What this is
- Light influences the expression of () in the (), which is critical for circadian rhythms.
- The study investigates how light and the regulate expression, particularly under different light/dark cycles.
- Findings indicate that while is primarily controlled by the , light also plays a significant modulatory role.
Essence
- expression in the is regulated by both light and the . Light can induce expression even in the absence of a functional , suggesting a direct role for light in regulating circadian rhythms.
Key takeaways
- expression rapidly adjusts to phase delays in light/dark cycles but shows limited response to phase advances. This indicates that the primarily governs 's molecular rhythm.
- Under varying photoperiods, expression expands with longer light periods but does not contract with shorter light periods, demonstrating a dominant circadian influence.
- In mice lacking functional circadian clocks, a low amplitude rhythm persists under light/dark conditions, confirming that light can drive expression independently of the circadian system.
Caveats
- The study primarily uses mouse models, which may limit the generalizability of the findings to other species, including humans.
- The effects of light on expression were observed under specific experimental conditions, which may not fully replicate natural light exposure scenarios.
Definitions
- prokineticin 2 (PK2): A clock-controlled gene involved in the regulation of circadian locomotor rhythms.
- suprachiasmatic nucleus (SCN): A brain region that serves as the master circadian clock, regulating daily rhythms in physiology and behavior.
- circadian clock: An internal biological mechanism that regulates physiological processes on a roughly 24-hour cycle.
AI simplified
Background
Light is the principal zeitgeber that entrains circadian rhythms of physiology and behaviour [,]. The major light input pathway to the suprachiasmatic nucleus (SCN) is the retinohypothalamic tract [], which arises from a population of retinal ganglion cells []. Recent studies have demonstrated that melanopsin-containing retinal ganglion cells, rods, and cones all convey photic information to the SCN, and mice lacking these photoreceptive systems cannot be entrained by light [-]. Excellent progress has been made in the understanding of circadian photic entrainment [-]. This includes light-induced transcriptional activation of core clock genes in the SCN, such asand, as well as immediate-early gene. Exposure to light pulses at night induces expression of these genes in the SCN, and this light induction mechanism has been suggested as a critical pathway for the resetting of circadian clock in response to changes in light/dark conditions [-]. Intercellular signalling mechanisms between SCN neurons are also important in circadian photic entrainment, as mice with mutation in a neuropeptide receptor for VIP (Vasoactive Intestinal Peptide) and PACAP (Pituitary Adenylate Cyclase Activating Peptide) are unable to sustain normal circadian behaviour and exhibit loss of sensitivity to light []. 1 2 3 4 5 11 12 15 16 19 20 Per1 Per2 c-fos
In addition to the effect of light on circadian entrainment, light also has a direct effect on physiology and behaviour, generally termed as "masking" [,]. For instance, light pulses given at night acutely suppress the locomotor behaviour of nocturnal rodents [,], and this can occur without functional clockwork [-]. Masking may account for the maintenance under normal light/dark conditions of wheel-running rhythms in cryptochrome-deficient () mice, which are behaviourally arrhythmic under constant darkness. The contribution of masking to normal locomotor activity rhythms is unclear, as is the participation of the SCN in masking effects of light. Vitaterna et al (1999) first observed a light-drivenrhythm in the SCN inmice, and have suggested that the light-driven molecular rhythm in the SCN may be related to the preservation of their locomotor rhythm []. 21 22 21 22 23 27 25 Cry1-/-Cry2-/- Per2 Cry1-/-Cry2-/-
