Effects of Monoamine Oxidase Inhibitors on Methamphetamine ...

Neurochemical Research, Vol. 30, No. 11, November 2005 (Ó 2005), pp. 1377 1385 DOI: 10.1007/s11064-005-8390-2

Effects of Monoamine Oxidase Inhibitors on Methamphetamine-Induced Stereotypy in Mice and Rats Tomohiro Tatsuta,1,2 Nobue Kitanaka,1 Junichi Kitanaka,1,3 Yoshio Morita,2 and Motohiko Takemura1 (Accepted September 7, 2005)

In male ICR mice, a single intraperitoneal administration of methamphetamine (METH) (10 mg/kg) induced stereotyped behavior such as continuous sniffing, circling, and nail biting, reaching a plateau level 20 min after the injection. Subcutaneous pretreatment with clorgyline, a monoamine oxidase (MAO)-A inhibitor, at a dose of 0.1 mg/kg 2 h prior to the drug challenge significantly decreased the initial (first 20 min) intensity of stereotypies and increased the latency to onset. The effect was not observed with either higher doses of clorgyline (1 and 10 mg/kg) or l-deprenyl, a MAO-B inhibitor, at doses of 0.1 10 mg/kg. In male Wistar rats, the inhibitory effect of clorgyline on METH-induced stereotypy was not observed. Pretreatment of the mice with clorgyline (0.1 mg/kg) had no effect on apparent serotonin and dopamine turnover in the striatum, although the higher doses of clorgyline (1 and 10 mg/ kg) significantly decreased the turnover. These results suggest that a low dose of clorgyline tends to increase the latency and decrease the intensity of stereotypies induced by METH in a dopamine metabolism-independent manner in mice. KEY WORDS: Clorgyline; methamphetamine; monoamine turnover; species difference; stereotypy.

considered a pharmacological model for amphetamine psychosis and, in some cases, for schizophrenia, because their symptoms closely resemble amphetamine-induced changes in human behavior (1 6). The mechanisms through which stereotypy is produced by amphetamines include limbic and striatal dopaminergic transmission (3,4). The activation of striatal dopamine receptors, both D1 and D2 subtypes, is considered to trigger the amphetamineproduced stereotypy (7 11). This hypothesis is supported by evidence that dopamine turnover (i.e., ratio of the amount of 3,4-dihydroxyphenylacetic acid (DOPAC), a dopamine metabolite formed by monoamine oxidase (MAO), to that of dopamine) markedly increased in the striatum and mesolimbic area of methamphetamine (METH)-sensitized rats,

INTRODUCTION In rodents, either repeated moderate doses or a single high dose of amphetamines produce increased motor activity and repetitive and compulsive behaviors called stereotypies, such as continuous sniffing, head-bobbing, nail-biting, and mouthing. Animals that display stereotyped behaviors are 1

2

3

Department of Pharmacology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan. Department of Neuropsychiatry, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo, 663-8501, Japan. Address reprint requests to: Junichi Kitanaka, Ph.D., Department of Pharmacology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Tel: +81 798 45 6333; Fax: +81 798 45 6332; E-mail: kitanaka-hyg@ umin.ac.jp

1377 0364-3190/05/1100 1377/0 Ó 2005 Springer Science+Business Media, Inc.

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suggesting increased dopaminergic transmission, leading to METH-produced stereotypy (3). To examine whether or not a modification of dopamine metabolism causes a change in stereotypy produced by METH, we tested the effects of pretreatment with MAO inhibitors on METH-produced stereotypy in mice and rats. MAO exists as two forms in the mammalian brain, namely MAO-A and -B, which differ in substrate specificity (12). In the mouse brain, dopamine is mainly metabolized by MAO-A under normal physiological conditions because of the predominance of expression of the isozyme (13). In the present study, clorgyline and l-deprenyl were used as a MAO-A and MAO-B inhibitor, respectively. Unexpectedly, the results suggested that pretreatment of the mice, but not rats, with a low dose of clorgyline tends to increase the latency and decrease the intensity of stereotypies induced by METH in a dopamine metabolism-independent manner.

