Mapping of Single Nuclear Transcriptomic Responses Uncovers Plasticity of Subpopulation of Purkinje Neurons Driving Motor Learning. Mapping of Single Nuclear Transcriptomic Responses Uncovers Plasticity of Subpopulation of Purkinje Neurons Driving Motor Learning

Cellular diversification is a fundamental feature of the brain that is critical for physiological functions of the nervous system including perception, motor control, and learning and memory. Advances in single cell RNA-sequencing have led to characterization of transcriptomic profiles of distinct major types of neurons in the brain. However, how transcriptomic profiles diversify within a specific population of neurons and their links to function remain poorly understood. Purkinje neurons represent some of the most iconic cells in the brain with decades of research characterizing their anatomy, cell biology, physiology, and roles in plasticity, learning and memory, as well as in diseases of the nervous system. In this study, we deployed an approach of isolating nuclei tagged in specific cell types followed by cell sorting and single nuclear RNA sequencing to profile Purkinje neurons and their response to motor activity and learning in adult mice. We uncovered the molecular map of two major subpopulations of Purkinje neurons, identified by the hallmark genes Aldoc and Plcb4, which bear distinct transcriptomic features. Remarkably, Plcb4+, but not Aldoc+, Purkinje neurons display robust plasticity of gene expression in mice subjected to sensorimotor and learning experience with downregulation of gene clusters related to chromatin regulation and synaptic organization and upregulation of gene clusters related to neuronal activity and synaptic transmission and neuron-immune interactions. Using in vivo calcium imaging and optogenetics perturbation approaches, we show that activation of Plcb4+ Purkinje neuron plays a crucial role in associative motor learning. Upon integrating single nuclear RNA-seq datasets with weighted gene network analysis, we also identify a motor activity and learning specific gene module that includes components of the FGFR2-SOS1-MAPK signaling pathway in Plcb4+ Purkinje neurons. Knockout of FGFR2 in Plcb4+ Purkinje neurons in adult mice by a CRISPR approach dramatically disrupts motor learning. Our findings provide a platform for identification of subpopulations of neurons and define how diversification of Purkinje neurons in the cerebellum links to their responses to motor learning. Our study, and by extension similar studies in the human brain, will provide the foundation for understanding the selective vulnerability of Plcb4+ Purkinje neurons to neurological diseases. Overall design: Exploration the diversification of Purkinje neurons in the cerebellum links to their responses to motor learning

细胞多样化是大脑的核心特征之一，对神经系统的感知、运动控制、学习记忆等生理功能至关重要。单细胞RNA测序（single cell RNA-sequencing）技术的进步，使得研究人员得以解析大脑中各类主要神经元的转录组特征。然而，特定神经元群体内部的转录组谱如何实现多样化，以及其与神经元功能的关联机制，目前仍未得到充分阐明。浦肯野神经元（Purkinje neurons）是大脑中最具标志性的细胞类群之一，数十年来的研究已对其解剖结构、细胞生物学特性、生理功能，以及在神经可塑性、学习记忆与神经系统疾病中的作用进行了充分阐释。本研究采用特定细胞类型标记细胞核分离、细胞分选结合单细胞核RNA测序（single nuclear RNA-sequencing）的方法，对成年小鼠的浦肯野神经元及其对运动活动与学习的响应开展转录组分析。本研究成功绘制了两类主要浦肯野神经元亚群的分子图谱，这两类亚群以标志性基因Aldoc与Plcb4为区分标志，各自具有独特的转录组特征。值得注意的是，在暴露于感觉运动与学习经历的小鼠中，Plcb4阳性（Plcb4+）而非Aldoc阳性（Aldoc+）的浦肯野神经元表现出显著的基因表达可塑性：其染色质调控与突触组织相关基因簇表达下调，而神经元活动、突触传递及神经元-免疫互作相关基因簇则表达上调。通过体内钙成像与光遗传学扰动实验，我们证实Plcb4+浦肯野神经元的激活在关联式运动学习中发挥关键作用。进一步将单细胞核RNA测序数据集与加权基因网络分析相结合，我们还在Plcb4+浦肯野神经元中鉴定出一个与运动活动及学习相关的特异性基因模块，该模块包含FGFR2-SOS1-MAPK信号通路的组成成分。通过CRISPR技术敲除成年小鼠Plcb4+浦肯野神经元中的FGFR2基因，可显著破坏运动学习能力。本研究的发现为神经元亚群的鉴定提供了研究框架，并阐明了小脑浦肯野神经元的多样化模式如何与其对运动学习的响应相关联。本研究以及后续在人类大脑中开展的类似研究，将为理解Plcb4+浦肯野神经元对神经系统疾病的选择性易感性奠定坚实基础。整体实验设计：探索小脑浦肯野神经元的多样化模式与其对运动学习的响应之间的关联。