Analyzing Synaptic Vesicle Recycling and Fluorescent Dye Uptake in Drosophila Larvae

Abstract

Source: Kopke, D. L., et al., FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation. J. Vis. Exp. (2018).

This video demonstrates a protocol to study synaptic vesicle recycling at neuromuscular junctions in channelrhodopsin-expressing Drosophila larvae using a fluorescent dye. Blue light activates the channelrhodopsins, causing calcium influx and synaptic vesicle exocytosis. The dye integrates into the neuronal membranes and is internalized during endocytosis, resulting in enhanced fluorescence. Further blue light stimulation induces exocytosis and dye release, which is shown by reduced fluorescence.

Protocol

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 (Table of Materials).
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 plexiglass dissection chamber (Figure 1, bottom) in preparation for the larval dissection.
4. Prepare the glue pipettes from borosilicate glass capillary using a standard microelectrode puller to obtai....

Results

Figure 1: Flowchart of FM1-43 dye loading protocol at the Drosophila NMJ. The larval glue dissection produces a flattened neuromusculature preparation, with the ventral nerve cord (VNC) projecting segmental nerves from the ventral midline (Vm) to t.......

Materials

List of materials used in this article

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
HRP:Alexa Fluor 647	Jackson ImmunoResearch	123-605-021	To label neuronal membranes
HEPES	Millipore Sigma	H3375	For saline
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
LSM 510 META laser-scanning confocal microscope	Zeiss		For imaging the fluorescent markers
Zen 2009 SP2 version 6.0	Zeiss		Software for imaging on confocal
HeNe 633nm laser	Lasos	To excite HRP:647 during imaging
Argon 488nm laser	Lasos		To excite the FM dye during imaging
Micro-Forge	WPI	MF200	To fire polish glass micropipettes
Stimulator	Grass	S48	To control the LED and electrical stimulation
All-trans Retinal	Millipore Sigma	R2500	Essential co-factor for ChR2
Zeiss Stemi Microscope with camera port	Zeiss	2000-C	Used during channelrhodopsin stimulation
LED 470nm	ThorLabs	M470L2	Used for ChR activation
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
40X Achroplan Water Immersion Objective	Zeiss	Used during electrical stimulation and confocal imaging