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.

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.

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.

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.

Waking experience modulates sleep need in mice.

BACKGROUND: Homeostatic regulation of sleep is reflected in the maintenance of a daily balance between sleep and wakefulness. Although numerous internal and external factors can influence sleep, it is unclear whether and to what extent the process that keeps track of time spent awake is determined by the content of the waking experience. We hypothesised that alterations in environmental conditions may elicit different types of wakefulness, which will in turn influence both the capacity to sustain continuous wakefulness as well as the rates of accumulating sleep pressure. To address this, we compared the effects of repetitive behaviours such as voluntary wheel running or performing a simple touchscreen task, with wakefulness dominated by novel object exploration, on sleep timing and EEG slow-wave activity (SWA) during subsequent NREM sleep. RESULTS: We find that voluntary wheel running is associated with higher wake EEG theta-frequency activity and results in longer wake episodes, as compared with exploratory behaviour; yet, it does not lead to higher levels of EEG SWA during subsequent NREM sleep in either the frontal or occipital derivation. Furthermore, engagement in a touchscreen task, motivated by food reward, results in lower SWA during subsequent NREM sleep in both derivations, as compared to exploratory wakefulness, even though the total duration of wakefulness is similar. CONCLUSION: Overall, our study suggests that sleep-wake behaviour is highly flexible within an individual and that the homeostatic processes that keep track of time spent awake are sensitive to the nature of the waking experience. We therefore conclude that sleep dynamics are determined, to a large degree, by the interaction between the organism and the environment.

Diazepam effects on local cortical neural activity during sleep in mice.

GABA-ergic neurotransmission plays a key role in sleep regulatory mechanisms and in brain oscillations during sleep. Benzodiazepines such as diazepam are known to induce sedation and promote sleep, however, EEG spectral power in slow frequencies is typically reduced after the administration of benzodiazepines or similar compounds. EEG slow waves arise from a synchronous alternation between periods of cortical network activity (ON) and silence (OFF), and represent a sensitive marker of preceding sleep-wake history. Yet it remains unclear how benzodiazepines act on cortical neural activity during sleep. To address this, we obtained chronic recordings of local field potentials and multiunit activity (MUA) from deep cortical layers of the primary motor cortex in freely behaving mice after diazepam injection. We found that the amplitude of individual LFP slow waves was significantly reduced after diazepam injection and was accompanied by a lower incidence and duration of the corresponding neuronal OFF periods. Further investigation suggested that this is due to a disruption in the synchronisation of cortical neurons. Our data suggest that the state of global sleep and local cortical synchrony can be dissociated, and that the brain state induced by benzodiazepines is qualitatively different from spontaneous physiological sleep.

Continuous and non-invasive thermography of mouse skin accurately describes core body temperature patterns, but not absolute core temperature.

Body temperature is an important physiological parameter in many studies of laboratory mice. Continuous assessment of body temperature has traditionally required surgical implantation of a telemeter, but this invasive procedure adversely impacts animal welfare. Near-infrared thermography provides a non-invasive alternative by continuously measuring the highest temperature on the outside of the body (Tskin), but the reliability of these recordings as a proxy for continuous core body temperature (Tcore) measurements has not been assessed. Here, Tcore (30 s resolution) and Tskin (1 s resolution) were continuously measured for three days in mice exposed to ad libitum and restricted feeding conditions. We subsequently developed an algorithm that optimised the reliability of a Tskin-derived estimate of Tcore. This identified the average of the maximum Tskin per minute over a 30-min interval as the optimal way to estimate Tcore. Subsequent validation analyses did however demonstrate that this Tskin-derived proxy did not provide a reliable estimate of the absolute Tcore due to the high between-animal variability in the relationship between Tskin and Tcore. Conversely, validation showed that Tskin-derived estimates of Tcore reliably describe temporal patterns in physiologically-relevant Tcore changes and provide an excellent measure to perform within-animal comparisons of relative changes in Tcore.

Global sleep homeostasis reflects temporally and spatially integrated local cortical neuronal activity.

Sleep homeostasis manifests as a relative constancy of its daily amount and intensity. Theoretical descriptions define 'Process S', a variable with dynamics dependent on global sleep-wake history, and reflected in electroencephalogram (EEG) slow wave activity (SWA, 0.5-4 Hz) during sleep. The notion of sleep as a local, activity-dependent process suggests that activity history must be integrated to determine the dynamics of global Process S. Here, we developed novel mathematical models of Process S based on cortical activity recorded in freely behaving mice, describing local Process S as a function of the deviation of neuronal firing rates from a locally defined set-point, independent of global sleep-wake state. Averaging locally derived Processes S and their rate parameters yielded values resembling those obtained from EEG SWA and global vigilance states. We conclude that local Process S dynamics reflects neuronal activity integrated over time, and global Process S reflects local processes integrated over space.

