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. 2009 Nov 18;29(46):14681–14686. doi: 10.1523/JNEUROSCI.3261-09.2009

Endogenous Rhythms in Period1 Mutant Suprachiasmatic Nuclei In Vitro Do Not Represent Circadian Behavior

Julie S Pendergast 1, Rio C Friday 1, Shin Yamazaki 1,✉
PMCID: PMC2806308  NIHMSID: NIHMS166801  PMID: 19923301

Abstract

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) controls daily rhythms of behavior and physiology. Lesions of the SCN cause arrhythmicity of locomotor activity, and transplants of fetal SCN tissue restore rhythmic behavior that is consistent with the periodicity of the donor's genotype, suggesting that the SCN determines the period of the circadian behavioral rhythm. While several studies have demonstrated that the circadian characteristics of in vitro SCN rhythms represent circadian behavior, others have shown that the periods of explanted SCN are not always congruent with locomotor activity. We find that the aberrant rhythms of ex vivo SCN lacking functional Period1 (Per1−/−) do not represent the behavioral rhythms of the mutant animals. Surprisingly, in C57BL/6J Per1−/− mice, the real-time circadian gene promoter activity rhythm is weak or absent in adult SCN slices in vitro even though the free-running wheel-running activity rhythm is indistinguishable from wild-type (Per1+/+) mice. While some neurons in Per1−/− SCN explants exhibit robust circadian rhythms, others have irregular and/or low-amplitude rhythms. Together, these data suggest that either a small population of rhythmic neurons in the Per1−/− SCN is sufficient to control wheel-running activity or that in vivo physiological factors can compensate for the aberrant endogenous rhythms of Per1−/− SCN.

Introduction

Circadian rhythms are self-sustained oscillations in physiology and behavior with endogenous periods of ∼24 h that can be entrained to environmental cues such as the light/dark cycle or temperature (Takahashi et al., 2001). In mammals, the master circadian clock, located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus, plays a critical role in the generation of daily rhythms. Transplanting an SCN graft into an SCN-lesioned hamster or mouse restores its locomotor activity rhythm with a period consistent with the donor animal's genotype, suggesting that the SCN controls the period of the circadian behavioral rhythm (Ralph et al., 1990; Silver et al., 1996; Sujino et al., 2003). In addition, averaging the period of the firing rhythms of individual dispersed SCN neurons from heterozygous and homozygous tau mutant hamsters matches the behavioral periods of the whole animals (Liu et al., 1997). Similarly, the lengthened behavioral period of locomotor activity in Clock mutant (Δ19) heterozygote mice matches the period of the spontaneous firing rate in dispersed SCN neurons and the period of circadian promoter-driven reporter expression in SCN explants from these mice (Herzog et al., 1998; Yoo et al., 2005). In contrast, other studies have shown that the period of the SCN and of locomotor activity may not always be congruent. For example, mice exposed to non-24 h T-cycles exhibit aftereffects, or period changes, in locomotor behavior and in the circadian gene expression rhythm in cultured SCN (Aton et al., 2004; Molyneux et al., 2008). However, the aftereffect periods of the SCN in vitro and of behavior are negatively correlated. Interestingly, a previous study found that even though individual, dispersed cells of circadian mutant SCN may not reflect the behavioral phenotype of the corresponding mutant mice, rhythms in whole SCN explants correlated strongly with circadian behavior (Liu et al., 2007).

The molecular mechanism of endogenous rhythm generation in the SCN is modeled as interlocking transcriptional and translational feedback loops of circadian gene expression (Ko and Takahashi, 2006). Two Period homologs (Per1 and Per2) are known to be negative components of the feedback loops. The functions of some circadian genes have been investigated by generating circadian gene knock-out mice, including mice with nonfunctional Per1 (Bae et al., 2001; Cermakian et al., 2001; Zheng et al., 2001). Unlike previous studies of Per1−/− mice on mixed genetic backgrounds (Cermakian et al., 2001; Zheng et al., 2001), in the current study we report that the periods of circadian behavior in Per1−/− mice congenic with the C57BL/6J strain are similar to Per1+/+ mice. Surprisingly, though, we find that the endogenous rhythms in Per1−/− SCN explants are not congruent with the behavioral phenotypes of the mice, suggesting that in vivo factors may compensate for aberrant rhythms in mutant SCN.

