Introduction

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The daily variations in biological functions such as the secretions of glands, and the synthesis of RNA and protein are suggested to be an additional variable influencing the susceptibility to a drug. In other words, the variation in efficiency or toxicity of many drugs, at least in part, is due to the time of administration (Ohdo et al., 1988, 1997, 2001).

A typical neuroleptic, haloperidol is a dopamine receptor antagonist for the treatment of schizophrenia. This drug has a high affinity (about 80%) for blockade of dopamine D₂ receptor and an appreciable affinity (about 1%) for blockade of dopamine D₁ receptor (Hyttel et al., 1985; Hurley et al., 1996; Manglapus et al., 1999). Circadian changes in several behavioral effects induced by neuroleptic dopamine antagonists, including haloperidol, have been reported (Nagayama et al., 1979, 1987; Kumar et al., 1981; Campbell et al., 1982). One of these, catalepsy, the animal equivalent of Parkinsonism, is a quantifiable effect of neuroleptic agents and was suggested to indicate antagonism of dopamine receptors in the extrapyramidal regions such as the striatum (Merchant et al., 1994; Coppens et al., 1995). In a recent study from our laboratory, under a 12:12-h light/dark cycle, there was a diurnal rhythm in the catalepsy responses to haloperidol. The maximum and minimum catalepsy responses were detected at mid-light and mid-dark, respectively.

Tolerance to haloperidol-induced catalepsy following long-term administration in rodent has been observed in several studies (Ezrin-Waters and Seeman, 1977; Carey and de Veaugh-Geiss, 1984). It was suggested that increases in the function of D₂ dopamine receptor (Kashihara et al., 1986; Filtz et al., 1994) and the levels of D₂ receptor mRNA (Creese et al., 1976) lead to the development of dopaminergic supersensitivity, which partially corresponds to the development of a tolerance to catalepsy. Nevertheless, the effect of dosing time on the development of tolerance has not been well delineated. It is suggested that the repeated haloperidol administration at different times may lead to the different rates of tolerance development. To investigate this idea, mice were administered haloperidol daily at times nearly corresponding to the maximal or minimal catalepsy, and the duration of catalepsy was monitored on a daily basis. Moreover, the time dependence in number and function of D₂ dopamine receptors was studied to explain this phenomenon.

	Materials and Methods

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Animals and Treatments. Five-week-old male ICR mice purchased from Charles River Japan (Kanagawa, Japan) were used. The animals were housed prior to experiments at 10 per cage with free access to food and water, under controlled lighting; 12:12 light/dark (7:00 AM, lights on; 7:00 PM, lights off), constant temperature (24°C ± 1°C), and controlled humidity (60 ± 10%). All mice were kept under these conditions for 1 week before use. Haloperidol (Serenace, 5-mg/ml ampule; Dai Nippon Seiyaku Co., Ltd., Tokyo, Japan) diluted in saline was administered by i.p. injection at the doses and times specified below. The volumes of injection were standardized to 1 ml/10 g of body weight.

Catalepsy Test. The catalepsy response of one mouse placed in an observation box was measured from the duration of an abnormal posture in which the forelimbs of the mouse were placed on a horizontal 0.2-mm-diameter wire bar suspended 7.5 cm above a platform. The catalepsy test ended when the forelimbs touched the bottom or the wall of the box or when the mouse climbed onto the bar. Animals were injected and evaluated behaviorally in a room under the above-mentioned conditions.

RNA Isolation from Mouse Striatum and RT-PCR Amplification of D₂ Receptor. Total RNA from the striatum was extracted using Trizol solution (Life Technologies, Rockville, MD). Finally, RNA was resuspended in diethylpyrocarbonate-treated water and kept at 80°C. A one-step RT-PCR system (Life Technologies) was used for reverse transcription of 150 ng of RNA. In the present experiment, a PCR primer was designed (D₂-5', 5'-TCG CCA TTG TCT GGG TCC TGT-3'; D₂-3', 5'-TGC CCT TGA GTG GTG TCT TCA-3'; GAPDH-5', 5'-GAC CTC AAC TAC ATG GTC TAC A-3'; GAPDH-3', 5'-ACT CCA CGA CAT ACT CAG CAC-3') to amplify D₂ receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA in a single tube. The size of the PCR products of D₂ receptor and GAPDH were 241 and 178 base pairs, respectively. PCR amplification was conducted in a temperature-controlled system (PC 700; ASTEC, Fukuoka, Japan) for 25 cycles under the following cycle conditions: 2 min at 94°C for denaturation, 1 min at 57°C for annealing, and 2 min at 72°C for extension and at 15°C for holding. PCR products were run on 3% agarose gels. The analysis of the band intensity of D₂ receptor RNA was accomplished using NIH image software on a Macintosh computer, and their amounts were measured versus GAPDH.

