Background The suprachiasmatic nucleus (SCN) contains the master circadian clock that regulates daily rhythms of many physiological and behavioural processes in mammals. Previously we have shown that prokineticin 2 ( PK2 ) is a clock-controlled gene that may function as a critical SCN output molecule responsible for circadian locomotor rhythms. As light is the principal zeitgeber that entrains the circadian oscillator, and  PK2  expression is responsive to nocturnal light pulses, we further investigated the effects of light on the molecular rhythm of  PK2  in the SCN. In particular, we examined how  PK2  responds to shifts of light/dark cycles and changes in photoperiod. We also investigated which photoreceptors are responsible for the light-induced  PK2  expression in the SCN. To determine whether light requires an intact functional circadian pacemaker to regulate  PK2 , we examined  PK2  expression in cryptochrome1,2-deficient ( Cry1-/-Cry2-/- ) mice that lack functional circadian clock under normal light/dark cycles and constant darkness.
Background
The suprachiasmatic nucleus (SCN) contains the master circadian clock that regulates daily rhythms of many physiological and behavioural processes in mammals. Previously we have shown that prokineticin 2 ( PK2 ) is a clock-controlled gene that may function as a critical SCN output molecule responsible for circadian locomotor rhythms. As light is the principal zeitgeber that entrains the circadian oscillator, and  PK2  expression is responsive to nocturnal light pulses, we further investigated the effects of light on the molecular rhythm of  PK2  in the SCN. In particular, we examined how  PK2  responds to shifts of light/dark cycles and changes in photoperiod. We also investigated which photoreceptors are responsible for the light-induced  PK2  expression in the SCN. To determine whether light requires an intact functional circadian pacemaker to regulate  PK2 , we examined  PK2  expression in cryptochrome1,2-deficient ( Cry1-/-Cry2-/- ) mice that lack functional circadian clock under normal light/dark cycles and constant darkness.PK2PK2PK2PK2PK2PK2PK2Cry1-/-Cry2-/-Results Upon abrupt shifts of the light/dark cycle,  PK2  expression exhibits transients in response to phase advances but rapidly entrains to phase delays. Photoperiod studies indicate that  PK2  responds differentially to changes in light period. Although the phase of  PK2  expression expands as the light period increases, decreasing light period does not further condense the phase of  PK2  expression. Genetic knockout studies revealed that functional melanopsin and rod-cone photoreceptive systems are required for the light-inducibility of  PK2 . In  Cry1-/-Cry2-/-  mice that lack a functional circadian clock, a low amplitude  PK2  rhythm is detected under light/dark conditions, but not in constant darkness. This suggests that light can directly regulate  PK2  expression in the SCN.
Results
Upon abrupt shifts of the light/dark cycle,  PK2  expression exhibits transients in response to phase advances but rapidly entrains to phase delays. Photoperiod studies indicate that  PK2  responds differentially to changes in light period. Although the phase of  PK2  expression expands as the light period increases, decreasing light period does not further condense the phase of  PK2  expression. Genetic knockout studies revealed that functional melanopsin and rod-cone photoreceptive systems are required for the light-inducibility of  PK2 . In  Cry1-/-Cry2-/-  mice that lack a functional circadian clock, a low amplitude  PK2  rhythm is detected under light/dark conditions, but not in constant darkness. This suggests that light can directly regulate  PK2  expression in the SCN.PK2PK2PK2PK2PK2Cry1-/-Cry2-/-PK2PK2Conclusion These data demonstrate that the molecular rhythm of  PK2  in the SCN is regulated by both the circadian clock and light.  PK2  is predominantly controlled by the endogenous circadian clock, while light plays a modulatory role. The  Cry1-/-Cry2-/-  mice studies reveal a light-driven  PK2  rhythm, indicating that light can induce  PK2  expression independent of the circadian oscillator. The light inducibility of  PK2  suggests that in addition to its role in clock-driven rhythms of locomotor behaviour,  PK2  may also participate in the photic entrainment of circadian locomotor rhythms.
Conclusion
These data demonstrate that the molecular rhythm of  PK2  in the SCN is regulated by both the circadian clock and light.  PK2  is predominantly controlled by the endogenous circadian clock, while light plays a modulatory role. The  Cry1-/-Cry2-/-  mice studies reveal a light-driven  PK2  rhythm, indicating that light can induce  PK2  expression independent of the circadian oscillator. The light inducibility of  PK2  suggests that in addition to its role in clock-driven rhythms of locomotor behaviour,  PK2  may also participate in the photic entrainment of circadian locomotor rhythms.PK2PK2Cry1-/-Cry2-/-PK2PK2PK2PK2

Background Light is the principal zeitgeber that entrains circadian rhythms of physiology and behaviour [ 1 , 2 ]. The major light input pathway to the suprachiasmatic nucleus (SCN) is the retinohypothalamic tract [ 3 ], which arises from a population of retinal ganglion cells [ 4 ]. 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 [ 5 - 11 ]. Excellent progress has been made in the understanding of circadian photic entrainment [ 12 - 15 ]. This includes light-induced transcriptional activation of core clock genes in the SCN, such as  Per1  and  Per2 , as well as immediate-early gene  c-fos . 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 [ 16 - 19 ]. 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 [ 20 ]. In addition to the effect of light on circadian entrainment, light also has a direct effect on physiology and behaviour, generally termed as "masking" [ 21 , 22 ]. For instance, light pulses given at night acutely suppress the locomotor behaviour of nocturnal rodents [ 21 , 22 ], and this can occur without functional clockwork [ 23 - 27 ]. Masking may account for the maintenance under normal light/dark conditions of wheel-running rhythms in cryptochrome-deficient ( Cry1-/-Cry2-/- ) 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-driven  Per2  rhythm in the SCN in  Cry1-/-Cry2-/-  mice, and have suggested that the light-driven molecular rhythm in the SCN may be related to the preservation of their locomotor rhythm [ 25 ]. We previously found that prokineticin 2 ( PK2 ) 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 [ 28 ]. 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 [ 28 , 29 ]. Interestingly, we also observed that  PK2  expression is rapidly induced by light pulses administered at night [ 28 ], 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 of  PK2  expression in the SCN. In particular, we investigated the photoreceptive mechanisms responsible for the light-induced  PK2  expression in the SCN. Utilizing  Cry1-/-Cry2-/-  mice, we also determined whether light can drive  PK2  expression in the SCN independent of a functional circadian clock.
