- Email: [email protected]
Nonphotic phase-shifting in Clock mutant mice
Brain Research 859 Ž2000. 398–403 www.elsevier.comrlocaterbres
Short communication
Nonphotic phase-shifting in Clock mutant mice Etienne Challet
a, )
, Joseph S. Takahashi
a,b
, Fred W. Turek
a
a
b
Center for Circadian Biology and Medicine, Department of Neurobiology and Physiology, Northwestern UniÕersity, 2153 North Campus DriÕe, EÕanston, IL 60208, USA Howard Hughes Medical Institute and National Science Foundation Center for Biological Timing, Northwestern UniÕersity, 2153 North Campus DriÕe, EÕanston, IL 60208, USA Accepted 11 January 2000
Abstract Nonphotic phase-shifting was studied in mice bearing the Clock mutation. First, free-running mice heterozygous for Clock and wild-type mice were induced to become active through a 4-h confinement to a novel running over 3 days. Second, mice exposed to light–dark cycle received daily hypocaloric food during 2 weeks, before being transferred to constant darkness and fed ad libitum. Behavioral activation during the mid-subjective day induced 40-min phase advances in the locomotor activity rhythm of wild-type mice, whereas it produced 50-min phase delays in the circadian behavior of Clockrq mice. Calorie restriction phase-advanced by 80 min the locomotor activity rhythm in wild-type mice, but not in Clockrq mice. Therefore, the response of the Clockrq mice to nonphotic phase shifting differs from that of wild-type mice. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Suprachiasmatic nucleus; Circadian rhythm; Photic synchronization; Light-entrainable pacemaker; Clock gene; Locomotor behavior
Circadian rhythms are behavioral or physiological endogenous rhythms driven by an internal timing system with a period close to, but generally not equal to, 24 h. In mammals, the master circadian clock is located in the suprachiasmatic nuclei ŽSCN. of the hypothalamus w11x. The molecular core of the mammalian circadian oscillator is thought to involve a negative autoregulatory feedback loop implicating several genes, including the Clock gene w19x. The Clock mutation, identified in the mouse from a chemical mutagenesis screen, lengthens the free-running period of circadian rhythms by approximately 1 h in Clockrq mice and 4 h in ClockrClock mice, the latter losing circadian rhythmicity after days or weeks in constant darkness w23x. CLOCK has been identified as a transcriptional regulatory protein that is expressed in the SCN as well as in many other cells and tissues w10x. Three mammalian homologs of the Drosophila clock gene period Ž per . have been cloned in the mouse: mPer1, mPer2 and mPer3. These three genes are expressed in the SCN wherein their mRNA levels show circadian oscillations, suggesting an involvement of mPer genes in the )
Corresponding author. Department of Neurobiology of Rhythmic and Seasonal Functions, CNRS-UMR7518, Louis Pasteur University, 12 rue de l’Universite, ´ F-67000 Strasbourg, France. Fax: q33-3-88-24-04-61; e-mail: [email protected]
feedback loop of the clock w1,5,15,18,20,24x. Together with BMAL1, another transcription factor, CLOCK forms heterodimers that activate transcription of mPer1, so that CLOCK and BMAL1 may be positive regulators of the clock transcriptional loop w4,6,8x. Moreover, expression of mPer genes is markedly reduced in Clock mutant mice, suggesting that CLOCK–BMAL1 complexes positively regulate expression of mPer w4,8x. The daily light–dark cycle is the main environmental cue that entrains Žsynchronizes. the SCN clock to the 24 h. In addition to lengthening the free-running period, the heterozygous state of the Clock mutation is associated with increased light-induced circadian phase shifts w22x. Furthermore, light-induced expression of mPer1 and mPer2 is reduced in the homozygous Clock mutant mice w14x. These findings suggest that CLOCK participates in the photoentrainment in the SCN. Besides light, the phase of the light-entrainable circadian clock can be shifted by a variety of nonphotic factors, including behavioral stimuli Že.g., confinement to a novel running wheel., pharmacological cues Že.g., triazolam treatment., or saline injection w7,17,21x. Moreover, some nonphotic events, like behavioral and metabolic cues, may modulate the circadian responses to light w2,12x. Nonphotic factors are considered to act on the circadian clock via transduction pathways different from those medi-
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 2 0 4 0 - 0
E. Challet et al.r Brain Research 859 (2000) 398–403
ating photoentrainement w7,17x. Very little is known about how nonphotic signals phase-shift the circadian oscillator at the molecular level or the role of the recently discovered circadian clock genes in these responses. In this context, we determined whether the Clock mutation affects the response of the circadian system to two different nonphotic phase-shifting cues. Seven Clockrq heterozygous mice, derived from a C57BLr6J founder animal w23x, and seven qrq adult C57BLr6J mice were bred at Northwestern University. For both groups of mice, four males and three females were housed singly in cages equipped with running wheels Ždiameter: 11 cm. in a temperature-controlled room Ž23 " 18C. with a light–dark 12:12 h cycle Žlights on at 0500 h.. During daytime, light intensity was about 200 lx at cage level. Food Žlaboratory chow, Harlan Teklad, WI. and water were available ad libitum, unless otherwise stated. Wheel-running activity was continuously recorded ŽChronobiology Kit, Stanford Software Systems, Stanford, CA.. Mice were phenotyped according to the circadian period Žt . as previously described w23x. Mice