Abstracts

Rationale: Alterations in thalamic T-type calcium channel currents play a major role in rebound burst firing that drives spike-wave seizure activity in Childhood Absence Epilepsy (CAE). Our lab has recently shown that abnormal cortical input to thalamic relay neurons results in elevated thalamic T-currents prior to the onset of spike-wave seizures in several monogenic mouse models of CAE, including the P/Q-type calcium channel mutant tottering. To explore the basis for downstream T-current remodeling in thalamic neurons of tottering mice, we performed single-cell resolution transcriptional analysis of Cacna1g, the only T-channel α-subunit encoding gene expressed in thalamic relay nuclei.

Methods: Highly sensitive, fluorescent in situ hybridization RNAscope technology (ACD Bio.) was utilized to target mRNA transcripts of thalamic T-type α-subunit Cacna1g in sex-matched wildtype (WT) and homozygous tottering (tg) littermates. To compare expression levels before and after seizure onset, cohorts were taken at two time points (P14, P120-180). High magnification images (40x) of thalamic relay nuclei were quantified using HALO analysis software (Indica Labs). Further phenotypes were explored by detecting the percentage of cells positive for probes or markers of interest.

Results: Expression of Cacna1g in thalamic relay cells of tottering mice prior to seizure onset (P14) showed a 2-fold increase in mRNA copy number/cell when compared to wildtype (p-value: < 0.001, t-test with Welch’s correction). There is a significant difference in percentage of cells with low Cacna1g expression (WT= 27.5, Tg=15.8; Two-way ANOVA, p-value: 0.0006) and high Cacna1g expression (WT= 18.5 Tg=33.4; Two-way ANOVA, p-value:< 0.0001). Similarly, when the same regions were compared in adult (P120-180) tottering mice, there was a significant decrease in the percentage of cells with no Cacna1g expression (WT= 25.3, Tg=10.3; Two-way ANOVA, p-value: 0.003) and a 3-fold increase in high Cacna1g expression (WT= 6.2 Tg=18.7; Two-way ANOVA, p-value: 0.0148). Analysis of these mice also revealed a significant increase in the number of DAPI-positive nuclei within areas of identical size in thalamic relay regions in tottering mice at both P14 (WT= 1894 Tg=2345; t-test , p-value: < 0.0001) and 4-6 month time points (WT= 1749 Tg=2084; t-test , p-value: < 0.0001).