sUPRa is a dual-color reporter for unbiased quantification of the unfolded protein response with cellular resolution.

The unfolded protein response (UPR) maintains proteostasis upon endoplasmic reticulum (ER) stress, and is initiated by a range of physiological and pathological processes. While there have been advances in developing fluorescent reporters for monitoring individual signaling pathways of the UPR, this approach may not capture a cell's overall UPR activity. Here we describe a novel sensor of UPR activity, sUPRa, which is designed to report the global UPR. sUPRa displays excellent response characteristics, outperforms reporters of individual UPR pathways in terms of sensitivity and kinetics, and responds to a range of different ER stress stimuli. Furthermore, sUPRa's dual promoter and fluorescent protein design ensures that both UPR-active and inactive cells are detected, and controls for reporter copy number. Using sUPRa, we reveal UPR activation in layer 2/3 pyramidal neurons of mouse cerebral cortex following a period of sleep deprivation. sUPRa affords new opportunities for quantifying physiological UPR activity with cellular resolution.

Ultrasonic vocalisation rate tracks the diurnal pattern of activity in winter phenotype Djungarian hamsters (Phodopus sungorus).

Vocalisations are increasingly being recognised as an important aspect of normal rodent behaviour yet little is known of how they interact with other spontaneous behaviours such as sleep and torpor, particularly in a social setting. We obtained chronic recordings of the vocal behaviour of adult male and female Djungarian hamsters (Phodopus sungorus) housed under short photoperiod (8 h light, 16 h dark, square wave transitions), in different social contexts. The animals were kept in isolation or in same-sex sibling pairs, separated by a grid which allowed non-physical social interaction. On approximately 20% of days hamsters spontaneously entered torpor, a state of metabolic depression that coincides with the rest phase of many small mammal species in response to actual or predicted energy shortages. Animals produced ultrasonic vocalisations (USVs) with a peak frequency of 57 kHz in both social and asocial conditions and there was a high degree of variability in vocalisation rate between subjects. Vocalisation rate was correlated with locomotor activity across the 24-h light cycle, occurring more frequently during the dark period when the hamsters were more active and peaking around light transitions. Solitary-housed animals did not vocalise whilst torpid and animals remained in torpor despite overlapping with vocalisations in social-housing. Besides a minor decrease in peak USV frequency when isolated hamsters were re-paired with their siblings, changing social contexts did not influence vocalisation behaviour or structure. In rare instances, temporally overlapping USVs occurred when animals were socially-housed and were grouped in such a way that could indicate coordination. We did not observe broadband calls (BBCs) contemporaneous with USVs in this paradigm, corroborating their correlation with physical aggression which was absent from our experiment. Overall, we find little evidence to suggest a direct social function of hamster USVs. We conclude that understanding the effects of vocalisations on spontaneous behaviours, such as sleep and torpor, will inform experimental design of future studies, especially where the role of social interactions is investigated.

Methods to estimate body temperature and energy expenditure dynamics in fed and fasted laboratory mice: effects of sleep deprivation and light exposure.

Monitoring body temperature and energy expenditure in freely-moving laboratory mice remains a powerful methodology used widely across a variety of disciplines-including circadian biology, sleep research, metabolic phenotyping, and the study of body temperature regulation. Some of the most pronounced changes in body temperature are observed when small heterothermic species reduce their body temperature during daily torpor. Daily torpor is an energy saving strategy characterized by dramatic reductions in body temperature employed by mice and other species when challenged to meet energetic demands. Typical measurements used to describe daily torpor are the measurement of core body temperature and energy expenditure. These approaches can have drawbacks and developing alternatives for these techniques provides options that can be beneficial both from an animal-welfare and study-complexity perspective. First, this paper presents and assesses a method to estimate core body temperature based on measurements of subcutaneous body temperature, and second, a separate approach to better estimate energy expenditure during daily torpor based on core body temperature. Third, the effects of light exposure during the habitual dark phase and sleep deprivation during the light period on body temperature dynamics were tested preliminary in fed and fasted mice. Together, the here-published approaches and datasets can be used in the future to assess body temperature and metabolism in freely-moving laboratory mice.

Somnotate: A probabilistic sleep stage classifier for studying vigilance state transitions.

