Cell-mediated exon skipping normalizes dystrophin expression and muscle function in a new mouse model of Duchenne Muscular Dystrophy.

Cell therapy for muscular dystrophy has met with limited success, mainly due to the poor engraftment of donor cells, especially in fibrotic muscle at an advanced stage of the disease. We developed a cell-mediated exon skipping that exploits the multinucleated nature of myofibers to achieve cross-correction of resident, dystrophic nuclei by the U7 small nuclear RNA engineered to skip exon 51 of the dystrophin gene. We observed that co-culture of genetically corrected human DMD myogenic cells (but not of WT cells) with their dystrophic counterparts at a ratio of either 1:10 or 1:30 leads to dystrophin production at a level several folds higher than what predicted by simple dilution. This is due to diffusion of U7 snRNA to neighbouring dystrophic resident nuclei. When transplanted into NSG-mdx-Δ51mice carrying a mutation of exon 51, genetically corrected human myogenic cells produce dystrophin at much higher level than WT cells, well in the therapeutic range, and lead to force recovery even with an engraftment of only 3-5%. This level of dystrophin production is an important step towards clinical efficacy for cell therapy.

Mechanical loading and hyperosmolarity as a daily resetting cue for skeletal circadian clocks.

Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light and feeding). The presence of tissue niche-dependent physiological time cues has been proposed, allowing tissues the ability of circadian phase adjustment based on local signals. However, to date, such stimuli have remained elusive. Here we show that daily patterns of mechanical loading and associated osmotic challenge within physiological ranges reset circadian clock phase and amplitude in cartilage and intervertebral disc tissues in vivo and in tissue explant cultures. Hyperosmolarity (but not hypo-osmolarity) resets clocks in young and ageing skeletal tissues and induce genome-wide expression of rhythmic genes in cells. Mechanistically, RNAseq and biochemical analysis revealed the PLD2-mTORC2-AKT-GSK3ß axis as a convergent pathway for both in vivo loading and hyperosmolarity-induced clock changes. These results reveal diurnal patterns of mechanical loading and consequent daily oscillations in osmolarity as a bona fide tissue niche-specific time cue to maintain skeletal circadian rhythms in sync.

The mouse suprachiasmatic nucleus encodes irradiance via a diverse population of neurons monotonically tuned to different ranges of intensity.

Many neurons of the mammalian master circadian oscillator in the suprachiasmatic nuclei (SCN) respond to light pulses with irradiance-dependent changes in firing. Here, we set out to better understand this irradiance coding ability by considering how the SCN tracks more continuous changes in irradiance at both population and single unit level. To this end, we recorded extracellular activity in the SCN of anaesthetised mice presented with up + down irradiance staircase stimuli covering moonlight to daylight conditions and incorporating epochs with steady light or superimposed higher frequency modulations (temporal white noise (WN) and frequency/contrast chirps). Single unit activity was extracted by spike sorting. The population response of SCN units to this stimulus was a progressive increase in firing rate at higher irradiances. This relationship was symmetrical for up vs. down phases of the ramp in the presence of white noise or chirps but exhibited hysteresis for steady light, with firing systematically higher during increasing irradiance. Single units also showed a monotonic relationship between firing and irradiance but exhibited diversity not only in response polarity (increases vs. decreases in firing), but also in the sensitivity (EC50 ) and slope of fitted functions. These data show that individual SCN neurons exhibit monotonic relationships between irradiance and firing rate but differ in the irradiance range over which they respond. This property may help the SCN to encode the large differences in irradiance found in nature using neurons with a constrained range of firing rates. KEY POINTS: Daily changes in environmental light (irradiance) entrain the suprachiasmatic nucleus (SCN) circadian clock. The mouse SCN shows graded increases in neurophysiological activity with light pulses of increasing irradiance. We show that this monotonic relationship between firing rate and irradiance is retained at population and single unit level when probed with more naturalistic staircase increases and decreases in irradiance. The irradiance response is more reliable in the presence of ongoing higher temporal frequency modulations in light intensity than under steady light. Single units varied in sensitivity allowing the population to cover a wide range of irradiances. Irradiance coding in the SCN has characteristics of a sparse code with individual neurons tracking different portions of the natural irradiance range. This property may address the challenge of encoding a 109 -fold day:night difference in irradiance within the constrained range of firing rates available to individual neurons.

