Mice Deficient of Glutamatergic Signaling from Intrinsically Photosensitive Retinal Ganglion Cells Exhibit Abnormal Circadian Photoentrainment

Several aspects of behavior and physiology, such as sleep and wakefulness, blood pressure, body temperature, and hormone secretion exhibit daily oscillations known as circadian rhythms. These circadian rhythms are orchestrated by an intrinsic biological clock in the suprachiasmatic nuclei (SCN) of the hypothalamus which is adjusted to the daily environmental cycles of day and night by the process of photoentrainment. In mammals, the neuronal signal for photoentrainment arises from a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that send a direct projection to the SCN. ipRGCs also mediate other non-image-forming (NIF) visual responses such as negative masking of locomotor activity by light, and the pupillary light reflex (PLR) via co-release of neurotransmitters glutamate and pituitary adenylate cyclase-activating peptide (PACAP) from their synaptic terminals. The relative contribution of each neurotransmitter system for the circadian photoentrainment and other NIF visual responses is still unresolved. We investigated the role of glutamatergic neurotransmission for circadian photoentrainment and NIF behaviors by selective ablation of ipRGC glutamatergic synaptic transmission in mice. Mutant mice displayed delayed re-entrainment to a 6 h phase shift (advance or delay) in the light cycle and incomplete photoentrainment in a symmetrical skeleton photoperiod regimen (1 h light pulses between 11 h dark periods). Circadian rhythmicity in constant darkness also was reduced in some mutant mice. Other NIF responses such as the PLR and negative masking responses to light were also partially attenuated. Overall, these results suggest that glutamate from ipRGCs drives circadian photoentrainment and negative masking responses to light.     

Behavioral responses of Vipr2-/- mice to light

Vasoactive intestinal polypeptide and its receptor, VPAC2, play important roles in the functioning of the dominant circadian pacemaker, located in the hypothalamic suprachiasmatic nuclei (SCN). Mice lacking VPAC2 receptors (Vipr2-/-) show altered circadian rhythms and impaired synchronization to environmental lighting cues. However, light can increase phosphoprotein and immediate early gene expression in the Vipr2-/- SCN demonstrating that the circadian clock is readily responsive to light in these mice. It is not clear whether these neurochemical responses to light can be transduced to behavioral changes as seen in wild-type (WT) animals. In this study we investigated the diurnal and circadian wheel-running profile of WT (C57BL/6J) and Vipr2-/- mice under a 12-h light:12-h complete darkness (LD) lighting schedule and in constant darkness (DD) and used 1-h light pulses to shift the activity of mice in DD. Unlike WT mice, Vipr2-/- mice show grossly altered locomotor patterns making the analysis of behavioral responses to light problematic. However, analyses of both the onset and the offset of locomotor activity reveal that in a subset of these mice, light can reset the offset of behavioral rhythms during the subjective night. This suggests that the SCN clock of Vipr2-/- mice and the rhythms it generates are responsive to photic stimulation and that these responses can be integrated to whole animal behavioral changes.     

Melanopsin--shedding light on the elusive circadian photopigment

Circadian photoentrainment is the process by which the brain's internal clock becomes synchronized with the daily external cycle of light and dark. In mammals, this process is mediated exclusively by a novel class of retinal ganglion cells that send axonal projections to the suprachiasmatic nuclei (SCN), the region of the brain that houses the circadian pacemaker. In contrast to their counterparts that mediate image-forming vision, SCN-projecting RGCs are intrinsically sensitive to light, independent of synaptic input from rod and cone photoreceptors. The recent discovery of these photosensitive RGCs has challenged the long-standing dogma of retinal physiology that rod and cone photoreceptors are the only retinal cells that respond directly to light and has explained the perplexing finding that mice lacking rod and cone photoreceptors can still reliably entrain their circadian rhythms to light. These SCN-projecting RGCs selectively express melanopsin, a novel opsin-like protein that has been proposed as a likely candidate for the photopigment in these cells. Research in the past three years has revealed that disruption of the melanopsin gene impairs circadian photo- entrainment, as well as other nonvisual responses to light such as the pupillary light reflex. Until recently, however, there was no direct demonstration that melanopsin formed a functional photopigment capable of catalyzing G-protein activation in a light-dependent manner. Our laboratory has recently succeeded in expressing melanopsin in a heterologous tissue culture system and reconstituting a pigment with the 11-cis-retinal chromophore. In a reconstituted biochemical system, the reconstituted melanopsin was capable of activating transducin, the G-protein of rod photoreceptors, in a light-dependent manner. The absorbance spectrum of this heterologously expressed melanopsin, however, does not match that predicted by previous behavioral and electophysiological studies. Although melanopsin is clearly the leading candidate for the elusive photopigment of the circadian system, further research is needed to resolve the mystery posed by its absorbance spectrum and to fully elucidate its role in circadian photoentrainment.     

