THE NEURAL NETWORK INVOLVED IN TORPOR MAINTENANCE AND AROUSAL IN MICE

Introduction: Torpor is an energy-saving strategy characterized by a reduction in metabolism and body temperature (Tb) that allows many species to survive harsh winters. While our understanding of the neural network involved in torpor onset has recently advanced in mice, a species that exhibits daily torpor, little is known about the network underlying the arousal from torpor. Since arousal entails a steep rise in Tb and the Raphe Pallidus (RPa) is a key brain area in the central control of thermogenesis, the present study aimed at investigating the neural network which is active during the arousal from torpor in mice, focusing on its projections to the RPa. Materials and methods: Female C57BL/6J mice (18–23g), acclimated to Ta 21◦C, underwent surgery under general anesthesia for the injection of a monosynaptic retrograde tracer (Cholera Toxin B, CTb) into the RPa and the implantation of a wireless Tb monitoring device. After a 7-day recovery, mice were acutely food-deprived to induce torpor. The three following experimental conditions were studied: i) Torpor (n=7): mice were euthanized during stable torpor, at least three hours after its onset; ii) Arousal (n=6): mice were euthanized 90 min. after the onset of the arousal from stable torpor; iii) Torpor-resistant (n=4): normothermic mice, in which torpor did not occur despite fasting, were euthanized at least three hours after fasting onset (fasted controls). At the end of the experiment, the brain was harvested for immunohistochemical quantification of cFos, and CTb expression. Results: The results showed widespread cFos expression in the brain across the different experimental conditions. Notably, within the thermoregulatory network, in the Dorsomedial Hypothalamus (DMH) overall activity was significantlyincreased in “Torpor” compared to “Torporresistant” animals, and significantlydecreased in “Arousal” compared to "Torpor" ones. However, no significantchanges in the activity of neurons projecting from the DMH to the RPa was observed among the three conditions. Moreover, the number of active cells projecting from the Preoptic Area (POA) to the RPa was significantlylower in “Torpor” compared to “Torpor-resistant” animals, and further significantly decreased in “Arousal” compared to “Torpor” ones. The analysis of the brainstem and hypothalamic structures involved in wake promotion showed that overall activity was increased in both “Torpor” and “Arousal” animals compared to "Torpor-resistant" ones in both Dorsal Raphe and Locus Coeruleus, but no differences between “Torpor” and “Arousal” animals were observed. Conclusions: These findingshighlight the role of the central thermoregulatory network in torpor regulation, with distinct levels of activity during its maintenance and the subsequent arousal. In particular a specificrole in the process of arousal from torpor seems to be played by the DMH, as previously observed for torpor induction, and by the POA, with a specific reference to its functional relationship with the RPa. Acknowledgments: This research has been funded by the Ministero dell'Universita e della Ricerca – Italy: PNRR MNESYS (PE0000006) - A multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022).