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.

Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice.

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

Binge alcohol drinking alters the differential control of cholinergic interneurons over nucleus accumbens D1 and D2 medium spiny neurons.

Animals studies support the notion that striatal cholinergic interneurons (ChIs) play a central role in basal ganglia function by regulating associative learning, reward processing, and motor control. In the nucleus accumbens (NAc), a brain region that mediates rewarding properties of substance abuse, acetylcholine regulates glutamatergic, dopaminergic, and GABAergic neurotransmission in naïve mice. However, it is unclear how ChIs orchestrate the control of these neurotransmitters/modulators to determine the synaptic excitability of medium spiny neurons (MSNs), the only projecting neurons that translate accumbens electrical activity into behavior. Also unknown is the impact of binge alcohol drinking on the regulation of dopamine D1- and D2 receptor-expressing MSNs (D1- and D2-MSNs, respectively) by ChIs. To investigate this question, we optogenetically stimulated ChIs while recording evoked and spontaneous excitatory postsynaptic currents (sEPSCs) in nucleus accumbens core D1- and D2-MSN of ChAT.ChR2.eYFPxDrd1.tdtomato mice. In alcohol-naïve mice, we found that stimulating NAc ChIs decreased sEPSCs frequency in both D1- and D2-MSNs, presumably through a presynaptic mechanism. Interestingly, ChI stimulation decreased MSN synaptic excitability through different mechanisms in D1- vs. D2-MSNs. While decrease of ChI-mediated sEPSCs frequency in D1-MSNs was mediated by dopamine, the same effect in D2-MSNs resulted from a direct control of glutamate release by ChIs. Interestingly, after 2 weeks of binge alcohol drinking, optogenetic stimulation of ChIs enhanced glutamate release in D1-MSNs, while its effect on D2-MSNs remained unchanged. Taken together, these data suggest that cholinergic interneurons could be a key target for regulation of NAc circuitry and for alcohol consumption.

Methods for Detecting PER2:LUCIFERASE Bioluminescence Rhythms in Freely Moving Mice.

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. The LumiCycle In Vivo applies a background subtraction that corrects for effects of room temperature on photomultiplier tube (PMT) output. We tested delivery of d-luciferin via a subcutaneous minipump and in the drinking water. We demonstrate spikes in bioluminescence associated with drinking bouts. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than d-luciferin, and can support longer-term studies. A small difference in phase of the PER2::LUC bioluminescence rhythms, with females phase leading males, can be detected with this technique. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

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.

Binge Alcohol Drinking Alters Synaptic Processing of Executive and Emotional Information in Core Nucleus Accumbens Medium Spiny Neurons.

The nucleus accumbens (NAc) is a forebrain region mediating the positive-reinforcing properties of drugs of abuse, including alcohol. It receives glutamatergic projections from multiple forebrain and limbic regions such as the prefrontal cortex (PFCx) and basolateral amygdala (BLA), respectively. However, it is unknown how NAc medium spiny neurons (MSNs) integrate PFCx and BLA inputs, and how this integration is affected by alcohol exposure. Because progress has been hampered by the inability to independently stimulate different pathways, we implemented a dual wavelength optogenetic approach to selectively and independently stimulate PFCx and BLA NAc inputs within the same brain slice. This approach functionally demonstrates that PFCx and BLA inputs synapse onto the same MSNs where they reciprocally inhibit each other pre-synaptically in a strict time-dependent manner. In alcohol-naïve mice, this temporal gating of BLA-inputs by PFCx afferents is stronger than the reverse, revealing that MSNs prioritize high-order executive processes information from the PFCx. Importantly, binge alcohol drinking alters this reciprocal inhibition by unilaterally strengthening BLA inhibition of PFCx inputs. In line with this observation, we demonstrate that in vivo optogenetic stimulation of the BLA, but not PFCx, blocks binge alcohol drinking escalation in mice. Overall, our results identify NAc MSNs as a key integrator of executive and emotional information and show that this integration is dysregulated during binge alcohol drinking.

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.

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.

Deconstructing circadian disruption: Assessing the contribution of reduced peripheral oscillator amplitude on obesity and glucose intolerance in mice.