were also genotyped for the Clock locus with genomic DNA extracted from tail tips using simple sequence length polymorphisms w9x. Experiment 1 was performed to assess the phase-shifting effects of confinement to a novel running wheel in Clockrq and qrq mice. For that purpose, mice were given novel-wheel pulses according to a protocol developed in Syrian hamsters by Mrosovsky and colleagues w7x. After 10 days in constant darkness, all mice were confined singly into clean wheels for 4 h during the subjective day Ži.e., from circadian time wCTx 6 to CT10, with CT12 defined as the onset of locomotor activity.. These novelwheel pulses were given on three consecutive days. In experiment 2, the synchronizing effects of timed calorie restriction, previously shown to change the phase angle of entrainment in wild-type mice exposed to a light–dark cycle w3x, were studied in Clockrq mice. For that purpose, once experiment 1 was completed, the mice were transferred back to a daily light–dark cycle for 2 weeks. Daily food intake was found to be about 6 g of chowrday regardless of the genotype. Mice were calorierestricted for 2 weeks and were given 66% of daily food intake Ži.e., 4 g of chowrday. 2 h after the onset of light. Thereafter, all animals were transferred to constant darkness and fed ad libitum for 4 weeks. When switched to ad libitum feeding, calorie-restricted mice were provided with food beginning at the time when hypocaloric food was given previously. Data for 10 consecutive cycles were divided into 10-min bins of time. The onset of the nocturnal wheel-running activity was defined as the first 10-min bin in which 20% of maximal intensity for that cycle was followed by a similar level of activity in three out of the next six bins. This analysis was performed over two intervals in experiment 1: the last 10 days prior to the first novel-wheel pulse day and the first 10 days after the third Žlast. novel-wheel
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pulse day. A similar analysis was performed over three intervals in experiment 2: the last 5 days of baseline conditions, the last 5 days of the calorie restriction, and the first 5 days in constant darkness. Linear regression analysis of the onsets of the nocturnal activity was performed with TableCurve software ŽJandel Scientific, San Rafael, CA.. In experiment 1, the data for the days with a novel-wheel pulse Ždenoted as days 11, 12 and 13. were discarded from the analysis. Data were fitted to the following equations: wif t F 10, y s At q B; if t ) 13, y s Ct q D x, where t was the number of days, A was the slope and B the initial phase of the line fitted to the 10 days prior to the first novel-wheel pulse Žday 11., and C was the slope and D the initial phase of the line fitted to the 10 days following the last novel-wheel pulse Žday 13.. The magnitude of the phase shift was calculated as the difference between these two lines on day 14. In experiment 2, during baseline conditions or calorie restriction, the fitting equation was: y s w A x, where A was the mean onset of nocturnal activity. Phase angle of entrainment to the light–dark cycle was calculated as the number of minutes before Žpositive. or after Žnegative. the offset of light compared to the mean nocturnal onset. For the data under constant darkness conditions, the fitting equation was: y s At q B, where A was the slope of the line Žaccording to t . and B the initial phase of the nocturnal onset of locomotor activity in constant darkness. Circadian phase changes were expressed as the number of minutes before Žpositive. or after Žnegative. the time of light offset Žthe day before. projected to the first day in constant darkness. t was assessed using the x 2 periodogram ŽChronobiology Kit software. over 10 days in constant darkness. Data are means " S.E.M. Novel-wheel pulses induced significant phase shifts in locomotor activity rhythm, with a direction depending on the genotype Ž t-test; P - 0.01.. Three novel-wheel pulses applied during the mid-subjective day led to 41 " 15-min phase advances in qrq wild-type mice and to 47 " 23min phase delays in Clockrq mice ŽFigs. 1 and 2.. Mean t calculated over experiment 1 was longer in mice heterozygous for Clock than in wild-type mice Žtwo-way ANOVA with repeated measures; effect of the genotype: P - 0.001.. Moreover, t was markedly modified by novel-wheel pulses Žeffect of the treatment: P - 0.001.: t was lengthened after behavioral activation in heterozygous mice Ž24.1 " 0.05 vs. 24.6 " 0.06 h, respectively; P 0.05., but not in wild-type mice Ž23.7 " 0.08 vs. 23.6 " 0.1 h, respectively; P ) 0.1; Fig. 1.. The cumulative wheel revolutions performed during the 4-h novel-wheel pulses were similar in qrq mice and Clockrq mice Ž9780 " 860 vs. 10080 " 510 wheel turns per 4 h, respectively; t-test; P ) 0.1.. The phase of the nocturnal onset of locomotor activity was modified by both the genotype and the calorie restriction over experiment 2 Žtwo-way ANOVA with repeated measures; effect of the genotype: P - 0.01; effect of the
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Fig. 1. Novel-wheel pulses. Wheel-running activity of a wild-type Žqrq . mouse Žtop. and a mouse heterozygous for Clock Ž Clockrq; bottom. kept in constant darkness. To facilitate visualization of phase shifts and t changes, data were presented modulo t Žt before wheel pulsess 23.6 and 24.0 h in the qrq mouse and the Clockrq mouse, respectively.. Successive circadian cycles are double-plotted Žtwo cycles per horizontal time scale.. Animals were confined into novel wheels for 4 h from circadian time 6 to circadian 10 over 3 days.
