Characterization of the Decision Network for Wing Expansion in Drosophila Using Targeted Expression of the TRPM8 Channel

Environmental perturbation delays wing expansion

The behavioral pattern required for wing expansion in Drosophila has been described previously as consisting of two principal phases: perch selection, in which the fly walks to identify a site for wing expansion, and expansion itself, during which the stationary fly swallows air and simultaneously contracts its abdomen to drive hemolymph (i.e., blood) into the wings (Baker and Truman, 2002). These behavioral phases are executed immediately after emergence from the pupal case (a process called eclosion) and also have been extensively characterized in blowflies (Zdarek et al., 1984). Genetic data demonstrate that the expansion phase is absent in Drosophila bearing null mutations in the rickets gene, which encodes the receptor for the hormone bursicon (Baker and Truman, 2002; Luo et al., 2005). However, apart from the observation of Kimura and Truman (1990) that fruit flies will delay wing expansion if forced to eclose into an empty puparium from which they cannot escape, there has been little investigation of how the behavioral programs underlying perch selection and wing expansion respond to environmental stimuli.

To analyze the behavioral consequences of different environmental conditions, we therefore investigated perch selection and wing expansion in wild-type flies in three different paradigms. In the first, which involved minimal perturbation, the flies were kept in the culture vial in which they had pupated (volume, ∼30 ml) and were observed by eye after eclosion. This condition required intermittently approaching the vial with a magnifying lens or gently manipulating the vial to optimize viewing as a fly moved. In the second paradigm, flies were gently transferred into a thin cylindrical chamber (volume, 0.616 ml) immediately after eclosion. This configuration (supplemental Fig. S1, available at www.jneurosci.org as supplemental material) was optimized for videomicroscopy to obtain comprehensive behavioral records, as described in Materials and Methods. Flies in the videochamber were exposed to a constant visual environment. In the third paradigm, flies were tapped into an even smaller cylindrical minichamber with cotton endplugs (volume, 4.9 × 10⁻² ml) and their behavior was videorecorded. In this paradigm, multiple chambers were monitored simultaneously and flies in adjacent chambers were exposed to each others' movements. The three paradigms thus differed in the degree of handling experienced by the fly after eclosion, the size of—and type of environment within—the chamber, and the visual environment to which the fly was exposed. We did not examine the contributions of each of these variables individually to the behavior of the fly, but each is likely to exert some effect on posteclosion behavior (J. Pohl, N. Peabody, and B. White, unpublished observations). In the following, we refer to the different paradigms as the “low-perturbation,” “medium-perturbation,” and “high-perturbation” conditions to reflect the increasing degrees of stimulation and confinement experienced by the flies after emergence.

We found that the time required for flies to complete wing expansion varied considerably in the three different conditions, increasing progressively with the level of perturbation. In the low-perturbation condition, flies typically expanded their wings in approximately half an hour (32.8 ± 12.8 min, n = 20), whereas in the medium- and high-perturbation conditions they took nearly three and four times this long (83.8 ± 30.7 min, n = 40 and 119.3 ± 42.7 min, n = 44, respectively). Our preliminary analysis indicated that this difference was largely due to an extension of the perch selection phase, as flies in the smaller chambers persisted in walking for extended periods before finally engaging in wing expansion. This difference was most pronounced for flies in the high-perturbation condition, which sometimes walked for several hours. In addition, these flies frequently executed a distinct “digging” program (Fraenkel, 1935; Reid et al., 1987) when they encountered the cotton plugs at the ends of the chambers. They would attempt to push through the plug by expanding their ptilinum into the gap between the chamber wall and the plug while trying to propel themselves forward by abdominal, and sometimes leg, movements. Animals eclosing in the culture vials never displayed this behavior, and it was observed in the videochamber only on rare occasions when a small gap was accidentally left between the hard plastic ring and the glass plates that formed the chamber.