EXPERIMENTAL PROCEDURE Animals. Male ICR mice and male Wistar rats (each species of 5 weeks old at purchase; Japan SLC, Shizuoka, Japan) were housed in groups of 8 (cage size, 37  22  15 cm) and 4 (cage size, 33  28  17 cm), respectively, in a temperature- (22 ± 2°C) and humidity- (50 ± 10%) controlled environment under a 12-h light/dark cycle (lights on at 07:00 h) with food and water available ad libitum except during the observations of stereotyped behavior and measurements of locomotor activity using the Animex apparatus (see below). Animal handling and care were conducted according to the NIH guidelines (14) and all experiments were approved by the Institutional Animal Research Committee. Every effort was made to minimize the number of animals used and their suffering. After at least 6 days’ habituation in this facility, mice were used in the experiments as follows.

Locomotor Activities. Mice and rats were weighed (29 39 g and 115 152 g, respectively, on Day 1) and divided randomly into eight and four groups, respectively. As shown in Table I, all mice and rats were injected intraperitoneally (i.p.) with 0.1 ml/10 g and 0.5 ml/100 g of sterile saline, respectively, on Day 1. This procedure was required to reduce the variance of the data on locomotor activities on Day 2 (15 17). On Day 2, the mice and rats in each group were subjected to treatment as shown in Table I and measurements of locomotor activity. For Experiment 1, the mice or rats were pretreated with various doses of clorgyline as shown in Table I. For Experiment 2, the mice were pretreated with various doses of l-deprenyl as shown in Table I. The doses of drugs refer to the weight of salt. All drugs were dissolved in sterile saline. Clorgyline and l-deprenyl were administered subcutaneously (s.c.) in a volume of 0.05 ml/10 g and 0.25 ml/100 g of body weight of the mice and rats, respectively. The same volume of saline was used for the control. METH was administered i.p. in a volume of 0.1 ml/ 10 g and 0.5 ml/100 g of body weight of the mice and rats, respectively. The horizontal locomotor activity was measured in a transparent acrylic test box (30  30  35 cm) with ca. 25 g of wood chips on the Animex Auto apparatus (System MK-110; Muromachi Kikai Co., Ltd., Tokyo, Japan) in a quiet room as described previously (15). All experiments were conducted between 8:30 and 16:30. Stereotyped Behaviors. Animals in the transparent acrylic test box undergoing locomotion measurements were simultaneously observed for stereotypy as scored for 1 h after the drug administration by an observer blind to the treatments, using a rating scale for stereotypies described below. Behavior was broken down into 30-s bins, and a predominant behavior was recorded for each bin. Behaviors scored were quiet wake/sleeping, ambulating, rearing, vigorous grooming, head-bobbing, continuous sniffing with apparent exploratory behaviors, circling, and nail and/or wood chip biting, according to the method of Weinshenker et al. (18) with a slight modification (i.e. 30-s vs. 10-s bins). Ambulating and rearing were considered locomotor/exploratory behaviors and the last four were considered stereotypies. The cumulative numbers of bins in which stereotypies were observed for every 5 min are shown (maximal value = 10). Measurement of Levels of Monoamines and their Metabolites. After the behavioral analyses, the mice were sacrificed by cervical dislocation and decapitation 1 h after the drug injection. The

Table I. Schedule of Drug Administration Test day Experiment 1 Group (n for mice and rats) S/M (12 and 8) C0.1/M (15 and 6) C1/M (10 and 6) C10/M (10 and 6) Experiment 2 Group (n for mice) S/M (10) D0.1/M (10) D1/M (10) D10/M (10)

1

2

Saline Saline Saline Saline

(i.p.) (i.p.) (i.p.) (i.p.)

Saline (s.c.)/METH (i.p.) Clorgyline 0.1 mg (s.c.)/METH (i.p.) Clorgyline 1 mg (s.c.)/METH (i.p.) Clorgyline 10 mg (s.c.)/METH (i.p.)

Saline Saline Saline Saline

(i.p.) (i.p.) (i.p.) (i.p.)

Saline (s.c.)/METH (i.p.) l-Deprenyl 0.1 mg (s.c.)/METH (i.p.) l-Deprenyl 1 mg (s.c.)/METH (i.p.) l-Deprenyl 10 mg (s.c.)/METH (i.p.)