Binding Assay for D₂ Receptor in Mouse Striatum. Binding experiments were modified from the methods of previous studies (Naber et al., 1980; Inoue et al., 1997). Briefly, after homogenization of the striatum in 100 ml of 50 mM Tris-HCl buffer (pH 7.4), the sample was centrifuged at 15,000 rpm for 15 min. The pellet was resuspended in the same buffer and incubated at 37°C for 15 min to allow endogenous bound dopamine to dissociate from the receptors. Then the sample was centrifuged again. The supernatant was replaced by buffer to achieve a final concentration of 1 mg of protein/ml of buffer. The accuracy of this dilution was verified by measuring the protein content (Lowry et al., 1951). Then, 50 µl of 50 mM Tris-HCl and 100 µl of homogenate were incubated with 50 µl of [³H]spiperone (Amersham Pharmacia Biotech, Buckinghamshire, UK; specific activity 16.5 Ci/mmol) at five final concentrations ranging from 0.125 to 2 nM at 37°C for 30 min (total volume of reaction mixture, 200 µl). This ligand was used in this study according to its high binding affinity to D₂ receptors (about 80%), corresponding to the binding affinity of haloperidol (Hyttel et al., 1985). After incubation, the mixture was added to 200 µl of fetal bovine serum, and the sample was centrifuged at 10,000 rpm for 5 min. The supernatant was removed, and the pellet was dissolved in 10 ml of aqueous counting scintillant (ACSII; Amersham Pharmacia Biotech). After 6 h of incubation at room temperature, radioactivity was counted with a liquid scintillation counter (LSC-1000; Aloka Co., Tokyo, Japan). Specific binding was defined as the difference in the amount of [³H]spiperone bound with and without 2 µM (+)-butaclamol. The obtained dissociation constant (K_d) and the maximal binding capacity (B_max) for [³H]spiperone were calculated from Scatchard plots, which were generated by the least-square regression analysis method.

Statistical Analysis. Data analyses used Student's t test for two independent groups and ANOVA for the multiple comparison. The 5% level of probability was considered significant. All values have expressed as the mean ± S.E.M.

	Results

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Effect of Dosage on Catalepsy Responses after Single Haloperidol Administration. Within 15 min after haloperidol administration, mice were calm and showed slow spontaneous activity. The cataleptic behavior showed marked differences in responses to varied doses of haloperidol (Fig. 1). The highest and lowest catalepsy duration scores at the peak point were seen in the 1 and 0.25 mg/kg/day-treated groups, respectively. At the dosage of 1 mg/kg, the peak effect appeared at about 1 h postinjection, while at the lower dosages, the maximum of catalepsy responses were shifted to 3 h postinjection.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effect of dosage on catalepsy responses to single 0.25, 0.5, and 1 mg/kg haloperidol. Each point represents mean ± S.E.M. of 10 mice. Student's t test indicated the significant difference between the peak of 1 and 0.5 mg/kg (*P < 0.05) and between the peak of 0.5 and 0.25 mg/kg (*P < 0.05). , 0.25 mg/kg; , 0.5 mg/kg; , 1 mg/kg.

