Bo Duan, Ph.D. - US grants

Chronic itch is a severe clinical problem that afflicts a large number of humans and it is very difficult to treat. Understanding the chronic itch circuitry and molecular mechanisms is critical to developing new therapies for this intractable disease. Mechanical itch sensitization (alloknesis) is one common symptom in many of chronic itch patients. Our previous work has identified neuropeptide Y-positive (NPY+) spinal inhibitory interneurons that gates mechanical itch. Our findings raise two fundamental questions: 1) What are the specific excitatory neurons in the dorsal spinal cord that transmit mechanical itch? 2) Does dysregulation of this pathway lead to chronic itch? Recently we have identified that spinal excitatory interneurons expressing Urocortin 3::Cre (Ucn3+) are mechanical itch-transmission neurons, which do not transmit touch, pain and chemical itch. Retrograde rabies virus tracing showed that NPY+ neurons monosynaptically connect to Ucn3+ neurons in the dorsal spinal cord. The goal of this project is to elucidate the spinal circuits that transmit and gate mechanical itch, and to study how the circuits are altered in chronic itch conditions. Aim 1: Delineate the functional organization of the spinal microcircuit that processes mechanical itch. We will examine the functional connections from NPY+ neurons onto Ucn3+ neurons. We will map the sensory inputs from dorsal root ganglion (DRG) neurons onto Ucn3+ and NPY+ neurons. Aim 2: Determine the mechanisms of mechanical itch sensitization in chronic itch conditions. We will test the mechanical itch sensitization and spontaneous itch behaviors in various chronic itch conditions after ablating spinal Ucn3+ interneurons. We will characterize the electrical properties of Ucn3+ and NPY+ neurons and synaptic transmission in the spinal mechanical itch circuits in chronic itch conditions. We will investigate whether disinhibition of spinal mechanical itch circuits is a common mechanism of mechanical itch sensitization in various chronic itch conditions. Finally, we will determine the disinhibition mechanisms in chronic itch.

The ability to sense environmental temperature is essential for life. Molecular thermal sensors are a central player in thermosensation. These thermal receptors are expressed in cold-sensitive neurons/cells in the periphery. Work in the past two decades has identified a large number of TRP family channels as heat receptors that sense a full range of warm and hot temperatures, spanning from 33°C to over 53°C. This has led to a fairly clear understanding of how animals sense heat. By contrast, little is known about cold sensation. Thus far, only one cold receptor (TRPM8) has been identified. TRPM8 senses cool temperatures with an activation threshold at ~26°C and mediates cool sensation. As animals and humans are clearly capable of sensing temperatures below 26°C, and TRPM8 knockout mice show robust responses to noxious cold, unknown cold receptors, particularly those sensing noxious cold, must exist but remain to be identified. The nematode C. elegans is a popular genetic model organism for sensory biology research. Like mammals, C. elegans senses a full range of temperature cues. Importantly, sensory receptors and channels tend to be evolutionarily conserved in C. elegans. This, together with its short generation time (~3 days) and facile and rich genetic tools, makes C. elegans an ideal system for identifying novel cold receptors. We therefore designed and conducted an unbiased, activity-based genetic screen for cold-sensing mutants in C. elegans, using a real-time PCR thermocycler. We identified GLR-3, a kainate-type glutamate receptor homolog, as a novel type of cold receptor that mediates cold sensation in C. elegans. Strikingly, the GLR-3 homolog GluK2 from fish, mouse and human can all function as a cold receptor in heterologous systems. We also found that mouse GluK2 is expressed in the peripheral DRG sensory neurons. The activation threshold of GluK2 is below 20°C, suggesting that it mainly senses noxious cold rather than cool temperatures. As glutamate receptors are best known to transmit chemical signals across synapses in the central nervous system, these results present a striking case where a central chemical receptor, surprisingly, functions as a thermal receptor in the periphery. Despite these exciting observations, many unanswered questions remain, particularly regarding the role of mammalian GluK2 in cold sensation. For example, does GluK2 mediate cold sensation in mice? If so, how? Here, we propose to address these questions by testing several hypotheses. To do so, we will leverage the expertise from two research groups using a multidisciplinary approach combining molecular genetics, behavioral analysis, calcium imaging, and electrophysiology. The proposed research will not only provide novel insights into the mechanisms of cold sensation, but also unveil an unexpected role of glutamate receptors in the periphery. .