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Suppressive effect of cycloheximide on behavioral sensitization to methamphetamine in mice
European Journal of Pharmacology, 234 (1993) 67-75
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© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 52973
Suppressive effect of cycloheximide on behavioral sensitization to methamphetamine in mice K a z u a k i S h i m o s a t o ~,b a n d Taiichi Saito a a
Department of Pharmacology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-01, Japan and b Behat~ioral Pharmacology and Genetics Laboratory, Preclinical Pharmacology Branch, NIDA Addiction Research Center, P.O. Box 5180, Baltimore, MD 21224, USA Received 11 November 1992, revised MS received 22 December 1992, accepted 5 January 1993
The effect of a protein synthesis inhibitor, cycloheximide, on behavioral sensitization to methamphetamine was investigated in mice. As indicated by the sensitization tests, repeated injection of methamphetamine (2 mg/kg i.p.) at intervals of 3 and 4 days resulted in a progressive augmentation of the locomotor-stimulating effect of methamphetamine. This phenomenon, called locomotor sensitization, was attenuated by simultaneous treatment with cycloheximide (120 mg/kg i.p.) at the time of stimulant injection. In contrast, when mice were treated with cycloheximide 4 h after stimulant injection, locomotor activity was progressively augmented in the same way as observed in mice receiving repeated injections of methamphetamine alone. On challenge, it was noted that locomotor activity was significantly higher in mice injected repeatedly with the stimulant alone and in those mice treated with the inhibitor 4 h after the stimulant injection compared to the saline-treated control mice. However, mice that had been simultaneously treated with cycloheximide and methamphetamine showed almost the same locomotor activity as the saline-treated control mice. These observations indicated that the locomotor sensitization to methamphetamine was possibly suppressed by simultaneous treatment with cycloheximide. We then examined the dose- and time-dependent nature of the effect of cycloheximide on locomotor sensitization. The stimulation of locomotion observed after repeated injection of the stimulant at a dose of 1.5 mg/kg was significantly attenuated by simultaneous treatment with 120 or 240 mg/kg of cycloheximide, but not by treatment with 60 mg/kg of the inhibitor. However, all the treatments failed to suppress the development of locomotor sensitization elicited by 3 mg/kg of methamphetamine. The locomotor stimulation observed after repeated injection of 1.5 mg/kg methamphetamine was effectively suppressed by treatment with 120 mg/kg of cycloheximide given either simultaneously or 2 h after the stimulant injection. Treatment 4 h after methamphetamine did not suppress the stimulation of locomotion. These results suggest that the protein synthesis inhibitor has wsuppressive effect on the development of behavioral sensitization to repeated injections of stimulant drugs in a dose- and time-dependent manner. Locomotion; Sensitization; Methamphetamine; Cycloheximide; (Mouse)
I. Introduction
Repeated administration of stimulant drugs such as methamphetamine, amphetamine or cocaine results in a progressive increase in the locomotor and stereotyped responses to the drugs in animals (Tilson and Rech, 1973; Roy et al., 1978; Hirabayashi and Alam, 1981). This phenomenon, termed behavioral sensitization, has been used as an animal model of stimulant-induced psychosis in humans (Robinson and Becker, 1986). Many different mechanisms have been proposed to explain this phenomenon. Some studies have presented evidence for the involvement of stimulants or
Correspondence to: K. Shimosato, Behavioral Pharmacology and Genetics Laboratory, Preclinical Pharmacology Branch, NIDA Addiction Research Center, P.O. Box 5180, Baltimore, MD 21224, USA.
their metabolites accumulated in the cerebrum (Segal and Mandell, 1974; Shimosato and Watanabe, 1989), whereas other research has provided evidence against this interpretation (Browne and Segal, 1977). There have also been disputes concerning the neurochemical changes in the brain. Although it has been suggested that chronic injection with amphetamine or methamphetamine causes up-regulation of postsynaptic dopamine receptors (Kilbey and Ellinwood, 1977; Nishikawa et al., 1983), many studies have shown no evidence for increased postsynaptic receptor sensitivity after chronic injections (Robinson and Becker, 1986). Furthermore, it has been proposed that changes in presynaptic dopaminergic function account for the sensitization (Robinson et al., 1988; Ichikawa, 1988). In addition, some findings have indicated that conditioning to the cues associated with drug administration and behavioral testing may contribute to the phenomenon (Tilson
68 and Rech, 1973; Hirabayashi and Alam, 1981), while other results have suggested that this is not so (Browne and Segal, 1977; Robinson and Becker, 1986). Thus, the mechanisms underlying the phenomenon have not yet been elucidated. Conditioned drug effects have been defined as the effects which are conditioned to neutral stimuli attending drug administration and behavioral testing (Tilson and Rech, 1973). A possible explanation for conditioned drug effects is that optimal conditioning for learning is presented during behavioral testing, and the learned response is maintained by the reinforcing properties of the drugs. It has been proposed that many phenomena, such as learning or long-term memory, drug tolerance, kindling, and long-term potentiation, may be dependent upon de novo protein synthesis in the brain (Davis and Squire, 1984; Goelet et al., 1986). With regard to conditioned drug effects, it has also been reported that conditioned circling behavior is completely suppressed by treatment with cycloheximide, a protein synthesis inhibitor, in rats with a unilateral 6-hydroxydopamine lesion of the substantia nigra (Silverman et al., 1989). If the behavioral sensitization following repeated injection of stimulant drugs results from conditioned drug effects which are mediated by a process similar to learning or long-term memory, the development of behavioral sensitization may be prevented by treatment with protein synthesis inhibitors. In the present study, we investigated the effect of cycloheximide on the behavioral sensitization to the locomotor-stimulating effect of repeated administration of methamphetamine to mice. The results indicate that treatment with cycloheximide, when given either simultaneously or shortly after the stimulant injection, probably prevents the development of behavioral sensitization to methamphetamine in a dose- and time-dependent manner.
