Materials and Reagents

1. Sterile disposable filter (0.2 μm pore size, aPES membrane, 75 mm diameter) [such as Nalgene^TM Rapid-Flow^TM Sterile Disposable Filter Units (Thermo Fisher Scientific)]
   
2. 1 borosilicate glass capillary (OD 1.0 mm, ID 0.58 mm) (Harvard Apparatus)
   
3. 3 silver filaments (1 mm diameter, 10 cm length) [such as Electrode AG/AGCL 1 mm DIA (WPI)]
   
4. 1 ml/2 ml Syringe + 1 ml/2 ml Syringe with elongated tip [such as MEDI-PLUS 1ml without needle (KDL Medical Product Company)]
   
5. Syringe filter (0.22 μm pore size, suitable for a 4 mm syringe tip) with PVDF membrane [such as Millex-GV Syringe Driven Filter Unit (Merck Millipore Corporation)]
   
6. White-eyed Drosophila flies (available for purchase at Bloomington Drosophila Stock Center)
   Note: Red-eyed Drosophila flies are also suitable for ERG recordings, but a PDA cannot be induced in these flies.
   
7. Homemade low temperature melting wax composed of mixture of paraffin and bee wax
   
8. Redux cream for electrocardiography [such as Redux Electrolyte Crème (Parker Laboratories)]
   
9. Ringer’s solution (see Recipes)
   

Equipment

1. Horizontal Micropipette puller [such as programmable Flaming/Brown type micropipette puller (Sutter Instrument Company, model: P-97 )] with platinum filament. Less expensive pullers [such as Narishige vertical puller (NARISHIGE Group, model: PP-830 )] can also be used
   
2. Pulsed gas flow anesthesia system with injector [such as the Sleeper system (Inject+Matic Sleeper)] connected to CO₂ tank
3. Fly stand [such as Alnico Shallow Pot Magnet (Eclipse Magnetics)] (Figure 1, #2, Figure 2, #2 and Video 1)
   
4. Fine tweezers [such as tweezers (Biologie, model: number 5 )] and fine paint brush [such as paint brush (Rekab, model: series 40, number 3 )]
   
5. Homemade Wax filament heater + soldering iron composed of a platinum-iridium (0.25 mm diameter) filament and a holder (Figure 3)
   
6. Dissecting Stereoscopic zoom Microscope [such as Nikon Stereozoom Microscope (Nikon Corporation, model: SMZ-2 )]
   
7.  “Cold” illuminator [such as KL 1500 LCD (SCHOTT North America, model: KL 1500 LCD )] with heat and red filters [such as KG3 and RG620, respectively (SCHOTT North America, models: KG3 and RG620 )]
   
8. Computer with electric signal processor (such as Clampex) and data analysis program (such as Clampfit)
   
9. Stereo-microscope with at least 4 different magnification settings [such as the Wild M5 (Leica Microsystems) with 6, 12, 25 and 50 magnification settings] (see Figure 1, #3 and Video 1)
10. Vibration Isolated Table
    
11. Dark Faraday cage covered in black cloth (Figure 1, #6 and Video 1).
    
12. On/Off magnet block to provide convenient placement of the fly stand [such as complete Magnetic Base (Eclipse Magnetics, model: E905 )] (Figure 2, #5 and Video 1)
    
13. 4 triple axis coarse mechanical micromanipulators [such as the Three-Axis Compact Coarse Micromanipulator (Tritech Research, Narishige, model: M-2 )] (Figure 1, #4, Figure 2, #3 and Video 1)
    
14. 1 triple axis, fine, very stable mechanical micromanipulator for holding the recording electrode [such as the Leitz Mechanical Micromanipulator (Leica Microsystems)] (Figure 1, #8)
    
15. Light detector phototransistor for light monitoring (to be fixed on one of the micromanipulators) (Figure 1, #5, Figure 2, #4 and Video 1).
    
16. Shutter system [such as LS2 2 mm Uni-stable Shutters (Uniblitz, Vincent Associates^®)] + shutter driver [such as VCM-D1 Single Channel Uni-stable (Uniblitz, Vincent Associates^®)]
    
17. High-pressure ozone-free 75 W Xenon lamp (operating on 50 W) + Schott KG3 heat filter + 2 condenser lenses + fiber optic (3 mm diameter, 1.3 m long) conducting light into the Faraday cage for light stimulation
    
18. Lamp power supply (such as PTI LPS-220)
    
19. Data acquisition system [such as Digidata 1550 digitizer (Molecular Devices, model: Digidata 1550 digitizer )]
    
20. 2 glass electrode holders suitable for capillary O.D. 1 mm (Figure 1, #1, Figure 2, #1 and Video 1)
    
21. Microelectrode preamplifier system with head-stage impedance tester, at least x10 amplification [such as Extracellular Preamplifier Current Pump System (Dagan, model: 2400A )] (Figure 1, #7 and Video 1)
    
22. Pulse generator [such as Master 8 and Master 9 (A.M.P.I, models: Master 8 and Master 9 )]
    
23. Neutral Density (ND) filters [such as ND filters (Balzer) and color filters including Red (SCHOTT North America, model: RG610 ), Orange (SCHOTT North America, model: OG590 ) and Blue (SCHOTT North America, model: BG63 )]
    
    
    Figure 1. An overview of the ERG setup. #1-electrode holder, #2-fly stand, #3- stereo-microscope, #4-micromanipulator, #5-light detector phototransistor, #6- Faraday cage, #7-amplifier head-stage, #8-Leica-Leitz Mechanical Micromanipulator.
    
    
    Figure 2. A magnified view of the ERG setup. #1-electrode holder, #2-fly stand, #3- micromanipulator, #4-light detector probe, #5-magnet block.
    
    
    Figure 3. A homemade instrument for immobilizing the living fly. A platinum-iridium (0.25 mm diameter) filament is electrically connected to a power supply with a controlled current output. The filament is attached via insolating plastic to a copper rod holder (heater and soldering iron).
    
    
    
    Video 1. A general overview of the ERG set

Procedure