Drug solutions were prepared daily and administered to the mice by intraperitoneal (i.p.) and subcutaneous (s.c.) injection in a volume of 0.1 ml/10 g and 0.5 ml/100 g of body weight, respectively, and to the rats by i.p. and s.c. injection in a volume of 0.05 ml/10 g and 0.25 ml/ 100 g of body weight, respectively. METH, methamphetamine (10 mg/kg for mice and rats).

MAOI and METH-Induced Stereotypy brains were immediately removed, and the regions of the striatum and nucleus accumbens and the thalamus and hypothalamus were isolated, weighed, and frozen in liquid nitrogen. The brain regions were pooled together because of the technical limitation in terms of dissection. Tissue levels of monoamines and their metabolites were quantified in the supernatant by high-performance liquid chromatography (HPLC) with electrochemical detection as described previously (15 17) as follows: Each frozen brain sample was homogenized with a Teflon/glass homogenizer in 10 volumes (w/v) of ice-cold 0.1 N perchloric acid with 30 lM Na2EDTA containing 3,4-dihydroxybenzylamine hydrobromide and isoproterenol as internal standards for the catechols and for the indoles, respectively. The homogenates were centrifuged at 10,000  g for 10 min at 4°C and the supernatants were filtered through a 0.20-lm membrane filter (Millipore Co., Bedford, MA, USA). The filtrates (10 ll) were injected directly into an HPLC system (system controller, model SCL-10A; auto-injector, model SIL-10A; pump, model LC-10AD; Shimadzu Co., Kyoto, Japan) equipped with a reversed-phase ODS-column (MCM column 150; 4.6  150 mm; MC Medical, Inc., Osaka, Japan) and an electrochemical detector (Coulochem Model 5100A, ESA, Inc., Chelmsford, MA, USA). The column temperature was maintained at 24°C, and the detector potentials were set at +0.40 V, +0.15 V and )0.35 V on the conditioning cell, and Detectors 1 and 2, respectively. The mobile phase was a 1000:35.2:85.8 (v/v) mixture of a buffer (50 mM Na2HPO4, 50 mM citric acid, 4.4 mM 1-heptanesulfonic acid and 0.1 mM Na2EDTA, pH 3.0), acetonitrile and methanol, and the flow rate was set at 0.9 ml/min. Reagents. METH hydrochloride was purchased from Dainippon Pharmaceutical Co. (Osaka, Japan). N-Methyl-N-propargyl3-(2,4-dichlorophenoxy)propylamine hydrochloride (clorgyline), R-())-deprenyl hydrochloride (l-deprenyl), and all standard reagents for HPLC were from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used were of the highest purity commercially available. Statistical Analysis. Values are shown as means with the standard errors of the means (SEMs). Statistical analysis was performed using a one-way or two-way analysis of variance (ANOVA) followed by Scheffe´’s comparisons or Student’s t-test as indicated. A P value of less than 0.05 was considered a statistically significant difference.

RESULTS Stereotypy and Locomotor Activity In control animals treated with saline alone, a stereotypy-like continuous sniffing with apparent exploratory behavior was scored by the observer blind to the treatments between 0 and 5 min after the second saline injection (score = 2.13 ± 0.58, n = 8), but the behavioral response disappeared 10 min after the injection. As shown in Fig. 1, the stereotyped behavior was scored between 5 and 10 min after the administration of METH i.p., and reached a plateau level 20 min later in all the mice pretreated with 0, 0.1, 1, and 10 mg/kg of clorgyline (Experiment 1) and l-deprenyl (Experiment 2). The plateau level was maintained for at least 1 h after the

