Phase and period responses of the circadian system of mice (Mus musculus) to light stimuli of different duration

To understand entrainment of circadian systems to different photoperiods in nature, it is important to know the effects of single light pulses of different durations on the free-running system. The authors studied the phase and period responses of laboratory mice (C57BL6J//OlaHsd) to single light pulses of 7 different durations (1, 3, 4, 6, 9, 12, and 18 h) given once per 11 days in otherwise constant darkness. Light-pulse duration affected both amplitude and shape of the phase response curve. Nine-hour light pulses yielded the maximal amplitude PRC. As in other systems, the circadian period slightly lengthened following delays and shortened following advances. The authors aimed to understand how different parts of the light signal contribute to the eventual phase shift. When PRCs were plotted using the onset, midpoint, and end of the pulse as a phase reference, they corresponded best with each other when using the mid-pulse. Using a simple phase-only model, the authors explored the possibility that light affects oscillator velocity strongly in the 1st hour and at reduced strength in later hours of the pulse due to photoreceptor adaptation. They fitted models based on the 1-h PRC to the data for all light pulses. The best overall correspondence between PRCs was obtained when the effect of light during all hours after the first was reduced by a factor of 0.22 relative to the 1st hour. For the predicted PRCs, the light action centered on average at 38% of the light pulse. This is close to the reference phase yielding best correspondence at 36% of the pulses. The result is thus compatible with an initial major contribution of the onset of the light pulse followed by a reduced effect of light responsible for the differences between PRCs for different duration pulses. The authors suggest that the mid-pulse is a better phase reference than lights-on to plot and compare PRCs of different light-pulse durations.     

The effect of multiple pulses of light on the circadian phase response of the rat.

Multiple pulses of light administered to humans have been reported to result in type 0 phase responses. These results suggest the underlying pacemaker to be nonsimple. At present, results with this type of protocol have only been reported for humans. Therefore, multiple pulses of light were administered to rats. Rats were exposed to one, two, three, or four pulses of light for 5 h (1000 lux) at successive 24-h intervals. Results did not suggest a type 0 phase response. Nonetheless, results with a second, third, or fourth light exposure were not fully predictable from a phase response curve derived from a single light pulse.     

Photobiology of the Gonyaulax circadian system. I. Different phase response curves for red and blue light

Phase responses to red and blue light pulses were measured at different times during the circadian cycle (phase response curves, PRC) in the marine unicellular dinoflagellate Gonyaulaxpolyedra Stein. Pulses were given during a 24-h period of darkness; thereafter, cultures were released into constant dim red light for the assessment of phase and period. The results confirmed earlier findings that the Gonyaulax circadian system receives light signals via two distinct input pathways. During the subjective day and for the first 3 h of the subjective night, red and blue light pulses led to identical phase responses. For the rest of the circadian cycle, however, phase responses to pulses of either red or blue light differed drastically both in their amplitude and direction (advances or delays). Thus, the Gonyaulax light PRC is generated by two distinct light responses. One of these represents responses via a light input that is responsive both to red and blue light mainly producing small delays. The other represents responses of a primarily blue-sensitive input system leading to large advances restricted to the subjective night. Via feed-back, the blue-sensitive light input appears to be under the control of the circadian system. Received: 27 November 1996/Accepted: 30 January 1997     

Light pulses suppress responsiveness within the mouse photic entrainment pathway

We have characterized a decrease in photic responsiveness of the mammalian circadian entrainment pathway caused by light stimulation. Phase delays of the running-wheel activity rhythm were used to quantify the photic responsiveness of the circadian system in mice (C57BL/6J). In an initial experiment, the authors measured the responsiveness to single "saturating" light pulses ("white" fluorescent light; approximately 1876 [microW; 15 min). In two additional experiments, the authors measured responses to this stimulus at several time points following a saturating pulse at CT 14 or CT 16. Data from these experiments were analyzed in two manners. Experiment 2 was analyzed assuming that the phase of the circadian pacemaker was unchanged by an initial pulse, and Experiment 3 was analyzed assuming that the initial pulse induced an instantaneous phase delay. Results reveal a significant reduction in responsivity to light that persists for at least 2 h and possibly up to 4 h after the initial stimulus. Immediately after the stimulus, the responsiveness of the photic entrainment pathway was reduced to levels < or = 12% of normal. After 2 h, the responsiveness was < or = 42% of normal, and by 4 h, responsiveness had recovered to levels that were < or = 60% of normal (levels not statistically different from controls). By the following circadian cycle, responsiveness was more completely recovered, although the magnitude of some phase delays remained < or = 85% of normal. These major reductions in the magnitude of phase delays (and phase response curve amplitude) caused by saturating light pulses confound interpretations of two-pulse experiments designed to measure the rate of circadian phase delays. In addition, the time course for this reduced responsiveness may reflect the time course of cellular and molecular events that underlie light-induced resetting of the mammalian circadian pacemaker.     

