Halothane

To determine MH susceptibility to volatile halogenated anesthetics, mice were exposed to an air mixture containing halothane (Sigma-Aldrich, Italy) at concentrations sufficient to induce stage 3 anesthesia (2% halothane, with more added as necessary to induce and maintain this level of anesthesia) using an Isotec-3 evaporator (GE Healthcare, Milan, Italy), as previously described [15 (link), 29 (link)]. During halothane exposure, mice were kept in a chamber at a constant temperature (32°C) to avoid a drop in body temperature during anesthesia. The maximum exposure time to halothane was 1 hr, and surviving mice were then recovered by suspension of anesthetic administration.
To determine MH susceptibility to high environmental temperature, mice were subjected to a heat stress protocol, performed in an environmental chamber at 41°C for 1 hr while their internal temperature was recorded, as previously described [15 (link), 29 (link)]. Core body temperature was measured using a rectal thermometer (four channels thermometer TM-946, XS instruments, Modena, Italy) taped on the tails of the animals and recorded every 5 min throughout the duration of heat challenge, as in [15 (link), 29 (link)].