Introduction

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A core clock mechanism in the mouse suprachiasmatic nucleus (SCN) seems to involve a transcriptional feedback loop in which CLOCK and BMAL1 function as positive regulators and the three mPeriod (mPer) genes, Per1 (Sun et al., 1997; Tei et al., 1997), Per2 (Albrecht et al., 1997; Shearman et al., 1997; Takumi et al., 1998), and Per3 (Zylka et al., 1998) are involved in negative feedback (Dunlap, 1999). In addition, it was determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop (Kume et al., 1999). It is already well known that the SCN contains a master pacemaker that regulates behavioral and physiological circadian rhythms such as locomotor activity, body temperature, and endocrine release (Inouye and Shibata, 1994). Interestingly, expression of the Per gene occurs not only in the SCN but also in other brain areas such as the cerebral cortex, caudate/putamen (CPu), and cerebellum (Albrecht et al., 1997; Shearman et al., 1997). However, the function of clock genes outside of the SCN has not been fully elucidated.

Destruction of the SCN abolishes the circadian rhythms of many physiological functions (Inouye and Shibata, 1994). On the other hand, there are at least two oscillators outside the SCN: a food-associated oscillation entrained by daily restricted feeding (Mistlberger, 1994) and methamphetamine (MAP)-induced oscillation produced by its daily injection (Shibata et al., 1994, 1995). In addition, oral administration of MAP through drinking bottle initiates a circadian rhythm with a long free-running period even after SCN ablation (Honma et al., 1987). Based on these facts, it has been suggested that other circadian oscillators such as the MAP-induced rhythm exist in areas other than the SCN. Thus, it is possible that mPer mRNA outside of the SCN regulates the SCN-independent circadian rhythm, and rapid induction of mPer outside of the SCN by MAP may entrain the SCN-independent oscillation.

Recently it was reported that sensitization to repeated cocaine exposure, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for period, clock, cycle, and doubletime, but not in flies mutant for timeless (Andretic et al., 1999) We demonstrated that the MAP-induced free-running oscillation of rat locomotion with drinking application of MAP exhibits a sensitization phenomenon (Nikaido et al., 1999). Therefore, the next progressive step was to examine whether MAP induces Per expression in the CPu, and whether sensitization is involved in MAP-induced Per expression but not timeless expression.

Treatment with MAP is known to increase locomotor activity and Fos expression in the CPu (Graybiel et al., 1990). Pharmacological studies have further revealed that both MAP-induced hyperlocomotion and Fos expression in the CPu are attenuated by pretreatment with dopamine D1, D2, or NMDA receptor antagonists (Ujike et al., 1989; Kuribara and Uchihashi, 1993; Kuribara, 1994, 1995, 1996; Yoshida et al., 1995). Thus, it has been suggested that D1, D2, and NMDA receptors play an important role in the sensitization induced by repeated injection of MAP. This evidence suggests that MAP-induced Per expression may be involved in the activation of both dopamine and NMDA receptors. Therefore, in the first part of our present experiment, we examined the pharmacological characteristics of MAP-induced Per gene expression in the mouse CPu. Then, in the latter part, we examined the expression pattern of Per and timeless mRNA using animals sensitized to MAP.

	Materials and Methods

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Animals. In all experiments, we used 4- to 6-week-old male ddY mice (Takasugi, Saitama, Japan) maintained under a 12 h:12 h light/dark cycle. All animals were allowed free access to food and water and were treated in accordance with the Law no. 105 and Notification no. 6 of the Japanese Government.

Locomotor Activity Measurement. For assessment of the locomotor activity, mice were housed individually in transparent plastic cages (31 × 20 × 13 cm). Motor activity was measured using an infrared area sensor (F5B; Omron, Tokyo, Japan), and the activity count (number of movements) was recorded by computer and stored on disk at 5-min intervals.

Sample Preparation. Mice were deeply anesthetized with ether and intracardially perfused with 0.1 M phosphate buffer (PB), pH 7.4, containing 4% paraformaldehyde (PFA). Brains were removed, postfixed in 0.1 M PB containing 4% PFA for 24 h at 4°C, and transferred into 20% sucrose in PB for 72 h at 4°C. Brain slices (40 µm thick) including the CPu, accumbens, piriform cortex, and SCN were made using a cryostat (HM505E; Microm, Walldorf, Germany) and placed in 2× standard saline citrate until processing for hybridization.

