1beta3 receptors and the resulting expression of beta3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S alpha1beta3 complex representing functional GABA(A) receptors. Therefore, our studies detail residues that specify GABA(A) receptor alphabeta subunit interactions. This domain, which is conserved in all alpha subunit isoforms, will therefore play a critical role in the assembly of GABA(A) receptors composed of alphabeta and alphabetagamma subunits.     

Transient expression of GABAA receptor alpha2 and alpha3 subunits in differentiating cerebellar neurons

In the adult mammalian brain, synaptic transmission mediated by gamma-amino butyric acid (GABA) plays a role in inhibition of excitatory synaptic transmission. During brain development, GABA is involved in brain morphogenesis. To clarify how GABA exerts its effect on immature neurons, we examined the expression of the GABAA receptor alpha2 and alpha3 subunits, which are abundantly expressed before alpha1 and alpha6 subunits appear, in the developing mouse cerebellum using in situ hybridization. Proliferating neuronal precursors in the ventricular zone and external granular layer expressed neither alpha2 nor alpha3 subunits. Hybridization signals for the alpha2 and alpha3 subunit mRNAs first appeared in the differentiating zone at embryonic day 13 (E13). The alpha2 subunit was detected in the migrating and differentiating granule cells and cerebellar nucleus neurons until postnatal day 14 (P14). Hybridization signals for the alpha3 subunit mRNA, on the other hand, were localized in the developing Purkinje cells and cerebellar nucleus neurons, and disappeared from Purkinje cells by the end of first postnatal week. Taken together, this indicated that the alpha2 and alpha3 subunits were abundantly expressed in distinct types of cerebellar neurons after completing cell proliferation while forming the neural network. These results suggest that GABA might extrasynaptically activate the GABAA receptors containing alpha2 and/or alpha3 subunits on the differentiating neurons before finishing the formation of synapses and networks, and could be involved in neuronal differentiation and maturation in the cerebellum.     

Heterogeneity of gamma-aminobutyric acid type A receptors in mitral and tufted cells of the rat main olfactory bulb

Mitral and tufted cells of the olfactory bulb receive strong gamma-aminobutyric acid (GABA)-ergic input and express GABA(A) receptors containing the alpha1 or alpha3 subunit. The distribution of these subunits was investigated in rats via multiple immunofluorescence and confocal microscopy, by using gephyrin as a marker of GABAergic synapses. A prominent immunoreactivity was detected throughout the external plexiform layer (EPL) and glomerular layer (GL). However, although staining for the alpha1 subunit was uniform throughout the EPL, that of the alpha3 subunit was most intense in the outer one-third of this layer. All mitral cells were positive for the alpha1 subunit. In contrast, the alpha3 subunit was restricted to a subpopulation of mitral cells, many of which also expressed calretinin. Likewise, external tufted cells could be subdivided into distinct groups, either singly labeled for the alpha1 or alpha3 subunit or doubly labeled. At the subcellular level, staining for the alpha1 and alpha3 subunits was punctate, forming clusters partially colocalized with gephyrin. However, many alpha1- and alpha3-positive clusters lacked gephyrin, suggesting the existence of either nonsynaptic GABA(A) receptor clusters or synaptic receptors not associated with gephyrin. Quantitative analysis of colocalization among the three markers in the inner EPL, outer EPL, and GL revealed considerable heterogeneity, suggestive of a differential organization of GABA(A) receptor subtypes in the apical and basal dendrites of mitral and tufted cells. Together these results reveal a complex subunit organization of GABA(A) receptors in the olfactory bulb and suggest that mitral and tufted cells participate in different synaptic circuits controlled by distinct GABA(A) receptor subtypes.     

Functional analysis of GABA(A) receptors in nucleus tractus solitarius neurons from neonatal rats.

To gain insight into specific GABA(A) receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1-14 day old rats using whole-cell patch clamp recordings. GABA(A-) receptor-mediated currents were detected in all neurons tested, with an average EC(50) of 22.2 microM. GABA currents were consistently stimulated by diazepam (EC(50)=63 nM), zolpidem (EC(50)=85 nM), loreclezole (EC(50)=10.1 microM) and the neurosteroid 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn(2+) (IC(50)=33.6 microM) picrotoxin (IC(50)=2.4 microM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10-1000 microM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant alpha subunit isoform present in GABA(A) receptors of the NTS appears to be the alpha1 and/or alpha2 subunit. beta2 and/or beta3 subunits are the major beta isoform, while the predominant gamma subunit is likely gamma2. Our data suggest the contribution to NTS GABA currents by alpha3-alpha6, beta1, gamma1 and delta subunits, if present, is minor by comparison.     

