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.

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.].

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.

Mitochondrial respiration in rats during hypothermia resulting from central drug administration.

The ability to induce a hypothermia resembling that of natural torpor would be greatly beneficial in medical and non-medical fields. At present, two procedures based on central nervous pharmacological manipulation have been shown to be effective in bringing core body temperature well below 30 °C in the rat, a non-hibernator: the first, based on the inhibition of a key relay in the central thermoregulatory pathway, the other, based on the activation of central adenosine A1 receptors. Although the role of mitochondria in the activation and maintenance of torpor has been extensively studied, no data are available for centrally induced hypothermia in non-hibernators. Thus, in the present work the respiration rate of mitochondria in the liver and in the kidney of rats following the aforementioned hypothermia-inducing treatments was studied. Moreover, to have an internal control, the same parameters were assessed in a well-consolidated model, i.e., mice during fasting-induced torpor. Our results show that state 3 respiration rate, which significantly decreased in the liver of mice, was unchanged in rats. An increase of state 4 respiration rate was observed in both species, although it was not statistically significant in rats under central adenosine stimulation. Also, a significant decrease of the respiratory control ratio was detected in both species. Finally, no effects were detected in kidney mitochondria in both species. Overall, in these hypothermic conditions liver mitochondria of rats remained active and apparently ready to be re-activated to produce energy and warm up the cells. These findings can be interpreted as encouraging in view of the finalization of a translational approach to humans.

Automating cell counting in fluorescent microscopy through deep learning with c-ResUnet.

Counting cells in fluorescent microscopy is a tedious, time-consuming task that researchers have to accomplish to assess the effects of different experimental conditions on biological structures of interest. Although such objects are generally easy to identify, the process of manually annotating cells is sometimes subject to fatigue errors and suffers from arbitrariness due to the operator's interpretation of the borderline cases. We propose a Deep Learning approach that exploits a fully-convolutional network in a binary segmentation fashion to localize the objects of interest. Counts are then retrieved as the number of detected items. Specifically, we introduce a Unet-like architecture, cell ResUnet (c-ResUnet), and compare its performance against 3 similar architectures. In addition, we evaluate through ablation studies the impact of two design choices, (i) artifacts oversampling and (ii) weight maps that penalize the errors on cells boundaries increasingly with overcrowding. In summary, the c-ResUnet outperforms the competitors with respect to both detection and counting metrics (respectively, [Formula: see text] score = 0.81 and MAE = 3.09). Also, the introduction of weight maps contribute to enhance performances, especially in presence of clumping cells, artifacts and confounding biological structures. Posterior qualitative assessment by domain experts corroborates previous results, suggesting human-level performance inasmuch even erroneous predictions seem to fall within the limits of operator interpretation. Finally, we release the pre-trained model and the annotated dataset to foster research in this and related fields.

Be cool to be far: Exploiting hibernation for space exploration.

In mammals, torpor/hibernation is a state that is characterized by an active reduction in metabolic rate followed by a progressive decrease in body temperature. Torpor was successfully mimicked in non-hibernators by inhibiting the activity of neurons within the brainstem region of the Raphe Pallidus, or by activating the adenosine A1 receptors in the brain. This state, called synthetic torpor, may be exploited for many medical applications, and for space exploration, providing many benefits for biological adaptation to the space environment, among which an enhanced protection from cosmic rays. As regards the use of synthetic torpor in space, to fully evaluate the degree of physiological advantage provided by this state, it is strongly advisable to move from Earth-based experiments to 'in the field' tests, possibly on board the International Space Station.

[The impact of listening to music on orthopedic patients: a randomized controlled trial]. / Effetti dell'ascolto della musica in pazienti ortopedici: uno studio randomizzato controllato.

BACKGROUND: Music listening represents a gold standard in the evidence-based holistic nursing practice. However, music listening is seldom involved in orthopedic postoperative settings, and only a few related studies can be retrieved in literature. PURPOSE: The aim was to assess the effects of music during the orthopedic postoperative period, when patients frequently report pain and anxiety. METHOD: A randomized controlled trial on 56 patients, equally divided in an experimental group treated with music and a control group in standard care, was conducted during their first-day of recovery from orthopedic surgery. The primary outcome was the pain level assessed with the VAS scale and the Short Form-McGill Pain Questionnaire. Secondary outcomes assessed were anxiety level, blood pressure, heart and respiratory rates and oxygen saturation. Following surgery, when clinically stabilized and soon after their return to their ward room, patients listened to music from a personal programmed playlist using their smartphones for 30 minutes. RESULTS: In music group, the Short Form-McGill Pain Questionnaire score significantly improved in the sensorial dimension (p=0.006) and in the affective dimension (p=0.02). Patients showed a pleasant experience in listening to music (90%), found it useful in coping with pain (64%) and improving mood (86%). CONCLUSIONS: Music in the orthopedic post-surgical period induced significant improvement in pain relief, representing a useful complementary intervention to drug treatment. Music listening could be a safe treatment, inexpensive and simple to manage by nurses in orthopedic postoperative settings.

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.

Autonomic effects induced by pharmacological activation and inhibition of Raphe Pallidus neurons in anaesthetized adult pigs.

