Molecular characterisation of neurotransmitter receptors in the CNS of the stargazer mutant mouse
The mutant mouse stargazer shows both ataxia and absence epilepsy from P14 onwards. A PGR amplification strategy was utilised to identify adult (i.e. > 3 months) +/stg from +/+ mice, which share the same phenotype, for use for breeding purposes. The same technique was employed to identify +/+, +/stg and stg/stg neonates (i.e. < 7 days old) for cell culture purposes, since the stargazer phenotype is not apparent at this age. GABA(_A) receptor α(_6) subunit expression levels were significantly decreased in adult stargazer (stg/stg) cerebella when compared to control (+/+ and +/stg) cerebella. Interestingly, autoradiography using [(^3)H] Ro15-4513 revealed an apparent upregulation in α(_4)γ-containing receptors in the adult stargazer dentate gyrus. No significant differences in the expression of NMDAR subunits were detected between adult control and stargazer brain membranes. A significant decrease was observed in AMPAR subunit expression within the adult stargazer cerebellum, particularly with the GIuR2 subunit, which was reduced by 73 %. This decrease was replicated in cerebellar granule cells cultured from stargazer neonates, which also expressed at the cell surface only 18 % of the total GluR2 found in control granule cells. Inmunohistochemistry analyses using mouse anti-stargazin antibodies revealed stargazin to be found throughout the adult control brain, with highest levels of expression being within the hippocampus and cerebellum. Stargazin protein, however, was not expressed in adult stargazer forebrain nor in adult stargazer cerebellar membranes. Finally, immunoaffinity columns using the anti-stargazin antibodies were prepared and demonstrated that stargazin could be purified from adult control mouse brain extracts. Moreover, AMPAR subunits co-immunoprecipitated, indicating an association in vivo.