Definition of posteclosion behavioral phases

To quantify the differences between the behavioral patterns observed in the three paradigms, we required a baseline description of the post-emergence behavioral program and behavioral endpoints that could be used to define the phases. We therefore analyzed in detail the records of 10 flies observed in the videochamber where movements could be observed at the highest resolution. The pattern of behavior for all the flies was quite stereotyped, as previously observed by Baker and Truman (2002), and followed the progression shown in the ethogram of Figure 1 A. Emerging flies, which have compact, folded wings (Fig. 1 Ai) first walked extensively and then perched. Sustained walking was rarely resumed after perching, although flies occasionally pivoted or walked short distances. While perched (Fig. 1 Aii), the flies groomed intermittently and occasionally extended their abdomens or probosces. Eventually, the extension of both the proboscis and the abdomen became sustained, with the extended abdomen contracted radially and flexed downward (Fig. 1 Aiiia). During this interval, the wings expanded, initially adopting a downwardly cupped shape, but gradually flattening while angled slightly away from the body axis. As the flattened wings were brought together over the body, the abdomen ceased to be flexed, although the proboscis often remained extended for some time. Grooming continued throughout the period of wing expansion, and persisted for some time afterward.

The overall behavioral pattern divided naturally into three principal phases, which we defined with respect to easily observable behavioral endpoints. As shown in Figure 1 B, each phase is typically distinguished by a primary category of behavior. The perch selection phase, which we refer to as phase I, is dominated largely by walking and ends when the animal stops and then remains sedentary for at least 5 min. The expansion phase, which we refer to as phase III, is defined by the onset and offset of tonic abdominal contraction. The proboscis also is extended during this phase and in favorably oriented flies (n = 7/10), the cibarial pump, which drives air swallowing, was seen to be active (Fig. 1 B). In the low-perturbation condition, flies dissected immediately after this phase had large bubbles in the abdomen (Fig. 1 Aiiib; Table 1), confirming the ingestion of air. Although expansion of the wings typically occurs during phase III, this event is thought to be mediated solely by movement of the blood into the wings rather than by exertions of wing-attached muscles. As described below, wing expansion can under some circumstances fail even when phase III and its associated motor patterns (i.e., abdominal contraction and air swallowing) appear normal. Interposed between phases I and III is a period dominated by grooming (Fig. 1 A,B). We call this period, which also has been noted in blowflies (Cottrell, 1962), phase II. Twenty animals from the low-perturbation condition dissected during this behavioral phase were devoid of air in the gut, indicating that air ingestion is largely or completely restricted to phase III.

We also examined bursicon release as a function of behavioral phase for flies eclosing in their culture vials (i.e., the low-perturbation condition), where we could most readily collect sufficient numbers of similarly staged animals. Hemolymph samples collected from flies during phases I, II, and III, and ∼15 min after the end of phase III were analyzed by Western blot (Fig. 1 C). Bursicon was not detectable in the hemolymph of animals during phase I. It first appeared in phase II, and rose to its highest level during the expansion phase (phase III). Thus, in the low-perturbation condition, bursicon release appears to be initiated within 5 min of perch selection and to peak during wing expansion.

Environmental perturbation extends the perch selection phase

Having defined the behavioral phases, we compared their durations under the three environmental conditions (Fig. 2). This analysis confirmed that the differences in wing expansion time resulted primarily from a progressive increase in the duration of the perch selection phase. Mean phase I durations differed significantly between the three conditions (p < 0.001 by the Kruskal–Wallis test), rising 20-fold between the low- and high-perturbation conditions. In contrast, the mean duration of the expansion phase (III) was ∼15 min in all cases and did not differ significantly between perturbation conditions (p > 0.7 by Kruskal–Wallis test). The stereotyped duration of phase III suggests that the motor patterns underlying expansion, once triggered, run for a fixed time. Bursicon is the likely trigger, and, as noted above, its release begins during phase II. Although phase II durations in the three different conditions were significantly different (p ≤ 0.007 in pairwise comparisons using the Mann–Whitney test), they showed no obvious trend with respect to the level of perturbation. The observed variability in phase II durations, both within any given condition and across conditions, suggests that the timing or rate of bursicon release may differ considerably among individual animals.