Disturbing the circadian regulation of physiology by disruption of the rhythmic environment is associated with adverse health outcomes but the underlying mechanisms are unknown. Here, the response of central and peripheral circadian clocks to an advance or delay of the light-dark cycle was determined in mice. This identified transient damping of peripheral clocks as a consequence of an advanced light-dark cycle. Similar depression of peripheral rhythm amplitude was observed in mice exposed to repeated phase shifts. To assess the metabolic consequences of such peripheral amplitude depression in isolation, temporally chimeric mice lacking a functional central clock (Vgat-Cre+ Bmal1fl/fl ) were housed in the absence of environmental rhythmicity. In vivo PER2::LUC bioluminescence imaging of anesthetized and freely moving mice revealed that this resulted in a state of peripheral amplitude depression, similar in severity to that observed transiently following an advance of the light-dark cycle. Surprisingly, our mice did not show alterations in body mass or glucose tolerance in males or females on regular or high-fat diets. Overall, our results identify transient damping of peripheral rhythm amplitude as a consequence of exposure to an advanced light-dark cycle but chronic damping of peripheral clocks in isolation is insufficient to induce adverse metabolic outcomes in mice.

Maximising survival by shifting the daily timing of activity.

Maximising survival requires animals to balance the competing demands of maintaining energy balance and avoiding predation. Here, quantitative modelling shows that optimising the daily timing of activity and rest based on the encountered environmental conditions enables small mammals to maximise survival. Our model shows that nocturnality is typically beneficial when predation risk is higher during the day than during the night, but this is reversed by the energetic benefit of diurnality when food becomes scarce. Empirical testing under semi-natural conditions revealed that the daily timing of activity and rest in mice exposed to manipulations in energy availability and perceived predation risk is in line with the model's predictions. Low food availability and decreased perceived daytime predation risk promote diurnal activity patterns. Overall, our results identify temporal niche switching in small mammals as a strategy to maximise survival in response to environmental changes in food availability and perceived predation risk.

Tardiness Increases in Winter: Evidence for Annual Rhythms in Humans.

Annual rhythms in humans have been described for a limited number of behavioral and physiological parameters. The aim of this study was to investigate time-of-year variations in late arrivals, sick leaves, dismissals from class (attendance), and grades (performance). Data were collected in Dutch high school students across 4 academic years (indicators of attendance in about 1700 students; grades in about 200 students). Absenteeism showed a seasonal variation, with a peak in winter, which was more strongly associated with photoperiod (number of hours of daylight) compared with other factors assessed (e.g., weather conditions). Grades also varied with time of year, albeit differently across the 4 years. The observed time-of-year variation in the number of sick leaves was in accordance with the literature on the seasonality of infectious diseases (e.g., influenza usually breaks out in winter). The winter peak in late arrivals was unexpected and requires more research. Our findings could be relevant for a seasonal adaptation of school schedules and working environments (e.g., later school and work hours in winter, especially at higher latitudes where seasonal differences in photoperiod are more pronounced).

Clocks and meals keep mice from being cool.

Daily torpor is used by small mammals to reduce daily energy expenditure in response to energetic challenges. Optimizing the timing of daily torpor allows mammals to maximize its energetic benefits and, accordingly, torpor typically occurs in the late night and early morning in most species. However, the regulatory mechanisms underlying such temporal regulation have not been elucidated. Direct control by the circadian clock and indirect control through the timing of food intake have both been suggested as possible mechanisms. Here, feeding cycles outside of the circadian range and brain-specific mutations of circadian clock genes (Vgat-Cre+ CK1δfl/fl εfl/+ ; Vgat-Cre+ Bmal1fl/fl ) were used to separate the roles of the circadian clock and food timing in controlling the timing of daily torpor in mice. These experiments revealed that the timing of daily torpor is transiently inhibited by feeding, while the circadian clock is the major determinant of the timing of torpor. Torpor never occurred during the early part of the circadian active phase, but was preferentially initiated late in the subjective night. Food intake disrupted torpor in the first 4-6 h after feeding by preventing or interrupting torpor bouts. Following interruption, re-initiation of torpor was unlikely until after the next circadian active phase. Overall, these results demonstrate that feeding transiently inhibits torpor while the central circadian clock gates the timing of daily torpor in response to energetic challenges by restricting the initiation of torpor to a specific circadian phase.

Functionally Complete Excision of Conditional Alleles in the Mouse Suprachiasmatic Nucleus by Vgat-ires-Cre.

