Jeremy C. Borniger

Light at night, clocks and health: from humans to wild organisms

The increasing use of electric lights has modified the natural light environment dramatically, posing novel challenges to both humans and wildlife. Indeed, several biomedical studies have linked artificial light at night to the disruption of circadian rhythms, with important consequences for human health, such as the increasing occurrence of metabolic syndromes, cancer and reduced immunity. In wild animals, light pollution is associated with changes in circadian behaviour, reproduction and predator–prey interactions, but we know little about the underlying physiological mechanisms and whether wild species suffer the same health problems as humans. In order to fill this gap, we advocate the need for integrating ecological studies in the field, with chronobiological approaches to identify and characterize pathways that may link temporal disruption caused by light at night and potential health and fitness consequences.

Exposure to dim light at night during early development increases adult anxiety-like responses

Early experiences produce effects that may persist throughout life. Therefore, to understand adult phenotype, it is important to investigate the role of early environmental stimuli in adult behavior and health. Artificial light at night (LAN) is an increasingly common phenomenon throughout the world. However, animals, including humans, evolved under dark night conditions. Many studies have revealed affective, immune, and metabolic alterations provoked by aberrant light exposure and subsequent circadian disruption. Pups are receptive to entraining cues from the mother and then light early during development, raising the possibility that the early life light environment may influence subsequent behavior. Thus, to investigate potential influences of early life exposure to LAN on adult phenotype, we exposed mice to dim (~5 lux; full spectrum white light) or dark (~0 lux) nights pre- and/or postnatally. After weaning at 3 weeks of age, all mice were maintained in dark nights until adulthood (9 weeks of age) when behavior was assessed. Mice exposed to dim light in early life increased anxiety-like behavior and fearful responses on the elevated plus maze and passive avoidance tests. These mice also displayed reduced growth rates, which ultimately normalized during adolescence. mRNA expression of brain derived neurotrophic factor (BDNF), a neurotrophin previously linked to early life environment and adult phenotype, was not altered in the prefrontal cortex or hippocampus by early life LAN exposure. Serum corticosterone concentrations were similar between groups at weaning, suggesting that early life LAN does not elicit a long-term physiologic stress response. Dim light exposure did not influence behavior on the open field, novel object, sucrose anhedonia, or forced swim tests. Our data highlight the potential deleterious consequences of low levels of light during early life to development and subsequent behavior. Whether these changes are due to altered maternal behavior or persistent circadian abnormalities incurred by LAN remains to be determined.

Dim light at night does not disrupt timing or quality of sleep in mice.

Artificial nighttime illumination has recently become commonplace throughout the world; however, in common with other animals, humans have not evolved in the ecological context of chronic light at night. With prevailing evidence linking the circadian, endocrine, immune, and metabolic systems, understanding these relationships is important to understanding the etiology and progression of several diseases. To eliminate the covariate of sleep disruption in light at night studies, researchers often use nocturnal animals. However, the assumption that light at night does not affect sleep in nocturnal animals remains unspecified. To test the effects of light at night on sleep, we maintained Swiss-Webster mice in standard light/dark (LD) or dim light at night (DLAN) conditions for 8–10 wks and then measured electroencephalogram (EEG) and electromyogram (EMG) biopotentials via wireless telemetry over the course of two consecutive days to determine differences in sleep timing and homeostasis. Results show no statistical differences in total percent time, number of episodes, maximum or average episode durations in wake, slow-wave sleep (SWS), or rapid eye movement (REM) sleep. No differences were evident in SWS delta power, an index of sleep drive, between groups. Mice kept in DLAN conditions showed a relative increase in REM sleep during the first few hours after the dark/light transition. Both groups displayed normal 24-h circadian rhythms as measured by voluntary running wheel activity. Groups did not differ in body mass, but a marked negative correlation of body mass with percent time spent awake and a positive correlation of body mass with time spent in SWS was evident. Elevated body mass was also associated with shorter maximum wake episode durations, indicating heavier animals had more trouble remaining in the wake vigilance state for extended periods of time. Body mass did not correlate with activity levels, nor did activity levels correlate with time spent in different sleep states. These data indicate that heavier animals tend to sleep more, potentially contributing to further weight gain. We conclude that chronic DLAN exposure does not significantly affect sleep timing or homeostasis in mice, supporting the use of dim light with nocturnal rodents in chronobiology research to eliminate the possible covariate of sleep disruption.