Sleep and ageing: from human studies to rodent models.

Sleep duration and lifespan vary greatly across Animalia. Human studies have demonstrated that ageing reduces the ability to obtain deep restorative sleep, and this may play a causative role in the development of age-related neurodegenerative disorders. Animal models are widely used in sleep and ageing studies. Importantly, in contrast to human studies, evidence from laboratory rodents suggests that sleep duration is increased with ageing, while evidence for reduced sleep intensity and consolidation is inconsistent. Here we discuss two possible explanations for these species differences. First, methodological differences between studies in humans and laboratory rodents may prevent straightforward comparison. Second, the role of ecological factors, which have a profound influence on both ageing and sleep, must be taken into account. We propose that the dynamics of sleep across the lifespan reflect both age-dependent changes in the neurobiological substrates of sleep as well as the capacity to adapt to the environment.

Sleep homeostasis during daytime food entrainment in mice.

Twenty-four hour rhythms of physiology and behavior are driven by the environment and an internal endogenous timing system. Daily restricted feeding (RF) in nocturnal rodents during their inactive phase initiates food anticipatory activity (FAA) and a reorganization of the typical 24-hour sleep-wake structure. Here, we investigate the effects of daytime feeding, where food access was restricted to 4 hours during the light period ZT4-8 (Zeitgeber time; ZT0 is lights on), on sleep-wake architecture and sleep homeostasis in mice. Following 10 days of RF, mice were returned to ad libitum feeding. To mimic the spontaneous wakefulness associated with FAA and daytime feeding, mice were then sleep deprived between ZT3-6. Although the amount of wake increased during FAA and subsequent feeding, total wake time over 24 hours remained stable as the loss of sleep in the light phase was compensated for by an increase in sleep in the dark phase. Interestingly, sleep that followed spontaneous wake episodes during the dark period and the extended period of wake associated with FAA, exhibited lower levels of slow-wave activity (SWA) when compared to baseline or after sleep deprivation, despite a similar duration of waking. This suggests an evolutionary mechanism of reducing sleep drive during negative energy balance to enable greater arousal for food-seeking behaviors. However, the total amount of sleep and SWA accumulated during the 24 hours was similar between baseline and RF. In summary, our study suggests that despite substantial changes in the daily distribution and quality of wake induced by RF, sleep homeostasis is maintained.

Sleep- and Wake-Like States in Small Networks In Vivo and In Vitro.

Wakefulness and sleep are highly complex and heterogeneous processes, involving multiple neurotransmitter systems and a sophisticated interplay between global and local networks of neurons and non-neuronal cells. Macroscopic approaches applied at the level of the whole organism, view sleep as a global behaviour and allow for investigation into aspects such as the effects of insufficient or disrupted sleep on cognitive function, metabolism, thermoregulation and sensory processing. While significant progress has been achieved using such large-scale approaches, the inherent complexity of sleep-wake regulation has necessitated the development of methods which tackle specific aspects of sleep in isolation. One way this may be achieved is by investigating specific cellular or molecular phenomena in the whole organism in situ, either during spontaneous or induced sleep-wake states. This approach has greatly advanced our knowledge about the electrophysiology and pharmacology of ion channels, specific receptors, intracellular pathways and the small networks implicated in the control and regulation of the sleep-wake cycle. Importantly though, there are a variety of external and internal factors that influence global behavioural states which are difficult to control for using these approaches. For this reason, over the last few decades, ex vivo experimental models have become increasingly popular and have greatly advanced our understanding of many fundamental aspects of sleep, including the neuroanatomy and neurochemistry of sleep states, sleep regulation, the origin and dynamics of specific sleep oscillations, network homeostasis as well as the functional roles of sleep. This chapter will focus on the use of small neuronal networks as experimental models and will highlight the most significant and novel insights these approaches have provided.

Absent sleep EEG spindle activity in GluA1 (Gria1) knockout mice: relevance to neuropsychiatric disorders.

Sleep EEG spindles have been implicated in attention, sensory processing, synaptic plasticity and memory consolidation. In humans, deficits in sleep spindles have been reported in a wide range of neurological and psychiatric disorders, including schizophrenia. Genome-wide association studies have suggested a link between schizophrenia and genes associated with synaptic plasticity, including the Gria1 gene which codes for the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. Gria1-/- mice exhibit a phenotype relevant for neuropsychiatric disorders, including reduced synaptic plasticity and, at the behavioural level, attentional deficits leading to aberrant salience. In this study we report a striking reduction of EEG power density including the spindle-frequency range (10-15 Hz) during sleep in Gria1-/- mice. The reduction of spindle-activity in Gria1-/- mice was accompanied by longer REM sleep episodes, increased EEG slow-wave activity in the occipital derivation during baseline sleep, and a reduced rate of decline of EEG slow wave activity (0.5-4 Hz) during NREM sleep after sleep deprivation. These data provide a novel link between glutamatergic dysfunction and sleep abnormalities in a schizophrenia-relevant mouse model.

