Mouradian Gary C, Liu Pengyuan, Nakagawa Pablo, Duffy Erin, Gomez Vargas Javier, Balapattabi Kirthikaa, Grobe Justin L, Sigmund Curt D, Hodges Matthew R
Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, United States.
Neuroscience Research Center, Medical College of Wisconsin, Milwaukee, WI, United States.
Front Synaptic Neurosci. 2022 Jun 30;14:910820. doi: 10.3389/fnsyn.2022.910820. eCollection 2022.
Acute regulation of CO and pH homeostasis requires sensory feedback from peripheral (carotid body) and central (central) CO/pH sensitive cells - so called respiratory chemoreceptors. Subsets of brainstem serotonin (5-HT) neurons in the medullary raphe are CO 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 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 rats). Subsequently, the transcriptomic and pathway profiles of CO 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 sensitive 5-HT neurons. scRNAseq of these recorded neurons revealed 166 differentially expressed genes among CO sensitive and insensitive 5-HT neurons. Pathway analyses yielded novel predicted upstream regulators, including the transcription factor and . Additional bioinformatic analyses identified 6 candidate gene markers of CO sensitive 5-HT neurons, and 2 selected candidate genes ( and ) were both expressed in 5-HT neurons determined via mRNA hybridization. Together, these data provide novel insights into the transcriptional control of cellular chemoreception and provide unbiased candidate gene markers of CO 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 chemoreception, in a single cell to identify potential mechanisms underlying functional differences in otherwise similar cell types.
对心输出量（CO）和pH稳态的急性调节需要来自外周（颈动脉体）和中枢（中枢）CO/pH敏感细胞（即所谓的呼吸化学感受器）的感觉反馈。基于胚胎起源的差异，延髓中缝的脑干血清素（5-HT）神经元亚群对CO敏感或不敏感，这表明这些功能不同的亚群可能具有独特的转录谱。在这里，我们使用膜片钳测序（Patch-to-Seq）来确定脑干5-HT神经元中的CO反应是否与独特的转录谱和/或独特的分子标记及信号通路相关。首先，在表达T2/ePet-eGFP转基因的转基因Dahl SS（SSMcwi）大鼠的急性脑干切片中，测量荧光标记的5-HT神经元在高碳酸血症酸中毒时的放电频率变化，这些大鼠在表达Pet-1的（血清素）神经元中表达T2/ePet-eGFP转基因（SS大鼠）。随后，通过单细胞RNA测序（scRNAseq）和生物信息学分析确定并比较CO敏感和不敏感的5-HT神经元的转录组和信号通路谱。低基线放电频率是CO敏感的5-HT神经元的一个显著特征。对这些记录的神经元进行scRNAseq分析发现，CO敏感和不敏感的5-HT神经元之间有166个差异表达基因。信号通路分析产生了新的预测上游调节因子，包括转录因子 和 。进一步的生物信息学分析确定了6个CO敏感的5-HT神经元的候选基因标记，并且通过mRNA杂交确定在2个选定的候选基因（ 和 ）在5-HT神经元中均有表达。总之，这些数据为细胞化学感受的转录控制提供了新的见解，并提供了CO敏感的5-HT神经元的无偏候选基因标记。在方法学上，这些数据突出了膜片钳测序技术在将基因表达与特定功能（如CO化学感受）联系起来的实用性，即在单个细胞中识别原本相似细胞类型中功能差异的潜在机制。
