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，因为疼痛严重，已知的镇痛药临床疗效有限。近年来疼痛研究的重要发现之一是ATP激活的P2X4受体（P2X4Rs）和小胶质细胞与神经元的相互作用在神经性疼痛中的作用。在神经性疼痛过程中，P2X4R在疼痛通路背角的小胶质细胞中表达上调，P2X4R功能的降低可缓解神经性疼痛的症状。然而，许多基本方面仍未被探索，对小胶质细胞P2X4R的特性或其在神经性疼痛中的上调机制只有初步的了解。由于不可能从组织切片中识别和记录表达P2X4R的细胞，因此进展受到阻碍。在切片和体内鉴定P2X4R表达细胞的方法将能够严格测试健康中枢神经系统和疾病过程中有关小胶质P2X4Rs的机制假设。在这里，我们试图利用红色荧光蛋白tdTomato生成并彻底表征一只光学报告小鼠。这只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中P2X4R表达的细胞，从而探索决定P2X4R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，我们将生成并充分表征报告小鼠，并利用它来测量P2X4反应，使用荧光引导膜片钳记录来自小胶质细胞和神经元。在Aim 1中，我们将产生表达tdTomato荧光蛋白的P2X4R报告小鼠，并建立和表征转基因小鼠的不同始祖系，以确定表达P2X4R细胞的位置和身份。在Aim 2中，我们将使用膜片钳电生理学在报告小鼠的组织切片中研究P2X4R表达细胞。我们将直接验证P2X4R上调是背角疼痛通路中激活的小胶质细胞特异性的假设。总的来说，我们的方法将提供新颖的，具有良好特征的光学报告小鼠，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（如中枢神经系统切片）中表达P2X4R的细胞。这些新的报告小鼠将成为P2X、小胶质细胞和疼痛研究界有价值的通用工具。

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. PUBLIC HEALTH RELEVANCE: We will develop mouse models that will allow us and other researchers to identify and record from cells in the brain that express P2X4 receptors. The availability of these mice would constitute exceptional tools with which to study the role of P2X4 receptors in the normal healthy brain and in diseases of the nervous system, including the processes that lead to the development of neuropathic pain states.