Sleep Deprivation Does Not Influence Photic Resetting of Circadian Activity Rhythms in Drosophila.

Previous investigations in humans and rodent animal models have assessed the interplay of sleep in the circadian system's phase responses to nighttime light exposure. The resulting data have been mixed, but generally support a modulatory role for sleep in circadian photic resetting (not an absolute requirement). Drosophila have been historically used to provide important insights in the sleep and circadian sciences. However, no experiments to date have evaluated how immediate sleep need or recent sleep history affects their pacemaker's phase readjustments to light. We did so in the current study by (1) forcing separate groups of animals to stay awake for 1 or 4 h after they were shown a broadspectrum pulse (15 min during the first half of the night, 950 lux), or (2) placing them on a restricted sleep schedule for a week before light presentation without any subsequent sleep disruption. Forced sleep restriction, whether acute or chronic, did not alter the size of light-induced phase shifts. These data are consistent with observations made in other diurnal animals and raise the possibility, more broadly, that phototherapies applied during sleep-such as may be necessary during the winter months-may still be efficacious in individuals experiencing sleep-continuity problems such as insomnia.

Investigation of the aging clock's intermittent-light responses uncovers selective deficits to green millisecond flashes.

The central pacemaker of flies, rodents, and humans generates less robust circadian output signals across normative aging. It is not well understood how changes in light sensitivity might contribute to this phenomenon. In the present study, we summarize results from an extended data series (n = 5681) showing that the locomotor activity rhythm of aged Drosophila can phase-shift normally to intermittently spaced episodes of bright polychromatic light exposure (600 lx) but that deficits emerge in response to 8, 16, and 120-millisecond flashes of narrowband blue (λm, 452 nm) and green (λm, 525 nm) LED light. For blue, phase-resetting of the activity rhythm of older flies is not as energy efficient as it is in younger flies at the fastest flash-exposures tested (8 milliseconds), suggesting there might be different floors of light duration necessary to incur photohabituation in each age group. For green, the responses of older flies are universally crippled relative to those of younger flies across the slate of protocols we tested. The difference in green flash photosensitivity is one of the most salient age-related phenotypes that has been documented in the circadian phase-shifting literature thus far. These data provide further impetus for investigations on pacemaker aging and how it might relate to changes in the circadian system's responses to particular sequences of light exposure tuned for wavelength, intensity, duration, and tempo.

The Drosophila circadian phase response curve to light: Conservation across seasonally relevant photoperiods and anchorage to sunset.

Photic history, including the relative duration of day versus night in a 24-hour cycle, is known to influence subsequent circadian responses to light in mammals. Whether such modulation is present in Drosophila is currently unknown. To date, all photic phase-response curves (PRCs) generated from Drosophila have done so with animals housed under seasonally agnostic equatorial photoperiods with alternating 12-hour segments of light and darkness. However, the genus contains thousands of species, some of which populate high and low-latitude habitats (20-50° north or south of the Equator) where seasonal variations in the light-dark schedule are pronounced. Here, we address this disconnect by constructing the first high-resolution Drosophila seasonal atlas for light-induced circadian phase-resetting. Testing the light responses of over 4,000 Drosophila at 120 timepoints across 5 seasonally-relevant rectangular photoperiods (i.e., LD 8:16, 10:14, 12:12, 14:10, and 16:8; 24 hourly intervals surveyed in each), we determined that many aspects of the fly circadian PRC waveform are conserved with increasing daylength. Surprisingly though, irrespective of LD schedule, the start of the PRCs always remained anchored to the timing of subjective sunset, creating a differential overlap of the advance zone with the morning hours after subjective sunrise that was maximized under summer photoperiods and minimized under winter photoperiods. These data suggest that there may be differences in flies versus mammals as to how the photoperiod modulates the waveform and amplitude of the circadian PRC to light. On the other hand, they support the possibility that the lights-off transition determines the phase-positioning of photic PRCs across seasons and across species. More work is necessary to test this claim and whether it might factor into the timing of seasonal light responses in humans.

Resilience in the suprachiasmatic nucleus: Implications for aging and Alzheimer's disease.

Many believe that the circadian impairments associated with aging and Alzheimer's disease are, simply enough, a byproduct of tissue degeneration within the central pacemaker, the suprachiasmatic nucleus (SCN). However, the findings that have accumulated to date examining the SCNs obtained postmortem from the brains of older individuals, or those diagnosed with Alzheimer's disease upon autopsy, suggest only limited atrophy. We review this literature as well as a complementary one concerning fetal-donor SCN transplant, which established that many circadian timekeeping functions can be maintained with rudimentary (structurally limited) representations of the SCN. Together, these corpora of data suggest that the SCN is a resilient brain region that cannot be directly (or solely) implicated in the behavioral manifestations of circadian disorganization often witnessed during aging as well as early and late progression of Alzheimer's disease. We complete our review by suggesting future directions of research that may bridge this conceptual divide and briefly discuss the implications of it for improving health outcomes in later adulthood.

