Cellular heterogeneity in the dopaminergic system

中间脑多巴胺（mDA）神经元在广泛的脑功能调控中发挥核心作用，包括自主运动和行为过程。这些功能是由位于黑质致密部（SNpc）和腹侧被盖区（VTA）的mDA神经元的不同亚型提供的。然而，有几个原因导致我们质疑这种分类。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服从VTA或SNpc的解剖边界。第三，最近的报道表明这些神经元簇内的功能异质性，部分mDA神经元共同释放GABA、谷氨酸，有些神经元同时使用这三种神经递质。总之，这些证据表明，mDA神经元多样性的解剖分类是不充分的，并强调需要更好的mDA神经元分类。此外，这些亚种群应有的功能仍然不清楚。本提案的总体目标是确定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个具体目标：1-基于轴突投影识别mDAS， 2-揭示每个群体的分子和生理特征，3-揭示mDAS在小鼠行为中的功能作用。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将被标记为GFP，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除的大脑中的整个轴突投影（CLARITY）。标记的神经元也将使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切片中分离mDAS，并进行基因表达分析。这些实验将明确mDA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学方法来执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mDAS的活性。该实验将提供多巴胺能亚回路的完整网络图，并提供mDA神经元亚群的第一个分子和生理传记。这里产生的数据也将揭示mDA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.