Acute dim light at night increases body mass, alters metabolism, and shifts core body temperature circadian rhythms

The circadian system is primarily entrained by the ambient light environment and is fundamentally linked to metabolism. Mounting evidence suggests a causal relationship among aberrant light exposure, shift work, and metabolic disease. Previous research has demonstrated deleterious metabolic phenotypes elicited by chronic (>4 weeks) exposure to dim light at night (DLAN) (∼5 lux). However, the metabolic effects of short-term (<2 weeks) exposure to DLAN are unspecified. We hypothesized that metabolic alterations would arise in response to just 2 weeks of DLAN. Specifically, we predicted that mice exposed to dim light would gain more body mass, alter whole body metabolism, and display altered body temperature (Tb) and activity rhythms compared to mice maintained in dark nights. Our data largely support these predictions; DLAN mice gained significantly more mass, reduced whole body energy expenditure, increased carbohydrate over fat oxidation, and altered temperature circadian rhythms. Importantly, these alterations occurred despite similar activity locomotor levels (and rhythms) and total food intake between groups. Peripheral clocks are potently entrained by body temperature rhythms, and the deregulation of body temperature we observed may contribute to metabolic problems due to “internal desynchrony” between the central circadian oscillator and temperature sensitive peripheral clocks. We conclude that even relatively short-term exposure to low levels of nighttime light can influence metabolism to increase mass gain.

Neuroendocrine control of photoperiodic changes in immune function

Seasonal variation in immune function putatively maximizes survival and reproductive success. Day length (photoperiod) is the most potent signal for time of year. Animals typically organize breeding, growth, and behavior to adapt to spatial and temporal niches. Outside the tropics individuals monitor photoperiod to support adaptations favoring survival and reproductive success. Changes in day length allow anticipation of seasonal changes in temperature and food availability that are critical for reproductive success. Immune function is typically bolstered during winter, whereas reproduction and growth are favored during summer. We provide an overview of how photoperiod influences neuronal function and melatonin secretion, how melatonin acts directly and indirectly to govern seasonal changes in immune function, and the manner by which other neuroendocrine effectors such as glucocorticoids, prolactin, thyroid, and sex steroid hormones modulate seasonal variations in immune function. Potential future research avenues include commensal gut microbiota and light pollution influences on photoperiodic responses.

Cytotoxic chemotherapy increases sleep and sleep fragmentation in non-tumor-bearing mice

Sleep disruption ranks among the most common complaints of breast cancer patients undergoing chemotherapy. Because of the complex interactions among cancer, treatment regimens, and life-history traits, studies to establish a causal link between chemotherapy and sleep disruption are uncommon. To investigate how chemotherapy acutely influences sleep, adult female c57bl/6 mice were ovariectomized and implanted with wireless biotelemetry units. EEG/EMG biopotentials were collected over the course of 3days pre- and post-injection of 13.5mg/kg doxorubicin and 135mg/kg cyclophosphamide or the vehicle. We predicted that cyclophosphamide+doxorubicin would disrupt sleep and increase central proinflammatory cytokine expression in brain areas that govern vigilance states (i.e., hypothalamus and brainstem). The results largely support these predictions; a single chemotherapy injection increased NREM and REM sleep during subsequent active (dark) phases; this induced sleep was fragmented and of low quality. Mice displayed marked increases in low theta (5-7Hz) to high theta (7-10Hz) ratios following chemotherapy treatment, indicating elevated sleep propensity. The effect was strongest during the first dark phase following injection, but mice displayed disrupted sleep for the entire 3-day duration of post-injection sleep recording. Vigilance state timing was not influenced by treatment, suggesting that acute chemotherapy administration alters sleep homeostasis without altering sleep timing. qPCR analysis revealed that disrupted sleep was accompanied by increased IL-6 mRNA expression in the hypothalamus. Together, these data implicate neuroinflammation as a potential contributor to sleep disruption after chemotherapy.

A Role for Hypocretin/Orexin in Metabolic and Sleep Abnormalities in a Mouse Model of Non-metastatic Breast Cancer

We investigated relationships among immune, metabolic, and sleep abnormalities in mice with non-metastatic mammary cancer. Tumor-bearing mice displayed interleukin-6 (IL-6)-mediated peripheral inflammation, coincident with altered hepatic glucose processing and sleep. Tumor-bearing mice were hyperphagic, had reduced serum leptin concentrations, and enhanced sensitivity to exogenous ghrelin. We tested whether these phenotypes were driven by inflammation using neutralizing monoclonal antibodies against IL-6; despite the reduction in IL-6 signaling, metabolic and sleep abnormalities persisted. We next investigated neural populations coupling metabolism and sleep, and observed altered activity within lateral-hypothalamic hypocretin/orexin (HO) neurons. We used a dual HO-receptor antagonist to test whether increased HO signaling was causing metabolic abnormalities. This approach rescued metabolic abnormalities and enhanced sleep quality in tumor-bearing mice. Peripheral sympathetic denervation prevented tumor-induced increases in serum glucose. Our results link metabolic and sleep abnormalities via the HO system, and provide evidence that central neuromodulators contribute to tumor-induced changes in metabolism.