Mice with targeted gene disruption have provided important information about the molecular mechanisms of circadian clock function. A full understanding of the roles of circadian-relevant genes requires manipulation of their expression in a tissue-specific manner, ideally including manipulation with high efficiency within the suprachiasmatic nuclei (SCN). To date, conditional manipulation of genes within the SCN has been difficult. In a previously developed mouse line, Cre recombinase was inserted into the vesicular GABA transporter (Vgat) locus. Since virtually all SCN neurons are GABAergic, this Vgat-Cre line seemed likely to have high efficiency at disrupting conditional alleles in SCN. To test this premise, the efficacy of Vgat-Cre in excising conditional (fl, for flanked by LoxP) alleles in the SCN was examined. Vgat-Cre-mediated excision of conditional alleles of Clock or Bmal1 led to loss of immunostaining for products of the targeted genes in the SCN. Vgat-Cre+; Clockfl/fl; Npas2m/m mice and Vgat-Cre+; Bmal1fl/fl mice became arrhythmic immediately upon exposure to constant darkness, as expected based on the phenotype of mice in which these genes are disrupted throughout the body. The phenotype of mice with other combinations of Vgat-Cre+, conditional Clock, and mutant Npas2 alleles also resembled the corresponding whole-body knockout mice. These data indicate that the Vgat-Cre line is useful for Cre-mediated recombination within the SCN, making it useful for Cre-enabled technologies including gene disruption, gene replacement, and opto- and chemogenetic manipulation of the SCN circadian clock.

Desynchrony between brain and peripheral clocks caused by CK1δ/ε disruption in GABA neurons does not lead to adverse metabolic outcomes.

Circadian disruption as a result of shift work is associated with adverse metabolic consequences. Internal desynchrony between the phase of the suprachiasmatic nuclei (SCN) and peripheral clocks is widely believed to be a major factor contributing to these adverse consequences, but this hypothesis has never been tested directly. A GABAergic Cre driver combined with conditional casein kinase mutations (Vgat-Cre+CK1δfl/flεfl/+ ) was used to lengthen the endogenous circadian period in GABAergic neurons, including the SCN, but not in peripheral tissues, to create a Discordant mouse model. These mice had a long (27.4 h) behavioral period to which peripheral clocks entrained in vivo, albeit with an advanced phase (∼6 h). Thus, in the absence of environmental timing cues, these mice had internal desynchrony between the SCN and peripheral clocks. Surprisingly, internal desynchrony did not result in obesity in this model. Instead, Discordant mice had reduced body mass compared with Cre-negative controls on regular chow and even when challenged with a high-fat diet. Similarly, internal desynchrony failed to induce glucose intolerance or disrupt body temperature and energy expenditure rhythms. Subsequently, a lighting cycle of 2-h light/23.5-h dark was used to create a similar internal desynchrony state in both genotypes. Under these conditions, Discordant mice maintained their lower body mass relative to controls, suggesting that internal desynchrony did not cause the lowered body mass. Overall, our results indicate that internal desynchrony does not necessarily lead to metabolic derangements and suggest that additional mechanisms contribute to the adverse metabolic consequences observed in circadian disruption protocols.

Flexible clock systems: adjusting the temporal programme.

Under natural conditions, many aspects of the abiotic and biotic environment vary with time of day, season or even era, while these conditions are typically kept constant in laboratory settings. The timing information contained within the environment serves as critical timing cues for the internal biological timing system, but how this system drives daily rhythms in behaviour and physiology may also depend on the internal state of the animal. The disparity between timing of these cues in natural and laboratory conditions can result in substantial differences in the scheduling of behaviour and physiology under these conditions. In nature, temporal coordination of biological processes is critical to maximize fitness because they optimize the balance between reproduction, foraging and predation risk. Here we focus on the role of peripheral circadian clocks, and the rhythms that they drive, in enabling adaptive phenotypes. We discuss how reproduction, endocrine activity and metabolism interact with peripheral clocks, and outline the complex phenotypes arising from changes in this system. We conclude that peripheral timing is critical to adaptive plasticity of circadian organization in the field, and that we must abandon standard laboratory conditions to understand the mechanisms that underlie this plasticity which maximizes fitness under natural conditions.This article is part of the themed issue 'Wild clocks: integrating chronobiology and ecology to understand timekeeping in free-living animals'.

Lower school performance in late chronotypes: underlying factors and mechanisms.

