circadian clocks regulate animal behavior and physiology by generating daily rhythms and coordinating these with local time. If you sleep well and abstain from flying around the globe or working rotating shift schedules, this pervasive influence is easy to take for granted, a background rhythm of life, like respiration. If, on the other hand, your circadian clock has an unusually slow or fast periodicity or if you lack photic input (are truly blind) or if you do fly to new time zones or are forced to alternate between day work and night work, the troubles of circadian timing may on some days seem as severe as a breathing disorder. If the clock is out of phase with local time or with the sleep-wake schedule that daily obligations mandate, it may alternately impede sleep onset and continuity at scheduled bedtime and impair vigilance and cognitive throughput during obligatory wake time. Anybody who regularly struggles with delayed sleep-phase insomnia, jet lag, or shift work malaise can relate. What is the solution?
One strategy is to try to rapidly shift the circadian clock to a better alignment by exploiting its phase-dependent sensitivity to natural resetting stimuli (“zeitgebers”), as described by so-called phase-response curves (PRCs). A ubiquitous and powerful zeitgeber is the daily light-dark cycle; light (or its neurochemical mediator glutamate) early in the night resets the clock back (a delay or westward shift), whereas light late in the night resets it forward (an advance, or eastward shift). Light during the midday has little effect, but in at least some species, some stimuli, generically termed “nonphotic,” can induce respectably sized phase-advance shifts when applied at this time. Nonphotic stimuli with this property include stimulated running or arousal and injections of drug agonists for neuropeptide Y (NPY), serotonin (5-HT), GABA, or enkephalin receptors (8, 9, 28, 30, 37).
It seems, then, that we do have some tools to exploit, both natural and pharmacological, but a problem is that under most conditions, phase shifts to these stimuli are small, in the range of 1 or 2 h, depending on stimulus timing, intensity, and duration (and for light, its wavelength). Consequently, adjustments of circadian timing take time, e.g., ∼1 day for each time zone in hamsters or humans flown east. Combining zeitgebers (zweitgebers, as coined by Marty Zatz) has been shown to help, at least in some animal models (31), if not yet in humans (4), but additional means of amplifying shifts would be gratefully accepted.
In this issue of the American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, Knoch et al. (22) report their third in a recent series of studies in which they describe and pharmacologically characterize a marked potentiation of nonphotic phase shifting in Syrian hamsters by one or two nights of exposure to constant light (brief LL). The inspiration for testing this effect of light was empirically grounded, as follows. There is mixed evidence that nonphotic shifting is mediated by 5-HT (26). In one species, the Syrian hamster, arousal stimulated by running in a novel wheel (30) or by gentle handling (1) in the middle of the “subjective day” (the usual sleep period in nocturnal animals) induces phase-advance shifts in the 1- to 3-h range. These arousal procedures dramatically increase extracellular 5-HT in the suprachiasmatic nucleus (SCN), the site of the master circadian pacemaker in mammals, by two- to threefold (10, 14). Systemic injections of the 5-HT1A/7 agonist 8-(+)2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahyronapthalene (8-OH-DPAT) in the subjective day also induce phase-advance shifts (6, 36), but single injections directly into the SCN produce little or no shift and attempts to block arousal-induced shifts using lesions or a range of 5-HT antagonists have also met with failure (e.g., see Refs. 2 and 7). Nonetheless, if hamsters are pretreated with the 5-HT synthesis inhibitor parachlorophenylalanine, causing 5-HT postsynaptic supersensitivity, then 3-h intra-SCN perfusions via reverse microdialysis do induce shifts comparable in size to those induced by behavioral arousal (12). Given that locomotor activity appears to drive 5-HT release in the SCN (10), this suggests that acute or chronic inhibition of locomotor activity might result in upregulation of SCN responses to nonphotic inputs. This might explain one prior report that young hamsters housed under sedentary conditions (no activity wheel) can exhibit very large phase-advance shifts if their first wheel running experience is stimulated at the beginning of the subjective day (13).
To test this intriguing hypothesis, Knoch et al. (21) subjected hamsters to one or two days of brief LL, because brief LL potently suppresses activity in nocturnal rodents (34). This simple manipulation (which may simulate “unnatural” light exposure experienced during night work or transmeridian jet travel) appears to open wide the gateway for nonphotic shifting. Stimulation by gentle handling or systemic 8-OH-DPAT during the midsubjective day now induced phase-advance shifts two- to threefold larger in magnitude, whereas the same stimuli applied in the middle of the subjective night or at the beginning of the subjective day, normally “dead zones” in the nonphotic PRC, resulted in delay or advance shifts up to 12 h in magnitude. A phase transition plot of these data (new phase as a function of old phase) had a slope of 0, qualifying this as type 0 (or strong) resetting, rarely observed in mammals, and not previously for a neurochemical stimuli. Surprisingly, whereas brief LL did flatten the nocturnal peak of 5-HT and restoration of the peak by reverse 5-HT dialysis partially blocked shift potentiation, restoration of locomotor activity by overnight food deprivation did not affect potentiation. The inferred dissociation between prior activity levels and 5-HT responsivity remains unexplained; in Syrian hamsters, overnight food deprivation does not affect clock resetting by photic or nonphotic stimuli (29), but perhaps it does reduce the amount of 5-HT release that occurs in association with nocturnal activity. Alternatively, brief LL may attenuate 5-HT unit activity or 5-HT release within the SCN, independently of its effects on activity levels. If so, then simply being sedentary is not the key to unlocking strong resetting responses to nonphotic stimuli. This is consonant with observations that 2 days without a wheel does not potentiate nonphotic shifting in hamsters maintained in constant dark (27).