nutritional state: P - 0.001; genotype= nutritional status interaction: P - 0.01.. When mice were fed ad libitum under a light–dark cycle, the onset of nocturnal activity was close to the time of light off, albeit with a more negative phase angle of entrainment in Clockrq mice compared to that of qrq mice Žy50 " 6 vs. y20 " 4
Fig. 2. Phase shifts in circadian activity rhythms of wild-type and Clock rq mice. Positive and negative values are advances and delays, respectively. Phase shifts after novel-wheel pulses were assessed in constant darkness. Phase shifts after calorie restriction were calculated as the differences between the initial phase under light–dark baseline conditions and that in constant darkness. Values are means"S.E.M.; ns 7 per group.
min relative to light offset, respectively; Figs. 2 and 3.. Providing a daily hypocaloric diet 2 h after lights on led to a significant phase advance in the onset of activity in both Clockrq mice and qrq mice Žonset: 50 " 13 and 90 " 14 min relative to light offset, respectively.. After transfer to constant darkness, a phase advance of about 80 min in the circadian rhythm of locomotor activity was observed in wild-type mice Žonset: 62 " 19 min relative to light offset the day before.. In contrast, no phase advance was detectable in Clockrq individuals Žonset: y54 " 18 min relative to light offset the day before; Figs. 2 and 3., the nocturnal onset in constant darkness starting close to the initial phase under light–dark baseline conditions. Mean t assessed during the first 10 days in DD was 1 h longer in mice heterozygous for Clock than in wild-type mice Ž24.6 " 0.06 vs. 23.6 " 0.06 h, respectively; t-test, P - 0.001; Fig. 3.. No changes in t were detected over 4 weeks in constant darkness Ž P ) 0.05.. Body mass loss after the 2 weeks of calorie restriction was not significantly different between Clockrq and qrq mice Žy24.3 " 1.0 vs. y26.6 " 0.7%, respectively; t-test, P ) 0.1.. The present results indicate that the Clock mutation modifies the response of the circadian timing system to nonphotic cues. First, behavioral activation during the subjective day induced opposite phase shifts in Clock mutant mice compared to those in wild-type individuals housed in constant darkness. Second, modulation of the photic entrainment by a timed calorie restriction, while
E. Challet et al.r Brain Research 859 (2000) 398–403
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Fig. 3. Timed calorie restriction. Double-plotted wheel-running activity of a wild-type Žqrq . mouse Žtop. and a mouse heterozygous for Clock Ž Clockrq; bottom.. Days 1–8, initial interval of ad libitum feeding under light–dark conditions; days 9–22, timed calorie restriction ŽCR.; days 23–33, final interval of ad libitum in constant darkness ŽDD.. When mice were housed under a light–dark cycle, nighttime is indicated by black bar on abscissa. Vertical lines are projected time of light offset Ži.e., 1900 h.. Time when hypocaloric diet was provided during calorie restriction Ži.e., 2 h after light onset. is indicated by vertical arrow on abscissa.
altering the daily organization of locomotor activity in both qrq and Clockrq mice exposed to a light–dark cycle, led to phase advances of nocturnal pattern of activity Žassessed in constant darkness. in wild-type mice, but not in mice heterozygous for Clock. The molecular mechanisms by which nonphotic cues reset the clock are still poorly understood. A feature shared by most nonphotic cues is their ability to robustly phase shift the clock when presented during the middle of the subjective day w16x, that is, when expression of mPer genes is the highest Že.g., Refs. w15,18x.. This correlation raises the possibility that nonphotic cues are able to interact at this step of the clock transcriptional loop. For instance, nonphotic cues might inhibit the transcription of an mPer gene. Resetting the clock during the subjective day by nonphotic cues usually induces phase advances w16x, as we observed after novel-wheel pulses in wild-type mice. It should therefore be emphasized that similar nonphotic cues led to opposite phase shifts Ži.e., phase delays. in Clockrq mice. Because phase advances and delays are probably mediated in the SCN through different molecular mechanisms, the model of novel-wheel pulses in Clock mutants may be a useful tool to determine whether the transcriptional regulation of mPer and other clock-related
genes during the subjective day is differentially affected by nonphotic cues. In Clockrq mice, behavioral activation induced a clear change in t . One possibility is that behavioral activation acted to trigger these changes in t . In spite of strong homeostatic control, the stability of t can be subject to subtle environmental history-dependent modulations. For instance, single nonphotic pulses can modify t in constant darkness w13x. Although calculation of phase shifts might be affected by t changes, this methodological issue is still an open question w13x. In the present study, it is noteworthy that t was not affected by behavioral activation in wild-type mice, suggesting that the SCN function is more sensitive to these kinds of modulation in Clockrq mice compared to wild-type individuals. A spontaneous lengthening in t has been reported in Clockrq mice housed in constant darkness w23x. Because t values were not modified over a similar period in free-running mice studied in the absence of phase-shifting cues Žexperiment 2., behavioral cues that have shifted the clock may also have accelerated the process of t lengthening in Clock mutant mice Žexperiment 1.. Besides novelty-induced behavioral activity, Clockrq mice were also studied in another paradigm: timed calorie
