The amnesiac gene is involved in the regulation of thermal nociception in Drosophila melanogaster

Abstract

Nociception is a mechanism fundamental to the ability of animals to avoid noxious stimuli capable of causing serious tissue damage. It has been established that in the fruit fly Drosophila melanogaster, the transient receptor potential (TRP) channel encoded by the painless gene (pain) is required for detecting thermal and mechanical noxious stimuli. Little is known, however, about other genetic components that control nociceptive behaviors in Drosophila. The amnesiac gene (amn), which encodes a putative neuropeptide precursor, is important for stabilizing olfactory memory, and is involved in various aspects of other associative and nonassociative learning. Previous studies have indicated that amn also regulates ethanol sensitivity and sleep. Here the authors show that amn plays an additional critical role in nociception. Their data show that amn mutant larvae and adults are significantly less responsive to noxious heat stimuli (greater than approximately 40 degrees C) than their wild-type counterparts. The phenotype of amn mutants in thermal nociception, which closely resembles that of pain mutants, was phenocopied in flies expressing amn RNAi, and this phenotype was rescued by the expression of a wild-type amn transgene. These results provide compelling evidence that amn is a novel genetic component of the mechanism that regulates thermal nociception in Drosophila.

Figure 1

amn^28A mutant flies exhibit reduced responsiveness to high temperatures in the light-driven heat avoidance test. (A) Schematic of the light-driven heat avoidance test, which is carried out using a modified counter-current apparatus equipped with heat bands. In response to a light stimulus, a group of flies (presented as asterisks) are challenged to pass a heat band of the designated temperature. Shown are the steps involved in a single trial. After two consecutive trials, flies were collected into three proximal tubes and the Performance Indices (PIs) were calculated using the indicated formula, where N_i is the number of flies collected in the i^th tube. (B) The mean PIs (± SEM) at the designated temperature (25–50 °C) are shown for wild-type flies (CS) as well as for pain¹, pain³ and amn^28A mutant flies. N=3–5. *P < 0.05, **P < 0.01, ***P < 0.001, for comparison to the value for wild-type flies (one-way ANOVA followed by pairwise multiple comparison with the Holm-Sidak method).

Figure 2

amn mutants display a longer latency for behavioral responses to directly applied noxious heat. Distributions of response latency in larvae (A) and adult flies (C) are presented as percentage of total number of flies (N). The same data sets are presented as box plots, for larvae (B) and adult flies (D), with the median values indicated by solid horizontal bars and the ranges between the lower and upper quartiles are shown as gray boxes. *P < 0.05, for comparison to values for the wild-type (CS) flies (Krustal-Wallis ANOVA, followed by Dunn’s pairwise test for multiple comparisons).

Figure 3

RNAi-mediated down-regulation of amn causes defects in thermal nociception. (A) RT-PCR analyses revealing the transcript levels of amn in flies carrying da-GAL4 and UAS-amn RNAi transgenes. A representative result of the RT-PCR analysis (top). Ratios of signal intensity of samples (bottom). Average ± standard deviation (STD), N=3. The presence or absence of a transgene is indicated by + or −. The amn transcript levels were decreased in flies carrying both da-GAL4 and UAS-amn RNAi. Latencies of the responses to the noxious heat stimulus are shown as box plots, for both larvae (B) and adult flies (C). The response latency in animals carrying both da-GAL4 and UAS-amn RNAi transgenes was significantly longer than for any other flies. *P < 0.05 (Krustal-Wallis ANOVA, followed by Dunn’s pairwise test for multiple comparisons).

Figure 4

A wild-type amn⁺ gene whose expression is driven by amn^28A and amn^X8 GAL4 activities rescues the thermal nociception defect in amn mutants. Nociception assays were performed for animals trans-heterozygous for amn^28A and amn^X8, with or without the UAS-amn⁺ transgene. Latencies of the responses to a noxious heat stimulus are shown as box plots, for larvae (A) and adult flies (B). The amn^28A/amn^X8; UAS-amn⁺/+ flies displayed response latencies that were reduced in comparison to those of amn^28A/amn^X8 flies. ***P < 0.001 (Mann-Whitney Rank Sum Test).

Figure 5