Background
Light is the principal zeitgeber that entrains circadian rhythms of physiology and behaviour [ 1 , 2 ]. The major light input pathway to the suprachiasmatic nucleus (SCN) is the retinohypothalamic tract [ 3 ], which arises from a population of retinal ganglion cells [ 4 ]. 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 [ 5 - 11 ]. Excellent progress has been made in the understanding of circadian photic entrainment [ 12 - 15 ]. This includes light-induced transcriptional activation of core clock genes in the SCN, such as  Per1  and  Per2 , as well as immediate-early gene  c-fos . 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 [ 16 - 19 ]. 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 [ 20 ].Per1Per2c-fosIn addition to the effect of light on circadian entrainment, light also has a direct effect on physiology and behaviour, generally termed as "masking" [ 21 , 22 ]. For instance, light pulses given at night acutely suppress the locomotor behaviour of nocturnal rodents [ 21 , 22 ], and this can occur without functional clockwork [ 23 - 27 ]. Masking may account for the maintenance under normal light/dark conditions of wheel-running rhythms in cryptochrome-deficient ( Cry1-/-Cry2-/- ) 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-driven  Per2  rhythm in the SCN in  Cry1-/-Cry2-/-  mice, and have suggested that the light-driven molecular rhythm in the SCN may be related to the preservation of their locomotor rhythm [ 25 ].Cry1-/-Cry2-/-Per2Cry1-/-Cry2-/-We previously found that prokineticin 2 ( PK2 ) 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 [ 28 ]. 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 [ 28 , 29 ]. Interestingly, we also observed that  PK2  expression is rapidly induced by light pulses administered at night [ 28 ], 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 of  PK2  expression in the SCN. In particular, we investigated the photoreceptive mechanisms responsible for the light-induced  PK2  expression in the SCN. Utilizing  Cry1-/-Cry2-/-  mice, we also determined whether light can drive  PK2  expression in the SCN independent of a functional circadian clock.PK2PK2PK2PK2Cry1-/-Cry2-/-PK2Results PK2  responds differentially to the delay and advance of light/dark cycles We first examined the effects of abrupt shifts of light/dark cycles on  PK2  mRNA 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 measured  PK2  mRNA in the SCN of these animals to examine how quickly the  PK2  mRNA rhythm re-entrains to the shifted light/dark cycles. Under LD,  PK2  mRNA peaks during the day and remains low or undetectable during the night. During the first cycle of the delayed shift (6hrD), the  PK2  mRNA rhythm responds quickly: the rising phase of  PK2  expression adjusts rapidly to the delayed light/dark cycles, while the falling phase still resembles that of the unshifted light/dark cycles (Figure  1A ). In contrast, the  PK2  mRNA rhythm responds very little to a 6 hour advance shift (6hrA). During the first cycle of the advance shift, the  PK2  oscillation pattern remains similar to that of the unshifted LD (Figure  1B ). These changes in  PK2  expression during 6hrD or 6hrA shift indicate that the endogenous circadian clock exerts dominant control over the  PK2  rhythm, as  PK2  expression cannot respond immediately and completely to the shifts of light/dark cycles. 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 [ 30 , 31 ], we next examined the timecourse of shifts of the  PK2  rhythm to 6 hour phase advances and delays. Consistent with the animal's locomotor behaviour, the  PK2  mRNA rhythm reaches stable phase within 2 days of 6hrD shift (Figure  1C ). In contrast, only the rise of  PK2  reaches stable phase within 2 days of 6hrA shift, while the fall of  PK2  takes longer (Figure  1D ). Thus, we further examined whether the  PK2  rhythm is stably entrained after 6 days of 6hrA shift. As expected, the  PK2  rhythm is completely entrained to 6hrA shift after 6 days (Figure  1D ). Together, the differential responses of  PK2  rhythm to a 6hrD or 6hrA shift indicate that the endogenous circadian clock predominantly controls  PK2  rhythm, as circadian oscillators typically show rapid phase delays but advance with transients [ 31 , 32 ]. The entrainment patterns of  PK2  during phase shifts are consistent with behavioural studies in animals and human subjects [ 30 , 31 ]. PK2  rhythm is entrained by different photoperiods We next examined the effect of photoperiod on the  PK2  molecular rhythm in the SCN.  PK2  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,  PK2  mRNA is highly expressed during the 12 hour light phase with peak level at ZT4 (Figure  1A , Figure  3A ). Under 16L:8D,  PK2  mRNA expands to the entire 16 hour light phase and is essentially undetectable during the 8 hour dark period (Figure  2B ). However, the expression of  PK2  mRNA is not confined to the light phase of the shorter photoperiod (8L:16D), as  PK2  mRNA rises before lights on and persists after lights off (Figure  2A ). The temporal profile of  PK2  mRNA under this short photoperiod (8L:16D) is very similar to that observed under 12L:12D (Figure  1A , Figure  3A ) or constant darkness (2DD) [ 28 ]. Thus, although light can induce  PK2  mRNA and expand the duration of  PK2  expression, the phase angle of  PK2  expression is determined by the circadian clock, and its duration cannot be further compressed under shorter photoperiods. Interestingly, the peak of  PK2  mRNA expression was significantly higher in long days (16L:8D) than in shorter days (8L:16D) (Figure  2A–B ), further indicate the enhancing effect of light on  PK2  expression. However, a significant reduction in the  PK2  peak level is observed under a very long photoperiod (20L: 4D) (Figure  2C ). We also noticed that under 20L:4D,  PK2  mRNA is further expanded and becomes detectable even in dark phase (Figure  2C ). Under this long photoperiod (20L:4D), the difference between the peak and basal level of  PK2  is only about 4 fold (Figure  2C ). As it has been reported that the rhythms of  mPer1  and  mPer2  mRNAs in the SCN are also entrained with different phase angles under a variety of photoperiods [ 33 - 35 ], we have also examined  Per1  and  Per2  rhythm in our photoperiod studies (see  Additional file 1 ). The  Per1  and  Per2  rhythm we observed under these photoperiods are consistent with previous findings [ 35 ]. Taken together, these results indicate that changes in photoperiod alter  PK2  rhythm in the SCN, and the amplitudes of  PK2  mRNA oscillation are greatly reduced in very long photoperiods. Light inducibility of  PK2  is eliminated in mice that lack melanopsin, rod and cone phototransduction system ( Opn4-/-, Gnat1-/- Cnga3-/-  mice) As melanopsin has been implicated in circadian photoreception [ 5 - 11 ], we examined whether the  PK2  molecular rhythm is normally entrained in melanopsin-deficient ( Opn4-/- ) mice. Figure  3  shows that the oscillation profile of  PK2  in the SCN of  Opn4-/-  mice is essentially identical to that observed in the wild type mice under LD. This normal temporal profile of  PK2  mRNA corresponds with the normal locomotor rhythm of  Opn4-/-  mice under light/dark conditions [ 7 , 8 ]. As  Opn4-/-  mice display attenuated phase resetting in response to light pulses and exhibit impaired