In Situ Hybridization. The quantity of mPer1, mPer2, mPer3, or mTim mRNA expression in the various brain areas was studied by means of in situ hybridization. Slices were treated with 1 µg/ml proteinase K in 10 mM Tris-HCl buffer, pH 7.5, containing 10 mM EDTA for 10 min at 37°C followed by 0.25% acetic anhydride in 0.1 M triethanolamine and 0.9% NaCl for 10 min. The slices were then incubated in the hybridization buffer [60% formamide, 10% dextran sulfate, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.6 M NaCl, 1× Denhardt's solution (0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin), 0.2 mg/ml transfer RNA, 0.25% sodium dodecyl sulfate] containing ³³P-labeled cRNA probes for 16 h at 60°C. Radioisotope ([-³³P]UTP)-labeled antisense cRNA probes (PerkinElmer Life Sciences, Boston, MA) were made from restriction enzyme-linearized cDNA templates [nucleotide positions: mPer1 (538-1752), mPer2 (1-638), mPer3 (814-1955), mTim (236-909)] kindly provided by Dr. Okamura (Kobe University, Kobe, Japan). After a high-stringency posthybridization wash in 2× standard saline citrate/50% formamide, slices were treated with RNaseA (10 µg/ml) for 30 min at 37°C.

Immunohistochemistry of Fos Protein and Emulsion Autoradiography of mPer1. The slices were fixed with 4% PFA and processed for immunohistochemistry according to the avidin-biotin-peroxidase complex method. Primary antibody (anti-Fos, 1:5000; Cambridge Research Biochemical, Northwich, UK) was diluted in 0.1 M phosphate buffer containing 1% normal goat serum in 0.3% Triton X-100.

Drugs and Application Schedule. The drugs used in this experiment consisted of methamphetamine HCl (Dainippon Co., Tokyo, Japan); SCH23390 (Funakoshi, Tokyo, Japan); (+)-sulpiride (Sigma, St. Louis, MO); and MK-801, SKF38393, and quinpirole (RBI/Sigma, Natick, MA). All drugs were dissolved into the physiological saline. Drugs were injected during the daytime because spontaneous locomotor activity and mPer1 expression in the CPu were low at this time (data not shown). To examine the blocking effect of receptor antagonists on MAP-induced mPer1 expression, these receptor antagonists were injected 15 min before MAP injection.

Statistics. Results are expressed as the mean ± S.E.M. The significance of differences between groups was determined by two-way or one-way analysis of variance followed by Dunnett's test or Student's t test.

	Results

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Methamphetamine-Induced mPer1 Expression in the Various Brain Areas. It is well known that dopaminergic neurons innervate the CPu, accumbens, and piriform cortex. Therefore, we examined the amount of mPer1 expression in these brain areas. Figure 1A shows the representative brain areas responding to MAP and the sampling area for each brain slice. Basal level mPer1 expression was high in the piriform cortex but low in the CPu and accumbens (Fig. 1, A and B). Unrelated to basal expression, MAP significantly induced mPer1 expression in the CPu (P < 0.05, Student's t test) and piriform cortex (P < 0.05, Student's t test) but not in the accumbens (Fig. 1, A and B). Previous papers demonstrated a strong induction of Fos protein in the dorsal CPu (Yoshida et al., 1995); therefore, we examined the Fos expression and mPer1 induction using CPu slices. Interestingly, mPer1 mRNA and Fos immunoreactivity were coexpressed in the same striatal cells (Fig. 1C).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effect of 5 mg/kg of MAP on mPer1 expression in CPu, accumbens (NAcc), and piriform cortex (PC). Mice were decapitated 60 min after MAP injection. A, representative in situ hybridization autoradiograms of mPer1 on X-ray film. The brain areas surrounded by a broken line exhibit the NAcc, CPu, and PC areas. B, radioisotope in situ hybridization was performed for quantitative analysis purposes. Numbers in parentheses indicate the number of animals (*P < 0.05 in comparison with saline by Student's t test). C, emulsion autoradiograms of mPer1 in the dorsal caudate. A 5-mg/kg injection of MAP increased mPer1 expression (black dots, middle panel), and there were many dots on cells showing the Fos immunoreactivity (right panel). Fos immunoreactive cells were stained by a brown color.

