Dual regulation of clock gene Per2 expression ... - Wiley Online Library

European Journal of Neuroscience

European Journal of Neuroscience, Vol. 39, pp. 229–240, 2014

doi:10.1111/ejn.12400

BEHAVIORAL NEUROSCIENCE

Dual regulation of clock gene Per2 expression in discrete brain areas by the circadian pacemaker and methamphetamine-induced oscillator in rats Akiyo Natsubori,1,2 Ken-ichi Honma2 and Sato Honma1 1 2

Department of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan Department of Neuropharmacology, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Keywords: behavioral rhythm, brain dopaminergic system, extra-SCN oscillator, luciferase reporter

Abstract Behavioral rhythms induced by methamphetamine (MAP) treatment in rats are independent of the circadian pacemaker in the suprachiasmatic nucleus (SCN). To know the site and mechanism of an underlying oscillation (MAP-induced oscillator; MAO), extra-SCN circadian rhythms in the discrete brain areas were examined in rats with and without the SCN. To fix the phase of MAO, MAP was supplied in drinking water at a restricted time of day for 14 days (R-MAP) and subsequently given ad libitum (ad-MAP). Plain water was given to the controls at the same restricted time (R-Water). Clock gene Per2 expression was measured by a bioluminescence reporter in cultured brain tissues. In SCN-intact rats, MAO was induced by R-MAP and behavioral rhythms were phase-delayed from the restricted time under ad-MAP with relative coordination. Circadian Per2 rhythms in R-MAP rats were not affected in the SCN but were slightly phase-advanced in the olfactory bulb (OB), caudate–putamen (CPU) and substantia nigra (SN) as compared with R-Water rats. Following SCN lesion, R-MAP-induced MAO phase-shifted more slowly and did not show a sign of relative coordination. In these rats, circadian Per2 rhythms were significantly phase-shifted in the OB and SN as compared with SCN-intact rats. These findings indicate that MAO was induced by MAP given at a restricted time of day in association with phase-shifts of the extra-SCN circadian oscillators in the brain dopaminergic areas. The findings also suggest that these extra-SCN oscillators are the components of MAO and receive dual regulation by MAO and the SCN circadian pacemaker.

Introduction The circadian rhythms of physiology and behavior in mammals are controlled by a hierarchical multi-oscillator system, consisting of a central circadian pacemaker in the suprachiasmatic nucleus (SCN) and peripheral oscillators in a variety of tissues and organs (Reppert & Weaver, 2002; Mohawk et al., 2012). The SCN circadian pacemaker entrains to light–dark cycles (LD) and resets the peripheral oscillators. Intracellular mechanisms of the central and peripheral circadian oscillators are considered to be an autoregulatory molecular feedback loop involving several clock genes and their protein products. On the other hand, at least two oscillators in the circadian range are reported to be induced independent of the SCN circadian pacemaker (Honma & Honma, 2009). One is the methamphetamine (MAP)-induced oscillator (MAO) and the other is the food-entrainable oscillator (FEO). MAO is induced by chronic MAP treatment via drinking water (Honma et al., 1986a; Tataroglu et al., 2006) and desynchronises some extra-SCN oscillators in the brain as well as

Correspondence: Ken-ichi Honma, as above. E-mail: [email protected] Received 2 August 2013, revised 23 September 2013, accepted 26 September 2013

behavioral rhythm from the SCN circadian pacemaker (Masubuchi et al., 2000; Natsubori et al., 2013b). The MAP-induced behavioral rhythms are regarded as an animal model of the human sleep–wake cycle because they show characteristics specifically observed in the human sleep–wake cycle such as internal desynchronisation, circabidian (ca. 48 h) rhythms and non-photic entrainment. On the other hand, FEO is induced by restricted daily feeding (RF) and characterised by anticipatory activity prior to daily meals (Stephan et al., 1979). Restricted water supply (R-Water) induces anticipatory activity similar to RF (Johnson & Levine, 1973; Dhume & Gogate, 1982) but its effects are regarded as secondary to food restriction (Mistlberger & Rechtschaffen, 1985; Honma et al., 1986b). Other than the SCN independency, FEO and MAO share common characteristics such as non-photic entrainment and involvement of the central catecholaminergic systems (Honma et al., 1992; Honma & Honma, 1995; Yoshihara et al., 1996). However, our previous study indicated distinct brain mechanisms for FEO and MAO (Natsubori et al., 2013a). Daily treatment with MAP and RF at the same time of day induced similar responses in behaviors but substantially different phase responses of Per2 expression rhythms in the cultured brain tissues, especially in the caudate–putamen (CPU) and substantia nigra (SN). These findings suggest that oscillatory mechanisms underlying FEO and MAO are different. However, the difference

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd

230 A. Natsubori et al. could be due to differential effects of the SCN circadian pacemaker on the FEO and MAO, as the experiments were carried out in rats with the SCN circadian pacemaker intact. In the present study the effects of MAO and the SCN circadian pacemaker on behavior and circadian Per2 expression rhythms were examined in cultured tissues of discrete brain areas in rats with intact SCN and with bilateral SCN lesions. To fix the phase of MAO, MAP was supplied in drinking water at a restricted time of day. Subsequent free access to MAP revealed the induction of MAO. Here we demonstrate dual effects of the SCN circadian pacemaker and MAO on behavior and on Per2 expression in extra-SCN regions, and also suggest involvements of extra-SCN circadian oscillators of several brain areas in the organisation of MAO.

