Sc 20

During two-photon imaging the brain was continuously perfused with fly extracellular saline at 1.5 ml/min via a computer-controlled system (VC-6, Warner Instruments). Bath temperature was kept at 20°C with an inline-solution heater (SC-20, Warner Instruments) and a temperature controller (TC-324, Warner Instruments). LC neurons were imaged at 920nm using a Ti:Sapphire pulse laser (Coherent, Santa Clara, CA) controlled by SlideBook (Version 6, 3i, Boulder, CO). We imaged with a 20x water-immersion objective (W Plan Apochromat, 1.0 DIC, Zeiss) with three layers of blue filter (Indigo, Rosco, No. 59) to reduce bleed-through from the LED arena to the photomultipliers. Single plane images were taken at 10 frames/s with each frame at a x-y pixel resolution ranging from 150×256 to 168×212 and 0.2 to 0.3 μm pixel spacing. To record population responses of a given LC type, GCaMP6f responses were recorded in the LC output glomeruli where all terminals merge together. To record activity of single LC neurons, GCaMP6f responses were measured from individual dendritic neurites in the lobula.

For temperature stimulation, preparations were continuously perfused with Ca²⁺-containing AHL (as described above). AHL was gravity fed through a 3-way valve (Lee company, part # LHDA1231315H) and flow rate was adjusted through a flow regulator. Following the valve, temperature was precisely regulated through 2 in-line solution heater/coolers (Warner, cat. # SC-20) in parallel with by a dual channel bipolar temperature controller (Warner Instruments, Cl-200A). Excess heat produced by each SC-20 Peltier was dissipated through a liquid cooling system (Koolance, Cat. # EXT-1055). To circumvent changes in resistivity and voltage offsets from changing the temperature of the bathing solution, the reference Ag-Cl pellet electrode was placed in an isolated well adjacent to the recording chamber (Warner Instruments, Cat. # RC-24N), filled with identical AHL and connected via a borosilicate capillary tube filled by 2% agar in 3 M KCl. The bath temperature was precisely recorded using a custom Type T thermocouple with 1cm exposed tip (Physitemp, Cat. # T-384A) connected to a thermometer (BAT-12, Physitemp) with an analogue output connected to the digitizer and sampled at 10 kHz. The tip of thermocouple was threaded through a borosilicate capillary tube and precisely placed near the antennae using a micromanipulator (MP-225, Sutter Instruments).

The temperature in the imaging chamber was adjusted by a dual in-line heater/cooler probe SC-20 which was controlled by bipolar temperature controller CL-100 (Warner Instruments, Connecticut, USA). The temperature of the brain slice was changed either from 37 to 9°C or from 9 to 37°C in 4°C intervals. The temperature was automatically adjusted by a feedback thermometer which was located in the bath adjacent to the brain slice. Before taking any measurements, brain slices were kept 5 minutes at each temperature.

For temperature stimulation, preparations were continuously perfused with Ca²⁺-containing AHL (as described above). AHL was gravity fed through a 3-way valve (Lee company, part # LHDA1231315H) and flow rate was adjusted through a flow regulator. Following the valve, temperature was precisely regulated through 2 in-line solution heater/coolers (Warner, cat. # SC-20) in parallel with by a dual channel bipolar temperature controller (Warner Instruments, Cl-200A). Excess heat produced by each SC-20 Peltier was dissipated through a liquid cooling system (Koolance, Cat. # EXT-1055). To circumvent changes in resistivity and voltage offsets from changing the temperature of the bathing solution, the reference Ag-Cl pellet electrode was placed in an isolated well adjacent to the recording chamber (Warner Instruments, Cat. # RC-24N), filled with identical AHL and connected via a borosilicate capillary tube filled by 2% agar in 3 M KCl. The bath temperature was precisely recorded using a custom Type T thermocouple with an exposed tip (Physitemp, Cat. # T-384A) connected to a thermometer (BAT-12, Physitemp) with an analogue output connected to the digitizer and sampled at 10 kHz. The tip of thermocouple was threaded through a borosilicate capillary tube and precisely placed near the antennae using a micromanipulator (MP-225, Sutter Instruments).