light masking responses to bright light [ 36 ], we also examined whether light inducibility of  PK2  is blunted in  Opn4-/-  mice. Figure  3B  shows that light pulse-induced  PK2  in the SCN of  Opn4-/-  mice was significantly reduced by about 50% and 60%, one and two hours after the light pulse, respectively. The  Opn4-/-  light pulse studies show that a residual  PK2  expression is still present after a light pulse, suggesting that without melanopsin, other phototransduction system can still transmit light information to induce  PK2  expression. Thus, we decided to examine the light inducibility of  PK2  in triple knockout mice lacking melanopsin, rod and cone phototransduction systems ( Opn4-/- Gnat1-/- Cnga3-/-  mice), as these animals free run under light dark conditions (LD) and lack masking responses to light [ 10 ]. Figure  3C  shows that the light pulse-induced  PK2  in the SCN is completely eliminated in these triple knockout mice, consistent with their malfunctioning photoentrainment systems and their lack of masking responses to light [ 10 ]. In addition, we also observed that  PK2  mRNA followed the free-running locomotor rhythms in these triple knockout mice (Figure  3D ), with high levels of  PK2  during the inactive phase (CT3) and low levels during active phase (CT15). Together, these results suggest that melanopsin contributes to the light inducibility of  PK2 , and intact melanopsin with functional rod/cone phototransduction systems are required for the light inducibility of  PK2 . A low amplitude  PK2  rhythm is preserved in cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice under light/dark conditions Previous studies have shown that the light-regulated  Per2  rhythm is maintained in the SCN of cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice that lack functional circadian clock [ 25 , 37 ]. In order to determine whether the regulation of  PK2 ,  Per1 ,  Per2  and  Bmal1  expression by light requires an intact circadian pacemaker, we systematically assessed the temporal mRNA profiles of clockwork genes in  Cry1-/-Cry2-/-  mice under both light/dark (LD) and constant dark (DD) conditions. Figure  4  shows that the molecular rhythm of  Per2  remained largely intact in  Cry1-/-Cry2-/-  mice entrained under12L:12D, with levels about 4-fold higher during the light phase than the dark phase. This amplitude of the  Per2  oscillation profile was similar to that observed in wild type mice [ 18 , 38 ]. A low amplitude  Per1  rhythm in  Cry1-/-Cry2-/  mice was also apparent under LD, but not DD (Figure  4B ). We further detected a light-driven  Bmal1  rhythm in the SCN of  Cry1-/-Cry2-/-  mice under LD, but not DD (Figure  4C ). Interestingly, this  Bmal1  rhythm in  Cry1-/-Cry2-/-  mice peaked during light phase, opposite from the  Bmal1  rhythm in wild type mice and in phase with  Per1  [ 39 , 40 ]. As it has been suggested that PER2 can positively regulate  Bmal1  expression via inhibition of the orphan nuclear receptor REV-ERBα [ 41 , 42 ], it is possible that this  Bmal1  rhythm is secondary to the light-driven  Per2  rhythm. Further studies are required to clarify this observation. We also examined the molecular rhythm of  PK2  in  Cry1-/-Cry2-/-  mice. Figure  4D  shows that  PK2  mRNA rhythm in the SCN of  Cry1-/-Cry2-/-  mice was apparent under LD, with the presence of a low level  PK2  during light phase and absence of  PK2  during dark phase (see  Additional file 2 ). Similar to wild type mice, the peak level of this low amplitude  PK2  rhythm was around ZT4, although its peak was only about 8% of that observed in wild type mice (Figure.  4D , Figure  1A , Figure  3A ). No  PK2  rhythm was evident when  Cry1-/-Cry2-/-  mice were placed under DD (Figure  4D ). Furthermore, the inducibility of  PK2  to nocturnal light pulses is also maintained in  Cry1-/-Cry2-/-  mice.  PK2  mRNA increased one and two hours after a brief light pulse at ZT14 (Figure  4E ). Nevertheless, light-induced  PK2  was still detected in  Per1,2,3-/-  mice and  Clk-/-  mice that lack functional circadian clock (Cheng, Weaver & Zhou, unpublished observations). As  PK2  remains responsive to light in these clock mutant mice that lack functional circadian clock, it is likely that the low amplitude  PK2  rhythm in  Cry1-/-Cry2-/-  mice under LD is directly driven by light. In order to test whether this light-driven  PK2  rhythm may be related to the maintenance of behavioural rhythms observed in  Cry1-/-Cry2-/-  mice under LD, we studied the responses of  Cry1-/-Cry2-/-  mice 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 of  Cry1-/-Cry2-/-  mice adjusted rapidly to 6 hr advance (Figure  4F ). Such a rapid response is characteristic of masking. A correlative rapid adjustment of  PK2  was also observed in the SCN of  Cry1-/-Cry2-/-  mice (Figure  4G ). As  Cry1-/-Cry2-/-  mice lack functional circadian clock and their locomotor behaviour and  PK2  expression patterns are completely light driven, our results suggest that this low amplitude, light-driven rhythm of  PK2  may contribute to or underlie the masking of locomotor behaviour in these animals.
Results
PK2  responds differentially to the delay and advance of light/dark cycles We first examined the effects of abrupt shifts of light/dark cycles on  PK2  mRNA 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 measured  PK2  mRNA in the SCN of these animals to examine how quickly the  PK2  mRNA rhythm re-entrains to the shifted light/dark cycles. Under LD,  PK2  mRNA peaks during the day and remains low or undetectable during the night. During the first cycle of the delayed shift (6hrD), the  PK2  mRNA rhythm responds quickly: the rising phase of  PK2  expression adjusts rapidly to the delayed light/dark cycles, while the falling phase still resembles that of the unshifted light/dark cycles (Figure  1A ). In contrast, the  PK2  mRNA rhythm responds very little to a 6 hour advance shift (6hrA). During the first cycle of the advance shift, the  PK2  oscillation pattern remains similar to that of the unshifted LD (Figure  1B ). These changes in  PK2  expression during 6hrD or 6hrA shift indicate that the endogenous circadian clock exerts dominant control over the  PK2  rhythm, as  PK2  expression cannot respond immediately and completely to the shifts of light/dark cycles. 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 [ 30 , 31 ], we next examined the timecourse of shifts of the  PK2  rhythm to 6 hour phase advances and delays. Consistent with the animal's locomotor behaviour, the  PK2  mRNA rhythm reaches stable phase within 2 days of 6hrD shift (Figure  1C ). In contrast, only the rise of  PK2  reaches stable phase within 2 days of 6hrA shift, while the fall of  PK2  takes longer (Figure  1D ). Thus, we further examined whether the  PK2  rhythm is stably entrained after 6 days of 6hrA shift. As expected, the  PK2  rhythm is completely entrained to 6hrA shift after 6 days (Figure  1D ). Together, the differential responses of  PK2  rhythm to a 6hrD or 6hrA shift indicate that the endogenous circadian clock predominantly controls  PK2  rhythm, as circadian oscillators typically show rapid phase delays but advance with transients [ 31 , 32 ]. The entrainment patterns of  PK2  during phase shifts are consistent with behavioural studies in animals and human subjects [ 30 , 31 ].
