The overall goal of this experiment is to observe the endo and exocytosis cycling of synaptic vesicles on the live preparation of Drosophila larval neuromuscular junction using fluorescent FM dyes. This method can help answer key questions in the field of neuroscience, such as which genes are critical for the function of the synaptic vesicle cycle? The main advantage of this technique is that you can optically track vesicle cycling in a live preparation using three different types of stimuli, high potassium depolarization, electrical stimulation, or using optogenetics.
To being this procedure, add the anti-HRP antibody, conjugated to a lexoflor 647 to the dissecting saline. Next, add an elastomer coated cover slip to the dissection chamber and then add the dissecting solution with HRP antibody for labeling the NMJ presynaptic terminal during dissection. Using a fine paintbrush, transfer a wandering third instar from the food vial onto an elastomer-coated cover glass.
Then fill a glass micro pipette tip with a small volume of glue, using negative air pressure generated by mouth. Subsequently, position the larvae, dorsal side up, with forceps and glue the head to the elastomer-coated cover slip with a small drop of glue using positive air pressure by mouth. Repeat this procedure with the posterior end of the larvae, making sure that the animal is stretched taut between the two glue attachments.
Using scissors, make a one millimeter horizontal cut at posterior and vertical cut along the dorsal midline. Then gently remove the dorsal trachea, gut, fat body, and other internal organs, covering the musculature. Repeat the gluing procedure for the four body wall flaps, making sure to gently stretch the body while both horizontally and vertically.
Afterward, lift the VNC and carefully cut the motor nerves with the scissors and completely remove the VNC. Replace the dissection saline with calcium free saline to stop SV cycling. For electrical stimulation, prepare a suction pipette using the microelectrode puller to obtain the required taper and tip size.
Afterward, fire polish the microelectrode tip with a micro forge to create a tight fit when sucking up a single motor nerve. Slide the suction pipette onto the electrode holder on a micromanipulator, which is attached to a long, flexible plastic tube and a syringe. Next, put the preparation with FM dye on the microscope stage and raise the stage until the larvae and suction pipette are in focus.
Then suck up a loop of cut motor nerve, innervating the selected hemi segment with negative air pressure, generated by the syringe into the suction electrode. Subsequently, stimulate the motor nerve using the selected parameters to drive SV endocytosis and FM 143 dye uptake. The most critical step at this point in the procedure is to suck up a single motor axon into the suction electrode with a tight fit.
This may take some trial and error. Once an adequate suction electrode is found, use the same one for all conditions being compared to ensure consistent stimulation strain. For FM dye unloading, suck the same motor nerve into the same electrode and then stimulate the motor nerve to activate SV exocytosis and FM 143 dye release.
For channelrhodopsin stimulation, attach a blue LED to a programmable stimulator using a coaxial cable and place the LED into the camera port. Focus the blue LED light beam onto the preparation with FM dye using the microscope zoom function. The most critical step at the point in the procedure is to make sure the LED settings are kept constant and that you illuminate the same part of each animal for all conditions being compared to ensure consistent stimulation strain.
Start the light stimulation and track with the timer for the predetermined duration of the optogenetic stimulation period. When the timer stops, quickly remove the FM dye solution and replace it with calcium free saline to stop the SV cycling. Of FM dye unloading, start the light stimulation and track with a timer for the predetermined duration of the optogenetics stimulation period.
When the timer period ends, quickly remove the FM dye solution and place it with calcium free saline to stop the SV cycling. To quantify the fluorescent image, load it in image J and create a maximum intensity projection by clicking Image, Stacks, Zproject, then using the anti-HRP 647 channel, go to Image, Adjust, Threshold, and slide the top toolbar until just the NMJ is highlighted. Using the wand tool, click on the NMJ.
If the NMJ is discontinuous, hold the shift button and select all parts. Then change the image to the FM 143 dye channel and go to Analyze, Measure, to obtain the fluorescence measurement. Repeat these steps for the unloaded image from the same NMJ.
Fluorescent FM 143 can be photoconverted to an oxidized DAB electron dense precipitate for visualization via TEM. Here is a schematic diagram of the photo conversion method to label the drosophila NMJ following activity dependent FM 143 dye loading. This is a representative TEM image of a wild type synaptic bouton at rest, which shows a dense population of uniform sized SVs.
These images show the synaptic boutons that have been stimulated with high potassium saline depolarization for five minutes without photoconversion or with FM 143 photoconversion. The bulk end homo structures can be observed here. And these images show the synaptic boutons that have been electrically stimulated with the nerve suction electrode at 20 hertz for five minutes with no photoconversion or with FM 143 photoconversion.
Note that the dye-loaded vesicles appear much darker than adjacent unloaded vesicles. After watching this video, you should have a good understanding of how to perform a larval glue dissection as well as FM dye loading and unloading using high potassium depolarization, electrical stimulation and optogenetic channelrhodopsin stimulation. Since FM dyes were first synthesized by Fei-Mao, these versatile dyes have been used to study activity dependent mobilization of distinct vesicle pools to study the difference between and clathrin-mediated endocytosis, as well as study evoked, spontaneous and miniature synaptic activities.
There are many factors to consider throughout this experiment. To name a few, the external calcium concentration, the type of stimulation method that you use, as well as how long to load and unload the FM dye during the uptake and release steps. Please take these factors into careful consideration when planning your own experiment.
Good luck!