Cortical region-specific sleep homeostasis in mice: effects of time of day and waking experience.

Sleep-wake history, wake behaviors, lighting conditions, and circadian time influence sleep, but neither their relative contribution nor the underlying mechanisms are fully understood. The dynamics of electroencephalogram (EEG) slow-wave activity (SWA) during sleep can be described using the two-process model, whereby the parameters of homeostatic Process S are estimated using empirical EEG SWA (0.5-4 Hz) in nonrapid eye movement sleep (NREMS), and the 24 hr distribution of vigilance states. We hypothesized that the influence of extrinsic factors on sleep homeostasis, such as the time of day or wake behavior, would manifest in systematic deviations between empirical SWA and model predictions. To test this hypothesis, we performed parameter estimation and tested model predictions using NREMS SWA derived from continuous EEG recordings from the frontal and occipital cortex in mice. The animals showed prolonged wake periods, followed by consolidated sleep, both during the dark and light phases, and wakefulness primarily consisted of voluntary wheel running, learning a new motor skill or novel object exploration. Simulated SWA matched empirical levels well across conditions, and neither waking experience nor time of day had a significant influence on the fit between data and simulation. However, we consistently observed that Process S declined during sleep significantly faster in the frontal than in the occipital area of the neocortex. The striking resilience of the model to specific wake behaviors, lighting conditions, and time of day suggests that intrinsic factors underpinning the dynamics of Process S are robust to extrinsic influences, despite their major role in shaping the overall amount and distribution of vigilance states across 24 hr.

Effects of Aging on Cortical Neural Dynamics and Local Sleep Homeostasis in Mice.

Healthy aging is associated with marked effects on sleep, including its daily amount and architecture, as well as the specific EEG oscillations. Neither the neurophysiological underpinnings nor the biological significance of these changes are understood, and crucially the question remains whether aging is associated with reduced sleep need or a diminished capacity to generate sufficient sleep. Here we tested the hypothesis that aging may affect local cortical networks, disrupting the capacity to generate and sustain sleep oscillations, and with it the local homeostatic response to sleep loss. We performed chronic recordings of cortical neural activity and local field potentials from the motor cortex in young and older male C57BL/6J mice, during spontaneous waking and sleep, as well as during sleep after sleep deprivation. In older animals, we observed an increase in the incidence of non-rapid eye movement sleep local field potential slow waves and their associated neuronal silent (OFF) periods, whereas the overall pattern of state-dependent cortical neuronal firing was generally similar between ages. Furthermore, we observed that the response to sleep deprivation at the level of local cortical network activity was not affected by aging. Our data thus suggest that the local cortical neural dynamics and local sleep homeostatic mechanisms, at least in the motor cortex, are not impaired during healthy senescence in mice. This indicates that powerful protective or compensatory mechanisms may exist to maintain neuronal function stable across the life span, counteracting global changes in sleep amount and architecture.SIGNIFICANCE STATEMENT The biological significance of age-dependent changes in sleep is unknown but may reflect either a diminished sleep need or a reduced capacity to generate deep sleep stages. As aging has been linked to profound disruptions in cortical sleep oscillations and because sleep need is reflected in specific patterns of cortical activity, we performed chronic electrophysiological recordings of cortical neural activity during waking, sleep, and after sleep deprivation from young and older mice. We found that all main hallmarks of cortical activity during spontaneous sleep and recovery sleep after sleep deprivation were largely intact in older mice, suggesting that the well-described age-related changes in global sleep are unlikely to arise from a disruption of local network dynamics within the neocortex.

Stereotypic wheel running decreases cortical activity in mice.

Prolonged wakefulness is thought to gradually increase 'sleep need' and influence subsequent sleep duration and intensity, but the role of specific waking behaviours remains unclear. Here we report the effect of voluntary wheel running during wakefulness on neuronal activity in the motor and somatosensory cortex in mice. We find that stereotypic wheel running is associated with a substantial reduction in firing rates among a large subpopulation of cortical neurons, especially at high speeds. Wheel running also has longer-term effects on spiking activity across periods of wakefulness. Specifically, cortical firing rates are significantly higher towards the end of a spontaneous prolonged waking period. However, this increase is abolished when wakefulness is dominated by running wheel activity. These findings indicate that wake-related changes in firing rates are determined not only by wake duration, but also by specific waking behaviours.