Sleep deprivation soon after recovery from synthetic torpor enhances tau protein dephosphorylation in the rat brain.

Neuronal Tau protein hyperphosphorylation (PPtau) is a hallmark of tauopathic neurodegeneration. However, a reversible brain PPtau occurs in mammals during either natural or "synthetic" torpor (ST), a transient deep hypothermic state that can be pharmacologically induced in rats. Since in both conditions a high sleep pressure builds up during the regaining of euthermia, the aim of this work was to assess the possible role of post-ST sleep in PPtau dephosphorylation. Male rats were studied at the hypothermic nadir of ST, and 3-6 h after the recovery of euthermia, after either normal sleep (NS) or total sleep deprivation (SD). The effects of SD were studied by assessing: (i) deep brain temperature (Tb); (ii) immunofluorescent staining for AT8 (phosphorylated Tau) and Tau-1 (non-phosphorylated Tau), assessed in 19 brain structures; (iii) different phosphorylated forms of Tau and the main cellular factors involved in Tau phospho-regulation, including pro- and anti-apoptotic markers, assessed through western blot in the parietal cortex and hippocampus; (iv) systemic factors which are involved in natural torpor; (v) microglia activation state, by considering morphometric variations. Unexpectedly, the reversibility of PPtau was more efficient in SD than in NS animals, and was concomitant with a higher Tb, higher melatonin plasma levels, and a higher frequency of the microglia resting phenotype. Since the reversibility of ST-induced PPtau was previously shown to be driven by a latent physiological molecular mechanism triggered by deep hypothermia, short-term SD soon after the regaining of euthermia seems to boost the possible neuroprotective effects of this mechanism.

Corrigendum: Synthetic torpor triggers a regulated mechanism in the rat brain, favoring the reversibility of Tau protein hyperphosphorylation.

[This corrects the article DOI: 10.3389/fphys.2023.1129278.].

Neurogenic Fever after Subarachnoid Hemorrhage in Animal Models: A Systematic Review.

The observation of neurogenic fever resulting from subarachnoid hemorrhage (SAH) in animal models is a useful tool for the interpretation of its pathophysiology in humans, which is still a major challenge in the management of neurocritical patients. This systematic review aims to identify the prognostic factors and pathophysiological elements that determine the onset of neurogenic fever and its severity in animal models. In addition, our study aims to analyze which pharmacological treatments are most effective. All the articles available in Pubmed, Embase, and the Biological Science Collection until August 2021 concerning in vivo experimental studies on SAH animal models, including full texts and abstracts written in English and Italian, were considered. The risk of bias was assessed with SYRCLE's Risk of Bias tool. In total, 81 records were retrieved; after excluding duplicates, 76 records were potentially relevant. A total of 64 articles was excluded after title and abstract screening. The remaining 12 studies were evaluated as full texts, and 6 other studies were excluded (SAH-induced animal studies without a body temperature assessment). In one study, body temperature was measured after SAH induction, but the authors did not report temperature recording. Therefore, only five studies met the search criteria. The high methodological heterogeneity (different animal species, different temperature measurement methods, and different methods of the induction of bleeding) prevented meta-analysis. Synthesis methodology without meta-analysis (SWiM) was used for data analysis. The total number of animals used as controls was 87 (23 rabbits, 32 mice, and 32 rats), while there were 130 animals used as interventions (54 rabbits, 44 mice, and 32 rats). The presence of blood in the subarachnoid space, particularly red blood cells, is responsible for neurogenic fever; the role of hemoglobin is unclear. The mechanism is apparently not mediated by prostaglandins. The autonomic nervous system innervating brown adipose tissue is undoubtedly implicated in the onset of neurogenic fever. The activation of the central adenosine-1 receptor is effective in controlling the temperature of animals with neurogenic fever (by inhibiting thermogenesis of brown adipose tissue).

Ultrasonic vocalisations during rapid eye movement sleep in the rat.

Rats are known to use a 22-kHz ultrasonic vocalisation as a distress call to warn of danger to other members of their group. We monitored 22-kHz ultrasonic vocalisation emissions in rats (lean and obese) as part of a sleep deprivation study to detect the eventual presence of stress during the procedure. Unexpectedly, we detected ultrasonic vocalisation emission during rapid eye movement (REM) sleep, but not during non-REM (NREM) sleep, in all the rats. The event occurs during the expiratory phase and can take place singularly or as a train. No difference was detected in the number or duration of these events in lean versus obese rats, during the light versus the dark period, and after sleep deprivation. As far as we know, this is the first report showing that rats can vocalise during REM sleep.

