Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Abstract

Skeletal muscle thermogenesis provides a potential avenue for better understanding metabolic homeostasis and the mechanisms underlying energy expenditure. Surprisingly little evidence is available to link the neural, myocellular, and molecular mechanisms of thermogenesis directly to measurable changes in muscle temperature. This paper describes a method in which temperature transponders are utilized to retrieve direct measurements of mouse and rat skeletal muscle temperature. Remote transponders are surgically implanted within the muscle of mice and rats, and the animals are given time to recover. Mice and rats must then be repeatedly habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage. Muscle temperature can also be measured during prescribed physical activity (i.e., treadmill walking at a constant speed) to factor out changes in activity as contributors to the changes in muscle temperature induced by these stimuli. This method has been successfully used to elucidate mechanisms underlying muscle thermogenic control at the level of the brain, sympathetic nervous system, and skeletal muscle. Provided are demonstrations of this success using predator odor (PO; ferret odor) as a contextual stimulus and injections of oxytocin (Oxt) as a pharmacological stimulus, where predator odor induces muscle thermogenesis, and Oxt suppresses muscle temperature. Thus, these datasets display the efficacy of this method in detecting rapid changes in muscle temperature.

Figure 1:. Transponders and home cage temperature testing.

(A) Diagram of unilateral transponder placement for testing temperature in a mouse gastrocnemius. Once programmed and placed, the transponder-reader (DAS-8027-IUS, shown) can be used to measure temperature. (B) Left, photo of an open mesh stainless steel tea ball and a 5 cm × 5 cm towel. Right, enclosed tea ball, used to hold habituation and odor towels in home cage testing. (C) Schematic of risers constructed with PVC piping for home cage testing. (D) Workflow of home cage testing protocol. (E) Facility images of home cage testing area. Left, four mouse cages atop a riser. Magnetic strips are located on the adjacent wall, magnets, and surgical cloth are on the table. Right, covered mouse cages on risers. A, C, and D were created with Biorender.com.

Figure 2:. Activity-controlled temperature testing.

(A) Workflow of activity-controlled temperature testing with a pharmacological agent using treadmill walking. (B) Facility images of treadmills. Left, an image of full equipment setup. Right, a closer image of individual treadmills and shockers. A was created with Biorender.com.

Figure 3:. Analysis of muscle temperature during habituation for home cage temperature testing.

Mice unilaterally implanted with transponders in the right gastrocnemius were habituated to the testing procedure. Mice were measured in the animal housing room, ‘Before Move,’ in the testing room, ‘After Move,’ after acclimation for 1–2 h, ‘Baseline,’ then consecutively over 1 h. All statistical comparisons shown were made between trial 1 and trial 4, * p < 0.05, ** < 0.01 (t-test, N = 10); † < 0.05, †† < 0.01, ‡ < 0.001 main effect trial (ANOVA, N [trials] = 4). Error bars shown display the standard error of the mean (SEM).

Figure 4:. Muscle temperature during pharmacological stimulation of oxytocin in mice.

Habituated mice, unilaterally implanted with transponders, were given 2 mg/kg (i.p.) of either oxytocin or vehicle (sterile saline). Significant decreases in muscle temperature were observed at 5 min after injection of oxytocin and normalized by 60 min, ** < 0.01, *** < 0.001 (two-tailed paired t-test, N = 9). Error bars shown display the standard error of the mean.

Figure 5:. Predator-odor thermogenesis in rat home cage temperature testing.

Temperature measurements in rats with transponders implanted bilaterally in the gastrocnemius after exposure to predator (ferret) odor for 10 min. After exposure for 10 min, towels containing stimulus were removed, as indicated by the arrow. Rats maintained increased temperature 20 min after stimulus removal. Significantly greater than baseline temperature, * p < 0.05, ** < 0.01, *** < 0.001 (t-test, N = 4). Error bars shown display the standard error of the mean.

Figure 6:. Ferret odor induces a rapid rise in muscle temperature compared to control.

(A) Gastrocnemius temperature was significantly elevated after predator (ferret) odor compared with control exposure in male rats (two-tailed paired t-test, N = 8). (B) Novel, aversive, or fox odors did not significantly change muscle temperature compared to control. Temperature change induced by moderate stress quickly declined after 5 minutes. Ferret odor maintained a robust response, in comparison to other conditions, for the entirety of the test (ANOVA, N = 7). † p < 0.05, ferret odor > all other conditions; * p < 0.025, point comparison between ferret odor and moderate stress vs control odor. Adapted with permission from the Journal of Experimental Biology.