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restriction under exposure to a light–dark cycle. During baseline light–dark conditions, a more negative phase of photic synchronization was observed in Clockrq mice in accordance with their longer t . Compared to this initial phase when food was provided ad libitum, timed calorie restriction modified the phase angle of photic entrainment by advancing Žabout 1.5 h. the nocturnal onset in both qrq and Clockrq mice. As previously observed w3x, a 1-h phase advance of the nocturnal pattern of locomotor activity was still detectable in wild-type mice after they were placed in constant darkness and fed ad libitum. In contrast, no significant phase change was found in freerunning mice heterozygous for Clock. Light induces phase delays during the early subjective night and phase advances during late subjective night. Because t in wild-type C57BL mice is - 24 h, light-induced phase delays are critical for photic entrainment. The circadian responses to light are reduced during calorie restriction w3x, and other situations of reduced glucose availability w2x. Therefore, the phase advances observed in calorie-restricted qrq mice Žwith no changes in t . might allow more light to fall in their delay region, thus inducing phase delays sufficient for photic entrainment. In addition to a lengthening of the circadian period, the Clock mutation is associated with increased light-induced circadian phase shifts in animals fed ad libitum w22x. Thus, a possible reduction of photic resetting in calorie-restricted Clockrq mice would not be sufficient to significantly phase shift the clock after transfer to constant darkness. Alternatively, the stepŽs. in photic phase resetting that are sensitive to timed calorie restriction may involve CLOCK or CLOCK-controlled factors. Although light is the major synchronizer of the light-entrainable clock, the present study confirms that nonphotic cues can also influence the circadian timing system either in constant darkness Žbehavioral activation. or in the presence of a light–dark cycle Žtimed calorie restriction.. In both paradigms, the Clock mutation altered the typical response of the circadian system. Therefore, protocols using nonphotic cues may offer useful tools to further unravel the molecular basis of the SCN clockwork and its entrainment to environmental stimuli.
Acknowledgements The authors are grateful to Anne-Marie Chang and Sue Losee-Olson for expert assistance with genotyping. This work was supported by National Institute of Health Grants HLrMH-96015 and AG-11412 ŽFWT., US Army Research Office Grant DAAG55-98-1-0196 ŽFWT., NSF Center for Biological Timing Grant ŽJST. and an Unrestricted Research Grant in Neuroscience from BristolMyers Squibb ŽJST.. JST is an investigator in the Howard Hughes Medical Institute.
References w1x U. Albrecht, Z.S. Sun, G. Eichele, C.C. Lee, A differential response of two putative mammalian circadian regulators, mPer1 and mPer2, to light, Cell 91 Ž1997. 1055–1064. w2x E. Challet, S. Losee-Olson, F.W. Turek, Reduced glucose availability attenuates circadian responses to light in mice, Am. J. Physiol. 276 Ž1999. R1063–R1070. w3x E. Challet, L.C. Solberg, F.W. Turek, Entrainment in calorie-restricted mice: conflicting zeitgebers and free-running conditions, Am. J. Physiol. 274 Ž1998. R1751–R1761. w4x N. Gekakis, D. Staknis, H.B. Nguyen, F.C. Davis, L.D. Wilsbacher, D.P. King, J.S. Takahashi, C.J. Weitz, Role of the CLOCK protein in the mammalian circadian mechanism, Science 280 Ž1998. 1564– 1569. w5x M.H. Hastings, M.D. Field, E.S. Maywood, D.R. Weaver, S.M. Reppert, Differential regulation of mPER1 and mTIM proteins in the mouse suprachiasmatic nuclei: new insights into a core clock mechanism, J. Neurosci. 19 ŽRC11. Ž1999. 1–7. w6x S. Honma, M. Ikeda, H. Abe, Y. Tanahashi, M. Namihira, K. Honma, M. Nomura, Circadian oscillation of BMAL1, a partner of a mammalian clock gene Clock, in rat suprachiasmatic nucleus, Biochem. Biophys. Res. Commun. 250 Ž1998. 83–87. w7x D. Janik, N. Mrosovsky, Gene expression in the geniculate induced by a nonphotic circadian phase shifting stimulus, NeuroReport 3 Ž1992. 575–578. w8x X. Jin, L.P. Shearman, D.R. Weaver, M.J. Zylka, G.J. DeVries, S.M. Reppert, A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock, Cell 96 Ž1999. 57–68. w9x D.P. King, M.H. Vitaterna, A.M. Chang, W.F. Dove, L.H. Pinto, F.W. Turek, J.S. Takahashi, The mouse Clock mutation behaves as an antimorph and maps within the W 19 H deletion, distal of Kit, Genetics 146 Ž1997. 1049–1060. w10x D.P. King, Y.L. Zhao, A.M. Sangoram, L.D. Wilsbacher, M. Tanaka, M.P. Antoch, T.D.L. Steeves, M.H. Vitaterna, J.M. Kornhauser, P.L. Lowrey, F.W. Turek, J.S. Takahashi, Positional cloning of the mouse circadian Clock gene, Cell 89 Ž1997. 641–653. w11x D.C. Klein, R.Y. Moore, S.M. Reppert, Suprachiasmatic Nucleus. The Mind’s Clock, Oxford Univ. Press, New York, 1991. w12x R.E. Mistlberger, M.C. Antle, Behavioral inhibition of light-induced circadian phase resetting is phase and serotonin dependent, Brain Res. 786 Ž1998. 31–38. w13x N. Mrosovsky, Tau changes after single nonphotic events, Chronobiol. Int. 10 Ž1993. 