We previously found that prokineticin 2 () is a first order clock-controlled gene, whose expression in the SCN is regulated by CLOCK and BMAL1 acting on the E-boxes in the gene's promoter []. We have also demonstrated that PK2 may function as a SCN output molecule that transmits circadian locomotor rhythm via activation of a G protein-coupled receptor [,]. Interestingly, we also observed thatexpression is rapidly induced by light pulses administered at night [], a characteristic that is usually seen with core clockwork genes but not clock-controlled genes. Here we further investigated the light regulation of the rhythm ofexpression in the SCN. In particular, we investigated the photoreceptive mechanisms responsible for the light-inducedexpression in the SCN. Utilizingmice, we also determined whether light can driveexpression in the SCN independent of a functional circadian clock. PK2 PK2 PK2 PK2 Cry1-/-Cry2-/- PK2 28 28 29 28
Results
responds differentially to the delay and advance of light/dark cycles PK2
We first examined the effects of abrupt shifts of light/dark cycles onmRNA rhythm in the SCN. Animals were first entrained for two weeks under 12 hour light: 12 hour dark (LD), then subjected to either a 6 hour delay (6hrD) shift or 6 hour advance (6hrA) shift of light/dark cycles. We measuredmRNA in the SCN of these animals to examine how quickly themRNA rhythm re-entrains to the shifted light/dark cycles. Under LD,mRNA peaks during the day and remains low or undetectable during the night. During the first cycle of the delayed shift (6hrD), themRNA rhythm responds quickly: the rising phase ofexpression adjusts rapidly to the delayed light/dark cycles, while the falling phase still resembles that of the unshifted light/dark cycles (Figure). In contrast, themRNA rhythm responds very little to a 6 hour advance shift (6hrA). During the first cycle of the advance shift, theoscillation pattern remains similar to that of the unshifted LD (Figure). These changes inexpression during 6hrD or 6hrA shift indicate that the endogenous circadian clock exerts dominant control over therhythm, asexpression cannot respond immediately and completely to the shifts of light/dark cycles. PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 1A 1B
As it normally takes about 1–2 days for locomotor rhythms to stably entrain to phase delays and about 5–6 days to entrain to phase advances [,], we next examined the timecourse of shifts of therhythm to 6 hour phase advances and delays. Consistent with the animal's locomotor behaviour, themRNA rhythm reaches stable phase within 2 days of 6hrD shift (Figure). In contrast, only the rise ofreaches stable phase within 2 days of 6hrA shift, while the fall oftakes longer (Figure). Thus, we further examined whether therhythm is stably entrained after 6 days of 6hrA shift. As expected, therhythm is completely entrained to 6hrA shift after 6 days (Figure). Together, the differential responses ofrhythm to a 6hrD or 6hrA shift indicate that the endogenous circadian clock predominantly controlsrhythm, as circadian oscillators typically show rapid phase delays but advance with transients [,]. The entrainment patterns ofduring phase shifts are consistent with behavioural studies in animals and human subjects [,]. 30 31 1C 1D 1D 31 32 30 31 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2
rhythm is entrained by different photoperiods PK2
We next examined the effect of photoperiod on themolecular rhythm in the SCN.mRNA was measured in the SCN of mice entrained under different photoperiods: 8 hour light: 16 hour dark (8L:16D), 16 hour light: 8 hour dark (16L:8D), or 20 hour light: 4 hour dark (20L:4D). During 12L:12D,mRNA is highly expressed during the 12 hour light phase with peak level at ZT4 (Figure, Figure). Under 16L:8D,mRNA expands to the entire 16 hour light phase and is essentially undetectable during the 8 hour dark period (Figure). However, the expression ofmRNA is not confined to the light phase of the shorter photoperiod (8L:16D), asmRNA rises before lights on and persists after lights off (Figure). The temporal profile ofmRNA under this short photoperiod (8L:16D) is very similar to that observed under 12L:12D (Figure, Figure) or constant darkness (2DD) []. Thus, although light can inducemRNA and expand the duration ofexpression, the phase angle ofexpression is determined by the circadian clock, and its duration cannot be further compressed under shorter photoperiods. Interestingly, the peak ofmRNA expression was significantly higher in long days (16L:8D) than in shorter days (8L:16D) (Figure), further indicate the enhancing effect of light onexpression. However, a significant reduction in thepeak level is observed under a very long photoperiod (20L: 4D) (Figure). We also noticed