Single Nuclear Transcriptomes of Raphe Cell Types May be Altered with Repeated Seizures

Epilepsy affects millions of individuals globally and is one of the most common neurological disorders in the US. A proportion of these individuals who suffer from intractable epilepsy (unresponsive to current treatments) are at an elevated risk for SUDEP (Sudden Unexpected Death in Epileptic Patients), which is more common in males than females. There is general agreement that post‐ictal cardiorespiratory suppression progressively leads to SUDEP. However, the pathophysiology remains unclear. Research has shown neuroinflammation playing a role in seizures and epilepsy. Moreover, two key modulators of brainstem cardiorespiratory control centers, serotonin (5HT) and adenosine, have been shown to play roles in recurrent seizures and animal models of epilepsy. The link between neuroinflammation and these mediators has not been studied. We hypothesized that repeated seizures may lead to a progressive brainstem pathology initiated by neuroinflammation leading to a SUDEP event. We exposed SS kcnj16 −/− rats to daily sound‐induced seizures for 3, 7, or 10 days for comparison to naïve (no seizures) SS kcnj16 −/− rats. Brainstems were rapidly extracted, flash‐frozen, and tissue punches obtained from the Raphe Magnus and processed for extraction of cell nuclei. Single‐nuclear RNAseq (sn‐RNAseq) was performed using a 10x Genomics protocol and sequenced (NovaSeq). Results from the modest numbers of nuclei recovered (~3000 per sample) showed high map rates (80%) to the rat genome. Sequence data was processed through CellRangr and Seurat to generate tSNE and UMAP plots to show clustering of cell types, from which microglia (Arhgap15 expression), astrocytes (Slc4a4 expression), and serotonergic neurons (Tph2 expression) were identified. Additional differential expression analyses within these cell types across conditions (increasing seizure number) are ongoing. Finally, we show for the first time, application of sn‐RNAseq to identify brainstem cell types affected by seizures in a rat model, with the ultimate goal to link neuroinflammation and pathophysiology to gain additional insights into pathophysiology behind SUDEP. Support or Funding Information NIH HL 122358UMAP of clusters generated through Seurat package on R. 12 clusters were generated and identified by genes highly expressed in that specific cluster. Clusters noted here of note are the microglia population, astrocyte population and serotonergic neuron population. Different seizure conditions shown as well.