Materials and Methods

Animals.

mPer1ldc−/− mice (Bae et al., 2001) (provided by Dr. David Weaver, University of Massachusetts, Worcester, MA) (congenic with the 129/sv genetic background) were backcrossed with male and female wild-type C57BL/6J mice (The Jackson Laboratory) for 10–11 generations (C57BL/6J Per1−/− mice are available from The Jackson Laboratory, stock #10491). Per1+/− mice were then crossed with C57BL/6J Per1-luc transgenic mice (1-8L) (Herzog et al., 2004) to generate mice that were heterozygous for the Per1 gene and for the Per1-luc transgene. Per1+/− mice were then crossed with Per1+/− mice with the Per1-luc transgene to generate Per1+/+, Per1+/−, and Per1−/− mutant mice that expressed Per1-luc (N10 to N11) that were used for experiments. To assess the expression of the PERIOD2::LUCIFERASE fusion protein, Per1+/+ and Per1−/− mice were generated as described above except that Per1+/− mice were crossed with C57BL/6J PER2::LUC mice (Yoo et al., 2004). Genotype was determined by PCR amplification of tail DNA as previously described (Bae et al., 2001). The mice were bred and group housed in the Vanderbilt University animal facility in a 12 h light/12 h dark cycle [12L:12D (LD)] and provided food and water ad libitum. Male and female mice, aged 25–204 d (mean ± SD: 65.2 ± 38.4 d) at the beginning of the experiment, were used for assessing behavior and for preparing tissue explants. All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Vanderbilt University.

Analysis of wheel-running activity.

For experiments assessing wheel-running activity, mice were singly housed in cages (33 × 17 × 14 cm) with unlimited access to a running wheel (diameter: 11 cm), food, and water. The cages were placed in light-tight, ventilated boxes where the light intensity was 350 lux. Wheel running activity was monitored by a micro-switch-activated signal using the ClockLab system (Actimetrics) and was collected by computer every minute. Analysis was performed using ClockLab software. Free-running period was determined by using a χ2 periodogram for 15 d [days 1–15 in constant darkness (DD)]. The amplitude (Qp) of the wheel-running rhythm was the peak value of the χ2 periodogram. Total activity level was determined by counting the total number of wheel revolutions from days 1–15 in DD and then averaging them to determine daily activity level. The phase angle of entrainment was defined as the time difference between lights off and the predicted time of activity onset on the first day in DD. This was calculated by drawing a regression line to activity onset for days 1–5 in DD and then extending the regression line to the last day in LD. A negative phase angle was obtained when activity started before lights off and a positive phase angle was obtained when activity started after lights off.

Luminescence recording and imaging.

The detailed methods for real-time measurement of luminescence from ex vivo tissues have been described (Yamazaki and Takahashi, 2005). Coronal slices of the SCN (300 μm) were prepared by trimming away most extra-SCN tissue (small and middle sizes shown in supplemental Fig. S3C, available at www.jneurosci.org as supplemental material) and bioluminescence was measured using the LumiCycle apparatus (Actimetrics). Bioluminescence imaging was performed using a water-cooled CCD camera (ORCAIIERW, Hamamatsu). The SCN explant was placed on an x–y–z stage in a temperature-controlled dark box, and the image was processed through direct coupling of a non-infinity-corrected microscope objective (NPLM20X, Union Optical) to the camera with a 3-cm-long extension tube. Focus was adjusted by moving the motorized z-stage controlled from outside of the box. Twenty-nine-minute exposures were collected every 30 min. The images were analyzed for signal intensity using ImageJ software (National Institutes of Health). LumiCycle software (Actimetrics) was used to subtract the 24 h moving average from the raw luminescence data, to smooth the data by 0.5 h adjacent averaging, and to determine the amplitude of the first cycle of the baseline-subtracted, smoothed data. To determine period and phase, the baseline-subtracted and smoothed data were exported to ClockLab (Actimetrics). The period was determined by fitting a regression line to the acrophase of at least 3 d of the Per1-luc rhythm, and the phase was determined from the first peak of Per1-luc expression in vitro.

