Mapping of brain areas containing RNA homologous to cDNAs encoding the alpha and beta subunits of the rat GABAA gamma-aminobutyrate receptor.

Abstract

An in situ hybridization technique was used to determine the distribution in rat brain of RNA homologous to cDNA clones encoding the a and .8 subunits of the rat brain GABAA yaminobutyrate receptor. The subunit proteins were mapped in adjacent sections autoradiographically and immmunohistochemically. Many brain areas containing high densities of GABAA receptors showed strong hybridization signals with both the a-and the 1subunit antisense RNA probe-e.g., cerebral cortex, hippocampus, and cerebellum. On a cellular level, a dense dendritic localization of GABAA receptors was correlated with a strong hybridization in the corresponding somata-e.g., in mitral cells of the olfactory bulb, pyramidal cells of hippocampus, granule cells of the dentate gyrus, and Purkinje and granule cells of the cerebellum. In some brain areas-e.g., substantia nigra--the intensity of the hybridization signal with the ,&subunit probe was much weaker than that with the a-subunit probe, whereas the inverse ratio of hybridization intensity was found in others-e.g., in bed nucleus. This regional heterogeneity in the hybridization pattern may reflect regional differences in RNA stability, transcription rate, or subunit composition. The results open the way for studies on the regulation of GABAA-receptor gene expression in normal and pathological brain in situ.The most abundant inhibitory neurotransmitter in the brain, ytaminobutyrate (GABA), facilitates chloride conductance of neuronal membranes by means ofGABAA receptors (1, 2). The function of GABAA receptors can be allosterically modulated, in particular by drugs acting at the benzodiazepine receptor (BZR) (3, 4); these drugs are widely used in the treatment of anxiety, insomnia, and epilepsy. The GABAA receptor complex is a heterooligomer consisting of a and p subunits (5-7) that not only contain the binding sites for GABA, BZR ligands, and other drugs (5-9) but also form the chloride channel (10).The identification of neurons that express the GABAA receptor is of therapeutic interest, since these cells propagate the drug effects elicited at the BZR. Such neurons have been identified electrophysiologically by their response to GABA and BZR ligands (1, 2). In addition, the distribution of the receptor protein helped to identify some GABA-recipient neurons (11, 12). However, an extensive mapping of neurons that express the GABAA receptor complex has not been possible. In situ hybridization histochemistry was therefore used to study the expression of mRNA homologous to the aand p-subunit cDNAs of the GABAA receptor in rat brain. The hybridization pattern was compared to the known regional, cellular, and subcellular distribution of the GABAA receptor protein in the rat central nervous system (11)(12)(13)(14).MATERIALS AND METHODS Preparation of Tissue. Male rats [specific pathogen-free (SPF) albino, Fullinsdorf, Switzerland] weighing 120-130 g were killed by transcardiac perfusion for 20-30 min with ice-cold phosphate-buffered saline containing as fixative either 4% (wt/vol) paraformal...

Pharmacological characterization and region‐specific expression in brain of the β2‐ and β3‐subunits of the rat GABAA receptor

“…No specific hybridization was observed in myelinated regions such as the corpus callosum; in addition, no labeling above background was detected using message sense probes as controls (data not shown). Our findings concur with, and extend those of others [9,24,25] in that mRNA encoding GABAA receptor subunits is detectable in brain regions that also contain benzodiazepine and muscimol binding sites. ~yl mRNA was prominently expressed throughout the CNS, including neurons of the olfactory bulb, anterior olfactory nuclei, islands of Calleja, neocortex, globus pallidus, CAl-3 hippocampal pyramidal cells, granule layer of the dentate gyrus, thalamic nuclei, inferior colliculus, geniculate nuclei, amygdala, pontine nuclei, cerebellum (granular, Purkinje and molecular cell layers) and cerebellar nuclei.…”

Photolabeled tryptic degradation products of benzodiazepine‐binding proteins are glycopeptides Implications for localization of cleavage sites

“…However, the preservation of the BZ-binding site which is thought to be located in the extracellular domain supports this conclusion ( Recently, a structural basis for the heterogeneity of the GABA* receptor has become apparent through the isolation of additional cDNA clones encoding proteins closely related to the a-subunit [ 16,181. The deduced amino acid sequences of the additional cDNAs [18] encompass more than two potential sites for N-linked glycosylation.…”

“…The a-subunit of the GABA/BZR from bovine [6] and rat brain [16,17] is believed to consist of a large extracellular N-terminus followed by four transmembrane regions (Ml-M4) and a short extracellular C-terminus region. The main BZbinding protein (53 kDa) in pig brain seems to share homologies with the bovine and rat cysubunits as shown by its size.…”

Regional expression of a MCD‐peptide and dendrotoxin I‐sensitive voltage‐dependent potassium channel in rat brain

“…This cDNA (called PCR3) was cloued into the Smul site of bluescript KS (Stratagene) in both orientation in order to generate single-stranded RNA probes sense and anti-sense using the T7 polymcrase (RNA transcription kit from Stratagene). Highly [3*S]thiotriphosphate labelled RNA probes were obtained following the Stratagene instructions and were purified and subjected to limited alkaline hydrolysis as described [13].…”

“…A series of mammalian brain K' channels have been cloned recently [7-lo] ferentiating the presence of mRNA populations having a certain degree of homology [13].…”