A Multimodal Assay to Infer Body Temperature Set-Point Shifts in Freely Moving Mice

Homeothermic animals maintain a stable core body temperature (Tc) at a set-point around 37 °C. Thermoregulatory homeostasis maintains Tc around the set-point, even when ambient temperature varies considerably. While most studies of thermoregulation focus on laboratory animals' systemic responses to changes in ambient temperature, little is known about how the central nervous system controls the Tc set-point. To facilitate such research, we developed an experimental strategy to infer Tc set-point shifts in freely moving mice. The core assay is based on the relationship between Tc and preferred environmental temperature (Tp), measured simultaneously within a thermal gradient apparatus (15-50 °C), thereby enabling evaluation of inferred Tc set-point changes through integrated physiological and behavioral responses in a single experiment. To support the interpretation of inferred set-point shifts, complementary thermoregulatory readouts were incorporated, including brown adipose tissue (BAT) surface temperature (TBAT) as an indirect proxy for thermogenesis and tail skin temperature (Ttail) as a measure of cutaneous heat loss. To demonstrate the utility of this assay, we bidirectionally manipulated the neuronal activity of EP3 receptor-expressing neurons in the medial preoptic area of the hypothalamus (MPA^EP3R neurons). Chemogenetic activation was performed in hM3Dq-expressing mice (n = 7), whereas inhibition was performed in hM4Di-expressing mice (n = 6). Deschloroclozapine and vehicle treatments were administered using a randomized within-subject design with a 24 h washout interval. Chemogenetic activation of MPA^EP3R neurons reduced Tc, along with coordinated cold-seeking behavior, reduced thermogenesis, and enhanced heat-loss responses, consistent with an inferred downward shift in the Tc set-point. Conversely, inhibition of these neurons induced warm-seeking behavior, enhanced thermogenesis, and heat-conservation responses, accompanied by elevated Tc, suggesting an inferred upward shift in the Tc set-point. Overall, this strategy provides a practical and reproducible approach for inferring Tc set-point dynamics in mice and will facilitate mechanistic studies of thermoregulation.