Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes.
Integration of electrophysiological recordings with single-cell RNA-seq data identifies neuronal subtypes.
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DOI：
10.1038/nbt.3443
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发表时间：
2016-02
影响因子：
46.9
通讯作者：
Harkany T
中科院分区：
文献类型：
--
作者：
Fuzik J;Zeisel A;Máté Z;Calvigioni D;Yanagawa Y;Szabó G;Linnarsson S;Harkany T
传统上，神经学家通过细胞的位置、形态、连通性和兴奋性来定义神经元的身份。然而，这些参数和分子表型之间的直接关系在很大程度上仍未得到探索。在这里，我们提出了一种方法，在小鼠脑片上进行全细胞膜片钳记录后，从单个新皮质锥体细胞和中间神经元获得完整的转录组数据。在我们称为Patch-seq的方法中，膜片钳刺激方案之后是整个体细胞室被吸入到记录吸管中，RNA的逆转录，包括添加独特的分子识别符，cDNA扩增，Illumina文库的制备和测序。我们发现Patch-seq揭示了电生理特征、对急性化学挑战的反应以及神经递质受体和通道的RNA表达之间的密切联系。此外，它还区分了与已建立的和我们所知的迄今未描述的神经元亚型相对应的神经元亚群。我们的发现证明了Patch-seq能够精确地映射神经元亚型并预测它们在大脑中的网络贡献。
Traditionally, neuroscientists have defined the identity of neurons by the cells' location, morphology, connectivity and excitability. However, the direct relationship between these parameters and the molecular phenotypes has remained largely unexplored. Here, we present a method for obtaining full transcriptome data from single neocortical pyramidal cells and interneurons after whole-cell patch-clamp recordings in mouse brain slices. In our approach, termed Patch-seq, a patch-clamp stimulus protocol is followed by the aspiration of the entire somatic compartment into the recording pipette, reverse transcription of RNA including addition of unique molecular identifiers, cDNA amplification, Illumina library preparation and sequencing. We show that Patch-seq reveals a close link between electrophysiological characteristics, responses to acute chemical challenges and RNA expression of neurotransmitter receptors and channels. Moreover, it distinguishes neuronal subpopulations that correspond to both well-established and, to our knowledge, hitherto undescribed neuronal subtypes. Our findings demonstrate the ability of Patch-seq to precisely map neuronal subtypes and predict their network contributions in the brain.