Flies were raised at 25°C on a standard cornmeal/molasses diet in 40 ml vials, under a 12:12 hr dark:light cycle. Imaging experiments were performed between ZT0–14, although time of day was not a factor in our experimental design or analysis. We imaged 1–7 day old female flies expressing either UAS-GCaMP6s (Chen et al., 2013) for dendritic regions or UAS-sytGCaMP6s (Cohn et al., 2015) for axon terminals, together with UAS-tdTomato (Shaner et al., 2004) for image registration (for genotypes see Appendix 2—table 1.). Flies were cold anesthetized and mounted on a custom fly holder, modified from Weir et al., 2016, with the head pitched forward so that its posterior surface was approximately horizontal (Figure 1—figure supplement 1A). Surfaces of the fly holder visible to the fly were covered in matte white paint (Citadel) and roughened to reduce confounding reflected polarized light cues (Foster et al., 2018). We fixed the fly to the holder using UV-curing glue (Fotoplast) around the posterior-dorsal cuticle of the head and at the base of the wings on either side of the thorax. To reduce movement of the brain, we fixed the legs, abdomen, and proboscis with beeswax. We used forceps to remove the cuticle and air-sacs above the optic lobe or central brain, depending on the recording site, and cut muscle 1 (Demerec, 1950) to reduce movement. Physiological saline (103 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 4 mM MgCl2, 26 mM NaHCO3, 1 mM NaH2PO4, 10 mM trehalose, 10 mM glucose, 5 mM TES, 2 mM sucrose) was perfused continuously over the brain at 1.5 ml/min via a gravity drip system and the bath was maintained at 22°C for the duration of experiments by an inline solution heater/cooler (SC-20, Warner Instruments) connected to a temperature controller (TC-324, Warner Instruments).

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