Non-photic modulation of phase shifts to long light pulses

Circadian rhythms can be reset by both photic and non-photic stimuli. Recent studies have used long light exposure to produce photic phase shifts or to enhance non-photic phase shifts. The presence or absence of light can also influence the expression of locomotor rhythms through masking; light during the night attenuates locomotor activity, while darkness during the day induces locomotor activity in nocturnal animals. Given this dual role of light, the current study was designed to examine the relative contributions of photic and non-photic components present in a long light pulse paradigm. Mice entrained to a light/dark cycle were exposed to light pulses of various durations (0, 3, 6, 9, or 12 h) starting at the time of lights-off. After the light exposure, animals were placed in DD and were either left undisturbed in their home cages or had their wheels locked for the remainder of the subjective night and subsequent subjective day. Light treatments of 6, 9, and 12 h produced large phase delays. These treatments were associated with decreased activity during the nocturnal light and increased activity during the initial hours of darkness following light exposure. When the wheels were locked to prevent high-amplitude activity, the resulting phase delays to the light were significantly attenuated, suggesting that the activity following the light exposure may have contributed to the overall phase shift. In a second experiment, telemetry probes were used to assess what effect permanently locking the wheels had on the phase shift to the long light pulses. These animals had phase shifts fully as large as animals without any form of wheel lock, suggesting that while non-photic events can modulate photic phase shifts, they do not play a role in the full phase-shift response observed in animals exposed to long light pulses. This paradigm will facilitate investigations into non-photic responses of the mouse circadian system.     

Chronic ethanol intake alters circadian period-responses to brief light pulses in rats

Although chronic alcohol intake is associated with widespread disruptions of sleep-wake cycles and other daily biological rhythms in both human alcoholics and experimental animals, the extent to which the chronobiological effects of alcohol are mediated by effects on the underlying circadian pacemaker remains unknown. Nevertheless, recent studies indicate that both adult and perinatal ethanol treatments may alter the free-running period and photic responsiveness of the circadian pacemaker. The present experiment was designed to further characterize the effects of chronic ethanol intake on the response of the rat circadian pacemaker to brief light pulses. Ethanol-treated and control animals were exposed to 15-min light pulses during either early or late subjective night on the first day of constant darkness following entrainment to a 12:12 light-dark cycle. Relative to pulses delivered during early subjective night and to “no-pulse” conditions, light pulses delivered during late subjective night resulted in period-shortening after-effects under constant darkness, but only in control animals, not in ethanol-treated animals. These results indicate that chronic ethanol intake reduces the responsiveness of the circadian pacemaker to acute photic stimulation, and suggest that the chronobiological disruptions seen in human alcoholics are due in part to alterations in circadian pacemaker function.     

Functioning of the rat circadian system is modified by light applied in critical postnatal days

Lighting conditions influence biological clocks. The present experiment was designed to test the presence of a critical window of days during the lactation stage of the rat in which light has a decisive role on the development of the circadian system. Rats were exposed to 4, 8, or 12 days of constant light (LL) during the first days of life. Their circadian rhythm was later studied under LL and constant darkness. The response to a light pulse was also examined. Results show that the greater the number of LL days during lactation, the stronger the rhythm under LL and the smaller the phase shift due to the light pulse. These responses are enhanced when rats are exposed to LL days around postnatal day 12. A mathematical model was built to explain the responses of the circadian system with respect to the timing of LL during lactation, and we deduced that between postnatal days 10 to 20 there is a critical period of sensitivity to light; consequently, exposure to LL during this time modifies the circadian organization of the motor activity.     

