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

Acute regulation of CO₂ and pH homeostasis requires sensory feedback from peripheral (carotid body) and central (central) CO₂/pH sensitive cells – so called respiratory chemoreceptors. Subsets of brainstem serotonin (5-HT) neurons in the medullary raphe are CO₂ 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 CO₂ 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 CO₂ 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 CO₂ sensitive 5-HT neurons. scRNAseq of these recorded neurons revealed 166 differentially expressed genes among CO₂ 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 CO₂ 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 CO₂ 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 CO₂ chemoreception, in a single cell to identify potential mechanisms underlying functional differences in otherwise similar cell types.