that under 20L:4D,mRNA is further expanded and becomes detectable even in dark phase (Figure). Under this long photoperiod (20L:4D), the difference between the peak and basal level ofis only about 4 fold (Figure). As it has been reported that the rhythms ofandmRNAs in the SCN are also entrained with different phase angles under a variety of photoperiods [-], we have also examinedandrhythm in our photoperiod studies (see). Theandrhythm we observed under these photoperiods are consistent with previous findings []. Taken together, these results indicate that changes in photoperiod alterrhythm in the SCN, and the amplitudes ofmRNA oscillation are greatly reduced in very long photoperiods. PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 mPer1 mPer2 Per1 Per2 Per1 Per2 PK2 PK2 1A 3A 2B 2A 1A 3A 28 2A–B 2C 2C 2C 33 35 Additional file 1 35
Light inducibility ofis eliminated in mice that lack melanopsin, rod and cone phototransduction system (mice) PK2 Opn4-/-, Gnat1-/- Cnga3-/-
As melanopsin has been implicated in circadian photoreception [-], we examined whether themolecular rhythm is normally entrained in melanopsin-deficient () mice. Figureshows that the oscillation profile ofin the SCN ofmice is essentially identical to that observed in the wild type mice under LD. This normal temporal profile ofmRNA corresponds with the normal locomotor rhythm ofmice under light/dark conditions [,]. Asmice display attenuated phase resetting in response to light pulses and exhibit impaired light masking responses to bright light [], we also examined whether light inducibility ofis blunted inmice. Figureshows that light pulse-inducedin the SCN ofmice was significantly reduced by about 50% and 60%, one and two hours after the light pulse, respectively. 5 11 3 7 8 36 3B PK2 Opn4-/- PK2 Opn4-/- PK2 Opn4-/- Opn4-/- PK2 Opn4-/- PK2 Opn4-/-
Thelight pulse studies show that a residualexpression is still present after a light pulse, suggesting that without melanopsin, other phototransduction system can still transmit light information to induceexpression. Thus, we decided to examine the light inducibility ofin triple knockout mice lacking melanopsin, rod and cone phototransduction systems (mice), as these animals free run under light dark conditions (LD) and lack masking responses to light []. Figureshows that the light pulse-inducedin the SCN is completely eliminated in these triple knockout mice, consistent with their malfunctioning photoentrainment systems and their lack of masking responses to light []. In addition, we also observed thatmRNA followed the free-running locomotor rhythms in these triple knockout mice (Figure), with high levels ofduring the inactive phase (CT3) and low levels during active phase (CT15). Together, these results suggest that melanopsin contributes to the light inducibility of, and intact melanopsin with functional rod/cone phototransduction systems are required for the light inducibility of. Opn4-/- PK2 PK2 PK2 Opn4-/- Gnat1-/- Cnga3-/- PK2 PK2 PK2 PK2 PK2 10 3C 10 3D
A low amplituderhythm is preserved in cryptochrome-deficient () mice under light/dark conditions PK2 Cry1-/-Cry2-/-
Previous studies have shown that the light-regulatedrhythm is maintained in the SCN of cryptochrome-deficient () mice that lack functional circadian clock [,]. In order to determine whether the regulation of,,andexpression by light requires an intact circadian pacemaker, we systematically assessed the temporal mRNA profiles of clockwork genes inmice under both light/dark (LD) and constant dark (DD) conditions. Figureshows that the molecular rhythm ofremained largely intact inmice entrained under12L:12D, with levels about 4-fold higher during the light phase than the dark phase. This amplitude of theoscillation profile was similar to that observed in wild type mice [,]. A low amplituderhythm inmice was also apparent under LD, but not DD (Figure). We further detected a light-drivenrhythm in the SCN ofmice under LD, but not DD (Figure). Interestingly, thisrhythm inmice peaked during light phase, opposite from therhythm in wild type mice and in phase with[,]. As it has been suggested that PER2 can positively regulateexpression via inhibition of the orphan nuclear receptor REV-ERBα [,], it is possible that thisrhythm is secondary to the light-drivenrhythm. Further studies are required to clarify this observation. Per2 Cry1-/-Cry2-/- PK2 Per1 Per2 Bmal1 Cry1-/-Cry2-/- Per2 Cry1-/-Cry2-/- Per2 Per1 Cry1-/-Cry2-/ Bmal1 Cry1-/-Cry2-/- Bmal1 Cry1-/-Cry2-/- Bmal1 Per1 Bmal1 Bmal1 Per2 25 37 4 18 38 4B 4C 39 40 41 42
