Cellular heterogeneity in the dopaminergic system

中间脑多巴胺(MDA)神经元在调节广泛的大脑功能，包括自主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)的不同亚型的丙二醛神经元提供。然而，有几个原因导致我们对这种分类提出了质疑。首先，丙二醛神经元支配多个靶区。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，这些簇内的功能不同，一部分丙二醛神经元共同释放GABA和谷氨酸，一些神经元使用所有这三种神经递质。总之，这些证据表明，对丙二醛神经元多样性的解剖学分类是不充分的，并突出了更好的丙二醛神经元分类的必要性。此外，这些亚群应有的功能仍然不清楚。这项建议的总体目标是确定丙二醛神经元亚群(MDA)，提供其输出电路的详细分析，并揭示它们的功能相关性。为了实现这一目标，我们有三个具体目标：1-根据MDA的轴突投射识别MDA，2-揭示每个种群的分子和生理特征，3-揭示MDAS对小鼠行为的功能作用。为了鉴定MDAS，我们将使用一种高度选择性的交叉遗传方法，这种方法依赖于Cre和FLP重组酶的重叠组合来选择性地操纵特定的种群。使用这种方法，MDAS将被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透明大脑中的整个轴突投影(Clarity)。标记的神经元也将用免疫标记和多重荧光原位杂交来表征。我们还将使用激光捕获显微解剖技术从新鲜脑片中分离MDA，并进行基因表达分析。这些实验将定义丙二醛神经元的多样性，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它们的电生理表征，并使用药物遗传学选择性地激活或抑制这些MDA的活性。这些实验将提供多巴胺能亚回路的完整网络图，并提供第一个丙二醛神经元亚群的分子和生理学传记。这里产生的数据还将揭示丙二醛神经元亚群的功能作用。

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.