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Dissociation of Per1 and Bmal1 circadian rhythms in PNAS PLUS the in parallel with behavioral outputs

Daisuke Onoa,1,2, Sato Honmab,1,3, Yoshihiro Nakajimac, Shigeru Kurodad, Ryosuke Enokia,b,e, and Ken-ichi Honmab

aPhotonic Bioimaging Section, Research Center for Cooperative Projects, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan; bDepartment of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan; cHealth Research Institute, National Institute of Advanced Industrial and (AIST), Takamatsu, Kagawa 761-0395, Japan; dResearch Institute for Electronic Science, Hokkaido University, Sapporo, 001-0020, Japan; and ePrecursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan

Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved March 28, 2017 (received for review August 11, 2016) The temporal order of physiology and behavior in mammals is The expression of Per in the SCN is activated by a timed primarily regulated by the circadian pacemaker located in the exposure to light, which phase shifts the circadian pacemaker (8, hypothalamic suprachiasmatic nucleus (SCN). Taking advantage of 9). The phase-dependent phase shifts of clock expression bioluminescence reporters, we monitored the circadian rhythms of are regarded as a key mechanism by which the circadian pace- the expression of clock genes Per1 and Bmal1 in the SCN of freely maker is entrained to a LD . Light signals from the retina moving mice and found that the rate of phase shifts induced by a stimulate the expression of Per genes, perturbing the core loop single light pulse was different in the two rhythms. The Per1-luc dynamics to produce a phase-dependent phase shift (8). How- rhythm was phase-delayed instantaneously by the light presented ever, the mechanisms by which light-induced phase-shift signals at the subjective evening in parallel with the activity onset of be- from the core loop are transduced to circadian rhythms in phys- havioral rhythm, whereas the Bmal1-ELuc rhythm was phase- iology and behavior are not well understood. delayed gradually, similar to the activity offset. The dissociation On the behavioral level, the onset and offset of an activity was confirmed in cultured SCN slices of mice carrying both Per1-luc band (activity onset and offset) of circadian behavioral rhythm and Bmal1-ELuc reporters. The two rhythms in a single SCN slice are known to respond differentially to a phase-shifted LD cycle showed significantly different periods in a long- (3 wk) cul- (10) and to a single light pulse under continuous darkness (DD) ture and were internally desynchronized. Regional specificity in in nocturnal rodents (11). In addition, the phase relation between the SCN was not detected for the of Per1-luc and Bmal1- the activity onset and offset is known to change under different ELuc rhythms. Furthermore, neither is synchronized with circadian photoperiods (12). Furthermore, the two phases of behavioral + intracellular Ca2 rhythms monitored by a calcium indicator, rhythm occasionally split under the constant light condition (12). GCaMP6s, or with firing rhythms monitored on a multielectrode From these findings, the two oscillator hypothesis was advanced to array dish, although the coupling between the circadian firing and explain behavioral in nocturnal rodents (13). Ca2+ rhythms persisted during culture. These findings indicate that the expressions of two key clock genes, Per1 and Bmal1, in the SCN Significance are regulated in such a way that they may adopt different phases and free-running periods relative to each other and are respec- The in the suprachiasmatic nucleus (SCN) regu- tively associated with the expression of activity onset and offset. lates seasonality in physiology and behavior, which is best characterized by the change in the activity of behavioral clock gene | in vivo recording | suprachiasmatic nucleus | rhythms. In nocturnal rodents, the activity time was shortened photic phase resetting | E and M oscillators in long summer days and lengthened in short winter days be- cause of the change in the phase relationship of activity onset n mammals, the circadian pacemaker in the hypothalamic and offset, for which different circadian oscillators are predicted. Isuprachiasmatic nucleus (SCN) entrains to a light–dark (LD) Taking advantage of in vivo monitoring of clock gene expres- cycle and regulates circadian rhythms of behavior and physiology sion in freely moving mice, we demonstrated that the circadian (1, 2). The circadian in the SCN is autonomous, and rhythms of Per1 and Bmal1 in the SCN are associated differen- the clock genes Per1, Per2, Cry1, Cry2, Clock, and Bmal1 play tially with the phase shifts of activity onset and offset, respec- crucial roles (3). A heterodimer of Clock and Bmal1 tively, suggesting the existence of two with different (CLOCK/BMAL1) activates the transcription of Per and Cry molecular mechanisms in timing of circadian behavior. genes; in turn, the products of these genes suppress their own transactivation by CLOCK/BMAL1, closing a feedback Author contributions: D.O., S.H., and K.H. designed research; D.O. performed research; loop. One turn of the auto-feedback loop (core loop) takes D.O., S.H., Y.N., S.K., and R.E. contributed new reagents/analytic ; D.O. and Y.N. ∼ constructed a method of simultaneous measurement of bioluminescence; D.O., S.H., and 24 h. On the other hand, Bmal1 expression is enhanced by R.E. constructed a method of fluorescence imaging; S.K. made an analytical program for RAR-related orphan (ROR) and is repressed imaging; D.O., S.H., and K.H. analyzed data; and D.O., S.H., and K.H. wrote the paper. by an orphan nuclear receptor in the RevErb family (α, β) The authors declare no conflict of interest. through ROR response element (4, 5). The expressions of ROR This article is a PNAS Direct Submission. and the RevErb family are enhanced in turn by a BMAL1/CLOCK 1To whom correspondence may be addressed. Email: [email protected] or heterodimer via an upstream E-box. Thus, the Bmal1 circadian [email protected]. rhythm is auto-regulated by a feedback loop (the Bmal1 loop) 2Present address: Department of Neuroscience II, Research Institute of Environmental which is interlocked with the core loop, maintaining an antiphasic Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. phase relationship with Per1 rhythm. This interlocked Bmal1 loop 3Present address: Research and Education Center for Brain Science, Hokkaido University, has been considered to contribute to stabilization and fine tuning Sapporo 060-8638, Japan.

of the core loop (4, 6) in addition to the regulation of downstream This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NEUROSCIENCE pathways (7). 1073/pnas.1613374114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1613374114 PNAS | Published online April 17, 2017 | E3699–E3708 Downloaded by guest on September 25, 2021 Local time

A 0 12 0 12 0 Per1-luc (SCN) Behavior (counts/min) 120 80 0

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Days 30 20 RLU (counts/min) 0 0 1 2 3 45678 91011121314 15 14 Days B Local time

0 12 0 12 0 Bmal1-ELuc (SCN) Behavior (counts/min) 80 0 120

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60 40 7 Days 30 20

RLU (counts/min) 0 0 1 2 3 45678 91011121314 15 14 Days Light pulse

C Local time Local time 6012 18 612186012 18 61218 1 1

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D Days after light pulse Days after light pulse Days after light pulse -1 0 123456789 -1 0 123456789 -1 0 1234 567 89 0 0 0 Per1-luc Per1-luc Bmal1-ELuc -2 Bmal1-ELuc -2 -2

