Wenqin Luo - US grants

Touch sensation, including the perception of form and texture, is essential for our daily lives. Subpopulations of trigeminal and dorsal root ganglion (DRG) neurons are primary sensory neurons mediating this process, and are classified as either rapidly (RA) or slowly adapting (SA) mechanoreceptors. Their molecular identities, unique functions in the context of form and texture perception, and mechanisms of development are largely unknown. Recently, I discovered that a small population of DRG neurons that express the receptor tyrosine kinase Ret are the elusive RA mechanoreceptors and that Ret signaling is essential for their development. In this application, I propose experiments to elucidate the mechanisms by which Ret and TrkB signaling control survival, peripheral end organ formation, central projections, and physiological properties of RA mechanoreceptors. In addition, I propose to develop new tactile discrimination behavior assays and a somatosensory neuron specific diphtheria toxin (DTA) mouse line to test the in vivo functions of RA mechaoreceptors during tactile descrimination. Specific Aim 1: To test the hypothesis that Ret and TrkB are redundant for survival of RA mechanoreceptors and that TrkB functions in RA mechanosensory DRG neurons autonomously for Meissner corpuscle formation. In this aim, Ret and TrkB double knock out mice and TrkB conditional knock out mice will be generated to test my hypothesis. Specific Aim 2: To address the mechanism by which Ret signaling directs central projections of RA mechanoreceptors. For this purpose, I will visualize the central axonal projections of individual Ret null RA mechanoreceptors during early development to establish the nature of the primary axonal projection deficit. In addition, I will identify the endogenous ligand and co-receptor of Ret, and their sources, to establish the mechanism of action. Specific Aim 3: To determine whether Ret signaling modulates the physiological properties of RA mechanoreceptors. In this aim, physiological properties of RA mechanoreceptors in which Ret is either ablated early during development or acutely activated or inhibited in adults will be examined. Aim 4: To begin to establish the functional roles of RA mechanoreceptors in vivo. To study the unique functions of various mechanoreceptors, I will develop new behavioral assays to test tactile discrimination in mice. In addition, I will generate a somatosensory neuron specific conditional DTA mouse line. Together, these new assays and reagent will enable me to address the role of RA mechanoreceptors and, in the future additional populations of DRG neurons during tactile discrimination.

Nociception, or the sense of noxious stimuli, is essential for our daily lives. Normal acute nociception prevents us from potential damage or repetitive injuries, while distorted neural circuits in pathological conditions gener- ate chronic pain, which is a huge human health problem. At present, our understanding of neural circuits in mediating and modulating pain sensation under normal and pathological conditions is surprisingly incomplete. We proposed to study a population of inhibitory dorsal spinal cord interneurons, which express the receptor tyrosine kinase RET neonatally and makes up about one third of inhibitory interneurons in laminae III to V (deep layer). Our preliminary study showed that these deep layer early RET+ inhibitory interneurons are unique and their circuits and functions in nociception have not been defined before. Aim 1. Define molecular, physiological, and anatomical properties of deep layer early RET+ inhibitory interneurons. In this aim, we will genetically label deep layer early RET+ inhibitory interneurons to study their gross anatomy, identities of inhibitory neural transmitter, physiological properties, and single neuron morpholo- gy. Our anticipated results will reveal unique features of deep layer early RET+ inhibitory interneurons and pro- vide an insight into their potential connections and functions. Aim 2. Dissect neural circuits associated with deep layer early RET+ inhibitory interneurons. In this aim, we will use both light/electronic microscopy imaging and spinal cord slice recording coupled with electric and optical stimuli to determine input and output of deep layer early RET+ inhibitory interneurons. Together, our work will reveal functional connections associated with this new population of DH inhibitory interneurons. Aim 3. Determine functions of deep layer early RET+ inhibitory interneurons in acute pain and chronic pain. In this aim, we will either ablate deep layer early RET+ inhibitory interneurons using toxin or acutely acti- vate them using optogenetic and pharmacological approach and test mouse nociceptive behavioral responses under acute and chronic pain conditions. With these experiments, we anticipate revealing important functions of deep layer early RET+ inhibitory interneurons in modulating acute and chronic pain. In short, our proposed study will elucidate circuits and function of a new population of DH inhibitory interneu- rons in governing the transmission and modulation of nociceptive information. Our work would lead to a better understanding about DH circuits and provide potential new thoughts for chronic pain treatment.

PROJECT SUMMARY Rodent models are highly valuable for elucidating the molecular and cellular mechanisms of chronic pain. Because rodents cannot articulate their sensation, ?pain-like? behaviors have been used as the proxy. However, sensitivity and specificity of many existing methods for measuring rodent ?pain? sensation, especially ?chronic pain?, are uncertain. Here we propose to explore the feasibility of a largely automated and data-driven behavioral assay for identifying spontaneous pain in freely behaving mice. Specifically, we will take advantage of recent advances in 3D motion analysis, which enable precise and robust measurements of movements without human intervention, to extract movement features from freely moving mice in various pain states (baseline, induced acute pain, chronic pain, and with painkiller treatment). We will generate a database of movement features of control mice and mice with induced acute cheek/leg pain or chronic neuropathic cheek/leg pain, using both sexes of two mouse strains. We will then use machine-learning algorithms to identify the best combination of movement features for predicting the pain state (a ?mouse chronic pain scale?). These efforts are expected to produce a novel and objective method to assess spontaneous pain, a characteristic feature of chronic pain, in mice. This method can supplement our recent method in measurements of evoked responses (a ?mouse acute pain scale?) to provide efficient, robust, and comprehensive assessments of pain-related rodent behaviors and facilitate mechanistic investigations of brain circuits in mediating and modulating pain. Our interdisciplinary team is well suited to complete these Aims, utilizing combined expertise in mouse somatosensory/pain system (PI Luo), behavioral, systems and computational neuroscience (PI Ding), and 3D imaging and computer vision (PI Park).