Materials and methods Animals and housing Female rats of the Wistar strain carrying a Period2-dLuciferase (Per2-dLuc) reporter system were used (Natsubori et al., 2013a,b). The rats were born and raised in our animal quarters under controlled environmental conditions (LD, 12 : 12 h with lights on at 06:00 h, 50–200 lux, temperature 22 2 °C, humidity 60 5%). They were weaned at the age of 3 weeks and housed together with three or four littermates in a polycarbonate cage (24 9 30 9 17.5 cm) until the experiments were begun at the age of 2–3 months. Rats were fed commercial rat chow and tap water ad libitum unless otherwise stated. The present experiments were ethically approved by Animal Research Committee of Hokkaido University (permission number 12-0064), and performed following the Guide for the Care and Use of Laboratory Animals in Hokkaido University and the guidelines laid down by the NIH in the US regarding the care and use of animals for experimental procedures. SCN lesion Electrolytic lesions were sterotaxically made in the bilateral SCN under pentobarbital anesthesia by passing a 3.0-mA direct current into each nucleus for 28 s through a stainless-steel electrode (0.4 mm diameter with an uninsulated tip of 0.1 mm in length). In the SCN-lesioned rats, aperiodism in behavior was confirmed by v2 periodogram analysis for at least 4 weeks after the operation. The lesions were histologically examined at the end of the experiments. The coronal brain slices containing the hypothalamus were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for at least 1 week, and cryoprotected in 30% sucrose in 0.1 M PB for 3 days. They were frozen on dry ice and serially sectioned (50 lm thick) on a cryostat. The sections were stained with Cresyl Violet. In the case of incomplete SCN lesion the results were excluded from further analyses. Measurement of behavioral rhythms Rats were transferred to an individual cage (24 9 30 9 35 cm) equipped with a running wheel (30 cm in diameter) in a light-proof and air-conditioned box (60 9 60 9 60 cm). Spontaneous movement was also measured by a thermal sensor located on the ceiling of the box. The LD of the box was the same as that in the animal quarter and the light intensity was ~300 lux at the bottom of the cage. The numbers of spontaneous movements and wheel revolutions were registered every minute on a hard disk by computer

software (The CHRONOBIOLOGY KIT; Stanford Software System, Stanford, CA, USA). Throughout the experiments, spontaneous movement and wheel-running activity were recorded simultaneously from each rat. Experimental protocols Thirty SCN-intact and 39 SCN-lesioned rats were used. The SCNintact and SCN-lesioned rats were each divided into two groups, one subjected to restricted-MAP drinking (R-MAP) and the other to R-Water. Among 30 SCN-intact rats, 15 rats were used for each experiment, six for the measurement of behavioral rhythms and nine for the measurement of Per2 expression rhythms in cultured brain tissues. Among 39 SCN-lesioned rats, 22 were used for R-MAP and 17 for R-Water experiments. Twelve rats in the R-MAP group and eight in the R-Water group were used for the measurement of behavioral rhythms, and 10 rats in the R-MAP group and nine in the R-Water group were used for the measurement of Per2 expression rhythms. Methamphetamine-HCl (Dainippon, Osaka, Japan) dissolved in drinking water at a concentration of 0.005% was administered to the R-MAP group daily from 10:00 to 14:00 h for 14 successive days. Plain water was supplied to the R-Water group from 10:00 to 14:00 h for 14 days. Food pellets were available all the time. Following the last MAP or water supply on the 14th day of the restricted schedule, MAP-containing water (0.005%) was given ad libitum to both the R-MAP and the R-Water group for 10 days (ad-MAP). For the measurement of Per2 expression rhythms, the brain was sampled on the 14th day of the restricted schedule at 15:00–18:00 h. The amount of water intake during the restricted time (10:00–14:00 h) as well as in the whole day was measured for 2 days immediately before the start of R-MAP or R-Water (pre-restriction; pre-R) and on all days of the restricted schedule. The amount of food intake in a day was measured for 2 days during pre-R and twice during the restricted schedule (days 3 and 4 and days 12 and 13). The body weight was measured on the day before the start of the restricted schedule and on the day of brain sampling. Measurement of bioluminescence rhythms Brains were removed immediately after decapitation and coronally sectioned (300 lm) using a microslicer (D.S.K, Kyoto, Japan). Four slices were obtained from one brain, one each including the SCN, olfactory bulb (OB), CPU/parietal cortex (PC) and substantia nigra (SN). These areas were dissected out in ice-cold Hanks’ balanced salt solution with a surgical knife under a stereoscopic microscope as described elsewhere (Natsubori et al., 2013a). A dissected tissue was placed on a membrane (Millicell-CM, Millipore, MA, USA; pore size 0.4 lm) in a 35-mm Petri dish, and cultured in air at 37 °C with 1300 lL of DMEM (Invitrogen, CA, USA) supplemented with: HEPES, 10 lM; NaHCO3, 2.7 mM; kanamycin (Gibco, NY, USA), 20 mg/L; apo-transferrin (Sigma, St. Louis, USA), 100 lg/mL; insulin (Sigma), 5 lg/mL; putrescine (Sigma), 100 lM; progesterone (Sigma), 20 nM; sodium selenite (Gibco), 30 nM; and D-Luciferin K salt (Dojindo, Kumamoto, Japan), 0.1 mM. Bioluminescence from each tissue was measured for 1 min at 10-min intervals for 5 successive days with a photomultiplier tube (Lumicycle, Actimetrics or Kronos, Atto, Tokyo, Japan). The discrete brain areas examined were major parts of the brain dopaminergic system. The brain tissue containing the SN included functionally different structures such as the SN pars compacta, SN