Rapid Phase Resetting of a Mammalian Orcadian Rhythm by Brief Light Pulses

The phase-shift (Δψ) responses of the circadian rhythm in the field mouse Mus booduga to brief light pulses (LPs) of 15 minutes duration and 1000 lux intensity were measured in 90 experiments. In each experiment, a resetting light pulse LP₁ was administered at CT14 (CT, circadian time), and a scanning light pulse LP₂ was then variously administered in separate experiments at CT16, CT20, and CT22 in the same and in the next circadian cycle. The Δψ obtained in all these two-pulse experiments did not differ significantly from theoretical values computed on the assumption that LP₁ reset the phase response curve (PRC) rapidly. In each case, the steady-state Δψ observed after LP₁ and LP₂ differed significantly from the Δψ obtained at the same CT in determination of the single-pulse PRC (control) and also differed significantly from the values on the assumption of no Δψ in the PRC following LP₁. These results indicate that the circadian pacemaker of M. booduga, as measured by its PRC, is substantially reset within 2h after a light pulse at CT14. (Chronobiology International 14(6), 537–548, 1997)     

Phase shifting the hamster circadian clock by 15-minute dark pulses

The mammalian circadian pacemaker can be phase shifted by exposure to a period of darkness interrupting otherwise continuous light. Circadian phase shifting by dark pulses was interpreted originally as reflecting a photic mirror-image mechanism, but more recent observations suggest that dark pulse-induced phase shifting may be mediated by a nonphotic, behavioral state-dependent mechanism. The authors recently presented evidence indicating that the dark-pulse phase response curve (PRC) is in fact a complex function, reflecting both photic mirror image and nonphotic mechanisms at different phases of the circadian cycle. Previous studies of dark pulse-induced phase shifting have universally employed relatively long (2 to 6 h) pulses, which complicates PRC analysis due to the extended segment of the underlying PRC spanned by such a long pulse. The present study was therefore designed to examine the phase-shifting effects of brief 15-min dark pulses presented at both mid-subjective day and subjective dusk, and to explore the possible activity dependence of these effects by using physical restraint to prevent evoked locomotor activity. The results indicate that 15-min dark pulses are effective phase-shifting stimuli at both midday and dusk. Furthermore, as with longer dark pulses, phase shifting by 15-min dark pulses is completely blocked by physical restraint during subjective day but combines in a simple additive manner with the independent phase-shifting effect of restraint at subjective dusk.     

Photoperiod differentially modulates photic and nonphotic phase response curves of hamsters

Circadian pacemakers respond to light pulses with phase adjustments that allow for daily synchronization to 24-h light-dark cycles. In Syrian hamsters, Mesocricetus auratus, light-induced phase shifts are larger after entrainment to short daylengths (e.g., 10 h light:14 h dark) vs. long daylengths (e.g., 14 h light:10 h dark). The present study assessed whether photoperiodic modulation of phase resetting magnitude extends to nonphotic perturbations of the circadian rhythm and, if so, whether the relationship parallels that of photic responses. Male Syrian hamsters, entrained for 31 days to either short or long daylengths, were transferred to novel wheel running cages for 2 h at times spanning the entire circadian cycle. Phase shifts induced by this stimulus varied with the circadian time of exposure, but the amplitude of the resulting phase response curve was not markedly influenced by photoperiod. Previously reported photoperiodic effects on photic phase resetting were verified under the current paradigm using 15-min light pulses. Photoperiodic modulation of phase resetting magnitude is input specific and may reflect alterations in the transmission of photic stimuli.     