PK2  responds differentially to the delay and advance of light/dark cycles
PK2We first examined the effects of abrupt shifts of light/dark cycles on  PK2  mRNA 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 measured  PK2  mRNA in the SCN of these animals to examine how quickly the  PK2  mRNA rhythm re-entrains to the shifted light/dark cycles. Under LD,  PK2  mRNA peaks during the day and remains low or undetectable during the night. During the first cycle of the delayed shift (6hrD), the  PK2  mRNA rhythm responds quickly: the rising phase of  PK2  expression adjusts rapidly to the delayed light/dark cycles, while the falling phase still resembles that of the unshifted light/dark cycles (Figure  1A ). In contrast, the  PK2  mRNA rhythm responds very little to a 6 hour advance shift (6hrA). During the first cycle of the advance shift, the  PK2  oscillation pattern remains similar to that of the unshifted LD (Figure  1B ). These changes in  PK2  expression during 6hrD or 6hrA shift indicate that the endogenous circadian clock exerts dominant control over the  PK2  rhythm, as  PK2  expression cannot respond immediately and completely to the shifts of light/dark cycles.PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2As 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 [ 30 , 31 ], we next examined the timecourse of shifts of the  PK2  rhythm to 6 hour phase advances and delays. Consistent with the animal's locomotor behaviour, the  PK2  mRNA rhythm reaches stable phase within 2 days of 6hrD shift (Figure  1C ). In contrast, only the rise of  PK2  reaches stable phase within 2 days of 6hrA shift, while the fall of  PK2  takes longer (Figure  1D ). Thus, we further examined whether the  PK2  rhythm is stably entrained after 6 days of 6hrA shift. As expected, the  PK2  rhythm is completely entrained to 6hrA shift after 6 days (Figure  1D ). Together, the differential responses of  PK2  rhythm to a 6hrD or 6hrA shift indicate that the endogenous circadian clock predominantly controls  PK2  rhythm, as circadian oscillators typically show rapid phase delays but advance with transients [ 31 , 32 ]. The entrainment patterns of  PK2  during phase shifts are consistent with behavioural studies in animals and human subjects [ 30 , 31 ].PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2  rhythm is entrained by different photoperiods We next examined the effect of photoperiod on the  PK2  molecular rhythm in the SCN.  PK2  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,  PK2  mRNA is highly expressed during the 12 hour light phase with peak level at ZT4 (Figure  1A , Figure  3A ). Under 16L:8D,  PK2  mRNA expands to the entire 16 hour light phase and is essentially undetectable during the 8 hour dark period (Figure  2B ). However, the expression of  PK2  mRNA is not confined to the light phase of the shorter photoperiod (8L:16D), as  PK2  mRNA rises before lights on and persists after lights off (Figure  2A ). The temporal profile of  PK2  mRNA under this short photoperiod (8L:16D) is very similar to that observed under 12L:12D (Figure  1A , Figure  3A ) or constant darkness (2DD) [ 28 ]. Thus, although light can induce  PK2  mRNA and expand the duration of  PK2  expression, the phase angle of  PK2  expression is determined by the circadian clock, and its duration cannot be further compressed under shorter photoperiods. Interestingly, the peak of  PK2  mRNA expression was significantly higher in long days (16L:8D) than in shorter days (8L:16D) (Figure  2A–B ), further indicate the enhancing effect of light on  PK2  expression. However, a significant reduction in the  PK2  peak level is observed under a very long photoperiod (20L: 4D) (Figure  2C ). We also noticed that under 20L:4D,  PK2  mRNA is further expanded and becomes detectable even in dark phase (Figure  2C ). Under this long photoperiod (20L:4D), the difference between the peak and basal level of  PK2  is only about 4 fold (Figure  2C ). As it has been reported that the rhythms of  mPer1  and  mPer2  mRNAs in the SCN are also entrained with different phase angles under a variety of photoperiods [ 33 - 35 ], we have also examined  Per1  and  Per2  rhythm in our photoperiod studies (see  Additional file 1 ). The  Per1  and  Per2  rhythm we observed under these photoperiods are consistent with previous findings [ 35 ]. Taken together, these results indicate that changes in photoperiod alter  PK2  rhythm in the SCN, and the amplitudes of  PK2  mRNA oscillation are greatly reduced in very long photoperiods.
PK2  rhythm is entrained by different photoperiods
PK2We next examined the effect of photoperiod on the  PK2  molecular rhythm in the SCN.  PK2  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,  PK2  mRNA is highly expressed during the 12 hour light phase with peak level at ZT4 (Figure  1A , Figure  3A ). Under 16L:8D,  PK2  mRNA expands to the entire 16 hour light phase and is essentially undetectable during the 8 hour dark period (Figure  2B ). However, the expression of  PK2  mRNA is not confined to the light phase of the shorter photoperiod (8L:16D), as  PK2  mRNA rises before lights on and persists after lights off (Figure  2A ). The temporal profile of  PK2  mRNA under this short photoperiod (8L:16D) is very similar to that observed under 12L:12D (Figure  1A , Figure  3A ) or constant darkness (2DD) [ 28 ]. Thus, although light can induce  PK2  mRNA and expand the duration of  PK2  expression, the phase angle of  PK2  expression is determined by the circadian clock, and its duration cannot be further compressed under shorter photoperiods. Interestingly, the peak of  PK2  mRNA expression was significantly higher in long days (16L:8D) than in shorter days (8L:16D) (Figure  2A–B ), further indicate the enhancing effect of light on  PK2  expression. However, a significant reduction in the  PK2  peak level is observed under a very long photoperiod (20L: 4D) (Figure  2C ). We also noticed that under 20L:4D,  PK2  mRNA is further expanded and becomes detectable even in dark phase (Figure  2C ). Under this long photoperiod (20L:4D), the difference between the peak and basal level of  PK2  is only about 4 fold (Figure  2C ). As it has been reported that the rhythms of  mPer1  and  mPer2  mRNAs in the SCN are also entrained with different phase angles under a variety of photoperiods [ 33 - 35 ], we have also examined  Per1  and  Per2  rhythm in our photoperiod studies (see  Additional file 1 ). The  Per1  and  Per2  rhythm we observed under these photoperiods are consistent with previous findings [ 35 ]. Taken together, these results indicate that changes in photoperiod alter  PK2  rhythm in the SCN, and the amplitudes of  PK2  mRNA oscillation are greatly reduced in very long photoperiods.PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2mPer1mPer2Per1Per2Per1Per2PK2PK2Light inducibility of  PK2  is eliminated in mice that lack melanopsin, rod and cone phototransduction system ( Opn4-/-, Gnat1-/- Cnga3-/-  mice) As melanopsin has been implicated in circadian photoreception [ 5 - 11 ], we examined whether the  PK2  molecular rhythm is normally entrained in melanopsin-deficient ( Opn4-/- ) mice. Figure  3  shows that the oscillation profile of  PK2  in the SCN of  Opn4-/-  mice is essentially identical to that observed in the wild type mice under LD. This normal temporal profile of  PK2  mRNA corresponds with the normal locomotor rhythm of  Opn4-/-  mice under light/dark conditions [ 7 , 8 ]. As  Opn4-/-  mice display attenuated phase resetting in response to light pulses and exhibit impaired light masking responses to bright light [ 36 ], we also examined whether light inducibility of  PK2  is blunted in  Opn4-/-  mice. Figure  3B  shows that light pulse-induced  PK2  in the SCN of  Opn4-/-  mice was significantly reduced by about 50% and 60%, one and two hours after the light pulse, respectively. The  Opn4-/-  light pulse studies show that a residual  PK2  expression is still present after a light pulse, suggesting that without melanopsin, other phototransduction system can still transmit light information to induce  PK2  expression. Thus, we decided to examine the light inducibility of  PK2  in triple knockout mice lacking melanopsin, rod and cone phototransduction systems ( Opn4-/- Gnat1-/- Cnga3-/-  mice), as these animals free run under light dark conditions (LD) and lack masking responses to light [ 10 ]. Figure  3C  shows that the light pulse-induced  PK2  in the SCN is completely eliminated in these triple knockout mice, consistent with their malfunctioning photoentrainment systems and their lack of masking responses to light [ 10 ]. In addition, we also observed that  PK2  mRNA followed the free-running locomotor rhythms in these triple knockout mice (Figure  3D ), with high levels of  PK2  during the inactive phase (CT3) and low levels during active phase (CT15). Together, these results suggest that melanopsin contributes to the light inducibility of  PK2 , and intact melanopsin with functional rod/cone phototransduction systems are required for the light inducibility of  PK2 .
