Monaural conductive hearing loss alters the expression of the GluA3 AMPA and glycine receptor α1 subunits in bushy and fusiform cells of the cochlear nucleus.

The impact of conductive hearing loss (CHL), the second most common form of hearing loss, on neuronal plasticity in the central auditory pathway is unknown. After short-term (1 day) monaural earplugging, the GluA3 subunits of the AMPA receptor (AMPAR) are upregulated at auditory nerve synapses on the projection neurons of the cochlear nucleus; glycine receptor α1 (GlyRα1) subunits are downregulated at inhibitory synapses in the same neuronal population. These data suggest that CHL affects receptor trafficking at synapses. We examined the impact of 7 days of CHL on the general expression of excitatory and inhibitory receptors by quantitative biochemistry and immunohistochemistry, using specific antibodies to detect AMPAR subunits (GluA1, GluA2, GluA2/3, and GluA4), GlyRα1, and the GABA(A) receptor subunits ß2/3. Following monaural earplugging and an elevation of the hearing threshold by approximately 35 dB, the immunolabeling of the antibody for the GluA2/3 subunits but not the GluA2 subunit increased on bushy cells (BCs) and fusiform cells (FCs) of the ipsilateral ventral and dorsal cochlear nuclei. These same cell types showed a downregulation of the GlyRα1 subunit. Similar results were observed in the contralateral nuclei. The expression levels of GABA(A) ß2/3 were unchanged. These findings suggest that, following longer periods of monaural conductive hearing loss, the synthesis and subsequent composition of specific glutamate and glycine receptors in projection neurons and their synapses are altered; these changes may contribute to abnormal auditory processing.

Clustered and non-clustered GABAA receptors in cultured hippocampal neurons.

In cultured hippocampal neurons, gamma2 subunit-containing GABA(A) Rs form large postsynaptic clusters at GABAergic synapses and small clusters outside GABAergic synapses. We now show that a pool of non-clustered gamma2 subunit-containing GABA(A) Rs are also present at the cell surface. We also demonstrate that myc- or EGFP-tagged gamma2, alpha2, beta3 or alpha1 subunits expressed in these neurons assemble with endogenous subunits, forming GABA(A) Rs that target large postsynaptic clusters, small clusters outside GABAergic synapses or a pool of non-clustered surface GABA(A) Rs. In contrast, myc- or EGFP-tagged delta subunits only form non-clustered GABA(A) Rs, which can be induced to form clusters by antibody capping. A myc-tagged chimeric gamma2 subunit possessing the large intracellular loop (IL) of the delta-subunit IL (myc gamma2S/delta-IL) assembled into GABA(A) Rs, but it did not form clusters, therefore behaving like the delta subunit. Thus, the large intracellular loops of gamma2 and delta play an important role in determining the synaptic clustering/non-clustering capacity of the GABA(A) Rs.

Biochemical analysis of GABA(A) receptor subunits alpha 1, alpha 5, beta 1, beta 2 in the hippocampus of patients with Alzheimer's disease neuropathology.

Alzheimer's disease (AD) is characterized by selective vulnerability of specific neuronal populations within particular brain regions. For example, hippocampal glutamatergic cell populations within the CA1/subicular pyramidal cell fields have been found to be particularly vulnerable early in AD progression. In contrast, hippocampal GABA-ergic neurons and receptors appear resistant to neurodegeneration. Despite relative sparing of GABA(A) receptors in AD, it is possible that the specific subunit composition of these receptors may undergo alterations with disease progression. In order to address this issue, we employed quantitative Western blot analysis to examine protein levels of GABA(A) receptor subunits alpha 1, alpha 5, beta 1, beta 2 in the hippocampus of subjects displaying increasing severity of AD neuropathology. Subjects were categorized into three groups based upon Braak staging pathologic criteria: pathologically mild (stages I/II, n=9); moderate (stages III/IV, n=8); and severe (stages V/VI, n=7). Across all subject groups, levels of subunit protein were heterogeneously distributed throughout the five hippocampal subregions analyzed (subiculum, CA1-3, dentate gyrus). Statistical analyses revealed differential preservation of GABA(A) receptor subunits in AD. In particular, alpha 1, beta 1, and beta 2 displayed little difference in protein levels among pathologically mild, moderate, and severe subject groups. In contrast, although relatively modest, protein levels of the alpha 5 subunit were significantly reduced between subjects with severe neuropathology compared with pathologically mild subjects (13.5% reduction). Collectively, our data provide evidence for heterogeneous distribution and relative sparing of GABA(A) receptor subunits in the hippocampus of AD patients.

