Detecting Physiological Responses from Sensory Neurons in Drosophila

1. Sample Preparation

1. Cross the Gal4 driver line to UAS-GCaMP5G Drosophila melanogaster flies (w;P{20XUAS-IVS-GCaMP5G}attP40). Allow the cross to grow at 25 °C.
   NOTE: Generating flies with multiple copies of the Gal4 or UAS-GCaMP transgenes can help to enhance the fluorescent signal intensity. An earlier version of the UAS-GCaMP transgene (UAS-GCaMP3) showed very weak fluorescence intensity when expressed under the control of the Gr68a-Gal4 driver.
   1. Collect and separate by sex newly eclosed adult flies and raise at 25 °C.
      ​NOTE: The baseline level of GCaMP5G fluorescence is optimal in flies aged between 14 to 28 days. Isolating flies by sex precludes the possibility of social interactions that may influence the neural responses.
2. Anesthetize a fly under a mild stream of CO₂.
3. Attach the fly to a glass coverslip by first placing a small drop of nail polish in the center of a 0.17 mm coverslip. Gently place a fly on its side on the drop of clear nail polish and ensure that the fly covers the entire drop.
4. Perform steps 1.5 and 1.6 under a dissecting stereomicroscope, used at 8X magnification.
5. Use a wet paintbrush to extend the fly's foreleg. Secure the foreleg in position by placing two thin strips of tape over the first and fifth tarsal segments (Figure 1A). This will expose tarsal segments T2-T4 for imaging (Figure 1B).
6. To image neurons on the proboscis, place a thin strip of tape over the rostrum of the proboscis to expose the labellum (Figure 1C, D).
7. Draw a hydrophobic barrier around the fly with a PAP (Peroxidase-antiperoxidase) pen to prevent the solution from flowing over the coverslip surface. Measure within 30 minutes of mounting.

2. Preparation of Stimulus Solution

1. Dilute lipophilic ligands (such as CH₅0₃, used in this study) in a PBST (Phosphate-Buffered Saline with Triton X-100) solution: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.1% Triton X-100 (v/v); pH 7.4.
   1. Make a 1 mg/ml stock solution of the ligand using PBST. Prepare all subsequent dilutions of the stock solution using PBST.
      NOTE: Depending on the solubility of the ligand of interest, different solvents may be needed to dissolve it. Table 1 describes the solubility of CH₅0₃ in various solvents. Unlike other solvents, PBST did not increase fluorescence.

3. Stimulation of Gustatory Neurons and Image Acquisition

1. Using a 10 µl pipettor, place 10 µl of PBST onto the tarsal segments.
   NOTE: This step moistens the leg and prevents the tarsal segments from drifting.
2. Use an optical system that has a camera of sufficient speed and sensitivity to achieve a good fluorescence signal at high time resolution.
   NOTE: For this study, we used a spinning disk confocal microscope and a sCMOS camera (see Materials List).
3. Choose the specific tarsal segment and neurons to be imaged. Acquire three pre-stimulation confocal Z-stacks.
   1. Ideally, use acquisition settings that are fast enough to allow maximal time resolution while still capturing the entire cell volume. Example acquisition settings are: 200 msec exposure, binning 2 x 2 and 6 x 0.5 µm2 optical sections, captured every 2 sec.
      ​NOTE: Immobilized legs have been imaged successfully for up to 10 min, at 30 sec intervals, with no discernible bleaching of the GCaMP signal. For longer recording times, ensure that the legs are held very firmly in place on the coverslip.
   2. Optimize laser power for the highest signal-to-noise achievable with minimal photobleaching during the entire imaging regime. For the experiments described here, use a 491 nm diode laser (100 mW) at 30% transmission to image both Gr68a and ppk23 gustatory neurons on the legs and proboscis. Use 2 x 2 binning to allow for shorter exposure times while still achieving sufficient spatial resolution.
      NOTE: The laser power settings were optimized to detect fluorescent changes ranging from a 1.2- to 4.5-fold increase.
4. Pipette 10 µl of the stimulus solution onto the tarsal segments. Add another 10 µl of PBST for control experiments. Immediately acquire 117 post-stimulation images.
   NOTE: The number of post-stimulation images acquired is based on the time it took to reach the maximal change in fluorescence (approximately 2 min).
   1. CRITICAL STEP: When pipetting solution on the preparation, do not touch the fly with the pipette tip, as this will cause the foreleg to move out of position.
      NOTE: Alternatively, use a micromanipulator to accurately position a glass capillary containing stimulus close to the tarsal segment to be imaged. Deliver controlled volumes of stimulus using an intracellular microinjection dispenser according to the manufacturer's instructions.

Figure 1: Mounting a live fly for imaging of the foreleg or proboscis neurons. (A) Mounting the foreleg: a 3.2X image of a live male fly, showing the forelegs fastened to the coverslip with tape. (B) A 40X image of the fly shows each of the tarsal segments (T1-5) and the placement of tape above the first tarsal segment and on the fifth tarsal segment. (C) Mounting the proboscis: a piece of tape is placed over the rostrum to expose the tip of the proboscis. (D) The pipette tip for stimulus addition is held slightly above the fly and does not make direct contact with the preparation.

Table 1: Solubility of CH503 in various solvents. DMSO: Dimethyl sulfoxide. Ethanol, hexane, and DMSO solutions were diluted with dH2O (v/v). The solubility of CH503  in 0.2% DMSO was limited only to 250 ng/ml. The increase in ΔF/F with solvent alone was equivalent to that observed upon stimulation with pheromone.