Statistical analysis.

Statistical analysis was performed using SigmaStat (Systat Software). One-way ANOVA followed by post hoc Fisher's least significant difference (LSD) tests was used for comparison of more than two groups, and independent t tests (two-tailed) were used to compare two groups except when data were not normally distributed or variances were not homogeneous. The Kolmogorov–Smirnov test (with Lilliefors' correction) was used to test data for normality. For nonparametric analyses, the Kruskal–Wallis one-way ANOVA on ranks followed by post hoc Dunn's method was used. Significance was ascribed at p < 0.05.

Results

Circadian wheel-running behavior of Per1−/− mice congenic with the C57BL/6J strain is similar to Per1+/+ mice

Previous studies have demonstrated that Per1−/− mice on a mixed genetic background have shortened free-running periods (Cermakian et al., 2001; Zheng et al., 2001) and those isogenic with the 129/sv strain sometimes become arrhythmic in DD (Bae et al., 2001). To determine whether these phenotypes were due to the genetic background of the mice, we generated Per1−/− mice congenic with the C57BL/6J strain by backcrossing mPer1ldc−/− mutants (Bae et al., 2001) for 10–11 generations with C57BL/6J wild-type mice (The Jackson Laboratory). We found that the free-running periods of C57BL/6J Per1−/− mice did not differ from Per1+/+ and Per1+/− mice (F(2,26) = 0.98, p = 0.39) (Fig. 1A–D). The phase angle of entrainment was similar between Per1+/+, Per1+/−, and Per1−/− mice (F(2,26) = 0.74, p = 0.49) (Fig. 1E). Total activity levels (supplemental Table S1, available at www.jneurosci.org as supplemental material) and the amplitude (Qp; F(2,26) = 3.03, p = 0.07) (Fig. 1F) of wheel-running activity in Per1−/− mice were indistinguishable from Per1+/+ and Per1+/− mice. None of the mice in our study became arrhythmic even after 3 weeks in DD.

Figure 1.

Figure 1.

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Circadian behavior does not differ between C57BL/6J Per1+/+, Per1+/−, and Per1−/− mice. Representative double-plotted actograms of wheel-running activity in Per1+/+ (A), Per1+/− (B), and Per1−/− (C) mice maintained in LD (lights on at 0 h and lights off at 12 h) for 7 d and then released into DD. The free-running period was determined by using a χ2 periodogram for days 1–15 in DD (D). The phase angle of entrainment (E) was determined by drawing a regression line to activity onset for days 1–5 in DD and then extending the regression line to the last day in LD. The phase angle was negative when activity started before lights off and positive when activity started after lights off. The amplitude (Qp; F) was the peak value of the χ2 periodogram. Data are the mean ± SD.

Per1-luc expression in C57BL/6J Per1−/− SCN slices in vitro is not congruent with locomotor activity

