In Praise of Masking: Behavioural Responses of Retinally Degenerate Mice to Dim Light

There are two ways in which animals compartmentalize their activities into day or night: they can have an endogenous clock which is synchronized each day by a light-dark cycle or there can be a direct response to light such as the decrease in activity by a nocturnal rodent. In the first case activity is said to be entrained by light. In the second case activity is said to be masked by light. Any demonstration of entrainment by periodic presentation of a stimulus must show that activity occurring in phase with that stimulus is not simply a direct response to the stimulus but represents control of phase by an endogenous clock. Thus masking has come to be something to be avoided and excluded in experiments on circadian rhythms. This has led chronobiologists into displaying a lack of interest or a negative attitude toward masking, though there are some exceptions (e.g., refs. 1-5).     

Circadian modulation of light-induced locomotion responses in Drosophila melanogaster

The relationship between light and the circadian system has long been a matter of discussion. Many studies have focused on entrainment of light with the internal biological clock. Light also functions as an environmental stimulus that affects the physiology and behaviour of animals directly. In this study, we used light as an unexpected stimulus for flies at different circadian times. We found that wildtype flies showed circadian changes in light-induced locomotion responses. Elevation of locomotor activity by light occurred during the subjective night, and performance in response to light pulses declined to trough during the subjective day. Moreover, arrhythmic mutants lost the rhythm of locomotion responses to light, with promotion of activity by light in timeless(01)mutants and inhibition of activity by light in Clock(ar)mutants. However, neither ablation of central oscillators nor disturbance of the functional clock inside compound eyes was sufficient to disrupt the rhythm of light responses. We show that, compound eyes, which have been identified as the control point for normal masking (promotion of activity by light), are sufficient but not necessary for paradoxical masking (suppression of activity by light) under high light intensity. This, taken together with the clear difference of light responses in wildtype flies, suggests that two different masking mechanisms may underlie the circadian modulation of light-induced locomotion responses.     

Nocturnal activity in a diurnal rodent (Arvicanthis niloticus): the importance of masking

It is known that day-active Nile grass rats, Arvicanthis niloticus, increase the amount of activity in the night relative to that in the day when provided with running wheels. This was confirmed in the present study. Animals without a wheel displayed 69.0% of their general activity in the L phase of a 12:12 h light-dark cycle; animals provided with wheels had only 48.6% of their wheel revolutions in the light. The contribution of direct (masking) responses to light to the increased nocturnality of animals with wheels was examined in two experiments. In experiment 1, masking was tested by exposing the animals to repeated cycles of 30 min of entraining light and 30 min of a different, usually dimmer light, during the L phase of a 12:12 h light-dark cycle. For animals with wheels, there was more running during the 30-min pulses of dim light or darkness than during the 30-min periods of entraining light. In contrast, for animals without wheels, there was more general activity during the 30-min periods of entraining light than during the 30-min pulses of dim light or darkness. In experiment 2, the animals were first exposed to a 12:12 h light-dark cycle and then put on a 1:10:1:12 h LDLD skeleton photoperiod. Animals with wheels increased their running during the subjective day of the skeleton photoperiod compared to that in the actual day of the 12:12 h light-dark cycle. Animals without wheels showed similar levels of general activity during the subjective day of the skeleton photoperiod and the actual day of the 12:12 h cycle. These experiments demonstrate that when Nile rats have running wheels, their increased nocturnal activity is associated with an increased suppression of locomotion in direct response to light. It is possible that changes in masking responses to light may be an essential and integral component of switching between diurnal and nocturnal activity profiles.     

The circadian <Emphasis Type="Italic">Clock</Emphasis> mutant mouse: impaired masking response to light

Synchronization of an internal clock (entrainment) and a direct response to light (masking) are complementary ways of restricting activity of an animal to day or night. The protein CLOCK has an important role in the oscillatory mechanism of mammalian pacemakers. Our data show that it is also involved in masking responses. Mice with the Clock/Clock mutation reduced their wheel running less than wildtypes when given 1-h light pulses of light (2–1,600 lx) in the night. With dimmer lights (<2 lx), there were no significant differences between mutant and wildtype mice. Impaired masking responses to light in Clock/Clock mice were confirmed in tests with ultradian light–dark cycles (3.5:3.5 h and 1:1 h). Tests with pulses of light longer than 1 h revealed that, although the mutants responded more slowly to light, they sustained the suppression of activity over the course of the 3-h tests better than wildtypes.     

