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

プロトコル

1. Larval Glue Dissection

1. Thoroughly mix 10 parts of silicone elastomer base with 1 part of silicone elastomer curing agent from the elastomer kit.
2. Coat 22 x 22 mm glass coverslips with the elastomer and cure on a hot plate at 75 ˚C for several hours (until no longer sticky to the touch).
3. Place a single elastomer-coated glass coverslip into the custom-made plexi glass dissection chamber for the larval dissection.
4. Prepare the glue pipettes from borosilicate glass capillary using a standard microelectrode puller to obtain the desired taper and tip size.
5. Gently break off the micropipette tip, and to the other end, attach 2 ft of flexible plastic tube (1/32" interior diameter, ID; 3/32" outside diameter, OD; 1/32" wall; with mouth fitting (P2 pipette tip).
6. In preparation for the larval dissection, fill a small container (0.6 mL Eppendorf tube cap) with a small volume (~20 µL) of glue.
7. 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.
8. 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.
9. Using a fine paintbrush (size 2), remove a wandering third instar from the food vial and place it onto the elastomer-coated cover glass.
10. Fill the glass micropipette tip with a small volume of glue using negative air pressure generated by the mouth with attachment (step 1.5).
11. 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.
12. Repeat this procedure with the larva's posterior end, ensuring the animal is stretched taut between the two glue attachments.
13. Using scissors (blades 3 mm), make a horizontal cut (~1 mm) at the posterior and a vertical cut all along the dorsal midline.
14. Using fine forceps (#5), gently remove the dorsal trachea, gut, fat body, and other internal organs covering the musculature.
15. Repeat the gluing procedure for the four body wall flaps; gently stretch the body wall horizontally and vertically.
16. Lift the ventral nerve cord (VNC) using forceps, carefully cut the motor nerves with the scissors, and then completely remove the VNC.
17. To stop SV cycling, replace the dissection saline with Ca²⁺-free saline (the same as the above dissection saline without the CaCl₂).

2. Electrical Stimulation FM Dye Loading

1. Prepare a suction pipette using the microelectrode puller to obtain the required taper and tip size.
2. Fire-polish the microelectrode tip with a micro-forge until a single motor nerve can be sucked up with a tight fit.
3. Slide the suction pipette onto the electrode holder on a micromanipulator and attach it to the long flexible plastic tube and a syringe.
4. Set stimulator parameters (e.g., 15 V, 20 Hz frequency, 20 ms duration, and time of 5 min or 1 min.
5. Replace the Ca²⁺-free saline in the larval preparation with the above FM1-43 saline (4 µM; 1 mM CaCl₂) in the electrophysiology rig.
6. Put the preparation on the microscope stage and raise the stage until the larva and suction pipette are focused (40X water immersion objective).
7. Suck up a loop of cut motor nerve innervating the selected hemisegment with negative air pressure generated by the syringe into the suction electrode.
8. Test the suction electrode function with a short burst of stimulation while visually monitoring for muscle contraction in the selected hemisegment.
9. Stimulate the motor nerve using selected parameters (step 2.4) to drive SV endocytosis and FM1-43 dye uptake.
10. Wash quickly with Ca²⁺-free saline (5x for 1 min) to remove the FM1-43 dye solution completely.
11. Maintain the larval preparation in fresh Ca²⁺-free saline for immediate imaging using the confocal imaging protocol from above.
12. Take careful note of the NMJ image (segment, side, and muscle) to ensure access to the same NMJ after FM dye unloading.

資料

Name	Company	Catalog Number	Comments
SylGard 184 Silicone Elastomer Kit	Fisher Scientific	NC9644388	To put on cover glass for dissections
Microscope Cover Glass 22x22-1	Fisherbrand	12-542-B	To put SylGard on for dissections
Aluminum Top Hot Plate Type 2200	Thermolyne	HPA2235M	To cure the SylGard
Plexi glass dissection chamber	N/A	N/A	Handmade
Borosilicate Glass Capillaries	WPI	1B100F-4	To make suction and glue micropipettes
Laser-Based Micropipette Puller	Sutter Instrument	P-2000	To make suction and glue micropipettes
Tygon E-3603 Laboratory Tubing	Component Supply Co.	TET-031A	For mouth and suction pipette
P2 pipette tip	USA Scientific	1111-3700	For mouth pipette
0.6-mL Eppendorf tube cap	Fisher Scientific	05-408-120	Used to put glue in for dissection
Vetbond 3M	WPI	vetbond	Glue used for dissections
Potassium Chloride	Fisher Scientific	P-217	Forsaline
Sodium Chloride	Millipore Sigma	S5886	For saline
Magnesium Chloride	Fisher Scientific	M35-500	For saline
Calcium Chloride Dihydrate	Millipore Sigma	C7902	For saline
Sucrose	Fisher Scientific	S5-3	For saline
HEPES	Millipore Sigma	H3375	For saline
HRP:Alexa Fluor 647	Jackson ImmunoResearch	123-605-021	To label neuronal membranes
Paintbrush	Winsor & Newton	94376864793	To manipulate the larvae
Dumont Dumostar Tweezers #5	WPI	500233	Used during dissection
7 cm McPherson-Vannas Microscissors (blades 3 mm)	WPI	14177	Used during dissection
FM1-43	Fisher Scientific	T35356	Fluorescent styryl dye
Digital Timer	VWR	62344-641	For timing FM dye load/unload
Micro-Forge	WPI	MF200	To fire polish glass micropipettes
20mL Syringe Slip Tip	BD	301625	To suck up the axon for electrical stimulation.
Micro Manipulator (magnetic base)	Narishige	MMN-9	To control the suction electrode for electrical stimulation
Stimulator	Grass	S48	To control the LED and electrical stimulation
Zeiss Axioskop Microscope	Zeiss		Used during electrical stimulation.
40X Achroplan Water Immersion Objective	Zeiss		Used during electrical stimulation and confocal imaging
Zeiss Stemi Microscope with camera port	Zeiss	2000-C	Used during channelrhodopsin stimulation
T-Cube LED Driver	ThorLabs	LEDD1B	To control the LED
LED Power Supply	Cincon Electronics Co.	TR15RA150	To power the LED
Optical Power and Energy Meter	ThorLabs	PM100D	To measure LED intensity