Electrophysiological Recording of Voltage Responses of Drosophila Retinal Photoreceptors to Light Stimuli

1. Recording from R1-R6 Photoreceptors

1. Always be grounded when operating the microelectrode amplifier (for example by touching the metal surface of the Faraday cage or antivibration table), as this precludes one from accidentally delivering a static charge to the head-stage, which could damage the circuitry.
2. Illuminate the fly preparation platform pole from above with two gooseneck light guides (Figure 1A) (with the cold light source inside the Faraday cage) so that the fly-holder can be placed on the pole in the preferred position under close visual control.
3. Mount the fly-holder (with the fly in it!) on the fly preparation platform pole. Rotate the fly-holder so that the fly's left eye is directly facing the investigator (Figure 1B).
4. Insert the blunt reference electrode gently through the fly's ocelli into the head capsule using a small coarse micromanipulator while observing the preparation through the stereomicroscope (Figure 1C). Do not push the electrode too deep, as this can damage the fly brain.
   1. Alternatively, insert the reference electrode into the back of the thorax. Always ensure that the fly appears healthy (moves its antennae), and its eyes are intact, not accidentally damaged. If the preparation looks less than immaculate, prepare a new fly for the experiments.
5. Drive the sharp recording microelectrode into the left eye through a petroleum jelly covered small opening. Use high magnification in the stereomicroscope and move the light guides and the focal plane so that the electrode tip location becomes apparent in 3D by its reflectance patterns.
   NOTE: Figure 1D shows how the fly's head should be positioned (with respect to the angle at which the recording microelectrode enters the eye) for photoreceptor recordings. Driving the electrode into the eye without breaking it is the most difficult phase of the experiment. If the electrode tip misses the small opening in the eye, hitting the cornea, it typically breaks.
6. Turn on the microelectrode amplifier once both electrodes are firmly inside the preparation, in electrical contact with the fly's body fluids.
7. Turn off the cold-light source (inside the Faraday cage) and unplug it from the mains. Connect its plug to the central ground to minimize ground-loop induced electrical noise and move the goose-neck light guides away so the Cardan-arm system can be freely moved around the fly. Switch off the room lights to ensure that the fly preparation is now in relative darkness.
8. Measure the resistance of the recording electrode in the eye. Use only recording electrodes in which resistance is 100 – 250 MΩ.
   NOTE: Achieving high-quality intracellular recordings by <70 MΩ electrode is virtually impossible. If the resistance is <80 MΩ, it is likely that the electrode tip is broken. In this case, switch off the amplifier and change the recording electrode.
9. Set the amplifier to current-clamp (CC) or bridge recording mode. Cancel out any arbitrary voltage difference between the recording and reference electrodes, as both are now resting in the electrically interconnected extracellular space, by setting the signal offset (recording voltage) to zero. Follow the signal offset changes using the amplifier's display readout or an oscilloscope screen.
10. Wait 2-3 min for the fly eye to dark-adapt.
11. Drive the recording electrode tip deeper into the eye with small 0.1 to 1-micron steps. Do this with an x-axis piezo-stepper of a remote-controlled micromanipulator or by gently rotating the fine-resolution knob of a manual manipulator.
12. Stimulate the fly eye with brief (1 – 10 msec) light flashes as the recording electrode is being advanced in the tissue.
    NOTE: If the recording electrode is positioned in the retina and the eye functions normally, each light flash will cause a brief and small drop in the voltage (0.2 – 5 mV hyperpolarization), called the electroretinogram (ERG). This change in the field potential of the extracellular space is caused by the retinal cells' collective response to light.