Neurophysiological and metabolic regulation of spontaneous and synthetic torpor: a translational perspective

Torpor is an energy-saving physiological state characterized by a transient and reversible decrease in metabolic rate and core temperature, which occurs in different species in conditions of scarce food availability. At present, the mechanism underlying torpor occurrence is unknown. The attempt to imitate natural torpor is pursued in clinical practice, in order to overcome the severe side effects that follow the induction of therapeutic hypothermia. Several attempts to induce a torpor-like state (synthetic torpor) by manipulating central nervous activity have been made in rodents. Most promising are the activation of central adenosine type-1 receptors and the pharmacological inhibition of the Raphe Pallidus (RPa). Aims of the present project were: i) to unravel the neural pathway of spontaneous torpor in mice, a species that enters daily torpor spontaneously; ii) to understand the possible mechanism of metabolic rate reduction in spontaneous and synthetic torpor, in mice and rats, respectively, by evaluating mitochondrial activity during deep hypothermia; iii) to explore the possibility to induce synthetic torpor in a large mammal, the swine, by the central manipulation of the RPa. In summary, the results showed that: i) Paraventricular and Dorsomedial Hypothalamic nuclei showed a specific neural activation at the entrance in torpor; ii) liver mitochondria showed a reduction in maximum respiration rate in spontaneous, but not in synthetic torpor, while no major changes occurred in kidney and brain; iii) central manipulation of the RPa in swine induced physiological modifications similar to those observed in rats

Neurons in the dorsomedial hypothalamus promote, prolong, and deepen torpor in the mouse

Torpor is a naturally occurring, hypometabolic, hypothermic state engaged by a wide range of animals in response to imbalance between the supply and demand for nutrients. Recent work has identified some of the key neuronal populations involved in daily torpor induction in mice, in particular, projections from the preoptic area of the hypothalamus to the dorsomedial hypothalamus (DMH). The DMH plays a role in thermoregulation, control of energy expenditure, and circadian rhythms, making it well positioned to contribute to the expression of torpor. We used activity-dependent genetic TRAPing techniques to target DMH neurons that were active during natural torpor bouts in female mice. Chemogenetic reactivation of torpor-TRAPed DMH neurons in calorie-restricted mice promoted torpor, resulting in longer and deeper torpor bouts. Chemogenetic inhibition of torpor-TRAPed DMH neurons did not block torpor entry, suggesting a modulatory role for the DMH in the control of torpor. This work adds to the evidence that the preoptic area of the hypothalamus and the DMH form part of a circuit within the mouse hypothalamus that controls entry into daily torpor. SIGNIFICANCE STATEMENT Daily heterotherms, such as mice, use torpor to cope with environments in which the supply of metabolic fuel is not sufficient for the maintenance of normothermia. Daily torpor involves reductions in body temperature, as well as active suppression of heart rate and metabolism. How the CNS controls this profound deviation from normal homeostasis is not known, but a projection from the preoptic area to the dorsomedial hypothalamus has recently been implicated. We demonstrate that the dorsomedial hypothalamus contains neurons that are active during torpor. Activity in these neurons promotes torpor entry and maintenance, but their activation alone does not appear to be sufficient for torpor entry

Turn it off and on again: characteristics and control of torpor [version 1; peer review: 1 approved, 2 approved with reservations, 1 not approved]

Torpor is a hypothermic, hypoactive, hypometabolic state entered into by a wide range of animals in response to environmental challenge. This review summarises the current understanding of torpor. We start by describing the characteristics of the wide-ranging physiological adaptations associated with torpor. Next follows a discussion of thermoregulation, control of food intake and energy expenditure, and the interactions of sleep and thermoregulation, with particular emphasis on how those processes pertain to torpor. We move on to take a critical view of the evidence for the systems that control torpor entry, including both the efferent circulating factors that signal the need for torpor, and the central processes that orchestrate it. Finally, we consider how the putative circuits responsible for torpor induction integrate with the established understanding of thermoregulation under non-torpid conditions and highlight important areas of uncertainty for future studies

Wake-sleep, thermoregulatory, and autonomic effects of cholinergic activation of the lateral hypothalamus in the rat: a pilot study

