Cerebellar granule cells in vitro recapitulate the in vivo pattern of GABAA-receptor subunit expression.
GABAA-receptor heterogeneity is based on the combinatorial assembly of a family of 15 subunits (alpha 1-6, beta 1-3, gamma 1-3, delta, rho 1-2) into multiple receptor subtypes. The factors regulating the differential expression of GABAA-receptor subtypes in the CNS are largely unknown. In the present study, we have used primary cultures of rat cerebellar granule cells as model system to analyze to which extent the expression, subunit composition, and subcellular localization of GABAA-receptors depend on signals available in the cerebellum in vivo, in particular GABAergic input which is lost in vitro. GABAA-receptor subunits were visualized immunohistochemically with subunit-specific antibodies and their subcellular localization was investigated by confocal laser microscopy with selective markers for dendritic proteins (microtubule-associated protein 2, non-phosphorylated neurofilaments) and synaptic vesicles (synaptophysin). Triple immunofluorescence staining experiments revealed that dissociated granule cells taken from 7-day-old rats and maintained in culture for 9 days co-expressed the subunits alpha 1 alpha 6 beta 2,3 gamma 2 delta, a subunit repertoire which is identical to that seen in vivo in the adult cerebellum. GABAA-receptor subunits were localized selectively in granule cell somata and in proximal neurites immunopositive for MAP-2. No staining was detected in distal neurites immunopositive for neurofilaments. GABAA-receptor subunits frequently were aggregated in clusters closely apposed to synaptophysin-immunoreactive varicosities, suggesting a post-synaptic localization. Thus, major functional determinants of GABAA-receptors in granule cells (subunit repertoire, subcellular segregation and clustering in post-synaptic sites) develop in vitro, indicating that they are regulated to a large extent by intrinsic factors, independently of GABAergic input.
D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008297:Male; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis; D016900:Neurofilament Proteins / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000096:biosynthesis*; D016708:Synaptophysin / Q000096:biosynthesis
D008869:Microtubule-Associated Proteins; D016900:Neurofilament Proteins; D011963:Receptors, GABA-A; D016708:Synaptophysin