All flies were grown on corn meal-molasses medium and maintained at 25°C in a constant 12 hour light-dark cycle. Wild type flies (Canton-S strain) were from the Bloomington Stock Center (Indiana University, Bloomington, IN). The Gal4 driver lines used in this study were generous gifts of the following: yw; CCAP-Gal4; + and w; +; CCAP-Gal4
(Park et al., 2003
, John Ewer); w; c929-Gal4; +
(O’Brien and Taghert, 1998
, Paul Taghert). The elavC155
-Gal4;+;+, yw;2XUAS-EGFP; 2XUAS-EGFP, and 1X, 2X, and 3X EKO lines, which carry one, two, and three copies of the EKO transgene have been described previously (White et al., 2001
Generation of UAS-TRPM8 constructs and fly lines
The coding sequence of the TRPM8 cDNA (McKemy et al., 2002
) was amplified by polymerase chain reaction using primers that introduced an EcoRI restriction site and an optimized translation initiation motif (with sequence CAAA, Cavener, 1987
) immediately before the ATG start codon and a Kpn I restriction site just after the stop codon. The amplified fragment was then subcloned into the pUAST plasmid for P-element transformation, using the unique EcoRI and KpnI restriction sites in the multiple cloning site. P-element injections and isolation of transformants was performed by Genetic Services, Inc. (Cambridge, MA). Flies with inserts of UAS-TRPM8 on the 2nd
(yw; C4-D; +
) and 3rd
(yw; +; C4-A
and yw; +; C1-A2
) chromosomes were used in this paper, separately or in combination.
Behavioral Observations and Analysis
Behavior of newly eclosed flies was observed by one of three methods as described in the Results. In all cases, flies were observed for at least 90 minutes after eclosion and wing expansion phenotypes were assessed at least 24 hours after eclosion.
For observations in the low perturbation condition, the vials used were either cultured at low density and cleared of any adults prior to observation of the first fly to eclose, or 3rd instar larvae in the wandering stage were transferred individually to new culture vials to pupate and were observed after eclosion. Most observations were made by eye, though in a few cases flies were videotaped and behavior was scored from the video. The onset and offset of walking and abdominal flexion were scored to determine the three principal phases defined below.
For observations in the medium perturbation condition, individual flies were transferred within 1-3 min of eclosion to a cylindrical chamber (1.4 cm diameter, 0.4 cm thick) formed by placing a plastic ring between two glass cover slides, which were held together by magnets glued to each slide. In some cases, an acetate sheet was attached to one of the glass slides to provide a better substrate for perching. Flies were videorecorded in this chamber using a Sony DCR-PC115 Digital Video Recorder, mounted on an Olympus SZX-12 stereomicroscope, with the video signal streamed to an external hard drive using a Firestore FS-1 (Focus Enhancements, Campbell, CA) for storage (see Figure S1
for a full description of the setup). Videorecords for 40 animals were analyzed for sustained “state” behaviors (e.g. walking, grooming, abdominal contraction, proboscis extension, etc.) using Observer Video-Pro Version 5.0 software (Noldus Information Techology, Wageningen, The Netherlands). Detailed ethograms were constructed for 10 flies, scoring individual movements of the legs, wings, abdomen, and proboscis. Cibarial pumping, which appeared as pulses of light reflected from the base of the proboscis could not be resolved in all flies depending on their orientation relative to the camera.
For the high perturbation condition, flies were transferred after eclosion into glass tubes (0.3 cm diameter) plugged on each end to form a small chamber 0.7 cm in length. Flies confined within the tubes were videorecorded using a Sony HDR-FX7 digital videocamera, and records were scored for walking, grooming, abdominal flexion, abdominal contraction, and wing expansion.
