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Patch‐to‐Seq Reveals Unique Transcriptomic Profiles of Chemosensitive Serotonergic Neurons
Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO2 levels. Prior data indicate that a sub‐population of brainstem serotonin (5‐HT) neurons are likely central respiratory chemoreceptors. However, it rem...
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Published in: | The FASEB journal 2021-05, Vol.35 (S1), p.n/a, Article fasebj.2021.35.S1.04448 |
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Main Authors: | , , , , |
Format: | Article |
Language: | English |
Online Access: | Get full text |
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Summary: | Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO2 levels. Prior data indicate that a sub‐population of brainstem serotonin (5‐HT) neurons are likely central respiratory chemoreceptors. However, it remains unclear which 5‐HT neurons develop chemosensitivity and what molecular markers may identify this unique sub‐population of 5‐HT neurons. Here we employed the “patch‐to‐seq” technique to measure action potential firing rate responses of 5‐HT neuron to hypercapnic acidosis using cell‐attached patch‐clamp electrophysiology followed by isolation of that neurons’ intracellular RNA content for single cell RNA Sequencing. Acute brainstem slices (200 µm) from young (P18‐23) transgenic rats expressing eGFP in all 5‐HT neurons (SSeGFP) were recorded while superfused with artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D‐AP5, 20 mM Gabazine) bubbled with either 5% CO2 (bal. O2; pH= 7.36; 5 min) or 15% CO2 (bal. O2; pH= 7.10). The Chemosensitivity Index (C.I.) determined cell phenotypes. pH‐sensitive eGFP+ neurons (n=48) had an average C.I. of 169.8± 8.25 SEM whereas pH‐insensitive eGFP+ neurons (n=44) had an average C.I. of 100.3 ± 1.4 (P < 0.0001; t‐test). Cell extracts from 11 chemosensitive and 10 insensitive recorded cells were subjected to single cell RNA Sequencing (scRNA‐Seq), from which an average of 48.3M reads were generated with a quality score of ~32.7 and ~75% mapping rate per sample with an average of ~8,000 genes detected. Purity of the isolated samples was confirmed by significantly more expression (Log2(FPKM+1)) of 6 serotonergic vs 7 neuronal and 8 glial gene markers, and more expression of 7 neuronal vs 8 known glial gene markers. There were 166differentially expressed genes (q < 0.05) between pH‐sensitive and ‐insensitive 5‐HT neuron populations none of which have a known contribution to pH regulation or sensing. Predicted upstream regulators of 352 DEGs (p |
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ISSN: | 0892-6638 1530-6860 |
DOI: | 10.1096/fasebj.2021.35.S1.04448 |