Social influences on mammalian circadian rhythms: animal and human studies

While light is considered the dominant stimulus for entraining (synchronizing) mammalian circadian rhythms to local environmental time, social stimuli are also widely cited as 'zeitgebers' (time-cues). This review critically assesses the evidence for social influences on mammalian circadian rhythms, and possible mechanisms of action. Social stimuli may affect circadian behavioural programmes by regulating the phase and period of circadian clocks (i.e. a zeitgeber action, either direct or by conditioning to photic zeitgebers), by influencing daily patterns of light exposure or modulating light input to the clock, or by associative learning processes that utilize circadian time as a discriminative or conditioned stimulus. There is good evidence that social stimuli can act as zeitgebers. In several species maternal signals are the primary zeitgeber in utero and prior to weaning. Adults of some species can also be phase shifted or entrained by single or periodic social interactions, but these effects are often weak, and appear to be mediated by social stimulation of arousal. There is no strong evidence yet for sensory-specific nonphotic inputs to the clock. The circadian phase-dependence of clock resetting to social stimuli or arousal (the 'nonphotic' phase response curve, PRC), where known, is distinct from that to light and similar in diurnal and nocturnal animals. There is some evidence that induction of arousal can modulate light input to the clock, but no studies yet of whether social stimuli can shift the clock by conditioning to photic cues, or be incorporated into the circadian programme by associative learning. In humans, social zeitgebers appear weak by comparison with light. In temporal isolation or under weak light-dark cycles, humans may ignore social cues and free-run independently, although cases of mutual synchrony among two or more group-housed individuals have been reported. Social cues may affect circadian timing by controlling sleep-wake states, but the phase of entrainment observed to fixed sleep-wake schedules in dim light is consistent with photic mediation (scheduled variations in behavioural state necessarily create daily light-dark cycles unless subjects are housed in constant dark or have no eyes). By contrast, discrete exercise sessions can induce phase shifts consistent with the nonphotic PRC observed in animal studies. The best evidence for social entrainment in humans is from a few totally blind subjects who synchronize to the 24 h day, or to near-24 h sleep-wake schedules under laboratory conditions. However, the critical entraining stimuli have not yet been identified, and there are no reported cases yet of social entrainment in bilaterally enucleated blind subjects. The role of social zeitgebers in mammalian behavioural ecology, their mechanisms of action, and their utility for manipulating circadian rhythms in humans, remains to be more fully elaborated.     

ELF3 modulates resetting of the circadian clock in Arabidopsis

The Arabidopsis early flowering 3 (elf3) mutation causes arrhythmic circadian output in continuous light, but there is some evidence of clock function in darkness. Here, we show conclusively that normal circadian function occurs with no alteration of period length in elf3 mutants in dark conditions and that the light-dependent arrhythmia observed in elf3 mutants is pleiotropic on multiple outputs normally expressed at different times of day. Plants overexpressing ELF3 have an increased period length in both constant blue and red light; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by using light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and constitutive overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. The phase of ELF3 function correlates with its peak expression levels in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting.     

Lesions of the Intergeniculate Leaflet Lead to a Reorganization in Circadian Regulation and a Reversal in Masking Responses to Photic Stimuli in the Nile Grass Rat

Light influences the daily patterning of behavior by entraining circadian rhythms and through its acute effects on activity levels (masking). Mechanisms of entrainment are quite similar across species, but masking can be very different. Specifically, in diurnal species, light generally increases locomotor activity (positive masking), and in nocturnal ones, it generally suppresses it (negative masking). The intergeniculate leaflet (IGL), a subdivision of the lateral geniculate complex, receives direct retinal input and is reciprocally connected with the primary circadian clock, the suprachiasmatic nucleus (SCN). Here, we evaluated the influence of the IGL on masking and the circadian system in a diurnal rodent, the Nile grass rat (Arvicanthis niloticus), by determining the effects of bilateral IGL lesions on general activity under different lighting conditions. To examine masking responses, light or dark pulses were delivered in the dark or light phase, respectively. Light pulses at Zeitgeber time (ZT) 14 increased activity in control animals but decreased it in animals with IGL lesions. Dark pulses had no effect on controls, but significantly increased activity in lesioned animals at ZT0. Lesions also significantly increased activity, primarily during the dark phase of a 12:12 light/dark cycle, and during the subjective night when animals were kept in constant conditions. Taken together, our results suggest that the IGL plays a vital role in the maintenance of both the species-typical masking responses to light, and the circadian contribution to diurnality in grass rats.     

