Variation in activity state, axonal projection, and position define the transcriptional identity of individual neocortical projection neurons.

Single-cell RNA sequencing has generated the first catalogs of transcriptionally defined neuronal subtypes of the brain. However, the cellular processes that contribute to neuronal subtype specification and transcriptional heterogeneity remain unclear. By comparing the gene expression profiles of a subset of single layer 6 corticothalamic neurons in somatosensory cortex, we show that transcriptional subtypes primarily reflect axonal projection pattern, laminar position within the cortex, and neuronal activity state. Pseudotemporal ordering of 1023 cellular responses to sensory manipulation demonstrates that changes in expression of activity-induced genes both reinforced cell-type identity and contributed to increased transcriptional heterogeneity within each cell type. This is due to cell-type biased choices of transcriptional states following manipulation of neuronal activity. These results reveal that axonal projection pattern, laminar position, and activity state define significant axes of variation that contribute both to the transcriptional identity of individual neurons and to the transcriptional heterogeneity within each neuronal subtype. Overall design: 1023 single cell RNA-Seq and 6 bulk RNA-seq

单细胞RNA测序（single-cell RNA sequencing）已构建出首个基于转录特征定义的大脑神经元亚型图谱。然而，促成神经元亚型特化与转录异质性的细胞生物学过程仍未明确。本研究通过对比体感皮层内第6层皮质丘脑神经元的一个亚群的基因表达谱，发现转录亚型主要反映轴突投射模式、皮层内分层位置以及神经元活动状态。对1023个响应感觉操纵的细胞开展拟时序分析（pseudotemporal ordering）后发现，活动诱导基因的表达变化既强化了细胞类型身份，又提升了每种细胞类型内的转录异质性。该现象源于神经元活动被操纵后，不同细胞类型会偏向性选择特定的转录状态。本研究结果表明，轴突投射模式、分层位置与活动状态构成了重要的变异维度，既塑造了单个神经元的转录特征身份，又促成了同一神经元亚型内部的转录异质性。实验整体设计：1023个单细胞RNA测序样本与6个批量RNA测序（bulk RNA-seq）样本。