Molecular Deconstruction of the Medullary Raphe Magnus in the rat: Transcriptional Responses to Repeated Seizures

Sudden Unexpected Death in Epilepsy (SUDEP) is a leading cause of death in patients with epilepsy and is thought to result from dysfunctional and/or failure of cardiorespiratory control systems. Post-mortem brainstem tissue analyses in human SUDEP cases point to reductions in markers of the brainstem serotonin (5-HT) system, which is known as a brainstem center that provides excitatory neuromodulation. We have previously shown in a knockout rat model (SS kcnj16-/- rats) that repeated seizures led to progressively greater ventilatory inhibition in the post-seizure period, seizure-associated mortality, and reduced brainstem 5-HT and tryptophan hydroxylase (Tph) particularly within the Raphe Magnus (RMg). Here, we account for the cellular constituency and local transcriptional responses to repeated seizures in male SS kcnj16-/- rats that experienced daily seizures for 3, 5, 7, or 10 consecutive days using single nuclear RNA sequencing (snRNA seq) from brainstem tissue biopsies including the RMg (-12.12 mm to -10.30 mm caudal to Bregma) two hours post-seizure. Unbiased cluster analysis identified 18 cell major clusters that were by identified by the expression of known gene markers, with most cells being oligodendrocytes. However, local RMg neurons showed the greatest numbers of differentially expressed genes with seizures compared to all other cell types. Further re-clustering of neuronal cell types yielded 14 distinct RMg neuron subpopulations, including 5 types of GABAergic neurons, 2 glutamatergic clusters, and 2 groups of 5-HT neurons which all had unique expression profiles. Nearly all DEGs across neuronal subtypes were increased following seizures, and a large fraction of which were common across seizure days and across neuron type suggesting uniformity in cellular response to seizures in this region. These studies provide foundational information regarding the cellular constituency of the RMg region in the rat, and altered neuronal function following repeated seizures in the absence of changes in other cell types in this key region of cardiorespiratory control.