Study of the Cholesystokinin (CCK) Molecular Subsets in the Mouse and Macaque Dorsal Horn

项目概要： 持续性疼痛是一种长期的状况，对患者的心理、身体和社会方面产生不利影响。 患者目前的治疗方法严重依赖阿片类药物和抗炎药物，这些药物可能会失去疗效 或者产生有害的副作用。持续性疼痛患者经常会经历静态或动态 机械性异常性疼痛是由点状刺激引起的疼痛感觉， 皮肤的压痕或在皮肤表面上的轻刷。将轻触转换为疼痛的机制， 损伤发生在脊髓背角内。识别相关的神经回路， 治疗靶向它的方法引起了强烈的兴趣。我们的实验室最近发现了 胆囊收缩素（CCK）表达兴奋性中间神经元，这些神经元对于传递静态和 动态机械性异常性疼痛这些神经元位于低阈值机械感觉区（板IIi-IV） 背角它们在由炎症和神经病理性诱发的两种形式的机械性异常性疼痛中的作用 使用病毒靶向的设计者受体，这些神经元的急性沉默证明了损伤 由设计师药物独家激活（DREADD）。炎症模型中的热超敏反应也是 颠倒了有趣的是，神经元短暂表达囊泡谷氨酸转运蛋白3（VGLUT 3）， 出生后的前两周发育是CCK Ⅲ-Ⅳ层细胞群的一个亚群，非常重要 仅用于传递动态异常性疼痛。为了更精确地识别哪个CCK子集负责不同的 持续性疼痛的形式，我们正在利用小鼠背角的单细胞转录组研究， CCK群体由Maf、Cpne 4和Trh标记的三个分子上不同的亚群组成。在 在初步实验中，我已经确定了我们用DREADD靶向的CCK神经元， 表达Trh或Cpne 4。由于所有的Trh+神经元都包括在目标群体中，因此在Aim中， 1a，我将确定Trh+神经元在持续性疼痛中的作用。由于瞬时VGLUT 3神经元是一种 CCK神经元的亚群，只需要动态异常性疼痛，在目标1b中，我将类似地 确定这些细胞的身份。为了更好地了解临床相关的持续性疼痛回路， 实验室希望将用于小鼠的相同工具应用于猕猴，并最终应用于人类。因此在 目的二，确定猕猴背角CCK的分子特性和层结构 亚群作为每个板层内兴奋性神经元的百分比。分子谱的保存和 小鼠和非人灵长类动物之间细胞类型的解剖学分布将提示 保持其功能作用。这一工作将为新型病毒的设计提供重要信息 策略，以靶细胞类型在灵长类动物背角研究疼痛电路，以及开发新的，非 用于机械性异常性疼痛的基于阿片样物质的治疗剂。

Project Summary: Persistent pain is a long-term condition that adversely affects psychological, physical and social aspects of patients. Current treatment methods rely heavily on opioids and anti-inflammatory drugs which can lose efficacy over time or produce deleterious side effects. Persistent pain patients often experience static or dynamic mechanical allodynia which is a painful sensation caused by innocuous stimuli that arises from punctate indentation of the skin or light brushing across the skin surface. Mechanisms that convert light touch into pain in the setting of injury occur within the spinal dorsal horn. Identifying the neural circuitry involved and developing ways to target it therapeutically are of intense interest. Our laboratory recently identified a subset of cholecystokinin (CCK) expressing excitatory interneurons that are important for conveying both static and dynamic mechanical allodynia. These neurons reside in the low threshold mechanosensory zone (lamina IIi-IV) of the dorsal horn. Their role in both forms of mechanical allodynia induced by inflammatory and neuropathic injuries was demonstrated by acute silencing of these neurons using a virally targeted Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Heat hypersensitivity in the inflammatory model was also reversed. Interestingly, neurons that transiently express the vesicular glutamate transporter 3 (VGLUT3) during the first two weeks of postnatal development are a subset of the lamina III-IV CCK population and are important only for conveying dynamic allodynia. To identify more precisely which CCK subset is responsible for the different forms of persistent pain, we are utilizing a single-cell transcriptome study of mouse dorsal horn, which reported that the CCK population is composed of three molecularly distinct subsets marked by Maf, Cpne4 and Trh. In preliminary experiments, I have identified the CCK neurons that we have targeted with the DREADD as expressing either Trh or Cpne4. Since all of the Trh+ neurons were included in the targeted population, in Aim 1a, I will determine the role of Trh+ neurons in persistent pain. Since the transient VGLUT3 neurons are a subpopulation of the CCK neurons and are required only for dynamic allodynia, in Aim 1b, I will similarly determine the identity of these cells. To better understand clinically relevant persistent pain circuitry, the laboratory would like to apply the same tools used in mouse to the macaque and eventually to human. Thus, In Aim 2, I will determine in the macaque dorsal horn, the molecular identity and laminar organization of the CCK subsets as a percentage of excitatory neurons within each lamina. Conservation of molecular profiles and anatomic distribution of cell-types between the mouse and non-human primate would be suggestive of a conservation of their functional roles. The work will provide important information for the design of novel viral strategies to target cell-types in the primate dorsal horn to study pain circuits as well as develop novel, non- opioid based therapeutics for mechanical allodynia.