Photic resetting and entrainment in CLOCK-deficient mice

Mice lacking the CLOCK protein have a relatively subtle circadian phenotype, including a slightly shorter period in constant darkness, differences in phase resetting after 4-hour light pulses in the early and late night, and a variably advanced phase angle of entrainment in a light-dark (LD) cycle. The present series of experiments was conducted to more fully characterize the circadian phenotype of Clock(-/-) mice under various lighting conditions. A phase-response curve (PRC) to 4-hour light pulses in free-running mice was conducted; the results confirm that Clock(-/-) mice exhibit very large phase advances after 4-hour light pulses in the late subjective night but have relatively normal responses to light at other phases. The abnormal shape of the PRC to light may explain the tendency of CLOCK-deficient mice to begin activity before lights-out when housed in a 12-hour light:12-hour dark lighting schedule. To assess this relationship further, Clock(-/-) and wild-type control mice were entrained to skeleton lighting cycles (1L:23D and 1L:10D:1L:12D). Comparing entrainment under the 2 types of skeleton photoperiods revealed that exposure to 1-hour light in the morning leads to a phase advance of activity onset (expressed the following afternoon) in Clock(-/-) mice but not in the controls. Constant light typically causes an intensity-dependent increase in circadian period in mice, but this did not occur in CLOCK-deficient mice. The failure of Clock(-/-) mice to respond to the period-lengthening effect of constant light likely results from the increased functional impact of light falling in the phase advance zone of the PRC. Collectively, these experiments reveal that alterations in the response of CLOCK-deficient mice to light in several paradigms are likely due to an imbalance in the shape of the PRC to light.     

PHASE AND PERIOD RESPONSES OF THE JERBOA JACULUS ORIENTALIS TO SHORT LIGHT PULSES

The phase and period responses to short light pulses were studied in the jerboa, a seasonal, hibernating, nocturnal rodent from the Atlas region in Morocco. The jerboa, which is a saltatory species, showed precise activity onsets and offsets under a light-dark (LD) cycle using infrared captors to record locomotor activity. When released into constant darkness (DD), the majority of animals showed a circadian period (τ) <24?h (mean τ?=?23.89?±?0.13?h) and a lengthening of the activity span, α. Animals were subsequently exposed to up to eight 15-min light pulses, each separated by at least 2 wks, for up to 160 days in DD. During this span, most individuals maintained robust circadian rhythmicity, with clearly defined activity onsets and offsets, similar levels of total activity, duration of α, and percent activity occurring during the subjective night. The phase response curve (PRC) is typical of other nocturnal rodents, with light eliciting delays during late subjective day and early subjective night (CT8–CT19) and advances during late subjective night to early subjective day (CT19–CT2). A dead zone, when light had no effect on phase, is observed during mid-subjective day (CT3–CT8). A few individuals showed large (>9?h) Type 0 phase resetting near the singularity region (CT19) that resulted in a complete phase reversal, but otherwise displayed normal phase-shifting responses at other CT times. The τ response curve showed a decrease in period from early to late subjective night with increases at other times, but these changes were small (maximum <9?min) and highly variable. There was a distinct tendency for animals that had an initial short τ in DD to conserve a short τ during the series of light pulses and, inversely, for animals with long τ to conserve a long τ. This suggests possible constraints on the plasticity of variation of τ in relation to the endogenous period of the animal. (Author correspondence: howard.cooper@inserm.fr)     

GIGANTEA regulates phytochrome A-mediated photomorphogenesis independently of its role in the circadian clock

GIGANTEA (GI) is a nuclear protein involved in the promotion of flowering by long days, in light input to the circadian clock, and in seedling photomorphogenesis under continuous red light but not far-red light (FR). Here, we report that in Arabidopsis (Arabidopsis thaliana) different alleles of gi have defects in the hypocotyl-growth and cotyledon-unfolding responses to hourly pulses of FR, a treatment perceived by phytochrome A (phyA). This phenotype is rescued by overexpression of GI. The very-low-fluence response of seed germination was also reduced in gi. Since the circadian clock modulates many light responses, we investigated whether these gi phenotypes were due to alterations in the circadian system or light signaling per se. In experiments where FR pulses were given to dark-incubated seeds or seedlings at different times of the day, gi showed reduced seed germination, cotyledon unfolding, and activity of a luciferase reporter fused to the promoter of a chlorophyll a/b-binding protein gene; however, rhythmic sensitivity was normal in these plants. We conclude that while GI does not affect the high-irradiance responses of phyA, it does affect phyA-mediated very-low-fluence responses via mechanisms that do not obviously involve its circadian functions.     