1379 administration. We have examined the behavioral profile of METH-induced stereotypy in mice. The data indicated that a single administration of METH (10 mg/kg, i.p.) induced a stereotypy consisting of nail/wood chip biting (52% of the total stereotypic score), vigorous grooming (45%), and sniffing (3%; n = 22 of Group S/M in Experiments 1 and 2). There was a significant difference between Groups S/M and C0.1/M (0.1 mg/kg of clorgyline pretreatment) (pretreatment  time interaction, F(33,516) = 1.647, P < 0.05, two-way ANOVA followed by Scheffe´’s comparisons). At 5 and 10 min after the drug challenge, a significant decrease in intensity of the stereotypies was observed in Group C0.1/M, compared with Group S/M (Fig. 1a). As for l-deprenyl pretreatment (Experiment 2 in Table I), the time course of expression of stereotypies showed no statistical difference among the mice pretreated with 0, 0.1, 1, and 10 mg/kg of l-deprenyl (Fig. 1b). There was no effects of MAO inhibitors alone on observed behaviors (data not shown). In Experiment 1, total horizontal locomotor activity in mice was measured and values for the first 20 min after the drug administration are shown (Fig. 2). There was a significant difference between Groups S/M and C0.1/M (0.1 mg/kg of clorgyline pretreatment) (F(33,516) = 3.967, P < 0.05, oneway ANOVA followed by Scheffe´’s comparisons). In rats, the same dose of METH as that used in mice (10 mg/kg,i.p.) was chosen, since a single administration of the rats with 5 mg/kg (i.p.) of METH induced hyperlocomotion predominantly, but not stereotypy with a plateau level of scores, in our preliminary experiments (data not shown). Pretreatment of the rats with clorgyline (0, 0.1, 1, and 10 mg/ kg, s.c.) had no effect on METH (10 mg/kg, i.p.)induced stereotypy (Fig. 3) (pretreatment x time interaction, F(33,240) = 0.2169, P = 0.999, twoway ANOVA followed by Scheffe´’s comparisons). Levels of Monoamines and their Metabolites As shown in Table II, the levels of L-dihydroxyphenylalanine (L-DOPA), dopamine, DOPAC, and norepinephrine did not differ among the four groups in the striatum and nucleus accumbens (one-way ANOVA followed by Scheffe´’s comparisons, F(3,43) = 2.179, P = 0.1043, F(3,43) = 1.951, P = 0.1356, F(3,43) = 1.324, P = 0.2790, and F(3,43) = 2.405, P = 0.0805, respectively). The levels of 3-methoxytyramine (3-MT) and 5-hydroxyindolacetic acid (5-HIAA) significantly increased and

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Fig. 1. Stereotypic scores after a single administration of METH (10 mg/kg, i.p.) in mice that were pretreated for 2 h with various doses of clorgyline (a, Experiment 1) and l-deprenyl (b, Experiment 2) on Day 2. All mice were injected with 0.1 ml/10 g (i.p.) of saline on Day 1 to reduce the variance of the data on locomotor activity on Day 2. *P < 0.05, **P < 0.01, compared with Group S/M (control) (Student’s t-test).

decreased, respectively, in the striatum and nucleus accumbens in Groups C1/M and C10/M, as compared with Group S/M (F(3,43) = 24.004, P < 0.001 and

Fig. 2. Horizontal locomotor activity (total counts for first 20 min) after a single administration of METH (10 mg/kg, i.p.) in mice pretreated with various doses of clorgyline (Experiment 1 in Table I). *P < 0.05, compared with Group S/M (control) (Student’s t-test).

F(3,43) = 17.685, P < 0.001, respectively). The striatal content of homovanillic acid (HVA) decreased significantly in Group C10/M, as compared with Group S/M (F(3,43) = 13.275, P < 0.001). The striatal content of 5-hydroxytryptamine (5-HT) increased significantly in Group C1/M, as compared with Group S/M (F(3,43) = 3.735, P < 0.05). 3-Methoxy-4-hydroxyphenylglycol (MHPG) was not detected in the striatum or nucleus accumbens in any of the four groups. In the thalamus and hypothalamus, the levels of L-DOPA, dopamine, and MHPG did not differ among the four groups (F(3,43) = 1.070, P = 0.3719, F(3,43) = 3.613, P = 0.1206, and F(3,43) = 3.643, P = 0.1199, respectively). The levels of DOPAC, 3-MT, and 5-HT increased significantly in this region in Groups C1/M and C10/M, as compared with Group S/M (F(3,43) = 26.370, P < 0.001, F(3,43) = 81.062, P < 0.001, and F(3,43) = 37.064, P < 0.001, respectively), although the levels of HVA and 5-HIAA decreased significantly in the thalamus and hypothalamus in Groups

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Fig. 3. Stereotypic scores after a single administration of METH (10 mg/kg, i.p.) in rats that were pretreated for 2 h with various doses of clorgyline on Day 2. All rats were injected with 0.5 ml/100 g (i.p.) of saline on Day 1 to reduce the variance of the data on locomotor activity on Day 2.