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Figure 2.

Environmental perturbation selectively extends the duration of the perch selection phase. Box-and-whisker plots show the distribution of phase durations for flies observed under the low (L)-, medium (M)-, and high (H)-perturbation conditions described in the text. Each graph shows the durations of one of the three principal behavioral phases. The duration of the perch selection phase (phase I) increases incrementally with perturbation level, while the expansion phase (phase III) is unaffected by environmental conditions. The grooming phase (phase II) does not vary systematically with perturbation. For each plot, the lower and upper boxes span the second and third quartiles of the data, with the division between them defining the median. The whiskers extend to the minimum and maximum values of the data except when outliers are present (see Materials and Methods), in which case the outliers are represented by individual plus signs. Diamonds represent mean values. Double asterisks indicate very significant differences (p ≤ 0.01) as determined by the Mann–Whitney test. For the low-, medium-, and high-perturbation conditions, n = 20, 40, and 44, respectively.

The perch selection phase is insensitive to N_CCAP suppression

The above data demonstrate that environmental perturbations delay wing expansion primarily by extending the perch selection phase. The perch selection program is thus modulated by the sensory pathways that mediate environmental perturbation, but it is possible that this modulation is indirect and requires the circuitry underlying the expansion program. For example, the expansion program may be the primary target of environmental modulation, and the perch selection program could simply be active until inhibited by initiation of the expansion program. If the expansion program never starts up, perch selection should continue indefinitely. To directly test the possible involvement of the expansion program circuitry, we examined the effects of inhibiting activity in N_CCAP in the relative absence of environmental stimuli, i.e., in the low-perturbation condition.

We previously showed (Luan et al., 2006) that graded levels of N_CCAP suppression can be achieved by using the CCAP-Gal4 driver to express increasing copy numbers of the UAS-EKO suppressor transgene. Expression of three copies of the UAS-EKO transgene (i.e., 3× UAS-EKO) in N_CCAP completely blocks wing expansion and inhibits bursicon release into the hemolymph (Luan et al., 2006). As expected, we found that in the low-perturbation condition incremental N_CCAP suppression causes wing expansion deficits of increasing severity and frequency (Fig. 3 A, top panel). Consistent with the known requirement for bursicon in expansion behaviors (Baker and Truman, 2002), suppression of N_CCAP by 3× UAS-EKO completely eliminated both the tonic abdominal contraction that defines phase III (Fig. 3 A, bottom panel) and air swallowing (Table 1; see also supplemental Movie S2, available at www.jneurosci.org as supplemental material). Despite the absence of the expansion phase, however, the duration of phase I in these animals was indistinguishable from that of the control flies (Fig. 3 B). Indeed, there was no significant difference in the length of phase I for any of the experimental groups (p > 0.2 by Kruskal–Wallis test), and almost all the flies completed perch selection in <10 min regardless of the level of N_CCAP inhibition. Our results demonstrate that N_CCAP neurons are not involved in the termination of perch selection, and therefore in the transition to phase II, but that they are essential for the subsequent activation of the expansion program.

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Figure 3.