Meta-analysis of light and circadian timekeeping in rodents.

We conducted a meta-analysis of papers published over the past half-century (1964-2017) that quantified the phase-shifting effects of timed light exposure on rodent locomotor rhythms. Descriptive statistics were tabulated in order to explore the extent to which these studies were generalizable across species, sex, age, circadian timing, and light sources. Attempts at understanding photic resetting were primarily targeted at younger male animals, with particular emphases placed on characterizing the pacemaker systems of C57BL/6 mice and Syrian hamsters during the parts of their subjective night most sensitive to delivery of white-fluorescent light. With subsequent analyses restricted to these rodent models, we then assessed the relationship between luminous exposure (via broadspectrum emission) and phase-shifting through a series of linear regressions. Monotonically increasing illuminance-response functions were noted at most circadian times surveyed. In the aggregate, our results show that previous research conducted on light's regulation of circadian timekeeping has been skewed in design with respect to several important biological variables. This bias might limit translation of phototherapy-relevant data to women and older individuals.

Optimization of circadian responses with shorter and shorter millisecond flashes.

Recent work suggests that the circadian pacemaker responds optimally to millisecond flashes of light, not continuous light exposure as has been historically believed. It is unclear whether these responses are influenced by the physical characteristics of the pulsing. In the present study, Drosophila (n = 2199) were stimulated with 8, 16 or 120 ms flashes. For each duration, the energy content of the exposure was systematically varied by changing the pulse irradiance and the number of stimuli delivered over a fixed 15 min administration window (64 protocols surveyed in all). Results showed that per microjoule invested, 8 ms flashes were more effective at resetting the circadian activity rhythm than 16- and 120 ms flashes (i.e. left shift of the dose-response curve, as well as a higher estimated maximal response). These data suggest that the circadian pacemaker's photosensitivity declines within milliseconds of light contact. Further introduction of light beyond a floor of (at least) 8 ms leads to diminishing returns on phase-shifting.

Responses to Intermittent Light Stimulation Late in the Night Phase Before Dawn.

The circadian clock is comprised of two oscillators that independently track sunset (evening) and sunrise (morning), though little is known about how light responses differ in each. Here, we quantified the morning oscillator's responses to 19 separate pulse trains, collecting observations from over 1300 Drosophila at ZT23. Our results show that the advances in activity onset produced by these protocols depended on the tempo of light administration even when total exposure was conserved across a 15-min window. Moreover, patterns of stimulation previously shown to optimize the evening oscillator's delay resetting at ZT13 (an hour after dusk) were equally effective for the M oscillator at ZT23 (an hour before dawn), though the morning oscillator was by comparison more photosensitive and could benefit from a greater number of fractionation strategies that better converted light into phase-shifting drive. These data continue to build the case that the reading frames for the pacemaker's time-of-day estimates at dusk and dawn are not uniform and suggest that the "photologic" for the evening versus morning oscillator's resetting might be dissociable.

Longitudinal study of sleep and diurnal rhythms in Drosophila ananassae.

Mistiming of circadian rhythms impairs quality of life. The sleep fragmentation that results can lead to fatigue, mood alteration, and short-term memory problems. Unfortunately, this suite occurs in humans as we age. In the current study, we used high-resolution monitors to track how circadian patterns of locomotor activity change in female Drosophila ananassae as they enter mid-to-late life. This equipment is a more recent addition to the fly circadian field and has not been previously used for long-term activity tracking. At 2-3 days post-eclosion, D. ananassae were placed into climate-controlled vivariums for 60 days. Daily actograms were generated for each animal, along with a time series of activity across the observational period. Consistent with findings from older rodents and humans, older D. ananassae exhibited degraded patterns of wake and sleep that were fragmented-but still rhythmic-across the 24-h cycle. Overall levels of daily activity declined with age, with particular loss of circadian arousal in the wake-maintenance zone a few hours before bedtime. Interestingly, our high-resolution monitoring strategy was also able to document a sleep correlation previously associated with human aging in flies: displacement of sleep timing arising from possible changes in circadian and homeostatic regulation. Future experiments may determine whether the age-related impairments seen in the sleep-circadian system of D. ananassae can be mitigated through precision light treatment.

The circadian activity rhythm is reset by nanowatt pulses of ultraviolet light.

The circadian pacemaker synchronizes to the Earth's rotation by tracking step-by-step changes in illumination that occur as the sun passes the horizon. While twilight progressions of irradiance and colour are considered important stimuli in this process, comparably less thought has been given to the possibility that ultraviolet A (UVA) radiation might actually play a more formative role given its evolutionary significance in shaping 24 h timekeeping. Here, we show that Drosophila activity rhythms can be phase-shifted by UVA light at an energy range seated well below that of the visible spectrum. Because the energy threshold for this resetting matches the incident amount of UVA on the human retina at twilight, our results suggest that UVA light has the potential to function as a similar time cue in people.

