Figure 2. TIGAR knockout mice are resistant to tubocurare but not cyclopiazonic acid (CPA).

(A) Wild type (WT) and whole-body Tigar knockout (TKO) male mice (n = 6) were intraperitoneally injected with CPA (10 mg/kg body weight), and then core body temperature was measured every 15 min at room temperature. (B) WT and TKO male mice (n = 6) were intraperitoneally injected with CPA (10 mg/kg body weight) at room temperature, and then shifted to 4°C for 30 min, with core body temperature measured every 15 min. (C) WT and TKO male mice (n = 6) were intraperitoneally injected with tubocurare (0.4 mg/kg body weight), and then core body temperature was measured every 10 minutes at room temperature. (D) WT and TKO male mice (n = 6) were intraperitoneally injected with tubocurare (0.4 mg/kg body weight) at room temperature, and then 10 min later shifted to 4°C for 30 min, with core body temperature measured every 10 min. (E) WT and TKO female mice (n = 6) were intraperitoneally injected with tubocurare (0.15 mg/kg body weight), and then core body temperature was measured every 15 min at room temperature. (F) WT and TKO male mice (n = 10) were intraperitoneally injected with different tubocurare doses, and the LD₅₀ of curare was calculated using an online software LD₅₀ Calculator (AAT Bioquest, Inc, Sunnyvale, CA). (G) WT and TKO male mice (n = 10) were intraperitoneally injected with a lethal tubocurare dose (0.6 mg/kg body weight), and the number of mice undergoing complete paralysis was plotted as a function of time in seconds. (H) The time to death (absence of respiration) of the same mice was plotted as a function of time in minutes. Statistical analyses are described in ‘Method details,’ and the data are presented as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2—figure supplement 1. Changes in the expression of skeletal muscle nicotinic acetylcholine receptor subunits do not account for the differential sensitivity to tubocurare.

Relative mRNA levels of the nicotinic acetylcholine receptor subunits (A) α1 (Chrna1), (B) β1 (Chrnb1), (C) γ (Chrng), (D) δ (Chrnd), and (E) ε (Chrne) in the gastrocnemius muscle from male (n = 3–10) wild type (WT) and whole-body Tigar knockout (TKO) mice at ambient temperature or after 1 hr 4°C exposure. Statistical analyses are described in ‘Method details,’ and the data are presented as the mean ± SD.

Figure 2—video 1. Whole-body Tigar knockout (TKO) mice are resistant to the paralytic effects of tubocurare.

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Representative video clip of wild type (WT) and TKO male mice behavior under ambient temperature and at 4°C following intraperitoneal (IP) injection of tubocurare (0.4 mg/kg body weight). Rectal temperature recording of WT and TKO mice with curare injection was performed on February 12, 2019. The date of birth of both WT and TKO mice was October 15, 2018. The body weights of WT and TKO were 25.3 g and 27.6 g, respectively. The volume of curare saline solution (0.1 mg/ml in saline) for IP injection was 101.2 µl for the WT mouse and 110.4 µl for the TKO mouse. The mice were IP injected with curare, then caged at ambient temperature (21–23°C) for 10 min, and subsequently transferred into a pre-chilled cage in a cold chamber at 4°C for 20 min. The mice were taken back to the cages at ambient temperature (see the sketch below). Rectal temperature was recorded at the time of curare injection and every 10 min thereafter (see the table in text). The mouse in the left cage is WT, and the mouse in the right cage is TKO.