Dr Mikko Juusola: In vivo Drosophila preparation

For intracellular recordings a fly is mounted with its head protruding from the open tip of a conical holder, whose hollow copper core is shielded outside with a ceramic insulator. The fly is fixed to the copper tip from its back with a mixture of beeswax and heat sink paste and the proboscis stretched to eliminate vergence eye movements. This leaves the abdomen intact for ventilation, allowing the fly to survive for up to two days. A hole, the size of a few ommatidia, is cut manually in the dorsal cornea with a sharp razor edge (see the small white dot in the eye; Fig. 1), and sealed with vaseline. The holder is mounted on top of a ceramic recording platform, where its copper core fits tightly to a Peltier element with heat sink paste. Underneath the Peltier element, inside the ceramic cylinder, a large copper rod functions as a heat sink.
Recording temperature is controlled The fly's body temperature is measured with a thermocouple (the green-white cable in Fig. 2) mounted in the copper core next to the fly and can be changed in seconds from 10 to 35oC by a custom-designed feed-back controlled power source driving the Peltier temperature (Juusola & Hardie, 2001a,b).
Intracellular recordings Membrane potentials are recorded with the amplifier operating in the compensated current-clamp (CC) or balanced bridge mode. The recordings are carried out from green-sensitive R1-6 photoreceptor cells, which are the dominant input to the Drosophila visual system (Strausfeld, 1989), and from first-order interneurons (LMCs). Because we mainly use red-eyed flies, instead of the commonly used white-eyed mutations, which lack all the screening pigments, and provide the light stimuli through a small point source, the effects of extracellular field potentials on the photoreceptor recordings are minimal. The maximum extracellular field potentials evoked by saturating light flashes measured in the retina are typically < 5 mV. A successful photoreceptor penetration is seen as a 60-75 mV drop in the electrode potential and vigorous voltage responses (50-75 mV) to light pulses. The average input resistance of the photoreceptors in the dark is ~400 MW and in fully light adapted conditions, which depolarized the membrane 25-40 mV above the resting potential ~250 MW. These values are much higher than those previously reported from intracellular recordings (Wu and Pak, 1978; Johnson and Pak, 1986), but similar to those measured using patch clamp electrodes (Hevers and Hardie, 1995).	We believe that the correct way to study photoreceptor dynamics is to stimulate in vivo preparation with moving images, which are as natural as possible. Therefore we have modified our intracellular set-ups to allow natural moving images to be presented to the Drosophila eye with adequate frame rates. This presents us an opportunity to study and model photoreceptor signalling and experience dependent plasticity in naturalistic situations that may be very different from the response properties obtained in typical laboratory conditions.