Ta 29

Saline temperature was monitored continuously using a temperature controller (model CL-100, Warner Instruments) and altered during each experiment using an associated Peltier device and thermocouple (SC-20 and TA-29, Warner Instruments). Saline inflow to the nervous system was positioned within 1 cm of the STG so that the measured temperature at the point of inflow was approximately that of the ganglion somata.

For induction, mice were exposed to 2% halothane (Sigma-Aldrich, B4388, 2-bromo-2-chloro-1,1,1-trifluoroethane ≥ 99% pure) in a small induction chamber (EZ-177 Sure-Seal Mouse Chamber; E-Z Anesthesia). After 3 to 5 min, the anesthetized mouse, as indicated by a lack of response to gentle rocking, was removed from the small chamber and placed on a warmed platform set to 38 °C as described previously (5 (link)). Its limbs were taped in place and its head placed into a nose cone (Kent VetFlo-0305) delivering 1.5% halothane. A rectal temperature probe (Warner Instruments, TA-29) was inserted. The temperature was monitored continuously and recorded every 2 min for up to 60 min (TC-324C, Warner Instruments) or until the mouse became rigid and stopped spontaneous respiration. If the mouse survived 60 min, halothane was removed from the breathing circuit and the mouse allowed to recover and were observed in their home cage for 20 min. At the end of the experiment, the mice were euthanized by cervical dislocation and decapitation. A subset of the Ca_V1.1-R174W HET (4 mice) surviving their initial testing were subsequently exposed to the anesthesia protocol at a bed temperature of 38 to 39 °C.

The 4 to 8-month-old mice were placed in a restrainer (Kent Scientific HLD-MS-T) and a lubricated rectal probe with bead thermistor (Warner Instruments, TA-29) inserted rectally for the duration that the mouse was in the restraint. The temperature was monitored continuously with the probe connected to a Single Channel Temperature Controller (TC-324C; Warner Instruments) and recorded every 2 min. After 5 min at room temperature (RT) (∼24 °C), the mice were placed in a temperature-controlled chamber (38 °C; Thermo Scientific Forma Series II, Model # 1087) for a maximum of 60 min or until the mouse temperature exceeded 41 °C or the animal succumbed to the heat stress. After 60 min if the mouse survived it was removed from the heat chamber and returned to RT and observed for an additional 20 min, with the rectal temperature still being recorded every 2 min. At the end of the experiment, the mice were euthanized by cervical dislocation and decapitation.