Synthetic torpor triggers a regulated mechanism in the rat brain, favoring the reversibility of Tau protein hyperphosphorylation.

Introduction: Hyperphosphorylated Tau protein (PPTau) is the hallmark of tauopathic neurodegeneration. During "synthetic torpor" (ST), a transient hypothermic state which can be induced in rats by the local pharmacological inhibition of the Raphe Pallidus, a reversible brain Tau hyperphosphorylation occurs. The aim of the present study was to elucidate the - as yet unknown - molecular mechanisms underlying this process, at both a cellular and systemic level. Methods: Different phosphorylated forms of Tau and the main cellular factors involved in Tau phospho-regulation were assessed by western blot in the parietal cortex and hippocampus of rats induced in ST, at either the hypothermic nadir or after the recovery of euthermia. Pro- and anti-apoptotic markers, as well as different systemic factors which are involved in natural torpor, were also assessed. Finally, the degree of microglia activation was determined through morphometry. Results: Overall, the results show that ST triggers a regulated biochemical process which can dam PPTau formation and favor its reversibility starting, unexpectedly for a non-hibernator, from the hypothermic nadir. In particular, at the nadir, the glycogen synthase kinase-ß was largely inhibited in both regions, the melatonin plasma levels were significantly increased and the antiapoptotic factor Akt was significantly activated in the hippocampus early after, while a transient neuroinflammation was observed during the recovery period. Discussion: Together, the present data suggest that ST can trigger a previously undescribed latent and regulated physiological process, that is able to cope with brain PPTau formation.

Central circuit controlling thermoregulatory inversion and torpor-like state.

To maintain core body temperature in mammals, the CNS thermoregulatory networks respond to cold exposure by increasing brown adipose tissue and shivering thermogenesis. However, in hibernation or torpor, this normal thermoregulatory response is supplanted by "thermoregulatory inversion", an altered homeostatic state in which cold exposure causes inhibition of thermogenesis and warm exposure stimulates thermogenesis. Here we demonstrate the existence of a novel, dynorphinergic thermoregulatory reflex pathway between the dorsolateral parabrachial nucleus and the dorsomedial hypothalamus that bypasses the normal thermoregulatory integrator in the hypothalamic preoptic area to play a critical role in mediating the inhibition of thermogenesis during thermoregulatory inversion. Our results indicate the existence of a neural circuit mechanism for thermoregulatory inversion within the CNS thermoregulatory pathways and support the potential for inducing a homeostatically-regulated, therapeutic hypothermia in non-hibernating species, including humans.

In a model of SAH-induced neurogenic fever, BAT thermogenesis is mediated by erythrocytes and blocked by agonism of adenosine A1 receptors.

Neurogenic fever (NF) after subarachnoid hemorrhage (SAH) is a major cause of morbidity that is associated with poor outcomes and prolonged stay in the neurointensive care unit (NICU). Though SAH is a much more common cause of fever than sepsis in the NICU, it is often a diagnosis of exclusion, requiring significant effort to rule out an infectious source. NF does not respond to standard anti-pyretic medications such as COX inhibitors, and lack of good medical therapy has led to the introduction of external cooling systems that have their own associated problems. In a rodent model of SAH, we measured the effects of injecting whole blood, blood plasma, or erythrocytes on the sympathetic nerve activity to brown adipose tissue and on febrile thermogenesis. We demonstrate that following SAH the acute activation of brown adipose tissue leading to NF, is not dependent on PGE2, that subarachnoid space injection of whole blood or erythrocytes, but not plasma alone, is sufficient to trigger brown adipose tissue thermogenesis, and that activation of adenosine A1 receptors in the CNS can block the brown adipose tissue thermogenic component contributing to NF after SAH. These findings point to a distinct thermogenic mechanism for generating NF, compared to those due to infectious causes, and will hopefully lead to new therapies.