A major role in the wake-promoting effects of the activation of the lateral hypothalamus (LH) has been ascribed to a population of orexin (ORX)-containing neurons that send projections to central areas which regulate Wake-Sleep and autonomic function. Since, in the rat, a substantial amount of ORX neurons receive cholinergic projections from cells involved in Wake-Sleep regulation, the aim of this study was to assess the role played by LH cholinoceptive cells in Wake-Sleep and autonomic regulations. To this end, the effects of a microinjection of the cholinergic agonist Carbachol (CBL) into the LH were compared to those obtained through the activation of a wider cell population by the microinjection of the GABAA antagonist GABAzine (GBZ). The results of this pilot study showed that both drugs elicited the same behavioral and autonomic effects, those caused by GBZ being larger and longer-lasting than those following administration of CBL. Briefly, wakefulness was enhanced and sleep was depressed, and brain temperature and heart rate consistently increased, while mean arterial pressure showed only a mild increment. Surprisingly, the administration of the drug vehicle (SAL) elicited a similar pattern of Wake-Sleep effects which, although much smaller, were sufficient to mask any statistical significance between treatment and control data. In conclusion, the results of this work show that the arousal elicited by LH disinhibition by GABAzine is concomitant with autonomic responses set by the intervention of cold-defense mechanisms. Since the same response is elicited at a lower level by CBL administration, the hypothesis of an involvement of cholinoceptive ORX neurons in its generation is discussed

Reversible Tau Phosphorylation Induced by Synthetic Torpor in the Spinal Cord of the Rat

Tau is a key protein in neurons, where it affects the dynamicsof the microtubulesystem. The hyperphosphorylation of Tau (PP-Tau) commonlyleads to the formationof neurofibrillary tangles, as it occurs in tauopathies, a group of neurodegenerativediseases, including Alzheimer’s. Hypothermia-related accumulation of PP-Tau has beendescribed in hibernators and during synthetic torpor (ST),a torpor-like condition that hasbeen induced in rats, a non-hibernating species. Remarkably, in ST PP-Tau is reversibleand Tau de-phosphorylates within a few hours following the torpor bout, apparentlynot evolving into pathology. These observations have been limited to the brain, but inanimal models of tauopathies, PP-Tau accumulation also appears to occur in the spinalcord (SpCo). The aim of the present work was to assess whetherST leads to PP-Tauaccumulation in the SpCo and whether this process is reversible. Immunofluorescence(IF) for AT8 (to assess PP-Tau) and Tau-1 (non-phosphorylated Tau) was carried out onSpCo coronal sections. AT8-IF was clearly expressed in the dorsal horns (DH) duringST, while in the ventral horns (VH) no staining was observed.The AT8-IF completelydisappeared after 6h from the return to euthermia. Tau-1-IFdisappeared in both DH andVH during ST, returning to normal levels during recovery. Toshed light on the cellularprocess underlying the PP-Tau pattern observed, the inhibited form of the glycogen-synthase kinase 3β(the main kinase acting on Tau) was assessed using IF: VH (i.e., inmotor neurons) were highly stained mainly during ST, while in DH there was no staining.Since tauopathies are also related to neuroinflammation, microglia activation was alsoassessed through morphometric analyses, but no ST-inducedmicroglia activation wasfound in the SpCo. Taken together, the present results show that, in the DH of SpCo, STinduces a reversible accumulation of PP-Tau. Since during ST there is no motor activity,the lack of AT8-IF in VH may result from an activity-related process at a cellular level. Thus,ST demonstrates a newly-described physiological mechanism that is able to resolve theaccumulation of PP-Tau and apparently avoid the neurodegenerative outcome.Keywords: hypothermia, hibernation, microglia, tauopathies, GSK3β, motor neurons, adaptive respons

Western-blot results from Synthetic-torpor experiments conducted in 2019-2020

This dataset contains the original results for the western-blot (WB) determinations from experiments carried out in the "Physiological regulations in the wake-sleep cycle" lab, at the Department of Biomedical and Neuromotor Sciences - University of Bologna, Italy. The WB procedure, the bands acquisitions and their intensity quantifications were conducted at the “Centre for Applied Biomedical Research – CRBA” - University of Bologna, St. Orsola Hospital, Italy