Diurnal Rhythm of Catalepsy Responses after Single Haloperidol Administration. The variations in catalepsy responses resulted from the different administration times exhibited under 12:12 light/dark, with a maximal response around mid-light (9:00 AM to 1:00 PM) and a minimal response around mid-dark (9:00 PM-1:00 AM). In this experiment, the diurnal difference between the maximal and minimal catalepsy effect was 1.99-fold (P < 0.01; Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Diurnal rhythm of catalepsy to single 1 mg/kg haloperidol. Each point represents mean ± S.E.M. of 10 mice. ANOVA revealed the significant variation over the time (P < 0.01). The light and dark bar indicates the light and dark period, respectively.

Diurnal Rhythm of D₂ Receptor mRNA Levels in Mouse Striatum. The present experiment showed that mRNA levels of D₂ receptor in mouse striatum oscillated with diurnal rhythmicity (Fig. 3). However, the frequency of the rhythm tended to be greater than one cycle in 24 h. First, the strongest expression of D₂ receptor mRNA was observed around mid-light (1:00 PM) and the second expression, which was lesser than the former, was observed around mid-dark until late dark (1:00-5:00 AM).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Diurnal expression of D2 receptor mRNA in mouse striatum. Photographs illustrate the band intensity of RT-PCR products of D2 receptor and GAPDH mRNA at each of six sampling time points.

Effect of Dosage on Development of Tolerance to Repeated Haloperidol. Focusing on the effect of repeated administration on the catalepsy responses, after termination of haloperidol treatment for 14 days, all animals became tolerant to the cataleptic action of haloperidol. At the moderately high doses of 1 and 2 mg/kg/day, however, the occurrence of catalepsy following haloperidol administration did not decrease during 5 days of experiments as shown in Fig. 4, B and C. Interestingly, the catalepsy responses reached a peak on day 5 of the experiment course. In contrast, at the high dose of 4 mg/kg/day (Fig. 4D), the mice became considerably tolerant to haloperidol during day 2 of the repeated administration (data not shown). In the saline-treated group, the catalepsy duration did not change over the treatment period (Fig. 4A). The catalepsy duration scores for the haloperidol-treated groups were still higher after 14 days compared with the saline-treated group (P < 0.01). It was also noted that the catalepsy responses to the high dose (4 mg/kg/day) slightly changed after 14 days, compared with day 7. 

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effect of dosage on development of tolerance to repeated saline or haloperidol one time daily over 14 days. Data showed the duration of catalepsy on day 1, 5, 7, and 14 of treatment. A-D, catalepsy responses to saline, 1, 2, and 4 mg/kg/day haloperidol, respectively. Each point represents mean ± S.E.M. of five mice. At 60 min after injection, Student's t test indicated the significant difference between catalepsy responses of day 1 and that of day 14 (*P < 0.05; **P < 0.01). , day 1; , day 5; , day 7; , day 14.

Effect of Dosing Time on Development of Tolerance to Repeated Haloperidol. Figure 5 shows the differences in the development of tolerance induced by 4 mg/kg haloperidol due to the different administration times. In addition, the present findings indicated that the tolerance was developed in two distinct phases; the rapid phase (day 1 to day 6), where the catalepsy tolerance was up to 55% of the initial maximum catalepsy response, and the slow phase (day 7 to day 14), where catalepsy responses were slightly changed. These findings extended those of a dose and time course study of tolerance to 4 mg/kg/day haloperidol. During the rapid phase, mice treated at 9:00 AM developed greater tolerance than those treated at 9:00 PM. The slopes of the rapid phase for treatment at 9:00 AM or 9:00 PM were 27.67 or 16.46, respectively. Moreover, the cataleptic behavior exhibited a significant difference, compared between the two dosing times (P < 0.01). During the slow phase, there was no evidence of a significant difference of catalepsy responses between the two groups, except on day 13 and day 14 of the experiment course (P < 0.05). Significant differences in catalepsy responses between the two dosing times in saline-treated mice did not appear (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of dosing time on development of tolerance to repeated 4 mg/kg/day haloperidol at 9:00 AM or 9:00 PM. Each point represents mean ± S.E.M. of 10 mice. Student's t test indicated the significant difference between the groups of mice treated at 9:00 AM and 9:00 PM (*P < 0.05; **P < 0.01). , haloperidol at 9:00 AM; , haloperidol at 9:00 PM.