Masking responses to light in period mutant mice

Masking is an acute effect of an external signal on an overt rhythm and is distinct from the process of entrainment. In the current study, we investigated the phase dependence and molecular mechanisms regulating masking effects of light pulses on spontaneous locomotor activity in mice. The circadian genes, Period1 (Per1) and Per2, are necessary components of the timekeeping machinery and entrainment by light appears to involve the induction of the expression of Per1 and Per2 mRNAs in the suprachiasmatic nuclei (SCN). We assessed the roles of the Per genes in regulating masking by assessing the effects of light pulses on nocturnal locomotor activity in C57BL/6J Per mutant mice. We found that Per1(-/-) and Per2(-/-) mice had robust negative masking responses to light. In addition, the locomotor activity of Per1(-/-)/Per2(-/-) mice appeared to be rhythmic in the light-dark (LD) cycle, and the phase of activity onset was advanced (but varied among individual mice) relative to lights off. This rhythm persisted for 1 to 2 days in constant darkness in some Per1(-/-)/Per2(-/-) mice. Furthermore, Per1(-/-)/Per2(-/-) mice exhibited robust negative masking responses to light. Negative masking was phase dependent in wild-type mice such that maximal suppression was induced by light pulses at zeitgeber time 14 (ZT14) and gradually weaker suppression occurred during light pulses at ZT16 and ZT18. By measuring the phase shifts induced by the masking protocol (light pulses were administered to mice maintained in the LD cycle), we found that the phase responsiveness of Per mutant mice was altered compared to wild-types. Together, our data suggest that negative masking responses to light are robust in Per mutant mice and that the Per1(-/-)/Per2(-/-) SCN may be a light-driven, weak/damping oscillator.     

Circadian photoreception in humans and mice

Circadian rhythms are the endogenous oscillations, occurring with a periodicity of approximately twenty-four hours, in the biochemical and behavioral functions of organisms. In mammals, the phase and period of the rhythm are synchronized to the daily light-dark cycle by light input through the eye. Certain retinal degenerative diseases affecting the photoreceptor cells, both rods and cones, in the outer retina reveal that classical opsins (i.e., rhodopsin and color opsins located in these cells) are essential for vision, but are not required for circadian photoreception. The mammalian cryptochromes and melanopsin (and possibly other opsin family pigments) have been proposed as circadian photoreceptor pigments that exist in the inner retina. Genetic analysis indicates that the cryptochromes, which contain flavin and folate as the light-absorbing cofactors, are the primary circadian photoreceptors. The classical photoreceptors in the outer retina, and melanopsin or other minor opsins in the inner retina, may perform redundant functions in circadian rhythmicity.     

Melanopsin-dependent nonvisual responses: evidence for photopigment bistability in vivo

