Faculty Collaboration Database

Patch-to-Seq and Transcriptomic Analyses Yield Molecular Markers of Functionally Distinct Brainstem Serotonin Neurons. Front Synaptic Neurosci 2022;14:910820

Date

07/19/2022

Pubmed ID

35844900

Pubmed Central ID

PMC9280690

DOI

10.3389/fnsyn.2022.910820

Scopus ID

2-s2.0-85134237459 (requires institutional sign-in at Scopus site)   2 Citations

Abstract

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.

Author List

Mouradian GC Jr, Liu P, Nakagawa P, Duffy E, Gomez Vargas J, Balapattabi K, Grobe JL, Sigmund CD, Hodges MR

Authors

Justin L. Grobe PhD Professor in the Physiology department at Medical College of Wisconsin
Matthew R. Hodges PhD Professor in the Physiology department at Medical College of Wisconsin
Gary C. Mouradian PhD Assistant Professor in the Physiology department at Medical College of Wisconsin
Pablo Nakagawa PhD Assistant Professor in the Physiology department at Medical College of Wisconsin
Curt Sigmund PhD Chair, Professor in the Physiology department at Medical College of Wisconsin