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

基本信息

批准号：
10707980
负责人：
H Richard Koerber
金额：
$ 59.99万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
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/10707980
关键词：
Acute Address Agonist American Brain Calcium Capsaicin Catalogs Cells Chemicals Chronic 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 Phenotype Physiological Population Preparation Process Property Quality of life Research Sensory Series Skin Spinal Spinal Cord Stimulus Testing Tetrodotoxin Thermal Hyperalgesias Transgenic Mice Vertebral column Visualization allodynia calcium indicator cell type cellular imaging central sensitization chronic pain dorsal horn effective therapy excitatory neuron experience heat stimulus imaging approach imaging study injured innovation insight mechanical stimulus nerve injury neural neural circuit new therapeutic target novel novel strategies pain chronification pain sensation pharmacologic programs receptor recruit response sensory input sensory integration sensory stimulus somatosensory spared nerve tool two-photon

项目摘要

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疾病，迫切需要安全、有效的治疗方法。患者会感到疼痛感增强，并且当受到无害刺激时常常会感到疼痛。然而，我们对这种敏感性增加的神经基础知之甚少。研究可能导致持续性疼痛的中枢过度兴奋的神经回路基础。结合新颖的生理学和药理学方法来实现我们的目标。在转基因小鼠品系中，我们可以在所有兴奋性脊髓神经元中表达钙指示剂GCamp6s。然后可以使用我们新颖的离体皮肤脊髓制剂和 2 光子钙成像来检查活性在幼稚小鼠和受伤后的小鼠中，脊髓背角活动对皮肤的刺激。方法将允许我们同时对许多（~200）神经元的反应进行成像，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识别程度现在，我们开发了一种新颖的方法来通过事后规避这一限制。细胞类型的药理学鉴定的基本概念是大多数神经元表达一种或多种。 Gq 偶联的 G 蛋白偶联受体 (GPCR) 的激活导致内部 Ca2+ 的释放在存在河豚毒素 (TTX) 来抑制神经元活动的情况下，唯一显示 Ca2+ 的神经元。对给定激动剂的瞬时反应是那些表达其受体的方法，我们有这种方法。称为 CICADA（通过药物激活通过 Ca2+ 偶联活性进行细胞类型识别），使我们能够根据细胞对一系列 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脊髓回路的功能特性如何在第一个目标中，我们将扩展并验证此分析的目标。开发可以在群体成像研究中识别的完整的背角亚型。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，但这种编码如何在第二个目标中，我们将对功能反应进行分类。在第三个目标中，我们将研究 CICADA 定义的细胞亚型群体的特性。辣椒素引起这些亚群的急性中枢敏化，疼痛的慢性化被认为是这样的。与维持痛觉过敏状态的中枢网络活动的长期变化有关。第四个目标，我们将识别 CICADA 定义的细胞类型，这些细胞类型在慢性疾病背景下表现出改变的活性我们在此提出的研究将开始使用幸存神经损伤（SNI）作为模型来识别疼痛。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工作的急性和慢性损伤情况下的改变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。