PROJECT SUMMARY The thirteen-lined ground squirrel is an obligate hibernator that seasonally drops its body temperature from 37° in the active state to 2-4°C during torpor. While such temperatures typically inhibit peripheral signaling in mammals, torpid squirrels remain sensitive to tactile cues and can be awakened by the sense of touch (mechanosensation). However, the neurobiological mechanisms involved remain unknown. The long-term goal of this study is to identify the cellular and molecular signatures that afford cold-resistant mechanosensation to support extreme thermal tolerance in mammals. Pressure stimuli are sensed by a specialized subset of neurons in the dorsal root ganglion (DRG) known as mechanoreceptors. Mechanically gated ion channels such as Piezo2 convert pressure into ionic current that activates voltage-gated ion channels, which relay the signal downstream through action potentials (APs). Piezo2 and voltage-gated ion channels have temperature-dependent dynamics ripe for evolutionary manipulations permitting thermal resistance. This study, which will be conducted at the laboratories of Drs. Elena Gracheva and Slav Bagriantsev at Yale University, assesses the central hypothesis that ion channel modifications in mechanoreceptors underlie cold-adapted mechanosensation in the thirteen-lined ground squirrel. Past work from the Gracheva and Bagriantsev labs has shown that this species shows constitutively low thermosensitivity and decreased function of nociceptive AP machinery during torpor. Our preliminary work also shows that mechanosensitive currents are preserved during torpor. Accordingly, we use a combination of in situ hybridization, RNA sequencing and electrophysiology to assess whether squirrel DRG neurons have selectively potentiated mechanosensitivity at the cost of other sensory modalities, relative to mouse DRG neurons (Aim 1). Moreover, cold exposure widens APs and disrupts voltage gradients in typical mammalian neurons due to slowed kinetics of the Na+/K+ ATPase pump. In contrast, our preliminary work shows that cold-exposed torpid neurons show decreased AP widening and intact resting membrane potential. In light of these data, we use whole-cell and ex vivo electrophysiology to assess whether squirrel mechanoreceptors have cold-resistant electrogenic machinery (Aim 2). To the best of our knowledge, this study represents the first attempt to dissect cellular mechanisms of cold- adapted mechanosensation in mammals. Elucidating neural function in extreme cold has the potential to provide novel targets for combatting neuronal damage following clinical hypothermic procedures.