Time Course of MAP-Induced Locomotion and mPer1 Expression. A 5-mg/kg injection of MAP significantly increased the locomotion, which was maintained for 4 h (Fig. 2A). Two-way analysis of variance revealed an interaction between drug treatment and time course in CPu mPer1 [F(3,23) = 13.1, P < 0.01]. Post hoc Dunnett's test demonstrated that the same dose of MAP increased mPer1 expression in the CPu 1 h (P < 0.05) and 2 h (P < 0.05) after MAP injection, but not 4 h after injection. On the other hand, MAP did not increase mPer2 expression in the CPu. In the SCN, MAP did not affect the expression of mPer1 or mPer2 at any time point after MAP injection. The increased basal level of mPer1 and mPer2 in the CPu and mPer2 in the SCN may reflect a circadian change in expression of these genes (Fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Time course of MAP-induced locomotor activity (A) and mPer expression in the CPu or SCN (B). A, MAP increased mouse locomotion for 4 h. , saline injection (n = 4); , 5 mg/kg MAP injection (n = 4) (*P < 0.05 in comparison with saline by Student's t test). B, relative value of mPer1 and mPer2 mRNA expression in the CPu. , saline injection; , 5 mg/kg MAP injection. Per expression observed in the saline group immediately after saline injection was set as 100%. Three to four animals made up each point (*P < 0.05 in comparison with saline by Student's t test; #P < 0.05, ##P < 0.01 in comparison with 0-min point by Dunnett's test).

Dose-Response Curve for MAP-Induced Locomotion and mPer mRNA. At a dose of 0.5 mg/kg, MAP did not change the locomotion or mPer expression in the CPu (Fig. 3). Two milligrams per kilogram was a sufficient dose to produce significant increase in locomotion (P < 0.05, Dunnett's test) and also mPer1 expression in the CPu [F(3,8) = 29.9, P < 0.01; P < 0.05, Dunnett's test]. In this experiment, MAP at any dose did not affect the expression of mPer2 [F(3,8) = 0.6, P > 0.05] or mPer3 [F(3,8) = 0.5, P > 0.05] in the CPu.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Dose-response of MAP-induced locomotor activity (A) and mPer expression in the CPu (B). A, vertical values exhibit total locomotor counts 3 h after MAP injection. Numbers in parentheses indicate the number of animals (*P < 0.05 in comparison with saline by Dunnett's test). B, relative value of mPer1, mPer2, and mPer3 mRNA expression in the CPu. Per expression observed in the saline group was set as 100%. Three animals made up each point (*P < 0.05 in comparison with saline by Dunnett's test).

Effect of DA and NMDA Receptor Antagonists on MAP-Induced Locomotion and mPer1 Expression in the CPu. Next, we examined the pharmacological profile of MAP-induced mPer1 expression in the CPu. MAP (5 mg/kg)-induced hyperlocomotion was significantly blocked by SCH23390 (P < 0.01, Dunnett's test) and sulpiride (P < 0.01) but not by MK-801 (P > 0.05) (Fig. 4A). Treatment with MAP produced a strong increase in the expression of mPer1 in the CPu (Fig. 4, B and C) that was completely attenuated by SCH23390 (P < 0.01, Dunnett's test), moderately attenuated by MK-801 (P < 0.01), and unaffected by sulpiride (P > 0.05) (Fig. 4, B and C). The basal level of mPer1 in the CPu was also significantly reduced by SCH23390 (P < 0.05, Dunnett's test) and MK-801 (P < 0.01) but increased by sulpiride (P < 0.05) (Fig. 4C).

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Effects of D1, D2, and NMDA receptor antagonists on MAP-induced locomotor activity (A) and mPer1 expression in the CPu (B and C). A, vertical values exhibit total locomotor counts 3 h after saline or MAP injection. Antagonists were injected 15 min before the 5-mg/kg MAP injection. Numbers in parentheses indicate the number of animals (**P < 0.01 in comparison with saline by Student's t test; ##P < 0.01 in comparison with MAP injection only by Dunnett's test). B, representative in situ hybridization autoradiograms of mPer1 were developed on X-ray film. The D1 receptor antagonist (SCH) and NMDA receptor antagonist (MK-801), but not the D2 receptor antagonist (SUL), antagonized MAP-induced mPer1 expression. Calibration on the saline panel is 1 mm. C, relative value of mPer1 mRNA expression in the CPu. Per expression observed in the saline group was set as 100%. Antagonists were injected 15 min before the 5-mg/kg MAP injection, and then mice were decapitated 60 min after MAP injection. Numbers in parentheses indicate the number of animals (**P < 0.01 in comparison with saline by Student's t test; ##P < 0.01 in comparison with MAP injection only; $P < 0.05, $$P < 0.01 in comparison with saline by Dunnett's test). SCH, SCH23390; SUL, sulpiride.