Electrophysiological recordings from freely behaving animals are a widespread and powerful mode of investigation in sleep research. These recordings generate large amounts of data that require sleep stage annotation (polysomnography), in which the data is parcellated according to three vigilance states: awake, rapid eye movement (REM) sleep, and non-REM (NREM) sleep. Manual and current computational annotation methods ignore intermediate states because the classification features become ambiguous, even though intermediate states contain important information regarding vigilance state dynamics. To address this problem, we have developed "Somnotate"-a probabilistic classifier based on a combination of linear discriminant analysis (LDA) with a hidden Markov model (HMM). First we demonstrate that Somnotate sets new standards in polysomnography, exhibiting annotation accuracies that exceed human experts on mouse electrophysiological data, remarkable robustness to errors in the training data, compatibility with different recording configurations, and an ability to maintain high accuracy during experimental interventions. However, the key feature of Somnotate is that it quantifies and reports the certainty of its annotations. We leverage this feature to reveal that many intermediate vigilance states cluster around state transitions, whereas others correspond to failed attempts to transition. This enables us to show for the first time that the success rates of different types of transition are differentially affected by experimental manipulations and can explain previously observed sleep patterns. Somnotate is open-source and has the potential to both facilitate the study of sleep stage transitions and offer new insights into the mechanisms underlying sleep-wake dynamics.

Penguins snatch seconds-long microsleeps.

Chinstrap penguins fall asleep thousands of times per day in the wild.

Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice.

Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools for remote control of targeted cell populations using chemical actuators that bind to modified receptors. Despite the popularity of DREADDs in neuroscience and sleep research, potential effects of the DREADD actuator clozapine-N-oxide (CNO) on sleep have never been systematically tested. Here, we show that intraperitoneal injections of commonly used CNO doses (1, 5, and 10 mg/kg) alter sleep in wild-type male laboratory mice. Using electroencephalography (EEG) and electromyography (EMG) to analyse sleep, we found a dose-dependent suppression of rapid eye movement (REM) sleep, changes in EEG spectral power during non-REM (NREM) sleep, and altered sleep architecture in a pattern previously reported for clozapine. Effects of CNO on sleep could arise from back-metabolism to clozapine or binding to endogenous neurotransmitter receptors. Interestingly, we found that the novel DREADD actuator, compound 21 (C21, 3 mg/kg), similarly modulates sleep despite a lack of back-metabolism to clozapine. Our results demonstrate that both CNO and C21 can modulate sleep of mice not expressing DREADD receptors. This implies that back-metabolism to clozapine is not the sole mechanism underlying side effects of chemogenetic actuators. Therefore, any chemogenetic experiment should include a DREADD-free control group injected with the same CNO, C21, or newly developed actuator. We suggest that electrophysiological sleep assessment could serve as a sensitive tool to test the biological inertness of novel chemogenetic actuators.

Scientists have developed ways to remotely turn on and off populations of neurons in the brain to test the role they play in behaviour. One technique that is frequently used is chemogenetics. In this approach, specific neurons are genetically modified to contain a special 'designer receptor' which switches cells on or off when its corresponding 'designer drug' is present. Recent studies have shown that the drug most commonly used in these experiments, clozapine-N-oxide (CNO), is broken down into small amounts of clozapine, an antipsychotic drug that binds to many natural receptors in the brain and modulates sleep. Nevertheless, CNO is still widely believed to not affect animals' sleep-wake patterns which in turn could influence a range of other brain activities and behaviours. However, there have been reports of animals lacking designer receptors still displaying unusual behaviours when administered CNO. This suggests that the breakdown of CNO to clozapine may cause off-target effects which could be skewing the results of chemogenetic studies. To investigate this possibility, Traut, Mengual et al. treated laboratory mice that do not have a designer receptor with three doses of CNO, and one dose of a new designer drug called compound-21 (C21) that is not broken down to clozapine. They found that high and medium doses of CNO, but also C21 altered the sleep-wake patterns of the mice and their brain activity during sleep. These findings show that CNO and C21 both have sleep-modulating effects on the brain and suggest that these effects are not only due to the production of clozapine, but the drugs binding to off-target natural receptors. To counteract this, Traut, Mengual et al. recommend optimizing the dose of drugs given to mice, and repeating the experiment on a control group which do not have the designer receptor. This will allow researchers to determine which behavioural changes are the result of turning on or off the neuron population of interest, and which are artefacts caused by the drug itself. They also suggest testing how newly developed designer drugs impact sleep before using them in behavioural experiments. Refining chemogenetic studies in these ways may yield more reliable insights about the role specific groups of cells have in the brain.

