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.

Acute Inhibition of Inflammation Mediated by Sympathetic Nerves: The Inflammatory Reflex.

In this review, we will try to convince the readers that the immune system is controlled by an endogenous neural reflex, termed inflammatory reflex, that inhibits the acute immune response during the course of a systemic immune challenge. We will analyse here the contribution of different sympathetic nerves as possible efferent arms of the inflammatory reflex. We will discuss the evidence that demonstrates that neither the splenic sympathetic nerves nor the hepatic sympathetic nerves are necessary for the endogenous neural reflex inhibition of inflammation. We will discuss the contribution of the adrenal glands to the reflex control of inflammation, noting that the neurally mediated release of catecholamines in the systemic circulation is responsible for the enhancement of the anti-inflammatory cytokine interleukin 10 (IL-10) but not of the inhibition of the pro-inflammatory cytokine tumour necrosis factor α (TNF). We will conclude by reviewing the evidence that demonstrates that the splanchnic anti-inflammatory pathway, composed by preganglionic and postganglionic sympathetic splanchnic fibres with different target organs, including the spleen and the adrenal glands, is the efferent arm of the inflammatory reflex. During the course of a systemic immune challenge, the splanchnic anti-inflammatory pathway is endogenously activated to inhibit the TNF and enhance the IL-10 response, independently, presumably acting on separate populations of leukocytes.

The endogenous inflammatory reflex inhibits the inflammatory response to different immune challenges in mice.

The splanchnic anti-inflammatory pathway, the efferent arm of the endogenous inflammatory reflex, has been shown to suppress the acute inflammatory response of rats to systemic lipopolysaccharide (LPS). Here we show for the first time that this applies also to mice, and that the reflex may be engaged by a range of inflammatory stimuli. Experiments were performed on mice under deep anaesthesia. Half the animals were subjected to bilateral section of the splanchnic sympathetic nerves, to disconnect the splanchnic anti-inflammatory pathway, while the remainder underwent a sham operation. Mice were then challenged intravenously with one of three inflammatory stimuli: the toll-like receptor (TLR)-4 agonist, LPS (60 µg/kg), the TLR-3 agonist Polyinosinic:polycytidylic acid (Poly I:C, 1 mg/kg) or the TLR-2 and -6 agonist dipalmitoyl-S-glyceryl cysteine (Pam2cys, 34 µg/kg). Ninety minutes later, blood was sampled by cardiac puncture for serum cytokine analysis. The splanchnic anti-inflammatory reflex action was assessed by comparing cytokine levels between animals with cut versus those with intact splanchnic nerves. A consistent pattern emerged: Tumor necrosis factor (TNF) levels in response to all three challenges were raised by prior splanchnic nerve section, while levels of the anti-inflammatory cytokine interleukin 10 (IL-10) were reduced. The raised TNF:IL-10 ratio after splanchnic nerve section indicates an enhanced inflammatory state when the reflex is disabled. These findings show for the first time that the inflammatory reflex drives a coordinated anti-inflammatory action also in mice, and demonstrate that its anti-inflammatory action is engaged, in similar fashion, by inflammatory stimuli mimicking a range of bacterial and viral infections.

Activation of orexin-A (hypocretin-1) receptors in the Raphe Pallidus at different ambient temperatures in the rat: effects on thermoregulation, cardiovascular control, sleep, and feeding behavior.

The Raphe Pallidus (RPa) is a brainstem nucleus containing sympathetic premotor neurons that control thermogenesis and modulate cardiovascular function. It receives inputs from various hypothalamic areas, including the Lateral Hypothalamus (LH), a heterogeneous region intricately involved in several autonomic and behavioral functions. A key subpopulation of neurons in the LH expresses orexin/hypocretin, a neuropeptide which is crucially involved in the regulation of the wake-sleep states and feeding behavior. The RPa receives orexinergic projections from the LH and orexinergic signalling in the RPa has been shown to enhance thermogenesis in the anaesthetized rat, but only in the presence of an already existing thermogenic drive, without significantly affecting cardiovascular function. The present work was aimed at exploring the effects on thermoregulation and autonomic function and the possible role in the modulation of the wake-sleep states and feeding behavior of orexin injection in the RPa in the free-behaving rat. In order to assess the influence of an already present thermogenic drive on orexinergic signalling in the RPa, animals were studied at three different ambient temperatures (Ta, 10°C, 24°C, and 32°C). We found that orexin injection into the RPa variably affected the wake-sleep states, autonomic functions, motor activity, and feeding behavior, at the different Tas. In particular, in the first post-injection hour, we observed an increase in wakefulness, which was large at Ta 24°C and Ta 10°C and rather mild at Ta 32°C. Deep brain temperature was increased by orexin injection at Ta 10°C, but not at either Ta 24°C or Ta 32°C. Moreover, an increase in mean arterial blood pressure occurred at Ta 24°C, which was probably masked by the high baseline levels at Ta 10°C and was completely absent at Ta 32°C. Finally, an enhancement in feeding behavior was observed at Ta 24°C and 10°C only. In accordance with what observed in anaesthetized rats, orexinergic signalling in the RPa seems to be ineffective in the absence of any thermogenic drive. Moreover, the effects observed on the wake-sleep states and feeding behavior introduce the RPa as a novel player in the central neural network promoting wakefulness and feeding.

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

Fluorescent Neuronal Cells v2: multi-task, multi-format annotations for deep learning in microscopy.

