Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish

We study the molecular and cell mechanism of synaptic transmission using neuromascular junction of larval zebrafish as a model system. We are interested in clarifying the regulatory moderation of the synapsis in response to various physiological and pathological conditions. This protocol allows us to visualize action potential-induced new transmission in larval zebrafish neuromuscular junction with improved sensitivity.

We can monitor the synaptic function elicited by moderate stimulation, which has not been tested previously. This imaging protocol can be performed under a conventional epifluorescence microscope and does not necessarily require a confocal laser scanning microscope. In other words, this method provides a cost-effective platform for imaging neuromuscular function compared to other experimental setups.

To prepare 200 milliliters of the extracellular solution, mix 6.4 milliliters of 3.5 molar sodium chloride, 0.4 milliliters of one molar potassium chloride, 1 milliliter of one molar HEPES, 0.4 milliliters of one molar calcium chloride, 0.2 milliliters of one molar magnesium chloride, and 360 milligrams of glucose. Then add ultrapure water to bring the total volume to 200 milliliters. Add 20 microliters of 15 millimolar D-tubocurarine stock solution to prepare 100 milliliters of extracellular solution with 3 micromolar D-tubocurarine.

To prepare 25 milliliters of the extracellular solution containing 0.02%tricaine, add 0.5 milliliters of 1%tricaine stock solution to 25 milliliters of solution. Using a micro pipette puller, pull the theta glass capillaries until the tip diameter measures within the range of 3 to 10 micrometers. Fill the pipette with the extracellular solution.

Insert a thin platinum wire into each opening of the capillary, and hold the theta glass pipette electrode onto the pipette holder of the motorized micro manipulator. Then connect the backend of each wire to the stimulus isolator. For 25 milliliters of extracellular solution containing 0.02%tricaine into a glass Petri dish, transfer a zebrafish larvae to the Petri dish for dissection.

Use two fine forceps to peel the skin of the larvae. Use the first forceps to hold the larvae in place and pinch the skin on the dorsal side of the swim bladder with the other forceps. Then using scalpel, remove the swim bladder, internal organs, and the head.

Use a glass pasteur pipette with a fire-polished tip to transfer the dissected zebrafish sample to the imaging chamber. Mechanically fix the sample with a nylon thread glued to a C-shaped platinum wire to orient it at an angle approximately parallel to the stimulating electrode. Perfuse the sample using a gravity flow system at a rate of 1 milliliter per minute with an extracellular solution containing 3 micromolar D-tubocurarine.

Next, insert the stimulation electrode into the spinal cord. Carefully position the electrode tip near the spinal motor neurons, identified as TagRFP-positive neurons located on the ventral side of the spinal cord. Advance the electrode at an oblique angle from the adjacent segment to the target position.

To begin on a computer, select the imaging region based on the live image of TagRFP fluorescence. Ensure the selected region includes multiple boutons and excludes those near the body segment boundary. Switch the fluorescence filter unit GFP fluorine fluorescence for imaging.

Next, adjust the intensity of the light source to obtain a bright image in 1 hertz time lapse mode without saturation. Enter the exposure time to 100 in the exposure milliseconds field of the micromanager window. Configure the image acquisition software and the digital input output device to synchronize image acquisition with electrical stimulation.

Next, determine the stimulation frequency, number of action potentials, and time delay between the start of image acquisition and stimulation delivery in the Arduino script. Based on these settings, calculate the total number of images required for acquisition and enter this value in the count field of the multidimensional acquisition window in micromanager. Set a 1-second interval in the interval field for 1 hertz time lapse imaging.

For electrical stimulation, configure the stimulus isolator to deliver 1-millisecond constant voltage pulses of 70 millivolts. Execute the digitizer command to begin image acquisition. Monitor the process to verify that there is no unacceptable image drift and that fluorine and responses are observable.

Modify the stimulation intensity, such as frequency in number of pulses, or adjust the temperature based on the experimental purpose. Save the time-lapse images as a TIFF time series stack. Synapses with large signal increases were selected for analysis, though all boutons showed detectable changes in the 20 hertz 100 action potential condition, Robust fluorine responses were observed upon high-frequency electrical stimulation with fluorescence changes indicating exocytosis during stimulation and subsequent decay, reflecting endocytosis and re-acidification of synaptic vesicles.

Fluorescence decay time constants varied with stimulus, intensity, and temperature with 20 hertz 25 action potentials showing at 16.6 seconds, 20 hertz 100 action potentials at 56.8 seconds, and 50 hertz 500 action potentials at 62.7 seconds.