Light inducibility of  PK2  is eliminated in mice that lack melanopsin, rod and cone phototransduction system ( Opn4-/-, Gnat1-/- Cnga3-/-  mice)
PK2Opn4-/-, Gnat1-/- Cnga3-/-As melanopsin has been implicated in circadian photoreception [ 5 - 11 ], we examined whether the  PK2  molecular rhythm is normally entrained in melanopsin-deficient ( Opn4-/- ) mice. Figure  3  shows that the oscillation profile of  PK2  in the SCN of  Opn4-/-  mice is essentially identical to that observed in the wild type mice under LD. This normal temporal profile of  PK2  mRNA corresponds with the normal locomotor rhythm of  Opn4-/-  mice under light/dark conditions [ 7 , 8 ]. As  Opn4-/-  mice display attenuated phase resetting in response to light pulses and exhibit impaired light masking responses to bright light [ 36 ], we also examined whether light inducibility of  PK2  is blunted in  Opn4-/-  mice. Figure  3B  shows that light pulse-induced  PK2  in the SCN of  Opn4-/-  mice was significantly reduced by about 50% and 60%, one and two hours after the light pulse, respectively.PK2Opn4-/-PK2Opn4-/-PK2Opn4-/-Opn4-/-PK2Opn4-/-PK2Opn4-/-The  Opn4-/-  light pulse studies show that a residual  PK2  expression is still present after a light pulse, suggesting that without melanopsin, other phototransduction system can still transmit light information to induce  PK2  expression. Thus, we decided to examine the light inducibility of  PK2  in triple knockout mice lacking melanopsin, rod and cone phototransduction systems ( Opn4-/- Gnat1-/- Cnga3-/-  mice), as these animals free run under light dark conditions (LD) and lack masking responses to light [ 10 ]. Figure  3C  shows that the light pulse-induced  PK2  in the SCN is completely eliminated in these triple knockout mice, consistent with their malfunctioning photoentrainment systems and their lack of masking responses to light [ 10 ]. In addition, we also observed that  PK2  mRNA followed the free-running locomotor rhythms in these triple knockout mice (Figure  3D ), with high levels of  PK2  during the inactive phase (CT3) and low levels during active phase (CT15). Together, these results suggest that melanopsin contributes to the light inducibility of  PK2 , and intact melanopsin with functional rod/cone phototransduction systems are required for the light inducibility of  PK2 .Opn4-/-PK2PK2PK2Opn4-/- Gnat1-/- Cnga3-/-PK2PK2PK2PK2PK2A low amplitude  PK2  rhythm is preserved in cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice under light/dark conditions Previous studies have shown that the light-regulated  Per2  rhythm is maintained in the SCN of cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice that lack functional circadian clock [ 25 , 37 ]. In order to determine whether the regulation of  PK2 ,  Per1 ,  Per2  and  Bmal1  expression by light requires an intact circadian pacemaker, we systematically assessed the temporal mRNA profiles of clockwork genes in  Cry1-/-Cry2-/-  mice under both light/dark (LD) and constant dark (DD) conditions. Figure  4  shows that the molecular rhythm of  Per2  remained largely intact in  Cry1-/-Cry2-/-  mice entrained under12L:12D, with levels about 4-fold higher during the light phase than the dark phase. This amplitude of the  Per2  oscillation profile was similar to that observed in wild type mice [ 18 , 38 ]. A low amplitude  Per1  rhythm in  Cry1-/-Cry2-/  mice was also apparent under LD, but not DD (Figure  4B ). We further detected a light-driven  Bmal1  rhythm in the SCN of  Cry1-/-Cry2-/-  mice under LD, but not DD (Figure  4C ). Interestingly, this  Bmal1  rhythm in  Cry1-/-Cry2-/-  mice peaked during light phase, opposite from the  Bmal1  rhythm in wild type mice and in phase with  Per1  [ 39 , 40 ]. As it has been suggested that PER2 can positively regulate  Bmal1  expression via inhibition of the orphan nuclear receptor REV-ERBα [ 41 , 42 ], it is possible that this  Bmal1  rhythm is secondary to the light-driven  Per2  rhythm. Further studies are required to clarify this observation. We also examined the molecular rhythm of  PK2  in  Cry1-/-Cry2-/-  mice. Figure  4D  shows that  PK2  mRNA rhythm in the SCN of  Cry1-/-Cry2-/-  mice was apparent under LD, with the presence of a low level  PK2  during light phase and absence of  PK2  during dark phase (see  Additional file 2 ). Similar to wild type mice, the peak level of this low amplitude  PK2  rhythm was around ZT4, although its peak was only about 8% of that observed in wild type mice (Figure.  4D , Figure  1A , Figure  3A ). No  PK2  rhythm was evident when  Cry1-/-Cry2-/-  mice were placed under DD (Figure  4D ). Furthermore, the inducibility of  PK2  to nocturnal light pulses is also maintained in  Cry1-/-Cry2-/-  mice.  PK2  mRNA increased one and two hours after a brief light pulse at ZT14 (Figure  4E ). Nevertheless, light-induced  PK2  was still detected in  Per1,2,3-/-  mice and  Clk-/-  mice that lack functional circadian clock (Cheng, Weaver & Zhou, unpublished observations). As  PK2  remains responsive to light in these clock mutant mice that lack functional circadian clock, it is likely that the low amplitude  PK2  rhythm in  Cry1-/-Cry2-/-  mice under LD is directly driven by light. In order to test whether this light-driven  PK2  rhythm may be related to the maintenance of behavioural rhythms observed in  Cry1-/-Cry2-/-  mice under LD, we studied the responses of  Cry1-/-Cry2-/-  mice 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 of  Cry1-/-Cry2-/-  mice adjusted rapidly to 6 hr advance (Figure  4F ). Such a rapid response is characteristic of masking. A correlative rapid adjustment of  PK2  was also observed in the SCN of  Cry1-/-Cry2-/-  mice (Figure  4G ). As  Cry1-/-Cry2-/-  mice lack functional circadian clock and their locomotor behaviour and  PK2  expression patterns are completely light driven, our results suggest that this low amplitude, light-driven rhythm of  PK2  may contribute to or underlie the masking of locomotor behaviour in these animals.