Detection of neuronal OFF periods as low amplitude neural activity segments.

BACKGROUND: During non-rapid eye movement sleep (NREM), alternating periods of synchronised high (ON period) and low (OFF period) neuronal activity are associated with high amplitude delta band (0.5-4 Hz) oscillations in neocortical electrophysiological signals termed slow waves. As this oscillation is dependent crucially on hyperpolarisation of cortical cells, there is an interest in understanding how neuronal silencing during OFF periods leads to the generation of slow waves and whether this relationship changes between cortical layers. A formal, widely adopted definition of OFF periods is absent, complicating their detection. Here, we grouped segments of high frequency neural activity containing spikes, recorded as multiunit activity from the neocortex of freely behaving mice, on the basis of amplitude and asked whether the population of low amplitude (LA) segments displayed the expected characteristics of OFF periods. RESULTS: Average LA segment length was comparable to previous reports for OFF periods but varied considerably, from as short as 8 ms to > 1 s. LA segments were longer and occurred more frequently in NREM but shorter LA segments also occurred in half of rapid eye movement sleep (REM) epochs and occasionally during wakefulness. LA segments in all states were associated with a local field potential (LFP) slow wave that increased in amplitude with LA segment duration. We found that LA segments > 50 ms displayed a homeostatic rebound in incidence following sleep deprivation whereas short LA segments (< 50 ms) did not. The temporal organisation of LA segments was more coherent between channels located at a similar cortical depth. CONCLUSION: We corroborate previous studies showing neural activity signals contain uniquely identifiable periods of low amplitude with distinct characteristics from the surrounding signal known as OFF periods and attribute the new characteristics of vigilance-state-dependent duration and duration-dependent homeostatic response to this phenomenon. This suggests that ON/OFF periods are currently underdefined and that their appearance is less binary than previously considered, instead representing a continuum.

Intracellular chloride regulation mediates local sleep pressure in the cortex.

Extended wakefulness is associated with reduced performance and the build-up of sleep pressure. In the cortex, this manifests as changes in network activity. These changes show local variation depending on the waking experience, and their underlying mechanisms represent targets for overcoming the effects of tiredness. Here, we reveal a central role for intracellular chloride regulation, which sets the strength of postsynaptic inhibition via GABAA receptors in cortical pyramidal neurons. Wakefulness results in depolarizing shifts in the equilibrium potential for GABAA receptors, reflecting local activity-dependent processes during waking and involving changes in chloride cotransporter activity. These changes underlie electrophysiological and behavioral markers of local sleep pressure within the cortex, including the levels of slow-wave activity during non-rapid eye movement sleep and low-frequency oscillatory activity and reduced performance levels in the sleep-deprived awake state. These findings identify chloride regulation as a crucial link between sleep-wake history, cortical activity and behavior.

Rodent models in translational circadian photobiology.

Over the last decades remarkable advances have been made in the understanding of the photobiology of circadian rhythms. The identification of a third photoreceptive system in the mammalian eye, in addition to the rods and cones that mediate vision, has transformed our appreciation of the role of light in regulating physiology and behavior. These photosensitive retinal ganglion cells (pRGCs) express the blue-light sensitive photopigment melanopsin and project to the suprachiasmatic nuclei (SCN)-the master circadian pacemaker-as well as many other brain regions. Much of our understanding of the fundamental mechanisms of the pRGCs, and the processes that they regulate, comes from mouse and other rodent models. Here we describe the contribution of rodent models to circadian photobiology, including both their strengths and limitations. In addition, we discuss how an appreciation of both rodent and human data is important for translational circadian photobiology. Such an approach enables a bi-directional flow of information whereby an understanding of basic mechanisms derived from mice can be integrated with studies from humans. Progress in this field is being driven forward at several levels of analysis, not least by the use of personalized light measurements and photoreceptor specific stimuli in human studies, and by studying the impact of environmental, rather than laboratory, lighting on different rodent models.