271–276. w14x L.P. Shearman, D.R. Weaver, Photic induction of Period gene expression is reduced in Clock mutant mice, NeuroReport 10 Ž1999. 613–618. w15x L.P. Shearman, M.J. Zylka, D.R. Weaver, L.F. Kolakowski, S.M. Reppert, Two Period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei, Neuron 19 Ž1997. 1261– 1269. w16x R.D. Smith, F.W. Turek, J.S. Takahashi, Two families of phase–response curves characterize the resetting of the hamster circadian clock, Am. J. Physiol. 262 Ž1992. R1149–R1153. w17x A. Sumova, F.J.P. Ebling, E.S. Maywood, J. Herbert, M.H. Hastings, Non-photic circadian entrainment in the Syrian hamster is not associated with phosphorylation of the transcriptional regulator CREB within the suprachiasmatic nucleus, but is associated with adrenocortical activation, Neuroendocrinology 59 Ž1994. 579–589. w18x Z.S. Sun, U. Albrecht, O. Zhuchenko, J. Bailey, G. Eichele, C.C. Lee, RIGUI, a putative mammalian ortholog of the Drosophila Period gene, Cell 90 Ž1997. 1003–1011. w19x J.S. Takahashi, Molecular neurobiology and genetics of circadian rhythms in mammals, Annu. Rev. Neurosci. 18 Ž1995. 531–553. w20x H. Tei, H. Okamura, Y. Shigeyoshi, C. Fukuhara, R. Ozawa, M. Hirose, Y. Sakaki, Circadian oscillation of a mammalian homologue of the Drosophila Period gene, Nature 389 Ž1997. 512–516.
E. Challet et al.r Brain Research 859 (2000) 398–403 w21x O. Van Reeth, F.W. Turek, Stimulated activity mediates phase shifts in the hamster circadian clock induced by dark pulses or benzodiazepines, Nature 339 Ž1989. 49–51. w22x M.H. Vitaterna, A.M. Chang, D.P. King, L.H. Pinto, F.W. Turek, J.S. Takahashi, Heterozygosity at the Clock locus alters phase response curves to light in mice, Soc. Res. Biol. Rhythms Abstr. 5 Ž1996. 127. w23x M.H. Vitaterna, D.P. King, A.M. Chang, J.M. Kornhauser, P.L.
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Lowrey, J.D. McDonald, W.F. Dove, L.H. Pinto, F.W. Turek, J.S. Takahashi, Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior, Science 264 Ž1994. 719–725. w24x M.J. Zylka, L.P. Shearman, D.R. Weaver, S.M. Reppert, Three Period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain, Neuron 20 Ž1998. 1103–1110.
Short communication
Nonphotic phase-shifting in Clock mutant mice Etienne Challet
a, )
, Joseph S. Takahashi
a,b
, Fred W. Turek
a
a
b
Center for Circadian Biology and Medicine, Department of Neurobiology and Physiology, Northwestern UniÕersity, 2153 North Campus DriÕe, EÕanston, IL 60208, USA Howard Hughes Medical Institute and National Science Foundation Center for Biological Timing, Northwestern UniÕersity, 2153 North Campus DriÕe, EÕanston, IL 60208, USA Accepted 11 January 2000
Abstract Nonphotic phase-shifting was studied in mice bearing the Clock mutation. First, free-running mice heterozygous for Clock and wild-type mice were induced to become active through a 4-h confinement to a novel running over 3 days. Second, mice exposed to light–dark cycle received daily hypocaloric food during 2 weeks, before being transferred to constant darkness and fed ad libitum. Behavioral activation during the mid-subjective day induced 40-min phase advances in the locomotor activity rhythm of wild-type mice, whereas it produced 50-min phase delays in the circadian behavior of Clockrq mice. Calorie restriction phase-advanced by 80 min the locomotor activity rhythm in wild-type mice, but not in Clockrq mice. Therefore, the response of the Clockrq mice to nonphotic phase shifting differs from that of wild-type mice. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Suprachiasmatic nucleus; Circadian rhythm; Photic synchronization; Light-entrainable pacemaker; Clock gene; Locomotor behavior
Circadian rhythms are behavioral or physiological endogenous rhythms driven by an internal timing system with a period close to, but generally not equal to, 24 h. In mammals, the master circadian clock is located in the suprachiasmatic nuclei ŽSCN. of the hypothalamus w11x. The molecular core of the mammalian circadian oscillator is thought to involve a negative autoregulatory feedback loop implicating several genes, including the Clock gene w19x. The Clock mutation, identified in the mouse from a chemical mutagenesis screen, lengthens the free-running period of circadian rhythms by approximately 1 h in Clockrq mice and 4 h in ClockrClock mice, the latter losing circadian rhythmicity after days or weeks in constant darkness w23x. CLOCK has been identified as a transcriptional regulatory protein that is expressed in the SCN as well as in many other cells and tissues w10x. Three mammalian homologs of the Drosophila clock gene period Ž per . have been cloned in the mouse: mPer1, mPer2 and mPer3. These three genes are expressed in the SCN wherein their mRNA levels show circadian oscillations, suggesting an involvement of mPer genes in the )
Corresponding author. Department of Neurobiology of Rhythmic and Seasonal Functions, CNRS-UMR7518, Louis Pasteur University, 12 rue de l’Universite, ´ F-67000 Strasbourg, France. Fax: q33-3-88-24-04-61; e-mail: [email protected]