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 229–240

Dual regulation by MAO and SCN circadian pacemaker 231 pars reticulata and ventral tegmental area. They were not separated in the present study. Data analysis and statistics Twenty-four-hour profiles of spontaneous movement and wheelrunning activity in individual rats were analysed in 1-h bins. The individual profiles were averaged for 2 days immediately before the restricted schedule (pre-R) and for the last 2 days (days 12 and 13) of the schedule. When the day corresponded to the estrus in the sexual cycle, the result on that day was replaced by those from the immediately prior proestrus or diestrus day. The group data were obtained by averaging these individual profiles. Circadian rhythmicity in behavior and its period were evaluated by v2 periodogram analysis in the range 10–40 h with a significance level of 0.01 (CLOCKLAB software, Actimetrics, Evanston, IL, USA). Behavior records in 5-min bins were used for this analysis. The preR records of the last 7 days and ad-MAP records of the first 10 days were used for the analyses in the SCN-lesioned rats. The ad-MAP records of the first 5 days were used in the SCN-intact rats. The onset, offset and midpoint of activity bands of behavioral rhythms were determined by ClockLab. Time series of bioluminescence data were detrended using a 24-h moving average subtraction method (Yamanaka et al., 2008) and smoothed with a five-point moving average method. A circadian peak was identified when a peak–trough difference in a single circadian range was > 2 SD of the values contained in the range. The circadian peak that appeared within 48 h after the start of culturing was regarded as the first circadian peak. When no clear peak appeared, the data were excluded from further analyses. The amplitude of circadian Per2-dLuc rhythm was standardised by dividing the difference between the first peak and following trough values by the peak value, as the difference was strongly correlated with the peak value (r = 0.95, P = 8.7 9 10 40 and r = 0.76, P = 1.3 9 10 15 for SCN-intact and SCN-lesioned rats, respectively). The damping rate of circadian Per2-dLuc rhythm was calculated as follows: a difference between the onsets of first and fourth peak was divided by the amplitude of first peak. Repeated-measure ANOVA with a post hoc Fisher’s Protected Least Significant Difference (PLSD) test (Excel Statistics) was used to statistically evaluate differences in the 24-h behavior profile between pre-R and R-MAP or R-Water, and changes in the amounts of water and food intake and body weight. Unpaired t-tests were used to evaluate differences in the phases of behavioral rhythm between two groups. Two-factor factorial ANOVA with a post hoc Fisher’s PLSD test was used to evaluate differences in the circadian peak phase, amplitude and damping rate of Per2-dLuc rhythms between the SCN-intact and SCN-lesioned rats, and between R-MAP and RWater groups.

Results Behavioral rhythms under R-Water and R-MAP in SCN-intact and SCN-lesioned rats Twenty-four-hour profiles of spontaneous movement and wheel-running activity were substantially modified by R-MAP in SCN-intact rats (Figs 1 and 3). The behavioral activities during the restricted time of MAP supply were enhanced and the nocturnal activities were suppressed in some rats but this was not statistically significant in the group (Fig. 3). Under subsequent ad-MAP, the activity components at the restricted time of MAP supply showed rapid phase-

delay shifts for the following 5 days, but the phase shifts slowed down when the activity onsets passed the middle of the dark phase. On the other hand, behavioral activity was enhanced by R-Water immediately prior to daily water supply (Fig. 3). Under subsequent ad-MAP, the nocturnal activities were enhanced and slightly phasedelayed. Circadian behavioral rhythms were abolished by bilateral SCNlesion (Fig. 2). In the R-MAP group, the behavioral activities were significantly enhanced during the restricted time of MAP supply, but such enhancement was not observed in the R-Water group (Fig. 3). Small but significant pre-drinking activity bouts were detected on the last few days of R-MAP and R-Water (Figs 2 and 3). Under subsequent ad-MAP, the enhanced activities during the restricted time of MAP supply showed steady phase-delay shifts without interruption by LD, indicating free-running of MAO. On the other hand, ad-MAP enhanced and consolidated behavioral activities in the R-Water group immediately after the previous restricted time of water supply, to form behavioral rhythms with a period close to 24 h. The phases of behavioral rhythms on the first day of ad-MAP were analysed in terms of activity band (Fig. 4A). In the SCN-intact rats, the midpoints of the activity bands in the R-MAP and R-Water groups were located in the dark phase, but were significantly phaseadvanced in the R-MAP group compared to the R-Water group in both spontaneous activity (21.4 1.0 vs. 25.3 h 0.4 h, mean SEM; t10 = 3.80, P = 0.003) and in wheel-running (21.3 0.9 vs. 24.7 0.3 h; t9 = 3.37, P = 0.008). On the other hand, in the SCN-lesioned rats the midpoint was located around the transition from light to dark phase in both R-MAP and R-Water, and significantly phase-advanced in R-MAP compared to R-Water in both spontaneous activity (14.9 h 0.5 vs. 18.2 1.0 h; t17 = 3.20, P = 0.005) and wheel-running (14.7 0.6 vs. 17.8 1.0 h; t16 = 2.68, P = 0.016). When compared between the SCN-intact and SCN-lesioned rats, the activity band was significantly phaseadvanced in the SCN-lesioned rats in both R-MAP (t16 = 6.48, P = 7.5 9 10 6 and t16 = 5.94, P = 2.1 9 10 5, respectively) and R-Water group (t11 = 6.11, P = 7.6 9 10 5 and t9 = 6.22, P = 1.6 9 10 4, respectively). The phase-shifting rate of behavioral rhythm per day under ad-MAP was analysed for the first 5 or 10 days (Fig. 4B). In the SCN-intact rats, the phase-shifting rate of spontaneous activity and wheel-running under the first 5 days of adMAP were significantly faster in the R-MAP group (2.4 0.7 and 2.3 0.8 h, respectively) than in the R-Water group (0.2 0.1 and 0.3 0.5 h; t10 = 3.02, P = 0.013 and t9 = 2.62, P = 0.028, respectively). In the SCN-lesioned rats, the phase-shifting rate of spontaneous activity and wheel-running for the first 10 days was also significantly faster in the R-MAP group (1.3 0.2 h and 1.3 0.2 h, respectively) than in the R-Water group (0.2 0.2 h; 0.0 0.1 h; t17 = 3.33, P = 0.004; t16 = 3.56, P = 0.003). The free-running period of spontaneous activity rhythm in the SCNlesioned rats was 25.3 0.2 h in the R-MAP group and 24.2 0.2 h in the R-Water group. There was no difference in the phase-shifting rate between the SCN-intact and SCN-lesioned rats in either the R-MAP (t16 = 1.83, P = 0.087; t16 = 1.61, P = 0.13) or the R-Water (t11 = 0.06, P = 0.95 and t9 = 0.83, P = 0.43, respectively) group. Water and food intakes during R-MAP and ad-MAP Daily water intake during R-MAP was significantly decreased in both the SCN-intact and SCN-lesioned rats (effect of time, F2,60 = 250.38, P = 7.6 9 10 30) but not different between the two groups (interaction between time and SCN-lesion, F2,60 = 0.48, P = 0.62; main effect