A low amplitude  PK2  rhythm is preserved in cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice under light/dark conditions
PK2Cry1-/-Cry2-/-Previous studies have shown that the light-regulated  Per2  rhythm is maintained in the SCN of cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice that lack functional circadian clock [ 25 , 37 ]. In order to determine whether the regulation of  PK2 ,  Per1 ,  Per2  and  Bmal1  expression by light requires an intact circadian pacemaker, we systematically assessed the temporal mRNA profiles of clockwork genes in  Cry1-/-Cry2-/-  mice under both light/dark (LD) and constant dark (DD) conditions. Figure  4  shows that the molecular rhythm of  Per2  remained largely intact in  Cry1-/-Cry2-/-  mice entrained under12L:12D, with levels about 4-fold higher during the light phase than the dark phase. This amplitude of the  Per2  oscillation profile was similar to that observed in wild type mice [ 18 , 38 ]. A low amplitude  Per1  rhythm in  Cry1-/-Cry2-/  mice was also apparent under LD, but not DD (Figure  4B ). We further detected a light-driven  Bmal1  rhythm in the SCN of  Cry1-/-Cry2-/-  mice under LD, but not DD (Figure  4C ). Interestingly, this  Bmal1  rhythm in  Cry1-/-Cry2-/-  mice peaked during light phase, opposite from the  Bmal1  rhythm in wild type mice and in phase with  Per1  [ 39 , 40 ]. As it has been suggested that PER2 can positively regulate  Bmal1  expression via inhibition of the orphan nuclear receptor REV-ERBα [ 41 , 42 ], it is possible that this  Bmal1  rhythm is secondary to the light-driven  Per2  rhythm. Further studies are required to clarify this observation.Per2Cry1-/-Cry2-/-PK2Per1Per2Bmal1Cry1-/-Cry2-/-Per2Cry1-/-Cry2-/-Per2Per1Cry1-/-Cry2-/Bmal1Cry1-/-Cry2-/-Bmal1Cry1-/-Cry2-/-Bmal1Per1Bmal1Bmal1Per2We also examined the molecular rhythm of  PK2  in  Cry1-/-Cry2-/-  mice. Figure  4D  shows that  PK2  mRNA rhythm in the SCN of  Cry1-/-Cry2-/-  mice was apparent under LD, with the presence of a low level  PK2  during light phase and absence of  PK2  during dark phase (see  Additional file 2 ). Similar to wild type mice, the peak level of this low amplitude  PK2  rhythm was around ZT4, although its peak was only about 8% of that observed in wild type mice (Figure.  4D , Figure  1A , Figure  3A ). No  PK2  rhythm was evident when  Cry1-/-Cry2-/-  mice were placed under DD (Figure  4D ). Furthermore, the inducibility of  PK2  to nocturnal light pulses is also maintained in  Cry1-/-Cry2-/-  mice.  PK2  mRNA increased one and two hours after a brief light pulse at ZT14 (Figure  4E ). Nevertheless, light-induced  PK2  was still detected in  Per1,2,3-/-  mice and  Clk-/-  mice that lack functional circadian clock (Cheng, Weaver & Zhou, unpublished observations). As  PK2  remains responsive to light in these clock mutant mice that lack functional circadian clock, it is likely that the low amplitude  PK2  rhythm in  Cry1-/-Cry2-/-  mice under LD is directly driven by light.PK2Cry1-/-Cry2-/-PK2Cry1-/-Cry2-/-PK2PK2PK2PK2Cry1-/-Cry2-/-PK2Cry1-/-Cry2-/-PK2PK2Per1,2,3-/-Clk-/-PK2PK2Cry1-/-Cry2-/-In order to test whether this light-driven  PK2  rhythm may be related to the maintenance of behavioural rhythms observed in  Cry1-/-Cry2-/-  mice under LD, we studied the responses of  Cry1-/-Cry2-/-  mice 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 of  Cry1-/-Cry2-/-  mice adjusted rapidly to 6 hr advance (Figure  4F ). Such a rapid response is characteristic of masking. A correlative rapid adjustment of  PK2  was also observed in the SCN of  Cry1-/-Cry2-/-  mice (Figure  4G ). As  Cry1-/-Cry2-/-  mice lack functional circadian clock and their locomotor behaviour and  PK2  expression patterns are completely light driven, our results suggest that this low amplitude, light-driven rhythm of  PK2  may contribute to or underlie the masking of locomotor behaviour in these animals.PK2Cry1-/-Cry2-/-Cry1-/-Cry2-/-Cry1-/-Cry2-/-PK2Cry1-/-Cry2-/-Cry1-/-Cry2-/-PK2PK2Discussion Our studies indicate that the molecular rhythm of  PK2  in 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 the  PK2  rhythm. While  PK2  expression re-entrained rapidly to phase delays, it takes several cycles of transients for  PK2  to be stably entrained to phase advances (Figure  1 ). The rate of re-entrainment of  PK2  molecular rhythms to these shifts is consistent with that of behavioural adaptation of animals and human subjects [ 30 , 31 ]. Our photoperiod studies indicate that  PK2  expression in the SCN responds differentially to changes in photoperiod length (Figure  2 ). Although increasing light period can induce  PK2  expression and expand the duration of  PK2  rhythm (Figure  2B ), shortening of the light period does not lead to corresponding reduction of the duration of  PK2  expression (Figure  2A ). It appears that a minimal duration of  PK2  expression is maintained under short photoperiod (Figure  2A ) and constant darkness [ 28 ], which further indicate the dominant control of  PK2  expression by the circadian clock. Interestingly, the amplitude of the  PK2  oscillation was greatly reduced under very long photoperiod (20L:4D) (Figure  2C ). As the amplitude of both  Per1  and  Per2  rhythms were also reduced during 20L:4D (see  Additional file 1 ), it is likely that these depressed rhythms of clockwork genes may contribute to the depressed  PK2  rhythm observed. Whether reduction in the amplitude of expression in any of these genes is related to arrhythmicity in LL deserves further examination. Our studies with  Cry1-/-Cry2-/-  mice revealed the presence of a light-driven  PK2  molecular rhythm in the SCN under LD, indicating that light can drive  PK2  rhythm independent of functional circadian clock. Interestingly, the molecular rhythms of some clockwork genes such as  Per2 ,  Per1 , and  Bmal1  were also partially maintained in the SCN of  Cry1-/-Cry2-/-  mice under LD (Figure  4 ). 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-regulated  Per2  molecular rhythm in the SCN of  Cry1-/-Cry2-/-  mice, and suggested the term of "light-driving" effect [ 25 ]. As  Cry1-/-Cry2-/-  mice lack functional circadian clocks and their locomotor behaviour remains rhythmic under LD, but not under DD conditions [ 24 , 25 ], it is likely that these light-driven molecular rhythms may drive the locomotor rhythms in these animals. As we have previously shown that  PK2  may be a critical output molecule responsible for circadian locomotor rhythms, the presence of this light-driven  PK2  rhythm in  Cry1-/-Cry2-/-  mice 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 [ 43 ]. The role of the SCN in masking of locomotor activity by light is controversial, with similar studies having produced contradictory results [ 23 , 44 ]. 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  PK2 -deficient mice will be necessary to resolve the exact role of PK2 in the light-driven locomotor rhythms. The light inducibility of  PK2  in 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  PK2  (Figure  3 ). The same photoreceptive system has been shown responsible for the entrainment of locomotor rhythm [ 5 - 11 ]. The light inducibility of  PK2  may be related to the presence of a putative cyclic AMP response element (CRE) in the promoter of the  PK2  gene [ 28 ]. CRE-dependent activation is critical for light-induced gene expression in the SCN [ 45 - 48 ]. The reduced light inducibility of  PK2  in mutant mice that lack functional clock may indicate that CRE-dependent pathway and CLK/BMAL1 transcriptional factors may interact in the light-induced  PK2  expression in the SCN. Accumulative data have implicated the photic regulation of the transcription of clock genes such as  Per1  and  Per2  in the entrainment of behavioural rhythms [ 30 , 34 ]. 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 the  Per1  promoter, whose activation in the SCN shifts rapidly when the LD cycle is advanced [ 31 ],  PK2  exhibits transients during phase advance, more similar to those of  Cry1  and  Cry2  [ 30 , 31 ]. This is consistent with the role for PK2 as a clock-controlled gene and thus is downstream from the light-regulated expression of  Per1  or  Per2 . The presence of E box motifs in the  PK2  promoter suggests that light-regulated  Per1  (and perhaps  Per2 ) expression can influence  PK2  expression. However, the light inducibility of  PK2  indicates 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 [ 49 ]. As receptor for PK2 is highly expressed in the SCN [ 28 ] and activation of the PK2 receptor triggers similar signalling pathways as that of glutamate receptors [ 29 ], 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 [ 50 ], 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 of  PK2  may be relevant to both the phase resetting of the core circadian loops and critical SCN output signals.
