Cl 100

Cold temperature stimulation was applied by passing saline through an in-line solution cooler (SC-20, Warner Instruments, Hamden, CT, United States) connected to the controller device (CL-100, Warner Instruments, Hamden, CT, United States) (Figure 1). To apply a fast temperature change, the superfusion path was quickly switched to run saline through SC-20 for the time of stimulation. To apply a slow temperature change, a command ramp waveform was created by the acquisition software Spike2 and was fed to the controller CL-100.

Ca²⁺ imaging experiments were performed using an Olympus IX-51 inverted microscope equipped with a DeltaRAM excitation light source (Photon Technology International, PTI), as described earlier (Badheka et al., 2017 (link)). HEK293 cells were transfected with hTRPM3α2-GFP or its mutants using the Effectene reagent (Qiagen). Cells were loaded with 1 μM fura-2 AM (Invitrogen) for 40 min before the measurements at 37°C, and dual-excitation images at 340 and 380 nm excitation wavelengths were detected at 510 nm with a Roper Cool-Snap digital CCD camera. Measurements were conducted at room temperature in extracellular solution containing 137 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES and 10 mM glucose, pH 7.4. PregS, and primidone were applied with a gravity-driven whole chamber perfusion system. Temperature stimulation was performed using a custom-built system as described earlier (Badheka et al., 2017 (link)) by pushing bath solution through a spiral tubing immersed in hot water using a 60 ml syringe while monitoring the temperature of the perfusion chamber using a CL-100 Warner Instruments temperature controller. The analogue signal from the CL-100 unit was fed into the Digidata digitizer and the temperature curve was collected in Clampex. Data analysis was performed using the Image Master 5 software (PTI).

For both imaging and electrophysiology experiments, we exposed the dorsal surface of the fly’s brain by cutting a rectangular window in the head capsule using a 30-gauge syringe (BD PrecisionGlide). We perfused the brain with an artificial extracellular saline solution^{75 (link)} bubbled with carbogen (95% CO₂ / 5% O₂). The composition of the saline solution, in mM, was 103 NaCl, 3 KCl, 5 N-Tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid, 10 trehalose, 10 glucose, 2 sucrose, 26 NaHCO₃, 1 NaH₂PO₄, 1.5 CaCl₂, and 4 MgCl₂. All chemicals were sourced from Sigma Aldrich. The solution’s osmolarity was measured to be ~280 mOsm, and after carbogen bubbling, the solution’s pH was close to 7.3. The saline was delivered to the brain using a gravity-fed perfusion system. Using a Peltier device (SC-20, Warner Instruments) regulated by a closed-loop temperature controller (CL-100, Warner Instruments), we set the saline’s temperature, measured in the bath, to 22°C for calcium imaging experiments and 25°C for electrophysiology experiments.

Segments of the SMA were pressurized and perfused in a custom-built bath chamber using a variable hydrostatic pressure column connected to a motorized set of concentric glass pipettes (Wangemann Instruments, Kansas State University, KS) mounted on an inverted microscope (Axiovert 200, Carl Zeiss, Göttingen, Germany) [12 (link)]. Briefly, arteries were held by a holding pipette and luminally perfused with a perfusion pipette at one end while the other end was occluded using a blunt glass pipette. All pipettes were prepared using a custom-built micro-forge. The pressurized vessel was superfused in the bath with either HEPES-buffered PSS at a rate of 1.6 ml/min, permitting one complete exchange of the bath volume (~70 μl) within ~3 s. Experiments were conducted at 37 °C. Bath temperature was maintained by a triple heating system consisting of regulating the temperatures of the superfusate (8-line heater, CL-100, Warner Instruments, Hamden, CT, USA), the bath chamber (TC 324B, Warner Instruments) and the microscope objective (TC 324B, Warner Instruments).

Whole-cell voltage-clamp recordings were made from dissociated DRG neurons using patch pipettes pulled from Model P-1000 (Sutter Instruments). Patch pipettes had a resistance of 3–5 MΩ when filled with an internal solution containing the following (in mM): 140 CsF, 10 NaCl, 1.1 EGTA,.1 CaCl₂, 10 HEPES, and 2.5 MgATP, pH with CsOH to 7.2. Seals and whole-cell configuration were obtained in an external solution containing the following (in mM): 145 NaCl, 5 KCl, 10 HEPES, 10 glucose, 2 CaCl₂, 2 MgCl₂, pH 7.3 with NaOH, osmolarity ~320 mOsm. Series resistance ranged from 6 to 11 MΩ and was compensated by 70–80%. Voltage errors were not directly assessed in current-clamp recordings. To isolate whole-cell sodium currents during voltage-clamp experiments, a modified external solution was applied containing the following (in mM): 15 NaCl, 130 TEA-Cl, 10 HEPES, 2 BaCl₂, 13 glucose, 0.03 CdCl₂, pH 7.3 with NaOH, osmolarity ~320 mOsm. Voltage-clamp recordings were performed at RT and current-clamp recordings were conducted at 37°C. Bath temperature was controlled and monitored using CL-100 (Warner Instruments).