232 A. Natsubori et al.

Fig. 1. Behavioral rhythms of the SCN-intact rats subjected to R-Water and R-MAP. Representative double-plotted actographs are shown of the SCN-intact rats subjected to R-Water (upper traces) and to R-MAP (lower traces), with spontaneous movement on the left and wheel-running activity on the right. Levels of behavioral activity are indicated by black columns. Red-shaded areas indicate the restricted time of MAP or water supply (10:00–14:00 h). Blue-shaded areas indicate the period of ad-MAP. The horizontal bar at the top of each panel represents the light cycle, the light phase with open bars and the dark phase with filled bars.

of SCN-lesion, F1,60 = 1.49, P = 0.23; Fig. 5A). Daily water intake during R-Water was significantly decreased in both the SCN-intact and the SCN-lesioned rats (effect of time, F2,42 = 38.56, P = 3.1 9 10 10) but not different between the two groups (interaction between time and SCN-lesion, F2,42 = 0.18, P = 0.83; main effect of SCN-lesion, F1,42 = 2.22, P = 0.15). When compared between the R-MAP and R-Water groups, daily water intake during the restricted schedule was significantly higher during R-Water than during R-MAP in both the SCN-intact (interaction between time and treatment, F2,48 = 0.37, P = 0.70; main effect of treatment, F1,48 = 5.68, P = 0.025) and SCN-lesioned rats (interaction between time and treatment, F2,54 = 0.33, P = 0.72; main effect of treatment, F1,54 = 9.36, P = 0.005). Daily food intake during R-MAP was significantly decreased in both the SCN-intact and SCN-lesioned rats (effect of time, F2,48 = 60.17, P = 8.4 9 10 14) but did not differ between the two groups (interaction between time and SCN-lesion, F2,48 = 0.18, P = 0.84; main effect of SCN-lesion, F1,48 = 0.87, P = 0.36; Fig. 5B). Daily food intake in the SCN-intact rats was slightly but significantly decreased during the early stage of R-Water (days 3 and 4: interaction between time and SCN-lesion, F2,30 = 10.22, P = 4.1 9 10 4; main effect of SCN-lesion, F1,30 = 0.73, P = 0.41; Fisher’s PLSD test, F5,45 = 3.29, P = 0.032), but recovered at the

late stage of the schedule (days 12 and 13). Daily food intake during R-Water was not changed in the SCN-lesioned rats. The body weight in the SCN-intact rats significantly decreased during R-MAP by 32.3 4.2 g and during R-Water by 15.9 3.0 g (interaction between time and treatment, F1,16 = 10.24, P = 0.006; main effect of treatment, F1,16 = 10.24, P = 0.006; Fisher’s PLSD test, F3,32 = 36.17, P = 1.2 9 10 4), and that in the SCN-lesioned rats decreased during R-MAP by 27.8 6.9 g while it increased during R-Water by 14.4 2.7 g (interaction between time and treatment, F1,17 = 29.74, P = 4.3 9 10 5; main effect of treatment, F1,17 = 29.74, P = 4.3 9 10 5; Fisher’s PLSD test, F3,34 = 21.18, P = 5.7 9 10 9). The amount of MAP intake was calculated from daily water intake. The daily mean of MAP intake during R-MAP was slightly but significantly larger in the SCN-intact (2.3 0.1 mg/kg body weight) than in the SCN-lesioned rats (2.0 0.1 mg/kg body weight; t35 = 2.36, P = 0.024). The daily mean of MAP intake during ad-MAP was not different in the R-MAP group between the SCN-intact (3.9 0.4 mg/kg body weight) and the SCN-lesioned (3.2 0.2 mg/kg body weight; t16 = 1.50, P = 0.15) rats, but was significantly different in the R-Water group between the SCN-intact (4.7 0.5 mg/kg body weight) and the SCN-lesioned (2.6 0.3 mg/kg body weight; t12 = 3.62, P = 0.004) rats.


Dual regulation by MAO and SCN circadian pacemaker 233

Fig. 2. Behavioral rhythms in the SCN-lesioned rats subjected to R-Water and R-MAP. Representative double-plotted actographs are shown of the SCNlesioned rats subjected to R-water (upper traces) and to R-MAP (lower traces). v2 periodograms for respective behavioral rhythms before the restricted-schedule (pre-R) and under ad-MAP are shown underneath each actograph. Arrows in the right margin of the actograph indicate the days used for periodogram analysis. An oblique line in a periodogram indicates a significance level (P = 0.01), and a number in a panel indicates the primary period. For the details, see the legend of Fig. 1.

