Functional and molecular heterogeneity of D2R neurons along dorsal ventral axis in the striatum

Action control is a key brain function determining the survival of animals in their environment. In mammals, neurons expressing dopamine D2 receptors (D2R) in the dorsal striatum (DS) and the nucleus accumbens (Acb) jointly but differentially contribute to the fine regulation of movement. However, their region-specific molecular features are presently unknown. By combining RNAseq of striatal D2R neurons and histological analyses, we identified hundreds of novel region-specific molecular markers, which may serve as tools to target selective subpopulations. As a proof of concept, we characterized the molecular identity of a subcircuit defined by Wfs1 neurons and evaluated multiple behavioral tasks after its temporally-controlled deletion of D2R. Consequently, conditional D2R knockout mice displayed a significant reduction in digging behavior and an exacerbated hyperlocomotor response to amphetamine. Thus, targeted molecular analyses reveal an unforeseen heterogeneity in D2R-expressing striatal neuronal populations, underlying specific D2R’s functional features in the control of specific motor behaviors. D2R-Cre mice were crossed with Ribotag-LoxP/LoxP mice to generate D2R-Cre/+:Ribotag-LoxP/+ mice that express the hemagglutinin (HA) epitope fused to the ribosomal protein rpl22 selectively in D2R-expressing cells. Cell type-specific tagged ribosomes from the dorsal striatum and nucleus accumbens were immunoprecipitated using anti-HA antibodies and their bound mRNAs -as well as the input fractions that contain the RNAs from all the cell types- were deeply sequenced in triplicate using Illumina HiSeq4000. In parallel, Wfs1-CreERT2 mice were crossed with Ribotag-LoxP/LoxP mice to generate Wfs1-Cre/+:Ribotag-LoxP/+ mice that express the HA epitope fused to the ribosomal protein rpl22 selectively in Wfs1-expressing cells. Cell type-specific tagged ribosomes from the nucleus accumbens were immunoprecipitated using anti-HA antibodies and their bound mRNAs -as well as the input fraction- were deeply sequenced in triplicate using Illumina HiSeq4000.动作控制是决定动物在所处环境中生存的核心脑功能。在哺乳动物体内，背侧纹状体（dorsal striatum, DS）与伏隔核（nucleus accumbens, Acb）中表达多巴胺D2受体（dopamine D2 receptors, D2R）的神经元，可共同且差异化地参与运动的精细调控。然而目前学界对这些神经元的脑区特异性分子特征仍知之甚少。本研究结合纹状体D2R神经元的RNA测序与组织学分析，鉴定出数百种全新的脑区特异性分子标记物，这类标记物可作为靶向特定神经元亚群的研究工具。作为概念验证，我们解析了由Wfs1神经元构成的子环路的分子身份，并在对其开展时序可控性D2R敲除后评估了多项行为学实验任务。结果显示，条件性D2R敲除小鼠的掘土行为显著减少，且对苯丙胺诱发的运动亢进反应出现加剧。综上，靶向性分子分析揭示了表达D2R的纹状体神经元群体中存在未曾被发现的异质性，这为D2R在特定运动行为调控中的独特功能特征提供了分子层面的解释依据。实验层面，我们将D2R-Cre小鼠与Ribotag-LoxP/LoxP小鼠杂交，获得可在表达D2R的细胞中特异性表达融合于核糖体蛋白rpl22的血凝素（hemagglutinin, HA）表位的D2R-Cre/+:Ribotag-LoxP/+小鼠。使用抗HA抗体免疫沉淀背侧纹状体与伏隔核中细胞类型特异性的标记核糖体，对其结合的mRNA以及包含所有细胞类型RNA的输入组分，均采用Illumina HiSeq4000平台进行三次重复的深度测序。与此同时，将Wfs1-CreERT2小鼠与Ribotag-LoxP/LoxP小鼠杂交，获得可在表达Wfs1的细胞中特异性表达融合于核糖体蛋白rpl22的HA表位的Wfs1-Cre/+:Ribotag-LoxP/+小鼠。使用抗HA抗体免疫沉淀伏隔核中细胞类型特异性的标记核糖体，对其结合的mRNA以及输入组分，均采用Illumina HiSeq4000平台进行三次重复的深度测序。