Paget-Blanc Vincent, Pronot Marie, Pfeffer Marlene E, Angelo Maria Florencia, Herzog Etienne
Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, Bordeaux, France.
Methods Mol Biol. 2025;2910:87-104. doi: 10.1007/978-1-0716-4446-1_6.
The central nervous system contains a complex intermingled network of neuronal, glial, and vascular cells, and for several decades, neurobiologists have used subcellular fractionation methods to analyze the molecular structure and functional features of the different cell populations. Biochemists have optimized fractionation protocols that enrich specific compartments such as synapses (called "synaptosomes") and synaptic vesicles to reduce this complexity. However, these approaches suffered from a lack of specificity and purity, which is why we previously extended the conventional synaptosome preparation to purify fluorescent synaptosomes from VGLUT1 knock-in mice on a cell sorter. We adapted our previous protocol to sort from single neuronal projections and small target regions of the brain as we did in the present example by labeling dopaminergic projections to the striatum. We proved that our newest method allows a steep enrichment in fluorescent dopaminergic synaptosomes containing presynaptic varicosities and associated postsynaptic elements and a substantial depletion in glial contaminants. Here we propose a detailed procedure for implementing projection-specific fluorescence-activated synaptosome sorting.
中枢神经系统包含一个由神经元、神经胶质细胞和血管细胞组成的复杂交织网络，几十年来，神经生物学家一直使用亚细胞分级分离方法来分析不同细胞群体的分子结构和功能特征。生物化学家优化了分级分离方案，以富集特定的区室，如突触（称为“突触体”）和突触小泡，以降低这种复杂性。然而，这些方法缺乏特异性和纯度，这就是为什么我们之前扩展了传统的突触体制备方法，以便在细胞分选仪上从VGLUT1基因敲入小鼠中纯化荧光突触体。我们调整了之前的方案，以便从单个神经投射和大脑的小目标区域进行分选，就像我们在本示例中通过标记多巴胺能投射到纹状体所做的那样。我们证明，我们的最新方法能够大幅富集含有突触前膨体和相关突触后元件的荧光多巴胺能突触体，并大量去除神经胶质污染物。在此，我们提出了一种实施投射特异性荧光激活突触体分选的详细程序。