The behavioral data in the text and in Figures and are presented as mean values ± standard error of the mean. For box and whisker plots, the boxes represent the interquartile range of the data separated by the median value, and the “whiskers” extend to the minimum and maximum values of the data, with the exception of outliers. Outliers are defined as values that lie more than three times the interquartile range above the upper quartile value (i.e. top of the upper box). Data were analyzed using non-parametric statistics, due to significant heteroskedacity based on the Levene test. Comparisons of more than two groups were performed using the Kruskal-Wallis test, followed by repeated post-hoc Mann-Whitney U tests with Bonferroni corrections for multiple comparisons. Pairs of groups were compared with Mann-Whitney U tests. All statistical analysis was performed with SPSS version 13.0 (SPSS Inc., Chicago, IL)
Posteclosion behavior in Drosophila consists of three phases with distinct patterns of behavior
Stimulation of NCCAP using UAS-TRPM8 drives rapid wing expansion and bursicon release in animals in the high-perturbation condition
Definitions of Behavioral Phases
Posteclosion behavior was divided into three phases, similar to those described previously in blowflies (Cottrell, 1962
; Zdarek et al., 1984
), defined by the following beginning and end points:
Phase I: From eclosion (or placement in the videorecording chamber) to first perch. A perch is defined as the cessation of walking for more than five minutes. Flies sometimes commenced walking and reperched, but such episodes were typically brief compared to the initial walking bout.
Phase II: From first perch to the start of sustained abdominal flexion, a condition defined by the elongation and downward flexion of the abdomen. Although short pulses of abdominal flexion could be observed in Phase II, sustained abdominal flexion typically lasted 10-15 min.
Phase III: From the onset to the cessation of abdominal flexion.
Quantification of air swallowing
Air swallowing was quantified by measuring the volume of air in the gut of fly. To do so, flies were briefly immersed in 100% ethanol to free the cuticle and bristles of any air, then pinned down through the head and anus in a Sylgard-treated culture dish filled with glycerol. The legs and wings were removed under a SZX-12 Olympus dissection microscope outfitted with a Nikon camera, and the gut was exposed by an incision extending up the midline of the fly. The air inside the gut was then released by gently tearing the gut membrane with forceps and, once liberated, formed a spherical bubble that rose slowly in the glycerol. This bubble was then photographed and its volume calculated from the value of the diameter measured using ImageJ software (Rasband, W.S. U. S. National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/
) calibrated with a 2 mm micrometer. Typically, air volume was measured at the end of Phase III or, in cases where this phase was absent, 90 minutes after the eclosion time.
Testing UAS-TRPM8 and Electrophysiology
The efficacy of the TRPM8 channel was tested using a pan-neural driver (elavC155-Gal4;+;+) to express one, two, or three copies of UAS-TRPM8. Up to 10 flies were placed in a large chamber (either a 35 mm culture dish or for the menthol experiments an empty food vial) and then either subjected to a temperature shift or exposed to saturating menthol vapor. The number of flies no longer standing (i.e. “flies down”) was scored every 2.5 minutes, typically from videorecords or in some cases by eye. Electrophysiological recordings from adult flight muscle were made as follows: Flies were anesthetized with CO2 just long enough to remove the wings and place them onto a temperature-controlled stage, where they were held in place with vacuum. To record UAS-TRPM8 activated motoneuron activity, electrolytically-sharpened tungsten recording electrodes were placed in dorsal longitudinal muscles (DLM) and reference electrodes in the abdomen. Signals were amplified with Dagan IX2-700 (Dagan Corporation, Minneapolis, MN) or A-M Systems 1700 (A-M Systems, Carlsborg, WA) differential amplifiers. The temperature-controlled stage consisted of a hollow brass disc through which water of the appropriate temperature was circulated. Temperature changes of >10°C/min could be achieved by switching between two water baths maintained at target temperatures. Temperature was monitored with a thermocouple probe (T-type, Physitemp Instruments Inc., Clifton, NJ) placed adjacent to the fly, with the signal transduced by a temperature controller (CNI-3252-DC, Omega Engineering Inc., Stamford, CT). Electrophysiological and temperature data were recorded using pClamp ver 8.2 (Molecular Devices, Union City, CA).