-4 -4 -4 * * * Phase shifts(h) Phase shifts (h) -6 Phase shifts (h) -6 -6 Activity onset Activity offset

Fig. 1. Light pulse-induced phase-delay shifts of circadian rhythms in the SCN and behavioral rhythms in freely moving adult mice. (A and B) Typical examples of phase response at CT11.5 are illustrated for the Per1-luc (A) and Bmal1-ELuc (B) rhythms in the SCN with a behavioral rhythm (black histogram) (Left)in double-plotting, in which the colored area indicates bioluminescence larger than the minimum value of a series, and in sequential plotting (Right), in which broken lines indicate raw data and solid lines indicate 4-h moving-averaged values (Per1-luc,blue;Bmal1-ELuc, green). The number of behavioral activities in 1-min intervals is indicated by black vertical bars. A yellow vertical bar indicates the time of the light pulse. (C) Mean acrophases ± SEM (horizontal bar) are illustrated for Per1-luc (blue circles) (Left) and Bmal1-ELuc (green circles) (Right) together with the mean activity onsets (gray triangles) and offsets (black squares). A yellow horizontal bar indicates the time of a light pulse. A horizontal gray and black bar at the top of each panel indicates the LD cycle to which mice had been entrained. (D) Daily phase shifts are reported by mean ± SEM (n = 4) for Per1-luc (blue circles) and Bmal1-ELuc (green circles) (Left) rhythms, for Per1-luc and two phase markers (onset and offset) of behavioral rhythm (Center), and for Bmal1-ELuc and the two phase markers (Right). The abscissa indicates the number of days after a light pulse. An asterisk indicates statistically significant difference (P < 0.05, two-way ANOVA with a post hoc t test) between Per1-luc and Bmal1-ELuc (Left), between Per1-luc and activity offset (Center), and between Bmal1-ELuc and activity onset (Right). RLU, relative luminescence units.

Namely, one oscillator, designated an evening (E) oscillator, reg- These findings indicate that there are at least two circadian ulates the activity onset, and the other oscillator, designated a pacemakers in the SCN, which have different molecular mech- morning (M) oscillator, controls the activity offset. Previously, re- anisms and differentially regulate behavioral outputs. gional differences were reported in the circadian rhythms of clock in the SCN (14–17), and the phase relation of the Results two rhythms changed under different photoperiods in parallel with Differential Responses of Per1-luc and Bmal1-ELuc Circadian Rhythms the change in activity time of behavioral circadian rhythm. These in the SCN in Vivo. We continuously measured the expression of findings suggested the existence of the E and M oscillators in the the clock genes Per1 and Bmal1 in the SCN of freely moving SCN. However, the molecular mechanisms underlying the E and transgenic mice carrying a bioluminescence reporter (Per1-luc or M oscillators are totally unknown. Recently, we developed an in Bmal1-ELuc) under DD. Spontaneous locomotor activity was vivo method for monitoring clock gene expression in the SCN of a monitored simultaneously by an infrared thermal sensor. Bio- freely moving mouse (18, 19), enabling us to compare the SCN luminescence emitted from the SCN was collected with an circadian rhythms with physiological and behavioral rhythms. implanted optical fiber connected to a cooled photo- In the study, to obtain insight into the molecular multiplier tube (PMT), as described previously (18, 19). mechanism of light entrainment, we continuously monitored Phase responses of Per1-luc and Bmal1-ELuc circadian circadian rhythms in clock gene Per1 and Bmal1 expression in the rhythms in the SCN were examined when a single light pulse of SCN together with behavioral rhythms. Surprisingly, the rhythms 9 h was given at circadian time (CT) 11.5 to make the of Per1 and Bmal1 expression responded differentially to the largest phase-delay shift according to a previous study (20), in light pulse. The dissociation between the two circadian rhythms which the activity onset of behavioral circadian rhythm was de- was confirmed in the cultured SCN slices from double-transgenic fined as CT12 (Fig. 1 and Fig. S1A). We also examined the effect mice carrying luciferase reporters for Per1 and Bmal1 expression. of a light pulse of the same duration given at CT21.5, when

E3700 | www.pnas.org/cgi/doi/10.1073/pnas.1613374114 Ono et al. Downloaded by guest on September 25, 2021 phase-advance shifts were expected (Fig. 2 and Fig. S1B). Data rhythm was compared with behavioral rhythms, the amount of PNAS PLUS of the in vivo experiments before a phase-delaying light pulse phase shift in the Per1-luc rhythm was not different from the (n = 4 for Per1 and n = 4 for Bmal1) were used in a previous phase shift of activity onset but was significantly different from study (18) to calculate the free-running period and to estimate the phase shift of activity offset (two-way ANOVA, post hoc the circadian and ultradian phases of behavioral and clock gene t test, P < 0.05) (Fig. 1D, Center). The amount of phase shift in expression rhythms. the Bmal1-ELuc rhythm was not different from the amount of In response to a light pulse at CT11.5, the activity onset of the phase shift in activity offset but was significantly different from behavioral rhythm was phase delayed immediately by 4.0 ± 0.4 h the amount of phase shift in the activity onset (two-way ANOVA, on average in Per1-luc mice (mean ± SD, n = 4) and by 3.5 ± post hoc t test, P < 0.01) (Fig. 1D, Right). 0.7 h on average in Bmal1-ELuc mice (n = 4). In contrast, the When a single light pulse was given at CT21.5, the amount of activity offset was gradually phase delayed over four or five cycles phase shift was small, and the dissociation between the Per1-luc by 4.4 ± 0.3 h in Per1-luc mice and by 3.6 ± 0.8 h in Bmal1-ELuc and Bmal1-ELuc circadian rhythms was not detected at statisti- mice before reaching a free-running steady state (Fig. 1 C and cally significant levels (Fig. 2 and Fig. S1B). Nevertheless, an D). The amount of phase shift in either behavioral marker was association of the circadian peak in clock gene expression with not significantly different in the two reporter mice. Thus, the the behavioral marker was observed, similar to that observed activity band was temporarily compressed after the light pulse with a light pulse at CT11.5. These results indicate that the cir- (Fig. 1C). On the other hand, the circadian peak of Per1-luc cadian rhythms of Per1 and Bmal1 expression are dissociable rhythm was immediately phase delayed in parallel with the ac- when an external perturbation produces a large phase-delay shift tivity onset (Fig. 1 C, Left and D), and the circadian peak of in the SCN circadian rhythm in vivo. Bmal1-ELuc rhythm was gradually phase delayed in parallel with the activity offset (Fig. 1 C, Right and D). The amount of phase Dissociation of Per1 and Bmal1 Oscillations in the SCN Slice. To test shift in the two bioluminescent rhythms on the after whether the dissociation of Per1 and Bmal1 circadian oscillation the light pulse was significantly different (two-way ANOVA, post really occurs, we made SCN slices from the double-transgenic hoc t test, P < 0.05) (Fig. 1D, Left). When each bioluminescent mice carrying reporters for both Per1 and Bmal1 expression and