Per2-dLuc rhythms in discrete brain areas In the SCN-intact rats, significant circadian rhythms in Per2-dLuc were observed in cultured brain slices of the SCN, OB, CPU, PC and SN in the R-MAP and R-Water groups (Fig. 6). The SCN and OB showed robust circadian Per2-dLuc rhythms with high amplitudes but those in

the OB were substantially damped within several cycles. On the other hand, the circadian rhythms in the CPU and PC were noisy and were damped within a few cycles. Most of the PC slices in the R-MAP group failed to show circadian rhythms (except for one slice) so they were excluded from the further analyses. In the SCN-lesioned rats, significant circadian rhythms in the OB, CPU, PC and SN were observed.


234 A. Natsubori et al. A

B

Fig. 3. Twenty-four hour profiles of behavior under R-Water and R-MAP. Twenty-four-hour profiles of behavior are shown of (A) SCN-intact and (B) SCNlesioned rats subjected to R-Water (upper) and to R-MAP (lower), respectively; spontaneous movement is on the left and wheel-running activity on the right. Behavior levels are expressed by the mean and SEM hourly activity counts in rats under pre-R (gray columns) and R-MAP or R-Water (black columns). Shaded areas indicate the time of restricted MAP or water supply. The horizontal bars at the top of each panel represent the light cycle (open, light; filled, dark). The number of rats examined was as follows: the SCN-intact rats, R-Water, 6; R-MAP, 6; and in SCN-lesioned rats, R-Water, 8; R-MAP, 12. *P < 0.05, **P < 0.01, pre-R vs. R-MAP or R-Water (repeated-measure ANOVA with post hoc Fisher’s PLSD test).

The mean as well as individual phases of first circadian peak were plotted against time of day (Fig. 7). In the SCN-intact rats, the peak phase in the SCN did not differ between the R-MAP and R-Water groups (Fig. 7B). The peak phases in three brain areas (OB, CPU and SN) differed slightly but significantly between the R-MAP and R-Water groups (interaction between brain area and treatment, F2,44 = 0.72, P = 0.49; main effect of treatment, F1,44 = 7.53, P = 0.009). In the SCN-lesioned rats, the peak phases in four brain areas (OB, CPU, PC and SN) were significantly different between the R-MAP and R-Water groups (interaction between brain area and treatment, F3,60 = 6.35, P = 8.3 9 10 4; main effect of treatment, F1,60 = 4.65, P = 0.035; Fig. 7C). A significant difference was revealed in the CPU and SN by a post hoc Fisher’s PLSD test (F7,60 = 8.05, P = 0.003 for CPU; P = 0.003 for SN). When compared between the SCN-intact and SCN-lesioned rats (Fig. 7D), the peak phases in the three brain areas (OB, CPU and SN) were significantly different under R-MAP (interaction between brain area

and SCN-lesion, F2,46 = 15.14, P = 8.9 9 10 6; main effect of SCN-lesion, F1,46 = 26.73, P = 5.0 9 10 6). A post hoc Fisher’s PLSD test revealed a significant difference in the OB and SN (F5,46 = 12.26, P = 0.013 for OB; P = 8.0 9 10 9 for SN). Under R-Water (Fig. 7E), the peak phases in the four brain areas examined were significantly different between the SCN-intact and SCN-lesioned rats (interaction between brain area and SCN-lesion, F3,55 = 2.98, P = 0.039; main effect of SCN-lesion, F1,55 = 23.59, P = 1.0 9 10 5). A significant difference was revealed in the CPU and PC by a post hoc Fisher’s PLSD test (F7,55 = 12.99, P = 4.2 9 10 5 for CPU; P = 0.010 for PC). The amplitude of first circadian peak in the SCN-intact rats (Fig. 8A) differed significantly among the four brain areas (effect of brain area, F3,60 = 54.19, P = 4.5 9 10 17) but not between the R-MAP and R-Water groups (interaction between brain area and treatment, F3,60 = 0.70, P = 0.56; main effect of treatment, F1,60 = 1.15, P = 0.29). The amplitude in the SCN-lesioned rats differed signifi-


Dual regulation by MAO and SCN circadian pacemaker 235 A

B

Fig. 4. Phase position of behavioral rhythms and daily phase-shifts under ad-MAP. (A) Phase position of activity band in (left) spontaneous movement and (right) wheel-running activity are shown in the SCN-intact (upper) and SCN-lesioned (lower) rats. The activity band was measured on the first day of ad-MAP (open, R-Water; filled, R-MAP). The horizontal bars at the tops of panels represent the light cycle (open, light; filled, dark). The activity onset and offset are expressed as mean SEM. Significant differences in the midpoint of the activity band are expressed as follows: *P < 0.05, **P < 0.01 for R-Water vs. RMAP and ††P < 0.01 for SCN-intact vs. SCN-lesioned (unpaired t-test) rats. (B) The rates of phase-shift of behavioral rhythm in the first 5 days under ad-MAP are shown for the SCN-intact rats (upper) and those in the first 10 days for the SCN-lesioned rats (lower) (open, R-Water; filled, R-MAP). The number of rats examined was as follows: for the SCN-intact, R-Water, 6; R-MAP, 6; and for SCN-lesioned, R-Water, 7; R-MAP, 12 in spontaneous movement. In wheel-running activity, SCN-intact, R-Water, 5; R-MAP, 6; and SCN-lesioned, R-Water, 6; R-MAP, 12. Values are expressed as mean SEM. *P < 0.05, **P < 0.01, R-Water vs. R-MAP (unpaired t-test).