We also examined the molecular rhythm ofinmice. Figureshows thatmRNA rhythm in the SCN ofmice was apparent under LD, with the presence of a low levelduring light phase and absence ofduring dark phase (see). Similar to wild type mice, the peak level of this low amplituderhythm was around ZT4, although its peak was only about 8% of that observed in wild type mice (Figure., Figure, Figure). Norhythm was evident whenmice were placed under DD (Figure). Furthermore, the inducibility ofto nocturnal light pulses is also maintained inmice.mRNA increased one and two hours after a brief light pulse at ZT14 (Figure). Nevertheless, light-inducedwas still detected inmice andmice that lack functional circadian clock (Cheng, Weaver & Zhou, unpublished observations). Asremains responsive to light in these clock mutant mice that lack functional circadian clock, it is likely that the low amplituderhythm inmice under LD is directly driven by light. PK2 Cry1-/-Cry2-/- PK2 Cry1-/-Cry2-/- PK2 PK2 PK2 PK2 Cry1-/-Cry2-/- PK2 Cry1-/-Cry2-/- PK2 PK2 Per1,2,3-/- Clk-/- PK2 PK2 Cry1-/-Cry2-/- 4D Additional file 2 4D 1A 3A 4D 4E
In order to test whether this light-drivenrhythm may be related to the maintenance of behavioural rhythms observed inmice under LD, we studied the responses ofmice to a 6 hour advance of lighting schedule. In contrast to the transients of entrainment of locomotor rhythms in wild type mice (which takes about 4-5 days to re-entrain to phase advance), the locomotor activity ofmice adjusted rapidly to 6 hr advance (Figure). Such a rapid response is characteristic of masking. A correlative rapid adjustment ofwas also observed in the SCN ofmice (Figure). Asmice lack functional circadian clock and their locomotor behaviour andexpression patterns are completely light driven, our results suggest that this low amplitude, light-driven rhythm ofmay contribute to or underlie the masking of locomotor behaviour in these animals. PK2 Cry1-/-Cry2-/- Cry1-/-Cry2-/- Cry1-/-Cry2-/- PK2 Cry1-/-Cry2-/- Cry1-/-Cry2-/- PK2 PK2 4F 4G
Discussion
Our studies indicate that the molecular rhythm ofin the SCN is predominantly controlled by the circadian clock, with light playing a modulatory role. Abrupt shifts of light/dark cycles significantly altered the phase of therhythm. Whileexpression re-entrained rapidly to phase delays, it takes several cycles of transients forto be stably entrained to phase advances (Figure). The rate of re-entrainment ofmolecular rhythms to these shifts is consistent with that of behavioural adaptation of animals and human subjects [,]. Our photoperiod studies indicate thatexpression in the SCN responds differentially to changes in photoperiod length (Figure). Although increasing light period can induceexpression and expand the duration ofrhythm (Figure), shortening of the light period does not lead to corresponding reduction of the duration ofexpression (Figure). It appears that a minimal duration ofexpression is maintained under short photoperiod (Figure) and constant darkness [], which further indicate the dominant control ofexpression by the circadian clock. Interestingly, the amplitude of theoscillation was greatly reduced under very long photoperiod (20L:4D) (Figure). As the amplitude of bothandrhythms were also reduced during 20L:4D (see), it is likely that these depressed rhythms of clockwork genes may contribute to the depressedrhythm observed. Whether reduction in the amplitude of expression in any of these genes is related to arrhythmicity in LL deserves further examination. PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 PK2 Per1 Per2 PK2 1 30 31 2 2B 2A 2A 28 2C Additional file 1