Local time

A 0 Per1-luc (SCN) Behavior (counts/min) 0 12 12 0 180 80 0 150 60 120 90 40 7 60 Days 20 30 RLU (counts/min) 0 0 1 2 3 45678 91011121314 15 14 Days B Local time

0 12 0 12 0 Bmal1-ELuc (SCN) Behavior (counts/min) 180 80 0 150 60 120 90 40 7 60 Days 20 30 RLU (counts/min) 0 0 1 2 3 45678 91011121314 15 14 Days Light pulse

C Local time Local time 6012 18 612186012 18 61218 1 1 Per1-luc Bmal1-ELuc

7 7 Days Days

14 14 D 5 5 5 Per1-luc Per1-luc Bmal1-ELuc 4 4 4 Bmal1-ELuc 3 3 3 2 2 2 1 1 1 * Phase shifts(h) Phase shifts (h) 0 Phase shifts (h) 0 0 -1 0 123456789 -1 0 123456789 -1 0 123456789 Days after light pulse Days after light pulse Days after light pulse Activity onset Activity offset

Fig. 2. Light pulse-induced phase-advance shifts of circadian rhythms in the SCN and behavioral rhythm in freely moving adult mice. (A and B) Typical examples of a light-induced phase response at CT21.5 are illustrated for the Per1-luc (A) and Bmal1-ELuc (B) rhythms in the SCN together with a behavioral rhythm in double plotting (Left) and sequential plot (Right) as in Fig. 1 A and B.(C) Mean acrophases are illustrated for Per1-luc (Left) and Bmal1-ELuc (Right) rhythms together with the mean activity onsets and offsets of behavioral rhythms. Also see the legend of Fig. 1C.(D) The amount of phase shifts after a light

pulse are shown as the mean and SEM (n = 3) for Per1-luc (blue circles) and Bmal1-ELuc (green circles) (Left), for Per1-luc and two phase markers (activity onset NEUROSCIENCE and offset) of behavioral rhythm (Center), and for Bmal1-ELuc and the phase markers (Right). Also see the legend of Fig. 1D.

Ono et al. PNAS | Published online April 17, 2017 | E3701 Downloaded by guest on September 25, 2021 monitored Per1-luc and Bmal1-ELuc simultaneously from the luc; these periods were significantly different (n = 9, paired t test, same SCN slice. Circadian Per1-luc and Bmal1-ELuc rhythms in P < 0.01) (Fig. 3D). The periods determined by other methods, the cultured SCN were separated successfully, as previously such as a linear regression line fitted to the consecutive cycle reported (21, 22), and persisted at least for 3 wk (Fig. S2). peaks, confirmed the difference (Fig. S3B). As a result, the phase In the neonatal SCN (Fig. 3 A–C), double-plotted circadian relation between the Per1-luc and Bmal1-ELuc circadian rhythms rhythms in Per1-luc and Bmal1-ELuc showed internal dissocia- changed gradually and significantly during culturing (Fig. 3C and tion, with a pattern similar to relative coordination in which the Fig. S3A), regardless of the circadian phase marker used [i.e., the phase relation of two rhythms changed continuously in the acrophase of a fitted cosine curve (Fig. 3I and Fig. S3C, Left)or course of free running (23). Similar patterns also were detected the peak of a detrended circadian rhythm (Fig. S3C, Right)]. To in other neonatal SCN slices (Fig. S3A). The mean circadian know whether the changes in the phase relationship between the period of Bmal1-ELuc mice was significantly shorter than that of two circadian rhythms reflected gradual and systematic alterations Per1-luc mice. The circadian period determined by χ2 periodo- of the rhythm shape, we compared the shape of rhythmicity on day gram was 22.7 ± 0.4 h for Bmal1-ELuc and 23.1 ± 0.4 h for Per1- 3 and day 15 of culture and did not find any difference (Fig. S3D).

Per1-luc Bmal1-ELuc RLU (1,000 counts/15sec) A B 5 20

Per1-luc 0 0 X Bmal1-ELuc -5 -20

RLU (1,000 counts/15sec) 0 5 10 15 20 Days in culture SCN slice C Local time D Per1-luc 0 12 0 12 0 Long pass Bmal1-ELuc 0 24.0 filter ** 5 23.5 10 PMT 23.0

15 Period (h) 22.5 Days in culture 20 Per1-luc Bmal1-ELuc 22.0

Per1-luc Bmal1-ELuc RLU (1,000 counts/15sec) E F 5 20

Per1-luc 0 0 X Bmal1-ELuc -5 -20 RLU (1,000 counts/15sec) 0 5 10 15 20 Days in culture SCN slice G Local time H Per1-luc 0 12 0 12 0 Long pass Bmal1-ELuc 0 24.0 filter ** 5 23.5 10 PMT 23.0

15 Period (h) 22.5 Days in culture 20 Per1-luc Bmal1-ELuc 22.0 I 20 18 16 14 12 Neonate SCN Adult SCN 10 * 8 * * ** ** ** Phase difference (h) Phase difference ( Per1-luc - Bmal1-ELuc ) 024 6 81012141618 Days in culture

Fig. 3. Simultaneous measurement of Per1-luc and Bmal1-ELuc rhythms in the cultured SCN slice. (A and E) Experimental schemes of simultaneous mea- surement of Per1-luc and Bmal1-ELuc expression in neonatal (A) and adult (E) SCN slices. (B, C, F, and G) Sequential plots and double plots of Per1-luc (blue) and Bmal1-ELuc (green) circadian rhythms in a neonatal (B and C) and an adult (F and G) SCN slice in culture. In double-plotting, the in which the bioluminescence is higher than the mean value of detrended data in a series is indicated by colored horizontal bars. Colored circles in a double plot (C and G) are acrophases. (D and H) Mean circadian periods calculated by χ2 periodogram (± SD) (21 d) of Per1-luc and Bmal1-ELuc in the neonatal (n = 9) (D) and adult (n = 5) (H) SCN slices are indicated by colored bars. Double asterisks (**) indicate a statistically significant difference between Per1-luc and Bmal1-ELuc (P < 0.01, paired t test). (I) Mean daily phase differences in terms of acrophase between Per1-luc and Bmal1-ELuc circadian rhythms in the neonatal (red, n = 9) and adult (black, n = 5) SCN slices in culture for 17 d. Two-way repeated-measure ANOVA revealed significant differences between the neonatal and adult SCN; **P < 0.01, *P < 0.05 (post hoc t test).

