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

项目摘要 了解脊髓感觉处理的细胞生物学和神经生理学， 这是推进慢性和急性疼痛疾病治疗中的医疗干预的基础。的 目前对脊髓回路的神经生理学的理解是建立在实验性的单一单元上的， 在麻醉动物和体外研究的电生理学，但有限的数据存在从体内功能 感觉信号的回路这种试验所面临的部分挑战是， 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中的活动 参与疼痛信号传递的神经元在体内仍是未知的。最近，我们发现了一种疼痛 由响应于主要伤害感受的光遗传学激活的舔行为组成的检测测定 在表达视紫红质2（ChR 2）的头部受限转基因小鼠中的外周传入神经纤维， 瞬时受体电位阳离子通道亚家族V成员1（TRPV 1）含有神经元。在这个模型中， 训练小鼠以提供对后爪的光诱发伤害性刺激的检测的舔报告。 我们的伤害感受舔报告检测试验能够对毫秒级单细胞， 清醒行为动物中枢神经系统疼痛处理的神经动力学基础。 此外，我们已经开发了一种“背包驱动器”，以提供多位点慢性细胞外记录， 背角神经元来源于浅层II-III。不幸的是，这种电生理学不能被 用于在记录期间确定细胞亚类。在这里，我们将重点推进我们的能力， 背角中细胞类型特异性活性响应于光激活的TRPV 1， 外围我们将开发一种可靠的方法，在特定的细胞中实现GCaMP 6家族表达的一致性。 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和表征钙动力学的神经元亚型在背角 在行为任务中。这项工作将建立一种方法来收集大类的时间动态， 神经元在背角的反应时间锁定，空间精确，和调幅输入在 导致对急性疼痛状况的理解得到改善。

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.