cantly among the four brain areas (effect of brain area, F3,61 = 17.81, P = 2.0 9 10 8; interaction between brain area and treatment, F3,61 = 3.43, P = 0.023; main effect of treatment, F1,61 = 3.99, P = 0.050). A post hoc Fisher’s PLSD test revealed a significant difference between the R-MAP and R-Water groups in the OB and PC (F7,61 = 9.67, P = 0.006 for OB; P = 0.031 for PC). When compared between the SCN-intact and SCN-lesioned rats, the amplitudes did not differ in the R-MAP group (interaction between brain area and SCN-lesion, F2,46 = 1.33, P = 0.28; main effect of SCN-lesion, F1,46 = 2.54, P = 0.12) but did significantly differ in the R-Water group (interaction between brain area and SCN-lesion, F3,55 = 15.86, P = 1.5 9 10 7; main effect of SCN-lesion, F1,55 = 14.00, P = 4.4 9 10 4). A post hoc Fisher’s PLSD test revealed a significant decrease in the OB (F7,55 = 22.20, P = 5.2 9 10 10). The damping rate of Per2-dLuc rhythms did not significantly differ among the brain areas in both the SCN-intact (effect of brain area, F3,60 = 7.05, P = 3.9 9 10 4) and SCN-lesioned (effect of brain area, F3,61 = 2.50, P = 0.068) rats, and they did not differ between the R-MAP and R-Water groups in either SCN-intact rats (interaction between brain area and treatment, F3,60 = 0.91, P = 0.44; main effect of treatment, F1,60 = 3.3 9 10 4, P = 0.99) or SCN-lesioned rats (interaction between brain area and treatment, F2,46 = 0.22, P = 0.81; main effect of treatment, F1,46 = 0.21, P = 0.65 for SCN-lesion; Fig. 8B). When compared between the SCN-intact and SCN-lesioned rats, the damping rates did not differ in either the R-MAP group (interaction between brain area and SCN-lesion, F2,46 = 0.22, P = 0.81; main effect of SCN-lesion, F1,46 = 0.21, P = 0.65) or the R-Water group (interaction between brain area and SCN-lesion, F3,55 = 1.92, P = 0.14; main effect of SCN-lesion, F1,55 = 0.95, P = 0.33).

The numbers of slices examined were as follows: (i) in the SCNintact rats: SCN, R-Water, 9; R-MAP, 9; OB, R-Water, 9; R-MAP, 9; CPU, R-Water, 7; R-MAP, 8; PC, R-Water, 5; R-MAP, 1; and SN, R-Water, 9; R-MAP, 8, and (ii) in the SCN-lesioned rats: OB, R-Water, 9; R-MAP, 10; CPU, R-Water, 8; R-MAP, 9; PC, R-Water, 8; R-MAP, 8; and SN, R-Water, 9; R-MAP, 8.

Discussion The present study clearly demonstrates that restricted MAP drinking at a restricted time of day not only induced MAO in behavior but also entrained it. The free-running of MAO under ad-MAP was modified by the SCN circadian pacemaker entraining to LD. MAO was also expressed in the circadian Per2 rhythms in several extraSCN brain areas. The Per2 rhythms were phase-shifted by R-MAP. The phase shifts were accelerated by the SCN lesion, especially in the OB and SN, indicating dual regulation of the extra-SCN circadian oscillators in the brain by the SCN and MAO. In the absence of the SCN circadian pacemaker, R-Water also induced circadian oscillation which was not identical with MAO. The oscillatory mechanism underlying MAP-induced behavioral rhythm (i.e., MAO) is suggested as consisting of several extra-SCN oscillators in the brain (Masubuchi et al., 2000) but the exact mechanism is not well understood. A success of ex vivo analysis of MAO (Natsubori et al., 2013a,b) opened a new experimental approach to this issue, and the fixation of the MAO phase by R-MAP in the present study enabled us to analyse the phase relationships among extra-SCN oscillators in the brain more precisely. The induction of MAO by R-MAP was revealed by subsequent ad-MAP, where the



B

Fig. 5. Amounts of water and food intake. (A) Daily amounts of water intake are shown for the SCN-intact (open bars) and SCN-lesioned (filled bars) rats subjected to R-MAP (left) and R-Water (right). Gray columns superimposed at the bottom of pre-R columns indicate the amounts of water intake from 10:00 to 14:00 h, the time of restricted MAP or water supply. The number of rats examined was as follows: R-Water, SCN-intact, 13; SCN-lesioned 10; and R-MAP, SCN-intact, 13; SCN-lesioned, 19. (B) Daily amounts of food intake are shown of the SCN-intact (open bars) and SCN-lesioned (filled bars) rats subjected to R-MAP (left) and R-Water (right). The number of rats examined was as follows: R-Water, SCN-intact, 8; SCN-lesioned, 9; and R-MAP, SCN-intact, 8; SCNlesioned, 18. Values are expressed as mean + SEM. *P < 0.05 (repeated-measure ANOVA with post hoc Fisher’s PLSD test).

