A Circadian Rhythm in Mice that is Unaffected by the Period Mutation at Clock

A Circadian Rhythm in Mice that is Unaffected by thePeriod Mutation at Clock

David J. Coward, Sean W. Cain and Martin R. Ralph

Departments of Psychology and Zoology, University of Toronto, Canada

Abstract

Circadian rhythms of locomotor behavior were induced in mice using chronic d-amphetamine. The periods of the rhythms at various doses of amphetamine wereunaffected by a mutation at the clock locus that lengthens circadian period. Amphetamine-induced rhythms were superimposed on the host’s natural circadianrhythm that is driven by the pacemaker in the suprachiasmatic nucleus. The resultsdemonstrate the existence of an alternative mechanism for generating circadianrhythms that does not require the activity of canonical clock genes.

Keywords: suprachiasmatic nucleus, SCN, amphetamine, dopamine, food entrainableoscillator, period, tau.

Introduction

In mammals, circadian rhythms of behavior and physiology are driven by a biologi-cal clock located in the hypothalamic suprachiasmatic nucleus (SCN) (Moore 1995;Ralph & Hurd 1995). While the primacy of the SCN as the orchestrator of circadianrhythmicity is not in dispute, there is a growing body of evidence for the existenceof circadian oscillators outside this nucleus. In particular, the circadian oscillator inthe mammalian retina displays all the requirements of an independently functioningclock (Tosini & Menaker, 1996, 1998) including sensitivity to the circadian periodmutation, tau (Ralph & Menaker, 1988). In addition, the molecular components thatare thought to define the mammalian circadian pacemaker are found in tissues otherthan the SCN, both within and outside the central nervous system (for rPER2, seeSakamoto et al., 1998; Oishi et al., 1998a; for mPER1, see Sun et al., 1997; forBMAL1, see Oishi et al., 1998b; for mTIM, see Zylka et al., 1998; Takumi et al., 1999;for CLOCK, see Steeves et al., 1999).

Clock genes have been identified across a broad phylogenetic range. Not only arethe structures of these genes conserved, but the functions of their protein products,

Address correspondence to: Martin R. Ralph, Departments of Psychology and Zoology, University ofToronto, 100 St. George Street, Toronto, M5S 3G3, Canada.

Biological Rhythm Research 0165-0424/01/3202-233$16.002001, Vol. 32, No. 2, pp. 233–242 © Swets & Zeitlinger

particularly their interactions with each other, are also similar (Wilsbacher & Takahashi, 1998; Vitaterna et al., 1999; Lowrey et al., 2000). This has raised the inter-esting possibility that circadian rhythmicity requires the activity of a distinct set ofgenes and proteins, and conversely, that their presence or activity in tissues indicatesthe capacity of those cells to support circadian oscillations.

However, while essentially all CNS tissue shows daily rhythms of activity undernormal conditions, few sites show strong expression of the canonical clock genes;and where peripheral expression is found, the phase of the rhythm tends to be delayedby up to 6 hours with respect to that of the SCN. To operate rhythmically withoutthese clock genes, two different types of organization are possible: (1) Non-expressing tissues might be driven passively by central pacemakers, or (2) oscilla-tions might be generated by alternative molecular means.

In rodents, as in all mammals tested, the pacemaker cells in the SCN are at the topof the circadian hierarchy (Ralph et al., 1990). Lesions of this nucleus result in thepermanent loss of rhythms in physiology and behavior. However, rhythmicity can berestored in the absence of the SCN with chronic exposure to amphetamine deriva-tives (Honma et al., 1987; Honma et al., 1989). Because these induced oscillationsdo not appear to be self-sustaining, we hypothesized that the chemically-inducibleoscillator (CIO) involves a neurochemical mechanism that is different from that whichunderlies the generation of self-sustaining rhythms in the SCN. To test this, we exam-ined the effect of chronic d-amphetamine (AMPH) on locomotor activity in mice. Wefirst determined that an oscillation could be induced in the mouse, as has been shownfor the rat (Honma et al., 1987; Honma et al., 1988). Then we compared the rhythmsproduced in wild type animals with those produced in siblings carrying the periodmutation at the clock locus.

