Cellular heterogeneity in the dopaminergic system

点击翻译按钮获取中文摘要

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.