Genetic, Molecular and Anatomical Characterization of VTA Cell Types Involved in Pain and Addiction

摘要 VTA 是两种常见病理状况（慢性疼痛和阿片类药物成瘾）的中心枢纽。在 在这两种情况下，VTA 神经元的生理特性都会发生显着改变。最近 有证据表明，在阿片类药物成瘾的情况下，并非所有 VTA 神经元都会做出相同的反应，并且在 疼痛时，不同的 VTA 神经元表现出互补的反应。清晰描述 VTA 中的细胞类型及其 同源预测和输入，以及其中哪些可能涉及这两种情况尚未得到很好的了解 阐明，并且对于理解这些病理状况至关重要。在此，我们建议确定 VTA 细胞类型的细胞、分子和解剖学景观，以及这些细胞在慢性疼痛和慢性疼痛中如何改变 成瘾模型。 VTA 显示出巨大的细胞异质性，由 DA 神经元、GABA 能神经元组成 和谷氨酸能神经元，以及非神经元细胞。此外，在 DA 神经元中还有额外的 异质性层，至少有四种 VTA DA 亚型已被证明。这种巨大的异质性 提出了理解 VTA 电路及其在这些条件下的变化的问题。在这里，我们的目标 的目的是将小鼠 VTA 分解成其组成细胞成分，以了解这些成分如何 疼痛和成瘾的个体成分会发生改变。在具体目标 1 中，我们将使用单细胞转录组学 分析慢性疼痛、吗啡给药或两者兼而有之的情况下的 VTA 细胞。我们期望首先细分 将 VTA 分解为其组成细胞类型，然后评估每种细胞类型中的分子变化 条件。这一目标将有助于发现治疗慢性疼痛的新分子靶点。在 具体目标2，使用新开发的交叉遗传工具来获取VTA细胞类型，我们将确定 几类 VTA DA 和非 DA 神经元的投射。接下来，我们将开发交叉狂犬病 病毒标记方法确定不同 VTA 细胞类型的输入。该目标将提供神经解剖学 理解参与这些病理状况的回路的基础。最后，在具体目标 3 中，指导 根据目标 1 和 2 的数据，我们将识别并验证特定 DA 细胞类型中的新靶点，目标是 逆转慢性疼痛特征的低多巴胺能状态。 这些目标的结果将为理解提供分子、细胞和解剖学框架 VTA 细胞类型，这将与本 P50 中的所有项目相关。此外，发现和验证 不同 VTA 细胞类型中的候选受体，将为开发提供一个极好的切入点 治疗慢性疼痛的阿片类药物的替代品。

Abstract The VTA is a central hub for two prevalent pathological conditions - chronic pain and opioid addiction. In both these conditions, the physiological properties of neurons in the VTA are significantly altered. Recent evidence suggests that in the case of opioid addiction, not all VTA neurons respond equally, and in the case of pain, different VTA neurons display complementary responses. A clear description of cell types in the VTA, their cognate projections and inputs, and which of these might be involved in these two conditions has not been well elucidated, and is essential to understand these pathological conditions. Here, we propose to determine the cellular, molecular and anatomical landscape of VTA cell types and how these are altered in chronic pain and addiction models. The VTA displays enormous cellular heterogeneity, being comprised of DA neurons, GABAergic neurons and Glutamatergic neurons, as well as non-neuronal cells. Further, among the DA neurons there is additional layer of heterogeneity, and at least four VTA DA subtypes have been demonstrated. This immense heterogeneity presents a problem for understanding VTA circuitry as well as its alterations in these conditions. Here, our goal is to disentangle the murine VTA into its constituent cellular components, towards understanding how these individual components are altered in pain and addiction. In Specific Aim 1, we will use single cell transcriptomics to analyze VTA cells in conditions of chronic pain, morphine administration, or both. We expect to first subdivide the VTA into its constituent cell types, and then evaluate the molecular changes within each cell type, in each condition. This aim will facilitate discovery of new molecular targets towards treatment of chronic pain. In Specific Aim 2, using newly developed intersectional genetic tools to access VTA cell types, we will determine the projections of several classes of VTA DA and non-DA neurons. Next, we will develop an intersectional rabies virus labeling approach to determine the inputs of distinct VTA cell types. This aim will provide a neuroanatomical foundation for understanding circuits involved in these pathological conditions. Finally, in Specific Aim 3, guided by data from Aim 1 and 2, we will identify and validate novel targets in specific DA cell types, with the goal of reversing the hypo-dopaminergic state that is characteristic of chronic pain. The results of these Aims will provide a molecular, cellular and anatomical framework for understanding VTA cell types, that will be relevant to all projects in this P50. Additionally, the discovery and validation of candidate receptors in distinct VTA cell types, will provide an excellent entry point towards developing alternatives to opioids in the management of chronic pain.