Part 1: Animal care and genetic crosses
Part 2: Rig setup
Part 3: Dissection
Part 4: Muscle recording and blue light stimulation
Representative Results:
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.
Material Name | Type | Company | Catalogue Number | Comment |
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 | AD instruments | www.adinstruments.com | ||
Grass stimulator | Grass instruments | www.grasstechnologies.com | ||
Desktop Computer | Dell | www.dell.com | ||
Dissecting Scope | Leica | www.leica-microsystems.com | ||
Light Source | Dolan-Jenner | 41446-062 | www.dolan-jenner.com | |
Fly Media | ||||
All-Trans-Retinal | Sigma-Aldrich | 116-31-4 | www.sigmaaldrich.com | |
OK-371 Gal4 Flies | Aberle lab, Griffith lab, 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 |
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LED Heat Sink | Thor Labs | 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 Leitz | ACS01 | www.leica-microsystems.com | |
Electrode Holder | Axon Instruments | www.axon.com | ||
Borosilicate Glass | FHC | www.fh-co.com/p14-15.pdf | ||
Electrode Puller | Sutter Instruments | 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 |
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Micro-Dissection Tools | Fine Science Tools | www.finescience.com |