enhanced behavior components at the time of restricted MAP supply showed phase-delay shifts with a period > 24 h. Acceleration and deceleration of phase-delay shifts in MAP-induced behavioral rhythm were observed in the SCN-intact rats but not in the rats with bilateral SCN lesions (Figs 1 and 2). The rate of phase-delay shifts in the SCN-lesioned rats was 1.3 h/day on average and corresponded to a free-running period of 25.3 h. The above-mentioned findings indicate that the modification of phase-delay shifts of MAP-induced behavioral rhythm (or MAO) is due to the SCN circadian pacemaker entrained by LD and depends on the phase relation between the two oscillations. A similar modification has previously been reported in MAP-induced behavioral rhythms under ad lib MAP drinking (Natsubori et al., 2013b). The phase shift was decelerated when the MAP-induced behavioral rhythm was located outside the subjective night and accelerated when it was inside. The phenomenon is called relative coordination and is taken as evidence for two interacting oscillators with different periods (Aschoff, 1965). In this respect, it is of interest to note that in the SCN-intact rats circadian Per2 rhythms in extra-SCN brain areas were only slightly phase-shifted by R-MAP in the present study (Fig. 7B) whereas the circadian rhythms in some brain areas were markedly phase-shifted by ad lib MAP in the previous studies (Masubuchi et al., 2000; Natsubori et al., 2013b). These seemingly inconsistent results could be explained by the phase relation between the SCN circadian pacemaker and MAO. In the previous studies, MAP-induced behavioral rhythms were 180° out of the subjective

night, which might reduce the influence of the SCN circadian pacemaker on MAO. On the other hand, the activity band of MAPinduced behavioral rhythm in the present study was located close to the subjective night (Fig. 4A), and therefore the influence of the SCN circadian pacemaker would be large. R-MAP-induced phase shifts of Per2 rhythms depended on the brain areas examined and also on the presence or absence of the SCN circadian pacemaker (Fig. 7D). R-MAP did not affect the circadian oscillation in the SCN at all. The phase shifts in the OB and SN were significantly larger in the absence of the SCN than in the presence. The findings indicate that the SCN circadian pacemaker exerted a strong influence on these extra-SCN oscillations even in the presence of MAO. The extent of influence was different among the extra-SCN oscillations, the largest being on the SN oscillation and the smallest on the CPU, of those regions so far examined. Several important insights into the oscillation mechanism of MAO are provided by these findings. Firstly, the extra-SCN oscillations in certain brain areas such as OB, PC and SN are regulated by both the SCN circadian pacemaker and MAO. Many brain areas exhibit independent circadian oscillations which are usually under the control of the SCN circadian pacemaker (Abe et al., 2002; Abraham et al., 2005), and not all of them are affected by MAP (Masubuchi et al., 2000). Secondly, effects of MAP on the extra-SCN oscillations are different depending on the brain areas. The influence is large in the OB and SN and small in the CPU, and this is also supported by previous results (Natsubori et al., 2013b). In addition, the direction of


Dual regulation by MAO and SCN circadian pacemaker 237

Fig. 6. Circadian Per2-dLuc rhythms in discrete brain areas. Representative circadian Per2-dLuc rhythms in the SCN, OB, CPU, PC and SN are illustrated in the SCN-intact (far left, left) and SCN-lesioned (right, far right) rats subjected to R-Water and R-MAP, respectively. The circadian rhythms were detrended by the 24-h moving-average subtraction method. They were not collected from the same animals. The ordinates indicate relative light units (RLU) of bioluminescence. The abscissae indicate culture days. Vertical solid and broken lines in each panel indicate 06:00 and 18:00 h in local time, respectively. Open and filled horizontal bars at the top on day 0 in each panel represent the light cycle on the day of the slice preparation (open, light; filled, dark).

phase shift of extra-SCN oscillation is different depending on the brain areas. Without the SCN circadian pacemaker, the circadian oscillation in the OB was phase-advanced whereas that in the SN was phase-delayed, again confirming previous results (Natsubori et al., 2013b). Finally, the phase shifts of extra-SCN oscillators in the OB and SN but not in the CPU were accelerated by the SCN lesion in parallel with the phase shift of the activity band of the MAP-induced behavioral rhythm. Although the circadian rhythm in the CPU was not significantly phase-shifted by R-MAP as compared with that by R-Water, this does not necessarily indicate that MAP did not affect the circadian oscillator in this structure. As R-Water affected the circadian oscillation in the CPU in the absence of the SCN, R-Water might be inappropriate as a control for R-MAP. When compared with the circadian phases under ad lib feeding and drinking (Natsubori et al., 2013a), a small but statistically significant phase-advance was detected in the CPU by R-MAP. Thus, R-MAP could also influence the circadian oscillation in the CPU. The above considerations lead us to the hypothesis that MAO is a complex or population oscillator consisting of multiple extra-SCN

circadian oscillators (Fig. 9). Chronic MAP treatment reorganises the networks of these extra-SCN oscillators to build-up MAO. The circadian oscillators in the OB, PC, SN and probably CPU are important components but the involvement of these in other parts of the brain is not excluded in MAO (Model 1). The structures examined in the present study are the major components of the brain dopaminergic system, and it is highly possible that these circadian oscillators in some of these structures are directly affected by MAP treatment, as MAP is an antagonist of the dopamine transporter and activates the dopaminergic system in the brain. Alternatively, the extra-SCN circadian oscillators in the OB and SN are not components of MAO but slave oscillators located downstream of MAO (Model 2). MAO is located somewhere else. This alternative is less probable because the extent and direction of phase shifts by R-MAP were different among the extra-SCN brain oscillators. Feedback effects from behavior on phasing of the extra-SCN oscillators are possible but also less likely, because the phase responses were different depending on the area examined and the treatment given (Natsubori et al., 2013a) even though MAP-induced behavior enhancement was not much different among them.



