Abstracts

Rationale: Mutations of CACNA1H were discovered in patients with childhood absence epilepsy (CAE). In vitro, the mutations increase T-type Ca2 channel activity by altering trafficking and voltage gating. Yet, the in vivo effects on seizure susceptibility and neural circuit function remain undefined. We created transgenic mice with a full length CACNA1H gene carrying either of two epilepsy-associated mutations (V831M and C456S) and a C-terminal flag tag to characterize their effects on native T-type Ca2 currents, circuit properties, and seizure risk during postnatal development. Methods: Spontaneous and induced seizures were measured by EEG and behavior at baseline and following i.p. bicarbonate and quantified as reported (Zhou et al. Nature Medicine 2009). Whole cell patch-clamp of hippocampal subiculum pyramidal neurons was performed in voltage-clamp and current-clamp mode in transverse hippocampal (300 ?m) slices. Results: T-type Ca2 currents were recorded by stepping from a holding potential of -100 mV to a set of test potentials. As reported for Cav3.2 cDNA in vitro, T-type Ca2 currents in transgenic mice displayed a left shifted voltage-dependent activation curve relative to controls (-43 1 vs. -36 1 mV, p<0.001). Interestingly, T-type Ca2 current density increased during the period of postnatal circuit developmental. At P8-10, current density in C456S mutant mice and wild type mice was not significantly different (4.6 0.4 vs. 4.5 0.4 pA/pF). However, by P17-19, current density increased and was magnified increase in mutant (8.0 0.6 vs. 5.6 0.3 pA/pF, P < 0.01). By contrast, in P17-19 Cav3.2 knockout neurons, current density remained low 4.6 1.1 pA/pF significantly lower than mutant at P17-19 (P < 0.05). The probability of firing in response to a brief (5 ms) EPSC-like stimulus of 500 pA was 100% in C456S (n = 4) compared to only 33% in wild-type (n = 3). A more prolong 1 sec current injection elicited firing at a lower threshold in C456S compared to wild type (27.3 3.0 vs. 55.6 8.0 pA, P < 0.01). Surprisingly, at P17-19, C456S mutant neurons displayed spontaneous firing at baseline: 86% in C456S mutant (n = 7 cells), but only 14% in wild-type (n = 7 cells), while resting membrane potential was unaffected (-67 1.3 vs. -67.1 0.9 mV, respectively). Hyperventilation triggers cortical spike-wave discharge in CAE patients with Cav3.2 mutations. Sodium bicarbonate injections (to induced a metabolic alkalosis mimicking the respiratory alkalosis; increasing HCO3-/CO2 ratio) epileptiform activity in frontal cortex selectively in C456S mutant mice 75% (12/16) compare to 0% of control littermates (0/12).