Time-of-Day Dictates Transcriptional Inflammatory Responses to Cytotoxic Chemotherapy

Many cytotoxic chemotherapeutics elicit a proinflammatory response which is often associated with chemotherapy-induced behavioral alterations. The immune system is under circadian influence; time-of-day may alter inflammatory responses to chemotherapeutics. We tested this hypothesis by administering cyclophosphamide and doxorubicin (Cyclo/Dox), a common treatment for breast cancer, to female BALB/c mice near the beginning of the light or dark phase. Mice were injected intravenously with Cyclo/Dox or the vehicle two hours after lights on (zeitgeber time (ZT2), or two hours after lights off (ZT14). Tissue was collected 1, 3, 9, and 24 hours later. Mice injected with Cyclo/Dox at ZT2 lost more body mass than mice injected at ZT14. Cyclo/Dox injected at ZT2 increased the expression of several pro-inflammatory genes within the spleen; this was not evident among mice treated at ZT14. Transcription of enzymes within the liver responsible for converting Cyclo/Dox into their toxic metabolites increased among mice injected at ZT2; furthermore, transcription of these enzymes correlated with splenic pro-inflammatory gene expression when treatment occurred at ZT2 but not ZT14. The pattern was reversed in the brain; pro-inflammatory gene expression increased among mice injected at ZT14. These data suggest that inflammatory responses to chemotherapy depend on time-of-day and are tissue specific.

Time-Restricted Feeding Alters the Innate Immune Response to Bacterial Endotoxin

An important entraining signal for the endogenous circadian clock, independent of light, is food intake. The circadian and immune systems are linked; forced desynchrony of the circadian clock via nighttime light exposure or genetic ablation of core clock components impairs immune function. The timing of food intake affects various aspects of the circadian clock, but its effects on immune function are unknown. We tested the hypothesis that temporal desynchrony of food intake alters innate immune responses. Adult male Swiss Webster mice were provided with food during the night, the day, or ad libitum for 4 wk, followed by administration of LPS prior to the onset of either the active phase (zeitgeber time [ZT]12: Experiment 1) or the inactive phase (ZT0: Experiment 2). Three hours after LPS administration, blood was collected, and serum was tested for bacteria-killing capacity against Escherichia coli, as a functional assay of immune function. Additionally, cytokine expression was examined in the serum (protein), spleen, and hypothalamus (mRNA). Day-fed mice suppressed bacteria-killing capacity and serum cytokine responses to LPS during the active phase (ZT12). Night-fed mice increased bactericidal capacity, as well as serum and hypothalamic mRNA responses of certain proinflammatory cytokines during the active phase. Only day-fed mice enhanced serum cytokine responses when LPS challenge occurred during the inactive phase (ZT0); this did not result in enhanced bactericidal capacity. These data suggest that mistimed feeding has functional relevance for immune function and provide further evidence for the integration of the circadian, metabolic, and immune systems.

Enduring effects of perinatal nicotine exposure on murine sleep in adulthood

The long-term consequences of early life nicotine exposure are poorly defined. Approximately 8-10% of women report smoking during pregnancy, and this may promote aberrant development in the offspring. To this end, we investigated potential enduring effects of perinatal nicotine exposure on murine sleep and affective behaviors in adulthood (~13-15 wk of age) in C57Bl6j mice. Mothers received a water bottle containing 200 µg/ml nicotine bitartrate dihydrate in 2% wt/vol saccharin or pH-matched 2% saccharin with 0.2% (vol/vol) tartaric acid throughout pregnancy and before weaning. Upon reaching adulthood, offspring were tested in the open field and elevated plus maze, as well as the forced swim and sucrose anhedonia tests. Nicotine-exposed male (but not female) mice had reduced mobility in the open field, but no differences were observed in anxiety-like or depressive-like responses. Upon observing this male-specific phenotype, we further assessed sleep-wake states via wireless EEG/EMG telemetry. Following baseline recording, we assessed whether mice exposed to nicotine altered their homeostatic response to 5 h of total sleep deprivation and whether nicotine influenced responses to a powerful somnogen [i.e., lipopolysaccharides (LPS)]. Males exposed to perinatal nicotine decreased the percent time spent awake and increased time in non-rapid eye movement (NREM) sleep, without changes to REM sleep. Nicotine-exposed males also displayed exaggerated responses (increased time asleep and NREM spectral power) to sleep deprivation. Nicotine-exposed animals additionally had blunted EEG slow-wave responses to LPS administration. Together, our data suggest that perinatal nicotine exposure has long-lasting effects on normal sleep and homeostatic sleep processes into adulthood.