2.6. Stress‐induced hyperthermia

Young adult (8–12 weeks) male C57BL/6J mice were evaluated in the SIH test, as described previously (Wydeven, Marron Fernandez de Velasco, et al., 2014). Mice were single‐housed a day prior to testing, a condition that has been shown to increase basal anxiety (Adriaan Bouwknecht et al., 2007; Groenink, Vinkers, van Oorschot, & Olivier, 2009). Mice were assigned randomly to different treatment groups and were injected i.p. with 10 ml·kg⁻¹ of a microsuspension of 0‐, 3‐, 10‐, or 30‐mg·kg⁻¹ VU0810464 in 20% β‐hydroxypropyl cyclodextrin in sterile water, 30 min before testing (Gad, Cassidy, Aubert, Spainhour, & Robbe, 2006). Subsequently, an initial rectal temperature (T₁) was determined using a RET‐3 rectal probe (Physitemp Instruments, Clifton, NJ). The probe was dipped into silicon oil, inserted ∼1.5 cm into the rectum, and held until a stable rectal temperature was observed (∼20 s). Mice were then returned to their home cage for 10 min, followed by a second rectal temperature measurement (T₂). The difference between the second and first temperature measurements was determined (ΔT = T₂ − T₁) and evaluated across treatment groups. Experimenters were blinded to the dose treatment while performing and analysing the data. To determine if the response was K_ir3 dependent, SIH tests were conducted using a cohort of male and female wild‐type and Kcnj3 ^−/− siblings (8–12 weeks) generated in our colony. A low initial temperature (T₁ < 35°C) of the animals may affect the ΔT and thus these animals were excluded for further analysis.