B

C

D

E

Fig. 7. Peak phases of circadian Per2-dLuc rhythms in the first cycle of culture. The mean (B–E) as well as individual (A) circadian peak phases in the first cycle of culture are plotted against time of day in the SCN, OB, CPU, PC and SN obtained from the SCN-intact and SCN-lesioned rats. Open and filled black circles indicate the results of R-Water and R-MAP, respectively, in the SCN-intact rats. Open and filled red circles indicate the results of R-Water and R-MAP, respectively, in the SCN-lesioned rats. Comparisons between R-Water and R-MAP in SCN-intact (B) and SCN-lesioned (C) rats, and between the SCN-intact and SCN-lesioned rats under R-MAP (D) and R-Water (E) are illustrated. Horizontal bars at the bottom of panels B–E indicate the activity bands of spontaneous movement rhythms on the first day of ad-MAP (open and filled black bars, R-Water and R-MAP, respectively, in SCN-intact rats; open and filled red bars, R-Water and R-MAP, respectively, in SCN-lesioned rats; see Fig. 4). Shaded area indicates the time of restricted MAP or water supply. Open and filled horizontal bars at the top of each panel represent the light cycle (open, light; filled, dark). The numbers in parentheses in the right margin of panel A indicate the numbers of slices examined (R-Water : R-MAP). Values are expressed as mean SEM. *P < 0.05, **P < 0.01 (two-factor factorial ANOVA with post hoc Fisher’s PLSD test). The data in the PC of SCN-intact rat lacks SEM because there was only a single result.

On the other hand, ad-MAP revealed behavioral rhythms in the R-Water group when the bilateral SCN was lesioned. The behavioral rhythms started to free-run from the phase immediately after the daily water supply (Fig. 2), indicating that R-Water induced behavioral rhythms in the absence of the SCN circadian pacemaker. The free-running period was close to 24 h and significantly different from that of R-MAP-induced behavioral rhythm (Fig. 4B). The period was rather similar to FEO (Yoshihara et al., 1997). We tentatively call the R-Water-induced circadian oscillation the water-entra-

inable oscillator (WEO). WEO was accompanied by pre-drinking (anticipatory) activity prior to R-Water (Fig. 3B). In the absence of the SCN circadian pacemaker, the circadian Per2 rhythms in the CPU and PC were significantly phase-shifted by R-Water (Fig. 7E). In addition, the circadian rhythms in the CPU and SN were differentially shifted by R-MAP and R-Water (Fig. 7C). These findings suggest that MAO and WEO consist of different extra-SCN circadian oscillators in the brain. The finding may explain the different periods of behavioral rhythms induced by


Dual regulation by MAO and SCN circadian pacemaker 239 A

B

Fig. 8. Amplitudes of the first peak and damping rates of circadian Per2-dLuc rhythms. (A) Standardised amplitudes of circadian Per2-dLuc rhythms are shown in the SCN, OB, CPU, PC and SN obtained from the SCN-intact (left) and SCN-lesioned (right) rats. (B) The damping rates of circadian Per2-dLuc rhythms are shown in the SCN, OB, CPU, PC and SN obtained from the SCN-intact (left) and SCN-lesioned (right) rats. Open and filled columns indicate the results of R-Water and R-MAP, respectively. Values are expressed as mean + SEM. The numbers in parentheses in each column of panel A indicates the number of animals analysed. *P < 0.05, **P < 0.01 (two-factor factorial ANOVA with post hoc Fisher’s PLSD test). The data in the PC of SCN-intact rat lacks SEM because there was only a single result.

Fig. 9. Two alternative models for the relationship among MAO, the SCN circadian pacemaker and extra-SCN oscillators in discrete brain areas. Model 1 (left): R-MAP re-organises several extra-SCN circadian oscillators in discrete brain areas including the OB, PC, SN and possibly CPU to build up MAO. The SCN circadian pacemaker influences MAO through constituent extra-SCN oscillators. Model 2 (right): R-MAP induces MAO in somewhere other than these brain areas examined. Extra-SCN circadian oscillators in these brain areas are not the components of MAO, but are regulated by MAO and the SCN circadian pacemaker simultaneously.

R-MAP and R-Water. R-Water has been reported to induce the anticipatory activity immediately prior to the time of restricted water intake (Johnson & Levine, 1973; Dhume & Gogate, 1982). The effect of R-Water was interpreted as a secondary effect of the food

restriction which was accompanied by R-Water (Mistlberger & Rechtschaffen, 1985; Honma et al., 1986a). However, the present results do not support this interpretation because food intake was not decreased by R-Water in the SCN-lesioned rats (Fig. 5B), and


240 A. Natsubori et al. WEO phase-shifted the extra-SCN circadian oscillators differently from the food-entrainable circadian oscillator (FEO; Natsubori et al., 2013a). WEO and FEO may be different oscillators. In conclusion, MAO is induced and phase-set by restricted MAP supply at a fixed time of day in rats. The circadian rhythms in Per2 expression in discrete brain areas as well as in behavior receive dual regulation by the SCN circadian pacemaker and MAO. Restricted water supply at a fixed time of day induced a circadian oscillation which was not identical either with MAO or with FEO.

Acknowledgements We are grateful to Dr S. Hashimoto (Astellas Pharma, Inc.) and Professor Y. Shigeyoshi (Kinki University) for the supply of Per2-dLuc-transgenic rats. This study was financially supported by the Strategic Research Program for Brain Sciences (SRPBS) to K.H. and S.H. and a Grant-in Aid for Science from the MEXT (No. 20249010 to K.H.).

Abbreviations ad-MAP, ad libitum MAP drinking; CPU, caudate–putamen; FEO, foodentrainable oscillatior; Fisher’s PLSD test, Fisher’s Protected Least Significant Difference test; LD, light–dark cycles; MAP, methamphetamine; MAO, MAP-induced oscillator; OB, olfactory bulb; PC, parietal cortex; Per2-dLuc, Period2-dLuciferase; pre-R, pre-restriction; RF, restricted daily feeding; R-MAP, restricted-MAP drinking; R-Water, restricted water supply; SCN, suprachiasmatic nucleus; SN, substantia nigra; WEO, water-entrainable oscillator

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