Investigating the interplay between excitatory neurotransmission and GABAa receptor expression and trafficking in the stargazer mutant mouse

Abstract

The stargazer mutant mouse is an animal model of human absence epilepsy and cerebellar ataxia. The stargazer mutation results in the complete ablation of expression of TARPy2， an AMPA receptor (AMPAR) auxiliary subunit. Results of the current study provide evidence for the involvement of TARPy2 in the regulation of expression and trafficking of specific GABAa receptor (GABAR) subtypes in the cerebellum and hippocampal formation. Expression of total GABARs (γ2 and δ-containing) in stargazer cerebellum was found to be reduced to 70 士 10% of control levels. In situ [(^3)H] muscimol autoradiography revealed that this loss of GABARs was entirely restricted to the cerebellar granule cell (CGC) layer. Total numbers of Y2-containing GABARs were only slightly reduced in the stargazer cerebellum (90 士 6% of control levels), which was entirely attributable to a reduction in the benzodiazepine- insensitive (BZ-IS) subtype of γ2 containing GABARs, conferred by α6βγ2 receptors which are located entirely within the CGC layer (to 78 ± 10% of control levels). The benzodiazepine-sensitive subtype of GABAR, largely represented by α1βγ2 receptors, was unaffected. This data implied that the extrasynaptic α6βδ GABARs were dramatically affected by the mutation. This was further supported by semi-quantitative immunoblotting analysis which demonstrated a reduction in GABAR а6 and δ subunit expression to 39 ±^ 22% and 36 ±21% of control levels respectively. Cultured cerebellar granule cells (CGCs) from control (AMPAR and kainite receptor-competent) and stargazer (AMPAR-incompetent but kainate receptor- competent) mice were employed as a model system to investigate how the stargazer mutation evoked aberrant α6βδ GABAR expression and trafficking. The studies described herein demonstrated that AMPAR activation resulted in L- type voltage-gated calcium channel-dependent up-regulation of GABAR δ subunit expression and cell surface trafficking which was not detected in CGCs derived from stargazer animals. This offers a partial explanation for the abnormalities observed in vivo. This investigation also provides evidence of a switch in GABAR expression in response to hyper-excitability in the dentate gyrus of the stargazer mutant, where a combination of in situ autoradiographical, immunohistochemical and immunoblotting analyses indicated that a.4J3y2 GABARs arose at the expense of the a.4J3o subtype. Hence, there is a potential switch from extrasynaptic GABARs in favour of synaptic GABARs, which may reflect an adaptive response to high frequency spike-wave discharges that enter the DG in the stargazer mutant.Overall the results of this study support the hypothesis that neural networks in both the cerebellum and the hippocampus are able to undergo homeostatic GABAergic plasticity in response to alterations in excitatory inputs.