Discussion
Our studies indicate that the molecular rhythm of  PK2  in 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 the  PK2  rhythm. While  PK2  expression re-entrained rapidly to phase delays, it takes several cycles of transients for  PK2  to be stably entrained to phase advances (Figure  1 ). The rate of re-entrainment of  PK2  molecular rhythms to these shifts is consistent with that of behavioural adaptation of animals and human subjects [ 30 , 31 ]. Our photoperiod studies indicate that  PK2  expression in the SCN responds differentially to changes in photoperiod length (Figure  2 ). Although increasing light period can induce  PK2  expression and expand the duration of  PK2  rhythm (Figure  2B ), shortening of the light period does not lead to corresponding reduction of the duration of  PK2  expression (Figure  2A ). It appears that a minimal duration of  PK2  expression is maintained under short photoperiod (Figure  2A ) and constant darkness [ 28 ], which further indicate the dominant control of  PK2  expression by the circadian clock. Interestingly, the amplitude of the  PK2  oscillation was greatly reduced under very long photoperiod (20L:4D) (Figure  2C ). As the amplitude of both  Per1  and  Per2  rhythms were also reduced during 20L:4D (see  Additional file 1 ), it is likely that these depressed rhythms of clockwork genes may contribute to the depressed  PK2  rhythm observed. Whether reduction in the amplitude of expression in any of these genes is related to arrhythmicity in LL deserves further examination.PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2PK2Per1Per2PK2Our studies with  Cry1-/-Cry2-/-  mice revealed the presence of a light-driven  PK2  molecular rhythm in the SCN under LD, indicating that light can drive  PK2  rhythm independent of functional circadian clock. Interestingly, the molecular rhythms of some clockwork genes such as  Per2 ,  Per1 , and  Bmal1  were also partially maintained in the SCN of  Cry1-/-Cry2-/-  mice under LD (Figure  4 ). 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-regulated  Per2  molecular rhythm in the SCN of  Cry1-/-Cry2-/-  mice, and suggested the term of "light-driving" effect [ 25 ]. As  Cry1-/-Cry2-/-  mice lack functional circadian clocks and their locomotor behaviour remains rhythmic under LD, but not under DD conditions [ 24 , 25 ], it is likely that these light-driven molecular rhythms may drive the locomotor rhythms in these animals. As we have previously shown that  PK2  may be a critical output molecule responsible for circadian locomotor rhythms, the presence of this light-driven  PK2  rhythm in  Cry1-/-Cry2-/-  mice 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 [ 43 ]. The role of the SCN in masking of locomotor activity by light is controversial, with similar studies having produced contradictory results [ 23 , 44 ]. 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  PK2 -deficient mice will be necessary to resolve the exact role of PK2 in the light-driven locomotor rhythms.Cry1-/-Cry2-/-PK2PK2Per2Per1Bmal1Cry1-/-Cry2-/-Per2Cry1-/-Cry2-/-Cry1-/-Cry2-/-PK2PK2Cry1-/-Cry2-/-PK2The light inducibility of  PK2  in 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  PK2  (Figure  3 ). The same photoreceptive system has been shown responsible for the entrainment of locomotor rhythm [ 5 - 11 ]. The light inducibility of  PK2  may be related to the presence of a putative cyclic AMP response element (CRE) in the promoter of the  PK2  gene [ 28 ]. CRE-dependent activation is critical for light-induced gene expression in the SCN [ 45 - 48 ]. The reduced light inducibility of  PK2  in mutant mice that lack functional clock may indicate that CRE-dependent pathway and CLK/BMAL1 transcriptional factors may interact in the light-induced  PK2  expression in the SCN. Accumulative data have implicated the photic regulation of the transcription of clock genes such as  Per1  and  Per2  in the entrainment of behavioural rhythms [ 30 , 34 ]. 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 the  Per1  promoter, whose activation in the SCN shifts rapidly when the LD cycle is advanced [ 31 ],  PK2  exhibits transients during phase advance, more similar to those of  Cry1  and  Cry2  [ 30 , 31 ]. This is consistent with the role for PK2 as a clock-controlled gene and thus is downstream from the light-regulated expression of  Per1  or  Per2 . The presence of E box motifs in the  PK2  promoter suggests that light-regulated  Per1  (and perhaps  Per2 ) expression can influence  PK2  expression. However, the light inducibility of  PK2  indicates 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 [ 49 ]. As receptor for PK2 is highly expressed in the SCN [ 28 ] and activation of the PK2 receptor triggers similar signalling pathways as that of glutamate receptors [ 29 ], 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 [ 50 ], 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 of  PK2  may be relevant to both the phase resetting of the core circadian loops and critical SCN output signals.PK2PK2PK2PK2PK2PK2Per1Per2Per1PK2Cry1Cry2Per1Per2PK2Per1Per2PK2PK2PK2Conclusion Our studies demonstrate that  PK2  is predominantly driven by the circadian clock, as  PK2  expression exhibits circadian transients in response to phase advances. Furthermore, shortening of the light period does not result in corresponding reduction of the phase of  PK2  rhythm, also consistent with the dominant control from the circadian clock on  PK2  expression. However, light also modulates  PK2  rhythm. Nocturnal light pulses can directly induce  PK2  expression in the SCN. Studies with  Cry1-/-Cry2-/-  mice revealed that light can drive a low amplitude  PK2  molecular rhythm in the SCN in the absence of functional circadian oscillators. These studies demonstrate that  PK2  molecular rhythm in the SCN is controlled by dual mechanisms: dominantly by the circadian transcriptional loops but also directly by light. The light inducibility of  PK2  in 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.
Conclusion
Our studies demonstrate that  PK2  is predominantly driven by the circadian clock, as  PK2  expression exhibits circadian transients in response to phase advances. Furthermore, shortening of the light period does not result in corresponding reduction of the phase of  PK2  rhythm, also consistent with the dominant control from the circadian clock on  PK2  expression. However, light also modulates  PK2  rhythm. Nocturnal light pulses can directly induce  PK2  expression in the SCN. Studies with  Cry1-/-Cry2-/-  mice revealed that light can drive a low amplitude  PK2  molecular rhythm in the SCN in the absence of functional circadian oscillators. These studies demonstrate that  PK2  molecular rhythm in the SCN is controlled by dual mechanisms: dominantly by the circadian transcriptional loops but also directly by light. The light inducibility of  PK2  in 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.PK2PK2PK2PK2PK2PK2Cry1-/-Cry2-/-PK2PK2PK2Methods 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 examine  PK2  expression 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 examine  PK2  mRNA expression in the SCN [ 28 ]. Antisense and sense riboprobes containing the coding region of mouse  PK2  (accession number AF487280 1-528 nt), mouse  Per1  (accession number AF022992 340-761nt), mouse  Per2  (accession number AF035830 9-489 nt) and mouse  Bmal1  (accession number AB015203 864-1362 nt) were generated. 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 ( Opn4-/- ) mice (on C57BL/6:129 hybrid background) [ 5 ] were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). For light pulse studies, wild type,  Opn4-/-  mice and triple knockouts ( Opn4-/- Gnat1-/- Cnga3-/-  mice) that lack melanopsin, rod and cone phototransduction systems were used [ 10 ]. 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. Studies of cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice Cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice on a C57BL/6:129 hybrid background were kindly provided by Dr. Aziz Sancar (University of North Carolina at Chapel Hill). Wild type and  Cry1-/-Cry2-/-  mice were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). A second group of  Cry1-/-Cry2-/-  mice were placed into two days of constant darkness (2DD) (Circadian time, CT, CT1-22). The mRNA levels of  PK2 ,  Per2 ,  Per1  and  Bmal1  were measured in the SCN. For light pulse experiments,  Cry1-/-Cry2-/-  mice received a 15 min light pulse (~400 lux) at ZT14, and sampled one or two hours after light pulse. Dark control  Cry1-/-Cry2-/-  mice did not receive a light pulse. For the shifting experiments, wildtype and  Cry1-/-Cry2-/-  mice 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.
