This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.
A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions
In This Article
Summary
Abstract
The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.
Protocol
Part 1: Animal care and genetic crosses
- Maintain UAS-ChR2 and OK371-GAL4 fly lines in separate bottles containing standard fly media 8.
- Collect virgins of OK371-GAL4 fly lines and males of UAS-ChR2 lines.
- Put both males and females in a vial containing standard fly media mixed with 1 mM all-trans retinal (ATR). ATR food should be made by first melting regular fly media in a microwave for ~1 minute. Once melted, allow to cool for about 30 seconds to one minute and then add 100 μl of 100 mM ATR in 100% ETOH for every 10 ml of fly media. Place vials on ice, then store in a dark area at 4°C.
- Let f....
Access restricted. Please log in or start a trial to view this content.
Discussion
Critical steps involve both the initial dissection and the entering of muscle cells. If nerves are cut or muscle is damaged during the initial dissection it is difficult to continue the rest of the experiment. During dissection, one must be very careful to angle dissecting scissors upwards as much as possible during dorsal incision. During the second crucial step, entering a muscle cell, one must watch for a hyperpolarization past ~30 mV. Values above -30 mV indicate that the electrode is either not properly within a mus.......
Access restricted. Please log in or start a trial to view this content.
Disclosures
We have no conflicts of interest to disclose.
Acknowledgements
This work was supported by National Institutes of Health grants RO1GM-33205 and MH-067284 to L.C. Griffith and by a Brandeis University summer undergraduate research scholarship to N. J. Hornstein. Preliminary experiments for this technique were performed at the Marine Biological Laboratory as part of the 2008 Neural Systems and Behavior summer course (NIMH grant: R25 MH059472) in Woods Hole, MA.
....Access restricted. Please log in or start a trial to view this content.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Sylgard | Ellsworth Adhesives | 4019862 | www.ellsworth.com |
| Minutens Pins | Fine Science Tools | 26002-10 | www.finescience.com |
| Dissecting Dish | Fisher Scientific | www.fishersci.com | |
| Neuroprobe Intracellular Amplifier and Head Stage | A-M Systems | 680100 | www.a-msystems.com |
| Powerlab 4/30 data acquisition system |  ADInstruments | www.adinstruments.com | |
| Grass stimulator | Grass Technologies | www.grasstechnologies.com | |
| Desktop Computer | Dell | www.dell.com | |
| Dissecting Scope | Leica Microsystems | www.leica-microsystems.com | |
| Light Source | Dolan-Jenner Industries | 41446-062 | www.dolan-jenner.com |
| Fly Media | |||
| All-Trans-Retinal | Sigma-Aldrich | 116-31-4 | www.sigmaaldrich.com |
| OK-371 Gal4 Flies | Bloomington Stock center | ||
| UAS-ChR2 Flies | Fiala lab, Griffith lab | ||
| LED controller circuit | Built in Griffith lab | http://www.ledsupply.com http://www.futureelectronics.com Composed of: 1. 200 mA Buck Puck 2. Blue LED 3. Insulated wire 4. Circuit bread board | |
| LED Heat Sink | Thorlabs Inc. | http://www.thorlabs.com/ | |
| Air Table | TMC | http://www.techmfg.com/products/accessories/intro3.html | |
| Faraday Cage | Built in Griffith lab | ||
| Leica Leitz M Micro-Manipulator | Leica Microsystems | ACS01 | www.leica-microsystems.com |
| Electrode Holder | Axon Instruments | www.axon.com | |
| Borosilicate Glass | FHC, Inc. | www.fh-co.com/p14-15.pdf | |
| Electrode Puller | Sutter Instrument Co. | www.sutter.com | |
| HL 3.1 Saline with 0.8mM Ca2+ | Contents (mM): NaCl:70 KCl:5 CaCl2: 0.8 MgCl2:4Sucrose:115 NaHCO3: 10 Trehalose: 5 HEPES | ||
| Micro-Dissection Tools | Fine Science Tools | www.finescience.com |
References
- Keshishian, H., Broadie, K., Chiba, A., Bate, M. The Drosophila neuromuscular junction: a model system for studying synaptic development and function. Annu Rev Neurosci. 19, 545(1996).
- Collins, C. A., DiAntonio, A. Synaptic development: insights from Dros....
Access restricted. Please log in or start a trial to view this content.
Reprints and Permissions