Compensatory alteration of inhibitory synaptic circuits in cerebellum and thalamus of gamma-aminobutyric acid type A receptor alpha1 subunit knockout mice

Targeted deletion of the alpha1 subunit gene results in a profound loss of gamma-aminobutyric acid type A (GABA(A)) receptors in adult mouse brain but has only moderate behavioral consequences. Mutant mice exhibit several adaptations in GABA(A) receptor subunit expression, as measured by Western blotting. By using immunohistochemistry, we investigated here whether these adaptations serve to replace the missing alpha1 subunit or represent compensatory changes in neurons that normally express these subunits. We focused on cerebellum and thalamus and distinguished postsynaptic GABA(A) receptor clusters by their colocalization with gephyrin. In the molecular layer of the cerebellum, alpha1 subunit clusters colocalized with gephyrin disappeared from Purkinje cell dendrites of mutant mice, whereas alpha3 subunit/gephyrin clusters, presumably located on dendrites of Golgi interneurons, increased sevenfold, suggesting profound network reorganization in the absence of the alpha1 subunit. In thalamus, a prominent increase in alpha3 and alpha4 subunit immunoreactivity was evident, but without change in regional distribution. In the ventrobasal complex, which contains primarily postsynaptic alpha1- and extrasynaptic alpha4-GABA(A) receptors, the loss of alpha1 subunit was accompanied by disruption of gamma2 subunit and gephyrin clustering, in spite of the increased alpha4 subunit expression. However, in the reticular nucleus, which lacks alpha1-GABA(A) receptors in wild-type mice, postsynaptic alpha3/gamma2/gephyrin clusters were unaffected. These results demonstrate that adaptive responses in the brain of alpha1(0/0) mice involve reorganization of GABAergic circuits and not merely replacement of the missing alpha1 subunit by another receptor subtype. In addition, clustering of gephyrin at synaptic sites in cerebellum and thalamus appears to be dependent on expression of a GABA(A) receptor subtype localized postsynaptically.     

Sex differences in GABAAergic system in rat substantia nigra pars reticulata

The substantia nigra pars reticulata (SNR) is involved in the control of movement disorders including seizures through its GABAergic neurons. Microinfusions of muscimol (a GABA(A) receptor agonist) produce specific effects on seizures depending on sex, infusion site (SNR(anterior) or SNR(posterior)) and age. To assess whether these effects are due to sex differences in GABAergic indices within the SNR we analyzed the expression of alpha(1) subunit mRNA of the GABA(A) receptor and the levels of GABA immunoreactivity (IR) of male and female rats at postnatal day 15 (PN15) and PN30. In each age, within the same SNR region, expression of alpha(1) subunit mRNA and intensity of GABA IR per neuron was higher in females compared to males. At PN15, in both sexes, there were no regional differences in expression of alpha(1) subunit mRNA and intensity of GABA IR. However, at PN30 in both sexes, expression of alpha(1) subunit mRNA and intensity of GABA IR per cell was higher in SNR(anterior) than in SNR(posterior). These results demonstrate that expression of alpha(1) subunit mRNA for GABA(A) receptor and levels of GABA IR in the SNR are sex- and site-specific, which may contribute to sex-, regional- and age-related differences in the expression of movement disorders and seizures.     

Evidence for coassembly of mutant GABAC rho1 with GABAA gamma2S, glycine alpha1 and glycine alpha2 receptor subunits in vitro

Functional coassembly of gamma-aminobutyric acid (GABA)C rho1 subunits with GABAA (alpha1, beta2, and gamma2S) or glycine (alpha1, alpha2, and beta) subunits was examined using two-electrode voltage-clamp recordings in the Xenopus laevis oocyte expression system. To facilitate this study, we took advantage of the unique gating and pharmacological properties of two mutant rho1 subunits, rho1(T314A) and rho1(T314A/L317A). When the rho1(T314A) subunit was coexpressed with GABA gamma2S, glycine alpha1 or glycine alpha2 subunits, GABA response properties were different from those of homomeric rho1(T314A) receptors. Additionally, the sensitivity of heteromeric rho1(T314A) and gamma2S receptors to picrotoxinin (PTX) blockade of GABA-evoked responses was altered compared to that of homomeric rho1(T314A) receptors. Changes in GABA response properties and picrotoxinin sensitivity were also observed when rho1(T314A) subunits were coexpressed with wild-type rho1 subunits. When rho1(T314A/L317A) subunits were coexpressed with GABA gamma2S, glycine alpha1 or glycine alpha2 subunits, suppression by GABA of spontaneously active current was reduced compared to that of homomeric rho1(T314A/L317A) receptors. Recovery of the spontaneous current from inhibition by GABA for GABA rho1(T314A/L317A)/gamma2S heteromeric receptors displayed an additional component. Coinjection of wild-type rho1 with gamma2S cRNAs at a ratio of 1 : 1 resulted in a > 10-fold reduction in GABA-evoked current. Furthermore, coexpression of wild-type rho1 and gamma2S subunits was found to shift the GABA dose-response curve. Our results provide functional evidence that the GABAC rho1 subunit can coassemble with the GABAA gamma2S subunit, and, at least in its mutated form, rho1 can also form heteromeric receptors with glycine alpha1 or alpha2 subunits in vitro.