Molecular deconstruction of the pre-Bötzinger Complex/Nucleus Ambiguus (preBötC/NA) region: cellular constituencies and transcriptional responses to repeated seizures in the rat hindbrain

Abstract

Epilepsy affects millions worldwide, but a significant portion suffers from uncontrollable epilepsy. Repeated seizures have many consequences, including a high risk of post-ictal cardiorespiratory failure and Sudden Unexpected Death in Epilepsy (SUDEP). Major risk factors for SUDEP include biological sex in addition to the occurrence of generalized tonic-clonic seizures (GTCSs). How repeated seizures lead to cardiorespiratory dysfunction remains unknown. A key factor in many neurological diseases is neuroinflammation, predominantly mediated by microglia and astrocytes that become dysfunctional. Mechanistically, questions remain how they affect neuronal function in epilepsy and contribute to cardiorespiratory dysfunction and increased SUDEP risk. Previously, we have shown that repeated seizures in our novel rat model with genetic mutations in kcnj16, an inwardly rectifying K+ channel, in the Dahl salt sensitive rat (SSkcnj16-/-) led to increased neuroinflammation in key ventilatory regions at 3 and 5 days of seizures. Specifically, there was increased recruitment of various inflammatory mediators, increased recruitment of activated microglia, with improvement in post-ictal respiratory dysfunction and mortality with usage of anti-inflammatory agents. Here we tested the hypothesis that repeated seizures lead to differential neuroinflammatory activation after repeated seizures in CNS regions of ventilatory control. Male SSkcnj16-/- rats were subjected to 0 (Naive), 3, 7 or 10 days of seizure, and subsequently, the pre-Botzinger Complex/Nucleus Ambiguus (preBotC/NA) was isolated and sent for nuclei isolation and sequencing. Seurat was utilized to filter and process the data, integrate across conditions and allow for differential gene expression (DEG) analysis. Afterwards, pathways enrichment analysis was performed allowing for determination of unique pathways recruited across cell types for each seizure condition. Overall, we were able to identify 18 unique cell types based on transcriptomic signatures, with 8 different neuronal populations, grouped based on Type 1, Type 2 or a mixed Type 1 & Type 2 genetic expression, indicating rhythm generation or pattern generation, respectively. We found that majority of the neuronal clusters were Type 1 or mixed type, indicating predominantly rhythmogenic neuronal populations. Importantly, these critical neuronal populations showed significant upregulation in various metabolic and neurological disease pathways at the 3 and 7 Day timepoints. Furthermore, we identified various glial cells, including microglia and astrocytes and saw increased recruitment in various Inflammatory pathways, Metabolic pathways and Chemokine related pathways after 3 and 7Days of seizures, confirming our previous results. Consequently, our results show for the first time, transcriptomic characterization of crucial rhythmogenic neuronal populations after repeated seizures and the changes that may underlie their dysfunction in SUDEP, mediated in part through the network change in upregulated inflammatory pathways in surrounding glial cells.