Optical reporter mice to study P2X4 receptors in microglia

描述（由申请人提供）：神经性疼痛是一种普遍的疾病，伴随着广泛的疾病，包括癌症、糖尿病、创伤性损伤和艾滋病。这是一个重要的临床问题，因为疼痛是严重的，已知的止痛药具有有限的临床疗效。近年来疼痛研究的重要发现之一是ATP激活的P2 X4受体（P2 X4 Rs）和小胶质细胞-神经元相互作用在神经病理性疼痛中的作用。在神经性疼痛期间，P2 X4 R表达在位于疼痛通路的背角中的小胶质细胞中上调，并且降低P2 X4 R功能加重了该病症的啮齿动物模型中的神经性疼痛症状。然而，许多基本的方面仍然没有探索，只有一个初步的了解小胶质细胞P2 X4 R的属性或参与其上调神经性疼痛的机制。进展受到阻碍，因为不可能从组织切片中鉴定和记录表达P2 X4 R的细胞。用于在切片和体内鉴定P2 X4 R表达细胞的方法将使得能够严格测试关于健康CNS中和在疾病过程中的小胶质细胞P2 X4 R的机制假设。在这里，我们试图产生和彻底表征光学报告小鼠使用红色荧光蛋白tdTomato。这种小鼠的可用性将使研究人员能够直接观察和记录组织切片内表达P2 X4 R的细胞，从而探索决定P2 X4 R上调的细胞机制，从而为理解神经性疼痛过程中疼痛通路的机制和可塑性变化做出重大贡献。在两个目标的范围内，我们将产生和充分表征报告小鼠，并利用它来测量P2 X4的反应，使用荧光引导膜片钳记录从小胶质细胞和神经元。在目标1中，我们将产生表达tdTomato荧光蛋白的P2 X4 R报告小鼠，以及建立和表征转基因小鼠的不同建立系，以定位P2 X4 R表达细胞的位置和身份。在目标2中，我们将使用膜片钳电生理学在来自报告小鼠的组织切片中研究P2 X4 R表达细胞。我们将直接测试的假设，P2 X4 R上调是特定的激活小胶质细胞在背角疼痛通路。总的来说，我们的方法将提供新的，充分表征的光学报告小鼠，这将使我们和其他研究人员能够识别和记录完整组织结构（如CNS切片）中的P2 X4 R表达细胞。这些新的报告小鼠将成为P2 X，小胶质细胞和疼痛研究社区的宝贵工具。

DESCRIPTION (provided by applicant): Neuropathic pain is a prevalent disorder that accompanies a wide spectrum of diseases including cancer, diabetes, traumatic injuries and AIDS. It is a significant clinical problem because the pain is severe and known analgesics have limited clinical efficacy. One of the important discoveries in pain research in the last few years has been the role of ATP activated P2X4 receptors (P2X4Rs) and microglia-neuron interactions in neuropathic pain. During neuropathic pain P2X4R expression is upregulated in microglia located in the dorsal horn of the pain pathway, and reducing P2X4R function alleviates symptoms of neuropathic pain in rodent models of the disorder. However, many fundamental aspects remain unexplored and there is only a rudimentary understanding of microglial P2X4R properties or the mechanisms involved in their upregulation in neuropathic pain. Progress has been hindered because it is not possible to identify and record from P2X4R expressing cells in tissue slices. A method for identifying P2X4R expressing cells in slices and in vivo would enable the rigorous testing of mechanistic hypotheses regarding microglial P2X4Rs in the healthy CNS and during disease processes. Here we seek to generate and thoroughly characterize an optical reporter mouse using the red fluorescent protein tdTomato. The availability of this mouse will allow researchers to directly visualize and record from P2X4R expressing cells within tissue slices and thus explore the cellular mechanisms that determine P2X4R upregulation and thus contribute significantly to the understanding of mechanisms and plasticity changes in the pain pathway during neuropathic pain. Within the scope of two aims we will generate and fully characterize the reporter mouse and exploit it to measure P2X4 responses using fluorescence guided patch-clamp recordings from microglia and neurons. In Aim 1 we will generate P2X4R reporter mice expressing tdTomato fluorescent proteins as well as establish and characterize different founder lines of transgenic mice to map the location and identity of P2X4R expressing cells. In Aim 2 we will study P2X4R expressing cells using patch-clamp electrophysiology in tissue slices from reporter mice. We will directly test the hypothesis that P2X4R upregulation is specific to activated microglia in the dorsal horn pain pathway. Overall, our approach will provide novel, well characterized optical reporter mice that will allow us and other researchers to identify and record from P2X4R expressing cells within intact tissue structures such as slices of CNS. These new reporter mice will be valuable general tools for the P2X, microglia and pain research communities.