E3702 | www.pnas.org/cgi/doi/10.1073/pnas.1613374114 Ono et al. Downloaded by guest on September 25, 2021 In addition, the skewness of circadian rhythm was not significantly relation between the dorsal and ventral regions was not changed PNAS PLUS different between day 3 and day 15 for either Per1-luc or Bmal1- on day 13 or 14 of culture in either the Per1-luc or the Bmal1- ELuc. Furthermore, to exclude a possible effect of rhythm damping ELuc rhythm (paired t test, Per1-luc, P = 0.841; Bmal1-ELuc, P = during culture on the determination of rhythm phase, we en- 0.932) (Fig. 4 J and L). We also analyzed the distribution of hanced the rhythm amplitude by replacing the culture medium circadian peaks in the dorsal and ventral regions of the SCN with fresh medium; this replacement is known to increase the using Rayleigh plots (Fig. 4 K and M and Fig. S5 C and D). The robustness of rhythmicity (Fig. S4). However, because the medium mean vector of the circadian rhythm was not changed in the exchange also is known to shift the circadian rhythm of the neo- dorsal and ventral regions of the SCN during culturing for either natal SCN depending on the time of replacement (24), we se- Per1-luc (Fig. 4K)orBmal1-ELuc (Fig. 4M), suggesting that the lected the time to minimize the phase shifts. The phase relation internal synchrony of cellular circadian rhythms remained un- between the two rhythms was kept essentially as it had been after changed during culture. These results indicate that the dissoci- medium exchange, and there was no systematic relation between ation between Per1-luc and Bmal1-ELuc rhythms is not caused the rhythm amplitude and phase difference. by a regional difference in the SCN neural network. A difference in the circadian period of the two clock gene rhythms also was detected in the adult SCN slice (Fig. 3 E–H and Simultaneous Measurement of Circadian Per1, Bmal1, Calcium, and Fig. S3). The circadian period of Per1-luc rhythm was 23.4 ± Spontaneous Firing in the Cultured SCN. To understand further 0.1 h, and that of Bmal1-ELuc rhythm was 23.2 ± 0.1 h (n = 5), a the relationships among the circadian firing rhythm, the core significant difference (paired t test, P < 0.01) (Fig. 3H). How- feedback loop, and the interlocked Bmal1 loop, we simulta- ever, the amount of dissociation in the adult SCN was less robust, neously measured Per1 and Bmal1 gene expression, spontaneous and the phase difference between the two rhythms after day 11 of firing, and the intracellular calcium level from single cultured culture was significantly smaller in the adult than in the neonatal SCN slices. The SCN of double-transgenic mice (Per1-luc and SCN (P < 0.01, two-way repeated-measure ANOVA) (Fig. 3I). Bmal1-ELuc) expressing the calcium indicator GCaMP6s was cultured on a MED probe, and the bioluminescence of firefly Spontaneous Firing Rhythms in the Cultured SCN Are Dissociated (F)-luc for Per1-luc and of E-Luc for Bmal1-ELuc, the fluores- from Per1 and Bmal1 Oscillations. Spontaneous firing is regarded cence of GCaMP6s, and spontaneous firing were monitored (Fig. as an important output signal from a core loop in the SCN (25, 5 A and B, Fig. S7, and Movie S1). In addition to an endogenous 26) and is well correlated with behavioral circadian rhythms (27). circadian oscillation as an output of the core feedback loop, However, the mechanism by which spontaneous firings in the intracellular calcium in the SCN is known to be controlled by the SCN regulate behavioral circadian rhythms remains unknown. input signals from the SCN neural network (29, 30). Plotting of To understand the relationship between the circadian firing rhythm the acrophase or of the circadian peak in each cycle revealed that and activity onset or offset, or between spontaneous firing and the circadian rhythms in Bmal1-ELuc, spontaneous firing, and clockgeneexpressionintheSCN,wemeasuredPer1-luc or Bmal1- intracellular calcium were gradually phase advanced relative to ELuc expression simultaneously with spontaneous firing in the the Per1-luc circadian rhythms in the course of culturing (one- neonatal SCN slices using a multielectrode array dish (MED) and way repeated-measure ANOVA, P < 0.05 and post hoc Tukey– a CCD camera (Fig. 4 A and B and Fig. S5 A and B). For this Kramer test) (Fig. 5 C–H). Interestingly, the calcium circadian experiment, we used single-transgenic mice carrying a bio- rhythm was essentially phase-locked to the circadian firing luminescence reporter for Per1-luc or Bmal1-ELuc expression. rhythms, keeping a stable phase difference of about 1 h for more The characteristics of the bioluminescent circadian rhythms in than 10 d (Fig. 5 D and H). Thus, the phase relations of circadian these single-transgenic mice were similar to those in double- rhythms in Per1-luc, Bmal1-ELuc, and firing changed gradually transgenic mice (Per1-luc,23.3± 0.1 h, n = 7; Bmal1-ELuc, during culturing. Although the phase relations among these cir- 22.7 ± 0.3 h, n = 7; Student’s t test, P < 0.01) (Fig. S6). cadian rhythms changed substantially during culture, the phase The circadian peak of Per1-luc rhythm in the neonatal SCN relation of the Per1-luc and Bmal1-ELuc circadian rhythms slice on the MED was gradually phase delayed relative to that of (Δphase) in the dorsal and ventral SCN regions did not change the spontaneous firing rhythms (Fig. 4 C–E). On the other hand, (day 2–3, 1.7 ± 0.3 h; day13–14, 2.1 ± 0.9 h; paired t test, P = the circadian peak of Bmal1-ELuc rhythm was gradually phase 0.283) (Fig. 5 F and G and Fig. S7C), suggesting that the change advanced relative to that of the firing rhythms (Fig. 4 C–E). The in the phase relationship of the two rhythms occurred at the phase difference between the firing rhythms and Per1 or Bmal1 same rate in both regions. The mean vector in these circadian expression rhythms was significantly larger at day 15 of culture rhythms was not different between day 2–3 and day 13–14, than that at the beginning of culture (Student’s t test, P < 0.01) again suggesting that the internal synchrony of cellular circadian (Fig. 4F). The circadian periods of Per1-luc and Bmal1-ELuc rhythms was maintained during culture (Fig. S7D). On the other rhythms also were significantly different (Student’s t test, P < hand, the mean vector of the Bmal1-ELuc circadian rhythm was 0.05) (Fig. 4G). These results indicate that the firing rhythms and slightly but significantly shorter than those of the other circadian Per1 or Bmal1 rhythms in the cultured SCN are dissociable and rhythms, suggesting differential internal synchrony and/or slop- that the firing rhythms are not a direct consequence of the cir- piness of the Bmal1-ELuc circadian rhythms. cadian oscillation of either the core feedback loop or the inter- locked Bmal1 loop. Discussion In the present study we found that the Per1-luc and Bmal1-ELuc Spatiotemporal Features of Per1 and Bmal1 Expression in the Cultured circadian rhythms were transiently dissociated when freely moving SCN. To know whether the dissociation between the Per1 and mice under DD experienced a phase-shifting light pulse. The Bmal1 circadian rhythms is caused by region-specific gene ex- gene-expression rhythms were differentially associated with the pression in the SCN, we analyzed the spatiotemporal features of activity onset and offset of behavioral circadian rhythm. Similar Per1-luc and Bmal1-ELuc expression in the SCN during culturing dissociation of Per1 and Bmal1 oscillation was observed in the using an automated rhythm analysis with time-series images on cultured SCN slices of mice carrying a dual reporter system. Such the pixel level (28). In both Per1-luc and Bmal1-ELuc rhythms, a transient dissociation between the circadian rhythms was de- the mean circadian phase on day 2 or 3 of culture was signifi- tected only by continuous and simultaneous measurement of cantly delayed in the ventral region relative to the dorsal region, multiple rhythms from a single SCN. The dual reporter system