Effect of Light Intensity on the Phase and Period Response Curves in the Nocturnal Field Mouse Mus booduga

The effect of light intensity on the phase response curve (PRC) and the period response curve (τRC) of the nocturnal field mouse Mus booduga was studied. PRCs and τRCs were constructed by exposing animals free-running in constant darkness (DD), to fluorescent light pulses (LPs) of 100 lux and 1000 lux intensities for 15min duration. The waveform of the PRCs and τRCs evoked by high light intensity (1000 lux) stimuli was significantly different compared to those constructed using low light intensity (100 lux). Moreover, a weak but significant correlation was observed between phase shifts and period changes when light stimuli of 1000 lux intensity were used; however, the phase shifts and period changes in the 100 lux PRC and τRC were not correlated. This suggests that the intensity of light stimuli affects both phase and period responses in the locomotor activity rhythm of the nocturnal field mouse M. booduga. These results indicate that complex mechanisms are involved in entrainment of circadian clocks, even in nocturnal rodents, in which PRC, τRC, and dose responses play a significant role.     

Circadian Period Modulation and Masking Effects Induced by Repetitive Light Pulses in Locomotor Rhythms of the Cricket, Gryllus bimaculatus

Effects of 15 min light pulses given at various intervals (every 1, 2, 4, 6, 8 and 12 hr) under constant darkness on the locomotor rhythm were investigated in the adult male cricket, Gryllus bimaculatus. A single pulse per 24 hr induced period modulation in a circadian phase dependent manner, yielding a period modulation curve (PMC): the 15 min light pulse lengthened the period in the early subjective night (CT11-16) and shortened it during the late subjective night to the early subjective day (CT20-5). Frequent light pulses modulated the freerunning period of the rhythm dependent on the interval of the pulses: when compared with the freerunning period in DD (23.74 +/- 0.03 hr) the period was significantly shorter in intervals of 2 and 4 hr, but lengthened when the interval was 1 and 12 hr. Frequent light pulses also resulted in entrainment of the rhythm to run with the period of 24 hr and the ratio of the entrained animals varied from 12% to 72% depending on the interval of the light pulses. The period modulation and the entrainment by the repetitive light pulses could be interpreted according to the PMC. In about 15% of animals, the light pulses induced a rhythm dissociation, suggesting that the bilaterally paired circadian pacemakers have their own sensitivity to the entraining photic information. The light pulse caused a masking effect, i.e., an intense burst of activity. The magnitude of the light induced responses was dependent on the circadian phase. The strongest masking effect was observed in the subjective night. The phase of the prominent period modulation and of the marked masking effects well coincides with the previously reported sensitive phase of the photoreceptive system.     

The Relationships between Phase and Period Responses to Light Pulses

Current theories of stable circadian entrainment postulate phase delays should be associated with period lengthening, while phase advances should be associated with period shortening. While characterising features of the rat PRC to light, we noted substantial numbers of responses that displayed the opposite pattern. Forty-eight rats provided data for 192 phase responses. Limiting our analysis to phase shifts greater than 1 hour, we found 44 displayed the expected predicted relationship, and 33 displayed the contrary paradoxical relationship. Paradoxical responders possessed significantly shorter initial activity periods, compared to predicted responders. Activity was significantly lengthened by paradoxical responders and shortened by predicted responders following light pulse exposure. These results suggest a second mode of stable entrainment. Additionally, these results indicate entrainment mode, predicted or paradoxical, is based upon activity period duration. Short activity period durations will be associated with paradoxical responses, long durations will be associated with predicted responses. We argue that, given the dynamic changes in photoperiod, both modes of entrainment are necessary to provide stable entrainment across the year.     

