Comprehensive Phenotyping of Specific Populations of Spinal Neurons Processing Cutaneous Information Before and After Injury

摘要 慢性疼痛是一种使人虚弱的疾病，迫切需要安全有效的治疗方法。这些 患者会感受到更强的痛感，当无害的刺激被 呈上了。然而，这种敏感度增加的神经基础却鲜为人知。在此，我们建议 研究可能导致持续性疼痛的中枢性过度兴奋的神经回路基础。我们会的 结合新的生理学和药理学方法来解决我们的目标。使用现有的 在转基因小鼠系中，我们可以在所有兴奋性脊髓神经元表达钙指示剂GCamp6s。我们 然后可以使用我们的新型体外皮肤-脊髓准备和双光子钙成像来检查活动 在幼稚小鼠和损伤后小鼠的脊髓背角对皮肤刺激的活动。虽然这件事 这种方法将允许我们同时成像许多(~200)个神经元的反应，但它不允许我们 识别不同类型的神经元，从而严重阻碍了数据的可获得性 翻译过来了。现在，我们已经开发了一种新的方法来绕过这一限制，通过后自组织 细胞类型的药理学鉴定。其基本概念是，大多数神经元表达一个或多个 Gq偶联G蛋白偶联受体(GPCRs)，其激活导致内源性钙释放 商店。在河豚毒素(TTX)沉默神经元活动的情况下，唯一显示钙离子的神经元 对某一特定激动剂的瞬时反应是那些表达其受体的受体。这种方法，我们有 被称为蝉(通过药物激活的钙偶联活性来识别细胞类型)，允许我们 明确地根据细胞对一系列GPCR激动剂的反应来定义细胞类型。现在我们是唯一的 准备解决有关脊髓回路功能的具体问题以及脊髓回路的功能特性 这些回路在受伤后会发生改变。在第一个目标中，我们将使用目标扩展和验证这一分析 开发一套完整的背角亚型曲目，可以在种群成像研究中识别。 虽然神经元对不同类型的感觉刺激(例如，热、冷和机械)做出反应，但这种编码是如何 在神经细胞群体中是否明显存在尚不清楚。在第二个目标中，我们将对功能反应进行分类 蝉定义的细胞亚型种群间的特性。在第三个目标中，我们将检查 辣椒素可引起这些亚群的急性中枢敏感化。疼痛的时代化被认为是 与中枢网络活动的长期变化有关，从而使痛觉过敏状态永久化。在 第四个目标，我们将确定蝉定义的细胞类型，这些细胞类型在慢性阻塞性肺疾病的背景下显示出活动的改变 以备用神经损伤(SNI)作为疼痛模型。我们在这里提出的研究将开始确定 参与中枢敏化的特定脊髓回路，并研究这些特定微回路是如何 在急性和慢性损伤的情况下发生变化。这一知识可能会阐明新的治疗靶点 治疗疼痛，这是我们项目研究的长期目标。

ABSTRACT Chronic pain is a debilitating condition for which there is a pressing need for safe, effective treatments. These patients experience enhanced pain sensations and often experience pain when innocuous stimuli are presented. However, the neural basis for this increased sensitivity is poorly understood. Here, we propose to investigate the neural circuit basis for central hyperexcitability that may contribute to persistent pain. We will be combining novel physiological, and pharmacological approaches to address our goals. Using existing transgenic mouse lines, we can express the calcium indicator GCamp6s in all excitatory spinal neurons. We can then use our novel ex vivo skin-spinal cord preparation and 2-photon calcium imaging to examine activity in the spinal dorsal horn activity to cutaneous stimulation in naïve mice and those following injury. While this approach will allow us to image the responses of many (~200) neurons simultaneously, it does not allow us to identify different types of neurons, thereby severely hampering the degree to which the data can be interpreted. Now, we have developed a novel approach to circumvent this limitation through post hoc pharmacological identification of cell types. The underlying concept is that most neurons express one or more Gq-coupled G-protein coupled receptors (GPCRs) whose activation results in the release of Ca2+ from internal stores. In the presence of tetrodotoxin (TTX) to silence neuronal activity, the only neurons that show a Ca2+ transient in response to a given agonist are those that express its receptor. This approach, which we have termed CICADA (Cell-type Identification by Ca2+-coupled Activity through Drug Activation), allows us to unambiguously define cell types based on their responses to a series of GPCR agonists. Now we are uniquely poised to address specific questions about the function of spinal circuitry and how the functional properties of these circuits are altered following injury. In the first Aim we will extend and validate this analysis with the goal of developing a complete repertoire dorsal horn subtypes that can be identified in population imaging studies. While neurons respond to different types of sensory stimuli (e.g., heat, cold, and mechanical), how this coding is manifest across neuronal populations is unclear. In the second Aim we will catalog functional response properties across populations of CICADA-defined cell subtypes. In the third Aim we will examine the effects of capsaicin induced acute central sensitization on these subpopulations. The chronification of pain is thought to be associated with long-term changes in central network activity that perpetuate hyperalgesic states. In the fourth Aim we will identify the CICADA-defined cell types that show altered activity in the context of chronic pain using the spared nerve injury (SNI) as a model. The studies we are proposing here will begin to identify specific spinal circuitry involved in central sensitization and investigate how these specific microcircuits are altered in conditions of acute and chronic injury. This knowledge may elucidate new therapeutic targets for the treatment of pain, which is the long-term goal of research of our program.