Evaluation of the functional roles of painless and dTRPA1 in chemical nociception in Drosophila. Samantha J Mandel, Madison L Shoaf, Pam A Fazio, Jason T Braco, Wayne L Silver, Erik C Johnson. Wake Forest University, Winston-Salem, NC.

   The detection of harmful chemical irritants is vital for the evasion of potential life threatening compounds. Among vertebrates, the trigeminal nerve is an important site of chemical nociception in its direct response to an array of chemical compounds. One of the many targets for multiple trigeminal stimulates is the TRPA1 channel. Drosophila possess multiple homologs of mammalian TRPA1 channels, two of which are painless and dTRPA1. However, there is disagreement regarding the role of painless in the behavioral aversion to allyl isothiocyanate (AITC). Previous studies showed that heterologous expression of painless failed to confer AITC-elicited changes, supporting the hypothesis that painless may not have a role in mediating aversion to chemical irritants. We have analyzed the behavioral phenotypes of painless and dTRPA1 mutants using a two-choice feeding assay and proboscis extension reflex (PER) assay, both of which pointed to the requirement for each channel in the aversion to AITC. Concerning the PER assay, we are standardizing the assay in order to determine whether responses are due to averse behaviors, starvation sensitivity, or sugar preference. Preliminary results support the conclusion that both pain and dTRPA1 are critical for averse behaviors. We have evaluated the expression patterns of painless and dTRPA1 to determine if there is any colocalization, and so far, there has been none observed in central or peripheral populations. To evaluate painless and dTRPA1 cell excitability we employed the GCaMP transgene to observe fluctuations of calcium levels. Both painless and dTRPA1- expressing cells showed significant changes in fluorescence following application of AITC. Collectively, our results provide further insight into the contribution of each of these channels in chemical nociception. Application of future directions includes investigation into pyrexia, another homologue to the mammalian TRPA1 channel, and whether it has a role in chemical nociception as well as examination of the neural circuits and how these channels interconnect with one another.