Spatiotemporal Coding in the Pain Circuit Along the Spine-brain Continuum

项目概要 了解脊髓感觉处理的细胞生物学和神经生理学是 对于推进治疗慢性和急性疼痛的医疗干预至关重要。的 目前对脊髓回路神经生理学的理解是建立在实验单单元的基础上的 麻醉动物的电生理学和体外研究，但体内功能数据有限 感觉信号的电路。这种实验面临的部分挑战是能力有限 监测清醒动物的电生理信号并诱导疼痛纤维的可靠激活。 因此，特定神经元亚型在传播兴奋性和抑制性信号方面的活性 体内疼痛信号传递的参与尚不清楚。最近，我们出现了一种疼痛 检测分析包括响应主要伤害感受的光遗传学激活的舔行为 表达通道视紫红质 2 (ChR2) 的头部受限转基因小鼠的周围传入神经纤维 瞬时受体电位阳离子通道亚家族 V 成员 1 (TRPV1) 含有神经元。在这个模型中， 训练小鼠提供舔报告以检测光诱发的后爪伤害性刺激。 我们的伤害性舔报告检测分析能够对毫秒、单细胞、 清醒行为动物中枢神经系统疼痛处理的神经动力学。 此外，我们还开发了一种“背包驱动器”，可以提供多位点慢性细胞外记录 背角神经元源自浅层 II-III。不幸的是，这种电生理学不能 用于在记录期间确定细胞亚类。在这里，我们将重点提高我们的记录能力 背角中含有光激活TRPV1神经元的细胞类型特异性活动 外围。我们将开发一种可靠的方法来在特定的环境中实现一致的 GCaMP6 家族表达 神经元细胞类型（例如 CaMKII、PV）通过我们的光遗传学参与疼痛信号的特异性激活 刺激实验设计。我们将优化脊柱光学窗口以执行清醒钙成像 在时间锁定的触觉输入期间表征背角神经元亚型的钙动态 在行为任务期间。这项工作将建立一种方法来收集大类的时间动态 背角神经元响应时间锁定、空间精确和幅度调制的输入 外围导致更好地了解急性疼痛状况。

PROJECT SUMMARY Understanding the cellular biology and neurophysiology of sensory processing in the spinal cord is fundamental to advancing medical intervention in the treatment of chronic and acute pain conditions. The current understanding of the neurophysiology of spinal cord circuitry is founded on experimental single-unit electrophysiology on anesthetized animals and in-vitro studies, but limited data exist from in-vivo functional circuitry of sensory signals. Part of the challenge to such experimentation has been the limited capacity for monitoring electrophysiologic signals in awake animals and inducing reliable activation of pain fibers. Consequently, the activity of specific neuronal subtypes in propagating excitatory and inhibitory signals involved in the transmission of pain signals remains unknown in-vivo. Recently, we have developed a pain detection assay consisting of a lick behavior in response to optogenetic activation of predominantly nociceptive peripheral afferent nerve fibers in head-restrained transgenic mice expressing Channelrhodopsin 2 (ChR2) in transient receptor potential cation channel subfamily V member 1 (TRPV1) containing neurons. In this model, mice are trained to provide lick reports to the detection of light-evoked nociceptive stimulation to the hind paw. Our nociceptive lick-report detection assay enables a host of investigations into the millisecond, single-cell, neural dynamics underlying pain processing in the central nervous system of awake behaving animals. Further, we have developed a “backpack drive” to provide multi-site chronic extracellular recordings from dorsal horn neurons derived from superficial laminas II-III. Unfortunately, such electrophysiology cannot be used to determine cellular subclasses during recording. Here, we will focus on advancing our ability to record cell-type-specific activity in the dorsal horn in response to light-activated TRPV1 containing neurons in the periphery. We will develop a reliable method for achieving consistent GCaMP6-family expression in specific neuronal cell types (e.g. CaMKII, PV) involved in the specific activation of pain signals through our optogenetic stimulation experimental design. We will optimize a spinal optical window to perform awake Calcium imaging during time-locked tactile input and characterize calcium dynamics in neuronal subtypes in the dorsal horn during behavioral tasks. This work will establish a methodology to collect temporal dynamics of large classes of neurons in the dorsal horn in response to time-locked, spatially-precise, and amplitude-modulated input in the periphery leading to improved understanding of acute pain conditions.