The Raphe Pallidus (RPa) is a region of the brainstem that was shown to modulate the sympathetic outflow to many tissues and organs involved in thermoregulation and energy expenditure. In rodents, the pharmacological activation of RPa neurons was shown to increase the activity of the brown adipose tissue, heart rate, and expired CO2 , whereas their inhibition was shown to induce cutaneous vasodilation and a state of hypothermia that, when prolonged, leads to a state resembling torpor referred to as synthetic torpor. If translatable to humans, this synthetic torpor-inducing procedure would be advantageous in many clinical settings. A first step to explore such translatability, has been to verify whether the neurons within the RPa play the same role described for rodents in a larger mammal such as the pig. In the present study, we show that the physiological responses inducible by the pharmacological stimulation of RPa neurons are very similar to those observed in rodents. Injection of the GABAA agonist GABAzine in the RPa induced an increase in heart rate (from 99 to 174 bpm), systolic (from 87 to 170 mm Hg) and diastolic (from 51 to 98 mm Hg) arterial pressure, and end-tidal CO2 (from 49 to 62 mm Hg). All these changes were reversed by the injection in the same area of the GABAA agonist muscimol. These results support the possibility for RPa neurons to be a key target in the research for a safe and effective procedure for the induction of synthetic torpor in humans.

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.

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.

Effects of Virtual Reality in Patients Undergoing Dialysis: Study Protocol.

Dialysis is often considered slow, repetitive, and with programmed intervals. Patients often perceive it as time taken from their lives with a sense of ineluctability and emptiness, engendering a negative emotional and cognitive perception of the world and one's place in it. Today, it is possible to improve the quality of life of patients during hemodialysis using virtual reality (VR). This creation of a true multisensory experience may absorb the patient's perceptions during hemodialysis, improving his/her quality of life. An Italian multicenter, longitudinal experimental study will be conducted with a randomized, pre-post test design, with balanced allocation 1:1, in parallel groups with a control group in the standard care of patients diagnosed with chronic renal failure who are, undergoing hemodialysis treatment. A sample of 186 patients calculated with sample size (power = 80%, ß = 0.2, α = 0.05) will be randomized into an experimental group exposed to VR, and a control group in standard care. The 2 groups will be studied over a period of 1 month, with 12 applications of VR and with measurements of the following outcomes: anxiety, fatigue, pruritus, arterial pressure, heart rate, respiration rate, and duration of the session at each hemodialysis session. This is the first international experimental protocol that examines the application of VR in patients undergoing hemodialysis. If the results show statistically and clinically significant differences, the VR could be an additional holistic intervention, which is evidence based, linked to the humanization of chronic, repetitive interventions, complementary to and synergistic with standard of care.

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.

Hibernation and Radioprotection: Gene Expression in the Liver and Testicle of Rats Irradiated under Synthetic Torpor.

Hibernation has been proposed as a tool for human space travel. In recent years, a procedure to induce a metabolic state known as "synthetic torpor" in non-hibernating mammals was successfully developed. Synthetic torpor may not only be an efficient method to spare resources and reduce psychological problems in long-term exploratory-class missions, but may also represent a countermeasure against cosmic rays. Here we show the preliminary results from an experiment in rats exposed to ionizing radiation in normothermic conditions or synthetic torpor. Animals were irradiated with 3 Gy X-rays and organs were collected 4 h after exposure. Histological analysis of liver and testicle showed a reduced toxicity in animals irradiated in torpor compared to controls irradiated at normal temperature and metabolic activity. The expression of ataxia telangiectasia mutated (ATM) in the liver was significantly downregulated in the group of animal in synthetic torpor. In the testicle, more genes involved in the DNA damage signaling were downregulated during synthetic torpor. These data show for the first time that synthetic torpor is a radioprotector in non-hibernators, similarly to natural torpor in hibernating animals. Synthetic torpor can be an effective strategy to protect humans during long term space exploration of the solar system.

Effects of Listening to Live Singing in Patients Undergoing Hemodialysis: A Randomized Controlled Crossover Study.

BACKGROUND: Participation in music therapy is associated with improved psychological and physical indices among chronically ill patients. Listening to music during hemodialysis treatments positively affects patients' hemodynamics, laboratory values, quality of life, and physical symptoms. The effect of live singing during hemodialysis treatments, however, has not previously been studied. METHODS: A total of 24 participants with a diagnosis of end-stage kidney disease participated in the study. The vocalist was a musically trained dialysis nurse. Twelve of the patients listened to 15 min of live singing during 6 consecutive hemodialysis sessions, while the other 12 underwent standard hemodialysis. After a washout period of 2 days, the two groups were reversed. RESULTS: Listening to live music was associated with improvements in systolic and diastolic blood pressure, better quality of sleep, fewer cramps, and reduced anxiety/depression, pain, and itching ( p < .05, all values). CONCLUSIONS: Listening to live music during hemodialysis is an effective and potentially low-cost therapy for the dialysis care team to employ during hemodialysis treatments.

A Conceptual Framework Encompassing the Psychoneuroimmunoendocrinological Influences of Listening to Music in Patients With Heart Failure.

Patients with heart failure have been traditionally treated with a pharmacology-based approach, diet, exercise, and rehabilitation for reducing symptoms, hospitalizations, and mortality. We have developed a solid conceptual framework for music listening-based protocols, showing how music may have a broad range of positive effects on cardiovascular health through psychoneuroimmunoendocrinological pathways.