We next assessed Per1-luc expression in cultured SCN explanted from Per1−/− mice (Fig. 2). Surprisingly, we found that the rhythm of Per1-luc expression in Per1−/− whole SCN in vitro was severely affected, such that most SCN (8 of 12) were arrhythmic after a single peak (Fig. 2C). Sometimes (4 of 12) multiple, low-amplitude rhythmic components were present and significant periods were detected by χ2 periodogram analysis. In these cases, the period of Per1-luc expression in Per1−/− SCN was significantly longer than Per1+/+, but not Per1+/−, SCN (F(2,15) = 4.35, p = 0.04, LSD p < 0.05) (Fig. 2D). Since there was one peak of Per1-luc expression in Per1−/− SCN explants, we were able to determine the circadian phase. The phase of Per1-luc expression in Per1−/− SCN was significantly delayed by ∼2 h compared with Per1+/+ and Per1+/− SCN (F(2,23) = 13.68, p < 0.001, LSD p < 0.001) (Fig. 2E). The first peak of Per1-luc expression in Per1−/− SCN may reflect the in vivo phase, since the timing of the peak was not affected by the time of culture preparation (supplemental Fig. S1A, available at www.jneurosci.org as supplemental material). The amplitude of the first cycle of Per1-luc expression in vitro in Per1−/− SCN explants was significantly smaller than Per1+/−, but not Per1+/+, SCN (H = 7.91, p = 0.009, Dunn's p < 0.05) (Fig. 2F). Interestingly, one arrhythmic Per1−/− SCN explant spontaneously became rhythmic after 5 d in culture (supplemental Fig. S2A, available at www.jneurosci.org as supplemental material), and another Per1−/− SCN slice had two to three cycles of rhythmic Per1-luc expression after a temperature pulse (supplemental Fig. S2B, available at www.jneurosci.org as supplemental material).

Figure 2.

Figure 2.

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Per1-luc expression in SCN explants from C57BL/6J Per1−/− mice is arrhythmic or has low-amplitude, irregular rhythms. Representative baseline-subtracted bioluminescence rhythms in SCN explants from Per1+/+ (A), Per1+/− (B), and Per1−/− (C) mice. The lighting condition of the previous light/dark cycle is indicated for the first day; open bars are light and black bars are dark. The scale of the y-axis in (C) is different from in A and B. D, The period was determined by fitting a regression line to the acrophase of at least three cycles of the Per1-luc rhythm in all SCN except in Per1−/− explants, when χ2 periodograms were used to determine whether there was a significant period. The period of Per1-luc expression in Per1−/− SCN explants was determined from four SCN that had significant periods by χ2 analysis (8 of 12 SCN were arrhythmic and no significant period was detected by χ2 analysis). One of four Per1−/− SCN had two significant components by χ2 analysis (24.5 h and 32.4 h), but only the 24.5 h component that is in the range of circadian rhythmicity was used to calculate the mean period. E, The phase was designated as the first peak of Per1-luc expression in vitro and is plotted relative to the light-dark cycle before culture, where 0 h is lights on and 12 h is lights off. F, The amplitude was determined from the first cycle of Per1-luc expression in vitro. Data are the mean ± SD; *p < 0.05, ***p < 0.001.

We were surprised by our findings in Per1−/− SCN explants because they differ markedly from a previous study (Liu et al., 2007). Liu et al. (2007) reported that PER2::LUC expression was “persistently rhythmic” in Per1−/− SCN explants. Furthermore, the period of PER2::LUC expression in Per1−/− SCN explants was similar to Per1+/+ SCN in their study. We next investigated the differences between our study and that of Liu et al. (2007) that could account for the contradictory results. To determine whether the use of different circadian reporters [we used Per1-luc while Liu et al. (2007) used PER2::LUC] could account for the disparate findings, we generated Per1−/− mice carrying the PER2::LUC transgene. Consistent with our previous results, we found that PER2::LUC expression was arrhythmic or had irregular, low-amplitude rhythms in Per1−/− SCN explants (supplemental Fig. S3A, available at www.jneurosci.org as supplemental material). The luminescence data presented by Liu et al. (2007) showed PER2::LUC expression in Per1−/− SCN starting on the eighth day in culture after a media change. Therefore, we changed the media of Per1−/− SCN explants on day 7 in vitro. We found that Per1-luc expression remained arrhythmic in Per1−/− SCN explants even after a media change (supplemental Fig. S3B, available at www.jneurosci.org as supplemental material). We next prepared SCN explants of varying sizes to determine whether extra-SCN tissue could confer robustness to the Per1−/− SCN explants. Once again, we found that circadian reporter gene expression was arrhythmic in Per1−/− SCN explants regardless of their size (supplemental Fig. S3C, available at www.jneurosci.org as supplemental material).

