Project Summary/Abstract Nociception is the mechanism by which animals mediate protective behavioral responses to noxious stimuli, including dangerous high and low temperatures, harmful chemicals, and physically damaging mechanical insults. Noxious stimuli are typically transduced by high-threshold sensory neurons (“nociceptors”), and ultimately elicit protective behaviors. Nociceptors are often multimodal responding to more than one sensory stimulus type; for example, vertebrate C fibers (a class of unmyelinated nociceptive neuron) can transduce innocuous mechanical and thermal stimuli, among others. Elucidating how nervous systems integrate complex information in order to produce relevant behaviors is a fundamental question in neuroscience, and understanding multimodality can have benefits for human health, as the inability to discriminate between noxious and innocuous stimuli can underlie chronic neuropathic pain. Many instances of neuropathic pain present as altered thermosensation (often cold sensing), which is present across organisms, and makes use of highly conserved mechanisms. We have previously demonstrated that Drosophila melanogaster Class III (CIII) sensory neurons are multimodal, and drive distinct, stereotyped behaviors in response to innocuous touch and noxious cold. Further, we have shown that these neurons make use of Transient Receptor Potential (TRP) channels, much like vertebrate nociceptors. However, it is presently unknown how CIII multimodal sensory neurons discriminately detect noxious cold stimuli to elicit nociceptive behavior. Preliminary discoveries have led us to hypothesize that TRP-mediated Ca2+ signaling contributes to CIII multimodality, and more specifically, that Anoctamin/TMEM16 family channels (a family of Ca2+-activated Cl- channel), in concert with chloride ion homeostasis mechanisms, function in a similar capacity in both Drosophila CIII neurons and vertebrate sensory neurons. The project aims and outcomes of this research will significantly advance our knowledge of cold nociception and molecular mechanisms by which multimodal sensory neurons discriminately encode neural activity to elicit stimulus-relevant behaviors. Capitalizing on this system and the genetic tractability of Drosophila, herein we combine neurogenetics, neurogenomics, molecular biology, cellular/functional imaging, optogenetics, electrophysiology, and behavioral analyses to significantly enhance our understanding of mechanisms important to behavior selection, multimodality, and thermosensory nociception.