The overall goal of this procedure is to measure the physiological responses of Drosophila gustatory neurons to lipid pheromones. This technique can be used to answer key questions in sensory neurobiology such as identifying the ligands for orphan gustatory receptors. The main advantage of this technique is that the responses of individual gustatory neurons can be studied in an intact, live animal.

After anesthetizing the fly, attach it to a 0.17 millimeter cover slip using a very small drop of clear nail polish. Attach the side of the fly to the slide using the entire drop. Next, under a dissecting microscope, use a wet paintbrush to extend a foreleg of the fly.

Then using two very thin strips of tape, secure the first and fifth tarsal segments to the cover slip. If neurons on the proboscis are to be imaged, then place another thin strip of tape over the rostrum of the proboscis to expose the labellum. Now, make a short wall around the fly using a PAP pen.

Let the anesthesia wear off for half an hour before taking measurements. For this protocol, prepare the necessary dilutions of the one milligram per milliliter stock ligand solution, using PBST. To begin the procedure, overlay 10 microliters of PBST onto the secured and exposed tarsal segments.

This moistens the leg and prevents it from drifting out of focus. Next, focus on the segments using an optical system which can achieve a good fluorescent signal with rapid time resolution such as a spinning disk confocal microscope with an sCMOS camera. Collect three prestimulation Z-stacks of the neurons of interest in the tarsal segment.

In this example, images are captured with 200 millisecond exposures and a two-by-two binning over a six micron by half-micron section every two seconds. Be sure to optimize the laser's power to minimize photo bleaching while still allowing a high signal-to-noise ratio. Here, a 491 nanometer 100-milliwatt laser is operated at 30%transmission.

The signal must be detectable prior to stimulation but not approach saturation. Now, pipette 10 microliters of stimulation solution onto the tarsal segment of interest. While pipetting, be very careful to avoid touching the leg or any of the fly's body or the tarsi will move out of position.

Then, immediately acquire the first post-stimulation images and continue to collect enough images to document the maximum change in fluorescence. For analysis, open a series of confocal Z-stacks using Fiji or ImageJ image analysis and processing software. Make a sum intensity projection of all the Z-slices for each of the time points.

Here, there are six slices and 120 time points. First, select the stack option under image and then select the Z-projection option. Enter one for the start slice and four for the stop slice.

For the projection type, enter sum slices and select all time frames. Now, define a region of interest, or ROI such as a cell body or neuronal projection. Use the freehand selection tool to draw around the structure's boundaries.

Then, click on the analyze label and select the ROI manager option under tools. Quantify the fluorescence of the ROI by first selecting the set measurements option under the analyze menu. Check the boxes for integrated density, area, mean and maximum grey value, and label.

Next, select the more option under the ROI manager menu and choose the multi-measure option. Now, calculate the maximum change in fluorescence for time point t. To quantify the post-stimulation fluorescence, measure the integrated density of the ROI.

For t equals zero, measure the prestimulation fluorescence from the prestimulus images of the ROI in the same manner. Average the values from three images for this t equals zero value. To quantify background fluorescence, measure the integrated density from a tissue area devoid of detectable neurons that has the same size and shape as the ROI.

Collect a unique background value for each time point. The GCamP calcium indicator was expressed using two different Gal4 expression patterns. Each pattern labels a distinct population of foreleg neurons and non-neuronal support cells.

For both expression patterns, ligand-specific responses to the lipophilic pheromone CH503 were observed. The relative change in fluorescence intensity of the GCamP signal increased with higher concentrations of CH503. Curiously, a five nanogram dose of CH503 may suppress the neuronal response.

The measured responses of the Gr68a and ppk23 neurons differed. The neuronal response of neurons labeled with the Gr68a Gal4 driver exhibited a tonic pattern whereas ppk23 neurons showed a phasic, oscillatory response in the leg. In the proboscis, ppk23 neurons showed a late-onset tonic response from the cell body and an oscillatory response from an axonal projection.

After watching this video, you should have a good understanding of how to measure the physiological responses of individual gustatory neurons to lipid pheromones and other hydrophobic ligands. This technique paves the way for researchers in the field of chemosensory biology to measure the responses of individual gustatory neurons in Drosophila melanogaster. Using this technique, neuronal activity can be silenced or activated using a temperature-controlled shibire or a TRiP a-one transgene by coupling the spinning disk microscope to a temperature-controlled onstage incubator.

Once mastered, this technique can be completed in five minutes, excluding the time taken by the fly to recover from anesthesia. While performing this method, it is important to fix the fly firmly on the cover slip without damaging its foreleg. While imaging, it is important to acquire images as soon as the foreleg is stimulated with pheromone solution.