Neuronal-spiking-based closed-loop stimulation during cortical ON- and OFF-states in freely moving mice.

The slow oscillation is a central neuronal dynamic during sleep, and is generated by alternating periods of high and low neuronal activity (ON- and OFF-states). Mounting evidence causally links the slow oscillation to sleep's functions, and it has recently become possible to manipulate the slow oscillation non-invasively and phase-specifically. These developments represent promising clinical avenues, but they also highlight the importance of improving our understanding of how ON/OFF-states affect incoming stimuli and what role they play in neuronal plasticity. Most studies using closed-loop stimulation rely on the electroencephalogram and local field potential signals, which reflect neuronal ON- and OFF-states only indirectly. Here we develop an online detection algorithm based on spiking activity recorded from laminar arrays in mouse motor cortex. We find that online detection of ON- and OFF-states reflects specific phases of spontaneous local field potential slow oscillation. Our neuronal-spiking-based closed-loop procedure offers a novel opportunity for testing the functional role of slow oscillation in sleep-related restorative processes and neural plasticity.

Tinnitus: at a crossroad between phantom perception and sleep.

Sensory disconnection from the environment is a hallmark of sleep and is crucial for sleep maintenance. It remains unclear, however, whether internally generated percepts-phantom percepts-may overcome such disconnection and, in turn, how sleep and its effect on sensory processing and brain plasticity may affect the function of the specific neural networks underlying such phenomena. A major hurdle in addressing this relationship is the methodological difficulty to study sensory phantoms, due to their subjective nature and lack of control over the parameters or neural activity underlying that percept. Here, we explore the most prevalent phantom percept, subjective tinnitus-or tinnitus for short-as a model to investigate this. Tinnitus is the permanent perception of a sound with no identifiable corresponding acoustic source. This review offers a novel perspective on the functional interaction between brain activity across the sleep-wake cycle and tinnitus. We discuss characteristic features of brain activity during tinnitus in the awake and the sleeping brain and explore its effect on sleep functions and homeostasis. We ask whether local changes in cortical activity in tinnitus may overcome sensory disconnection and prevent the occurrence of global restorative sleep and, in turn, how accumulating sleep pressure may temporarily alleviate the persistence of a phantom sound. Beyond an acute interaction between sleep and neural activity, we discuss how the effects of sleep on brain plasticity may contribute to aberrant neural circuit activity and promote tinnitus consolidation. Tinnitus represents a unique window into understanding the role of sleep in sensory processing. Clarification of the underlying relationship may offer novel insights into therapeutic interventions in tinnitus management.

Re-examining extreme sleep duration in bats: implications for sleep phylogeny, ecology, and function.

Bats, quoted as sleeping for up to 20 h a day, are an often used example of extreme sleep duration amongst mammals. Given that duration has historically been one of the primary metrics featured in comparative studies of sleep, it is important that species specific sleep durations are well founded. Here, we re-examined the evidence for the characterization of bats as extreme sleepers and discuss whether it provides a useful representation of the sleep behavior of Chiroptera. Although there are a wealth of activity data to suggest that the diurnal cycle of bats is dominated by rest, estimates of sleep time generated from electrophysiological analyses suggest considerable interspecific variation, ranging from 83% to a more moderate 61% of the 24 h day spent asleep. Temperature-dependent changes in the duration and electroencephalographic profile of sleep suggest that bats represent a unique model for investigating the relationship between sleep and torpor. Further sources of intra-specific variation in sleep duration, including the impact of artificial laboratory environments and sleep intensity, remain unexplored. Future studies conducted in naturalistic environments, using larger sample sizes and relying on a pre-determined set of defining criteria will undoubtedly provide novel insights into sleep in bats and other species.

Human lesions and animal studies link the claustrum to perception, salience, sleep and pain.

