Continuous sampling of cerebrospinal fluid from the third ventricle of the hypothalamus to identify accumulating or depleting metabolites during hibernation.

Hibernation is a seasonally regulated process dramatically reducing metabolic rate and subsequently core body temperature (Tb). Many hibernators repeatedly cycle between torpor and arousal (T-A cycling) throughout the hibernation season. How these cycles are regulated is still unresolved. The sensing of metabolites accumulating/depleting during torpor may regulate T-A cycling. Here, we report the development of a microdialysis protocol to sample at a high temporal resolution from the third ventricle (3V) of the hypothalamus, from an individual hibernating golden hamster (Mesocricetus auratus). Over multiple days and T-A cycles we saw no negative impact on hibernation physiology or dynamics during the microdialysis. We collected samples at 2 h resolution over two successive T-A cycles and measured metabolites by targeted and untargeted metabolomics. Finally, we developed an analysis pipeline to identify accumulating or depleting metabolites during torpor, providing a proof of concept that cerebrospinal fluid microdialysis from the 3V of a hibernating golden hamster can be used in future studies to understand regulation of T-A cycling.