The relationship between the golden spiny mouse circadian system and its diurnal activity: an experimental field enclosures and laboratory study

Examples of animals that switch activity times between nocturnality and diurnality in nature are relatively infrequent. Furthermore, the mechanism for switching activity time is not clear: does a complete inversion of the circadian system occur in conjunction with activity pattern? Are there switching centers downstream from the internal clock that interpret the clock differently? Or does the switch reflect a masking effect? Answering these key questions may shed light on the mechanisms regulating activity patterns and their evolution. The golden spiny mouse (Acomys russatus) can switch between nocturnal and diurnal activity. This study investigated the relationship between its internal circadian clock and its diurnal activity pattern observed in the field. The goal is to understand the mechanisms underlying species rhythm shifts in order to gain insight into the evolution of activity patterns. All golden spiny mice had opposite activity patterns in the field than those under controlled continuous dark conditions in the laboratory. Activity and body temperature patterns in the field were diurnal, while in the laboratory all individuals immediately showed a free-running rhythm starting with a nocturnal pattern. No phase transients were found toward the preferred nocturnal activity pattern, as would be expected in the case of true entrainment. Moreover, the fact that the free-running activity patterns began from the individuals' subjective night suggests that golden spiny mice are nocturnal and that their diurnality in their natural habitat in the field results from a change that is downstream to the internal clock or reflects a masking effect.     

The Relationship between the Golden Spiny Mouse Circadian System and Its Diurnal Activity: An Experimental Field Enclosures and Laboratory Study

Examples of animals that switch activity times between nocturnality and diurnality in nature are relatively infrequent. Furthermore, the mechanism for switching activity time is not clear: does a complete inversion of the circadian system occur in conjunction with activity pattern? Are there switching centers downstream from the internal clock that interpret the clock differently? Or does the switch reflect a masking effect? Answering these key questions may shed light on the mechanisms regulating activity patterns and their evolution. The golden spiny mouse (Acomys russatus) can switch between nocturnal and diurnal activity. This study investigated the relationship between its internal circadian clock and its diurnal activity pattern observed in the field. The goal is to understand the mechanisms underlying species rhythm shifts in order to gain insight into the evolution of activity patterns. All golden spiny mice had opposite activity patterns in the field than those under controlled continuous dark conditions in the laboratory. Activity and body temperature patterns in the field were diurnal, while in the laboratory all individuals immediately showed a free‐running rhythm starting with a nocturnal pattern. No phase transients were found toward the preferred nocturnal activity pattern, as would be expected in the case of true entrainment. Moreover, the fact that the free‐running activity patterns began from the individuals' subjective night suggests that golden spiny mice are nocturnal and that their diurnality in their natural habitat in the field results from a change that is downstream to the internal clock or reflects a masking effect.     

Impaired Masking Responses to Light in Melanopsin‐Knockout Mice

There are two ways in which an animal can confine its behavior to a nocturnal or diurnal niche. One is to synchronize an endogenous clock that in turn controls the sleep–wake cycle. The other is to respond directly to illumination with changes in activity. In mice, high illumination levels suppress locomotion (negative masking) and low illumination levels enhance locomotion (positive masking). To investigate the role of the newly discovered opsin‐like protein melanopsin in masking, we used 1h and 3h pulses of light given in the night, and also a 3.5:3.5h light–dark (LD) cycle. Mice lacking the melanopsin gene had normal enhancement of locomotion in the presence of dim lights but an impaired suppression of locomotion in the presence of bright light. This impairment was evident only with lights in the order of 10 lux or brighter. This suggests that melanopsin in retinal ganglion cells is involved in masking, as it is in pupil contraction and phase shifts. Melanopsin is especially important in maintaining masking responses over long periods.     