Circadian response reduction in light and response restoration in darkness: a "skeleton" light pulse PRC study in mice (Mus musculus)

Entrainment may involve responses to dawn, to dusk, and to the light in between these transitions. Previous studies showed that the circadian system responds to only 2 light pulses, one at the beginning and one at the end of the day, in a similar way as to a full photoperiod, as long as the photoperiod is less than approximately 1/2 tau. The authors used a double 1-h light pulse protocol with different intervals of darkness in between (1, 2, 4, 7, 10, and 16 h) to study the phase responses of mice. The phase response curves obtained were compared to full light pulse PRCs of corresponding durations. Up to 6 hours, phase responses induced by double light pulses are virtually the same as by a corresponding full light pulse. The authors made a simple phase-only model to estimate the response reduction due to light exposure and response restoration due to dark exposure of the system. In this model, they assumed a 100% contribution of the first 1-h light pulse and fitted the reduction factor for the second light pulse to yield the best fit to the observations. The results suggest that after 1 h of light followed by less than 4 h of darkness, there is a considerable reduction in response to the second light pulse. Full response restoration requires more than 10 h of darkness. To investigate the influence of the duration of light on the response saturation, the authors performed a second series of experiments where the duration of the 2 light pulses was varied from 4 to 60 min each with a fixed duration of the stimulus (4 h). The response to 2 light pulses saturates when they are between 30 and 60 min long. In conclusion, double pulses replace single full light pulses of a corresponding duration of up to 6 h due to a response reduction during light, combined with response restoration during darkness. By the combined response reduction and response restoration, mice can maintain stable entrainment to the external LD cycle without being continuously exposed to it.     

The Relationships between Phase and Period Responses to Light Pulses

Current theories of stable circadian entrainment postulate phase delays should be associated with period lengthening, while phase advances should be associated with period shortening. While characterising features of the rat PRC to light, we noted substantial numbers of responses that displayed the opposite pattern. Forty-eight rats provided data for 192 phase responses. Limiting our analysis to phase shifts greater than 1 hour, we found 44 displayed the expected predicted relationship, and 33 displayed the contrary paradoxical relationship. Paradoxical responders possessed significantly shorter initial activity periods, compared to predicted responders. Activity was significantly lengthened by paradoxical responders and shortened by predicted responders following light pulse exposure. These results suggest a second mode of stable entrainment. Additionally, these results indicate entrainment mode, predicted or paradoxical, is based upon activity period duration. Short activity period durations will be associated with paradoxical responses, long durations will be associated with predicted responses. We argue that, given the dynamic changes in photoperiod, both modes of entrainment are necessary to provide stable entrainment across the year.     

Light pulses entrain the circadian activity rhythm of a diurnal rodent (Ammospermophilus leucurus)

Circadian rhythms of wheel-running activity of the antelope ground squirrel (Ammospermophilus leucurus) were entrained by light-dark cycles (LD: 100 1x vs total darkness) with periods (T) between ca 23.75 and 24.75 hr. Two 1-hr light pulses per cycle ('skeleton photoperiods') with T = 24.25 hr as well as one 1-hr light pulse per cycle with Ts of 23.75 and 24.25 hr were effective in entraining the circadian activity rhythms in at least 50% of the antelope ground squirrels. Phase and period responses to single 1-hr light pulses were measured which depend on the initial phase and period of the rhythm. It is concluded that discrete (phasic) light input contributes to the mechanism of entrainment to LD cycles in diurnal rodents.     

Asymmetric re‐entrainment and phase response of the circadian activity rhythm of a nocturnal marsupial (Petaurus breviceps: Phalangeridae)

Abstract

Sugar Gliders (Petaurus breviceps) re‐entrain faster after 8‐h delay shifts of an LD 12:12 and an LD 8:16 (31–56:0.3 lux each) than after 8‐h advance shifts of these Zeitgeber cycles. In order to test whether this asymmetric re‐entrainment behavior is related to, or even caused by the phase response characteristics of the circadian system, the phase response of the activity rhythm to short and long light pulses was studied. Short light pulses (15 min of 31–56 lux against a background intensity of 0.3 lux) caused only relatively small delay shifts when applied around the onset, and more pronounced advance shifts when given at the end of the activity time (α). Onset and end of activity shifted by different amounts. Long light pulses produced by 8‐h advances and delays of one single lighttime of an LD 12:12 elicited pronounced phase delays when applied at the beginning of the activity time, but only minor phase advances when given at the posterior part of α. These results indicate that in Petaurus breviceps the phase response characteristics to long light pulses exerting parametric effects of light are responsible for the pronounced asymmetry effect in re‐entrainment. Differing phase responses of onset and end of activity point to a two‐oscillator structure of the circadian pacemaker system in this marsupial.