C1/Mand C10/M, as compared with Group S/M (F(3,43) = 7.44, P < 0.001 and F(3,43) = 34.831, P < 0.001, respectively). The hypothalamic content of norepinephrine increased significantly in Group C1/M, as compared with Group S/M (F(3,43) = 9.821, P < 0.001). Apparent Monoamine Turnover As shown in Fig. 4, the apparent monoamine turnover was evaluated by calculating the ratio of the tissue levels of monoamines and their metabolites shown in Table II. As for norepinephrine turnover, a marked decrease in the ratio of MHPG to norepinephrine was observed in the thalamus and hypothalamus of Group C1/M mice, as compared with Group S/M mice (one-way ANOVA followed by Scheffe´’s comparisons, F(3,43) = 7.172, P < 0.001) (Fig. 4b). As for dopamine turnover, a marked decrease in the ratio of DOPAC to dopamine was observed in the striatum and nucleus accumbens in Group C1/M, as compared with Group S/M (F(3,43) = 4.341, P < 0.01) (Fig. 4a). In the thalamus and hypothalamus, a marked increase in the ratio of DOPAC to dopamine was observed in Groups C0.1/M, C1/M, and C10/M, as compared with Group S/M (F(3,43) = 33.459, P < 0.001) (Fig. 4b). A marked increase in the ratio of 3-MT to dopamine was observed in the striatum and nucleus accumbens and the thalamus and hypothalamus in Groups C1/M and C10/M, as compared with Group S/M (F(3,43) = 50.143, P < 0.001 and F(3,43) = 36.724, P < 0.001, respectively) (Fig. 4a and b). A marked decrease of the ratio of HVA to dopamine was

observed in the striatum and nucleus accumbens in Groups C1/M and C10/M, as compared with Group S/M (F(3,43) = 20.703, P < 0.001) (Fig. 4a). In the thalamus and hypothalamus, a marked decrease in the ratio of HVA to dopamine was observed in Group C1/M, as compared with Group S/M (F(3,43) = 8.935, P < 0.001) (Fig. 4b). As for 5-HT turnover, a marked decrease in the ratio of 5-HIAA to 5-HT was observed in the striatum and nucleus accumbens and the thalamus and hypothalamus in Groups C1/M and C10/M, as compared with Group S/M (F(3,43) = 8.193, P < 0.001 and F(3,43) = 129.961, P < 0.001, respectively) (Fig. 4a and b).

DISCUSSION In the present study, the numbers of 30-s bins of behavior were presented (Fig. 1). The measure is appropriate because the acute administration of METH (10 mg/kg, i.p.) in male ICR mice and Wistar rats produced stereotypy with a time course similar to that reported previously in rats (3). Pretreatment of the mice with a low dose of clorgyline (0.1 mg/kg, s.c.), a MAO-A inhibitor (19), two hours prior to the drug challenge shifted the motor activity from stereotyped behavior to horizontal locomotor activity during the first 20 min (Fig. 1a and 2), resulting in an increased latency to onset. On the other hand, l-deprenyl, a MAO-B inhibitor, had no effect on the latency of the stereotypy in mice (Fig. 1b). Acute treatment of the rats with 1 and 10 mg/kg (s.c.) l-deprenyl inhibited brain MAO-B activity completely while ca. 90% of inhibition by 0.1 mg/kg (20). On the