The claustrum is the most densely interconnected region in the human brain. Despite the accumulating data from clinical and experimental studies, the functional role of the claustrum remains unknown. Here, we systematically review claustrum lesion studies and discuss their functional implications. Claustral lesions are associated with an array of signs and symptoms, including changes in cognitive, perceptual and motor abilities; electrical activity; mental state; and sleep. The wide range of symptoms observed following claustral lesions do not provide compelling evidence to support prominent current theories of claustrum function such as multisensory integration or salience computation. Conversely, the lesions studies support the hypothesis that the claustrum regulates cortical excitability. We argue that the claustrum is connected to, or part of, multiple brain networks that perform both fundamental and higher cognitive functions. As a multifunctional node in numerous networks, this may explain the manifold effects of claustrum damage on brain and behaviour.

Psilocin acutely alters sleep-wake architecture and cortical brain activity in laboratory mice.

Serotonergic psychedelic drugs, such as psilocin (4-hydroxy-N,N-dimethyltryptamine), profoundly alter the quality of consciousness through mechanisms which are incompletely understood. Growing evidence suggests that a single psychedelic experience can positively impact long-term psychological well-being, with relevance for the treatment of psychiatric disorders, including depression. A prominent factor associated with psychiatric disorders is disturbed sleep, and the sleep-wake cycle is implicated in the homeostatic regulation of neuronal activity and synaptic plasticity. However, it remains largely unknown to what extent psychedelic agents directly affect sleep, in terms of both acute arousal and homeostatic sleep regulation. Here, chronic electrophysiological recordings were obtained in mice to track sleep-wake architecture and cortical activity after psilocin injection. Administration of psilocin led to delayed REM sleep onset and reduced NREM sleep maintenance for up to approximately 3 h after dosing, and the acute EEG response was associated primarily with an enhanced oscillation around 4 Hz. No long-term changes in sleep-wake quantity were found. When combined with sleep deprivation, psilocin did not alter the dynamics of homeostatic sleep rebound during the subsequent recovery period, as reflected in both sleep amount and EEG slow-wave activity. However, psilocin decreased the recovery rate of sleep slow-wave activity following sleep deprivation in the local field potentials of electrodes targeting the medial prefrontal and surrounding cortex. It is concluded that psilocin affects both global vigilance state control and local sleep homeostasis, an effect which may be relevant for its antidepressant efficacy.

The hypothalamic link between arousal and sleep homeostasis in mice.

Sleep and wakefulness are not simple, homogenous all-or-none states but represent a spectrum of substates, distinguished by behavior, levels of arousal, and brain activity at the local and global levels. Until now, the role of the hypothalamic circuitry in sleep-wake control was studied primarily with respect to its contribution to rapid state transitions. In contrast, whether the hypothalamus modulates within-state dynamics (state "quality") and the functional significance thereof remains unexplored. Here, we show that photoactivation of inhibitory neurons in the lateral preoptic area (LPO) of the hypothalamus of adult male and female laboratory mice does not merely trigger awakening from sleep, but the resulting awake state is also characterized by an activated electroencephalogram (EEG) pattern, suggesting increased levels of arousal. This was associated with a faster build-up of sleep pressure, as reflected in higher EEG slow-wave activity (SWA) during subsequent sleep. In contrast, photoinhibition of inhibitory LPO neurons did not result in changes in vigilance states but was associated with persistently increased EEG SWA during spontaneous sleep. These findings suggest a role of the LPO in regulating arousal levels, which we propose as a key variable shaping the daily architecture of sleep-wake states.

Deletion of AMPA receptor GluA1 subunit gene (Gria1) causes circadian rhythm disruption and aberrant responses to environmental cues.