Precision Light for the Treatment of Psychiatric Disorders.

Circadian timekeeping can be reset by brief flashes of light using stimulation protocols thousands of times shorter than those previously assumed to be necessary for traditional phototherapy. These observations point to a future where flexible architectures of nanosecond-, microsecond-, and millisecond-scale light pulses are compiled to reprogram the brain's internal clock when it has been altered by psychiatric illness or advanced age. In the current review, we present a chronology of seminal experiments that established the synchronizing influence of light on the human circadian system and the efficacy of prolonged bright-light exposure for reducing symptoms associated with seasonal affective disorder. We conclude with a discussion of the different ways that precision flashes could be parlayed during sleep to effect neuroadaptive changes in brain function. This article is a contribution to a special issue on Circadian Rhythms in Regulation of Brain Processes and Role in Psychiatric Disorders curated by editors Shimon Amir, Karen Gamble, Oliver Stork, and Harry Pantazopoulos.

Circadian phase-shifting by light: Beyond photons.

Circadian entrainment to the solar light:dark schedule is thought to be maintained by a simple photon counting method. According to this hypothesis, the pacemaker adjusts the phase of the body's endogenous rhythms in accordance to the intensity and duration with which it encounters a perceived twilight signal. While previous data have generally supported the hypothesis, more recent analysis has codified other factors besides irradiance that influence the magnitude of resetting responses to light delivered within the same phase of the circadian cycle. In particular, the frequency with which light is alternated with darkness, or whether it's packaged in millisecond flashes versus continuous blocks, can significantly alter the dose-response relationship. Here, we used a drosophilid model to test whether circadian photon-counting trends can be broken with light administration protocols spanning just 15 minutes. In the early part of the delay zone, a 15-min continuous light pulse was fragmented until it could no longer produce a full-magnitude shift of the flies' locomotor activity rhythms. The remaining exposure was then reorganized along various fractionation schemes that employed pulses with different widths and interstimulus intervals. Our results suggest that the pacemaker integrates the phase-shifting effects of equiluminous light differently depending on the stimulus pattern with which light is made available. For example, despite having fewer photons, certain ratios of light and darkness could be optimized on a timescale of seconds and minutes so as to achieve pacemaker resetting close to par with steady luminance. These data provide further evidence that the circadian pacemaker's responses to light entail more than photon counting and motivate continued discussion on how phototherapy can be best optimized in clinical practice to improve conditions linked to circadian impairment.

Spontaneous alternation: A potential gateway to spatial working memory in Drosophila.

Despite their ubiquity in biomedical research, Drosophila have yet to be widely employed as model organisms in psychology. Many complex human-like behaviors are observed in Drosophila, which exhibit elaborate displays of inter-male aggression and female courtship, self-medication with alcohol in response to stress, and even cultural transmission of social information. Here, we asked whether Drosophila can demonstrate behavioral indices of spatial working memory in a Y-maze, a classic test of memory function and novelty-seeking in rodents. Our data show that Drosophila, like rodents, alternate their visits among the three arms of a Y-maze and spontaneously favor entry into arms they have explored less recently versus ones they have just seen. These findings suggest that Drosophila possess some of the information-seeking and working memory facilities mammals depend on to navigate through space and might be relevant models for understanding human psychological phenomena such as curiosity.

Glycogen synthase kinase 3, circadian rhythms, and bipolar disorder: a molecular link in the therapeutic action of lithium.

BACKGROUND: Bipolar disorder (BPD) is a widespread condition characterized by recurring states of mania and depression. Lithium, a direct inhibitor of glycogen synthase kinase 3 (GSK3) activity, and a mainstay in BPD therapeutics, has been proposed to target GSK3 as a mechanism of mood stabilization. In addition to mood imbalances, patients with BPD often suffer from circadian disturbances. GSK3, an essential kinase with widespread roles in development, cell survival, and metabolism has been demonstrated to be an essential component of the Drosophila circadian clock. We sought to investigate the role of GSK3 in the mammalian clock mechanism, as a possible mediator of lithium's therapeutic effects. METHODS: GSK3 activity was decreased in mouse embryonic fibroblasts (MEFs) genetically and pharmacologically, and changes in the cyclical expression of core clock genes--mPer2 in particular--were examined. RESULTS: We demonstrate that genetic depletion of GSK3 in synchronized oscillating MEFs results in a significant delay in the periodicity of the endogenous clock mechanism, particularly in the cycling period of mPer2. Furthermore, we demonstrate that pharmacological inhibition of GSK3 activity by kenpaullone, a known antagonist of GSK3 activity, as well as by lithium, a direct inhibitor of GSK3 and the most common treatment for BPD, induces a phase delay in mPer2 transcription that resembles the effect observed with GSK3 knockdown. CONCLUSION: These results confirm GSK3 as a plausible target of lithium action in BPD therapeutics, and suggest the circadian clock mechanism as a significant modulator of lithium's clinical benefits.