The brains were dissected 1 h after the drug challenge on Day 2. Values are expressed as nanograms per milligram of wet tissue (n = 12, 15, 10, and 10 for Groups S/M, C0.1/M, C1/ M, and C10/M, respectively). *P < 0.05, **P < 0.001, compared with Group S/M (one-way ANOVA followed by Scheffe´’s comparisons). S/M =10 mg/kg methamphetamine (METH) injection (i.p.) two hours after 0.05 ml/10 g saline injection (s.c.); C0.1/M = 10 mg/kg METH injection (i.p.) two hours after 0.1 mg/kg clorgyline injection (s.c.); C1/ M = 10 mg/kg METH injection (i.p.) two hours after 1 mg/kg clorgyline injection (s.c.); C10/M = 10 mg/kg METH injection (i.p.) two hours after 10 mg/kg clorgyline injection (s.c.). L-DOPA, L-dihydroxyphenylalanine; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; 3-MT, 3-methoxytyramine; HVA, homovanillic acid, NE, norepinephrine; MHPG, 3-methoxy-4-hydroxyphenylglycol; 5-HT, 5-hydroxytryptamine (serotonin); 5-HIAA, 5-hydroxyindolacetic acid. N.D., not detected.

0.032 0.042 0.017** 0.009** ± ± ± ± 0.411 0.387 0.081 0.044 0.07 0.13 0.13** 0.17** ± ± ± ± 1.48 1.75 3.16 2.73 0.036 ± 0.007 0.032 ± 0.009 0.012 ± 0.002 N.D. 0.11 0.16 0.16** 0.22 ± ± ± ± 2.11 2.32 3.30 2.77 0.176 ± 0.039 0.136 ± 0.024 0.063 ± 0.016** N.D 0.010 0.011 0.019** 0.023** ± ± ± ± 0.032 0.039 0.284 0.265 0.012 0.025 0.032** 0.022** ± ± ± ± 0.102 0.184 0.346 0.357 ± ± ± ± Thalamus S/M C0.1/M C1/M C10/M

and hypothalamus 0.029 ± 0.007 0.334 0.029 ± 0.011 0.302 0.061 ± 0.025 0.428 0.036 ± 0.014 0.319

± ± ± ± Striatum and nucleus accumbens S/M 0.359 ± 0.052 6.20 C0.1/M 0.398 ± 0.050 8.40 C1/M 0.282 ± 0.058 7.49 C10/M 0.216 ± 0.061 6.89

0.021 0.024 0.039 0.033

0.030 0.049 0.035** 0.012** ± ± ± ± 0.362 0.367 0.099 0.070 0.083 0.107 0.66* 0.13 ± ± ± ± 0.785 0.890 2.08 1.36 N.D. N.D. N.D. N.D. 0.049 0.100 0.063 0.13 ± ± ± ± 0.068 0.092 0.070 0.035** ± ± ± ± 0.053 0.14 0.26** 0.21** ± ± ± ± 0.017 0.042 0.026 0.030 ± ± ± ± 0.316 0.347 0.259 0.292

DA

0.25 0.82 0.78 0.76

DOPAC

0.680 1.29 2.30 2.46

3-MT

0.581 0.794 0.272 0.216

HVA

0.716 0.895 0.981 1.06

NE

MHPG

5-HT

5-HIAA

Tatsuta, Kitanaka, Kitanaka, Morita, and Takemura

L-DOPA

Table II. Tissue Levels of Monoamines and their Metabolites in the Striatum and Nucleus Accumbens and the Thalamus and Hypothalamus of the Mice (Experiment 1)