Regardless of the role of activity, the apparent role of 5-HT tone in setting the gain of SCN responses to serotonergics suggested the reasonable hypothesis that potentiated shifting was specific to this particular neurochemical input pathway to the clock. One possibility is that 5-HT receptor numbers are altered by brief LL, such that postsynaptic responses are enhanced and/or presynaptic autoinhibitory responses are attenuated. However, autoradiographic analyses of 8-OH-DPAT binding in the SCN and midbrain raphe have provided no evidence for upregulation or downregulation of 5-HT receptors by brief LL (11, 22). Changes at the level of second messengers remain possible, but there could be a more general mechanism at work. NPY has also been implicated as a mediator of arousal-induced shifts, and the supporting body of evidence is generally considered more internally consistent than is the evidence favoring 5-HT (30). When Knoch et al. (21) tested responsivity to NPY in brief LL-exposed hamsters, they found that shifts were also enhanced approximately twofold in the midsubjective day (they did not test the beginning of the subjective day when 8-OH-DPAT induces 12-h shifts).
What might explain increased responsivity to both 5-HT and NPY? Circadian clocks have been modeled successfully as limit cycle oscillators (20). Limit cycles can exhibit weak or strong resetting depending on the magnitude of the resetting stimulus or the amplitude of oscillation. In humans, critically timed bright light at night does appear to flatten the amplitude of overt rhythms and render the circadian clock amenable to strong resetting by a second or third night of light exposure (19, but see Ref. 5 for a contrary view). If brief LL also suppresses circadian pacemaker amplitude in hamsters, then resetting responses should be potentiated to any stimulus. However, tests of this hypothesis using probes other than NPY and 5-HT have so far provided no supporting evidence; NMDA (a glutamate agonist mimicking light), melatonin, and physical restraint each can shift circadian rhythms in hamsters, with unique PRCs, but brief LL does not affect the magnitude of resetting responses to these stimuli (Ref. 23; Glass JD, personal communication; Webb IQ, Landry GJ, and Mistlberger RE, unpublished data). It is conceivable (if not parsimonious) that brief LL does attenuate pacemaker amplitude but also desensitizes input pathways utilized by NMDA, melatonin, and restraint, thereby offsetting any gain in shift magnitude. A conservative position, therefore, is that the amplitude hypothesis is not yet decisively falsified.
Another approach to the problem is to look directly at the molecular gears of the circadian clock, to see how their amplitude of oscillation and magnitude of change in response to resetting stimuli is affected by brief LL. In the second of their reports, Glass and colleagues (11) measured the response of two so-called circadian clock genes, per1 and per2, which are thought to play a central role in the transcription-translation feedback loops at the core of the mammalian circadian clock (16). Per1 and 2 expression is high during the day and low at night. Photic stimuli appear to reset the loop by driving per expression up, which will delay or advance its cycle depending on whether expression is declining (early in the night) or rising (late in the night), respectively. Nonphotic stimuli do the opposite, and thus can induce advances by suppressing per expression when it is at its peak in the day (15, 18, 25). If brief LL were to tonically raise the level of per in the day, this would provide more room for a nonphotic stimulus to knock it back, and thus induce a larger phase shift. However, neither the basal level nor the magnitude of 8-OH-DPAT-induced suppression of per1 and 2 were enhanced by brief LL, and the only correlate of larger shifts was a significant suppression by 8-OH-DPAT of per1 in the core subregion of the SCN at the very beginning of the subjective day, which was not evident in hamsters not exposed to brief LL (11). In the midsubjective day, potentiation of 8-OH-DPAT-induced phase shifts by brief LL was not associated with larger changes in per1 or 2.
But perhaps we should not expect to see differences at the per gene level, because the per1 cycle has now been shown, in mper1-luc transgenic mice, to be unaffected by LL at the single cell level (33). Rather, it is the synchrony among SCN clock cells that is attenuated by LL and that correlates with changes in behavioral rhythms. LL may also affect coupling relations between putative oscillator ensembles (the so-called evening and morning oscillators, embedded in left-right, core-shell and/or rostral-caudal subdivisions of the bilateral SCN; see Refs. 3, 17, 24, and 35). In silico experiments suggest that altered coupling may be sufficient to effect a switch from weak to strong resetting (32), and this will be worth exploring as a mechanism by which brief LL potentiates nonphotic shifting.
In their latest set of experiments, Knoch and colleagues (22) used 5-HT antagonists and intra-SCN vs. systemic 8-OH-DPAT injections to reveal yet another surprising complication; strong resetting to 8-OH-DPAT or behavioral arousal at the beginning of the subjective day is mediated by intra-SCN 5-HT1A receptors, whereas resetting to these stimuli during the midday is mediated by 5-HT7 receptors outside of the SCN. Brief LL appears to open a 5-HT1A window to clock resetting that is not readily accessible under normal lighting conditions or in constant dark. These results underscore the potential importance of recent light history in setting not only the gain of resetting responses but also the avenues by which such responses can be reached. These analyses indicate that we clearly do not yet know everything about serotonergic clock resetting or the neurochemical basis of nonphotic shifting.
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