Materials and Methods

Animals

Mice carrying the long period mutation at the clock locus, along with their wild type siblings, were raised in the breeding colony at the University of Toronto, Department of Zoology. The mutation was expressed on a C57Bl6J background.Animals were between three and four months of age at the beginning of activity recording.

Data acquisition and analysis

For recording of locomotor activity, all animals were housed individually in cagesequipped with a 17 cm diameter running wheel. Animals had free access to food, waterand the wheel throughout each experiment. Activity was monitored continuouslyusing Dataquest III (Minimitter Co., Sunriver, OR). Activity was measured as wheelturns which were accumulated into 6 minute bins for analysis.

The data was analyzed using Dataquest III and a program (Ezpaste) written forthis purpose at the NSF Center for Biological Timing, University of Virginia. The

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periods of the circadian rhythms were calculated by periodogram analysis and veri-fied by visual inspection (eye-fit lines drawn through successive activity onsets).

Amphetamine administration

Amphetamine hydrochloride was dissolved in the drinking water at concentrations of75, 120 or 225mg/L. The amount of water consumed was determined by weighingthe water bottle each day. The body weight for each subject was measured immedi-ately prior to each experiment, so that an assessment of dosage could be obtained.

Results

Experiment 1. Dose-response relationship for wild type animals

For the first experiment, chronic AMPH did not affect the amount of water consumedper day, and the distribution of body weights was comparable across groups. Dosagetherefore, was directly proportional to concentration. Wild type animals consumed3.0mg per 5 days at 75mg/L (average = 16 ± 1mg/kg/day), 6.5mg per 5 days at 120mg/L (average = 31 ± 1mg/kg/day), and 11.3mg per 5 days at 225mg/L (average =54 ± 2mg/kg/day).

Locomotor rhythms with periods of about 29h were induced at all concentrations(Fig. 1). In many cases, especially at the lowest concentration, both the inducedrhythm and the SCN-driven rhythm were expressed simultaneously in the activityrecords (cf. Fig. 1a). While the latency to reach steady state was dose-dependent, thesteady state periods were the same for all concentrations (Fig. 2) (p = .68; Duncan’sMultiple Range Test).

Experiment 2. Effects of the long period mutation on AMPH induced rhythms

Comparable results were obtained using mice carrying the clock mutation (Fig. 3).For heterozygous clock mutants, the latency to reach steady state periodicitydecreased with increasing AMPH concentration but the eventual period was the samefor all groups (Fig. 4). Similarly, the homozygous mutants, which were arrhythmicin constant conditions prior to treatment, had induced rhythms with periods of about29h (Fig. 5). Body weight distributions and amounts of water consumed were com-parable to wild type animals.

Despite the lengthened circadian period characteristic of the heterozygous clock/+animals, the periods of the rhythms induced by AMPH in clock/clock and clock/+ micewere not different from the wild type (p = .39; Duncan’s Multiple Range Test).

Discussion

The period of the AMPH-induced rhythm, therefore, is not affected by the periodmutation at the clock locus. In fact, neither the period of the rhythm, nor the dose-

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Figure 1. AMPH-induced rhythmicity in mice. Locomotor activity records of mice that were exposed to chronic AMPH in the drinking water. Duration of AMPH treatment (vertical,hatched bar) along with calculated periods before, during and after treatment are indicated to the right of each record. Actograms are double-plotted so that each line presents 48h of continuous recording, with the second 24h repeated on the first half of the subsequent line.Responses to three different concentration of AMPH are shown. Top panel: 75 mg/L; Centerpanel: 120mg/L; Bottom panel: 225 mg/L.

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Figure 2. Dose-response relationships indicated by steady state period and latency to reachsteady state. Lower concentrations of AMPH take longer to produce the effect, but the endresult is independent of dose. Results are from a moving window periodogram analysis wheresuccessive windows of 7 days each (abcissa) are analyzed for periodicity.

dependent latency, nor the change in wheel running intensity (data not shown) in thepresence of AMPH were modified by clock genotype.