Success at school determines future career opportunities. We described a time-of-day specific disparity in school performance between early and late chronotypes. Several studies showed that students with a late chronotype and short sleep duration obtain lower grades, suggesting that early school starting times handicap their performance. How chronotype, sleep duration, and time of day impact school performance is not clear. At a Dutch high school, we collected 40,890 grades obtained in a variety of school subjects over an entire school year. We found that the strength of the effect of chronotype on grades was similar to that of absenteeism, and that late chronotypes were more often absent. The difference in grades between the earliest 20% and the latest 20% of chronotypes corresponds to a drop from the 55th to 43rd percentile of grades. In academic subjects using mainly fluid cognition (scientific subjects), the correlation with grades and chronotype was significant while subjects relying on crystallised intelligence (humanistic/linguistic) showed no correlation with chronotype. Based on these and previous results, we can expand our earlier findings concerning exam times: students with a late chronotype are at a disadvantage in exams on scientific subjects, and when they are examined early in the day.

The flexible clock: predictive and reactive homeostasis, energy balance and the circadian regulation of sleep-wake timing.

The Darwinian fitness of mammals living in a rhythmic environment depends on endogenous daily (circadian) rhythms in behavior and physiology. Here, we discuss the mechanisms underlying the circadian regulation of physiology and behavior in mammals. We also review recent efforts to understand circadian flexibility, such as how the phase of activity and rest is altered depending on the encountered environment. We explain why shifting activity to the day is an adaptive strategy to cope with energetic challenges and show how this can reduce thermoregulatory costs. A framework is provided to make predictions about the optimal timing of activity and rest of non-model species for a wide range of habitats. This Review illustrates how the timing of daily rhythms is reciprocally linked to energy homeostasis, and it highlights the importance of this link in understanding daily rhythms in physiology and behavior.

Diurnality as an energy-saving strategy: energetic consequences of temporal niche switching in small mammals.

Endogenous daily (circadian) rhythms allow organisms to anticipate daily changes in the environment. Most mammals are specialized to be active during the night (nocturnal) or day (diurnal). However, typically nocturnal mammals become diurnal when energetically challenged by cold or hunger. The circadian thermo-energetics (CTE) hypothesis predicts that diurnal activity patterns reduce daily energy expenditure (DEE) compared with nocturnal activity patterns. Here, we tested the CTE hypothesis by quantifying the energetic consequences of relevant environmental factors in mice. Under natural conditions, diurnality reduces DEE by 6-10% in energetically challenged mice. Combined with night-time torpor, as observed in mice under prolonged food scarcity, DEE can be reduced by ∼20%. The dominant factor determining the energetic benefit of diurnality is thermal buffering provided by a sheltered resting location. Compared with nocturnal animals, diurnal animals encounter higher ambient temperatures during both day and night, leading to reduced thermogenesis costs in temperate climates. Analysis of weather station data shows that diurnality is energetically beneficial on almost all days of the year in a temperate climate region. Furthermore, diurnality provides energetic benefits at all investigated geographical locations on European longitudinal and latitudinal transects. The reduction of DEE by diurnality provides an ultimate explanation for temporal niche switching observed in typically nocturnal small mammals under energetically challenging conditions. Diurnality allows mammals to compensate for reductions in food availability and temperature as it reduces energetic needs. The optimal circadian organization of an animal ultimately depends on the balance between energetic consequences and other fitness consequences of the selected temporal niche.

Timing of examinations affects school performance differently in early and late chronotypes.

Circadian clocks of adolescents typically run late-including sleep times-yet adolescents generally are expected at school early in the morning. Due to this mismatch between internal (circadian) and external (social) times, adolescents suffer from chronic sleep deficiency, which, in turn, affects academic performance negatively. This constellation affects students' future career prospects. Our study correlates chronotype and examination performance. In total, 4734 grades were collected from 741 Dutch high school students (ages 11-18 years) who had completed the Munich ChronoType Questionnaire to estimate their internal time. Overall, the lowest grades were obtained by students who were very late chronotypes (MSFsc > 5.31 h) or slept very short on schooldays (SDw < 7.03 h). The effect of chronotype on examination performance depended on the time of day that examinations were taken. Opposed to late types, early chronotypes obtained significantly higher grades during the early (0815-0945 h) and late (1000-1215 h) morning. This group difference in grades disappeared in the early afternoon (1245-1500 h). Late types also obtained lower grades than early types when tested at the same internal time (hours after MSFsc), which may reflect general attention and learning disadvantages of late chronotypes during the early morning. Our results support delaying high school starting times as well as scheduling examinations in the early afternoon to avoid discrimination of late chronotypes and to give all high school students equal academic opportunities.