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Patch‐to‐Seq Reveals Unique Transcriptomic Profiles of Chemosensitive Serotonergic Neurons
Author(s) -
Mouradian Gary,
Duffy Erin,
Liu Pengyuan,
GomezVargas Javier,
Hodges Matt
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.04448
Subject(s) - patch clamp , brainstem , electrophysiology , population , serotonergic , chemistry , slice preparation , neuron , hypercapnia , chemoreceptor , biology , cnqx , neuroscience , medicine , serotonin , acidosis , endocrinology , biochemistry , glutamate receptor , ampa receptor , receptor , environmental health
Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO 2 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<0.01) point to the importance of Egr2 activation, a developmental transcription factor previously shown to give rise to pH sensitive 5‐HT neurons in mice. Machine learning algorithms identified unique markers of CO 2 /pH sensitive 5‐HT neurons in rats, where supervised analyses (using Support Vector Machine learning and Recursive Feature Elimination (SVM‐RFE)) yielded several genes in which expression levels significantly (P<0.001) and accurately (%) predict the predefined phenotype from the DEGs ( CD46 , 68.7%; Iba57 , 71.2%; Chd5 , 72.0%; and Maz , 74.0%) and among the 1,000s of detected genes ( Hnmpk , 81.0%; Diablo , 87.9%) but the biological/function significance of these genes in CO2/pH sensing remains unclear. RNAScope confirmed 25% of Tph2 + neurons in the raphe midline co‐express 4 or more RNA molecules of CD46 or Iba57 RNA. Together, these data suggest: 1) 5‐HT pH sensitive neurons are transcriptionally distinct from insensitive 5‐HT neurons, and 2) novel molecular markers of 5‐HT pH sensitive rat neurons may be useful identifiers for this unique subpopulation.