Graded suppression of N_CCAP incrementally inhibits wing expansion and slows or blocks entry into the expansional phase. A , Inhibition of N_CCAP in the low-perturbation condition by expression of one to three copies of the suppressor transgene, UAS-EKO, incrementally increases wing expansion deficits (top graph) and decreases the percentage of animals that execute phase III (bottom graph). EW, PEW, and UEW represent expanded, partially expanded, and unexpanded wing phenotypes, respectively. All data were taken from animals tested in the low-perturbation condition. B , Box-and-whisker plot showing the distribution of phase durations for animals expressing 1–3× UAS-EKO in the low-perturbation condition. Definitions are as described in the legend of Figure 2. Animals expressing 3× UAS-EKO failed to enter phase III, and phase II was therefore of indeterminate length. Both phases are rendered as “not defined” (ND) for these animals. Phase II and phase III plots for animals expressing 2× UAS-EKO include data only from the 12 animals (of 25 total) that executed phase III. Also, the phase I data set for these animals included three outliers, only one of which is represented, because two of the animals walked for the entire observation period and therefore did not end phase I. Only a median value (and no mean) is therefore given for this plot. For 1× and 3× UAS-EKO conditions, n = 10 and 6, respectively. Plus signs and double asterisks are as defined in the legend of Figure 2. A single asterisk indicates a significant difference (p < 0.05) across conditions as determined by the Kruskal–Wallis test.

N_CCAP activity regulates initiation of the expansion program

The behavioral effects of N_CCAP suppression have implications not only for the regulation of the perch selection program, but also for the mechanisms governing the wing expansion-related motor patterns. In this regard, the results for animals expressing 2× UAS-EKO are of particular interest. These flies can be divided into two distinct categories: Approximately half resembled flies expressing 1× UAS-EKO, which exhibited a robust expansion phase that included tonic abdominal contraction (Fig. 3 A, bottom panel) and air swallowing (Table 1), while the rest resembled 3× UAS-EKO animals, which completely lack phase III behaviors (Fig. 3 A, bottom panel; Table 1). This observation implies that there is a threshold level of N_CCAP activity which must be exceeded if the expansion phase is to be initiated. Because the two behaviors required for wing expansion either occur or fail in tandem, our results also imply that the two motor patterns share a common mechanism of initiation.

The common mechanism of initiation of the phase III motor patterns most likely reflects their shared requirement for bursicon (Baker and Truman, 2002), and their coincident failure at higher levels of N_CCAP suppression presumably results from impaired hormone release. Impaired bursicon release is also likely to underlie the observation that animals expressing 1× UAS-EKO, as well as approximately one-half of animals expressing 2× UAS-EKO, initiate phase III only after a significant delay, as reflected in the extension of phase II (Fig. 3 B). Further work will be required to determine whether the extension of phase II, which appears to result from longer quiescent episodes between grooming bouts, is caused by the slowing of bursicon release or by its delay (or both), but the former possibility would conveniently explain the observed threshold in N_CCAP activity for wing expansion. If EKO-mediated suppression attenuates bursicon release, the threshold for N_CCAP activity would simply reflect a threshold requirement for bursicon, with expansion triggered only when bursicon reaches a critical level.

A final implication of the results shown in Figure 3 is that N_CCAP modulates not just the expansion program, but also the nonbehavioral processes necessary for wing expansion. This is again most obvious for flies expressing 2× UAS-EKO. Approximately half of these flies executed phase III (Fig. 3 A, bottom panel), but none of these expanded their wings fully, and 70% did not expand them at all (Fig. 3 A, top panel). In addition, most flies expressing 1× UAS-EKO in N_CCAP only partially expanded their wings, despite executing normal phase III behavior. While we cannot rule out subtle behavioral deficits in the animals that failed to expand their wings, our results suggest that N_CCAP regulates nonbehavioral processes involved in wing expansion, such as plasticizing the wing cuticle to render it pliable. These nonbehavioral processes may well be mediated by bursicon released into the hemolymph, as the blood-borne hormone is known to be required for somatic changes at the level of the wing cuticle (Reynolds, 1976, 1977; Dai et al., 2008). In contrast to motor-pattern deficits resulting from N_CCAP inhibition, which are all-or-none, wing expansion defects are often partial (Fig. 3 A, top panel) and change in a graded manner. The nonbehavioral deficits associated with N_CCAP suppression thus appear to lack a threshold.

