Effects of the T-type calcium channel CaV3.2 R1584P mutation on absence seizure susceptibility in GAERS and NEC congenic rats models

Abstract

Rationale: Low-voltage-activated or T-type Ca²⁺ channels play a key role in the generation of seizures in absence epilepsy. We have described a homozygous, gain of function substitution mutation (R1584P) in the Ca_V3.2 T-type Ca²⁺ channel gene (Cacna1h) in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). The non-epileptic control (NEC) rats, derived from the same original Wistar strains as GAERS but selectively in-breed not to express seizures, are null for the R1584P mutation. To study the effects of this mutation in rats who otherwise have a GAERS or NEC genetic background, we bred congenic GAERS-Cacna1hNEC (GAERS null for R1584P mutation) and congenic NEC-Cacna1hGAERS (NEC homozygous for R1584P mutation) and evaluated the seizure and behavioral phenotype of these strains in comparison to the original GAERS and NEC strains.

Methods: To evaluate seizure expression in the congenic strains, EEG electrodes were implanted in NEC, GAERS, GAERS^-Cacna1hNEC without the R1584P mutation, and NEC^{-Cacna1hGAERS} with the R1584P mutation rats. In the first study, continuous EEG recordings were acquired from week 4 (when seizures begin to develop in GAERS) to week 14 of age (when GAERS display hundreds of seizures per day). In the second study, the seizure and behavioral phenotype of GAERS and NEC^{-Cacna1hGAERS} strains were evaluated during young age (6 weeks of age) and adulthood (16 weeks of age) of GAERS, NEC, GAERS^-Cacna1hNEC and NEC^{-Cacna1hGAERS}. The Open field test (OFT) and sucrose preference test (SPT) were performed to evaluate anxiety-like and depressive-like behavior, respectively. This was followed by EEG recordings at 18 weeks of age to quantify the seizures, and spike-wave discharge (SWD) cycle frequency. At the end of the study, the whole thalamus was collected for T-type calcium channel mRNA expression analysis.

Results: GAERS had a significantly shorter latency to first seizures and an increased number of seizures per day compared to GAERS^-Cacna1hNEC. On the other hand, the presence of the R1584P mutation in the NEC^{-Cacna1hGAERS} was not enough to generate spontaneous seizures in their seizure-resistant background. 6 and 16-week-old GAERS and GAERS^-Cacna1hNEC rats showed anxiety-like behavior in the OFT, in contrast to NEC and NEC^{-Cacna1hGAERS}. Results from the SPT showed that the GAERS developed depressive-like in the SPT compared to GAERS^-Cacna1hNEC, NEC, and NEC^{-Cacna1hGAERS}. Analysis of the EEG at 18 weeks of age showed that the GAERS had an increased number of seizures per day, increased total seizure duration and a higher cycle frequency of SWD relative to GAERS^-Cacna1hNEC. However, the average seizure duration was not significantly different between strains. Quantitative real-time PCR showed that the T-type Ca²⁺ channel isoform Ca_V3.2 channel expression was significantly increased in GAERS compared to NEC, GAERS^-Cacna1hNEC and NEC^{-Cacna1hGAERS}. The presence of the R1584P mutation increased the total ratio of Ca_V3.2 + 25/-25 splice variants in GAERS and NEC^{-Cacna1hGAERS} compared to NEC and GAERS^-Cacna1hNEC.

Discussion: The data from this study demonstrate that the R1584P mutation in isolation on a seizure-resistant NEC genetic background was insufficient to generate absence seizures, and that a GAERS genetic background can cause seizures even without the mutation. However, the study provides evidence that the R1584P mutation acts as a modulator of seizures development and expression, and depressive-like behavior in the SPT, but not the anxiety phenotype of the GAERS model of absence epilepsy.

Keywords: Absence epilepsy; EEG; GAERS; Genetic absence epilepsy of Strasbourg; NEC.