Immunocytochemical localization of the beta(3) subunit of the gamma-aminobutyric acid(A) receptor in the rat brain.

A novel anti-beta(3) subunit-specific GABA(A) receptor (GABA(A)R) antibody has been prepared by immunizing a rabbit with a bacterial fusion protein of the large intracellular loop of the beta(3) subunit. The antiserum immunoprecipitated the solubilized GABA(A) receptor. The anti-beta(3) antibody was affinity purified on immobilized beta(3) large intracellular loop peptide. In immunoblots, the purified antibody reacted with a 57 KDa peptide. Immunocytochemistry with the affinity-purified antibody has revealed the localization of the beta(3) subunit in the rat brain. A comparative study with the immunocytochemical distribution of the beta(2) subunit has also been performed. There are areas of the brain and cell types where the distribution of beta(2) and beta(3) overlap (i.e., cerebral cortex, cerebellum,and most layers of the olfactory bulb). There are also clear differences in the expression of beta(3) and beta(2) in other brain areas and cell types. Thus, high beta(3) but low or no beta(2) expression was observed in the corpus striatum and in granule cells of the olfactory bulb. In the hippocampus the expression of beta(3) was considerably higher than that of beta(2), but some hippocampal interneurons showed high expression of beta(2). High beta(2) but little or no expression of beta(3) was observed in thalamic nuclei, substantia nigra, globus pallidus, inferior colliculus and the short axon cells of the olfactory bulb.

Normal electrophysiological and behavioral responses to ethanol in mice lacking the long splice variant of the gamma2 subunit of the gamma-aminobutyrate type A receptor.

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.

Dopamine D2 receptor effects on GABA(A) receptor expression may modify melanotrope peptide release.

Stimulation of melanotrope dopamine D2 receptors decreases mitotic rate, calcium channel activity, and the biosynthesis of several proteins. This study demonstrates that D2 receptor activation also affects GABA(A) receptor beta2/beta3 subunit immunoreactivity. Following chronic treatment with haloperidol, a D2 receptor antagonist, GABA(A) receptor immunoreactivity increased, whereas it decreased after chronic treatment with bromocriptine, a dopamine D2 receptor agonist. Thus, these data indicate that D2 function regulates GABA(A) receptor expression in melanotropes, a mechanism by which peptide release may be modified.

N-acetylaspartylglutamate stimulates metabotropic glutamate receptor 3 to regulate expression of the GABA(A) alpha6 subunit in cerebellar granule cells.

We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 microM) results in the transient increase in content of GABA(A) alpha6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine and (2S)-alpha-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in alpha6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, alpha2-adrenergic, muscarinic, and GABA(B) receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in alpha6 levels induced by 30 microM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the beta-adrenergic receptor agonist isoproterenol. The increase in alpha6 mRNA content is followed by a fourfold increase in alpha6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional alpha6-containing GABA(A) receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA(A) alpha6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA(A) receptor subunit composition.

Coexistence of two beta subunit isoforms in the same gamma-aminobutyric acid type A receptor.

Three novel subunit-specific antisera to the beta1, beta2, and beta3 subunits of rat gamma-aminobutyric acid type A (GABAA) receptors have been used to study the native receptor in the rat brain. Affinity-purified anti-beta1, anti-beta2, and anti-beta3 antibodies recognized in immunoblots protein bands of 57, 55, and 57 kDa, respectively. Quantitative immunoprecipitation of solubilized GABAA receptors from various rat brain regions showed that the beta2 subunit was the most abundant isoform in cerebellum (in 96% of the GABAA receptors) and cerebral cortex (64%) but that it was the least abundant isoform in hippocampus (44%). The beta3 subunit was found most abundant in hippocampus (64%) followed by cerebral cortex (48%) and cerebellum (33%). The beta1 subunit was present in a very small proportion of the cerebellar GABAA receptors (3%), but it was present in a high proportion of the GABAA receptors from the hippocampus (49%) and cerebral cortex (32%). Quantitative receptor immunoprecipitation or immunopurification followed by immunoblotting experiments have revealed the existence of colocalization of two different beta subunit isoforms in a significant proportion of the brain GABAA receptors. Thus, in the rat cerebral cortex 33% of the GABAA receptors have both beta2 and beta3 subunits, and 19% of the receptors have both beta1 and beta3 subunits. The extent of colocalization of beta subunit isoforms varied among brain regions, being highest in hippocampus and lowest in cerebellum. These and other results taken together suggest that the number of alpha, beta, and gamma subunits (stoichiometry) in the brain GABAA receptor pentamers might not be unique. It might vary depending on receptor type.