Effects of long-term individual housing of middle-aged female Octodon degus on spatial learning and memory in the Barnes maze task.

Introduction: Prolonged social isolation is a form of passive chronic stress that has consequences on human and animal behavior. The present study was undertaken to elucidate whether the long-term isolation would precipitate age-related changes in anxiety and spatial learning and memory in degus. Methods: We investigated the effects of long-term social isolation on anxiety levels in the light-dark test, and spatial orientation abilities in the Barnes maze. Middle-aged female Octodon degus were allocated to either group-housed (3 animals per cage) or individually-housed for 5 months. Results: Under this experimental condition, there were no significant group differences in the anxiety level tested in the light-dark test and in the motivation to escape from the Barnes maze. There were no significant differences in cortisol levels between individually- and group-housed animals. On the last acquisition training day of spatial learning, individually- housed animals had a significantly higher number of correct responses and a smaller number of reference and working memory errors than the group-housed animals. In addition, isolated animals showed a tendency for reference and working memory impairment on the retention trial, while group-housed degus showed improvement in these parameters. Discussion and conclusion: The present study indicates that prolonged social isolation during adulthood in female degus has a dual effect on spatial orientation. Specifically, it results in a significant improvement in acquisition skills but a slight impairment in memory retention. The obtained cognitive changes were not accompanied by modification in anxiety and cortisol levels.

The genomic basis of temporal niche evolution in a diurnal rodent.

Patterns of diel activity-how animals allocate their activity throughout the 24-h daily cycle-play key roles in shaping the internal physiology of an animal and its relationship with the external environment.1,2,3,4,5 Although shifts in diel activity patterns have occurred numerous times over the course of vertebrate evolution,6 the genomic correlates of such transitions remain unknown. Here, we use the African striped mouse (Rhabdomys pumilio), a species that transitioned from the ancestrally nocturnal diel niche of its close relatives to a diurnal one,7,8,9,10,11 to define patterns of naturally occurring molecular variation in diel niche traits. First, to facilitate genomic analyses, we generate a chromosome-level genome assembly of the striped mouse. Next, using transcriptomics, we show that the switch to daytime activity in this species is associated with a realignment of daily rhythms in peripheral tissues with respect to the light:dark cycle and the central circadian clock. To uncover selection pressures associated with this temporal niche shift, we perform comparative genomic analyses with closely related rodent species and find evidence of relaxation of purifying selection on striped mouse genes in the rod phototransduction pathway. In agreement with this, electroretinogram measurements demonstrate that striped mice have functional differences in dim-light visual responses compared with nocturnal rodents. Taken together, our results show that striped mice have undergone a drastic change in circadian organization and provide evidence that the visual system has been a major target of selection as this species transitioned to a novel temporal niche.

Slow vision: Measuring melanopsin-mediated light effects in animal models.

Mammalian retinas contain three specialized photoreceptors: the rods and cones in the outer retina, whose primary function is to support visual perception in dim and bright environments, respectively, and a small subset of retinal ganglion cells ("intrinsically photosensitive" retinal ganglion cells; ipRGCs), which are directly light-responsive owing to their expression of the photopigment melanopsin. Melanopsin photoreception is optimized to encode low-frequency changes in the light environment and, as a result, extends the temporal and spatial range over which light is detected by the retina. ipRGCs innervate many brain areas, and this allows melanopsin light responses to be used for diverse purposes, ranging from the synchronization of the circadian clock with the solar day to light's regulation of mood, alertness, and neuroendocrine and cognitive functions. In this review, we discuss the methods and findings that have contributed to our understanding of melanopsin across biology. We particularly focus on the approaches that allow melanopsin to be studied at a systems/whole animal level and how these methods have illuminated the role of melanopsin in diverse physiological outputs.

Daily electrical activity in the master circadian clock of a diurnal mammal.

