It 23

An ultrafine flexible temperature thermocouple (IT-23, Physitemp Instruments LLC, Clifton, NJ, USA) was placed at the bottom of an Eppendorf tube filled with 1 ml of F6H8 or saline solution, or empty of liquid (n = 4 observations per condition). Temperature was continuously recorded with a digital thermometer (BAT-12 Microprobe Thermometer, Physitemp Instruments LLC) (Figure 2D). Increases and decreases of temperature inside the tube were produced by a home-made temperature controller device whose Peltier cell was placed inside the tube. This device allows changing temperature between 15° and 50°C although only the temperature range close to the normal ocular surface temperature values were explored. From a resting Peltier temperature (T_Peltier) around 34°C, temperature was increased by 3°C in a single step at an approximate rate of 0.030°C·s⁻¹. After 8 min at 37°C, a 3°C cooling step was induced with the Peltier at a similar cooling rate. T_Peltier and temperature of the solution (T_F6H8, T_saline, or T_Air) were recorded simultaneously and stored in a computer using a micro1401 CED interface and Spike2 software (both from Cambridge Electronic Devices, Ltd., Milton, Cambridge, UK) for further off-line analysis. As in the case of human measurements, experiments were made at a room temperature of 23–24°C and a partial humidity around 40%.

The brain of one GCaMP 6f mouse was extracted and cut into 1 mm thick slices. The slices are immersed in a temperature-controlled water bath. The temperature was continuously monitored using a thermocouple (IT-23, Physitemp Instruments, Clifton, New Jersey) and recorded to a PC by means of a USB interface (NI 9213, National Instruments, Austin, Texas). Heating and subsequent cooling cycles are averaged together. Fluorescence was recorded using the same setup previously described in Sec. 2.1.

The experimenters were blinded to genotypes. A 60 mm dish containing approximately 1 ml distilled water (testing chamber) was placed on a temperature-controlled plate MATS-SPE (TOKAI HIT, Shizuoka, Japan) set at 44.5 °C to equilibrate the water temperature in a testing chamber to 35 ± 1 °C, which was continuously monitored using a T-type thermocouple wire IT-23 (Physitemp, NJ), USB-TC01 (National Instruments), and the NI Signal Express software (National Instruments, TX). Wandering third instar larvae expressing dTRPA1 by ppk1.9-GAL4 and/or DSK-2A-GAL4 were harvested to another 60 mm dish at room temperature (23–25°C), and gently transferred to the 35 °C testing chamber using a paintbrush. All experiments were performed and recorded under a binocular microscope with a camcorder, and the latency from the placement of larvae to rolling was measured offline for each larva.

A type A-K3 thermocouple (Ellab, Hillerød, Denmark) was used to measure skull temperature. The thermocouple was placed subcutaneously 2 mm lateral to the bregma in the irradiated zone. A burr hole was drilled under inhalation anesthesia (1% isoflurane at 1 L/min N₂O/O₂—70:30). To measure the brain surface temperature under the 1267 nm laser irradiation, the medial part of the left temporal muscle was detached from the skull bone, a small burr hole was drilled into the temporal bone, and a flexible thermocouple probe (IT-23, 0.23 mm diam, Physitemp Instruments LLC, NJ, USA) was introduced between the parietal bone and brain into the epidural space. Brain surface temperature was measured before and during the laser stimulation in 5 min increments using a handheld thermometer (BAT-7001H, Physitemp Instruments LLC, NJ, USA).