GABAA receptor subunit expression changes in the rat cerebellum and cerebral cortex during aging.

Significant aging-related decreased expression of various GABAAR subunit mRNAs (alpha 1, gamma 2, beta 2, beta 3 and sigma) was found in both cerebellum and cerebral cortex using quantitative dot blot and in situ hybridization techniques. Contrary to the other subunits, the alpha 6 mRNA expression was significantly increased in the aged cerebellum. Parallel age-related changes in protein expression for gamma 2 and beta 2/3 (decrease) and alpha 6 (increase) were revealed in cerebellum by quantitative immunocytochemistry. However, no significant changes in alpha 1 protein expression nor in the number or affinity of [3H]zolpidem binding sites were detected in cerebellum even though alpha 1 mRNA expression was significantly decreased in the aged rat. Age-related increased expression of alpha 6 mRNA and protein in the cerebellum was accompanied by no significant changes in the number of diazepam-insensitive [3H]Ro15-4513 binding sites. In the cerebral cortex, no changes in the protein expression of the main GABAA receptor subunits (alpha 1, gamma 2 and beta 2/3) were observed which contrasted with the age-related decreased expression of the corresponding mRNAs. No significant changes in the number or affinity of [3H]zolpidem binding sites were observed in the cerebral cortex. Thus, age-related changes in the mRNA expression of a particular subunit does not necessarily lead to similar changes in protein or assembly into mature GABAA receptors. The results reveal the existence of complex regulatory mechanisms of GABAA receptor expression, at the transcriptional, translational and post-translational and/or assembly levels, which vary with the subunit and brain area.

Aging-related subunit expression changes of the GABAA receptor in the rat hippocampus.

Aging-related changes in the subunit expression of some hippocampal GABAA receptors have been found. Quantitative in situ hybridization has revealed that alpha 1, subunit messenger RNA expression was significantly increased in the hippocampus (34%) of old rats. The largest increases were observed in the dentate gyrus (76%) and in the CA1 field (30%). Quantitative immunocytochemistry also showed increased protein expression of the alpha 1 subunit in the dentate gyrus (19%) and CA1 (14%) of old rats. The increased alpha 1 messenger RNA and protein expression led to increased proportions of assembled GABAA receptors that contained alpha 1 subunits, as revealed by quantitative immunoprecipitation of (3H)flunitrazepam and (3H)muscimol binding. In contrast, there were no significant changes in the expression of beta 2, beta 3 and total gamma 2 (gamma 2S + gamma 2L) subunits, although a slightly increased expression of gamma 2L peptide was detected in the hippocampus proper (7%), but not in the dentate gyrus. The results are consistent with the notion that in the rat hippocampus there is an aging-related change in the subunit composition of some GABAA receptors.

The alpha 1 and alpha 6 subunits can coexist in the same cerebellar GABAA receptor maintaining their individual benzodiazepine-binding specificities.

