A gamma-aminobutyric acidA (GABAA) receptor (GABAAR) gamma 2 subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage lambda gt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors lambda gt11 and pGEX-3X. Both beta-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the gamma 2IL were purified, and a rabbit antibody to the LacZ-gamma 2IL was made. The antibody reacted with the gamma 2IL of both LacZ and GST fusion proteins and immunoprecipitated the GABAAR/benzodiazepine receptor (GABAAR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABAAR/BZDR immunoblots with a wide peptide band of 44,000-49,000 M(r). Immunoprecipitation studies with the anti-gamma 2IL antibody suggest that in the cerebral cortex, 87% of the GABAARs with high affinity for benzodiazepines and 70% of the GABAARs with high affinity for muscimol contain at least a gamma subunit, probably a gamma 2. These results indicate that there are [3H]muscimol binding GABAARs that do not bind [3H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABAAR/BZDR antibodies indicate that the most abundant combination of GABAAR subunits in the cerebral cortex involves alpha 1, gamma 2 (or other gamma), and beta 2 and/or beta 3 subunits. These subunits coexist in > 60% of the GABAAR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20-25%) of the cerebellar GABAAR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain alpha 6, also contain gamma 2 (or other gamma) subunit(s). The alpha 1 and beta 2 or beta 3 subunits are also frequently associated with gamma 2 (or other gamma) and alpha 6 in these cerebellar receptors.

A selective age-related decrease in both the protein and mRNA levels of the most abundant GABAA receptor subunits has been revealed in the rat inferior colliculus. The number (not affinity) of the native and fully assembled GABAA receptors assayed by 3H-muscimol binding was also decreased (35-49%). The decrease in GABA receptors was accompanied by a decrease in the protein and mRNA of the GABA-synthesizing enzyme glutamic acid decarboxylase. No other region of the rat brain showed such large age-related changes in these GABAergic synaptic molecules. Specific antibodies and riboprobes in conjunction with a computerized image analysis system were used to quantify immunocytochemistry and in situ hybridization. In old Sprague-Dawley rats, the combination of beta 2 and beta 3 peptide subunits was reduced 55%, while the beta 2 and beta 3 mRNAs were decreased 31% and 22%, respectively. The gamma 2S and gamma 2L subunit proteins decreased 43% and 21%, respectively, while the gamma 2 mRNA, including both short and long forms, was reduced 61%. The alpha 1 subunit protein was decreased 28%, whereas the alpha 1 mRNA decreased 40%. The glutamic acid decarboxylase protein was reduced 62% while GAD65 mRNA decreased 42%. Similar age-related changes were also observed in the inferior colliculus of Fischer-344 rats. In contrast, no changes were observed in the level of expression of some glial and/or neuronal proteins such as S-100, glial fibrillary acidic protein, and 160 KDa neurofilament protein in the inferior colliculus.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of the short (r,J and long (y2J subunits of the GABA, receptors in the rat brain has been revealed by light microscopy immunocytochemistry with novel subunitspecific antibodies (anti-y,, and anti-y,,). We have also used other subunit-specific antibodies including anti-y,lL2 (which recognizes both rzS and Yap), anti-a,COOH, and the monoclonal antibody 62-3Gl to & for comparing the regional and cellular distribution of the most abundant GABA, receptor subunits in the rat brain. The distributions of -yzS and yzL immunoreactivities are similar throughout the brain although the relative intensity of both signals varies depending on the brain area and neuronal type. In the hippocampus, cerebral cortex, and olfactory bulb (particularly mitral, periglomerular, and tufted neurons), yzS was more abundant than 72L. In contrast, the inferior colliculus, medulla, and the cerebellar Purkinje cells displayed more yzL than yzS immunolabeling. An important difference in the distribution of the various subunits was found in cerebellum: ySS and 'yzL were predominantly localized in the molecular layer, whereas (Y, and @,,, were more abundant in the granular layer. In the thalamus, -rSL and rzS were less abundant than either 01, or & subunits. The results showed that there is colocalization of rzS and yzL subunits in some brain areas and neuronal types, as well as areas of mismatch. Colocalization and mismatches were also found among a,, &, and y2, probably resulting from the heterogeneity in the subunit composition of the GABA, receptors through the brain.

No abstract

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.