microelectrode-mediated fm dye loading and unloading in drosophila larvae nerve cells

Microelectrode-Mediated FM Dye Loading and Unloading in Drosophila Larvae Nerve Cells

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 microforge 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 larva 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 FM1-43 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 strength.
For FM dye unloading, suck the same motor nerve into the same electrode, and then stimulate the motor nerve to activate SV exocytosis and FM1-43 dye release.

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1. Larval Glue Dissection
<ol>
	<li>Thoroughly mix 10 parts of silicone elastomer base with 1 part of silicone elastomer curing agent from the elastomer kit.</li>
	<li>Coat 22 x 22 mm glass coverslips with the elastomer and cure on a hot plate at 75 &#730;C for several hours (until no longer sticky to the touch).</li>
	<li>Place a single elastomer-coated glass coverslip into the custom-made plexi glass dissection chamber for the larval dissection.</li>
	<li>Prepare the glue pipettes from borosilicate glass capillary using a standard microelectrode puller to obtain the desired taper and tip size.</li>
	<li>Gently break off the micropipette tip, and to the other end, attach 2 ft of flexible plastic tube (1/32&#34; interior diameter, ID; 3/32&#34; outside diameter, OD; 1/32&#34; wall; with mouth fitting (P2 pipette tip).</li>
	<li>In preparation for the larval dissection, fill a small container (0.6 mL Eppendorf tube cap) with a small volume (~20 &#181;L) of glue.</li>
	<li>Fill the chamber with saline (in mM): 128 NaCl, 2 KCl, 4 MgCl2, 1 CaCl2, 70 sucrose, and 5 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) pH 7.2.</li>
	<li>Add anti-horse radish peroxidase (HRP) antibody conjugated to Alexa Fluor 647 (anti-HRP:647; dilute 1:10 from a 1 mg/mL stock) for labeling the NMJ presynaptic terminal during dissection21,22.</li>
	<li>Using a fine paintbrush (size 2), remove a wandering third instar from the food vial and place it onto the elastomer-coated cover glass.</li>
	<li>Fill the glass micropipette tip with a small volume of glue using negative air pressure generated by the mouth with attachment (step 1.5).</li>
	<li>Position the larva dorsal side up with forceps and glue the head to the elastomer-coated coverslip with a small drop of glue using positive air pressure by mouth.</li>
	<li>Repeat this procedure with the larva&#39;s posterior end, ensuring the animal is stretched taut between the two glue attachments.</li>
	<li>Using scissors (blades 3 mm), make a horizontal cut (~1 mm) at the posterior and a vertical cut all along the dorsal midline.</li>
	<li>Using fine forceps (#5), gently remove the dorsal trachea, gut, fat body, and other internal organs covering the musculature.</li>
	<li>Repeat the gluing procedure for the four body wall flaps; gently stretch the body wall horizontally and vertically.</li>
	<li>Lift the ventral nerve cord (VNC) using forceps, carefully cut the motor nerves with the scissors, and then completely remove the VNC.</li>
	<li>To stop SV cycling, replace the dissection saline with Ca2+-free saline (the same as the above dissection saline without the CaCl2).</li>
</ol>
2. Electrical Stimulation FM Dye Loading
<ol>
	<li>Prepare a suction pipette using the microelectrode puller to obtain the required taper and tip size.</li>
	<li>Fire-polish the microelectrode tip with a micro-forge until a single motor nerve can be sucked up with a tight fit.</li>
	<li>Slide the suction pipette onto the electrode holder on a micromanipulator and attach it to the long flexible plastic tube and a syringe.</li>
	<li>Set stimulator parameters (e.g., 15 V, 20 Hz frequency, 20 ms duration, and time of 5 min or 1 min.</li>
	<li>Replace the Ca2+-free saline in the larval preparation with the above FM1-43 saline (4 &#181;M; 1 mM CaCl2) in the electrophysiology rig.</li>
	<li>Put the preparation on the microscope stage and raise the stage until the larva and suction pipette are focused (40X water immersion objective).</li>
	<li>Suck up a loop of cut motor nerve innervating the selected hemisegment with negative air pressure generated by the syringe into the suction electrode.</li>
	<li>Test the suction electrode function with a short burst of stimulation while visually monitoring for muscle contraction in the selected hemisegment.</li>
	<li>Stimulate the motor nerve using selected parameters (step 2.4) to drive SV endocytosis and FM1-43 dye uptake.</li>
	<li>Wash quickly with Ca2+-free saline (5x for 1 min) to remove the FM1-43 dye solution completely.</li>
	<li>Maintain the larval preparation in fresh Ca2+-free saline for immediate imaging using the confocal imaging protocol from above.</li>
	<li>Take careful note of the NMJ image (segment, side, and muscle) to ensure access to the same NMJ after FM dye unloading.</li>
</ol>

Source: Kopke, D. L., et al., <a href="https://app.jove.com/v/57765">FM dye cycling at the synapse: comparing high potassium depolarization, electrical and channelrhodopsin stimulation.</a> J. Vis. Exp. (2018)
In this video, Drosophila larvae nerve fibers are immersed in FM dye, which binds to cell membrane lipids. Suction microelectrode and electrical pulses stimulate the endocytosis process, incorporating the FM dye into newly formed endocytic vesicles. This method, which visualizes presynaptic stages of neurotransmission, is useful for studying synaptic function and neurotransmitter release.

1. Larval Glue Dissection

	Thoroughly mix 10 parts of silicone elastomer base with 1 part of silicone elastomer curing agent from the elastomer kit.
	...

Begin with Drosophila larvae, with exposed muscle and nerve fibers immersed in a solution containing vesicles of fluorescent membrane dye, or FM dye.
The FM dye binds to the lipids in the cell membrane, forming a fluorescent coating over it.
Using a micromanipulator unit, position the suction microelectrode tip under a microscope.
Focus on the nerve fibers and the microelectrode.
Use negative pressure to suck a loop of the nerve fiber into the microelectrode.
Apply current pulses to stimulate the nerve fiber.
This triggers the neuromuscular junction, where a motor neuron connects to a muscle fiber, to initiate synaptic vesicle endocytosis.
During this process, the cell engulfs the FM dye-coated membrane to form stained endocytic vesicles, ideal for visualizing presynaptic stages of neurotransmission.
Wash off the FM dye and replace the external solution.
Apply current pulses to unload the FM dye-stained vesicles out of the cell.

9 Views. Microelectrode-Mediated FM Dye Loading and Unloading in Drosophila Larvae Nerve Cells

Microelectrode-Mediated FM Dye Loading and Unloading in Drosophila Larvae Nerve Cells

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