In mammals, nonvisual responses to light have been shown to involve intrinsically photosensitive retinal ganglion cells (ipRGC) that express melanopsin and that are modulated by input from both rods and cones. Recent in vitro evidence suggests that melanopsin possesses dual photosensory and photoisomerase functions, previously thought to be a unique feature of invertebrate rhabdomeric photopigments. In cultured cells that normally do not respond to light, heterologous expression of mammalian melanopsin confers light sensitivity that can be restored by prior stimulation with appropriate wavelengths. Using three different physiological and behavioral assays, we show that this in vitro property translates to in vivo, melanopsin-dependent nonvisual responses. We find that prestimulation with long-wavelength light not only restores but enhances single-unit responses of SCN neurons to 480-nm light, whereas the long-wavelength stimulus alone fails to elicit any response. Recordings in Opn4-/- mice confirm that melanopsin provides the main photosensory input to the SCN, and furthermore, demonstrate that melanopsin is required for response enhancement, because this capacity is abolished in the knockout mouse. The efficiency of the light-enhancement effect depends on wavelength, irradiance, and duration. Prior long-wavelength light exposure also enhances short-wavelength-induced phase shifts of locomotor activity and pupillary constriction, consistent with the expression of a photoisomerase-like function in nonvisual responses to light.     

A quadruple photoreceptor mutant still keeps track of time

Time measurement and light detection are inextricably linked. Cryptochromes, the blue-light photoreceptors shared between plants and animals, are critical for circadian rhythms in flies and mice [1-3]. WC-1, a putative blue-light photoreceptor, is also essential for the maintenance of circadian rhythms in Neurospora [4]. In contrast, we report here that in Arabidopsis thaliana the double mutant lacking the cryptochromes cry1 and cry2, and even a quadruple mutant lacking the red/ far-red photoreceptor phytochromes phyA and phyB as well as cry1 and cry2, retain robust circadian rhythmicity. Interestingly, the quadruple mutant was nearly blind for developmental responses but perceived a light cue for entraining the circadian clock. These results indicate that cryptochromes and phytochromes are not essential components of the central oscillator in Arabidopsis and suggest that plants could possess specific photosensory mechanisms for temporal orientation, in addition to cryptochromes and phytochromes, which are used for both spatial and temporal adaptation.     

Plasticity of circadian behavior and the suprachiasmatic nucleus following exposure to non-24-hour light cycles

Period aftereffects are a form of behavioral plasticity in which the free-running period of circadian behavior undergoes experience-dependent changes. It is unclear whether this plasticity is age dependent and whether the changes in behavioral period relate to changes in the SCN or the retina, 2 known circadian pacemakers in mammals. To determine whether these changes vary with age, Per1-luc transgenic mice (in which the luciferase gene is driven by the Period1 promoter) of different ages were exposed to short (10 h light: 10 h dark, T20) or long (14 h light: 14 h dark, T28) light cycles (T cycles). Recordings of running-wheel activity in constant darkness (DD) revealed that the intrinsic periods of T20 mice were significantly shorter than of T28 mice at all ages. Aftereffects following the shorter light cycle were significantly smaller in mice older than 3 months, corresponding with a decreased ability to entrain to T20. Age did not diminish entrainment or aftereffects in the 28-h light schedule. The behavioral period of pups born in DD depended on the T cycle experienced in utero, showing maternal transference of aftereffects. Recordings of Per1-luc activity from the isolated SCN in vitro revealed that the SCN of young mice expressed aftereffects, but the periods of behavior and SCN were negatively correlated. Enucleation in DD had no effect on behavioral aftereffects, indicating the eyes are not required for aftereffects expression. These data show that circadian aftereffects are an age-dependent form of plasticity mediated by stable changes in the SCN and, importantly, extra-SCN tissues.     

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.     

Daily restricted feeding resets the circadian clock in the suprachiasmatic nucleus of CS mice

Circadian rhythms in clock gene expressions in the suprachiasmatic nucleus (SCN) of CS mice and C57BL/6J mice were measured under a daily restricted feeding (RF) schedule in continuous darkness (DD), and entrainment of the SCN circadian pacemaker to RF was examined. After 2-3 wk under a light-dark cycle with free access to food, animals were released into DD and fed for 3 h at a fixed time of day for 3-4 wk. Subsequently, they returned to having free access to food for 2-3 wk. In CS mice, wheel-running rhythms entrained to RF with a stable phase relationship between the activity onset and feeding time, and the rhythms started to free run from the feeding time after the termination of RF. mPer1, mPer2, and mBMAL1 mRNA rhythms in the SCN showed a fixed phase relationship with feeding time, indicating that the circadian pacemaker in the SCN entrained to RF. On the other hand, in C57BL/6J mice, wheel-running rhythms free ran under RF, and clock gene expression rhythms in the SCN showed a stable phase relation not to feeding time but to the behavioral rhythms, indicating that the circadian pacemaker in the SCN did not entrain. These results indicate that the SCN circadian pacemaker of CS mice is entrainable to RF under DD and suggest that CS mice have a circadian clock system that can be reset by a signal associated with feeding time.     