The simplest interpretation of these results is that AMPH-induced rhythmicity doesnot involve the activity of the clock gene. Extrapolating from this, it is reasonable toconclude that the known molecular structure of circadian clocks is not producingthese rhythms. Perhaps more importantly, it indicates that circadian rhythms can beproduced through alternative means that do not require the activity of canonical clockgenes.

The data do not explicitly eliminate all possible involvement of clock, but it is stillthe case that if it were involved, its function would have to be altered substantially.This assertion is based on the known role that clock plays in the production of circa-dian rhythms in different systems. As a participant in transcription regulation, theCLOCK protein interacts with BMAL1, TIM, mPER, and the CRY proteins to controlits own transcription as well as that of other genes that are regulated through E-boxinteractions. If CLOCK participates in the CIO, then its activities must be signifi-

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Figure 3. Effects of AMPH on clock phenotypes. Top 2 panels: clock/+; Bottom 2 panels:clock/clock. Duration of AMPH administration (hatched vertical bar) and periods derivedbefore, during and after AMPH administration are show to the right of each panel. Actogramshave been double plotted as in Figure 1.

cantly altered so that the period mutation becomes inconsequential. This seemsunlikely.

A more parsimonious and testable model is that the CIO is a neural circuit thatincludes one or more of the AMPH-sensitive dopamine (DA) pathways. AscendingDA projections from the substantial nigra and ventral tegmental area participate inthe control of general locomotion and signaling of reward. Activity in these pathwaysis regulated by negative feedback from forebrain targets.

AMPH derivatives that alter DA signaling could either (1) induce the feedbackloop to oscillate, or (2) increase the amplitude of an oscillation that already exists. In either case, the period of the oscillation is close enough to 24h to suggest that under natural conditions, it might produce a highly damped rhythm that is set into motion by a daily signal from the SCN. The increased latency to reach steady state periodicity at low AMPH doses is likely to be due to a sensitization toAMPH that occurs during lengthy exposure. In the presence of AMPH, the influenceof the SCN is less significant, as locomotor activity becomes associated with the inducible oscillator rather than remaining gated by the SCN. This is reflected in the reduced presence of the natural circadian oscillation as AMPH concentrationincreases.

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Figure 4. Dose-response relationship for clock/+ mice indicated by steady state period andlatency to reach steady state.

The natural function of the CIO is not clear. A similar, if not the same, oscillatoris entrainable by scheduled food availability (Stephan et al., 1979; Mistleberger &Rechtschaften, 1983; Honma et al., 1988; Honma et al., 1992). It is thought that thismechanism might provide organisms with a means of learning to anticipate food (orperhaps other) reward at different times of day. In general, time-of-day learning couldbe the result of associating reward with a phase of this inducible cycle. This couldoccur with or without the host’s SCN.

Moreover, an adaptive advantage may be gained from using non-pacemaker tissuesthat are tuned to respond over a circa-24 h period to periodic signals from the circa-dian clock. Such an arrangement might reduce the burden on the clock itself toprovide continuous regulation of all targets. At the same time, it would eliminate theneed for target tissues to maintain highly regulated circadian oscillations. A relativelysimple response system such as this would not need a mechanism for self-initiationof successive cycles.

The data presented here suggest that circadian organization extends well beyondthat which may defined by the presence and activity of clock proteins and genes. The

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Figure 5. Response of the three clock genotypes to middle doses (120 mg/L) of AMPH. No significant differences among groups were found after two weeks (window 6) at this concentration.

vertebrate brain is likely to deal with the natural cyclicity of the environment in manydifferent ways. The mechanisms for these types of oscillator may not be as conservedas that of the central clock, and could be highly divergent.

Acknowledgements

The authors wish to thank Drs. J.S. Takahashi and M.H. Vitaterna for providing theclock mutant breeders for this study. Supported by a grant from the Natural Sciencesand Engineering Council of Canada.

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