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.