Drosophila TRPA1 channel mediates chemical avoidance in gustatory receptor neurons

Abstract

Mammalian sweet, bitter, and umami taste is mediated by a single transduction pathway that includes a phospholipase C (PLC)beta and one cation channel, TRPM5. However, in insects such as the fruit fly, Drosophila melanogaster, it is unclear whether different tastants, such as bitter compounds, are sensed in gustatory receptor neurons (GRNs) through one or multiple ion channels, as the cation channels required in insect GRNs are unknown. Here, we set out to explore additional sensory roles for the Drosophila TRPA1 channel, which was known to function in thermosensation. We found that TRPA1 was expressed in GRNs that respond to aversive compounds. Elimination of TRPA1 had no impact on the responses to nearly all bitter compounds tested, including caffeine, quinine, and strychnine. Rather, we found that TRPA1 was required in a subset of avoidance GRNs for the behavioral and electrophysiological responses to aristolochic acid. TRPA1 did not appear to be activated or inhibited directly by aristolochic acid. We found that elimination of the same PLC that leads to activation of TRPA1 in thermosensory neurons was also required in the TRPA1-expressing GRNs for avoiding aristolochic acid. Given that mammalian TRPA1 is required for responding to noxious chemicals, many of which cause pain and injury, our analysis underscores the evolutionarily conserved role for TRPA1 channels in chemical avoidance.

Fig. 1.

Generation and expression of a trpA1^GAL4 reporter and mutant. (A) Schematic of the trpA1 genomic locus. The trpA1 exons are indicated by blue rectangles. The targeted trpA1^GAL4 was generated by ends-out homologous recombination (49), resulting in deletion of 185 nucleotides spanning the trpA1 translation initiation codon (ATG). The green and red rectangles indicate the GAL4 and miniwhite genes, respectively. The arrows indicate the primers used for PCR confirmation of the targeting and deletion in trpA1. (B) PCR analyses of genomic DNA to verify the targeting of the left and right arms and the deletion in trpA1. The size markers indicate kilobases. (C) Spatial distribution of the trpA1 reporter in a dissected labellum. A UAS-mCD8::GFP transgene was crossed into a trpA1^GAL4/+ background and the tissue was stained with anti-GFP. The Inset (lower right) shows a magnified view of a GRN from an s6 sensillum. (D) RT-PCR analyses performed using RNA prepared from the indicated flies. (E) Coexpression of the trpA1 reporter with a subset of GR93a-expressing GRNs. The labellum was dissected from UAS-GFP;trpA1/+ flies and stained with anti-GFP and anti-GR93a.

Fig. 2.

trpA1 mutation caused impaired behavioral avoidance of aristolochic acid. (A) Survey of behavioral response to bitter compounds in trpA1¹ flies. Binary food-choice assays were performed using 1 mM sucrose alone versus 5 mM sucrose alone or 5 mM sucrose with 10 mM caffeine, 1 mM quinine, 0.3 mM denatonium, 0.1 mM berberine, 0.3 mM lobeline, 2 mM papaverine, 0.5 mM strychnine, or 10 mM aristolochic acid (n ≥ 4). (B–D) Flies were allowed to select 1 mM sucrose alone or 5 mM sucrose plus 10 mM aristolochic acid. (B) Survey of trp mutants for avoidance to aristolochic acid. (C) Verification of aristolochic acid behavioral avoidance in three trpA1 mutant alleles: trpA1¹, trpA1^ins, and trpA1^GAL4. (D) Rescue of trpA1 impairment by expressing UAS-trpA1 under control of the trpA-GAL4 (trpA1^GAL4) or the Gr66a-GAL4. Asterisks denote statistical significance (P < 0.05) using unpaired Student's t test. The error bars represent SEMs. See Tables S1–S4 for detailed statistics.

Fig. 3.

trpA1 is required for aristolochic acid-induced action potentials. (A) Tip recordings were performed on s6 bristles except for the sucrose recordings, which were performed on l4 and l6. Shown are the average frequencies of action potentials after application of 50 mM sucrose, 10 mM caffeine, 1 mM quinine, 1 mM denatonium, 0.1 mM berberine, 1 mM lobeline, 1 mM papaverine, 1 mM strychnine, or 1 mM aristolochic acid (n ≥ 7). (B) Sample tip recordings after application of recording pipettes with buffer alone, caffeine, or aristolochic acid. (C) Impairment of aristolochic acid-induced action potentials in multiple trpA1 alleles and rescue of the defect by expressing UAS-trpA1 under control of either trpA1-GAL4 (trpA1^GAL4) or Gr66a-GAL4 (n ≥ 7). The error bars represent SEMs. The asterisks indicate significant differences from wild type (P < 0.05). Detailed statistics are presented in Tables S5 and S6.

Fig. 4.

Aristolochic acid did not activate or inhibit Drosophila TRPA1 in vitro. (A) Two-electrode voltage clamp recordings from Xenopus oocytes expressing TRPA1. Aristolochic acid (0.1 mM) (23 °C) did not activate TRPA1 (ΔI ± SEM = −0.0004 ± 0.0002 μA, n = 3). Lower concentrations of aristolochic acid (dropwise addition of 0.1 mM solution) to the bath did not induce channel activation (ΔI = −0.0002 ± 0.0002 μA, n = 4). Heated (37 °C) ND96 buffer resulted in the strong activation of TRPA1 (ΔI = −2.662 ± 0.418 μA, n = 6). (B) Aristolochic acid did not inhibit TRPA1. Voltage-clamped oocytes were preincubated in 0.25 mM aristolochic acid for 5 min and then warmed in the presence of aristolochic acid (ΔI = −2.190 ± 0.591 μA, n = 4).

Fig. 5.

Requirement for PLC for sensing aristolochic acid. (A) Binary food-choice assays were performed using 1 mM sucrose alone vs. 5 mM sucrose plus 10 mM aristolochic acid. Two independent norpA alleles, norpA^P24 and norpA^P54 showed reduced avoidance to aristolochic acid. Expression of UAS-norpA using the trpA1-GAL4 (trpA1^GAL4/+) rescued the phenotype. (B) Tip recording analyses showed that both norpA allele mutants had reduced frequencies of aristolochic acid-induced action potentials. This deficit was fully restored by the introduction of UAS-norpA and trpA1^GAL4. The error bars represent SEMs. The asterisks indicate significant differences from wild type (P < 0.05). Detailed statistics are presented in Tables S7 and S8.