Purification of cell-type specific synaptic material using virally-expressed tags

项目总结 到目前为止，大脑是最复杂的器官，有数百甚至数千种不同的细胞类型混合在一起 在一个组织中，乍一看，似乎是相当均匀的。这给生物化学带来了一个问题。 对特定细胞类型的研究，因为特定细胞类型的蛋白质含量很难或不可能从其中提纯 周围的组织。例如，在纹状体中，直接路径与间接路径的中等棘神经元 (dMSN与iMSN)表达不同的分子标记，细胞类型特异的激活或沉默产生 相反的行为输出。在吸毒成瘾的背景下，沉默iMSN增强了自我- 管理药物，同时沉默dMSN减少寻求药物的行为。治疗方法可能会 纠正药物滥用引起的广泛分子改变是很有希望的治疗策略。 然而，考虑到iMSN和dMSN相反的行为输出，这是一种减少药物寻求的药物 通过一条途径的行为可以通过另一条途径增强相同的行为。细胞类型的鉴定 特定的靶点将增加调节特定纹状体途径和行为的可能性，但我们的 由于无法提纯IMSN与dMSN蛋白，因此无法进行这种探索性实验。在此，我们建议 开发一种基于常见免疫学技术--磁细胞分选(MACS)的分选方法。我们 将在imsn或dmsn神经元的树突棘中表达一种惰性的、细胞外被识别的“标签”。 由高度特异的商用MACS分选试剂制成。细胞类型的特异性将通过使用 病毒双注射策略，其中将注射包含反向标签构造的DIO/FLEX载体 进入纹状体，逆行Cre病毒将被注射到腹侧被盖区(VTA)或 腹侧苍白球(VP)分别标记直接通路和间接通路神经元。排序将是 使用金属结合抗体和磁化柱完成，允许快速分离大鼠 生物材料的数量。在目标1中，我们开发并验证了大鼠的标签系统。在目标2中，我们将iMSN标记为 和dMSN，并使用扩展的间歇性接触自我给药范式来产生 表现出低水平和高水平的成瘾者行为(以及对控制的食物奖励)的一组大鼠。我们 然后，将从表现高和低成瘾者样行为的大鼠中分离出iMSN和dMSN，并使用 鉴定iMSN或iMSN差异表达蛋白质的定量质谱学方法 大鼠的DMSN表现出高水平与低水平的成瘾者行为。超范围的后续工作 格兰特将探索在表达成瘾样蛋白的大鼠中调节这些蛋白表达的可能性 行为，目的是通过特定靶向IMSN或dMSN的细胞来减少寻求药物的行为 路径。更广泛地说，我们的标记和分类方法可以广泛应用于细胞类型的研究。 特定蛋白质在大脑中的表达。

PROJECT SUMMARY The brain is by far the most complex organ, with hundreds or even thousands of distinct cell types intermingled in a tissue that appears, at first glance, to be fairly homogeneous. This presents a problem for biochemical studies of specific cell types, since the protein content of a given cell type is difficult or impossible to purify from the surrounding tissue. For example, in the striatum, medium spiny neurons of the direct vs. indirect pathway (dMSNs vs. iMSNs) express different molecular markers, and cell-type-specific activation or silencing produces opposing behavioral outputs. In the context of drug addiction, silencing iMSNs enhances the motivation to self- administer drugs, while silencing dMSNs reduces drug-seeking behavior. Treatment approaches that could correct the extensive molecular alterations caused by drugs of abuse are promising therapeutic strategies. However, given the opposing behavioral outputs of iMSNs vs. dMSNs, a drug that reduces drug-seeking behavior via one pathway could enhance the same behavior via the other pathway. Identification of cell-type specific targets would increase the possibility of modulating specific striatal pathways and behaviors, but our inability to purify iMSN vs. dMSN protein precludes such exploratory experiments. Here, we propose to develop a sorting approach based on a common Immunology technique, Magnetic Cell Sorting (MACS). We will express, in the dendritic spines of iMSN or dMSN neurons, an inert, extracellular “TAG” that is recognized by highly specific, commercially available MACS sorting reagents. Cell-type-specificity will be achieved using a viral dual-injection strategy, in which a DIO/FLEX vector containing an inverted TAG construct will be injected into the striatum, and a retrograde Cre virus will be injected into the Ventral Tegmental Area (VTA) or the Ventral Pallidum (VP) to label neurons of the direct or indirect pathway, respectively. Sorting will be accomplished using metal-conjugated antibodies and a magnetized column, allowing rapid isolation of a large amount of biomaterial. In aim 1, we develop and validate the TAG system in the rat. In Aim 2, we label iMSNs and dMSNs in rats, and use an extended intermittent access herion self-administration paradigm to produce groups of rats that express low- and high-levels of addict-like behavior (as well as food reward for control). We will then isolate iMSNs and dMSNs from rats expressing high and low addict-like behavior, and use a quantitative mass spectrometry approach to identify proteins that are differentially expressed in iMSNs or dMSNs in rats showing high- vs. low-levels of addict-like behavior. Follow-up work beyond the scope of the grant will explore the potential of modulating the expression of these proteins in rats expressing addict-like behavior, with the goal of reducing drug-seeking behavior by specifically targeting cells of the iMSN or dMSN pathway. More generally, our tagging-and-sorting approach could be widely applicable to the study of cell-type- specific protein expression in the brain.