Proceedings of The Physiological Society

Sleep Sleep and Circadian Rhythms (London, UK) (2018) Proc Physiol Soc 42, C21

Poster Communications

Torpor Preferentially Induces c-Fos Expression in Dorsomedial and Posterior Hypothalamus in Mice

M. T. Ambler1, M. Cerri2, A. Pickering1

1. Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, Bristol, United Kingdom. 2. Department of biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy.

Torpor is the naturally occurring hypothermic, hypometabolic and hypoactive component of hibernation(1). It is an adaptive, controlled reduction in temperature and metabolic demand in response to reduced availability of substrate. If such a centrally-driven hypothermic and hypometabolic state could be mimicked in a clinical setting it may represent an improved strategy for therapeutic hypothermia(2). The purpose of this study was to identify regions of the brain active during torpor in the mouse. Female mice (C57BL/6J, Charles River) were maintained on a 12-hour reversed light/dark cycle, and acclimatised to an ambient temperature of 30 °Cfor 5-7 days. Torpor was induced by reducing the ambient temperature to 18°C at lights off, then after 24 hours cold acclimatisation, food was removed for 12 hours(3). Torpor was detected by monitoring surface temperature changes using a thermal imaging camera (Flir C2, ResearchIR 4 software), and defined as a surface temperature greater than 2 standard deviations below the mean during the 24 hours prior to fasting. Controls were fasted at 30°C for 12 hours, or exposed to 18°C ambient temperature with access to food. No control mice entered torpor, and all mice exposed to cooling and fasting entered torpor. The mean nadir surface temperature for torpid mice was 24.2 +/- 0.9°C, 27.8 +/-0.06°C in the cooled controls (p < 0.001) and 34.2+/-0.2°C in the fasted controls (p<0.0001) (one-way ANOVA). Two hours after torpor entry (n=3), at the end of a 12 hour fast (n=4), or after 36 hours cold exposure (n=4), mice were terminally anaesthetised with pentobarbitone (175mg/kg i.p), then transcardially-perfused with formalin. Brains were post-fixed for 24 hours at 4°C before being cryoprotected in 30% sucrose for 48 hours. Sagittal sections of subcortical structures and brainstem (40μm) were cut, blocked with 5% normal donkey serum, and incubated overnight at room temperature in 1:2000 anti-c-fos primary (Cell Signalling Technologies, 2250s) followed by a fluorescent secondary antibody (donkey anti-mouse, AlexaFluor488, 1:1000). Sections were tile-scan imaged using a Leica DMI6000 Widefield fluorescence microscope. Images were analysed using ImageJ software with a semi-automated image processing protocol to count c-fos positive nuclei. Figure 1 shows an example plot of surface temperature and activity of a mouse entering several short torpor bouts of increasing depth. Figure 2 shows that cooling increased c-fos expression in the medial preoptic, parabrachial nucleus, and bed nucleus of the stria terminalis. Fasting induced c-Fos expression in the arcuate, the dorsomedial, posterior, and paraventricular hypothalamus. The dorsomedial and posterior hypothalamus increased c-fos expression during torpor compared to either cold or fasting alone, and as such they represent potential nodes within the torpor induction circuitry.

Where applicable, experiments conform with Society ethical requirements