Two GABAA receptor subunit-specific antibodies anti-alpha 6 and anti-alpha 1 have been used for elucidating the relationship between the presence of alpha 1 and/or alpha 6 subunits in the cerebellar GABAA receptors and the benzodiazepine-binding specificity. Receptor immunoprecipitation with the subunit-specific antibodies shows that 39% of the cerebellar GABAA receptors have alpha 6, whereas 76% of the receptors have alpha 1 as determined by [3H]-muscimol binding. Results show that 42-45% of the receptors having alpha 6 also have alpha 1, whereas 13-15% of the receptors that contain alpha 1 also have alpha 6. The immunoprecipitation results as well as immunopurification and immunoblotting experiments reveal the existence of three types of cerebellar GABAA receptors; i.e., one has both alpha 1 and alpha 6 subunits, a second type has alpha 1 but not alpha 6, and a third type has alpha 6 but not alpha 1 subunits. The results also show that receptors where alpha 1 and alpha 6 subunits coexist have two pharmacologically different benzodiazepine-binding properties, each associated with a different alpha subunit. The alpha 1 subunit contributes the high-affinity binding of [3H]Ro 15-1788 (flumazenil) and the diazepam-sensitive binding of [3H]Ro 15-4513. The alpha 6 subunit contributes the diazepam-insensitive binding of [3H]Ro 15-4513, but it does not bind [3H]Ro 15-1788 with high affinity. Thus, in the cerebellar alpha 1- alpha 6 GABAA receptors, there is no dominance of the pharmacology of one alpha subunit over the other.

Brain GABAA receptors studied with subunit-specific antibodies.

Brain GABAA/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl- channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl- channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl- channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABAA receptor subunits and the relationships between subunit composition, ligand binding, and Cl- channel properties. Nevertheless, little is known about the complete subunit composition of the native GABAA receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (no reconstituted) brain GABAA receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABAA receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABAA receptors and that have fewer side effects.

The gamma subunits of the native GABAA/benzodiazepine receptors.

Subunit-specific antibodies to all the gamma subunit isoforms described in mammalian brain (gamma(1), gamma(2S), gamma(2L), and gamma(3) have been made. The proportion of GABA(A) receptors containing each gamma subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the gamma(1) and gamma(3) subunits are present in considerable smaller proportion of GABA(A) receptor than the gamma(2) subunit. Immunocytochemistry shows that gamma(1) immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, gamma(1) immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, gamma(3) immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in gamma(2S). In the cerebellum, gamma(1)++ immunolabeling was localized on the Bergmann glia. The gamma(2S) and gamma(2L) subunits are differentially expressed in various brain regions. Thus the gamma(2S) is highly expressed in the olfactory bulb and hippocampus whereas the gamma(2L) is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The gamma(2S) and gamma(2L) coexist in some brain areas and cell types. Moreover, the gamma(2S) and gamma(2L) subunits can coexist in the same GABA(A) receptor pentamer. We have shown that this is the case in some GABA(A) receptors expressed in cerebellar granule cells. These GABA(A) receptors also have alpha and beta subunits forming the pentamer. Immunoblots have shown that the rat gamma(1), gamma(2S), gamma(2L) and gamma(3) subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the gamma(2S) and gamma(2L) subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA(A) receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various gamma subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA(A) receptors in brain.

Distribution of immunoreactivity for the beta 2 and beta 3 subunits of the GABAA receptor in the mammalian spinal cord.

The localization of GABAA receptors in cat and rat spinal cord was analyzed using two monoclonal antibodies specific for an epitope shared by the beta 2 and beta 3 subunits of the receptor. beta 2/beta 3-subunit immunoreactivity was the most intense in inner lamina II, lamina III, and lamina X, and it was the least intense in lamina IX. In laminae I-III, generally, the staining had a rather diffuse appearance, but the surfaces of small cell bodies in these laminae were outlined clearly by discrete labeling, as were many cell bodies and dendrites in deeper laminae. Rhizotomy experiments and ultrastructural observations indicated that beta 2/beta 3-subunit immunoreactivity in the dorsal horn was largely localized in intrinsic neuropil elements rather than in the terminals of primary afferent fibers, even though labeling overlapped with the terminal fields of different types of primary afferents and was also detected on the membranes of dorsal root ganglion neurons. With few exceptions (most notably, a highly immunoreactive group of dorsolaterally located cells in the cat lumbar ventral horn), motoneurons expressed low levels of beta 2/beta 3-subunit immunoreactivity. Labeling of neuronal membranes was fairly continuous, but focal accumulations of beta 2/beta 3-subunit immunoreactivity were also detected using immunofluorescence. Focal "hot spots" correlated ultrastructurally with the presence of synaptic junctions. Dual-color immunofluorescence revealed that focal accumulations of beta 2/beta 3-subunit immunoreactivity were frequently apposed by glutamic acid decarboxylase (GAD)-immunoreactive terminals. However, the density of continuous-membrane beta 2/beta 3 immunolabeling and GAD terminal density were not correlated in many individual neurons. The results suggest the existence of "classical" (synaptic) and "nonclassical" (paracrine) actions mediated via spinal cord GABAA receptors. The study also revealed the relative paucity of beta 2/beta 3-subunit immunoreactivity postsynaptic to certain GABAergic terminals, particularly those presynaptic to motoneurons or primary afferent terminals.