by ca. 1 h for Per1-luc (paired t test, P < 0.05) and by ca. 3 h for used in the present study enabled us to identify the change in the NEUROSCIENCE Bmal1-ELuc (paired t test, P < 0.05) (Fig. 4 J and L). The phase phase relation of two different rhythms. However, because the

Ono et al. PNAS | Published online April 17, 2017 | E3703 Downloaded by guest on September 25, 2021 4 21 A C Firing rate (Hz) Per1-luc Bmal1-ELuc 18 15 SCN slice 12

Per1-luc 0 9 MED 6 RLU,

lens (x100 counts/h) 3 -4 0 CCD camera 3 18 15 Firing rate (Hz) B Per1-luc Bmal1-ELuc 12 0 9 d d Bmal1-ELuc 6

(x100 counts/h) 3 RLU, -3 0 v v 0 51015 20 Days

D Local time E Days F 0 12 0 12 0 0 3 6912 15 18 21 6 ** 0 0 -2 4 5 -4 2 -6 10 -8 0 Days 15 -10 -12 Per1-luc -2 hase difference (h) P 20 Phase difference (h) -14 Neuronal activity Per1-luc (vs. Neuronal activity) -4 Local time G 24.5 * 0 12 0 12 0 17 Bmal1-ELuc 0 15 5 13 10 11 Period (h) Days 15 9 23.0 7 Phase difference (h)

20 (vs. Neuronal activity) Bmal1-ELuc 0 3 6912 15 18 21 Neuronal activity Per1-luc Days Bmal1-ELuc HIPer1-luc Bmal1-ELuc Slice A Slice B Slice C Slice D Slice A Slice B Slice C Slice D d d d d d d d d

v v Day2-3 Day2-3 v v v v v v

-12h -12h Day13-14 Day13-14 +12h +12h

JKdorsal ventral LMdorsal ventral 8.0 1.0 n.s. 1.0 n.s. 8.0 1.0 1.0 n.s. n.s. n.s. 0.8 0.8 0.8 0.8 6.0 6.0

n.s. 0.6 0.6 0.6 0.6 4.0 4.0 0.4 0.4 0.4 0.4

ventral-dorsal 2.0 ventral-dorsal 2.0 0.2 0.2 0.2 0.2 Mean vector (r) Mean vector (r) Mean vector (r) Mean vector (r) Phase difference (h) Phase difference Phase difference (h) Phase difference 0 0 0 0 0 0

Day2-3 Day2-3 Day2-3 Day2-3 Day2-3 Day2-3 Day13-14 Day13-14 Day13-14 Day13-14 Day13-14 Day13-14

Fig. 4. Simultaneous measurement of Per1-luc and Bmal1-ELuc together with spontaneous firing in the cultured SCN. (A) Experimental scheme of simul- taneous measurement of Per1-luc, Bmal1-ELuc, and spontaneous firing in the neonatal SCN. (B) Bioluminescence images of Per1-luc (Left) and Bmal1-ELuc (Right) on a MED probe. (Scale bars: 100 μm.) (C) Sequential plots of Per1-luc (Upper, blue) and Bmal1-ELuc (Lower, green) rhythms with firing rhythm (black) from the entire area of the SCN. (D) Double-plotted circadian rhythms of Per1-luc (Upper, blue) and Bmal1-ELuc (Lower, green) with respective acrophases (closed circles) are illustrated together with double-plotted circadian rhythms of spontaneous firing (gray bars) with acrophase (black circles). The colored zones indicate the time when bioluminescence was higher than the mean value of detrended data in a series. Also see the legend of Fig. 3. (E) Daily acrophase differences in Per1-luc (Upper)orBmal1-ELuc (Lower) rhythms and firing rhythms in four SCNs; SCNs are shown in different colors. One SCN partially lacks firing data. (F) Phase difference (mean ± SD) between days 13–14 and days 2–3 in culture in the Per1-luc (Left) and Bmal1-ELuc (Right) circadian rhythms. Negative values indicate phase delay, and positive values indicate phase advance (**P < 0.01, Student’s t test). (G) Circadian period determined by χ2 periodogram (mean ± SD) using records of Per1-luc and Bmal1-ELuc at day 14 in culture (*P < 0.05, Student’s t test). (H and I) Acrophase maps of Per1-luc (H) and Bmal1-ELuc (I) at days 2–3(Upper) and 13–14 (Lower) in culture (n = 4). The mean acrophase was adjusted to 0 h. The color key indicates phase dis- tribution in . (J–M) The phase difference (mean ± SD) in Per1 (J)orBmal1 (L) circadian rhythms on the pixel level between the ventral and dorsal regions and the length of the mean vector of the respective rhythm (K and M) in the dorsal and ventral region at days 2–3 and 13–14 in culture.

method is based on luciferin–luciferase reaction and the sepa- from the circadian rhythm of the intracellular calcium level. ration of emitted light by filtering, these findings should be These findings indicate that the Bmal1 loop has an oscillatory confirmed by more direct measurement of the transcription or nature similar to that of the core loop and behaves as a consti- translation of clock genes in . Most of the current direct tutional subunit of the circadian system in the SCN. methods are still difficult to apply because of insufficient time We demonstrated the dissociation of circadian rhythms in resolution resulting from individual differences and use of a clock gene expression of freely moving mice when the circadian large number of animals. rhythms were on the way to steady-state free running after the Interestingly, the circadian rhythm in spontaneous firing was light pulse (Fig. 1). A similar dissociation of the circadian rhythms dissociated from the Per1 and Bmal1 circadian rhythms but not of clock gene expression has been observed previously with shifts