Effect of D1 and D2 Receptor Agonist on mPer Expression in the CPu. Because MAP-induced mPer1 expression in the CPu was attenuated by the D1 receptor antagonist and facilitated by the D2 receptor antagonist, we examined the effect of D1 and D2 receptor agonists on the basal level of mPer1 expression. Application of D1 receptor agonist SKF38393 did not affect mPer1 and mPer2 expression in the CPu (Fig. 5), whereas the D2 receptor agonist, quinpirole, slightly reduced mPer1 and mPer2 expression (Fig. 5).

View larger version (79K): [in this window] [in a new window]   Fig. 5.   Effect of the D1 (SKF38393) and D2 (quinpirole) receptor agonists on mPer1 and mPer2 expression in the CPu. Mice were decapitated 60 min after each agonist injection. Columns represent the relative value of mPer1 mRNA expression in the CPu. Per expression observed in the saline group was set as 100%. Numbers in parentheses indicate the number of animals.

Sensitized Expression of Locomotion and mPer mRNA in the CPu with Repeated Injection of MAP. In preparing the four experimental groups, MAP or saline was injected initially into all mice, then half of the MAP- (5 mg/kg) or saline-injected groups received another injection of MAP (0.5 mg/kg) or saline. Figure 6, A and B demonstrates the time course of locomotion (Fig. 6A) after injection of saline or MAP (0.5 mg/kg) and total locomotor counts (Fig. 6B) 60 min after injection, respectively. Small doses of MAP at 0.5 mg/kg did not increase locomotion in the saline-pretreated group but significantly increased locomotion in MAP (5 mg/kg)-pretreated mice [F(3,12) = 6.9, P < 0.01] (Fig. 6B).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Sensitization of locomotor activity induced by repeated application of MAP. A, time course of locomotor activity. M-M, MAP (5 mg/kg) injection followed by MAP (0.5 mg/kg) injection with 7-day withdrawal; S-M, saline injection followed by MAP (0.5 mg/kg) injection with 7-day interval; M-S, MAP (5 mg/kg) injection followed by saline injection with 7-day withdrawal; S-S, saline injection followed by saline injection with 7-day interval (*P < 0.05 in comparison with S-M group by Student's t test). B, total activity counts over a duration of 60 min (*P < 0.05 in comparison with S-M group by Dunnett's test). Numbers in parentheses indicate the number of animals.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Sensitization of mPer1, but not of mPer2 or mTim, induced by repeated application of MAP. A, representative in situ hybridization autoradiograms of mPer1 developed on X-ray film. M-M, MAP (5 mg/kg) injection followed by MAP (0.5 mg/kg) injection with 7-day withdrawal; S-M, saline injection followed by MAP (0.5 mg/kg) injection with 7-day interval; M-S, MAP (5 mg/kg) injection followed by saline injection with 7-day withdrawal; S-S, saline injection followed by saline injection with 7-day interval. As shown in panel M-M, a small doses of MAP (0.5 mg/kg) augmented mPer1 expression in the dorsal caudate of the MAP (5 mg/kg)-pretreated mouse. Calibration on the S-S panel is 1 mm. B, relative value of mPer1, mPer2, or mTim mRNA expression in the CPu. Per expression observed in the S-S group was set as 100%. Mice were decapitated 60 min after the last MAP or saline injection. Numbers in parentheses indicate the number of animals (**P < 0.01 in comparison with S-S by Dunnett's test).

	Discussion

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In the present experiment, we demonstrated that MAP dose dependently induces the expression of mPer1 in the CPu and piriform cortex but not in the accumbens or SCN, whereas MAP did not affect the levels of mPer2 and mPer3 in these brain areas. Coadministration of D1 receptor antagonist SCH23390 or NMDA receptor antagonist MK-801 significantly attenuated MAP-induced expression of mPer1, but D2 receptor antagonist sulpiride did not block this expression. Interestingly, Fos induction in the CPu by MAP injection was reportedly attenuated by either SCH23390 or MK-801 (Konradi et al., 1994, 1996; Ohno et al., 1994; Yoshida et al., 1995). Treatment with MK-801 attenuated MAP-induced mPer1 expression in the CPu. Previous reports demonstrated that MK-801 inhibited amphetamine-induced glutamate release in the ventral tegmental area (Wolf and Xue, 1999), MAP-induced striatal dopamine release (Finnegan and Taraska, 1996), and striatal Fos expression (Ohno et al., 1994). Thus, NMDA receptor mechanisms are also involved in MAP-induced biochemical responses such as mPer1 and Fos expression. From a pharmacological point of view, above-mentioned articles have suggested that there may be common neural mechanisms between the expression of Fos and of mPer1 induced by MAP. The present double-staining experiment demonstrated a dense expression of both Fos and mPer1 mRNA in the dorsomedial regions of the CPu, which also support the above possibility.