In contrast to our contradictory results in the SCN, we found that Per1-luc expression in peripheral tissues from C57BL/6J Per1−/− mice was arrhythmic (supplemental Fig. S4, available at www.jneurosci.org as supplemental material), consistent with Liu et al. (2007).

Individual SCN neurons in C57BL/6J Per1−/− SCN slices have variable Per1-luc expression

We found that Per1-luc expression was often arrhythmic in whole SCN explants from C57BL/6J Per1−/− mice. There are two possible explanations for this observation. First, the Per1−/− SCN could be composed of individual neurons that are arrhythmic. Second, it is possible that some or all of the individual SCN neurons are rhythmic, but they have widely distributed phases of Per1-luc expression. To assess these possibilities, we imaged Per1-luc luminescence in Per1−/− SCN explants (Fig. 3). In the absence of functional Per1, we found that some neurons exhibited robust circadian rhythmicity, while others had irregular and/or low-amplitude rhythms of Per1-luc expression (Fig. 3H). This is in contrast to Per1+/+ SCN explants, where every cell analyzed had a robust rhythm of Per1-luc expression and the phases of individual neurons were synchronized (Fig. 3D). When bioluminescence from 10 individual neurons (Fig. 3D or Fig. 3H) was averaged, the resulting trace (Fig. 3C or Fig. 3G) strongly resembled the actual luminescence measured from the whole slice (Fig. 3B or Fig. 3F), suggesting that the neurons analyzed faithfully represented the whole SCN.

Figure 3.

Figure 3.

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Imaging analysis of Per1-luc expression in SCN explanted from Per1+/+ and Per1−/− mice. Bioluminescence (raw) was measured from the whole SCN (A, B, E, F) and from individual SCN neurons (D, H) in Per1+/+ (A–D) and Per1−/− (E–H) mice. C, G, The mean ± SEM (SEM shown in gray shading) of the rhythms from the 10 Per1+/+ neurons (C) or from the 10 Per1−/− neurons (G) shown in D or H, respectively.

Discussion

Surgical destruction of the SCN abolishes most circadian rhythms, including rhythmic locomotor activity (Moore and Eichler, 1972; Stephan and Zucker, 1972; Rusak and Zucker, 1979). Numerous studies suggest that the SCN encodes the period of rhythmic behavior. For example, transplanting an SCN graft into an SCN-lesioned animal restores its locomotor activity rhythm with a period identical to the donor animal (Ralph et al., 1990; Silver et al., 1996; Sujino et al., 2003), and the period of neural activity or circadian gene expression in SCN explanted from circadian mutants correlates with their free-running periods of wheel-running activity (Liu et al., 1997, 2007; Herzog et al., 1998; Yoo et al., 2005; Meng et al., 2008). While a recent study demonstrated that disrupting coupling between SCN neurons from Per1−/− or Cry1−/− mice by dispersing them in culture reveals that individual cells are arrhythmic, whole SCN explants have always been found to be rhythmic if locomotor activity was rhythmic in DD (Liu et al., 2007). In contrast, we find that wheel-running activity in C57BL/6J Per1−/− mice is indistinguishable from Per1+/+ mice, but circadian gene promoter-driven luciferase activity is arrhythmic or has an irregular, low-amplitude rhythm in whole Per1−/− SCN slices cultured in vitro. While we cannot account for the discrepancy between our findings and those of Liu et al. (2007), it is possible that the genetic background of the Per1−/− mice differed between the studies. Liu et al. (2007) used the same line of Per1−/− mice that we used (Bae et al., 2001), but they did not report the strain of mice used for their studies. Previous studies have demonstrated that genetic background affects circadian behavior (Ebihara et al., 1978; Possidente and Hegmann, 1982; Schwartz and Zimmerman, 1990; Shimomura et al., 2001), so it is possible that the phenotype of in vitro SCN explants could also vary among different strains of mice.