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 dynamics of the microtubule system. The hyperphosphorylation of Tau (PP-Tau) commonly leads to the formation of neurofibrillary tangles, as it occurs in tauopathies, a group of neurodegenerative diseases, including Alzheimer's. Hypothermia-related accumulation of PP-Tau has been described in hibernators and during synthetic torpor (ST), a torpor-like condition that has been induced in rats, a non-hibernating species. Remarkably, in ST PP-Tau is reversible and Tau de-phosphorylates within a few hours following the torpor bout, apparently not evolving into pathology. These observations have been limited to the brain, but in animal models of tauopathies, PP-Tau accumulation also appears to occur in the spinal cord (SpCo). The aim of the present work was to assess whether ST leads to PP-Tau accumulation 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 on SpCo coronal sections. AT8-IF was clearly expressed in the dorsal horns (DH) during ST, while in the ventral horns (VH) no staining was observed. The AT8-IF completely disappeared after 6 h from the return to euthermia. Tau-1-IF disappeared in both DH and VH during ST, returning to normal levels during recovery. To shed light on the cellular process underlying the PP-Tau pattern observed, the inhibited form of the glycogen-synthase kinase 3ß (the main kinase acting on Tau) was assessed using IF: VH (i.e., in motor neurons) were highly stained mainly during ST, while in DH there was no staining. Since tauopathies are also related to neuroinflammation, microglia activation was also assessed through morphometric analyses, but no ST-induced microglia activation was found in the SpCo. Taken together, the present results show that, in the DH of SpCo, ST induces 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 the accumulation of PP-Tau and apparently avoid the neurodegenerative outcome.

Median preoptic area neurons are required for the cooling and febrile activations of brown adipose tissue thermogenesis in rat.

Within the central neural circuitry for thermoregulation, the balance between excitatory and inhibitory inputs to the dorsomedial hypothalamus (DMH) determines the level of activation of brown adipose tissue (BAT) thermogenesis. We employed neuroanatomical and in vivo electrophysiological techniques to identify a source of excitation to thermogenesis-promoting neurons in the DMH that is required for cold defense and fever. Inhibition of median preoptic area (MnPO) neurons blocked the BAT thermogenic responses during both PGE2-induced fever and cold exposure. Disinhibition or direct activation of MnPO neurons induced a BAT thermogenic response in warm rats. Blockade of ionotropic glutamate receptors in the DMH, or brain transection rostral to DMH, blocked cold-evoked or NMDA in MnPO-evoked BAT thermogenesis. RNAscope technique identified a glutamatergic population of MnPO neurons that projects to the DMH and expresses c-Fos following cold exposure. These discoveries relative to the glutamatergic drive to BAT sympathoexcitatory neurons in DMH augment our understanding of the central thermoregulatory circuitry in non-torpid mammals. Our data will contribute to the development of novel therapeutic approaches to induce therapeutic hypothermia for treating drug-resistant fever, and for improving glucose and energy homeostasis.

Author Correction: Neural control of fasting-induced torpor in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neural control of fasting-induced torpor in mice.

Torpor is a peculiar mammalian behaviour, characterized by the active reduction of metabolic rate, followed by a drop in body temperature. To enter torpor, the activation of all thermogenic organs that could potentially defend body temperature must be prevented. Most of these organs, such as the brown adipose tissue, are controlled by the key thermoregulatory region of the Raphe Pallidus (RPa). Currently, it is not known which brain areas mediate the entrance into torpor. To identify these areas, the expression of the early gene c-Fos at torpor onset was assessed in different brain regions in mice injected with a retrograde tracer (Cholera Toxin subunit b, CTb) into the RPa region. The results show a network of hypothalamic neurons that are specifically activated at torpor onset and a direct torpor-specific projection from the Dorsomedial Hypothalamus to the RPa that could putatively mediate the suppression of thermogenesis during torpor.

Parabrachial Complex: A Hub for Pain and Aversion.

The parabrachial nucleus (PBN) has long been recognized as a sensory relay receiving an array of interoceptive and exteroceptive inputs relevant to taste and ingestive behavior, pain, and multiple aspects of autonomic control, including respiration, blood pressure, water balance, and thermoregulation. Outputs are known to be similarly widespread and complex. How sensory information is handled in PBN and used to inform different outputs to maintain homeostasis and promote survival is only now being elucidated. With a focus on taste and ingestive behaviors, pain, and thermoregulation, this review is intended to provide a context for analysis of PBN circuits involved in aversion and avoidance, and consider how information of various modalities, interoceptive and exteroceptive, is processed within PBN and transmitted to distinct targets to signal challenge, and to engage appropriate behavioral and physiological responses to maintain homeostasis.

Phosphorylation and Dephosphorylation of Tau Protein During Synthetic Torpor.

