2P/3P imaging preparations

All animals used for imaging experiments were male flies with indicated genotypes kept at 25°C incubators and maintained on conventional cornmeal-agar-molasses medium. Flies used for chronic functional experiments were 2–7 days old, and flies used for short-term functional experiments were 1–4 days old. To perform through-cuticle brain imaging, flies were first head fixed in a 40 mm weigh dish (VWR#76299–236) with a hole made with forceps. A drop of UV curable resin (Liquid plastic welder, Bondic) was applied to the head and thorax, which was then cured with blue light (~470 nm) and fused to a cover glass. The fly antennae are ensured to be fully exposed after curing. Fly proboscises were immobilized with blue light curable resin to minimize head motion caused by muscle contractions. Video 1 explains the imaging preparation.

The dorsal head air sacs were repositioned to the posterior most portion of the head. This was done by deeply anesthetizing the flies on ice for ~5 min. The flies were placed into a modified pipette, allowing their head to stick out of the tip. Dental wax was wrapped around the head stabilizing it to the pipette. A sharpened glass capillary held in a micromanipulator was used to make a small incision just medial to the eye on the dorsal posterior area of the fly’s head. A sharpened tungsten needle curved into a micro hook, held in a micro manipulator was inserted into the incision, and run just under the cuticle to hook the dorsal air sac. The hook was pulled to the rear of the head, bringing the air sacs with it. The hook was then manipulated to release the air sac. The procedure was repeated on the other side. The incisions were closed using a very small amount of UV curable resin over the incision site. The flies were then allowed to recover for 24 hr at 25 degrees on conventional food. Flies used in Figure 4, Figure 2—figure supplement 1, Figure 3—figure supplement 2, Figure 3—figure supplement 4 went through the air sac removal surgery.

Flies were anesthetized on ice for ~1 min then placed into a holder made from a 0.02 mm thick carbon steel sheet with a small hole cut to allow the dorsal thorax and dorsal part of the head to protrude through the sheet. The flies were fixed to the imaging chamber using a UV curable resin (Bondic) around the perimeter of the hole in the sheet. Approximately 500 μl of adult artificial hemolymph was placed on the imaging chamber and the head cuticle was removed using a 20-gauge needle to cut along the medial perimeter of the eyes, the dorsal posterior extent of the head between the eyes, and just posterior to the antenna along the front of the head. Any air sacs, fat bodies, or trachea on top of the exposed brain were removed with fine forceps.