Fluorescent Neuronal Cells v2 is a collection of fluorescence microscopy images and the corresponding ground-truth annotations, designed to foster innovative research in the domains of Life Sciences and Deep Learning. This dataset encompasses three image collections wherein rodent neuronal cell nuclei and cytoplasm are stained with diverse markers to highlight their anatomical or functional characteristics. Specifically, we release 1874 high-resolution images alongside 750 corresponding ground-truth annotations for several learning tasks, including semantic segmentation, object detection and counting. The contribution is two-fold. First, thanks to the variety of annotations and their accessible formats, we anticipate our work will facilitate methodological advancements in computer vision approaches for segmentation, detection, feature extraction, unsupervised and self-supervised learning, transfer learning, and related areas. Second, by enabling extensive exploration and benchmarking, we hope Fluorescent Neuronal Cells v2 will catalyze breakthroughs in fluorescence microscopy analysis and promote cutting-edge discoveries in life sciences.

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.

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.

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.].

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.

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.

Hericium erinaceus Improves Mood and Sleep Disorders in Patients Affected by Overweight or Obesity: Could Circulating Pro-BDNF and BDNF Be Potential Biomarkers?

Epidemiological data indicate that subjects affected by obesity have an increased risk of developing mood disorders. The relationship between obesity and mood disorders is bidirectional. We assessed whether a Hericium erinaceus treatment improved depression, anxiety, sleep, and binge eating disorders after 8 weeks of supplementation in subjects affected by overweight or obesity under a low calorie diet regimen. Looking for a possible clinical biomarker, we assessed the serum balance between brain-derived neurotrophic factor (BDNF) and its precursor pro-BDNF before and after H. erinaceus supplementation. Seventy-seven volunteers affected by overweight or obesity were recruited at the offices of the Department of Preventive Medicine, Luigi Devoto Clinic of Work, Obesity Centre, at the IRCCS Foundation Policlinico Hospital of Milan (Italy). Patients were recruited only if they had a mood and/or sleep disorder and/or were binge eating as evaluated through self-assessment questionnaires. We used two different enzyme-linked immunosorbent assays kits to discriminate circulating levels of pro-BDNF and BDNF. Eight weeks of oral H. erinaceus supplementation decreased depression, anxiety, and sleep disorders. H. erinaceus supplementation improved mood disorders of a depressive-anxious nature and the quality of the nocturnal rest. H. erinaceus increased circulating pro-BDNF levels without any significant change in BDNF circulating levels.

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.

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.

Hericium erinaceus Improves Recognition Memory and Induces Hippocampal and Cerebellar Neurogenesis in Frail Mice during Aging.

Frailty is a geriatric syndrome associated with both locomotor and cognitive decline, implicated in both poor quality of life and negative health outcomes. One central question surrounding frailty is whether phenotypic frailty is associated with the cognitive impairment during aging. Using spontaneous behavioral tests and by studying the dynamic change during aging, we demonstrated that the two form of vulnerability, locomotor and recognition memory decline, develop in parallel and therefore, integration of the motoric and cognitive evaluations are imperative. We developed an integrated frailty index based on both phenotypic and recognition memory performances. Hericium erinaceus (H. erinaceus) is a medicinal mushroom that improves recognition memory in mice. By using HPLC-UV-ESI/MS analyses we obtained standardized amounts of erinacine A and hericenones C and D in H. erinaceus extracts, that were tested in our animal model of physiological aging. Two-month oral supplementation with H. erinaceus reversed the age-decline of recognition memory. Proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) immunohistochemistry in the hippocampus and cerebellum in treated mice supported a positive effect of an H. erinaceus on neurogenesis in frail mice.

The Gut-Brain Axis in Alzheimer's Disease and Omega-3. A Critical Overview of Clinical Trials.

Despite intensive study, neurodegenerative diseases remain insufficiently understood, precluding rational design of therapeutic interventions that can reverse or even arrest the progressive loss of neurological function. In the last decade, several theories investigating the causes of neurodegenerative diseases have been formulated and a condition or risk factor that can contribute is described by the gut-brain axis hypothesis: stress, unbalanced diet, and drugs impact altering microbiota composition which contributes to dysbiosis. An altered gut microbiota may lead to a dysbiotic condition and to a subsequent increase in intestinal permeability, causing the so-called leaky-gut syndrome. Herein, in this review we report recent findings in clinical trials on the risk factor of the gut-brain axis in Alzheimer's disease and on the effect of omega-3 supplementation, in shifting gut microbiota balance towards an eubiosis status. Despite this promising effect, evidences reported in selected randomized clinical trials on the effect of omega-3 fatty acid on cognitive decline in Alzheimer's disease are few. Only Mild Cognitive Impairment, a prodromal state that could precede the progress to Alzheimer's disease could be affected by omega-3 FA supplementation. We report some of the critical issues which emerged from these studies. Randomized controlled trials in well-selected AD patients considering the critical points underlined in this review are warranted.

Dietary Supplementation of Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes), and Spatial Memory in Wild-Type Mice.

Hericium erinaceus is an edible and medicinal mushroom with potential neuroprotective effects. The study of H. erinaceus has attracted considerable attention during the past 10 years, particularly with regard to its potential utility in the treatment of motor dysfunction, Alzheimer disease, and other forms of dementia. We previously determined that oral supplementation with H. erinaceus results in significant improvements in novelty-seeking behavior and novel object recognition in mice. In this study, H. erinaceus was added to the diets of wild-type mice for 2 months, and effects on spatial memory were evaluated by means of a Y maze and an object location task. We found that H. erinaceus increased general locomotor activity but had no effect on spatial memory. Thus, oral supplementation with H. erinaceus yields specific and selective improvements in recognition memory without altering spatial working memory, which supports the hypothesis that recognition memory can be modeled as a dual process. In this model, the perirhinal cortex supports the recognition of individual items as part of a circuit involved in familiarity with an encountered stimulus, whereas the hippocampus supports recollected associations and relationships between stimuli.