Circadian rhythms in mammals are orchestrated by a central clock within the suprachiasmatic nuclei (SCN). Our understanding of the electrophysiological basis of SCN activity comes overwhelmingly from a small number of nocturnal rodent species, and the extent to which these are retained in day-active animals remains unclear. Here, we recorded the spontaneous and evoked electrical activity of single SCN neurons in the diurnal rodent Rhabdomys pumilio, and developed cutting-edge data assimilation and mathematical modeling approaches to uncover the underlying ionic mechanisms. As in nocturnal rodents, R. pumilio SCN neurons were more excited during daytime hours. By contrast, the evoked activity of R. pumilio neurons included a prominent suppressive response that is not present in the SCN of nocturnal rodents. Our modeling revealed and subsequent experiments confirmed transient subthreshold A-type potassium channels as the primary determinant of this response, and suggest a key role for this ionic mechanism in optimizing SCN function to accommodate R. pumilio's diurnal niche.

Behavioral and Thermoregulatory Responses to Changes in Ambient Temperature and Wheel Running Availability in Octodon degus.

Octodon degus is primarily a diurnal species, however, in laboratory conditions, it can switch from diurnal to nocturnal in response to wheel running availability. It has been proposed that this activity inversion obeys thermoregulatory constraints induced by vigorous physical exercise. Thus, its activity shifts to the night as the ambient temperature is lower.Here, we investigate the relationship between thermoregulation and the activity phase-inversion in response to wheel-running in this species. We measured behavioral activity and body temperature rhythms in diurnal naïve animals under 12 h light: 12 h dark cycles at four different ambient temperatures (spanning from ~26°C to 32°C), and following access to running wheels while maintained under high ambient temperature.Our results show that naïve degus do not shift their diurnal activity and body temperature rhythms to a nocturnal phase when subjected to sequential increases in ambient temperature. However, when they were provided with wheels under constant high-temperature conditions, all animals inverted their diurnal phase preference becoming nocturnal. Both, negative masking by light and entrainment to the dark phase appeared involved in the nocturnalism of these animals. Analysis of the thermoregulatory response to wheel running revealed some differences between masked and entrained nocturnal chronotypes.These data highlight the importance of the coupling between wheel running availability and ambient temperature in the nocturnalism of the degus. The results support the view that an innate "protective" pre-program mechanism (associating darkness and lower ambient temperature) may change the timing of behavioral activity in this species to reduce the potential risk of hyperthermia.

Timed daily exercise remodels circadian rhythms in mice.

Regular exercise is important for physical and mental health. An underexplored and intriguing property of exercise is its actions on the body's 24 h or circadian rhythms. Molecular clock cells in the brain's suprachiasmatic nuclei (SCN) use electrical and chemical signals to orchestrate their activity and convey time of day information to the rest of the brain and body. To date, the long-lasting effects of regular physical exercise on SCN clock cell coordination and communication remain unresolved. Utilizing mouse models in which SCN intercellular neuropeptide signaling is impaired as well as those with intact SCN neurochemical signaling, we examined how daily scheduled voluntary exercise (SVE) influenced behavioral rhythms and SCN molecular and neuronal activities. We show that in mice with disrupted neuropeptide signaling, SVE promotes SCN clock cell synchrony and robust 24 h rhythms in behavior. Interestingly, in both intact and neuropeptide signaling deficient animals, SVE reduces SCN neural activity and alters GABAergic signaling. These findings illustrate the potential utility of regular exercise as a long-lasting and effective non-invasive intervention in the elderly or mentally ill where circadian rhythms can be blunted and poorly aligned to the external world.

Bright daytime light enhances circadian amplitude in a diurnal mammal.

Mammalian circadian rhythms are orchestrated by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN), which receives information about the 24 h light-dark cycle from the retina. The accepted function of this light signal is to reset circadian phase in order to ensure appropriate synchronization with the celestial day. Here, we ask whether light also impacts another key property of the circadian oscillation, its amplitude. To this end, we measured circadian rhythms in behavioral activity, body temperature, and SCN electrophysiological activity in the diurnal murid rodent Rhabdomys pumilio following stable entrainment to 12:12 light-dark cycles at four different daytime intensities (ranging from 18 to 1,900 lx melanopic equivalent daylight illuminance). R. pumilio showed strongly diurnal activity and body temperature rhythms in all conditions, but measures of rhythm robustness were positively correlated with daytime irradiance under both entrainment and subsequent free run. Whole-cell and extracellular recordings of electrophysiological activity in ex vivo SCN revealed substantial differences in electrophysiological activity between dim and bright light conditions. At lower daytime irradiance, daytime peaks in SCN spontaneous firing rate and membrane depolarization were substantially depressed, leading to an overall marked reduction in the amplitude of circadian rhythms in spontaneous activity. Our data reveal a previously unappreciated impact of daytime light intensity on SCN physiology and the amplitude of circadian rhythms and highlight the potential importance of daytime light exposure for circadian health.

