Abstracts

Rationale: Stargazer mice are mutated in the Cacng2 gene and have frequent spontaneous absence seizures. To determine if deletions of other CACNG subunits can lead to absence seizures, we have focused our studies on the CACNG3 and CACNG4 proteins. These proteins are closely related to CACNG2 and are expressed in brain regions associated with absence seizures, namely the cortex and thalamus. Previously we have characterized the Cacng4 targeted mutant, and here we compare these results with our recent findings from the targeted mutation in the Cacng3 gene. Both single and double mutant studies were pursued to determine if the deletion of the Cacng3 gene could result in absence seizures. Methods: The targeted mutation in Cacng3^tm1Dgen mutant was generated by Deltagen. The first exon of the Cacng3 gene was disrupted with a LacZ-cassette. Brain sections were stained with X-Gal to reveal the regions with LacZ activity. EEGs were recorded to determine if the Cacng3^tm1Dgen mouse had spontaneous spike-wave discharges. The Cacng3^tm1Dgen strain was crossed to the waggler and stargazer3J mice, both Cacng2 mutants, to observe if the double homozygotes showed seizure activity. Results: The Cacng3 gene was correctly targeted as the Cacng3^tm1Dgen mutant was missing the Cacng3 transcript. CACNG3 was predominantly expressed in the cortex and hippocampus. (In contrast, our previous results showed that CACNG4 expression was missing from the cortex but was highly expressed in the thalamus.) The homozygous Cacng3^tm1Dgen mutant appeared overtly normal and had no spike-wave discharges. These results mirrored our previous results with the Cacng4 targeted (Cacng4^tm1) mice. In the double mutant studies, the waggler:Cacng3^tm1Dgen double homozygotes failed to survive beyond three weeks of age. (In contrast, waggler:Cacng4^tm1 double homozygotes did survive and breed.) We were only able to construct double mutants that survived to adulthood between Cacng3 ^tm1Dgen and the stargazer3J mutation, the mildest allele of stargazer. EEGs from these double mutant mice revealed spike-wave discharges, not previously observed in either parental strain separately. Conclusions: The single Cacng3^tm1Dgen mutant revealed no obvious phenotype, including no incidence of seizures. However when this mutation was introduced onto the stargazer3J mutant background, the double homozygotes showed absence seizure activity, indicating that the CACNG3 protein does have a role in seizure suppression but this could only be revealed in a compromised Cacng2 background. A similar result had been observed for the Cacng^4tm1 mutant. Interestingly, CACNG4 expression is localized to the thalamus and CACNG3 expression is predominantly in the cortex, but a mutation in either gene can give rise to seizures when combined with the stargazer3J mutation, possibly implying that absence epilepsy can originate from the cortex and the thalamus. Finally the loss of CACNG3 expression had a more profound effect on double mutant viability than CACNG4. (VAL supported by NS32801).