Targeted expression of rat TRPM8 can be used to acutely activate neurons

The suppression experiments described above indicate that perch selection normally terminates without input from N_CCAP and that the bursicon released from N_CCAP during phase II initiates the expansion program. Initiation of expansion thus requires activation (or disinhibition) of N_CCAP, and these neurons are probable targets of modulation by circuits that mediate the perch selection program or environmental inputs. To determine whether N_CCAP neurons are the sole targets of such modulation, or whether downstream neurons that mediate the execution of the expansion program are also subject to regulation, we sought to artificially activate N_CCAP during perch selection. We reasoned that if neurons downstream of N_CCAP are normally inhibited during perch selection, either by the circuitry underlying this program or by negative environmental signals, activation of these neurons would fail to initiate the expansion motor patterns. If, on the other hand, artificial activation of N_CCAP readily induced expansion, this would provide strong evidence that neural substrates of the expansion program downstream of N_CCAP were unlikely regulatory targets of the circuits underlying perch selection and/or environmental inputs. To determine which of these two possibilities might be the case, and more generally to determine whether N_CCAP activity is sufficient to initiate expansion behavior, we sought to acutely stimulate these neurons in post-eclosion flies.

Efforts to use the light-sensitive Channelrhodopsin-2 protein (Nagel et al., 2003), which has been used successfully before in Drosophila for acute neuronal activation (Schroll et al., 2006; Suh et al., 2007), proved ineffective in newly emerged adults, perhaps due to poor light penetration through the cuticle or depletion of the retinal-A cofactor during metamorphosis. We therefore sought to develop an alternative, and simpler, system for stimulating neurons in the live animal. To this end, we generated transgenic flies that can express the rat TRPM8 gene, which encodes a nonselective cation channel expressed in mammalian sensory neurons (McKemy et al., 2002; Peier et al., 2002). This channel is responsible for the sensation of mild cold (Colburn et al., 2007; Dhaka et al., 2007) and activates in response to decrements in temperature in the range of ∼25°C to 10°C. The thermal sensitivity of the TRPM8 channel, which is compatible with the temperatures at which flies are normally active, makes it particularly suitable for manipulating activity in Drosophila. Because cold, unlike light, normally attenuates motor activity, neuronal activation using TRPM8 also has an advantage over optical techniques in minimizing the confounding behavioral effects of the activating stimulus.

We made transgenic fly lines that express the TRPM8 channel under the control of Gal4, and tested their efficacy by assaying for abnormal behavior when the channel was expressed pan-neuronally. Several lines expressed the TRPM8 channel at sufficiently high levels to give robust phenotypes (such as seizing, jumping, and eventually falling down) upon transfer to 15°C from room temperature. We combined the strongest inserts to make lines that bore from one to three copies of the UAS-TRPM8 transgene. As shown in Figure 4 A, flies expressing UAS-TRPM8 pan-neuronally fell down quickly upon transfer to 15°C, with animals expressing 3× UAS-TRPM8 displaying a more rapid and robust response. Electrophysiological recordings from flight muscles of these animals revealed sustained action potential generation in motoneurons in response a downward temperature ramp (Fig. 4 B). This effect was readily reversible. Males typically showed greater sensitivity than females to a given temperature shift, as can be seen from the responses of animals expressing 2× UAS-TRPM8 shifted to 18°C (Fig. 4 C). At this temperature, 60% of males eventually fell over and could not right themselves, but none of the females did so, even after 10 min. We also tested the sensitivity of flies expressing UAS-TRPM8 to menthol. Menthol potentiates the activity of TRPM8 by shifting its temperature sensitivity to more positive temperatures (McKemy et al., 2002). Accordingly, saturating menthol vapor administered to flies expressing UAS-TRPM8 pan-neuronally at room temperature (24°C) caused the animals to seize and fall over as they did in response to cold (Fig. 4 D). The response was slow and poorly penetrant with a single copy of the transgene, but most animals expressing 2× UAS-TRPM8 fell down within 5 min of menthol exposure and all were down by 10 min.