Widespread Doublecortin Expression in the Cerebral Cortex of the Octodon degus.

It has been demonstrated that in adulthood rodents show newly born neurons in the subgranular layer (SGL) of the dentate gyrus (DG), and in the subventricular zone (SVZ). The neurons generated in the SVZ migrate through the rostral migratory stream (RMS) to the olfactory bulb. One of the markers of newly generated neurons is doublecortin (DCX). The degu similarly shows significant numbers of DCX-labeled neurons in the SGL, SVZ, and RMS. Further, most of the nuclei of these DCX-expressing neurons are also labeled by proliferating nuclear antigen (PCNA) and Ki67. Finally, whereas in rats and mice DCX-labeled neurons are predominantly present in the SGL and SVZ, with only a few DCX neurons present in piriform cortex, the degu also shows significant numbers of DCX expressing neurons in areas outside of SVZ, DG, and PC. Many areas of neocortex in degu demonstrate DCX-labeled neurons in layer II, and most of these neurons are found in the limbic cortices. The DCX-labeled cells do not stain with NeuN, indicating they are immature neurons.

Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons.

There is no consensus on the best inhibitory optogenetic tool. Since Gi/o signalling is a native mechanism of neuronal inhibition, we asked whether Lamprey Parapinopsin ("Lamplight"), a Gi/o-coupled bistable animal opsin, could be used for optogenetic silencing. We show that short (405 nm) and long (525 nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, respectively, and that combining these wavelengths can be used to achieve intermediate levels of activity. These properties can be applied to produce switchable neuronal hyperpolarisation and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. Expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, with 405 and 525 nm stimuli producing responses of opposite sign in the output neurons of the retina. We conclude that bistable animal opsins can co-opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and reversible.

A circadian clock in the sinus node mediates day-night rhythms in Hcn4 and heart rate.

BACKGROUND: Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night. OBJECTIVE: The lower heart rate during sleep is assumed to be neural in origin, but here we tested whether a day-night difference in intrinsic pacemaking is involved. METHODS: In vivo and in vitro electrocardiographic recordings, vagotomy, transgenics, quantitative polymerase chain reaction, Western blotting, immunohistochemistry, patch clamp, reporter bioluminescence recordings, and chromatin immunoprecipitation were used. RESULTS: The day-night difference in the average heart rate of mice was independent of fluctuations in average locomotor activity and persisted under pharmacological, surgical, and transgenic interruption of autonomic input to the heart. Spontaneous beating rate of isolated (ie, denervated) sinus node (SN) preparations exhibited a day-night rhythm concomitant with rhythmic messenger RNA expression of ion channels including hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4). In vitro studies demonstrated 24-hour rhythms in the human HCN4 promoter and the corresponding funny current. The day-night heart rate difference in mice was abolished by HCN block, both in vivo and in the isolated SN. Rhythmic expression of canonical circadian clock transcription factors, for example, Brain and muscle ARNT-Like 1 (BMAL1) and Cryptochrome (CRY) was identified in the SN and disruption of the local clock (by cardiomyocyte-specific knockout of Bmal1) abolished the day-night difference in Hcn4 and intrinsic heart rate. Chromatin immunoprecipitation revealed specific BMAL1 binding sites on Hcn4, linking the local clock with intrinsic rate control. CONCLUSION: The circadian variation in heart rate involves SN local clock-dependent Hcn4 rhythmicity. Data reveal a novel regulator of heart rate and mechanistic insight into bradycardia during sleep.

Perforated Multi-Electrode Array Recording in Hypothalamic Brain Slices.

The ability to record ensemble action potential (AP) discharge frequencies from large populations of neurons over extended periods of time in vitro offers clear advantages in neuroscience and circadian biology research. Here, we provide an overview of a step-by-step method to perform multisite extracellular AP activity recordings in suprachiasmatic and mediobasal hypothalamic nuclei brain slices, using a state-of-the-art perforated multielectrode array system. Further, we describe in detail a setup architecture which systematically delivers stable, high-quality recordings with excellent anatomical accuracy and consistency. We also provide some procedural, technical, and methodological troubleshooting notes and examples of good quality recordings.

