Patch-to-Seq and Transcriptomic Analyses Yield Molecular Markers of Functionally Distinct Brainstem Serotonin Neurons

Acute regulation of CO(2) and pH homeostasis requires sensory feedback from peripheral (carotid body) and central (central) CO(2)/pH sensitive cells – so called respiratory chemoreceptors. Subsets of brainstem serotonin (5-HT) neurons in the medullary raphe are CO(2) sensitive or insensitive based on differences in embryonic origin, suggesting these functionally distinct subpopulations may have unique transcriptional profiles. Here, we used Patch-to-Seq to determine if the CO(2) responses in brainstem 5-HT neurons could be correlated to unique transcriptional profiles and/or unique molecular markers and pathways. First, firing rate changes with hypercapnic acidosis were measured in fluorescently labeled 5-HT neurons in acute brainstem slices from transgenic, Dahl SS (SSMcwi) rats expressing T2/ePet-eGFP transgene in Pet-1 expressing (serotonin) neurons (SS(ePet1–eGFP) rats). Subsequently, the transcriptomic and pathway profiles of CO(2) sensitive and insensitive 5-HT neurons were determined and compared by single cell RNA (scRNAseq) and bioinformatic analyses. Low baseline firing rates were a distinguishing feature of CO(2) sensitive 5-HT neurons. scRNAseq of these recorded neurons revealed 166 differentially expressed genes among CO(2) sensitive and insensitive 5-HT neurons. Pathway analyses yielded novel predicted upstream regulators, including the transcription factor Egr2 and Leptin. Additional bioinformatic analyses identified 6 candidate gene markers of CO(2) sensitive 5-HT neurons, and 2 selected candidate genes (CD46 and Iba57) were both expressed in 5-HT neurons determined via in situ mRNA hybridization. Together, these data provide novel insights into the transcriptional control of cellular chemoreception and provide unbiased candidate gene markers of CO(2) sensitive 5-HT neurons. Methodologically, these data highlight the utility of the patch-to-seq technique in enabling the linkage of gene expression to specific functions, like CO(2) chemoreception, in a single cell to identify potential mechanisms underlying functional differences in otherwise similar cell types.