Transcript (A) dTRPA1
Re-evaluation of the functional roles of painless and dTRPA1 in chemical nociception in Drosophila Pamela A. Fazio, Samantha J. Mandel, Madison L. Shoaf, Jason T. Braco, Wayne L. Silver, and Erik C. Johnson Department of Biology, Wake Forest University, Winston-Salem NC USA 1. Background 2. Behavioral Assays A Difference in Amount Consumed (uL) The detection of harmful chemical irritants is important for the avoidance of potential life threatening compounds. In vertebrates, the trigeminal nerve is an important anatomical site of chemical nociception, and the nerve directly responds to a variety of chemical compounds. A molecular target for many of these trigeminal stimulants is the TRPA1 channel. Drosophila possess multiple mammalian TRPA1 homologs, two of which are encoded by the painless and dTRPA1 genes. Both of these channels have been reported to be required for the detection and subsequent behavioral avoidance of noxious chemicals, such as allyl isothiocyanate (AITC); however, data have been conflicting. What’s in the Literature: B Painless dtrpA1 Figure 1. A. Painless is required for the aversive behaviors to AITC (Al-Anzi et al., 2006). B. In contrast, it was later reported that dTRPA1, not painless, is required for the behavioral avoidance to AITC (Kang et al., 2010). A painless HEK 293 Cell No Response 3a. A 2+ Ca influx B dTRPA1 Figure 4. A. Heterologous expression of painless did not result in AITC-evoked responses (Sokabe et al., 2008). B. However, heterologous expression of dTRPA1 did confer AITC-sensitivity (Kang et al., 2010). Therefore, the role of painless in the behavioral aversion to AITC is not clear. Xenopus oocyte Figure 2. PER data of varying sucrose concentrations with 2mM AITC. Results demonstrate a significant difference in aversion for W1118 but not for dTrpA1 or painless. 2+ Ca influx 3b. A Figure 3. CAFE results of 2 mM AITC in varying sucrose concentrations demonstrating that painless and dtrpA1 are not sufficient on its own and are affected by sucrose. measurements continued 5. Conclusion pain-GAL4/UAS-GCaMP; dTRPA1 Response Figure 6. Painless-expressing neurons exhibited activation in a dtrpA1 null background seen through the increase in fluorescence using the GCaMP. measurements from ventral nerve cord neurons trpA-GAL4; UAS-GCaMP B 4. 2+ Ca influx A C measurements from neuroendocrine AKH cells • Behavioral assays demonstrate that aversion to AITC is a complex behavior requiring both functional dTRPA1 and painless channels • The CAFE assay is a more quantitative measure of aversive behavior than PER • Calcium influx measurements show that both dTRPA1 and painless channels respond independently to AITC • Our current model for AITC aversion is that painless and dTRPA1 are additive; on their own neither are sufficient • Future directions include analyzing the circuitry and anatomical dissections. B pain-GAL4; UAS-GCaMP painless dTRPA1 Behavioral Aversion Figure 5. Cells in the adult ventral nerve cord (A) containing the GCaMP transgene were used to observe changes in calcium levels in painless- and dTRPA1-expressing neurons in response to AITC. Both (B) dTRPA1 and (C) painless-expressing neurons in the ventral nerve cord exhibited significant increases in fluorescence in response to AITC as compared to the control. Figure 6. Ectopically expressed (A) dTRPA1 and (B) painless in neuroendocrine AKH cells displayed activation in response to AITC, indicating each channel’s ability to act as a direct receptor. Acknowledgements: We would like to thank Wake Forest University for funding, Dan Tracey, Paul Garrity, and The Bloomington Stock Center for fly lines.