Our studies withmice revealed the presence of a light-drivenmolecular rhythm in the SCN under LD, indicating that light can driverhythm independent of functional circadian clock. Interestingly, the molecular rhythms of some clockwork genes such as,, andwere also partially maintained in the SCN ofmice under LD (Figure). Thus, light-driven molecular oscillations of clockwork or clock-controlled output genes exist in the absence of functional circadian clock. Vitaterna et al (1999) first noticed such light-regulatedmolecular rhythm in the SCN ofmice, and suggested the term of "light-driving" effect []. Asmice lack functional circadian clocks and their locomotor behaviour remains rhythmic under LD, but not under DD conditions [,], it is likely that these light-driven molecular rhythms may drive the locomotor rhythms in these animals. As we have previously shown thatmay be a critical output molecule responsible for circadian locomotor rhythms, the presence of this light-drivenrhythm inmice may thus contribute to or underlie masking as well as the free running behavioural rhythms in these animals. It is well established that an intact SCN is necessary for the preservation of free running locomotor rhythms []. The role of the SCN in masking of locomotor activity by light is controversial, with similar studies having produced contradictory results [,]. Thus, it is possible that there might be common signal molecule(s) that mediate(s) the light-masking and the circadian clock-controlled locomotor behaviour. Construction of-deficient mice will be necessary to resolve the exact role of PK2 in the light-driven locomotor rhythms. Cry1-/-Cry2-/- PK2 PK2 Per2 Per1 Bmal1 Cry1-/-Cry2-/- Per2 Cry1-/-Cry2-/- Cry1-/-Cry2-/- PK2 PK2 Cry1-/-Cry2-/- PK2 4 25 24 25 43 23 44
The light inducibility ofin the SCN is an unusual characteristic for a clock-controlled gene. Our results demonstrate that melanopsin-positive retinal ganglion cells, in conjunction with rods and cones, are responsible for the light-inducibility of(Figure). The same photoreceptive system has been shown responsible for the entrainment of locomotor rhythm [-]. The light inducibility ofmay be related to the presence of a putative cyclic AMP response element (CRE) in the promoter of thegene []. CRE-dependent activation is critical for light-induced gene expression in the SCN [-]. The reduced light inducibility ofin mutant mice that lack functional clock may indicate that CRE-dependent pathway and CLK/BMAL1 transcriptional factors may interact in the light-inducedexpression in the SCN. Accumulative data have implicated the photic regulation of the transcription of clock genes such asandin the entrainment of behavioural rhythms [,]. The phase of the core SCN clock gene expression determines the timing of clock-controlled SCN output signals that ultimately regulate physiology and behaviour. Unlike thepromoter, whose activation in the SCN shifts rapidly when the LD cycle is advanced [],exhibits transients during phase advance, more similar to those ofand[,]. This is consistent with the role for PK2 as a clock-controlled gene and thus is downstream from the light-regulated expression ofor. The presence of E box motifs in thepromoter suggests that light-regulated(and perhaps) expression can influenceexpression. However, the light inducibility ofindicates that PK2 may have a more direct and central role in entrainment in addition to its putative role as an SCN output signal. In other words, whether PK2 functions completely outside the central circadian loops or partly within them has yet to be determined. It is well established that the activation of glutamate receptor and its downstream actions are critical for the retinohypothalamic inputs of light to the SCN []. As receptor for PK2 is highly expressed in the SCN [] and activation of the PK2 receptor triggers similar signalling pathways as that of glutamate receptors [], it is possible that the circadian clock and/or light-driven PK2 may feed back to the core circadian loops in the SCN. In addition, PK2 has recently been shown to excite neurons that express PK2 receptor [], further suggesting that PK2 may activate the firing of SCN neurons, and thus possibly participate in the synchronization of the circadian clock. Thus, the light inducibility ofmay be relevant to both the phase resetting of the core circadian loops and critical SCN output signals. PK2 PK2 PK2 PK2 PK2 PK2 Per1 Per2 Per1 PK2 Cry1 Cry2 Per1 Per2 PK2 Per1 Per2 PK2 PK2 PK2 3 5 11 28 45 48 30 34 31 30 31 49 28 29 50
Conclusion