Ultraviolet light provides a major input to non-image-forming light detection in mice

The change in irradiance at dawn and dusk provides the primary cue for the entrainment of the mammalian circadian pacemaker. Irradiance detection has been ascribed largely to melanopsin-based phototransduction [1-5]. Here we examine the role of ultraviolet-sensitive (UVS) cones in the modulation of circadian behavior, sleep, and suprachiasmatic nucleus (SCN) electrical activity. UV light exposure leads to phase-shifting responses comparable to those of white light. Moreover, UV light exposure induces sleep in wild-type and melanopsin-deficient (Opn4(-/-)) mice with equal efficacy. Electrical recordings from the SCN of wild-type mice show that UV light elicits irradiance-dependent sustained responses that are similar to those induced by white light, with characteristic fast transient components occurring at the light transitions. These responses are retained in Opn4(-/-) mice and preserved under saturating photopic conditions. The sensitivity of phase-shifting responses to UV light is unaffected by the loss of rods but is severely attenuated by the additional loss of cones. Our data show that UVS cones play an important role in circadian and sleep regulation in mice.     

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2^−/− mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2^−/− were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2^−/− mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light.     

Altered Phase-Relationship between Peripheral Oscillators and Environmental Time in Cry1 or Cry2 Deficient Mouse Models for Early and Late Chronotypes

The mammalian circadian system is composed of a light-entrainable central clock in the suprachiasmatic nuclei (SCN) of the brain and peripheral clocks in virtually any other tissue. It allows the organism to optimally adjust metabolic, physiological and behavioral functions to the physiological needs it will have at specific time of the day. According to the resonance theory, such rhythms are only advantageous to an organism when in tune with the environment, which is illustrated by the adverse health effects originating from chronic circadian disruption by jetlag and shift work. Using short-period Cry1 and long-period Cry2 deficient mice as models for morningness and eveningness, respectively, we explored the effect of chronotype on the phase relationship between the central SCN clock and peripheral clocks in other organs. Whereas the behavioral activity patterns and circadian gene expression in the SCN of light-entrained Cry1^-/- and Cry2^-/- mice largely overlapped with that of wild type mice, expression of clock and clock controlled genes in liver, kidney, small intestine, and skin was shown to be markedly phase-advanced or phase-delayed, respectively. Likewise, circadian rhythms in urinary corticosterone were shown to display a significantly altered phase relationship similar to that of gene expression in peripheral tissues. We show that the daily dissonance between peripheral clocks and the environment did not affect the lifespan of Cry1^-/- or Cry2^-/- mice. Nonetheless, the phase-shifted peripheral clocks in light-entrained mice with morningness and eveningness-like phenotypes may have implications for personalized preventive and therapeutic (i.e. chronomodulation-based) health care for people with early and late chronotypes.     

In search of the pathways for light-induced pacemaker resetting in the suprachiasmatic nucleus

Within the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is a circadian pacemaker that functions as a clock. Its endogenous period is adjusted to the external 24-h light-dark cycle, primarily by light-induced phase shifts that reset the pacemaker's oscillation. Evidence using a wide variety of neurobiological and molecular genetic tools has elucidated key elements that comprise the visual input pathway for SCN photoentrainment in rodents. Important questions remain regarding the intracellular signals that reset the autoregulatory molecular loop within photoresponsive cells in the SCN's retino-recipient subdivision, as well as the intercellular coupling mechanisms that enable SCN tissue to generate phase shifts of overt behavioral and physiological circadian rhythms such as locomotion and SCN neuronal firing rate. Multiple neurotransmitters, protein kinases, and photoinducible genes add to system complexity, and we still do not fully understand how dawn and dusk light pulses ultimately produce bidirectional, advancing and delaying phase shifts for pacemaker entrainment.