Tau protein is of primary importance for many physiological processes in neurons, where it affects the dynamics of the microtubule system. When hyperphosphorylated (PP-Tau), Tau monomers detach from microtubules and tend to aggregate firstly in oligomers, and then in neurofibrillary tangles, as it occurs in a group of neurodegenerative disorders named thauopathies. A hypothermia-related accumulation of PP-Tau, which is quickly reversed after the return to normothermia, has been shown to occur in the brain of hibernators during torpor. Since, recently, in our lab, a hypothermic torpor-like condition (synthetic torpor, ST) was pharmacologically induced in the rat, a non-hibernator, the aim of the present work was to assess whether ST can lead to a reversible PP-Tau accumulation in the rat brain. PP-Tau was immunohistochemically assessed by staining for AT8 (phosphorylated Tau) and Tau-1 (non-phosphorylated Tau) in 19 brain structures, which were chosen mostly due to their involvement in the regulation of autonomic and cognitive functions in relation to behavioral states. During ST, AT8 staining was strongly expressed throughout the brain, while Tau-1 staining was reduced compared to control conditions. During the following recovery period, AT8 staining progressively reduced close to zero after 6 h from ST. However, Tau-1 staining remained low even after 38 h from ST. Thus, overall, these results show that ST induced an accumulation of PP-Tau that was, apparently, only partially reversed to normal during the recovery period. While the accumulation of PP-Tau may only depend on the physicochemical characteristics of the enzymes regulating Tau phosphorylation, the reverse process of dephosphorylation should be actively regulated, also in non-hibernators. In conclusion, in this work a reversible and widespread PP-Tau accumulation has been induced through a procedure that leads a non-hibernator to a degree of reversible hypothermia, which is comparable to that observed in hibernators. Therefore, the physiological mechanism involved in this process can sustain an adaptive neuronal response to extreme conditions, which may however lead to neurodegeneration when particular intensities and durations are exceeded.

c-Fos expression in the limbic thalamus following thermoregulatory and wake-sleep changes in the rat.

A cellular degeneration of two thalamic nuclei belonging to the "limbic thalamus", i.e., the anteroventral (AV) and mediodorsal (MD) nuclei, has been shown in patients suffering from Fatal Familial Insomnia (FFI), a lethal prion disease characterized by autonomic activation and severe insomnia. To better assess the physiological role of these nuclei in autonomic and sleep regulation, c-Fos expression was measured in rats during a prolonged exposure to low ambient temperature (Ta, - 10 °C) and in the first hours of the subsequent recovery period at normal laboratory Ta (25 °C). Under this protocol, the thermoregulatory and autonomic activation led to a tonic increase in waking and to a reciprocal depression in sleep occurrence, which was more evident for REM sleep. These effects were followed by a clear REM sleep rebound and by a rebound of Delta power during non-REM sleep in the following recovery period. In the anterior thalamic nuclei, c-Fos expression was (1) larger during the activity rather than the rest period in the baseline; (2) clamped at a level in-between the normal daily variation during cold exposure; (3) not significantly affected during the recovery period in comparison to the time-matched baseline. No significant changes were observed in either the MD or the paraventricular thalamic nucleus, which is also part of the limbic thalamus. The observed changes in the activity of the anterior thalamic nuclei appear, therefore, to be more specifically related to behavioral activation than to autonomic or sleep regulation.

Reestablishment of Energy Balance in a Male Mouse Model With POMC Neuron Deletion of BMPR1A.

The regulation of energy balance involves complex processes in the brain, including coordination by hypothalamic neurons that contain pro-opiomelanocortin (POMC). We previously demonstrated that central bone morphogenetic protein (BMP) 7 reduced appetite. Now we show that a type 1 BMP receptor, BMPR1A, is colocalized with POMC neurons and that POMC-BMPR1A-knockout (KO) mice are hyperphagic, revealing physiological involvement of BMP signaling in anorectic POMC neurons in the regulation of appetite. Surprisingly, the hyperphagic POMC-BMPR1A-KO mice exhibited a lack of obesity, even on a 45% high-fat diet. This is because the brown adipose tissue (BAT) of KO animals exhibited increased sympathetic activation and greater thermogenic capacity owing to a reestablishment of energy balance, most likely stemming from a compensatory increase of BMPR1A in the whole hypothalamus of KO mice. Indeed, control animals given central BMP7 displayed increased energy expenditure and a specific increase in sympathetic nerve activity (SNA) in BAT. In these animals, pharmacological blockade of BMPR1A-SMAD signaling blunted the ability of BMP7 to increase energy expenditure or BAT SNA. Together, we demonstrated an important role for hypothalamic BMP signaling in the regulation of energy balance, including BMPR1A-mediated appetite regulation in POMC neurons as well as hypothalamic BMP-SMAD regulation of the sympathetic drive to BAT for thermogenesis.

