Abstracts

Rationale: Several mutations in genes encoding for GABA(A) receptor subunits have been associated with epilepsy. Neurotransmission via GABA is implicated in several developmental processes such as neuronal proliferation, migration, differentiation and synapse formation (Di Cristo, 2007). How different mutations in GABA(A) subunits affect distinct aspects of neural circuit development is not well understood. Here, we investigate the effects of three mutations of the GABRA1 gene (A322D, K353delins18X and D219N) on synapse formation in mouse neocortex. These mutations were found in a cohort of French Canadian families (Lachance-Touchette et al., 2011 and Cossette et al., 2002) with genetic generalized epilepsy (GGE).Methods: Cortical organotypic cultures were prepared from GABRA1^flox/flox mice and biolistically transfected with either GFP or GFP/CRE under the control of a promoter that drives gene expression in both GABAergic and pyramidal cells. This method was previously described by Chattopadhyaya et al. (2007). Neurons were transfected during the phase of synapse proliferation. CRE expression efficiently inactivated the GABRA1 gene in the transfected cells, allowing the surrounding neurons to produce normal levels of GABRA1. To compare the effects of each GABRA1 mutant on synapse formation, we expressed GFP/CRE in combination with either a mutant GABRA1 (A322D, K353delins18X or D219N) or the wild-type form. GFP-positive GABAergic and pyramidal neurons were imaged using a confocal microscope and 3-D reconstructed with Neurolucida software (MicroBrightField). For pyramidal cells we analyzed dendritic length, dendritic arbour complexity and spine density (total and per subtype mushroom, stubby and thin spines). For GABAergic cells, we analyzed terminal branches and perisomatic bouton density formed by the GABAergic axon around target neurons immuno-labeled with NeuN.Results: We found that different mutations have distinct effects on spine density and shape. In particular, GABRA1^-/- pyramidal cells expressing A322D-GABRA1 had a higher spine density. Furthermore, the number of mushroom spines, thought to represent more mature glutamatergic synapses, was significantly increased. In contrast, GABRA1^-/- pyramidal cells expressing K353delins18X-GABRA1 appear simpler compared to control cells. Characterization of GABAergic cell morphology following transfection of a mutated GABRA1 gene is currently in progress.Conclusions: The effects of different GABA(A)R mutations on synapse formation appear specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of cortical organotypic cultures will provide a better understanding of the specific and distinct neural circuit alterations caused by different GABRA1 mutations and will help define the pathophysiology of genetic generalized epilepsy syndromes.