GABA(B) receptor isoforms GBR1a and GBR1b, appear to be associated with pre- and post-synaptic elements respectively in rat and human cerebellum

Abstract

1. Metabotropic gamma-aminobutyric acid (GABA) receptors, GABA(B), are coupled through G-proteins to K+ and Ca2+ channels in neuronal membranes. Cloning of the GABAB receptor has not uncovered receptor subtypes, but demonstrated two isoforms, designated GBR1a and GBR1b, which differ in their N terminal regions. In the rodent cerebellum GABA(B) receptors are localized to a greater extent in the molecular layer, and are reported to exist on granule cell parallel fibre terminals and Purkinje cell (PC) dendrites, which may represent pre- and post-synaptic receptors. 2. The objective of this study was to localize the mRNA splice variants, GBR1a and GBR1b for GABA(B) receptors in rat cerebellum, for comparison with the localization in human cerebellum using in situ hybridization. 3. Receptor autoradiography was performed utilizing [3H]-CGP62349 to localize GABA(B) receptors in rat and human cerebellum. Radioactively labelled oligonucleotide probes were used to localize GBR1a and GBR1b, and by dipping slides in photographic emulsion, silver grain images were obtained for quantification at the cellular level. 4. Binding of 0.5 nM [3H]-CGP62349 demonstrated significantly higher binding to GABA(B) receptors in the molecular layer than the granule cell (GC) layer of rat cerebellum (molecular layer binding 200+/-11% of GC layer; P<0.0001). GBR1a mRNA expression was found to be predominantly in the GC layer (PC layer grains 6+/-6% of GC layer grains; P<0.05), and GBR1b expression predominantly in PCs (PC layer grains 818+/-14% of GC layer grains; P<0.0001). 5. The differential distribution of GBR1a and GBR1b mRNA splice variants for GABA(B) receptors suggests a possible association of GBR1a and GBR1b with pre- and post-synaptic elements respectively.

Figure 1

Autoradiographic images depicting total binding of 0.5 mM [³H]-CGP62349 in 10 μm sections of (a) rat cerebellum, and (b) human cerebellum. Non specific binding was equivalent to film background. Abbreviations: Gr, granule cell layer; Mol, molecular layer.

Figure 2

Autoradiographic images at macro- and microscopic levels depicting in situ hybridization of ³⁵S-dATP labelled oligonucleotides specific for GABA_B splice variants in 10 μm sections of rat cerebellum, in (a) and (b) GBR1pan, (c) and (d) GBR1a and (e) and (f) GBR1b. Abbreviations: Gr, granule cell layer; Mol, molecular layer; P, Purkinje cell.

Figure 3

Mean number of grains counted over Purkinje cells (filled bars) and granule cell layer (open bars) in rat cerebellum expressed as a percentage of grains counted in the granule cell layer (mean±s.e.mean, n=3) for (a) GRB1pan, (b) GBR1a and (c) GBR1b. Statistical analysis used Student's t-test (two tailed) where ^* represents P<0.05; ^*** represents P<0.0001.

Figure 4

Autoradiographic images at macro- and microscopic levels depicting in situ hybridization of ³⁵S-dATP labelled oligonucleotides specific for GABA_B splice variants in 10 μm sections of human cerebellar cortex, in (a) and (b) GBR1pan, (c) and (d) GBR1a and (e) and (f) GBR1b. The black areas at the top right of (e) represent artefact. Abbreviations: Gr, granule cell layer; Mol, molecular layer; P, Purkinje cell.

Figure 5

Number of grains counted over Purkinje cells (filled bars) and granule cell layer (open bars) of human cerebellum expressed as a percentage of grains counted in the granule cell layer (n=1) for (a) GBR1pan, (b) GBR1a and (c) GBR1b.