Abstracts

Rationale: An autosomal dominant mutation in the GABA_A receptor (GABA_AR) α1 subunit [α1(A322D), α1(AD)] causes juvenile myoclonic epilepsy (JME) in a large human kindred, and additional α1 subunit mutations are associated with other forms of genetic generalized epilepsy. Previously, we demonstrated that heterozygous deletion of the α1 subunit (Het-KO) caused absence seizures in mice at postnatal day 35 (P35), and that α1(AD) subunit caused a modest, but significant, dominant-negative effect on GABA_AR expression and physiology in vitro. Here, produced a knock-in mouse with the α1(AD) mutation (Het-AD) to determine the effects of the Het-KO and Het-AD on seizure phenotype, GABA_AR expression and GABA_AR physiology at two developmental timepoints. Methods: We generated the Het-AD mouse and mated Het-KO with Het-AD to produce wild type, Het-KO, and Het-AD mice in the same litter. We performed 24 hour video/EEG monitoring at P35 and P120. Western blots of frontal cortex tissue were used to determine the relative amounts of α1, α3, β2/3, and γ2 subunit at these different ages and immunohistochemistry determined the relative amounts of α1 and α3 subunit associated with the postsynaptic marker, gephyrin. Patch-clamp electrophysiology studies of wild type and Het-KO mice in layer II/III of the frontal cortex determined the effects of Het-KO on GABAergic miniature inhibitory postsynaptic currents (mIPSCs). Results: Both Het-KO and Het-AD experienced spontaneous spike-wave discharges (SWD) and absence seizures at ages P35 and P120; there was no difference in SWD frequency between the two mutant genotypes or at the two ages. In addition, both Het-KO and Het-AD mice exhibited spontaneous polyspike discharges (PSD); 10-15% of the PSDs were associated with visible myoclonic jerks (MJ). PSDs were significantly more frequent at P120 than P35 in both Het-KO and Het-AD mice. Low dose, repetitive pentylenetetrazol produced age-dependent PSDs and MJ in all three genotypes, but PSDs occurred with a shorter latency in the mutant compared with wild type mice. Het-KO and Het-AD mice had increased expression of the α3 subunit compared with wild type and the expression of α3, α1, β2/3 and γ2 subunits were reduced in P120 mice compared with P35 mice in all geneotypes. The relative clustering of α1 and α3 subunits with gephyrin did not vary with age or genotype. In layer II/III of motor cortex, Het-KO reduced mIPSC amplitude at both P35 and P120. Het-KO increased the mIPSC decay constant, τ, but τ was reduced at P120 compared with P35. Conclusions: Both Het-KO and Het-AD undergo an age-dependent change in epilepsy phenotype from exhibiting only absence seizures at P35 to absence and myoclonic seizures at P120. Therefore, these mice model the syndrome of childhood absence epilepsy evolving into JME. The evolution of the epilepsy phenotype is associated with a general decrease in frontal lobe GABA_AR expression and a shortening of the mIPSC duration. This developmentally-dependent increase in disinhibition may contribute to the JME-like phenotype that emerges at this age.