Our studies also highlight the importance of genetic background when analyzing the effect of gene knock-out on circadian behavior. Our behavioral results from Per1−/− mice on a C57BL/6J genetic background differ markedly from previous studies that examined Per1−/− mice on mixed or isogenic 129/sv backgrounds (Bae et al., 2001; Cermakian et al., 2001; Zheng et al., 2001). We find that the period, phase angle of entrainment, total daily activity, and amplitude (Qp) of wheel-running activity of C57BL/6J Per1−/− mice do not differ from Per1+/+ mice. Additionally, in contrast to previous studies (Bae et al., 2001; Zheng et al., 2001), none of the mice in our study became arrhythmic in DD. Our data therefore suggest that the C57BL/6J genetic background can compensate for aberrant endogenous rhythms in Per1−/− mice.

We find that circadian wheel-running behavior in C57BL/6J Per1−/− mice is indistinguishable from Per1+/+ mice, but circadian gene promoter-driven luciferase expression in whole Per1−/− SCN is arrhythmic or has a low-amplitude rhythm. The low-amplitude or absent rhythm in Per1−/− SCN slices could result if the phases of individual neurons are widely dispersed or if the individual neurons are themselves arrhythmic. We find that in Per1+/+ SCN explants, all neurons have robust rhythms that are in phase with each other. In contrast, in SCN explants from C57BL/6J Per1−/− mice, a subset of neurons maintains rhythmicity in vitro, albeit irregular, but the phases of the individual rhythms are not consolidated. These few rhythmic neurons may account for the low-amplitude rhythm of Per1-luc expression that we sometimes observe in Per1−/− SCN explants.

Limitations of our technique prevent us from analyzing regional differences in rhythmicity in our SCN slices. We prepare 300 μm SCN slices to preserve coupling within our explants. Since we measure light emission from one focal plane, only 10–15 cells are clearly in focus and can be analyzed for several days (resulting in fuzzy images of the SCN in Fig. 3). Therefore, there are not enough cells from each region to perform a comprehensive analysis. However, it should be noted that the cells that were analyzed were chosen without bias to a certain region and were distributed around the SCN. Future studies could assess regional differences in Per1−/− SCN by preparing thin (50–100 μm), sequential slices and compiling the data. For this study, we preferred to keep coupling intact in Per1−/− SCN to avoid any factors that could account for the low-amplitude, irregular rhythms that we observed.

We also find that the Per1-luc rhythm can be initiated in Per1−/− SCN explants when they are exposed to a temperature change (e.g., opening the incubator that contains the LumiCycle). A previous study demonstrated that a heat shock induced luciferase expression in PER2::LUC-expressing liver and lung explants (Kornmann et al., 2007). It is possible that the phases of the small subset of rhythmic neurons in Per1−/− SCN are synchronized following a temperature pulse.

Together, our data raise two critical questions regarding timekeeping by the mammalian circadian system. First, do circadian rhythms measured from ex vivo SCN and peripheral tissues accurately reflect the in vivo phenotype of the tissue? While mutant phenotypes may be readily revealed in vitro, our data suggest that in vivo factors, such as coupling with extra-SCN oscillators or feedback to the SCN, may compensate for the aberrant endogenous rhythms of mutant SCN. Second, is a small subset of rhythmic neurons adequate to drive behavior? A previous study showed that only a few vasoactive intestinal peptide-positive cells were present in grafts of cell suspensions from donor SCN that restored rhythmic locomotor behavior to SCN-lesioned hamsters (Silver et al., 1990). These data suggest that only a small population of SCN neurons may be necessary to produce behavioral rhythms. Since we find that only a few individual neurons in Per1−/− SCN explants have robust 24 h rhythms, it is possible that the ensemble of neurons in the SCN is not necessary to control behavior, but rather a small number of rhythmic neurons are sufficient to regulate wheel-running activity. Further analysis of the in vivo phenotype of C57BL/6J Per1−/− SCN is necessary to explore these issues.

Footnotes

This research was supported by a National Institutes of Health grant (NS051278 to S.Y.) and by the Vanderbilt University Summer Research Program (to R.C.F.). We thank David Weaver for mPer1ldc−/− mice and Akiko Hida and Hajime Tei for Per1-luc mice.

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