E3704 | www.pnas.org/cgi/doi/10.1073/pnas.1613374114 Ono et al. Downloaded by guest on September 25, 2021 A C PNAS PLUS CCD camera Per1-luc Bmal1-ELuc hSyn-GCaMP6s Neuronal activity Long pass 18 21 filter Emission 12 18

no-filter 15 Firing (Hz) LED 6 12 Excitation 0 Dichroic 9 -6 Per1-luc lens 6 X

Bmal1-ELuc MED RLU (x100 counts/h) -12 3 + hSyn-GCaMP6s SCN slice -18 0 0 2468101214 16 B Per1-luc Bmal1-ELuc Days D Local time E Local time 0 12 0 12 0 014 8162220 4 0 0 Acrophase 4 5 GCaMP6s 8 Peak phase Days Days 10 12 15 16 Per1-luc hSyn-GCaMP6s Bmal1-ELuc Neuronal activity

F G ⊿Phase (h) Bmal1 vs Per1 H Neuronal activity Phase difference map Bmal1-ELuc (Bmal1 vs Per1) Whole SCN dorsal SCN ventral SCN hSyn-GCaMP6s Day2-3 0 0 0 16 ** * ** * 14 0h d 18 6 18 6 6 18 12

12h Day2-3 10 v 12 12 12 24h 8 ⊿Phase (h) Bmal1 vs Per1 6 Day13-14 Whole SCN dorsal SCN ventral SCN * * ** * 0 0 0 4 * ** * 0h phase (h) vs Per1-luc * *

d ⊿ 2 * ***** 6 12h 18 6 18 6 18 0 v Day13-14 0 3 6912 15 24h 12 12 12 Days

Fig. 5. Simultaneous measurement of Per1-luc, Bmal1-ELuc, intracellular calcium, and spontaneous firing in the cultured SCN slice. (A) Experimental scheme of simultaneous multifunctional measurement in the neonate SCN slice. (B) Bioluminescence images of Per1-luc (Upper Left) and Bmal1-ELuc (Upper Right), fluorescence images of GCaMP6s (Lower Left), and a bright field image (Lower Right) of a cultured SCN slice on a MED probe. (Scale bars: 200 μm.) (C) Sequential plots of circadian Per1-luc, Bmal1-ELuc, GCaMP6s, and firing rhythms from the entire area of the SCN. Spontaneous firing was expressed as the mean firing rate from electrodes covered by bilateral SCN. (D) Double plots of circadian rhythms of four measures. Colored circles are acrophases of circadian Per1-luc, Bmal1-ELuc, GCaMP6s, and firing rhythms. Yellow bars indicate the time zone in which GCaMP6s fluorescence is higher than the mean value of detrended data in a series. Also see the legend of Fig. 4D.(E) Longitudinal plotting of daily acrophases and of cycle peaks of the four circadian rhythms demonstrated in C. The features of free running are almost identical regardless of the phase marker. (F) Phase-difference maps (Per1-luc vs. Bmal1-ELuc)at days 2–3(Upper) and 13–14 (Lower) in culture. The color key indicates the phase difference in hours. (G) Rayleigh plots of phase difference on the pixel level between Per1-luc and Bmal1-ELuc in the whole (Left), dorsal (Center), and ventral (Right) SCN are shown for days 2–3(Upper) and 13–14 (Lower) in culture. A red line in a Rayleigh circle indicates the mean phase. (H) The mean daily phase difference, in terms of acrophase, between the Per1-luc and three other circadian rhythms (n = 3). (*P < 0.05, vs. day 2, one-way repeated-measure ANOVA with post hoc Tukey–Kramer test).

in LD schedules (31, 32), but the relevance to behavioral outputs photoperiod in the SCN (14, 15). The pacemaker corresponding was not clear. Importantly, the phase shifts of Per1 and Bmal1 to the E oscillator is likely located in the rostral part of SCN, and rhythms in our study were closely associated with the phase shifts that corresponding to the M oscillator is likely located in the of either the activity onset or offset of behavioral rhythm. Pre- caudal part. The present findings further suggest that the two viously, Vansteensel et al. (33) reported the dissociation between circadian oscillations have different molecular mechanisms. the Per1-luc circadian rhythm in the SCN and behavioral rhythms Dissociation between the Per1 and Bmal1 oscillation also was that occurred following a shift in the LD cycle. The finding raised observed in the SCN ex vivo (Figs. 3–5). Because of a significant the possibility that the Per1-luc circadian rhythm reports only a difference in the circadian period, the phase relation between subset of SCN neurons. In agreement with this report, the present the Per1 and Bmal1 oscillation changed gradually during cul- findings suggest not only the existence of two coupled circadian turing; this change was not the result of systematic changes in the oscillations with different molecular mechanisms but also their shapes of circadian rhythms (Fig. S3 and Fig. S7). The core and differential association with the activity onset and offset of be- interlocked Bmal1 loop have been regarded as the origin of the havioral rhythms. According to Pittendrigh’s hypothesis (13), ac- circadian rhythms of Per1 and Bmal1 expression (3–5). The tivity onset and offset of nocturnal rodents are regulated by two present results indicate that the two loops are coupled to each different oscillators, the E and M oscillator, respectively. The other but exhibit ongoing differences in period and relative hypothesis was based on the findings of rhythm splitting under phase that suggest they may oscillate independently. They are constant light and differential responses to light (12). In agree- dissociable by a light pulse in vivo and by prolonged free running ment with the above hypothesis, Honma et al. (11) demonstrated ex vivo. The theoretical backgrounds of the present findings are a in rats that activity onset and offset showed different phase re- coupling of two oscillating feedback loops and a dependency of sponses to a single light pulse. Thus, the Per1-luc circadian rhythm the circadian period on the relative strength of interlocked loops in the SCN seems to associate with the E oscillator, and the as formulated in another associated loop (34). In addition, the Bmal1-ELuc circadian rhythm seems to associate with the M os- relative coordination between of Per1-luc and Bmal1-ELuc

cillator. Previous reports demonstrated separate regional pace- rhythms suggests that the core loop and Bmal1 loop are not in- NEUROSCIENCE makers which behaved differently under a long and a short dependent but are mutually interactive even under dissociation.

