To investigate how this seizure-prone state progresses, we will dissect the brain from Adar2 mutant pups and their wild-type siblings at various postnatal days before and during the period when they begin to have night seizures. One side of each brain will be processed for RNA to use for RNA Seq analysis, RT-qPCR studies on expression of Adar2 transcripts and on expression, editing and splicing of transcripts encoding glutamate receptors. The other side will be processed for protein samples to study glutamate receptor subunit expression levels by mass spectrometry analysis, followed by confirmational immunoblots against specific hit proteins. We anticipate that levels of Gria2 pre-mRNA should be near normal in Adar2 mutants before seizures at P10 but pre-mRNAs with unspliced intron 11, which is required for the Q/R editing event, will accumulate and mature Gria2 mRNA will be much lower than normal. In the Adar2 mutant brain as the seizure-prone state develops after P10, we expect that Gria2 transcription may decrease abnormally, as reported in other seizure models, and that changes in expression of other glutamate receptor transcripts will also appear gradually.

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