Immunocytochemical localization of the alpha 6 subunit of the gamma-aminobutyric acidA receptor in the rat nervous system.

The localization in the rat central nervous system and retina of the alpha 6 subunit peptide of the gamma-aminobutyric acid (GABAA) receptor has been studied by light microscopy immunocytochemistry with a specific anti-alpha 6 antibody. The alpha 6 subunit was present in the granule cells of the cerebellum, the granule cells of the dorsal cochlear nucleus, axons of the olfactory nerve including the glomerular endings, layer II of the dorsal horn of the spinal cord, and in the retinal synaptic layers, particularly the inner plexiform layer. Thus, contrary to the general belief, the alpha 6 subunit is not exclusively localized in the granule cells of the cerebellum. It is also expressed in some sensory neurons and other neurons involved in the initial processing of sensory information.

Altered expression of gamma 2L and gamma 2S GABAA receptor subunits in the aging rat brain.

Aging-related alterations in both protein and mRNA expression of gamma 2S and gamma 2L subunits of the GABAA receptors have been observed in several brain areas of Sprague-Dawley and Fischer 344 rats. Subunit-specific antibodies to gamma 2S and gamma 2L as well as a riboprobe to the large intracellular loop of gamma 2, which recognizes both gamma 2S and gamma 2L mRNAs, in conjunction with computerized image analysis were used for quantitative immunocytochemistry and in situ hybridization. In addition, specific oligonucleotide probes to gamma 2S or gamma 2L mRNA were used for quantitative dot blot hybridization. A large increase in the number of heavily immunostained neurons with the anti-gamma 2L antibody was detected in the cerebral cortex (115%) of old rats. However, only a small (but significant) aging-related increase in the density of gamma 2L immunostaining (7%) was observed throughout the cerebral cortex whereas no significant aging-related change in gamma 2L mRNA was detected in this brain region. Contrary to gamma 2L, the gamma 2S immunostaining did not show aging-related increased number of heavily immunostained neurons in cerebral cortex. Moreover, the density of gamma 2S immunostaining and the expression of gamma 2S mRNA were significantly decreased in the cerebral cortex (9-24%). Important aging-related changes were also found in the cerebellum of old rats where the expression of both gamma 2S and gamma 2L peptides was significantly decreased (24% and 23% respectively). This decrease in gamma 2 protein expression was accompanied by decreased expression of gamma 2S (16-38%) and gamma 2L (24%) mRNAs. Nevertheless, the most important decrease of gamma 2S (48%) and gamma 2L protein (20%) was revealed in the molecular layer of the cerebellum. In addition, the expression of gamma 2S protein was increased (14%) whereas the expression of gamma 2L was decreased (13%) in the granule cell layer. Therefore, the relative expression of gamma 2S protein in both layers was reversed in old animals. The observed aging-related changes in the expression of GABAA receptor subunits might lead to altered GABAA receptor/benzodiazepine receptor subunit composition.

The alpha 4 subunit of the GABAA receptors from rat brain and retina.

