In Situ Hybridization Localization of the GABA A Receptor β2S- and β2L-Subunit Transcripts Reveals Cell-Specific Splicing of Alternate Cassette Exons

Abstract

We have recently described two variants of the chicken GABA A receptor β2 subunit which arise by alternative splicing of the corresponding primary gene transcript. The long form of the β2 subunit ( β2L) differs from the short form ( β2S) by the insertion of an additional 17 amino acids, in the large presumed intracellular loop, between the third and fourth membrane-spanning domains. In this study, we have utilized in situ hybridization with transcript-specific oligonucleotide probes to determine the regional and cellular localizations of the β2S- and β2L-subunit messenger RNAs in the 1-day-old chick brain. We show that the β2-subunit gene is expressed in many brain areas that also transcribe the GABA A receptor α1- and γ2-polypeptide genes. We also demonstrate that while the β2S- and β2L-subunit messenger RNAs frequently co-localize in many brain areas, certain structures (e.g., the ectostriatum, the hippocampus, the nucleus solitarius, the nucleus isthmi, pars parvocellularis, the nucleus isthmi, pars magnocellularis, the paleostriatum primitivum, the Purkinje cell layer, and the deep cerebellar nuclei) exclusively or predominantly contain either the β2S- or the β2L-subunit transcript. The distributions of the β2S- and β2L-polypeptide messenger RNAs resemble those previously described for the chicken GABA A receptor γ2S- and γ2L-subunit transcripts, respectively, which are also generated by alternative splicing. Our results indicate that a major GABA A receptor subtype in the avian brain is comprised of α1, β2 and γ2 subunits. In addition, the data obtained reveal that many neurons in the chicken CNS are capable of producing more than one alternatively-spliced form of a given primary gene transcript. However, the avian brain also appears to contain two small populations of neurons that possess mechanisms that result in either the incorporation of alternate cassette exons into mature transcripts, or the exclusion of such exons from processed messenger RNAs.