Patch-to-Seq and Transcriptomic Analyses Yield Molecular Markers of Functionally Distinct Brainstem Serotonin Neurons.
Patch-to-Seq and Transcriptomic Analyses Yield Molecular Markers of Functionally Distinct Brainstem Serotonin Neurons.
复制标题
DOI：
10.3389/fnsyn.2022.910820
复制
发表时间：
2022
影响因子：
3.7
通讯作者：
Hodges, Matthew R.
中科院分区：
文献类型：
--
作者：
Mouradian, Gary C., Jr.;Liu, Pengyuan;Nakagawa, Pablo;Duffy, Erin;Gomez Vargas, Javier;Balapattabi, Kirthikaa;Grobe, Justin L.;Sigmund, Curt D.;Hodges, Matthew R.
关键词：
二氧化碳和pH动态平衡的急性调节需要外周(颈动脉小体)和中央(中央)对CO2/pH敏感的细胞--即所谓的呼吸化学感受器--的感觉反馈。延髓中缝中的脑干5-羟色胺(5-HT)神经元亚群对二氧化碳敏感或不敏感，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Patch-to-Seq来确定脑干5-羟色胺神经元中的二氧化碳反应是否与独特的转录图谱和/或独特的分子标记和途径相关。首先，检测了转基因Dahl SS(SSMcwi)大鼠(SSePet1-EGFP大鼠)在表达5-羟色胺(Pet-1)神经元中表达T2/ePet-EGFP转基因大鼠(SSePet1-EGFP大鼠)的急性脑干切片中5-羟色胺(5-HT)神经元高碳酸血症时放电频率的变化。随后，通过单细胞RNA(ScRNAseq)和生物信息学分析，测定和比较了CO2敏感和不敏感的5-羟色胺神经元的转录和通路特征。低基线放电频率是CO2敏感的5-羟色胺神经元的一个显著特征。这些记录的神经元的scRNAseq显示，在二氧化碳敏感和不敏感的5-羟色胺神经元中，有166个差异表达基因。通路分析产生了新的预测的上游调控因子，包括转录因子Egr2和瘦素。进一步的生物信息学分析确定了6个CO2敏感的5-羟色胺神经元的候选基因标记，通过原位mRNA杂交确定了2个候选基因(CD46和Iba57)在5-羟色胺神经元中均有表达。总之，这些数据为细胞化学接收的转录控制提供了新的见解，并为二氧化碳敏感的5-羟色胺神经元提供了无偏见的候选基因标记物。在方法学上，这些数据突出了补丁到序列技术的效用，使基因表达与单个细胞中的特定功能，如二氧化碳化学接收，能够联系起来，以确定潜在的机制，在其他类似的细胞类型的功能差异。
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 (SSePet1–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.
登录
查看更多内容
影响因子：
46.9
作者：
Fuzik J;Zeisel A;Máté Z;Calvigioni D;Yanagawa Y;Szabó G;Linnarsson S;Harkany T
通讯作者：
Harkany T
影响因子：
2.3
作者：
Corcoran, Andrea E.;Hodges, Matthew R.;Wu, Yuanming;Wang, Wengang;Wylie, Christie J.;Deneris, Evan S.;Richerson, George B.
通讯作者：
Richerson, George B.
DOI：
10.1126/science.1190721
发表时间：
2010-07-30
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gourine AV;Kasymov V;Marina N;Tang F;Figueiredo MF;Lane S;Teschemacher AG;Spyer KM;Deisseroth K;Kasparov S
通讯作者：
Kasparov S
影响因子：
46.9
作者：
Cadwell CR;Palasantza A;Jiang X;Berens P;Deng Q;Yilmaz M;Reimer J;Shen S;Bethge M;Tolias KF;Sandberg R;Tolias AS
通讯作者：
Tolias AS
影响因子：
2.5
作者：
Iceman, Kimberly E.;Richerson, George B.;Harris, Michael B.
通讯作者：
Harris, Michael B.