Aberrant cerebellar granule cell-specific GABAA receptor expression in the epileptic and ataxic mouse mutant, Tottering

Abstract

The Tottering (cacna1atg) mouse arose as a consequence of a spontaneous mutation in cacna1a, the gene encoding the pore-forming subunit of the pre-synaptic P/Q-type voltage-gated calcium channel (VGCC, CaV2.1). The mouse phenotype includes ataxia and intermittent myoclonic seizures which have been attributed to impaired excitatory neurotransmission at cerebellar granule cell (CGC) parallel fiber–Purkinje cell (PF–PC) synapses [Zhou YD, Turner TJ, Dunlap K (2003) Enhanced G-protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca2+-channel mutant mouse, tottering. J Physiol 547:497–507]. We hypothesized that the expression of cerebellar GABAA receptors may be affected by the mutation. Indeed, abnormal GABAA receptor function and expression in the cacna1atg forebrain has been reported previously [Tehrani MH, Barnes EM Jr (1995) Reduced function of gamma-aminobutyric acid A receptors in tottering mouse brain: role of cAMP-dependent protein kinase. Epilepsy Res 22:13–21; Tehrani MH, Baumgartner BJ, Liu SC, Barnes EM Jr (1997) Aberrant expression of GABAA receptor subunits in the tottering mouse: an animal model for absence seizures. Epilepsy Res 28:213–223]. Here we show a deficit of 40.2±3.6% in the total number of cerebellar GABAA receptors expressed (γ2+δ subtypes) in adult cacna1atg relative to controls. [3H]Muscimol autoradiography identified that this was partly due to a significant loss of CGC-specific α6 subunit-containing GABAA receptor subtypes. A large proportion of this loss of α6 receptors was attributable to a significantly reduced expression of the CGC-specific benzodiazepine-insensitive Ro15-4513 (BZ-IS) binding subtype, α6βγ2 subunit-containing receptors. BZ-IS binding was reduced by 36.6±2.6% relative to controls in cerebellar membrane homogenates and by 37.2±3.7% in cerebellar sections. Quantitative immunoblotting revealed that the steady-state expression level of α6 and γ2 subunits was selectively reduced relative to controls by 30.2±8.2% and 38.8±13.1%, respectively, α1, β3 and δ were unaffected. Immunohistochemically probed control and cacna1atg cerebellar sections verified that α6 and γ2 subunit expression was reduced and that this deficit was restricted to the CGC layer. Thus, we have shown that abnormal cerebellar P/Q-type VGCC activity results in a deficit of CGC-specific subtype(s) of GABAA receptors which may contribute to, or may be a consequence of the impaired cerebellar network signaling that occurs in cacna1atg mice.