Dysfunction of the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 subunit and deficits in synaptic plasticity are implicated in schizophrenia and sleep and circadian rhythm disruption. To investigate the role of GluA1 in circadian and sleep behaviour, we used wheel-running, passive-infrared, and video-based home-cage activity monitoring to assess daily rest-activity profiles of GluA1-knockout mice (Gria1-/-). We showed that these mice displayed various circadian abnormalities, including misaligned, fragmented, and more variable rest-activity patterns. In addition, they showed heightened, but transient, behavioural arousal to light→dark and dark→light transitions, as well as attenuated nocturnal-light-induced activity suppression (negative masking). In the hypothalamic suprachiasmatic nuclei (SCN), nocturnal-light-induced cFos signals (a molecular marker of neuronal activity in the preceding ~1-2 h) were attenuated, indicating reduced light sensitivity in the SCN. However, there was no change in the neuroanatomical distribution of expression levels of two neuropeptides-vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP)-differentially expressed in the core (ventromedial) vs. shell (dorsolateral) SCN subregions and both are known to be important for neuronal synchronisation within the SCN and circadian rhythmicity. In the motor cortex (area M1/M2), there was increased inter-individual variability in cFos levels during the evening period, mirroring the increased inter-individual variability in locomotor activity under nocturnal light. Finally, in the spontaneous odour recognition task GluA1 knockouts' short-term memory was impaired due to enhanced attention to the recently encountered familiar odour. These abnormalities due to altered AMPA-receptor-mediated signalling resemble and may contribute to sleep and circadian rhythm disruption and attentional deficits in different modalities in schizophrenia.

Dim light in the evening causes coordinated realignment of circadian rhythms, sleep, and short-term memory.

Light provides the primary signal for entraining circadian rhythms to the day/night cycle. In addition to rods and cones, the retina contains a small population of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). Concerns have been raised that exposure to dim artificial lighting in the evening (DLE) may perturb circadian rhythms and sleep patterns, and OPN4 is presumed to mediate these effects. Here, we examine the effects of 4-h, 20-lux DLE on circadian physiology and behavior in mice and the role of OPN4 in these responses. We show that 2 wk of DLE induces a phase delay of ∼2 to 3 h in mice, comparable to that reported in humans. DLE-induced phase shifts are unaffected in Opn4-/- mice, indicating that rods and cones are capable of driving these responses in the absence of melanopsin. DLE delays molecular clock rhythms in the heart, liver, adrenal gland, and dorsal hippocampus. It also reverses short-term recognition memory performance, which is associated with changes in preceding sleep history. In addition, DLE modifies patterns of hypothalamic and cortical cFos signals, a molecular correlate of recent neuronal activity. Together, our data show that DLE causes coordinated realignment of circadian rhythms, sleep patterns, and short-term memory process in mice. These effects are particularly relevant as DLE conditions-due to artificial light exposure-are experienced by the majority of the populace on a daily basis.

A role for the cortex in sleep-wake regulation.

Cortical and subcortical circuitry are thought to play distinct roles in the generation of sleep oscillations and global state control, respectively. Here we silenced a subset of neocortical layer 5 pyramidal and archicortical dentate gyrus granule cells in male mice by ablating SNAP25. This markedly increased wakefulness and reduced rebound of electroencephalographic slow-wave activity after sleep deprivation, suggesting a role for the cortex in both vigilance state control and sleep homeostasis.

The relationship between fasting-induced torpor, sleep, and wakefulness in laboratory mice.

STUDY OBJECTIVES: Torpor is a regulated and reversible state of metabolic suppression used by many mammalian species to conserve energy. Whereas the relationship between torpor and sleep has been well-studied in seasonal hibernators, less is known about the effects of fasting-induced torpor on states of vigilance and brain activity in laboratory mice. METHODS: Continuous monitoring of electroencephalogram (EEG), electromyogram (EMG), and surface body temperature was undertaken in adult, male C57BL/6 mice over consecutive days of scheduled restricted feeding. RESULTS: All animals showed bouts of hypothermia that became progressively deeper and longer as fasting progressed. EEG and EMG were markedly affected by hypothermia, although the typical electrophysiological signatures of non-rapid eye movement (NREM) sleep, rapid eye movement (REM) sleep, and wakefulness enabled us to perform vigilance-state classification in all cases. Consistent with previous studies, hypothermic bouts were initiated from a state indistinguishable from NREM sleep, with EEG power decreasing gradually in parallel with decreasing surface body temperature. During deep hypothermia, REM sleep was largely abolished, and we observed shivering-associated intense bursts of muscle activity. CONCLUSIONS: Our study highlights important similarities between EEG signatures of fasting-induced torpor in mice, daily torpor in Djungarian hamsters and hibernation in seasonally hibernating species. Future studies are necessary to clarify the effects on fasting-induced torpor on subsequent sleep.