feedback loop of the clock w1,5,15,18,20,24x. Together with BMAL1, another transcription factor, CLOCK forms heterodimers that activate transcription of mPer1, so that CLOCK and BMAL1 may be positive regulators of the clock transcriptional loop w4,6,8x. Moreover, expression of mPer genes is markedly reduced in Clock mutant mice, suggesting that CLOCK–BMAL1 complexes positively regulate expression of mPer w4,8x. The daily light–dark cycle is the main environmental cue that entrains Žsynchronizes. the SCN clock to the 24 h. In addition to lengthening the free-running period, the heterozygous state of the Clock mutation is associated with increased light-induced circadian phase shifts w22x. Furthermore, light-induced expression of mPer1 and mPer2 is reduced in the homozygous Clock mutant mice w14x. These findings suggest that CLOCK participates in the photoentrainment in the SCN. Besides light, the phase of the light-entrainable circadian clock can be shifted by a variety of nonphotic factors, including behavioral stimuli Že.g., confinement to a novel running wheel., pharmacological cues Že.g., triazolam treatment., or saline injection w7,17,21x. Moreover, some nonphotic events, like behavioral and metabolic cues, may modulate the circadian responses to light w2,12x. Nonphotic factors are considered to act on the circadian clock via transduction pathways different from those medi-
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 2 0 4 0 - 0
E. Challet et al.r Brain Research 859 (2000) 398–403
ating photoentrainement w7,17x. Very little is known about how nonphotic signals phase-shift the circadian oscillator at the molecular level or the role of the recently discovered circadian clock genes in these responses. In this context, we determined whether the Clock mutation affects the response of the circadian system to two different nonphotic phase-shifting cues. Seven Clockrq heterozygous mice, derived from a C57BLr6J founder animal w23x, and seven qrq adult C57BLr6J mice were bred at Northwestern University. For both groups of mice, four males and three females were housed singly in cages equipped with running wheels Ždiameter: 11 cm. in a temperature-controlled room Ž23 " 18C. with a light–dark 12:12 h cycle Žlights on at 0500 h.. During daytime, light intensity was about 200 lx at cage level. Food Žlaboratory chow, Harlan Teklad, WI. and water were available ad libitum, unless otherwise stated. Wheel-running activity was continuously recorded ŽChronobiology Kit, Stanford Software Systems, Stanford, CA.. Mice were phenotyped according to the circadian period Žt . as previously described w23x. Mice were also genotyped for the Clock locus with genomic DNA extracted from tail tips using simple sequence length polymorphisms w9x. Experiment 1 was performed to assess the phase-shifting effects of confinement to a novel running wheel in Clockrq and qrq mice. For that purpose, mice were given novel-wheel pulses according to a protocol developed in Syrian hamsters by Mrosovsky and colleagues w7x. After 10 days in constant darkness, all mice were confined singly into clean wheels for 4 h during the subjective day Ži.e., from circadian time wCTx 6 to CT10, with CT12 defined as the onset of locomotor activity.. These novelwheel pulses were given on three consecutive days. In experiment 2, the synchronizing effects of timed calorie restriction, previously shown to change the phase angle of entrainment in wild-type mice exposed to a light–dark cycle w3x, were studied in Clockrq mice. For that purpose, once experiment 1 was completed, the mice were transferred back to a daily light–dark cycle for 2 weeks. Daily food intake was found to be about 6 g of chowrday regardless of the genotype. Mice were calorierestricted for 2 weeks and were given 66% of daily food intake Ži.e., 4 g of chowrday. 2 h after the onset of light. Thereafter, all animals were transferred to constant darkness and fed ad libitum for 4 weeks. When switched to ad libitum feeding, calorie-restricted mice were provided with food beginning at the time when hypocaloric food was given previously. Data for 10 consecutive cycles were divided into 10-min bins of time. The onset of the nocturnal wheel-running activity was defined as the first 10-min bin in which 20% of maximal intensity for that cycle was followed by a similar level of activity in three out of the next six bins. This analysis was performed over two intervals in experiment 1: the last 10 days prior to the first novel-wheel pulse day and the first 10 days after the third Žlast. novel-wheel
399
pulse day. A similar analysis was performed over three intervals in experiment 2: the last 5 days of baseline conditions, the last 5 days of the calorie restriction, and the first 5 days in constant darkness. Linear regression analysis of the onsets of the nocturnal activity was performed with TableCurve software ŽJandel Scientific, San Rafael, CA.. In experiment 1, the data for the days with a novel-wheel pulse Ždenoted as days 11, 12 and 13. were discarded from the analysis. Data were fitted to the following equations: wif t F 10, y s At q B; if t ) 13, y s Ct q D x, where t was the number of days, A was the slope and B the initial phase of the line fitted to the 10 days prior to the first novel-wheel pulse Žday 11., and C was the slope and D the initial phase of the line fitted to the 10 days following the last novel-wheel pulse Žday 13.. The magnitude of the phase shift was calculated as the difference between these two lines on day 14. In experiment 2, during baseline conditions or calorie restriction, the fitting equation was: y s w A x, where A was the mean onset of nocturnal activity. Phase angle of entrainment to the light–dark cycle was calculated as the number of minutes before Žpositive. or after Žnegative. the offset of light