Integrated single-cell analysis reveals coupled molecular gradient and functional subnetworks in the thalamic reticular nucleus. Integrated single-cell analysis reveals coupled molecular gradient and functional subnetworks in the thalamic reticular nucleus

The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, is known to regulate thalamocortical interactions critical for sensory processing, attention and cognition. TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders. Currently, little is known about the organizational principles underlying its divergent functions. We performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that TRN cellular heterogeneity is characterized by a transcriptomic gradient of two negatively correlated gene expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core/shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections to the functionally distinct first-order and higher order thalamic nuclei to form molecularly defined TRN-thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. Taken together, our study provides a comprehensive atlas for TRN neurons at the single-cell resolution, and links molecularly defined subnetworks to the functional organization of the thalamo-cortical circuits. Overall design: The project involved 8 plates of TRN nuclei with NeuN selection, 9 plates of TRN nuclei without NeuN selection, 4 plates of TRN-adjacent (Thalamus and Globus Pallidus) nuclei, 7 plates hippocampus Pvalb+ nuclei, 2 plates somatosensory cortex Pvalb+ nuclei, 1 plate striatium Pvalb+ nuclei, 3 plates M2 cortex Pvalb+ nuclei, and 3 batches of Patch-seq samples. We used 96-well plates to collect and process nuclei for Smart-seq2 library construction.

丘脑网状核（thalamic reticular nucleus, TRN）作为丘脑抑制的主要来源，已知其可调控对感觉处理、注意力及认知至关重要的丘脑皮层交互过程。TRN功能异常已被证实与多种神经发育障碍中的感觉异常、注意力缺陷及睡眠紊乱存在关联。目前，学界对其多样功能背后的组织原则尚所知甚少。本研究开展整合分析，将小鼠TRN的单细胞分子与电生理特征，与其连接特性及系统级功能进行关联。研究发现，TRN的细胞异质性以两种负相关基因表达谱构成的转录组梯度为特征，每种谱均涵盖数百个基因。该转录组梯度两端的神经元表达相互排斥的标记物，呈现类似核心/外壳的解剖结构，并具备截然不同的电生理特性。这两种TRN亚群分别与功能迥异的一级及高阶丘脑核团形成差异性连接，从而构成分子特征定义的TRN-丘脑子网。在活体中对这两种子网进行选择性扰动实验，揭示了二者在调控睡眠过程中的差异化作用。综上，本研究提供了单细胞分辨率下TRN神经元的综合图谱，并将分子定义的子网与丘脑皮层环路的功能组织关联起来。
实验整体设计：本项目共包含8块经NeuN分选的TRN细胞核样本孔板、9块未进行NeuN分选的TRN细胞核样本孔板、4块TRN邻近组织（丘脑与苍白球）的细胞核样本孔板、7块海马体Pvalb+细胞核样本孔板、2块躯体感觉皮层Pvalb+细胞核样本孔板、1块纹状体Pvalb+细胞核样本孔板、3块M2皮层Pvalb+细胞核样本孔板，以及3批Patch-seq样本。我们采用96孔板收集并处理细胞核，用于构建Smart-seq2测序文库。