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衰弱的疾病，迫切需要安全有效的治疗方法。这些 患者经历增强的疼痛感，并且当无害刺激被施加时， 提出了然而，这种敏感性增加的神经基础知之甚少。在此，我们建议 研究可能导致持续性疼痛的中枢兴奋性的神经回路基础。我们将 结合新的生理学和药理学方法来实现我们的目标。使用现有 转基因小鼠品系，我们可以在所有兴奋性脊髓神经元中表达钙指示剂GCamp 6s。我们 然后可以使用我们的新的离体皮肤脊髓制备和双光子钙成像来检查活动 在脊髓背角活动的皮肤刺激在幼稚的小鼠和那些受伤后。虽然这 这种方法将允许我们同时成像许多（~200）神经元的反应，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可以被 翻译。现在，我们已经开发了一种新的方法来规避这一限制，通过事后 细胞类型的药理学鉴定。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（GPCR），其激活导致Ca 2+从细胞内释放， 店在河豚毒素（TTX）沉默神经元活动的存在下，只有神经元显示Ca 2 + 瞬时响应给定激动剂的是表达其受体的那些。这种方法，我们有 称为CICADA（通过药物活化的Ca 2+偶联活性进行细胞类型鉴定），使我们能够 基于细胞对一系列GPCR激动剂的反应，明确定义细胞类型。现在我们是独一无二的 准备解决有关脊髓回路功能的具体问题，以及 这些回路在受伤后会改变。在第一个目标中，我们将扩展并验证此分析，目标是 发展一个完整的背角亚型，可以在人口成像研究中确定。 虽然神经元对不同类型的感觉刺激（例如，热，冷，和机械），如何编码 在神经元群体中的表现尚不清楚。在第二个目标中，我们将分类功能反应 CICADA定义的细胞亚型群体之间的特性。在第三个目标中，我们将研究 辣椒素诱导这些亚群的急性中枢致敏。疼痛的慢性化被认为是 与中枢网络活动的长期变化有关，这些变化使痛觉过敏状态永久化。在 第四个目标：我们将鉴定CICADA定义的细胞类型，这些细胞类型在慢性炎症背景下显示出改变的活性。 疼痛使用备用神经损伤（SNI）作为模型。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，并研究这些特定的微回路是如何 在急性和慢性损伤条件下发生改变。这一知识可能阐明新的治疗靶点， 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.