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.

中脑多巴胺（mDA）神经元在调节广泛的脑功能，包括自主运动和行为过程中发挥核心作用。这些功能由位于黑质下丘脑腹侧被盖区（SNpc）和腹侧被盖区（VTA）的不同亚型的mDA神经元提供。然而，有几个原因导致我们对这种分类提出质疑。首先，mDA神经元支配多个靶区域。第二，大多数已知的基因表达模式并不完全服从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和一部分的mDA神经元共同释放GABA，谷氨酸，和一些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，并强调需要一个更好的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），提供其输出电路的详细分析，并揭示其功能相关性。为了实现这一目标，我们有3个具体的目标：1-识别mDAS的轴突投射的基础上，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。为了鉴定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将被标记与GFP，我们将使用我们最近开发的高分辨率光片显微镜映射他们的整个轴突投影在整个清除大脑（MANUITY）。标记的神经元也将使用免疫标记和多重荧光原位杂交的特点。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神经元的多样性，并建立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学的方法来进行其电生理特性和选择性激活或抑制这些mDAS的活性，利用药物遗传学。该实验将允许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生物学。这里产生的数据也将揭示mDA神经元亚群的功能作用。