Spectral sensitivity of cone vision in the diurnal murid Rhabdomys pumilio.

An animal's temporal niche - the time of day at which it is active - is known to drive a variety of adaptations in the visual system. These include variations in the topography, spectral sensitivity and density of retinal photoreceptors, and changes in the eye's gross anatomy and spectral transmission characteristics. We have characterised visual spectral sensitivity in the murid rodent Rhabdomys pumilio (the four-striped grass mouse), which is in the same family as (nocturnal) mice and rats but exhibits a strong diurnal niche. As is common in diurnal species, the R. pumilio lens acts as a long-pass spectral filter, providing limited transmission of light <400 nm. Conversely, we found strong sequence homologies with the R. pumilio SWS and MWS opsins and those of related nocturnal species (mice and rats) whose SWS opsins are maximally sensitive in the near-UV. We continued to assess in vivo spectral sensitivity of cone vision using electroretinography and multi-channel recordings from the visual thalamus. These revealed that responses across the human visible range could be adequately described by those of a single pigment (assumed to be MWS opsin) maximally sensitive at ∼500 nm, but that sensitivity in the near-UV required inclusion of a second pigment whose peak sensitivity lay well into the UV range (λmax<400 nm, probably ∼360 nm). We therefore conclude that, despite the UV-filtering effects of the lens, R. pumilio retains an SWS pigment with a UV-A λmax In effect, this somewhat paradoxical combination of long-pass lens and UV-A λmax results in narrow-band sensitivity for SWS cone pathways in the UV-A range.

Melatonin alleviates circadian system disruption induced by chronic shifts of the light-dark cycle in Octodon degus.

Modern 24-h society lifestyle is associated with experiencing frequent shifts in the lighting conditions which can negatively impact human health. Here, we use the degus, a species exhibiting diurnal and nocturnal chronotypes, to: (a) assess the impact of chronic shifts of the light:dark (LD) cycle in the animal's physiology and behaviour and (b) test the therapeutic potential of melatonin in enhancing rhythmicity under these conditions. Degus were subjected to a "5d + 2d" LD-shifting schedule for 19 weeks. This protocol aims to mimic lighting conditions experienced by humans during shift work: LD cycle was weekly delayed by 8h during 5 "working" days (Morning, Afternoon and Night schedule); during weekends (2 days), animals were kept under Morning schedule. After 9 weeks, melatonin was provided daily for 6h in the drinking water. The "5d + 2d" shifting LD schedule led to a disruption in wheel-running activity (WRA) and body temperature (Tb) rhythms which manifested up to three separate periods in the circadian range. This chronodisruption was more evident in nocturnal than in diurnal degus, particularly during the Afternoon schedule when a phase misalignment between WRA and Tb rhythms appeared. Melatonin treatment and, to a lesser extent, water restriction enhanced the 24-h component, suggesting a potential role in ameliorating the disruptive effects of shift work.

Light color importance for circadian entrainment in a diurnal (Octodon degus) and a nocturnal (Rattus norvegicus) rodent.

The central circadian pacemaker (Suprachiasmatic Nuclei, SCN) maintains the phase relationship with the external world thanks to the light/dark cycle. Light intensity, spectra, and timing are important for SCN synchronisation. Exposure to blue-light at night leads to circadian misalignment that could be avoided by using less circadian-disruptive wavelengths. This study tests the capacity of a diurnal Octodon degus and nocturnal Rattus norvegicus to synchronise to different nocturnal lights. Animals were subjected to combined red-green-blue lights (RGB) during the day and to: darkness; red light (R); combined red-green LED (RG) lights; and combined red-green-violet LED (RGV) lights during the night. Activity rhythms free-ran in rats under a RGB:RG cycle and became arrhythmic under RGB:RGV. Degus remained synchronised, despite the fact that day and night-time lighting systems differed only in spectra, but not in intensity. For degus SCN c-Fos activation by light was stronger with RGB-light than with RGV. This could be relevant for developing lighting that reduces the disruptive effects of nocturnal light in humans, without compromising chromaticity.