Activity Rhythms and Masking Response in the Diurnal Fat Sand Rat Under Laboratory Conditions

Daily rhythms are heavily influenced by light in two major ways. One is through photic entrainment of a circadian clock, and the other is through a more direct process, referred to as masking. Whereas entraining effects of photic stimuli are quite similar in nocturnal and diurnal species, masking is very different. Laboratory conditions differ greatly from what is experienced by individuals in their natural habitat, and several studies have shown that activity patterns can greatly differ between laboratory environment and natural condition. This is especially prevalent in diurnal rodents. We studied the daily rhythms and masking response in the fat sand rat (Psammomys obesus), a diurnal desert rodent, and activity rhythms of Tristram’s jird (Meriones tristrami), a nocturnal member of the same subfamily (Gerbillinae). We found that most sand rats kept on a 12?h:12?h light-dark (LD) cycles at two light intensities (500 and 1000?lux) have a nocturnal phase preferences of general activity and higher body temperature during the dark phase. In most individuals, activity was not as stable that of the nocturnal Tritram’s jirds, which showed a clear and stable nocturnal activity pattern under the same conditions. Sand rats responded to a 6-h phase advance and 6-h phase delay as expected, and, under constant conditions, all tested animals free ran. In contrast with the nocturnal phase preference, fat sand rats did not show a masking response to light pulses during the dark phase or to a dark pulse during the light phase. They did, however, have a significant preference to the light phase under a 3.5?h:3.5?h LD schedule. Currently, we could not identify the underlying mechanisms responsible for the temporal niche switch in this species. However, our results provide us with a valuable tool for further studies of the circadian system of diurnal species, and will hopefully lead us to understanding diurnality, its mechanisms, causes, and consequences.     

Impaired masking responses to light in melanopsin-knockout mice

There are two ways in which an animal can confine its behavior to a nocturnal or diurnal niche. One is to synchronize an endogenous clock that in turn controls the sleep-wake cycle. The other is to respond directly to illumination with changes in activity. In mice, high illumination levels suppress locomotion (negative masking) and low illumination levels enhance locomotion (positive masking). To investigate the role of the newly discovered opsin-like protein melanopsin in masking, we used 1h and 3h pulses of light given in the night, and also a 3.5:3.5h light-dark (LD) cycle. Mice lacking the melanopsin gene had normal enhancement of locomotion in the presence of dim lights but an impaired suppression of locomotion in the presence of bright light. This impairment was evident only with lights in the order of 10 lux or brighter. This suggests that melanopsin in retinal ganglion cells is involved in masking, as it is in pupil contraction and phase shifts. Melanopsin is especially important in maintaining masking responses over long periods.     

Behavioral responses to combinations of timed light, food availability, and ultradian rhythms in the common vole (Microtus arvalis)

Light is the main entraining signal of the central circadian clock, which drives circadian organization of activity. When food is made available during only certain parts of the day, it can entrain the clock in the liver without changing the phase of the central circadian clock. Although a hallmark of food entrainment is a behavioral anticipation of food availability, the extent of behavioral alterations in response to food availability has not been fully characterized. The authors have investigated interactions between light and temporal food availability in the timing of activity in the common vole. Temporally restricted food availability enhanced or attenuated re-entrainment to a phase advance in light entrainment when it was shifted together with the light or remained at the same time of day, respectively. When light-entrained behavior was challenged with temporal food availability cycles with a different period, two distinct activity components were observed. More so, the present data indicate that in the presence of cycles of different period length of food and light, an activity component emerged that appeared to be driven by a free-running (light-entrainable) clock. Because the authors have previously shown that in the common vole altering activity through running-wheel availability can alter the effectiveness of food availability to entrain the clock in the liver, the authors included running-wheel availability as a parameter that alters the circadian/ultradian balance in activity. In the current protocols, running-wheel availability enhanced the entraining potential of both light and food availability in a differential way. The data presented here show that in the vole activity is a complex of individually driven components and that this activity is, itself, an important modulator of the effectiveness of entraining signals such as light and food.     

Acute Behavioral Responses to Light and Darkness in Nocturnal Mus musculus and Diurnal Arvicanthis niloticus