Ono et al. PNAS | Published online April 17, 2017 | E3705 Downloaded by guest on September 25, 2021 The Bmal1 loop consists of a positive-feedback component of C57BL/6j background mice for more than seven generations. Mice were ROR and a negative-feedback component of RevErbα for Bmal1 reared in our animal quarters where environmental conditions were con- expression and potentially is capable of oscillating without os- trolled (lights-on 6:00–18:00; light intensity ∼100 lx at the cage bottom; ± cillation of the core loop, at least in theory (35). humidity 60 10%). Mice had free access to food pellets and water. Ex- periments were conducted in compliance with the rules and regulations The circadian oscillation in the dorsomedial region of the SCN established by the Animal Care and Use Committee of Hokkaido University was reported to be faster than that in the ventrolateral region under the ethical permission of the Animal Research Committee of Hokkaido (36). Regional differences also were reported in the intensity of University (approvals no. 13-0053 for in vivo experiments and no. 13-0064 for clock gene expression (37). Therefore the dissociation between ex vivo experiments). the two clock gene rhythms could be ascribed to regional dif- ferences in circadian oscillation. However, in the present study, Measurement of Behavioral Activity. Mice (nine males, four females) were the phase relation of the Per1 and Bmal1 expression rhythms was used in the in vivo study at 2–6 mo. Mice were individually housed in a not different in the dorsal and ventral SCN during culturing, polycarbonate cage (115 mm wide × 215 mm long × 300 mm high), which bringing into question the possibility that regional differences in was placed in a light-tight and air-conditioned box (40 × 50 × 50 cm). An – respective circadian oscillation might be a cause of dissociation. LED light source with an intensity of 150 250 lx on the ceiling of a box was turned on when a light pulse was given after 5 d exposure to DD. Spon- Recently, we found sharply differentiated clusters of cells that taneous movements were measured by a passive infrared sensor, which expressed the PER2 circadian rhythm with different periods in detects a change in thermal radiation from an animal caused by body the SCN of Cry1,2 double deficient mice (38). The cells in each movements (42). The amount of behavioral activity was recorded auto- of these cell clusters showed no regional specificity but instead matically every by a (The Kit; Stanford were distributed diffusely. The finding suggested that in the Software System). circadian system of the SCN a specific type of oscillating cell builds up a constitutional oscillator that does not necessarily Surgery. Surgery was performed under isoflurane anesthesia as previously show regional specificity. The Per1- and Bmal1-specific oscillator described (18, 19). To measure bioluminescence from the SCN in vivo, a cells could be located diffusely throughout the SCN. Alterna- handmade guide cannula (i.d. 1.12 mm; o.d. 1.48 mm) was stereotaxically tively, the dissociation of two clock gene rhythms could be inserted into the brain 0.2 mm posterior to the bregma, 0.2 mm lateral from caused by the dissociation of the core loop and the Bmal1 loop in the midline, and 3.0 mm from the surface of the skull and was fixed to the skull with dental resin. After a recovery period of more than 4 d, a polymethyl single SCN cells. If so, within a single cell, the core molecular methacrylate optical fiber (fiber diameter, 0.5 mm; surface cladding, 0.25 mm loop would be responsible for the activity onset and the Bmal1 thick) was inserted into the guide cannula aimed at the SCN at a 5.8-mm depth loop would be responsible for the activity offset of behavioral from the surface of the skull and was fixed to the skull with dental resin. rhythm. In this case, we should assume different output signals More than 4 d after the insertion of the optical fiber, an osmotic from a single cell. In any case, the coupling of two oscillations (model 2002; Alzet; flow speed, 0.5 μL/h, pump volume, 200 μL) containing was less strong in the neonatal SCN than in the adult SCN, D-luciferin, a substrate of luciferase, was implanted in the peritoneal cavity. suggesting a developmental change in the SCN circadian system. To deliver the substrate, the osmotic pump was filled with D-luciferin We had expected that the circadian firing rhythm in the SCN K (100 mM) dissolved in physiological saline. After each surgery, penicillin-G slice might synchronize with either of the two rhythms of clock (40 units/g of body weight) was administered i.m. to prevent infection. As- gene expression or show a splitting into two components. Con- pirin (120 mg/kg of body weight, administered orally) or buprenorphine (0.05–0.1 mg/kg of body weight, administered by s.c. injection) was used for trary to our expectation, the simultaneously determined circa- postoperative analgesia. dian firing rhythms dissociated from both of the circadian gene- expression rhythms (Figs. 4 and 5). However, a coupling between In Vivo Measurement of Bioluminescence. Three to five days after the im- the circadian firing and intracellular calcium rhythms persisted plantation of the osmotic pump, bioluminescence from the SCN was mea- for at least for 2 wk in culture, suggesting that the two overt sured in freely moving mice under DD. The measurement was performed rhythms are regulated by the same oscillatory mechanism, which every minute via an optical fiber connected to a photon-counting device (In could be neither the core loop nor the interlocked Bmal1 loop. vivo Kronos; Atto) (18, 19) equipped with a photomultiplier tube (Hama- The cellular circadian rhythms in the SCN are likely regulated by matsu Photonics). Recorded data were fed into a computer and analyzed. two circadian oscillations of different origins: the intracellular molecular feedback loop and the SCN network in which the cell Histological Examination. Once the measurements were completed, mice were is involved. The relative intensities of the two oscillations may anesthetized with ether and were intracardially perfused with physiological saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains determine the nature of the cellular circadian rhythms examined. were cryoprotected with 20% sucrose in 0.1 M PB and stored at −80 °C. Serial Circadian firing and calcium rhythms would be affected by 30-μm-thick coronal sections of the brain were made using a Leica cryostat and feedback loops and the SCN network (39). A strong coherence were stained with cresyl violet to identify the placement of the tip of the of the circadian firing rhythm to the intracellular calcium rhythm optical fiber. could be interpreted in terms of rather than oscillatory + coupling. Neither Ca2 nor firing rhythm in the SCN is associ- Preparation of SCN Slices for Culture. To measure the bioluminescence from ated directly with the onset or offset of circadian behavioral the SCN slice, mice were killed to harvest the SCN between 8:00 and 16:00 h. rhythm. The present findings indicate that the links between the To measure the level in tissue, 300-μm-thick coronal slices from the SCN of SCN circadian rhythms and behavior are more complicated than neonatal (postnatal day 7) mice were made with a tissue chopper (Mcllwain), previously thought (40). and slices from the SCN of adult (2- to 6-mo-old) mice were made with a microslicer (DTK-1000; Dosaka EM). The SCN tissue was dissected at the mid- Taken together, the present findings indicate that external rostrocaudal region, and paired SCN slices were cultured on a Millicell-CM perturbation, such as a single light pulse, transiently uncouples culture insert (Millipore Corporation) under culture conditions as described interlocked molecular loops that are separately associated with previously (43). Briefly, the slices were cultured in air at 36.5 °C with 1.2 mL the onset and offset of behavioral rhythms. DMEM (Invitrogen) with 0.1 mM D-luciferin K and 5% supplement solution.