REM Sleep and Endothermy: Potential Sites and Mechanism of a Reciprocal Interference.

Numerous data show a reciprocal interaction between REM sleep and thermoregulation. During REM sleep, the function of thermoregulation appears to be impaired; from the other hand, the tonic activation of thermogenesis, such as during cold exposure, suppresses REM sleep occurrence. Recently, both the central neural network controlling REM sleep and the central neural network controlling thermoregulation have been progressively unraveled. Thermoregulation was shown to be controlled by a central "core" circuit, responsible for the maintenance of body temperature, modulated by a set of accessory areas. REM sleep was suggested to be controlled by a group of hypothalamic neurons overlooking at the REM sleep generating circuits within the brainstem. The two networks overlap in a few areas, and in this review, we will suggest that in such overlap may reside the explanation of the reciprocal interaction between REM sleep and thermoregulation. Considering the peculiar modulation of thermoregulation by REM sleep the result of their coincidental evolution, REM sleep may therefore be seen as a period of transient heterothermy.

Thermoregulatory inversion: a novel thermoregulatory paradigm.

To maintain core body temperature in mammals, the normal central nervous system (CNS) thermoregulatory reflex networks produce an increase in brown adipose tissue (BAT) thermogenesis in response to skin cooling and an inhibition of the sympathetic outflow to BAT during skin rewarming. In contrast, these normal thermoregulatory reflexes appear to be inverted in hibernation/torpor; thermogenesis is inhibited during exposure to a cold environment, allowing dramatic reductions in core temperature and metabolism, and thermogenesis is activated during skin rewarming, contributing to a return of normal body temperature. Here, we describe two unrelated experimental paradigms in which rats, a nonhibernating/torpid species, exhibit a "thermoregulatory inversion," which is characterized by an inhibition of BAT thermogenesis in response to skin cooling, and a switch in the gain of the skin cooling reflex transfer function from negative to positive values. Either transection of the neuraxis immediately rostral to the dorsomedial hypothalamus in anesthetized rats or activation of A1 adenosine receptors within the CNS of free-behaving rats produces a state of thermoregulatory inversion in which skin cooling inhibits BAT thermogenesis, leading to hypothermia, and skin warming activates BAT, supporting an increase in core temperature. These results reflect the existence of a novel neural circuit that mediates inverted thermoregulatory reflexes and suggests a pharmacological mechanism through which a deeply hypothermic state can be achieved in nonhibernating/torpid mammals, possibly including humans.

Parabrachial complex links pain transmission to descending pain modulation.

The rostral ventromedial medulla (RVM) has a well-documented role in pain modulation and exerts antinociceptive and pronociceptive influences mediated by 2 distinct classes of neurons, OFF-cells and ON-cells. OFF-cells are defined by a sudden pause in firing in response to nociceptive inputs, whereas ON-cells are characterized by a "burst" of activity. Although these reflex-related changes in ON- and OFF-cell firing are critical to their pain-modulating function, the pathways mediating these responses have not been identified. The present experiments were designed to test the hypothesis that nociceptive input to the RVM is relayed through the parabrachial complex (PB). In electrophysiological studies, ON- and OFF-cells were recorded in the RVM of lightly anesthetized male rats before and after an infusion of lidocaine or muscimol into PB. The ON-cell burst and OFF-cell pause evoked by noxious heat or mechanical probing were substantially attenuated by inactivation of the lateral, but not medial, parabrachial area. Retrograde tracing studies showed that neurons projecting to the RVM were scattered throughout PB. Few of these neurons expressed calcitonin gene-related peptide, suggesting that the RVM projection from PB is distinct from that to the amygdala. These data show that a substantial component of "bottom-up" nociceptive drive to RVM pain-modulating neurons is relayed through the PB. While the PB is well known as an important relay for ascending nociceptive information, its functional connection with the RVM allows the spinoparabrachial pathway to access descending control systems as part of a recurrent circuit.

Hibernation, Hypothermia and a Possible Therapeutic "Shifted Homeostasis" Induced by Central Activation of A1 Adenosine Receptor (A1AR).

The positive outcome that hypothermia contributes to brain and cardiac protection following ischemia has stimulated research in the development of pharmacological approaches to induce a hypothermic/hypometabolic state. Pharmacological manipulation of central autonomic thermoregulatory circuits could represent a potential target for the induction of a hypothermic state. Here we present a brief description of the CNS thermoregulatory centers and how the manipulation of these circuits can be useful in the treatment of pathological conditions such as stroke or brain hemorrhage.