Temperature integration at the AC thermosensory neurons in Drosophila

Abstract

Temperature sensation has a strong impact on animal behavior and is necessary for animals to avoid exposure to harmful temperatures. It is now well known that thermoTRP (transient receptor potential) channels in thermosensory neurons detect a variable range of temperature stimuli. However, little is known about how a range of temperature information is relayed and integrated in the neural circuits. Here, we show novel temperature integration between two warm inputs via Drosophila TRPA channels, TRPA1 and Pyrexia (Pyx). The internal AC (anterior cell) thermosensory neurons, which express TRPA1, detect warm temperatures and mediate temperature preference behavior. We found that the AC neurons were activated twice when subjected to increasing temperatures. The first response was at ∼25°C via TRPA1 channel, which is expressed in the AC neurons. The second response was at ∼27°C via the second antennal segments, indicating that the second antennal segments are involved in the detection of warm temperatures. Further analysis reveals that pyx-Gal4-expressing neurons have synapses on the AC neurons and that mutation of pyx eliminates the second response of the AC neurons. These data suggest that AC neurons integrate both their own TRPA1-dependent temperature responses and a Pyx-dependent temperature response from the second antennal segments. Our data reveal the first identification of temperature integration, which combines warm temperature information from peripheral to central neurons and provides the possibility that temperature integration is involved in the plasticity of behavioral outputs.

Figure 1.

AC neurons receive temperature inputs from the antennae. A, B, The warmth responses of AC neurons were monitored with antenna (n = 7 neurons) (A) and without antenna (n = 11 neurons) (B) in w¹¹¹⁸ control flies. G-CaMP3.0 was expressed in the AC neurons using TrpA1 ^SH-Gal4. The AC neurons of the control flies were activated twice as the temperature increased (A). The bar graph shows the mean percentage of fluorescence increase (ΔF/F) of the AC neurons during the first response at ∼25°C (white bar) and during the second response at ∼27°C (black bar). The circles show the peak temperatures of the experiments in each category. The number of circles represents the number of assays. Error bars are the SEM. The line graph shows a representative experiment, comparing the fluorescence changes (ΔF/F) (blue line) and the temperature change (gray line). The background fluorescence was subtracted from the mean fluorescent intensity of the AC neurons. The diagrams represent the fly brains. AL, Antennal lobe. C, D, The comparison between the first (C) and second (D) responses of the AC neurons in the wild-type with (n = 7 neurons) and without antenna (n = 11 neurons). The same bar graph data from A and B were used in C and D. The bar graph shows the mean percentage of fluorescence increase (ΔF/F) of the AC neurons during the first response at ∼25°C (white bar, C) and during the second response at ∼27°C (black bar, D). Significance was determined using unpaired t test compared with and without antenna; ***p < 0.001. Error bars are the SEM.

Figure 2.

TRPA1 is necessary for the first response but not the second response of the AC neurons. A, B, G-CaMP3.0 was expressed in the AC neurons using TrpA1 ^SH-Gal4 in the WT and TrpA1^ins background. Warmth responses of AC neurons were monitored with antenna in WT (n = 7 neurons), without antennae in WT (n = 11 neurons), with antenna in TrpA1^ins (n = 10 neurons), and without antennae in TrpA1^ins (n = 14 neurons). The same WT data from Figure 1 were used in Figure 2. The bar graph shows the mean percentage of fluorescence increase (ΔF/F) of the AC neurons during the first response at ∼25°C (white bar, A) and during the second response at ∼27°C (black bar, B). One-way ANOVA with the Tukey's post hoc test was used to determine pairwise differences between groups; *p < 0.05, **p < 0.01, and ***p < 0.001. Error bars are the SEM.

Figure 3.

The second response of the AC neurons arises from the second antennal segments. A, B, G-CaMP3.0 was expressed in the AC neurons using TrpA1 ^SH-Gal4 in the WT background. The fly antenna is composed of three segments, as depicted in the diagrams (a, intact antenna, n = 7 neurons; b, third antenna removal, n = 7 neurons; c, second and third antennae removal, n = 7 neurons; d, no antenna, n = 11 neurons). The bar graph shows the mean percentage of fluorescence increase (ΔF/F) of the AC neurons during the first response at ∼25°C (white bar, A) and during the second response at ∼27°C (black bar, B). Each response was compared with the other responses of flies with various antenna ablations. One-way ANOVA with the Tukey's post hoc test was used to determine pairwise differences between groups; *p < 0.05 and **p < 0.01. Error bars are the SEM.