Our studies demonstrate thatis predominantly driven by the circadian clock, asexpression exhibits circadian transients in response to phase advances. Furthermore, shortening of the light period does not result in corresponding reduction of the phase ofrhythm, also consistent with the dominant control from the circadian clock onexpression. However, light also modulatesrhythm. Nocturnal light pulses can directly induceexpression in the SCN. Studies withmice revealed that light can drive a low amplitudemolecular rhythm in the SCN in the absence of functional circadian oscillators. These studies demonstrate thatmolecular rhythm in the SCN is controlled by dual mechanisms: dominantly by the circadian transcriptional loops but also directly by light. The light inducibility ofin the SCN suggest that in addition to PK2's role as a SCN output signal, PK2 may also participate in the photic entrainment of circadian clock and perhaps in masking. PK2 PK2 PK2 PK2 PK2 PK2 Cry1-/-Cry2-/- PK2 PK2 PK2
Methods
Experiments of light/dark cycle shifts
Male adult C57BL/6 mice (Taconic Farms, New York) were entrained under 12 hour light: 12 hour dark (12L:12D, lights on at 0700 h) cycle for two weeks with food and water available ad libitum. Light phase was either delayed by 6 hours (lights on at 1300 h) or advanced by 6 hours (lights on at 0100 h) and samples were taken every three hours for the 24 hour period (Zeitgeber time, ZT, ZT1-22). To examineexpression two days after the shift, animals were placed in two additional light/dark cycles and brain samples were collected. All animal procedures were approved by the Institutional Animal Care and Use Committee and consistent with Federal guidelines. In situ hybridization was used in all studies to examinemRNA expression in the SCN []. Antisense and sense riboprobes containing the coding region of mouse(accession number1-528 nt), mouse(accession number340-761nt), mouse(accession number9-489 nt) and mouse(accession number864-1362 nt) were generated. PK2 PK2 PK2 Per1 Per2 Bmal1 28 AF487280 AF022992 AF035830 AB015203
Photoperiod studies
Animals were initially entrained under 12L:12D for one week, followed by placement in different photoperiods (light intensity ~400 lux) for three to four weeks: 8 hour light:16 hour dark (8L:16D, lights on at 0900 h, lights off at 1700 h), 16 hour light: 8 hour dark (16L:8D, lights on at 0500 h, lights off at 2100 h). For the 20 hour light: 4 hour dark (20L:4D, lights on at 0300 h, lights off at 2300 h), animals were first placed in 14L:10D for one week, transferred to 16L:8D for another week, followed by two weeks in 20L:4D. All brain samples were taken every two hours throughout the 24 hour cycle, except the first and the last two time points which were sampled every three hours.
Studies of melanopsin-deficient mice and mice that lack melanopsin, rods and cones
Wild type and melanopsin-deficient () mice (on C57BL/6:129 hybrid background) [] were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). For light pulse studies, wild type,mice and triple knockouts (mice) that lack melanopsin, rod and cone phototransduction systems were used []. Animals received a 15 min light pulse (~200 lux) at ZT14 and brains were sampled one or two hours after light pulse. Dark control animals did not receive a light pulse. Opn4-/- Opn4-/- Opn4-/- Gnat1-/- Cnga3-/- 5 10
Studies of cryptochrome-deficient () mice Cry1-/-Cry2-/-
Cryptochrome-deficient () mice on a C57BL/6:129 hybrid background were kindly provided by Dr. Aziz Sancar (University of North Carolina at Chapel Hill). Wild type andmice were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). A second group ofmice were placed into two days of constant darkness (2DD) (Circadian time, CT, CT1-22). The mRNA levels of,,andwere measured in the SCN. For light pulse experiments,mice received a 15 min light pulse (~400 lux) at ZT14, and sampled one or two hours after light pulse. Dark controlmice did not receive a light pulse. For the shifting experiments, wildtype andmice were initially entrained under 12L:12D, then subjected to an acute 6 hour advance of lighting schedule. Running-wheel activities of these mice were monitored 10 days before and 10 days after the 6 hour advance shift. The 6 hour phase advance was then repeated and brains were collected at ZT4 and ZT16 on the day of the shift. Cry1-/-Cry2-/- Cry1-/-Cry2-/- Cry1-/-Cry2-/- PK2 Per2 Per1 Bmal1 Cry1-/-Cry2-/- Cry1-/-Cry2-/- Cry1-/-Cry2-/-
Authors' contributions
ELB sampled themice and performed behavior experiments onmice. SH sampled the melanopsin-deficient mice and triple knockout mice. MYC performed the tissue sectioning, in situ hybridizations and all quantitative analyses. MYC, ELB and QYZ drafted the manuscript. ELB, SH, MYC and QYZ designed the studies. All authors read and approved the final manuscript. Cry1-/-Cry2-/- Cry1-/-Cry2-/-