Department of Biology, Brandeis
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Hornstein, N. J., Pulver, S. R., Griffith, L. C. Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions. J. Vis. Exp. (25), e1133, doi:10.3791/1133 (2009).
Part 1: Animal care and genetic crosses
Part 2: Rig setup
Part 3: Dissection
Part 4: Muscle recording and blue light stimulation
Figure 1A shows a schematic of the recording setup and filleted preparation. Figure 1B shows typical EJP evoked by short light pulses. EJP amplitude shown is summed amplitude from two motor neurons known to both innervate M6. Lower light intensities only activated one motor unit (data not shown).
Figure 1: A) General schematic of an intracellular recording rig and with blue LED. Brain (Br) is removed to inhibit rhythmic activity in the ventral ganglion (VG). ChR2 is expressed in motor neurons using the GAL4-UAS system. B) Intracellular recording from a M6 muscle. 40 ms blue light pulses (at 127 µW / mm2) reliably evoke large synaptic potentials (asterisks) in M6.
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 muscle cell or in an unhealthy cell.
We have no conflicts of interest to disclose.
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.
|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|
|Dissecting Scope||Leica Microsystems||www.leica-microsystems.com|
|Light Source||Dolan-Jenner Industries||41446-062||www.dolan-jenner.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
1. 200 mA Buck Puck
2. Blue LED
3. Insulated wire
4. Circuit bread board
|LED Heat Sink||Thorlabs Inc.||http://www.thorlabs.com/|
|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
NaHCO3: 10 Trehalose: 5 HEPES
|Micro-Dissection Tools||Fine Science Tools||www.finescience.com|