Figure 4.

AC neurons receive projections from pyx-Gal4-expressing neurons. A, B, The flies were stained with anti-GFP (green) and anti-TRPA1 (red). AC neurons (arrowhead) were specifically labeled using anti-TRPA1 antisera. Orco-Gal4::UAS-mCD:GFP labels olfactory neurons (A) and F-Gal4::UAS-mCD:GFP labels mechanosensory neurons (B), but neither of these overlapped with the somas of the AC neurons. C, D, The flies showed overlapping of membrane RFP (green) or synaptic GFP (green) with AC neurons (red). C, c155-Gal4::UAS-SyteGFP (synaptic GFP) with anti-GFP (green) and anti-TRPA1 (red) (D1) pyx-Gal4:: UAS-mCD:RFP with anti-RFP (green) and anti-TRPA1 (red), (D2) pyx-Gal4:: UAS-syteGFP with anti-GFP (green) and anti-TRPA1 (red). E, pain-Gal4:: UAS-mCD:GFP with anti-GFP (green) did not overlap with AC neurons (red), but overlapped with the cells right next to the AC neurons. F, A schematic of the fly brain and antennae. The AC neurons are shown in blue. The soma belongs to neither mechanosensory neurons (F-Gal4) nor olfactory neurons (Orco-Gal4). AL, Antennal lobe; AN, antennal nerve; JO, Johnston's organ; AMMC, antennal mechanosensory and motor center.

Figure 5.

The second warmth response in AC neuron is Pyrexia dependent. A, B, Warmth responses of AC neurons were monitored in pyx³ mutants (n = 8 neurons) (A) and pain¹ mutants (n = 8 neurons) (B). G-CaMP3.0 was expressed in the AC neurons using TrpA1-Gal4. The bar graph shows the mean percentage of fluorescence increase (ΔF/F) of the AC neurons during the first response at ∼25°C (white bar) and during the second response at ∼27°C (black bar). The circles show the peak temperatures of the experiments in each category and the number of circles represents the number of assays. The line graph shows a representative experiment, comparing the fluorescence changes (ΔF/F) (black line) and the temperature change (gray line). C, D, The comparison of the first (C) and second (D) responses of the AC neurons in wild-type (n = 7 neurons), pyx³ (n = 8 neurons), pyx³/Df(3L)ED201 (n = 9 neurons), pyx³;pyxGe (pyx minigene with pyx³, n = 8 neurons), and pain¹ (n = 8 neurons) flies. One-way ANOVA with the Tukey's post hoc test was used to determine pairwise differences between groups; *p < 0.05 and ***p < 0.001. Error bars are the SEM.

Figure 6.

Disruption of pyrexia did not change temperature preference behavior. A, Mean preferred temperatures of wild-type (cs) (n = 11 independent assays), pyx³/+ (n = 5 independent assays), pyx³ (n = 6 independent assays), pyx³/Df(3L)ED201 (n = 7 independent assays), pyx³/Df(3L)Exel6084 (n = 6 independent assays), and TrpA1^ins mutant flies (n = 11 independent assays). pyx³ flies were backcrossed to Canton-S (cs). TrpA1^ins mutant flies preferred a significantly higher temperature compared with wild type. Significance was determined using unpaired t test compared WT and TrpA1^ins mutants, ***p < 0.001. Error bars are the SEM. The mean preferred temperatures of pyx³ mutant flies and two pyx³/Df flies were not significantly different compared with wild-type and pyx³/+ flies. One-way ANOVA with the Tukey's post hoc test was used to determine pairwise differences between groups. B, TNT expression driven by pyx-Gal4 (n = 6 independent assays) did not cause a significant change of the preferred temperature compared with two control flies pyx-Gal4/+ (n = 14 independent assays) and UAS-TNT/+ (n = 8 independent assays). One-way ANOVA was used, p = 0.1259.

Figure 7.

Temperature neural circuits from antennae to brain. Schematic of the temperature neural circuits based on this manuscript and on other studies (Hamada et al., 2008; Sun et al., 2009; Gallio et al., 2011). Based on the Gal4 expression data, Pyx is expressed in the second antennal segments, and Brv is expressed in the third antennal segments.