The term masking refers to immediate responses to stimuli that override the influence of the circadian timekeeping system on behavior and physiology. Masking by light and darkness plays an important role in shaping an organism's daily pattern of activity. Nocturnal animals generally become more active in response to darkness (positive masking) and less active in response to light (negative masking), and diurnal animals generally have opposite patterns of response. These responses can vary as a function of light intensity as well as time of day. Few studies have directly compared masking in diurnal and nocturnal species, and none have compared rhythms in masking behavior of diurnal and nocturnal species. Here, we assessed masking in nocturnal mice (Mus musculus) and diurnal grass rats (Arvicanthis niloticus). In the first experiment, animals were housed in a 12:12 light-dark (LD) cycle, with dark or light pulses presented at 6 Zeitgeber times (ZTs; with ZT0 = lights on). Light pulses during the dark phase produced negative masking in nocturnal mice but only at ZT14, whereas light pulses resulted in positive masking in diurnal grass rats across the dark phase. In both species, dark pulses had no effect on behavior. In the 2nd experiment, animals were kept in constant darkness or constant light and were presented with light or dark pulses, respectively, at 6 circadian times (CTs). CT0 corresponded to ZT0 of the preceding LD cycle. Rhythms in masking responses to light differed between species; responses were evident at all CTs in grass rats but only at CT14 in mice. Responses to darkness were observed only in mice, in which there was a significant increase in activity at CT 22. In the 3rd experiment, animals were kept on a 3.5:3.5-h LD cycle. Surprisingly, masking was evident only in grass rats. In mice, levels of activity during the light and dark phases of the 7-h cycle did not differ, even though the same animals had responded to discrete photic stimuli in the first 2 experiments. The results of the 3 experiments are discussed in terms of their methodological implications and for the insight they offer into the mechanisms and evolution of diurnality.     

Signaling in the mammalian circadian clock: the NO/cGMP pathway

Mammalian circadian rhythms are generated by a hypothalamic suprachiasmatic nuclei (SCN) clock. Light pulses synchronize body rhythms by inducing phase delays during the early night and phase advances during the late night. Phosphorylation events are known to be involved in circadian phase shifting, both for delays and advances. Pharmacological inhibition of the cGMP-dependent kinase (cGK) or Ca2+/calmodulin-dependent kinase (CaMK), or of neuronal nitric oxide synthase (nNOS) blocks the circadian responses to light in vivo. Light pulses administered during the subjective night, but not during the day, induce rapid phosphorylation of both p-CAMKII and p-nNOS (specifically phosphorylated by CaMKII). CaMKII inhibitors block light-induced nNOS activity and phosphorylation, suggesting a direct pathway between both enzymes. Furthermore, SCN cGMP exhibits diurnal and circadian rhythms with maximal values during the day or subjective day. This variation of cGMP levels appears to be related to temporal changes in phosphodiesterase (PDE) activity and not to guanylyl cyclase (GC) activity. Light pulses increase SCN cGMP levels at circadian time (CT) 18 (when light causes phase advances of rhythms) but not at CT 14 (the time for light-induced phase delays). cGK II is expressed in the hamster SCN and also exhibits circadian changes in its levels, peaking during the day. Light pulses increase cGK activity at CT 18 but not at CT 14. In addition, cGK and GC inhibition by KT-5823 and ODQ significantly attenuated light-induced phase shifts at CT 18. This inhibition did not change c-Fos expression SCN but affected the expression of the clock gene per in the SCN. These results suggest a signal transduction pathway responsible for light-induced phase advances of the circadian clock which could be summarized as follows: Glu-Ca2+-CaMKII-nNOS-GC-cGMP-cGK-->-->clock genes. This pathway offers a signaling window that allows peering into the circadian clock machinery in order to decipher its temporal cogs and wheels.     

Nonphotic enhancement of adjustment to new light-dark cycles: masking interpretation discounted

The adjustment of hamsters to advanced light-dark (LD) cycles can be greatly accelerated by scheduling a single 3-hr bout of extra activity in a novel running wheel, starting about 7 hr before the time when the animals become active in the preceding LD cycle. The present experiments were designed to provide stronger evidence that this effect depends on a shift in the pacemaker rather than on masking. It was shown that when hamsters were put into continuous darkness (DD) 1 day after the exercise-accelerated phase shift, their free-running rhythms took off from a time nearer to the onset of darkness in the new LD cycle than in the preceding LD cycle. An incidental finding was that in DD the free-running period of the hamsters with the accelerated phase shifts was longer than that of the control animals. Further evidence that the 3-hr exercise pulse had produced a greater phase advance than that occurring in undisturbed control animals was obtained by giving a light pulse at the same clock time to all animals after they had been in DD for 8 days. The animals that had previously exercised for the additional 3-hr phase-advanced in response to the light pulse, while the undisturbed control animals phase-delayed.     