In the present experiment, MAP increased the level of mPer1 but did not affect the levels of mPer2 and mPer3 in the CPu. Application of forskolin induced Per1 but not Per2 expression in Rat-1 cells with the induction of cyclic AMP-responsive element binding protein phosphorylation; then it initiated the oscillation of Per2 expression (Yagita and Okamura, 2000). Interestingly, we found that the promoter region of mPer1 contains a total of four cyclic AMP-responsive elements (Yamaguchi et al., 2000). This cyclic AMP-responsive element site may be responsible for the mPer1 induction that occurs with MAP application. In fact, not only D1 and NMDA receptor activation (Das et al., 1997) but also MAP application (Muratake et al., 1998) reportedly cause cyclic AMP-responsive element binding protein phosphorylation.

Light exposure strongly increases Per1 and Per2 but not Per3 expression in the SCN of mice, rats, and hamsters (Shigeyoshi et al., 1997; Yan et al., 1999; Horikawa et al., 2000); however, in our study, MAP failed to change mPer gene expression in the SCN. We found a significant circadian oscillation of Per1 and Per2 in the hamster SCN with a peak at subjective day (Horikawa et al., 2000). Local injection of NMDA into the SCN at subjective night causes the induction of Per1 in the hamster (Moriya et al., 2000). On the other hand, stimulation of the D1 receptor in the SCN produces Fos induction in early developmental rodents but not in adults (Viswanathan et al., 1994; Weaver and Reppert, 1995; Grosse and Davis, 1999). Thus, the reason why MAP failed to produce mPer1 expression in the SCN may be related to weak contribution of D1 receptors in the adult SCN. Changes in mPer1 mRNA levels of CPu detected by in situ hybridization exhibited a circadian rhythm with a peak at early subjective night (data not shown). Therefore, it is interesting to determine whether transiently induced mPer1 in the CPu at subjective day may cause a phase shift of circadian rhythm of mPer1 expression in the CPu. The answer requires an examination of circadian time course of mPer1 expression in the CPu subsequent to MAP treatment, and this important question should be the follow-up to this article.

In this experiment, SCH23390 and MK-801 administration alone lowered the expression of mPer1 in the CPu, suggesting a tonic activation of the mPer1 gene through D1 and/or NMDA receptors in the CPu. Actually, MK-801 decreased the MAP-induced mPer1 expression but slightly augmented the MAP-induced locomotion. On the contrary, sulpiride strongly attenuated the MAP-induced locomotion without affecting mPer1 expression. These results seemingly indicate that MAP-induced locomotor stimulation is not sufficient for induction of mPer1 by MAP. One of our previous articles supports this idea by showing that the D1 and NMDA receptor blockade abolished MAP-induced anticipatory behavior without attenuating its induction of hyperlocomotion (Shibata et al., 1995).

In the present experiments, repeated administration of MAP caused sensitization in mPer1 expression but not in mPer2, mPer3, or mTim expression in the CPu. Therefore, behavioral sensitization is associated with mPer1 expression in the CPu but not that in the SCN, suggesting the important role of mPer1 gene expression in MAP-induced behavioral sensitization. Furthermore, we demonstrated that the MAP-induced free-running oscillation of rat locomotion with drinking application of MAP exhibited a sensitization phenomenon (Nikaido et al., 1999). Interestingly, Andretic et al. (1999) reported that flies mutant for period, clock, cycle, and doubletime lack sensitization to repeated cocaine administration, but flies mutant for timeless do not. On the other hand, in contrast to Drosophila melanogaster, mutation of the Clock gene that regulates circadian rhythm in mice does not affect acute or sensitized responses to cocaine (Sidiropoulou et al., 2000). Thus, in mammals, the Clock gene is not required for the induction of behavioral sensitization to cocaine. Therefore, we should investigate whether the sensitized increase in mPer1 expression reflects the result or cause using mPer1 gene mutant mice. Taken together, the results seem to indicate that MAP-induced sensitized expression of mPer1 may be related, at least, to the sensitized phenomenon.

In conclusion, the present results demonstrate that the activation of both D1 and NMDA receptors plays an important role in causing MAP-induced expression of mPer1 in the CPu. Furthermore, behavioral sensitization induced by repeated MAP injection is associated with sensitization of MAP-induced mPer1 expression.