Procedures for animal preparation were based on earlier methods (Turner et al., 2008 (link); Murthy and Turner, 2010 ). Briefly, flies were transferred to a glass tube and anesthetized on ice until movement ceased (about 15 s). A female fly was then gently inserted into a rectangular hole (about 0.77 by 1.5 mm) cut into a piece of aluminum foil glued to the underside of the recording platform. The fly’s head was tilted forward to provide access to the posterior surface of the brain where the KC cell bodies are located. The olfactory organs point downwards in this preparation, allowing airborne odor delivery (Figure 1A ). The fly was fixed in place using fast-drying epoxy (Devcon 5-Minute Epoxy).
The bath surrounding the head capsule was continuously perfused with oxygenated saline (Wilson et al., 2004 (link)) and the cuticle at the back of the head was dissected away using sharpened forceps. We sometimes found it necessary to minimize brain motion by removing the pulsatile organ at the neck (care was taken to avoid damaging the gut) and the proboscis retractor muscles, which pass over the caudal aspect of the optic lobes. Air sacs and fat deposits occluding the MB were cleared from the brain’s surface. We did not purposefully attempt to remove the peri-neural sheath, as is needed for electrophysiological experiments. Flies remained healthy and active throughout the experiment, as evidenced by abundant voluntary leg movements. Many preparations were discarded due to excessive brain motion that prevented us from tracking individual neurons throughout the imaging session.
The bath surrounding the head capsule was continuously perfused with oxygenated saline (Wilson et al., 2004 (link)) and the cuticle at the back of the head was dissected away using sharpened forceps. We sometimes found it necessary to minimize brain motion by removing the pulsatile organ at the neck (care was taken to avoid damaging the gut) and the proboscis retractor muscles, which pass over the caudal aspect of the optic lobes. Air sacs and fat deposits occluding the MB were cleared from the brain’s surface. We did not purposefully attempt to remove the peri-neural sheath, as is needed for electrophysiological experiments. Flies remained healthy and active throughout the experiment, as evidenced by abundant voluntary leg movements. Many preparations were discarded due to excessive brain motion that prevented us from tracking individual neurons throughout the imaging session.