Animals were exposed to saturating menthol vapors in plastic vials (94mm long × 27mm diameter) as follows: flies were introduced into a vial capped by a foam plug in which menthol crystals were embedded. To test the effects of menthol on animals expressing UAS-TRPM8 pan-neuronally, one day old adults were transiently immobilized by CO2 and permitted one hour recovery in an empty vial prior to menthol exposure. Animals were observed and scored every two and a half minutes for 25 minutes. To test the effects of menthol on wing expansion time, animals were collected within 5 minutes of eclosion and immediately transferred into a menthol-saturated vial for 15 minutes. After 15 min, the plug was gently replaced with one lacking menthol crystals. Individual flies were scored for wing expansion phenotypes at one minute intervals.
Hemolymph Collection and Immunoblotting
For experiments on wildtype flies in the low perturbation condition, hemolymph was extracted (as described below) from flies transferred into individual culture vials directly after eclosion and continuously monitored until they had spent five minutes in Phases I, II, or III, or were at least 15 minutes past completion of Phase III. For experiments in which flies expressing UAS-TRPM8 in NCCAP
had been subjected to a temperature shift in the high perturbation condition, hemolymph was taken one hour after the onset of the temperature shift (i.e. 45 minutes after return of the flies to 25°C). Hemolymph was extracted in both cases by briefly anesthetizing flies with CO2
, piercing the thorax with a needle and centrifuging as described previously (Luan et al., 2006b
). Collected samples were mixed with HE Buffer (100mM KCL, 20mM HEPES-pH 7.5, 5% glycerol, 0.5M EDTA, 0.1% Triton X-100) containing 2X Halt protease inhibitor (Pierce, Rockford, IL) and then frozen on dry ice. They were then thawed in equal portions of Laemmli sample loading buffer containing 5% β-mercaptoethanol, and boiled for five minutes prior to electrophoresis on a 12% Tris-HCL gel (Bio-Rad, Hercules, CA). Gels were transferred to 0.2 μm nitrocellulose membranes using a Tris-Glycine-20%MeOH buffer, and immunoblotted. The primary rabbit anti-bursicon α-subunit antibody (Luan et al., 2006b
) was used at a dilution of 1:5000 and a goat anti-rabbit secondary antibody (10μg/ml, Pierce, Rockford, IL) at 1:2000. Blots were incubated with West Femto chemiluminescent substrate (Pierce, Rockford, IL) for five minutes before development on BioMax film (Eastman Kodak, Rochester, NY) for 10 minutes.
Immunohistochemistry and confocal analysis
For analysis of whole-mount nervous system preparations, freshly emerged adults (i.e. within 15 minutes of eclosion) or stage P15i pharate adults (Bainbridge and Bownes, 1981
) were dissected in PBS, and the excised nervous systems were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS for approximately 20 min, followed by postfixation in 4% paraformaldehyde/PBS plus 0.5% Triton X-100 (Sigma, St. Louis, MO) for 15 min. Procedures for immunostaining were as previously described (Luan et al., 2006b
). Rabbit anti-TRPM8 antibodies (Novus Biologicals, Littleton, CO) were used at 1:200 dilution. Secondary antibodies (AlexaFluor 594 goat anti-rabbit from Invitrogen, Carlsbad, CA) were used at 1:500 dilution. Confocal imaging was performed using a Nikon (Tokyo, Japan) C-1 confocal microscope. Z
-series through either the brain or ventral nerve cord of each sample were acquired in 1 μm increments using a 20X objective unless otherwise noted, using 488 nm and 543 nm laser emission lines for EGFP and fluorophore excitation, respectively. Unless otherwise noted, the images shown are maximal projections of the volume rendered Z-stacks of confocal sections taken through the entire nervous system.
The consensus pattern of CCAP-Gal4>UAS-TRPM8 expression was determined by analyzing the intensity and frequency of labeling of identified CCAP-expressing neurons in multiple wholemount preparations. CCAP-expressing neurons in each preparation were identified in confocal sections by expression of UAS-EGFP and the intensity of TRPM8-associated fluorescence was scored for each neuronal soma on a scale of 0–3. The consensus intensity value (I) for a given neuron was calculated by averaging all non-zero values for this neuron across preparations. The frequency (v) with which a given neuron was labeled was calculated by dividing the number of preparations in which that neuron had a non-zero labeling intensity by the total number of preparations.