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

损伤前后处理皮肤信息的脊髓神经元特定群体的综合表型

• 批准号: 10211006
• 负责人: H Richard Koerber
• 金额: $ 59.21万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-16 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211006
• 关键词: Acute; Address; Agonist; American; Brain; Calcium; Capsaicin; Catalogs; Cells; Chemicals; Chronic; Cicadas; Code; Coupled; Cutaneous; Data; G-Protein-Coupled Receptors; Goals; Hand; Health; Hyperalgesia; Image; Individual; Injury; Knowledge; Label; Measures; Mechanics; Modeling; Mus; Nerve; Neurons; Neuropathy; Nociception; Optics; Output; Pain; Pain management; Patients; Peripheral; Persistent pain; Pharmaceutical Preparations; Pharmacology; Phenotype; Physiological; Population; Preparation; Process; Property; Research; Sensory; Series; Skin; Spinal; Spinal Cord; Stimulus; Testing; Tetrodotoxin; Thermal Hyperalgesias; Transgenic Mice; allodynia; base; calcium indicator; cell type; central sensitization; chronic pain; dorsal horn; effective therapy; excitatory neuron; experience; heat stimulus; imaging approach; imaging study; injured; innovation; insight; nerve injury; neural circuit; new therapeutic target; novel; novel strategies; pain chronification; pain sensation; programs; receptor; recruit; relating to nervous system; response; sensory input; sensory stimulus; somatosensory; spared nerve; tool; two-photon

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.

抽象的 慢性疼痛是一种使人衰弱的状况，对安全有效的治疗迫切需要。这些 当无害的刺激时，患者会感到疼痛增强，并且经常会出现疼痛 提出。但是，敏感性提高的神经元基础知之甚少。在这里，我们建议 研究可能导致持续性疼痛的中心过度兴奋性的神经回路基础。我们会的 结合新颖的物理和药物方法来解决我们的目标。使用现有 转基因小鼠系，我们可以在所有兴奋性脊柱神经元中表达钙指示剂GCAMP6。我们 然后可以使用我们的新型外体皮肤脊髓制备和2光子钙成像进行检查活动 在幼稚的小鼠和受伤后的脊髓角性刺激的脊柱背角活性中。同时 方法将使我们能够简单地想象许多（〜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的程度 解释。现在，我们已经开发了一种新颖的方法来通过事后绕过这种限制 细胞类型的药理鉴定。基本概念是大多数神经元表达一个或多个 GQ耦合的G蛋白偶联受体（GPCR），其激活导致Ca2+从内部释放 商店。在存在四毒素（TTX）以沉默神经元活性的情况下，唯一显示Ca2+的神经元 响应给定激动剂的瞬态是表达接收器的激动剂。我们有这种方法 称为CICADA（通过Ca2+偶联活性通过药物激活鉴定细胞类型），使我们能够 明确定义的细胞类型基于它们对一系列GPCR激动剂的响应。现在我们是独特的 中毒以解决有关脊柱电路功能以及如何功能特性的特定问题 受伤后这些电路会改变。在第一个目的中，我们将扩展和验证该分析的目标 开发可以在人群成像研究中鉴定出的完整曲目背角亚型。 神经元对不同类型的感觉刺激（例如热，冷和机械）做出反应 在神经元种群中表现出来的尚不清楚。在第二个目标中，我们将分类功能响应 西卡达定义细胞亚型种群之间的特性。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心敏感性。人们认为疼痛的编回 与中央网络活动的长期变化有关，使痛苦状态永存。在 第四目的我们将确定蝉定义的细胞类型，这些细胞类型在慢性的背景下显示出改变活动的变化 使用幸免的神经损伤（SNI）作为模型疼痛。我们在这里提出的研究将开始识别 涉及中央灵敏度的特定脊柱回路，并研究了这些特定的微电路 急性和慢性损伤状况发生了改变。这些知识可以阐明针对 治疗疼痛，这是我们计划研究的长期目标。