compared to the mean nocturnal onset. For the data under constant darkness conditions, the fitting equation was: y s At q B, where A was the slope of the line Žaccording to t . and B the initial phase of the nocturnal onset of locomotor activity in constant darkness. Circadian phase changes were expressed as the number of minutes before Žpositive. or after Žnegative. the time of light offset Žthe day before. projected to the first day in constant darkness. t was assessed using the x 2 periodogram ŽChronobiology Kit software. over 10 days in constant darkness. Data are means " S.E.M. Novel-wheel pulses induced significant phase shifts in locomotor activity rhythm, with a direction depending on the genotype Ž t-test; P - 0.01.. Three novel-wheel pulses applied during the mid-subjective day led to 41 " 15-min phase advances in qrq wild-type mice and to 47 " 23min phase delays in Clockrq mice ŽFigs. 1 and 2.. Mean t calculated over experiment 1 was longer in mice heterozygous for Clock than in wild-type mice Žtwo-way ANOVA with repeated measures; effect of the genotype: P - 0.001.. Moreover, t was markedly modified by novel-wheel pulses Žeffect of the treatment: P - 0.001.: t was lengthened after behavioral activation in heterozygous mice Ž24.1 " 0.05 vs. 24.6 " 0.06 h, respectively; P 0.05., but not in wild-type mice Ž23.7 " 0.08 vs. 23.6 " 0.1 h, respectively; P ) 0.1; Fig. 1.. The cumulative wheel revolutions performed during the 4-h novel-wheel pulses were similar in qrq mice and Clockrq mice Ž9780 " 860 vs. 10080 " 510 wheel turns per 4 h, respectively; t-test; P ) 0.1.. The phase of the nocturnal onset of locomotor activity was modified by both the genotype and the calorie restriction over experiment 2 Žtwo-way ANOVA with repeated measures; effect of the genotype: P - 0.01; effect of the
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Fig. 1. Novel-wheel pulses. Wheel-running activity of a wild-type Žqrq . mouse Žtop. and a mouse heterozygous for Clock Ž Clockrq; bottom. kept in constant darkness. To facilitate visualization of phase shifts and t changes, data were presented modulo t Žt before wheel pulsess 23.6 and 24.0 h in the qrq mouse and the Clockrq mouse, respectively.. Successive circadian cycles are double-plotted Žtwo cycles per horizontal time scale.. Animals were confined into novel wheels for 4 h from circadian time 6 to circadian 10 over 3 days.
nutritional state: P - 0.001; genotype= nutritional status interaction: P - 0.01.. When mice were fed ad libitum under a light–dark cycle, the onset of nocturnal activity was close to the time of light off, albeit with a more negative phase angle of entrainment in Clockrq mice compared to that of qrq mice Žy50 " 6 vs. y20 " 4
Fig. 2. Phase shifts in circadian activity rhythms of wild-type and Clock rq mice. Positive and negative values are advances and delays, respectively. Phase shifts after novel-wheel pulses were assessed in constant darkness. Phase shifts after calorie restriction were calculated as the differences between the initial phase under light–dark baseline conditions and that in constant darkness. Values are means"S.E.M.; ns 7 per group.
min relative to light offset, respectively; Figs. 2 and 3.. Providing a daily hypocaloric diet 2 h after lights on led to a significant phase advance in the onset of activity in both Clockrq mice and qrq mice Žonset: 50 " 13 and 90 " 14 min relative to light offset, respectively.. After transfer to constant darkness, a phase advance of about 80 min in the circadian rhythm of locomotor activity was observed in wild-type mice Žonset: 62 " 19 min relative to light offset the day before.. In contrast, no phase advance was detectable in Clockrq individuals Žonset: y54 " 18 min relative to light offset the day before; Figs. 2 and 3., the nocturnal onset in constant darkness starting close to the initial phase under light–dark baseline conditions. Mean t assessed during the first 10 days in DD was 1 h longer in mice heterozygous for Clock than in wild-type mice Ž24.6 " 0.06 vs. 23.6 " 0.06 h, respectively; t-test, P - 0.001; Fig. 3.. No changes in t were detected over 4 weeks in constant darkness Ž P ) 0.05.. Body mass loss after the 2 weeks of calorie restriction was not significantly different between Clockrq and qrq mice Žy24.3 " 1.0 vs. y26.6 " 0.7%, respectively; t-test, P ) 0.1.. The present results indicate that the Clock mutation modifies the response of the circadian timing system to nonphotic cues. First, behavioral activation during the subjective day induced opposite phase shifts in Clock mutant mice compared to those in wild-type individuals housed in constant darkness. Second, modulation of the photic entrainment by a timed calorie restriction, while
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Fig. 3. Timed calorie restriction. Double-plotted wheel-running activity of a wild-type Žqrq . mouse Žtop. and a mouse heterozygous for Clock Ž Clockrq; bottom.. Days 1–8, initial interval of ad libitum feeding under light–dark conditions; days 9–22, timed calorie restriction ŽCR.; days 23–33, final interval of ad libitum in constant darkness ŽDD.. When mice were housed under a light–dark cycle, nighttime is indicated by black bar on abscissa. Vertical lines are projected time of light offset Ži.e., 1900 h.. Time when hypocaloric diet was provided during calorie restriction Ži.e., 2 h after light onset. is indicated by vertical arrow on abscissa.
