Flies were raised at 25°C on a standard cornmeal/molasses diet in bottles or vials, under a 12:12 hr dark:light cycle, and we dissected 3–4 day old female flies (for genotypes see Appendix 2—table 1.). For trans-Tango analyses we dissected 17–18 day old female flies raised at 18°C (Talay et al., 2017).

Immunohistochemical staining was conducted as previously described (Omoto et al., 2017; Omoto et al., 2018). Briefly, brains were dissected in phosphate buffered saline (PBS) and fixed in ice–cold 4% EM–grade paraformaldehyde in PBS for 2.5 hr. They were subsequently washed for 4 × 15 min in ice–cold PBS followed by cold ethanol dehydration (5 min washes in 5, 10, 20, 50, 70, 100% EtOH). After incubation for approximately 12 hr in 100% EtOH at −20°C, brains were subjected to a rehydration procedure with EtOH in the reverse sequence. Brains were then washed for 4 × 15 min in ice–cold PBS and 4 × 15 min in ice-cold 0.3% PBT (PBS with 0.3% Triton X–100), followed by 4 × 15 min in room temperature (RT) 0.3% PBT. They were then incubated in blocking buffer (10% Normal Goat Serum in 0.3% PBT) for 30 min at RT. Following this, the brains were incubated in primary antibodies, diluted in blocking buffer at 4°C for approximately three days. They were subsequently washed 4 × 15 min in RT 0.3% PBT and placed in secondary antibodies diluted in blocking buffer at 4°C for approximately 3 days. They were finally washed 4 × 15 min in RT 0.3% PBT and placed in VectaShield (Vector Laboratories) at 4°C overnight before imaging. trans–Tango and GRASP analyses required separate staining of neuropil and respective fluorophores due to different incubation times.

Processed brains were mounted on glass slides and imaged in either the anteroposterior (A–P) or dorsoventral (D–V) axis with a Zeiss LSM 700 Imager M2 using Zen 2009 (Carl Zeiss), with a 40× oil objective. Images were processed using Image J (FIJI) (Schindelin et al., 2012). Image stacks of the AOTU or EB were rotated slightly and interpolated to align the neuropil with the imaging plane. Background labeling was removed to improve visualization in some projections (Figure 2B,C, Figure 5A).

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