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Figure 4.

UAS-TRPM8 acutely activates neurons in response to temperature decrements. One to three copies of a UAS construct expressing the rat cold-and-menthol receptor, encoded by the TRPM8 gene, were targeted to neurons using the pan-neural elav-Gal4 driver. Flies were tested for aberrant behavior (i.e., falling down) or physiological activity in response to cold or menthol exposure. A , Graph of the percentage of flies that have fallen down (typically accompanied by seizing and jumping) in response to a temperature decrement from 24 to 15°C. The percentage of flies lying on their backs was measured at 2.5 min intervals over a 10 min observation period. Experimental animals expressed either one (1×) or three (3×) copies of UAS-TRPM8 and were compared with control animals bearing the 1× UAS-TRPM8 transgene, but no driver. B , Intracellular recordings of physiological activity in flight muscles elicited by stimulation of innervating motoneurons. Animals expressing 1× (top trace) or 3× (middle trace) UAS-TRPM8 pan-neuronally were exposed to a 3 min temperature ramp from 23 to 12°C (bottom trace). The time of onset and frequency of the temperature-elicited action potentials increased as a function of UAS-TRPM8 levels, and background activity (arrowhead), presumably deriving from action potentials distal to the recording site, could be seen almost immediately after the onset of the temperature drop. C , Males are typically more sensitive to UAS-TRPM8 activation than females as can be seen by the intermediate levels of channel activation. The percentage of elav-Gal4 > 2× UAS-TRPM8 males (solid line) that fall down over time in response to a temperature shift from 24 to 18°C is higher than that of females (dotted line), which are not affected by this manipulation. D , Menthol, which potentiates the temperature sensitivity of TRPM8, elicits behavioral effects in flies expressing UAS-TRPM8 at room temperature. The percentage of flies that fall down over time increases very slowly in animals expressing 1× UAS-TRPM8 (dotted line), but the rate is much faster in animals expressing 2× UAS-TRPM8 (solid line), consistent with higher expression levels in these animals.

Activation of N_CCAP using TRPM8 is sufficient to initiate wing expansion

To activate N_CCAP with stimuli of various magnitudes, we subjected flies expressing 1× and 2× UAS-TRPM8 in N_CCAP (see Fig. S3 for anti-TRPM8 immunostaining) to temperatures shifts of various magnitudes. Under conditions similar to our low-perturbation condition, newly eclosed flies were transferred into food vials at room temperature and then placed at temperatures ranging from 18°C to 12°C. Even the mildest stimulus tested gave robust results: CCAP-Gal4 > 1× UAS-TRPM8 animals shifted briefly to 18°C immediately after eclosion rapidly expanded their wings relative to controls bearing the 1× UAS-TRPM8 transgene without the CCAP-Gal4 driver (Fig. 5 A, left). Both the speed with which the experimental group expanded their wings and the low variance in expansion times implied strong and uniform stimulation of N_CCAP in these animals, effects that were also observed when wing expansion was induced with a 15 min exposure to saturating menthol vapor at room temperature (Fig. 5 A, right).

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Figure 5.