Mice lacking the PACAP type I receptor have impaired photic entrainment and negative masking

The retinohypothalamic tract (RHT) is a retinofugal neuronal pathway which, in mammals, mediates nonimage-forming vision to various areas in the brain involved in circadian timing, masking behavior, and regulation of the pupillary light reflex. The RHT costores the two neurotransmitters glutamate and pituitary adenylate cyclase activating peptide (PACAP), which in a rather complex interplay are mediators of photic adjustment of the circadian system. To further characterize the role of PACAP/PACAP receptor type 1 (PAC1) receptor signaling in light entrainment of the clock and in negative masking behavior, we extended previous studies in mice lacking the PAC1 receptor (PAC1 KO) by examining their phase response to single light pulses using Aschoff type II regime, their ability to entrain to non-24-h light-dark (LD) cycles and large phase shifts of the LD cycle (jet lag), as well as their negative masking response during different light intensities. A prominent finding in PAC1 KO mice was a significantly decreased phase delay of the endogenous rhythm at early night. In accordance, PAC1 KO mice had a reduced ability to entrain to T cycles longer than 26 h and needed more time to reentrain to large phase delays, which was prominent at low light intensities. The data obtained at late night indicated that PACAP/PAC1 receptor signaling is less important during the phase-advancing part of the phase-response curve. Finally, the PAC1 KO mice showed impaired negative masking behavior at low light intensities. Our findings substantiate a role for PACAP/PAC1 receptor signaling in nonimage-forming vision and indicate that the system is particularly important at lower light intensities.     

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.     

PACAP-Deficient Mice Exhibit Light Parameter–Dependent Abnormalities on Nonvisual Photoreception and Early Activity Onset

Background

The photopigment melanopsin has been suggested to act as a dominant photoreceptor in nonvisual photoreception including resetting of the circadian clock (entrainment), direct tuning or masking of vital status (activity, sleep/wake cycles, etc.), and the pupillary light reflex (PLR). Pituitary adenylate cyclase-activating polypeptide (PACAP) is exclusively coexpressed with melanopsin in a small subset of retinal ganglion cells and is predicted to be involved extensively in these responses; however, there were inconsistencies in the previous reports, and its functional role has not been well understood.

Methodology/Principal Findings

Here we show that PACAP-deficient mice exhibited severe dysfunctions of entrainment in a time-dependent manner. The abnormalities in the mutant mice were intensity-dependent in phase delay and duration-dependent in phase advance. The knockout mice also displayed blunted masking, which was dependent on lighting conditions, but not completely lost. The dysfunctions of masking in the mutant mice were recovered by infusion of PACAP-38. By contrast, these mutant mice show a normal PLR. We examined the retinal morphology and innervations in the mutant mice, and no apparent changes were observed in melanopsin-immunoreactive cells. These data suggest that the dysfunctions of entrainment and masking were caused by the loss of PACAP, not by the loss of light input itself. Moreover, PACAP-deficient mice express an unusually early onset of activities, from approximately four hours before the dark period, without influencing the phase of the endogenous circadian clock.

Conclusions/Significance

Although some groups including us reported the abnormalities in photic entrainments in PACAP- and PAC₁-knockout mice, there were inconsistencies in their results [1], [2], [3], [4]. The time-dependent dysfunctions of photic entrainment in the PACAP-knockout mice described in this paper can integrate the incompatible data in previous reports. The recovery of impaired masking by infusion of PACAP-38 in the mutant mice is the first direct evidence of the relationship between PACAP and masking. These results indicate that PACAP regulates particular nonvisual light responses by conveying parametric light information—that is, intensity and duration. The “early-bird” phenotype in the mutant mice originally reported in this paper supposed that PACAP also has a critical role in daily behavioral patterns, especially during the light-to-dark transition period.     

Thresholds for masking responses to light in three strains of retinally degenerate mice

Mutant mice with retinal degeneration (rd/rd) were given 1-h pulses of light of varying brightness at times of the night when they would normally be active. The mutant mice showed a significantly greater inhibition of locomotor activity to light (negative masking) than wildtype controls. Lack of impairment, or even enhancement of negative masking suggests that this response may depend on sparing in retinally degenerate mice of the same receptor type that mediates clock resetting, because synchronization of the circadian system is known to be unimpaired in these mutants. With very dim light pulses, mutants did not change their activity, but wildtypes actually became more active (positive masking). Positive and negative masking appear to depend on different sensory and central processes. Accepted: 11 May 1998