Contributions of the lateral habenula to circadian timekeeping.

Over the past 20years, substantive research has firmly implicated the lateral habenula in myriad neural processes including addiction, depression, and sleep. More recently, evidence has emerged suggesting that the lateral habenula is a component of the brain's intrinsic daily or circadian timekeeping system. This system centers on the master circadian pacemaker in the suprachiasmatic nuclei of the hypothalamus that is synchronized to the external world through environmental light information received directly from the eye. Rhythmic clock gene expression in suprachiasmatic neurons drives variation in their electrical activity enabling communication of temporal information, and the organization of circadian rhythms in downstream targets. Here, we review the evidence implicating the lateral habenula as part of an extended neural circadian system. We consider findings suggesting that the lateral habenula is a recipient of circadian signals from the suprachiasmatic nuclei as well as light information from the eye. Further we examine the proposition that the lateral habenula itself expresses intrinsic clock gene and neuronal rhythms. We then speculate on how circadian information communicated from the lateral habenula could influence activity and function in downstream targets such as the ventral tegmental area and raphe nuclei.

Circadian system functionality, hippocampal oxidative stress, and spatial memory in the APPswe/PS1dE9 transgenic model of Alzheimer disease: effects of melatonin or ramelteon.

Alzheimer disease (AD) is a neurodegenerative disorder that primarily causes ß-amyloid accumulation in the brain, resulting in cognitive and behavioral deficits. AD patients, however, also suffer from severe circadian rhythm disruptions, and the underlying causes are still not fully known. Patients with AD show reduced systemic melatonin levels. This may contribute to their symptoms, since melatonin is an effective chronobiotic and antioxidant with neuroprotective properties. Here, the authors critically assessed the effects of long-term melatonin treatment on circadian system function, hippocampal oxidative stress, and spatial memory performance in the APPswe/PS1 double transgenic (Tg) mouse model of AD. To test if melatonin MT1/MT2 receptor activation, alone, was involved, the authors chronically treated some mice with the selective MT1/MT2 receptor agonist ramelteon. The results indicate that many of the circadian and behavioral parameters measured, including oxidative stress markers, were not significantly affected in these AD mice. During the day, though, Tg controls (Tg-CON) showed significantly higher mean activity and body temperature (BT) than wild-type (WT) mice. Overall, BT rhythm amplitude was significantly lower in Tg than in WT mice. Although melatonin treatment had no effect, ramelteon significantly reduced the amplitude of the BT rhythm in Tg mice. Towards the end of the experiment, Tg mice treated with ramelteon (Tg-RAM) showed significantly higher circadian rhythm fragmentation than Tg-CON and reduced circadian BT rhythm strength. The free-running period (τ) for the BT and locomotor activity (LA) rhythms of Tg-CON was <24 h. Whereas melatonin maintained τ at 24 h for BT and LA in both genotypes, ramelteon treatment had no effect. In the behavioral tests, the number of approaches and time spent exploring novel objects were significantly higher in Tg-CON than WT controls. Brain tissue analysis revealed significant reduction in hippocampal protein oxidation in Tg-MEL and Tg-RAM compared with Tg-CON animals. These results suggest that not all aspects of the circadian system are affected in the APPswe/PS1 mice. Therefore, care should be taken when extending the results obtained in Tg mice to develop new therapies in humans. This study also revealed the complexity in the therapeutic actions of melatonin and ramelteon in this mouse model of AD.

Exercise and melatonin in humans: reciprocal benefits.

The aim of this review is to update the reader as to the association between physical exercise and melatonin, and to clarify how the melatonin rhythm may be affected by different types of exercise. Exercise may act as a zeitgeber, although the effects of exercise on the human circadian system are only now being explored. Depending on the time of the day, on the intensity of light, and on the proximity of the exercise to the onset or decline of the circadian production of melatonin, the consequence of exercise on the melatonin rhythm varies. Moreover, especially strenuous exercise per se induces an increased oxidative stress that in turn may affect melatonin levels in the peripheral circulation because indole is rapidly used to combat free radical damage. On the other hand, melatonin also may influence physical performance, and thus, there are mutually interactions between exercise and melatonin production which may be beneficial.