The mAb 62–3G1 to the GABAA receptor/benzodiazepine receptor/Cl- channel complex was used with light-microscopy immunocytochemistry for studying the localization of the GABAA receptors (GABAR) in the rat brain. The results have shown a receptor distribution identical to the one obtained by others using 3H-muscimol binding in combination with autoradiographic techniques. The external plexiform layer of the olfactory bulb, cerebral cortex, granule cell layer of the cerebellum, hippocampus, dentate gyrus, substantia nigra, dorsolateral and medium geniculate nuclei, and the lateral posterior thalamic nucleus, among other areas, were rich in GABAA receptor immunoreactivity. In the cerebellum the granule cell layer had more immunoreactivity than did the molecular layer. In the hippocampus the receptor was most abundant in the stratum oriens and in the molecular layer of the dentate gyrus. The immunocytochemical techniques have also allowed us to study the distribution of the GABAA receptor with high-resolution light microscopy. These studies have shown that the GABAA receptors are localized in neuronal membranes and concentrated in structures rich in GABAergic synapses, such as the cerebellar and olfactory glomeruli and the external plexiform layer of the olfactory bulb, the deep cerebellar nuclei, and the substantia nigra. The mAb 62–3G1 was generated by immunizing mice with the affinity-purified GABAA receptor/benzodiazepine receptor (BZDR) complex. This mAb bound to the 57,000 Mr peptide but not to the benzodiazepine binding 51,000 Mr peptide. The distribution of the GABAR immunoreactivity in the rat brain colocalized better with 3H-muscimol than with 3H-benzodiazepine binding. Therefore, it is suggested that (1) the 57,000 Mr peptide that is recognized by the mAb 62–3G1 is the muscimol (GABAA receptor agonist) binding subunit of the receptor complex, (2) there is an important population of brain GABAA receptors that is not functionally coupled to the benzodiazepine receptors, and (3) both the BZDR-coupled and uncoupled forms of the GABAA receptor are immunologically similar, if not identical.
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 cytoarchitecture of the cerebellar Bergmann fibers in the adult rat was investigated. Two monoclonal antibodies, one specific for the Bergmann fibers and astrocyte processes and the other specific for the cell bodies and dendrites of the Purkinje cells as well as an antiserum to the glial fibrillary acidic protein, were used in immunocytochemical peroxidase-antiperoxidase assays. The Bergmann fibers are revealed as columns organized in long vertical palisades parallel to the longitudinal plane of the folium. The palisades are not continuous; instead they are formed by sets of two to six aligned Bergmann fibers. Each of these sets of Bergmann fibers is separated from its longitudinally aligned neighbors by gaps. Each Bergmann fiber is formed by a bundle of two to four Bergmann glia processes which frequently show a helical organization. These results help to reconcile the different views on the organization of the .Bergmann fibers derived from the'studies done with the light microscope versus those done with the electron microscope. The Bergmann glia may play a fundamental role in directing the geometrical organization of the cerebellar constituents. 265 266
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.
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