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 examine  PK2  expression 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 examine  PK2  mRNA expression in the SCN [ 28 ]. Antisense and sense riboprobes containing the coding region of mouse  PK2  (accession number AF487280 1-528 nt), mouse  Per1  (accession number AF022992 340-761nt), mouse  Per2  (accession number AF035830 9-489 nt) and mouse  Bmal1  (accession number AB015203 864-1362 nt) were generated.
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 examine  PK2  expression 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 examine  PK2  mRNA expression in the SCN [ 28 ]. Antisense and sense riboprobes containing the coding region of mouse  PK2  (accession number AF487280 1-528 nt), mouse  Per1  (accession number AF022992 340-761nt), mouse  Per2  (accession number AF035830 9-489 nt) and mouse  Bmal1  (accession number AB015203 864-1362 nt) were generated.PK2PK2PK2Per1Per2Bmal1Photoperiod 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.
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 ( Opn4-/- ) mice (on C57BL/6:129 hybrid background) [ 5 ] were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). For light pulse studies, wild type,  Opn4-/-  mice and triple knockouts ( Opn4-/- Gnat1-/- Cnga3-/-  mice) that lack melanopsin, rod and cone phototransduction systems were used [ 10 ]. 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.
Studies of melanopsin-deficient mice and mice that lack melanopsin, rods and cones
Wild type and melanopsin-deficient ( Opn4-/- ) mice (on C57BL/6:129 hybrid background) [ 5 ] were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). For light pulse studies, wild type,  Opn4-/-  mice and triple knockouts ( Opn4-/- Gnat1-/- Cnga3-/-  mice) that lack melanopsin, rod and cone phototransduction systems were used [ 10 ]. 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-/-Studies of cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice Cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice on a C57BL/6:129 hybrid background were kindly provided by Dr. Aziz Sancar (University of North Carolina at Chapel Hill). Wild type and  Cry1-/-Cry2-/-  mice were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). A second group of  Cry1-/-Cry2-/-  mice were placed into two days of constant darkness (2DD) (Circadian time, CT, CT1-22). The mRNA levels of  PK2 ,  Per2 ,  Per1  and  Bmal1  were measured in the SCN. For light pulse experiments,  Cry1-/-Cry2-/-  mice received a 15 min light pulse (~400 lux) at ZT14, and sampled one or two hours after light pulse. Dark control  Cry1-/-Cry2-/-  mice did not receive a light pulse. For the shifting experiments, wildtype and  Cry1-/-Cry2-/-  mice 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.
Studies of cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice
Cry1-/-Cry2-/-Cryptochrome-deficient ( Cry1-/-Cry2-/- ) mice on a C57BL/6:129 hybrid background were kindly provided by Dr. Aziz Sancar (University of North Carolina at Chapel Hill). Wild type and  Cry1-/-Cry2-/-  mice were entrained to 12L:12D and sampled every three hours for the 24 hour period (ZT1-22). A second group of  Cry1-/-Cry2-/-  mice were placed into two days of constant darkness (2DD) (Circadian time, CT, CT1-22). The mRNA levels of  PK2 ,  Per2 ,  Per1  and  Bmal1  were measured in the SCN. For light pulse experiments,  Cry1-/-Cry2-/-  mice received a 15 min light pulse (~400 lux) at ZT14, and sampled one or two hours after light pulse. Dark control  Cry1-/-Cry2-/-  mice did not receive a light pulse. For the shifting experiments, wildtype and  Cry1-/-Cry2-/-  mice 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-/-PK2Per2Per1Bmal1Cry1-/-Cry2-/-Cry1-/-Cry2-/-Cry1-/-Cry2-/-Authors' contributions ELB sampled the  Cry1-/-Cry2-/-  mice and performed behavior experiments on  Cry1-/-Cry2-/-  mice. 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.
Authors' contributions
ELB sampled the  Cry1-/-Cry2-/-  mice and performed behavior experiments on  Cry1-/-Cry2-/-  mice. 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-/-Supplementary Material Additional File 1 Effect of different photoperiods on molecular rhythms in the SCN. Temporal profiles of Per1 (a) and Per2 (b) mRNA under 8L:16D, 16L:8D, 20L:4D. Open and filled bars indicate light and dark periods, respectively. The zeitgeber time (ZT) on the x-axis reflects the timescale for each photoperiod. Each value represents the mean ± SEM of 3–4 animals. Click here for file Additional File 2 PK2 mRNA expression in Cry1-/-Cry2-/- and wildtype mice. Representative autoradiograms of PK2 mRNA in the SCN of Cry1-/-Cry2-/- mice (Cry) and wild type mice (WT) under LD (ZT1-22) are shown (top and bottom row, respectively). Scale bar = 1 mm. Click here for file
Supplementary Material
Additional File 1
Effect of different photoperiods on molecular rhythms in the SCN. Temporal profiles of Per1 (a) and Per2 (b) mRNA under 8L:16D, 16L:8D, 20L:4D. Open and filled bars indicate light and dark periods, respectively. The zeitgeber time (ZT) on the x-axis reflects the timescale for each photoperiod. Each value represents the mean ± SEM of 3–4 animals.Click here for fileAdditional File 2
PK2 mRNA expression in Cry1-/-Cry2-/- and wildtype mice. Representative autoradiograms of PK2 mRNA in the SCN of Cry1-/-Cry2-/- mice (Cry) and wild type mice (WT) under LD (ZT1-22) are shown (top and bottom row, respectively). Scale bar = 1 mm.Click here for file


The Medical Library Association converts access to information into access to knowledge in a networked environment of digital resources

“I look forward to such an organization of the literary records of medicine that a puzzled worker in any part of the civilized world shall in an hour be able to gain the knowledge pertaining to a subject of the experience of every other man in the world.”
 —George Gould, first president of the Association of Medical Librarians (now the Medical Library Association), May 1898“I look forward to such an organization of the literary records of medicine that a puzzled worker in any part of the civilized world shall in an hour be able to gain the knowledge pertaining to a subject of the experience of every other man in the world.”“I look forward to such an organization of the literary records of medicine that a puzzled worker in any part of the civilized world shall in an hour be able to gain the knowledge pertaining to a subject of the experience of every other man in the world.”—George Gould, first president of the Association of Medical Librarians (now the Medical Library Association), May 1898For over 100 years, the Medical Library Association (MLA) has upheld the belief that quality information is essential for improved health and has worked to ensure that health sciences librarians have the skills, knowledge, and leadership necessary for the delivery of information in a biomedical setting. The association has also promoted the concept of unrestricted, affordable, and permanent access of health information worldwide. For example, MLA's peer-reviewed  Journal of the Medical Library Association
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MLA's statement on open access, found at  http://www.mlanet.org/government/info_access/openaccess_statement.html , defines the association's position on this important topic.Journal of the Medical Library Association(JMLA)Bulletin of the Medical Library Association (BMLA)BMLAHowever, open access increases the need for more sophisticated information management tools and systems, such as quality filtering and customization of clinical and research information at the point of need and decision-making. MLA is pursuing a number of initiatives that address the specific information needs of clinicians, healthcare students, biomedical researchers, and institutional leaders. Our members are excited to be in a unique position to develop tools, resources, and advice on how to find relevant information on the Internet. For example, MLA members have developed a  User's Guide to Finding and Evaluating Health Information on the Web  for the Pew Internet and American Life Project. 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