altering the daily organization of locomotor activity in both qrq and Clockrq mice exposed to a light–dark cycle, led to phase advances of nocturnal pattern of activity Žassessed in constant darkness. in wild-type mice, but not in mice heterozygous for Clock. The molecular mechanisms by which nonphotic cues reset the clock are still poorly understood. A feature shared by most nonphotic cues is their ability to robustly phase shift the clock when presented during the middle of the subjective day w16x, that is, when expression of mPer genes is the highest Že.g., Refs. w15,18x.. This correlation raises the possibility that nonphotic cues are able to interact at this step of the clock transcriptional loop. For instance, nonphotic cues might inhibit the transcription of an mPer gene. Resetting the clock during the subjective day by nonphotic cues usually induces phase advances w16x, as we observed after novel-wheel pulses in wild-type mice. It should therefore be emphasized that similar nonphotic cues led to opposite phase shifts Ži.e., phase delays. in Clockrq mice. Because phase advances and delays are probably mediated in the SCN through different molecular mechanisms, the model of novel-wheel pulses in Clock mutants may be a useful tool to determine whether the transcriptional regulation of mPer and other clock-related
genes during the subjective day is differentially affected by nonphotic cues. In Clockrq mice, behavioral activation induced a clear change in t . One possibility is that behavioral activation acted to trigger these changes in t . In spite of strong homeostatic control, the stability of t can be subject to subtle environmental history-dependent modulations. For instance, single nonphotic pulses can modify t in constant darkness w13x. Although calculation of phase shifts might be affected by t changes, this methodological issue is still an open question w13x. In the present study, it is noteworthy that t was not affected by behavioral activation in wild-type mice, suggesting that the SCN function is more sensitive to these kinds of modulation in Clockrq mice compared to wild-type individuals. A spontaneous lengthening in t has been reported in Clockrq mice housed in constant darkness w23x. Because t values were not modified over a similar period in free-running mice studied in the absence of phase-shifting cues Žexperiment 2., behavioral cues that have shifted the clock may also have accelerated the process of t lengthening in Clock mutant mice Žexperiment 1.. Besides novelty-induced behavioral activity, Clockrq mice were also studied in another paradigm: timed calorie
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restriction under exposure to a light–dark cycle. During baseline light–dark conditions, a more negative phase of photic synchronization was observed in Clockrq mice in accordance with their longer t . Compared to this initial phase when food was provided ad libitum, timed calorie restriction modified the phase angle of photic entrainment by advancing Žabout 1.5 h. the nocturnal onset in both qrq and Clockrq mice. As previously observed w3x, a 1-h phase advance of the nocturnal pattern of locomotor activity was still detectable in wild-type mice after they were placed in constant darkness and fed ad libitum. In contrast, no significant phase change was found in freerunning mice heterozygous for Clock. Light induces phase delays during the early subjective night and phase advances during late subjective night. Because t in wild-type C57BL mice is - 24 h, light-induced phase delays are critical for photic entrainment. The circadian responses to light are reduced during calorie restriction w3x, and other situations of reduced glucose availability w2x. Therefore, the phase advances observed in calorie-restricted qrq mice Žwith no changes in t . might allow more light to fall in their delay region, thus inducing phase delays sufficient for photic entrainment. In addition to a lengthening of the circadian period, the Clock mutation is associated with increased light-induced circadian phase shifts in animals fed ad libitum w22x. Thus, a possible reduction of photic resetting in calorie-restricted Clockrq mice would not be sufficient to significantly phase shift the clock after transfer to constant darkness. Alternatively, the stepŽs. in photic phase resetting that are sensitive to timed calorie restriction may involve CLOCK or CLOCK-controlled factors. Although light is the major synchronizer of the light-entrainable clock, the present study confirms that nonphotic cues can also influence the circadian timing system either in constant darkness Žbehavioral activation. or in the presence of a light–dark cycle Žtimed calorie restriction.. In both paradigms, the Clock mutation altered the typical response of the circadian system. Therefore, protocols using nonphotic cues may offer useful tools to further unravel the molecular basis of the SCN clockwork and its entrainment to environmental stimuli.
Acknowledgements The authors are grateful to Anne-Marie Chang and Sue Losee-Olson for expert assistance with genotyping. This work was supported by National Institute of Health Grants HLrMH-96015 and AG-11412 ŽFWT., US Army Research Office Grant DAAG55-98-1-0196 ŽFWT., NSF Center for Biological Timing Grant ŽJST. and an Unrestricted Research Grant in Neuroscience from BristolMyers Squibb ŽJST.. JST is an investigator in the Howard Hughes Medical Institute.
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