Stimulation of N_CCAP using UAS-TRPM8 drives rapid wing expansion and bursicon release in animals in the high-perturbation condition. A , Bar graphs show the time taken by flies expressing UAS-TRPM8 in N_CCAP to expand their wings after temperature shift to 18°C (ΔTemp) or exposure to saturating menthol vapor (Menthol). In each case, the time taken by CCAP-Gal4 > 1× UAS-TRPM8 animals (n = 31 and 18 for the ΔTemp and Menthol conditions, respectively) was compared with that of w¹¹¹⁸ control flies bearing a single copy of the UAS-TRPM8 transgene, but no driver (n = 30 and 20 for the ΔTemp and Menthol conditions, respectively). CCAP-Gal4 > 1× UAS-TRPM8 animals subjected neither to an 18°C temperature shift nor to menthol took 31 ± 26 min to expand their wings (n = 20). B , Bar graphs show the average duration of each behavioral phase for animals expressing 1× UAS-TRPM8 in N_CCAP (n = 15) compared with parallel control animals bearing the UAS-TRPM8 transgene, but no driver (n = 12). All animals were tested in the high-perturbation condition within 30 min of eclosion. Activation of N_CCAP using UAS-TRPM8 significantly shortened both phases I and II, but did not alter the duration of phase III. C , N_CCAP activation causes rapid tanning, as indicated by these pictures of representative flies expressing 1× UAS-TRPM8 in N_CCAP, one of which received (right, +ΔTemp), and one of which did not receive (left, −ΔTemp), a 15 min temperature shift to 18°C. Both flies were subjected in parallel to the high-perturbation condition and were photographed 3 h after the time of onset of the temperature pulse delivered to the experimental fly. All flies that received the temperature pulse (n = 20) expanded their wings and tanned, as indicated by the generally dark appearance of their cuticles, while control flies (n = 19) uniformly failed to expand their wings or tan and retained the whitish cuticle color seen at eclosion. D , N_CCAP activation also causes rapid release of bursicon into the hemolymph. Hemolymph samples extracted from CCAP-Gal4 > 1× UAS-TRPM8 flies that either had (right, +) or had not (left, −) been subjected to a 15 min temperature shift to 18°C were analyzed by Western blot probed with an antibody to the bursicon α-subunit. For each experimental condition, hemolymph was extracted from >40 flies that had been subjected to the high-perturbation condition. Hemolymph was collected 1 h after the onset of the temperature shift or an equivalent time in unshifted controls. Molecular weight markers (in kDa) are shown.

To determine whether strong induction of the perch selection program attenuated the ability of UAS-TRPM8 to drive wing expansion, we examined the response of flies in the high-perturbation condition. Newly eclosed animals were placed in minichambers and exposed to a 15 min temperature shift from 24°C to 18°C. We found that N_CCAP activation by UAS-TRPM8 resulted in a substantial acceleration of wing expansion in all animals (supplemental Movie S4, available at www.jneurosci.org as supplemental material). On average, these animals took 25 ± 3 min (n = 12) to expand their wings compared with 219 ± 25 min (n = 10) for control flies bearing the 1× UAS-TRPM8 transgene, but lacking the CCAP-Gal4 driver (Fig. 5 B). All behavioral phases were typically present in the experimental animals, although phase II was extremely brief. Most animals persisted in walking and digging through most of the 15 min temperature pulse, but then transitioned almost immediately into the expansion phase after perching. Consistent with our observations under other conditions, phase III was relatively uniform in duration and lasted on average ∼15 min. All animals remained sedentary during wing expansion, indicating that execution of the expansion program potently inhibits perch selection behaviors. In addition to performing the expansion motor programs, animals expressing 1× UAS-TRPM8 in the high-perturbation condition displayed normal cuticle tanning after N_CCAP activation (Fig. 5 C, compare left, right). N_CCAP activation thus appears to induce bursicon release into the hemolymph. We confirmed this directly by measuring hormone titers after the temperature shift by Western blot (Fig. 5 D).

Thus, under conditions that normally bias the animal to persist in perch selection behaviors, mild activation of N_CCAP initiates hormone release and induces both the behavioral and physiological hallmarks of bursicon action. These results indicate that the neural pathways mediating perch selection and/or environmental input regulate the wing expansion program at, or above, the level of N_CCAP, and that neurons downstream of N_CCAP are not targets of modulation. In addition, our results demonstrate that N_CCAP neurons are not only necessary for wing expansion but also sufficient to drive this entire process.