Effect of Dosing Time on D₂ Receptor in Mouse Striatum to Repeated Haloperidol Administration. The effect of dosing time on mRNA expression of D₂ receptor and [³H]spiperone binding following repeated treatment was observed in the mouse striatum. As shown in Fig. 6, increases in the levels of D₂ receptor mRNA were activated by 14 days of repeated haloperidol administration in both dosing times. However, the expression of D₂ receptor mRNA in the group of mice treated by haloperidol at 9:00 AM was higher than that in the group of mice treated by haloperidol at 9:00 PM. In the binding experiment, as shown in Fig. 7A, the amount of bound [³H]spiperone at 9:00 AM was significantly greater than that at 9:00 PM (P < 0.01) in both the saline and haloperidol groups for 14 days of treatment. At injection time 9:00 AM, the amount of specifically bound 0.125 nM [³H]spiperone was significantly increased by 72% in the haloperidol group compared with the saline group, whereas at injection time 9:00 PM, a significant increase by 54% was observed (data not shown). For binding parameters, as shown in Fig. 7B and Table 1, the obtained K_d values and B_max in the haloperidol group were significantly increased compared with those in the saline group at both times. When compared between injection times, B_max at 9:00 AM was significantly greater than that at 9:00 PM. In contrast, no difference in K_d was shown.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of dosing time on the levels of D2 receptor mRNA in striatum from mice that received repeated saline or 4 mg/kg/day haloperidol at 9:00 AM or 9:00 PM. Photographs illustrated the band intensity of RT-PCR products of D2 receptor and GAPDH mRNA. S, saline; H, haloperidol.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Effect of dosing time on [3H]spiperone binding in striatum from mice that received repeated saline or 4 mg/kg/day haloperidol at 9:00 AM or 9:00 PM. A, specific binding of [3H]spiperone. Each point represents mean ± S.E.M. of six mice. ANOVA indicated a significant difference between the groups of mice treated at 9:00 AM and 9:00 PM both in saline and haloperidol groups (*P < 0.05). B, Scatchard analysis of the specific binding of [3H]spiperone. , saline at 9:00 AM; , saline at 9:00 PM; , haloperidol at 9:00 AM; , haloperidol at 9:00 PM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Several recent studies have indicated significant rhythmic variables in behavioral phenomena, in the central nervous system, and in immune functions (Wirz-Justice, 1984; Ohdo et al., 1995; Koyanagi et al., 1997). In the present study, we demonstrated marked daily rhythms in catalepsy response to single haloperidol administrations that agreed with the findings of a previous study (Campbell and Baldessarini, 1981) and also indicated the importance of dosing time to drug responses. One possible explanation for the changes in the behavioral responses induced by neuroleptic drugs such as haloperidol is the pharmacodynamics of receptors (dopamine receptor availability or sensitivity). The findings of the present study suggested the expression of D₂ receptor mRNA also exhibited a diurnal rhythm, and the phase of the rhythm was similar to that of the catalepsy responses. However, the frequency of the rhythm was possibly greater than once daily since the strong expression of D₂ receptor mRNA was first observed around mid-light and a second expression, lesser than the former, was observed around mid-dark until late dark.

It was reported that there are daily changes in hepatic metabolism of many drugs, with the greatest activity during mid-dark and the smallest activity during mid-light (Radzialowski and Bousquet, 1968; Jori et al., 1971). Therefore, the pharmacokinetic rhythms contributing to changes in drug response should be considered. It also has been reported that marked changes in the rat brain and serum of haloperidol occur when they are administered at different times throughout 24 h (Campbell et al., 1982). The levels of the drug rose and fell in close correspondence with the rhythmicity of catalepsy responses observed in the present study. The present findings suggest that the metabolism or elimination of haloperidol may vary throughout the day, thus resulting, at least in part, in the daily variation in catalepsy responses.

It appears that decreases in catalepsy responses may result from increases in the activity of the rodent during the active period since opposite rhythms of spontaneous activity and of catalepsy responses were observed. However, according to a study of the circadian rhythms of catalepsy responses to haloperidol in rats (Campbell and Baldessarini, 1982), catalepsy rhythms under constant (24-h) light or dark did not change after 1 month. Moreover, whereas circadian rhythms of spontaneous activity were markedly phase-shifted by the manipulation of the lighting schedule, the pattern of the catalepsy rhythms resisted such changes. These findings suggest that the catalepsy responses are not merely a result of spontaneous activity.

