Name: channelrhodopsin2 mediated stimulation of synaptic potentials at drosophila neuromuscular junctions
Uploaded: 2009-03-16T00:00:00
Description: Watch this Scientific Journal Video about Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions at JoVE.com

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.

Part 1: Animal care and genetic crosses
<ol>
<li>Maintain UAS-ChR2 and OK371-GAL4 fly lines in separate bottles containing standard fly media 8.</li>
<li>Collect virgins of OK371-GAL4 fly lines and males of UAS-ChR2 lines.</li>
<li>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 mi...

<ol>
	<li>Keshishian, H., Broadie, K., Chiba, A., Bate, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+neuromuscular+junction:+a+model+system+for+studying+synaptic+development+and+function.">The Drosophila neuromuscular junction: a model system for studying synaptic development and function.</a> Annu Rev Neurosci. 19, 545 (1996).</li><li>Collins, C. A., DiAntonio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+development:+insights+from+Drosophila.">Synaptic development: insights from Drosophila.</a> Curr Opin Neurobiol. 17 (1), 35 (2007).</li><li>Lagow, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+a+hydrophobic+layer+by+a+point+mutation+in+syntaxin+1A+regulates+the+rate+of+synaptic+vesicle+fusion.">Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.</a> PLoS Biol. 5 (4), e72 (2007).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr Biol. 15 (24), 2279 (2005).</li><li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc Natl Acad Sci U S A. 100 (24), 13940 (2003).</li><li>Schroll, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+activation+of+distinct+modulatory+neurons+triggers+appetitive+or+aversive+learning+in+Drosophila+larvae.">Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae.</a> Curr Biol. 16 (17), 1741 (2006).</li><li>Lin, D. M., Auld, V. J., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+neuronal+cell+ablation+in+the+Drosophila+embryo:+pathfinding+by+follower+growth+cones+in+the+absence+of+pioneers.">Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers.</a> Neuron. 14 (4), 707 (1995).</li><li>Ralph Greenspan, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly Pushing: The Theory and Practice of Drosophila Genetics, 2nd ed. , (2004).</li></ol>

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...

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comment</td>
</tr>	
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Ellsworth adhesives </td>
<td>4019862</td>
<td><a href="http://www.ellsworth.com" target="_blank">www.ellsworth.com</a></td>
</tr>
<tr>
<td>Minutens Pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td><a href="http://www.finescience.com" target="_blank">www.finescience.com</a></td>
</tr>
<tr>
<td>Dissecting Dish</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td><a href="http://www.fishersci.com" target="_blank">www.fishersci.com</a></td>
</tr>
<tr>
<td>Neuroprobe Intracellular Amplifier and Head Stage</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>680100</td>
<td><a href="http://www.a-msystems.com" target="_blank">www.a-msystems.com</a></td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">Powerlab 4/30 data acquisition system</a></td>
<td>&nbsp;</td>
<td><a href="http://www.adinstruments.com" target="_blank">AD instruments</a></td>
<td>&nbsp;</td>
<td><a href="http://www.adinstruments.com" target="_blank">www.adinstruments.com</a></td>
</tr>
<tr>
<td>Grass stimulator</td>
<td>&nbsp;</td>
<td>Grass instruments</td>
<td>&nbsp;</td>
<td><a href="http://www.grasstechnologies.com" target="_blank">www.grasstechnologies.com</a></td>
</tr>
<tr>
<td>Desktop Computer</td>
<td>&nbsp;</td>
<td>Dell</td>
<td>&nbsp;</td>
<td><a href="http://www.dell.com" target="_blank">www.dell.com</a></td>
</tr>
<tr>
<td>Dissecting Scope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td><a href="http://www.leica-microsystems.com" target="_blank">www.leica-microsystems.com</a></td>
</tr>
<tr>
<td>Light Source</td>
<td>&nbsp;</td>
<td>Dolan-Jenner</td>
<td>41446-062</td>
<td><a href="http://www.dolan-jenner.com" target="_blank">www.dolan-jenner.com</a></td>
</tr>
<tr>
<td>Fly Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>All-Trans-Retinal</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>116-31-4</td>
<td><a href="http://www.sigmaaldrich.com" target="_blank">www.sigmaaldrich.com</a></td>
</tr>
<tr>
<td>OK-371 Gal4 Flies</td>
<td>&nbsp;</td>
<td>Aberle lab, Griffith lab, Bloomington stock center </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>UAS-ChR2 Flies</td>
<td>&nbsp;</td>
<td>Fiala lab, Griffith lab </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>LED controller circuit</td>
<td>&nbsp;</td>
<td>Built in Griffith lab</td>
<td>&nbsp;</td>
<td><a href="http://www.ledsupply.com" target="_blank">http://www.ledsupply.com</a> 
<a href="http://www.futureelectronics.com" target="_blank">http://www.futureelectronics.com</a> 
Composed of: 
1. 200 mA Buck Puck 
2. Blue LED 
3. Insulated wire 
4. Circuit bread board </td>
</tr>
<tr>
<td>LED Heat Sink</td>
<td>&nbsp;</td>
<td>Thor Labs</td>
<td>&nbsp;</td>
<td><a href="http://www.thorlabs.com/" target="_blank">http://www.thorlabs.com/</a></td>
</tr>
<tr>
<td>Air Table</td>
<td>&nbsp;</td>
<td>TMC</td>
<td>&nbsp;</td>
<td><a href="http://www.techmfg.com/products/accessories/intro3.html" target="_blank">http://www.techmfg.com/products/accessories/intro3.html</a></td>
</tr>
<tr>
<td>Faraday Cage</td>
<td>&nbsp;</td>
<td>Built in Griffith lab</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Leica Leitz M Micro-Manipulator</td>
<td>&nbsp;</td>
<td>Leica Leitz</td>
<td>ACS01</td>
<td><a href="http://www.leica-microsystems.com" target="_blank">www.leica-microsystems.com</a></td>
</tr>
<tr>
<td>Electrode Holder</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>&nbsp;</td>
<td><a href="http://www.axon.com" target="_blank">www.axon.com</a></td>
</tr>
<tr>
<td>Borosilicate Glass</td>
<td>&nbsp;</td>
<td>FHC</td>
<td>&nbsp;</td>
<td><a href="http://www.fh-co.com/p14-15.pdf" target="_blank">www.fh-co.com/p14-15.pdf</a></td>
</tr>
<tr>
<td>Electrode Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td><a href="http://www.sutter.com" target="_blank">www.sutter.com</a></td>
</tr>
<tr>
<td>HL 3.1 Saline with 0.8mM Ca2+</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Contents (mM): 
NaCl:70 KCl:5 CaCl2: 0.8 MgCl2:4Sucrose:115 
NaHCO3: 10 Trehalose: 5 HEPES
</td>
</tr>
<tr>
<td>Micro-Dissection Tools</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td><a href="http://www.finescience.com" target="_blank">www.finescience.com</a></td>
</tr>		
</tbody>
</table>

channelrhodopsin2 mediated stimulation of synaptic potentials at drosophila neuromuscular junctions

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.

Watch this Scientific Journal Video about Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions at JoVE.com

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

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.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

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