A novel anti-alpha 4 antibody has been used for the purification and characterization of the alpha 4-containing GABAA receptors in the rat brain and for studying the immunocytochemical distribution of the alpha 4 subunit peptide in rat brain and retina. The anti-alpha 4 antibody recognized a 66 kDa peptide in brain membranes and immunoprecipitated 10-28% of the brain GABAA receptors in various brain regions as determined by [3H]muscimol binding. The highest immunoprecipitation values were obtained in the thalamus and the lowest in the cerebellum. Surprisingly, the receptors immunoprecipitated by anti-alpha 4 showed little or no diazepam-insensitive or diazepam-sensitive [3H]Ro15-4513 binding sites in any brain region. In the cerebellum, where 25% of the [3H]Ro15-4513 binding is diazepam-insensitive, much of the latter was immunoprecipitated by an anti-alpha 6 antibody but not by the anti-alpha 4 antibody. Immunoblots of immunoaffinity-purified GABAA receptors from the cerebral cortex on immobilized anti-alpha 4 revealed molecular colocalization of alpha 4 and gamma 2. However, the absence of significant benzodiazepine binding in these GABAA receptors suggests that the assembly of the alpha 4 and gamma 2 subunits in the cerebral cortex and in other brain regions is such that they do not normally form diazepam-insensitive [3H]Ro15-4513 binding sites. This result contrasts with the presence of diazepam-insensitive [3H]Ro15-4513 binding sites in the GABAA receptors expressed in heterologous systems resulting from the combination of alpha 4, gamma 2 and beta 2 subunits. Immunocytochemistry has revealed the abundance of alpha 4 peptide immunoreactivity in the thalamus and dentate gyrus (mainly in the hilar neurons and the inner third of the granule cell layer). The alpha 4 immunoreactivity is also present in the external plexiform layer of the olfactory bulb and in all layers of the neocortex and pyriform cortex. In the retina, alpha 4 is concentrated on ganglion cells (including some giant ganglion cells), the inner plexiform layer and to a lesser extent in the outer plexiform layer.

Differential expression of GABAA/benzodiazepine receptor subunit mRNAs and ligand binding sites in mouse cerebellar neurons following in vivo ethanol administration: an autoradiographic analysis.

The gamma-aminobutyric acidA (GABAA)/benzodiazepine (BZ) receptor is a pentamer composed of subunits belonging to several classes (alpha 1-6, beta 1-4, gamma 1-4, delta, and rho 1 and rho 2). In situ hybridization, radioligand autoradiography, and immunocytochemistry were used to examine GABAA/BZ receptor alpha 1, alpha 6, beta 2, beta 3, and gamma 2 subunit expression in murine Purkinje, granule, and deep cerebellar neurons after in vivo ethanol exposure. Chronic ethanol treatment resulted in decreased alpha 1 subunit mRNA expression in each cell type, whereas the expression of alpha 6 and gamma 2 subunit mRNA levels increased; no changes were observed in the expression of beta 2 and beta 3 subunit mRNA. GABA and BZ agonist binding and antibody staining paralleled the changes in mRNA levels. Acute ethanol injection resulted in increased expression of alpha 1 and beta 3 mRNAs, whereas levels of alpha 6, beta 2, and gamma 2 mRNAs remained stable. Our results indicate that, in cerebellar neurons, the expression of specific GABAA/BZ receptor subunit mRNAs, polypeptides, and binding sites is independently regulated by in vivo administration of alcohol. The observed changes were not restricted to any one cerebellar cell type, because subunit expression in Purkinje, granule, and deep cerebellar cells was similarly affected.

Localization of GABAA receptor immunoreactivity in NO synthase positive myenteric neurones.

GABAA receptors were localized within laminar preparations of the rat distal colon myenteric plexus using a monoclonal antibody (mAb 62-3G1) to the affinity purified GABAA receptor/benzodiazepine receptor/Cl- channel complex. The immunofluorescence procedure showed that approximately half of the myenteric ganglion cells displayed extensive GABAA receptor labelling of their soma. This population was further characterised by treating some GABAA-receptor-labelled laminar preparations for the histochemical demonstration of nitric oxide (NO) synthase-related NADPH-dependent diaphorase activity. A subpopulation of the GABAA-receptor-immunoreactive cells (35%) were also found to display intense NO-synthase-related activity. These findings extend our understanding of the GABAA-receptor-related innervation of the rat gut wall herein referred to as 'A-GABAergic' and provides an anatomical basis for the pharmacologically-identified GABA-nitrergic pathway in the mammalian gut.

Monoclonal antibodies to the human gamma 2 subunit of the GABAA/benzodiazepine receptors.

The large intracellular loop (IL) of the gamma 2 subunit of the cloned human gamma-aminobutyric acidA (GABAA) receptor (gamma 2 IL) was expressed in bacteria as glutathione-S-transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the gamma 2 IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABAA/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000-49,000-M(r) peptide band in immunoblots of affinity-purified GABAA receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABAA receptors in various brain regions.