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other hand, non-specific inhibition (ca. 45%) by l-deprenyl of MAO-A activity was observed when the rats were treated with 10 mg/kg l-deprenyl (20). The degree of MAO-A inhibition was also attained when the rat were treated with 0.3 mg/kg clorgyline alone. In line with the observations as well as the present data, the observed effect of a low dose of clorgyline on METH-induced stereotypy is likely to be independent on MAO-A activity. The inhibitory effect of clorgyline on METH-induced stereotypy was species-specific; pretreatment of the rats with clorgyline had no effect on METH-induced stereotypy (Fig. 3). The differences of the properties and proportion of MAOA and MAO-B between mouse and rat brains have not been known (12). Thus, the species difference of the clorgyline effect might be attributed to mechanism(s) other than monoamine metabolism. Clorgyline at 1 and 10, but not 0.1 mg/kg (s.c.), had a typical pharmacological effect in the striatum and nucleus accumbens of the mouse; a significant decrease in the ratio of 5-HIAA to 5-HT and of HVA to dopamine (Fig. 4a). These dosages of clorgyline (1 and 10 mg/kg) had no effect on the latency of stereotypy (Fig. 1a), suggesting again that the low dose of clorgyline affected METH-produced stereotypy in a dopamine metabolism-independent manner. The lowest dose of clorgyline actually increased and decreased significantly the apparent dopamine and serotonin turnover, respectively, in the thalamus and hypothalamus (Fig. 4b). The effect was also attained when the mice were treated with 1 and 10 mg/ kg clorgyline, suggesting that the dopamine and/or serotonin turnover changes observed in the thalamus and hypothalamus have not effect on the unexpected response observed at the dose of 0.1 mg/kg of clorgyline (Fig. 1a). There is a possibility that the decrease in stereotypy under a lowest dose of clorgyline pretreatment was a non-specific effect of the fact that animals were moving more over during the first 20 min after METH administration (Fig. 2). However, the significant increase in locomotor activity could rate as hyperlocomotion rather than stereotypy based on our criteria. Previously, we demonstrated that METHinduced hyperlocomotion and behavioral sensitization were inhibited after treatment of the mice with 1.0 mg/ kg clorgyline (16,17). Failure to replicate the same clorgyline pretreatment effect in the three studies (i.e. METH-induced hyperlocomotion (16) and behavioral sensitization (17)), but the present study (Group S/M vs. C1/M in Fig. 2) raises the possibility that subtle or

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Fig. 4. Apparent monoamine turnover in the striatum and nucleus accumbens (a) and the thalamus and hypothalamus (b) of the mice (Experiment 1) one hour after the METH administration on Day 2. Each column represents the mean ± SEM. MHPG, 3-methoxy-4hydroxyphenylglycol; 3-MT, 3-methoxytyramine; NE, norepinephrine; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA; homovanillic acid; DA, dopamine; 5-HIAA, 5-hydroxyindolacetic acid; 5-HT, serotonin. **P < 0.01, ***P < 0.001, significantly different change in apparent monoamine turnover, compared with control animals (Group S/M) (one-way ANOVA followed by Scheffe´’s comparisons). N.D., not determined.

unintended methodological factor(s) could account for the unexpected pretreatment effect on stereotypy. Pretreatment of the rats with a combination of clorgyline and pargyline (1 mg/kg per day for 21 days) increased apomorphine (1 and 3 mg/kg,

i.p.)-produced stereotypy with no change in the number of [3H]spiroperidol-binding sites in the striatum (21). Pargyline is a relatively selective MAO-B inhibitor and apomorphine is a mixed D1/D2 dopamine agonist. The mechanism through which the

1384 effect of MAOs appeared was unclear. Clorgyline (1 mg/kg per day for 28 days via osmotic minipumps, or 1 mg/kg, s.c., twice weekly for a total of eight injections) is reported to shift the development of behavioral sensitization from a locomotor response to a mouthing response (‘‘oral stereotypy’’) to quinpirole in rats (22,23). Culver et al. (23) proposed a novel MAO inhibitor-displaceable quinpirole binding site; the site might be involved in the quinpirole-induced locomotor sensitization. Since the site is considered to be a different endogenous entity from MAO (i.e. imidazoline I2-binding site, which is considered to exist on MAO, at a location distinct from the catalytic site, refs. 24 and 25), there might be modes of action of clorgyline other than the inhibition of MAO (22,23). While the mechanism by which a low dose of clorgyline affects METHproduced stereotypy in mice is not clear, novel modes of action of clorgyline including the MAO inhibitordisplaceable quinpirole binding site should be taken into consideration. The possibility of a species difference in the actions of clorgyline toward the novel molecular target could not be excluded. Overall, the results presented in this study suggest that a low dose of clorgyline tends to increase the latency and decrease the intensity of the stereotypies induced by METH in a dopamine metabolism-independent manner in mice. Further investigation is required to clarify the exact mode of action of clorgyline.

ACKNOWLEDGMENTS NK was supported by a Grant-in-Aid for Researchers, Hyogo College of Medicine.

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