Detecting Physiological Responses from Sensory Neurons in Drosophila

Attach an anesthetized genetically modified Drosophila melanogaster on its side to a coverslip.

Extend its forelegs and secure them to expose the tarsal segments.

These segments have gustatory neurons expressing calcium indicator complexes that fluoresce in calcium-bound form.

Draw a hydrophobic barrier around the fly to retain the solution.

Allow the fly to recover and overlay a buffered saline solution onto the exposed tarsal segments to moisten them.

Under a confocal microscope, observe tarsal segments and image the gustatory neurons at various depths to obtain a pre-stimulation fluorescence.

Add a stimulation solution containing lipophilic ligands onto the segment.

These ligands bind to the gustatory neuron receptors, initiating signaling pathways and triggering the opening of calcium channels.

This allows calcium ions from the extracellular fluid to enter the cytoplasm and bind to the calcium indicator complexes, resulting in fluorescence emission.

When observed again, an increase in neuronal fluorescence confirms the neuronal physiological response to the ligand.

After anesthetizing the fly, attach it to a 0.17-millimeter coverslip, 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 coverslip.

If neurons in 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 1-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 fluorescence signal with rapid time resolution, such as a spinning disk confocal microscope with an sCMOS camera. Collect three pre-stimulation Z-stacks of the neurons of interest in the tarsal segment.

In this example, images are captured with 200-millisecond exposures and a 2-by-2 binning over a 6-micron-by-1/2-micron section every two seconds. Be sure to optimize the laser's power to minimize photobleaching, 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.