Transcriptional profiling of mammalian habenula using scRNAseq

The habenula complex is appreciated as a critical regulator of motivated and pathological behavioral states via its output to midbrain nuclei. Despite this, transcriptional definition of cell populations that comprise both the medial (MHb) and lateral habenular (LHb) subregions in mammals remain undefined. To resolve this, we performed single-cell transcriptional profiling and highly multiplexed in situ hybridization experiments of the mouse habenula complex in naïve mice and those exposed to an acute aversive stimulus. Transcriptionally distinct neuronal cell types identified within the MHb and LHb, were spatially defined, and differentially engaged by aversive stimuli. Cell types identified in mice, also displayed a high degree of transcriptional similarity to those previously described in zebrafish, highlighting the well conserved nature of habenular cell types across the phylum. These data identify key molecular targets within habenula cell types, and provide a critical resource for future studies. We applied scRNAseq to unbiasedly characterize mammalian habenula transcriptome. We obtained transcriptional dataset of 11,878 cells including 5,558 neuronal cells. Overall design: To characterize transcriptional identities of single cells in the mammalian habenula, we utilized droplet based chromium technology developed by 10x Genomics. We microdissected the habenula from brain live slices of adult male mice (P50-55), enzymatically digested the tissue to obtain well dissociated highly viable single cells (viability >80 %), captured single cell transcriptomes, and generated cDNA libraries for subsequent sequencing.

缰核复合体（habenula complex）通过向中脑核团发出投射，被认为是动机性与病理性行为状态的关键调控因子。尽管如此，哺乳动物内侧缰核（medial habenular, MHb）与外侧缰核（lateral habenular, LHb）两个亚区的细胞群的转录组定义仍未明确。为解决这一问题，我们对未受刺激的小鼠以及暴露于急性厌恶性刺激的小鼠的缰核复合体开展了单细胞转录组测序与多重原位杂交实验。我们在MHb与LHb内鉴定出转录特征各异的神经元细胞类型，明确了其空间分布特征，并发现这些细胞类型会被厌恶性刺激差异性激活。我们在小鼠中鉴定出的细胞类型，与此前在斑马鱼中报道的细胞类型具有高度的转录组相似性，这凸显了缰核细胞类型在整个动物门类中的高度保守性。本数据集不仅鉴定出缰核细胞类型中的关键分子靶点，同时为后续相关研究提供了重要的科研资源。我们采用单细胞RNA测序（single-cell RNA sequencing, scRNAseq）技术，无偏倚地表征了哺乳动物缰核的转录组。本次实验共获得11878个细胞的转录组数据，其中包含5558个神经元细胞。整体实验设计：为表征哺乳动物缰核内单细胞的转录组特征，我们采用了10x Genomics公司开发的微滴式Chromium单细胞测序技术。我们从成年雄性小鼠（P50-P55）的新鲜脑切片中显微解剖分离缰核组织，通过酶消化获得解离充分、活性优异的单细胞悬液（细胞存活率>80%），随后捕获单细胞转录组并构建cDNA文库用于后续测序。