Cellular heterogeneity in the dopaminergic system

多巴胺能系统的细胞异质性

• 批准号: RGPIN-2018-06262
• 负责人: Lévesque, Martin
• 金额: $ 3.06万
• 依托单位: Université Laval
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=688436
• 关键词: Cellular; heterogeneity; dopaminergic; system

Mesodiencephalic dopamine (mDA) neurons play central roles in the regulation of a wide range of brain functions, including voluntary movement and behavioural processes. These functions are served by distinct subtypes of mDA neurons located in the subtantia nigra pars compacta (SNpc) and the ventral tegmental area (VTA). However, several reasons led us to challenge this classification. First, mDA neurons innervate multiple target regions. Second, most known gene expression patterns do not exclusively obey the anatomical boundaries of the VTA or SNpc. Third, recent reports indicate functional heterogeneity within these clusters and a portion of mDA neurons co-release GABA, glutamate, and some use all three neurotransmitters. Together, these lines of evidence suggest that the anatomical classification of mDA neuron diversity is inadequate, and highlight the need for a better mDA neuron classification. In addition, the function deserved by these subpopulations remains obscure. The overall goal of this proposal is to identify mDA neuron subpopulations (mDAS), provide a detailed analysis of their output circuits and reveal their functional relevance. To achieve this goal, we have 3 specific objectives: 1- Identify mDAS based on their axonal projections, 2- Reveal the molecular and physiological signature for each population, 3- Uncover the functional role of mDAS on mouse behaviour. To identify mDAS, we will use a highly selective intersectional genetic approach, which relies on overlapping combinations of Cre and Flp recombinase to selectively manipulate specific populations. Using this method, mDAS will be labelled with GFP and we will use our recently developed high-resolution light-sheet microscope to map their entire axonal projections in the whole cleared brain (CLARITY). Labelled neurons will also be characterized using immunolabeling and multiplex fluorescent in situ hybridization. We will also isolate mDAS from fresh brain slices using laser capture microdissection and perform gene expression analysis. These experiments will define mDA neuron diversity and establish their relative proportion and anatomical location in the adult brain. Then, we will use intersectional genetic approach to perform their electrophysiological characterization and selectively activate or inhibit the activity of these mDAS using pharmacogenetics. The experiments will allow a complete network map of the dopaminergic sub-circuits and provide the first molecular and physiological biography of mDA neurons subsets. Data generated here will also reveal the functional role of mDA neuron subpopulations.

中二脑多巴胺 (mDA) 神经元在调节多种大脑功能（包括随意运动和行为过程）中发挥着核心作用。这些功能由位于黑质致密部 (SNpc) 和腹侧被盖区 (VTA) 的 mDA 神经元的不同亚型发挥作用。然而，有几个原因导致我们对这种分类提出质疑。首先，mDA 神经元支配多个目标区域。其次，大多数已知的基因表达模式并不完全遵循 VTA 或 SNpc 的解剖边界。第三，最近的报告表明这些簇内的功能异质性，部分 mDA 神经元共同释放 GABA、谷氨酸，有些神经元使用所有三种神经递质。总之，这些证据表明 mDA 神经元多样性的解剖学分类是不够的，并强调需要更好的 mDA 神经元分类。此外，这些亚群应有的功能仍然不清楚。该提案的总体目标是识别 mDA 神经元亚群 (mDAS)，对其输出电路进行详细分析并揭示其功能相关性。为了实现这一目标，我们有 3 个具体目标：1- 根据轴突投影识别 mDAS，2- 揭示每个群体的分子和生理特征，3- 揭示 mDAS 对小鼠行为的功能作用。为了识别 mDAS，我们将使用高度选择性的交叉遗传方法，该方法依赖于 Cre 和 Flp 重组酶的重叠组合来选择性地操纵特定群体。使用这种方法，mDAS 将用 GFP 标记，我们将使用我们最近开发的高分辨率光片显微镜来绘制它们在整个清晰大脑 (CLARITY) 中的整个轴突投影。标记的神经元也将使用免疫标记和多重荧光原位杂交进行表征。我们还将使用激光捕获显微切割从新鲜脑切片中分离 mDAS 并进行基因表达分析。这些实验将定义 mDA 神经元多样性，并确定它们在成人大脑中的相对比例和解剖位置。然后，我们将使用交叉遗传方法来进行其电生理学表征，并利用药物遗传学选择性激活或抑制这些 mDAS 的活性。这些实验将获得多巴胺能子回路的完整网络图，并提供 mDA 神经元子集的第一个分子和生理传记。这里生成的数据还将揭示 mDA 神经元亚群的功能作用。