Flies were anesthetized using ice and anchored to a wax support ventral side down. Two stimulating electrodes made of tungsten connected to a stimulator (Constant Voltage Isolated Stimulator, model DS2A-Mk.II, Digitimer) were placed into both eyes to activate the giant fiber system (GFS). Similarly, two tungsten stimulating electrodes were also placed in the middle of femur segment of the right (ipsilateral) or left (contralateral) leg to activate nociceptive GFS escape through the leg. For GFS through the eye, flies were given 20 single stimuli with a maximum stimulation intensity smaller than 15 V. For leg stimulation nociceptive escape, the maximum stimulation intensity was less than 60 V. For all experiments, stimulation duration was kept constantly at 10 μs. A tungsten ground electrode was placed into the fly abdomen. A tungsten recording electrode, sharpened in sodium hydroxide 5M (with a bench-top power supply, PSU 130-LASCAR), was placed into the left backside of the fly at the DLM fiber to record the PSPs. PSPs of at least nine flies for each group were recorded using Microelectrode AC Amplifier, Model 1800(A-M System) filtered at 0.5 kHz and digitized at 1 kHz. PSPs were analyzed using AxoGraph software (AxoGraph Scientific, Berkeley, CA). To determine whether the response measured by stimulating the leg was mediated by the CNS, a similar setup for recordings was used, with the head of the fly removed. Mann-Whitney rank sum test was used to determine differences in response latency and duration.

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