Materials and Methods Measurement of Bioluminescence in SCN Tissue. Bioluminescence in SCN tissue Animals. Male and female Per1-luc (heterozygous or homozygous) (14) and was measured using a Lumicycle (Actimetrics) or Kronos (Atto) PMT at 10-min Bmal1-ELuc (heterozygous) (41) reporter mice of C57BL/6j background were intervals with an exposure time of 1 min. For the simultaneous measurement used. Per1-luc and Bmal1-ELuc mice were produced in the YS New Tech- of Per1 and Bmal1 expression, the bioluminescence of Per1-luc and Bmal1-ELuc nology Institute and the National Institute of Advanced Industrial Science from the same SCN slice was monitored alternatively by a dish-type luminometer and Technology, respectively, by injecting the reporter construct into fer- with a turntable for eight recording dishes (Kronos, Atto). Bioluminescence was tilized eggs of C57BL/6j background. We back-crossed these mice with measured for 15 s in the presence of a 600-nm long-pass filter (R60 filter; Hoya),

E3706 | www.pnas.org/cgi/doi/10.1073/pnas.1613374114 Ono et al. Downloaded by guest on September 25, 2021 then in the presence of a 560-nm long-pass filter (O56 filter; Hoya), and then circadian period was determined by the slope of the regression line, and the PNAS PLUS without any filter. The measurement was repeated at 10-min intervals. The amount of daily phase shift was calculated from the phase difference be- intensities of Per1-luc and Bmal1-ELuc bioluminescence were calculated tween the phase on the day of light pulse and that on the following day. as described previously (21, 22). For bioluminescence calculation, we used a Three chronobiology experts (D.O., S.H., and K.H.) participated in the visual 600-nm long-pass filter. inspection. For the analyses of circadian bioluminescent rhythms in vivo and ex vivo, Simultaneous Recordings of Bioluminescence and Spontaneous Firing from an data obtained with a PMT were smoothed by a 4-h moving average for in vivo SCN Slice. An SCN slice from a 3- to 5-d-old pup was cultured on a MED with data and a 50-min moving average for ex vivo data. The smoothed data then 64 electrodes. The size of an electrode was 20 × 20 μm (MED-P210A). were detrended by a 24-h moving average subtraction method (18, 19). Spontaneous firings were recorded using a MED 64 system (Alpha MED Neuronal activity rhythms also were detrended by a 24-h moving average Scientific). Spike discharges with a signal-to-noise ratio >2.0 were collected subtraction method. To compare the peak phases of circadian rhythms in by Spike Detector software (Alpha MED Scientific) as described previously vivo and ex vivo, we used an acrophase obtained by ClockLab software (43). The number of spikes per minute was calculated for each electrode (Actimetrics) or simply the peak phase in a cycle. The circadian period in the covered by the SCN slice. SCN slice was calculated by a periodogram or from the slope of a regression The MED was placed in a mini-incubator installed on the stage of a mi- line fitted to consecutive peak phases by the least-squares method. The croscope (ECLIPSE TE2000-U or ECLIPSE E1000; Nikon). The culture conditions difference in phase angle between two circadian rhythms was calculated were as described previously (43). Bioluminescence was recorded with a CCD using the acrophase of a best-fitted cosine curve or the peak of a circadian camera (ORCA-ІІ; Hamamatsu Photonics) cooled at −60 °C. The pixel size was cycle. For time-lapse images obtained by a CCD camera, the properties of 4.3 × 4.3 μm. circadian rhythm in bioluminescence and fluorescence signals were ana- lyzed on the pixel level using custom-made software based on a cosine Simultaneous Recordings of Per1-luc, Bmal1-ELuc, Calcium, and Spontaneous curve-fitting method as described previously (39, 44). The dorsal and ven- Firing from an SCN Slice. An SCN slice from a 3- to 5-d-old pup of Per1-luc tral areas of the SCN were separated by a line drawn at the midpoint and Bmal1-ELuc pup was cultured on a Millicell-CM culture insert. Aliquots between the dorsal and ventral edges so that it crossed the third ventricle of adeno-associated virus (AAV) serotype rh10 harboring GCaMP6s, a ge- at a right angle. A Rayleigh plot was made using the Oriana4 software netically encoded calcium sensor under the control of human synapsin- (Kovach Computing Service). Data are expressed as mean ± SD unless 1 promotor (University of Pennsylvania Gene Therapy Program Vector Core), otherwise indicated. were inoculated onto the surface of the cultured SCN slice 3–5 d after slice preparation. On the day after AAV infection, the SCN slice was transferred Statistics. Student’s t test was used when two independent group means onto the MED probe. Simultaneous measurement of bioluminescence, were compared. A paired t test was used when two dependent group means – fluorescence, and neuronal activity began 7 10 d after the SCN was cultured. were compared. A one-way ANOVA with a post hoc Tukey–Kramer test was The MED was placed in a mini-incubator installed on the stage of a mi- used to analyze data of a single time series. A two-way ANOVA with a post croscope (ECLIPSE-80i; Nikon) equipped with an EM-CCD camera (ImagEM; hoc t test was used when the data of two independent time series were Hamamatsu Photonics). A fluorescent calcium sensor (GCaMP6s) was ex- compared (Statview or Statcel 3). cited at cyan color (475/28 nm) with an LED light source (Retra Light ; Lumencor) and was visualized with a 495-nm dichroic mirror and 520/35-nm ACKNOWLEDGMENTS. We thank H. Tei for providing the Per1-luciferase emission filters (SemLock). To measure F-luc and E-Luc bioluminescence reporter plasmid; M. Shimogawara and H. Kubota for the development of (Per1-luc and Bmal1-ELuc) separately, a long-pass filter (AT610, Chroma an in vivo bioluminescence measurement system; K. Baba for technical ad- Technology Corporation) was used. Bioluminescence with or without the vice; T. Ueda for the analysis program; M. Mieda for advice on AAV purifi- filter was measured every (exposure time, 29 min for each condition). cation; M. P. Butler for thorough discussions; and the Genetically-Encoded Per1-luc and Bmal1-ELuc bioluminescence were calculated on the pixel Neuronal Indicator and Effector (GENIE) Project and the Janelia Farm Re- level with the method used for PMT measurement. search Campus of the Howard Hughes Medical Institute for sharing GCaMP6s constructs. This work was supported in part by the Uehara Memorial Foun- dation; the Nakajima Foundation; the Project for Developing Innovation Data Analysis. For the analyses of behavioral rhythms in vivo, spontaneous Systems of the Ministry of Education, Culture, Sports, Science and Technol- movements obtained every minute were used. The number of phase shifts ogy (MEXT); the Creation of Innovation Centers for Advanced Interdisciplin- was determined on the basis of a double-plotted actograph by visual in- ary Research Areas Program, MEXT; and Japan Society for the Promotion of spection. A regression line was fitted to the succeeding activity onsets or Science Grants in Aid for Scientific Research (KAKENHI) Grants 15H04679, offsets during a steady-state free-run before and after the light pulse. The 26860156, and 15K12763.

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