We also investigated the dose and time course of the development of tolerance. The findings obtained confirmed the development of a catalepsy tolerance to all dosages of haloperidol and indicated that the tolerance takes place within 14 days of treatment, which supports the findings of other studies (Asper et al., 1973; Carey and de Veaugh-Geiss, 1982) that reported complete tolerance occurred in rats within 3 weeks. The decrease in the catalepsy response was seen during the 2 days of high-dose treatment (4 mg/kg/day). However, the decrease in catalepsy was not observed in the moderate high-dose (1 and 2 mg/kg/day)-treated groups during the first 5 days of treatment. In contrast, the catalepsy score reached a peak on day 5. These findings agree well with those of a previous study (Gyorgy et al., 1969) that did not find catalepsy tolerance in mice (3 mg/kg/day) or in rats (2 mg/kg/day) within 1 week. It is likely that the timing of tolerance can vary directly with the dose of the neuroleptic administered. The lower dosages lead to a lower concentration of the drug in plasma, including the central action of the drug in the brain, and possibly prevent the development of tolerance. For this reason, a longer treatment with a moderately high dose of haloperidol is required to produce a complete catalepsy tolerance. Moreover, learning and memory functions may alter the cataleptic effect of this drug (Hinson et al., 1982; de Graaf and Korf, 1986), since we found that in this study, including the time-dependent tolerance study, the catalepsy tolerance to high doses of haloperidol did not change after 14 days of treatment, compared with day 7.

In addition, we have shown the effect of dosing time on tolerance development after repeated treatment of haloperidol. In this situation, the time of administration was determined at a point closely corresponding to the maximal (9:00 AM) or minimal (9:00 PM) catalepsy responses to a single dose, and 4 mg/kg/day haloperidol was used, based on the dose and time course study. Mice treated at 9:00 AM showed significantly greater catalepsy responses and tolerance rates than those treated at 9:00 PM. It was, thus, suggested that the tolerance effect of haloperidol was significantly affected by the time of administration. The most likely hypothesis to explain the tolerance development after long-term treatment of neuroleptic drugs is, first, the increased receptor density of D₂ receptor and, second, the increased affinity of the dopaminergic receptors toward agonist binding in the brain. However, the mechanism underlying the time-dependent tolerance in the catalepsy effect of haloperidol has not been clarified. It is possible that there is a difference in the affinity or in the number of cerebral dopamine receptors at different times of the day after repeated administration. The repeated drug regimen during the lights-on period perhaps induces the greater number of dopamine receptors in mouse striatum and then leaves a greater number of these receptors unoccupied, which evokes a higher cataleptic tolerance. The present findings revealed that after 14 days of treatment the increase in D₂ receptor mRNA levels in mice treated by haloperidol at 9:00 AM was higher than that of mice treated by haloperidol at 9:00 PM. In addition, findings from the binding experiment indicated that repeated administration produced a significantly increasing number of [³H]spiperone binding sites at both times, and repeated injection at 9:00 AM induced a greater number of receptors compared with injection at 9:00 PM.

Another possible explanation of the differences in tolerance development is the pharmacokinetic rhythmicity of haloperidol, as mentioned above. Since higher dosages of haloperidol developed higher tolerance of catalepsy in the present study, the time dependence of haloperidol concentrations may also affect the tolerance.

In conclusion, the findings of the present study emphasize the importance of dosing time on haloperidol-induced tolerance development. We have discovered the relation of administration time to D₂ receptor change and tolerance in mice. Nevertheless, the pharmacokinetic effects such as the daily variation in drug metabolism should not be excluded. Both factors (pharmacodynamics of dopamine receptors and pharmacokinetics of haloperidol) may be synergistic, thus leading to daily changes in catalepsy responses and to differences in tolerance development. These findings may provide some clues to determining the administration time of neuroleptic drugs and produce a greater clinical benefit.