Optical reporter mice to study P2X4 receptors in microglia

描述(申请人提供)：神经性疼痛是一种普遍存在的疾病，伴随着多种疾病，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，因为疼痛很严重，而且已知的止痛药临床疗效有限。最近几年在疼痛研究中的重要发现之一是三磷酸腺苷激活的P2X4受体(P2X4Rs)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。在神经病理性疼痛过程中，位于疼痛通路背角的小胶质细胞中，P2X4R的表达上调，而降低P2X4R功能可以缓解该疾病啮齿动物模型中的神经病理性疼痛症状。然而，许多基本方面仍未被探索，对小胶质细胞P2X4R的特性或其在神经病理性疼痛中上调的机制仅有初步的了解。由于不可能从组织切片中鉴定和记录表达P2X4R的细胞，这一进展受到了阻碍。一种在切片和体内识别表达P2X4R的细胞的方法将使在健康中枢神经系统和疾病过程中关于小胶质细胞P2X4R的机制假说得到严格的检验。在这里，我们试图利用红色荧光蛋白tdTomato来培育和彻底鉴定一种光学报告小鼠。这只小鼠的出现将使研究人员能够直接可视化和记录组织切片中表达P2X4R的细胞，从而探索决定P2X4R上调的细胞机制，从而有助于理解神经病理性疼痛过程中疼痛通路的机制和可塑性变化。在两个目标的范围内，我们将产生并充分描述报告小鼠，并利用它来使用来自小胶质细胞和神经元的荧光引导膜片钳记录来测量P2X4反应。在目标1中，我们将建立表达td番茄荧光蛋白的P2X4R报告小鼠，并建立和鉴定不同的转基因小鼠系，以定位表达P2X4R的细胞的位置和身份。在目标2中，我们将使用膜片钳电生理学方法研究报告小鼠组织切片中表达P2X4R的细胞。我们将直接检验这一假设，即P2X4R上调是背角疼痛通路中激活的小胶质细胞所特有的。总体而言，我们的方法将提供新颖的、特征良好的光学报告小鼠，使我们和其他研究人员能够识别和记录在完整的组织结构中表达细胞的P2X4R，如中枢神经系统切片。这些新的报告小鼠将成为P2X、小胶质细胞和疼痛研究社区的宝贵通用工具。 与公共健康相关：我们将开发小鼠模型，使我们和其他研究人员能够识别和记录大脑中表达P2X4受体的细胞。这些小鼠的出现将构成研究P2X4受体在正常健康大脑和神经系统疾病中的作用的特殊工具，包括导致神经病理性疼痛状态发展的过程。

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.