2. Materials and methods
2. l. Animals and drugs
Male mice of the ICR strain (Charles River Japan, Inc.), weighing 25-32 g at the start of experiments, were used (N = 6-10 in each group). The animals were kept four per cage under our standard animal facility conditions on a constant light/dark cycle (illuminated at 7 : 00-21 : 00) and at 23 _+ 1°C. Food and water were freely available except during the behavioral test. d-Methamphetamine hydrochloride (Dainippon Pharmaceutical Co., Japan) was dissolved in physiological saline and injected i.p. at a dose of either 1.5, 2 or 3 mg/kg (0.005 m l / g body weight). Cycloheximide (Sigma Chemical Co., U.S.A.) was dissolved in saline
and administered i.p. in a dose of either 60, 120 or 240 mg/kg (0.01 m l / g body weight for 60 and 120 mg/kg, or 0.02 m l / g for 240 mg/kg). Control animals were injected with an equivalent volume of saline. 2.2. Behauioral assessment
The experiment consisted of three or five sensitization tests and a challenge test. Locomotor activity was measured with sensitivity-matched activity meters (Animex; Muromachi Kikai Co., Japan and LKB Farad, Sweden) in a quiet room (20-25°C). Measurements were made during the 1st, 3rd and 5th sensitization sessions and the challenge session. In the 2nd and 4th sessions, mice received drug injections as during the odd-numbered sessions but locomotor activity was not measured. By measuring locomotor activity only in the odd-numbered sessions and by staggering sets of mice, twice as many animals could be tested in the same period. For each session, at least 3 h before drug injection, mice were placed in individual transparent test boxes (30 × 40 × 30 cm) in order to become adjusted to the experimental surroundings. During this period, spontaneous locomotion was assessed for 20 min in each group. In the sensitization test of one experiment, each mouse was assigned to one of four groups and was injected i.p. with one of the following combinations of drugs: methamphetamine (2 mg/kg) + saline, methamphetamine + cycloheximide (120 mg/kg), saline + cycloheximide or saline alone. After administration, locomotor activity was measured at 10-min intervals over 180 min. Another group of mice was injected i.p. with methamphetamine (2 mg/kg) and then locomotor activity was measured for 180 min. Four hours after the injection, these mice were treated with cycloheximide (120 mg/kg). These administrations were repeated 5 times at intervals of 3 and 4 days. In the challenge test, mice from each group were challenged with the same dose of methamphetamine 7 days after the last sensitization test. In the dose-effect experiment, we examined the effect of 60, 120 and 240 mg/kg of cycloheximide on the locomotor sensitization elicited by 1.5 or 3 mg/kg of methamphetamine. Based on these results, we studied the time course of the suppression of locomotor sensitization using a treatment combination of 120 mg/kg of cycloheximide and 1.5 mg/kg of methamphetamine. The effect of various times of cycloheximide treatment were examined after its administration simultaneously with, or 2 or 4 h after injection with methamphetamine. 2.3. Statistics
The data were analyzed with repeated measures analysis of variance (ANOVA) techniques except when
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indicated otherwise. To evaluate the significance of difference among the factors, a post-hoe analysis was done with Scheff6's S-test. For the locomotion data, the analyses were carried out with data obtained during the initial 60-min and 120-min intervals. As there was no difference between the results for these two periods, only the statistics computed using the data for the initial 60-min interval are presented in the text.
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3. Results
3.1. Toxicity o
The general toxicity of cycloheximide was examined by evaluating its effects on weight gain and spontaneous locomotion during the acclimatization period. Administration of cycloheximide (120 m g / k g ) with either methamphetamine (2 m g / k g ) or saline produced no obvious signs of toxicity during behavioral assessment. There was no overall significant effect of the various drug injections on weight gain (F(4,28) = 1.217, P > 0.05) (fig. 1). In addition, the injections did not have a significant effect on spontaneous locomotion during the initial 20-min acclimatization period in each session (F(3,32)= 0.064, P > 0.05 for the sensitization test, repeated measures A_NOVA; F(4,40) = 0.329, P > 0.05 for the challenge test, one-way A.NOVA) (fig. 2). 3.2. Sensitization test
The effects of repeated administration of methamphetamine (2 m g / k g ) a n d / o r cycloheximide (120
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Fig. 1. Weight gain in mice. Animals were injected i.p. with either saline alone (o), MAP (2 m g / k g ) + s a l i n e (©), M A P + C X M (120 mg/kg) (zx), or saline + CXM ( • ) . Another group of mice were injected i.p. with methamphetamine and then treated with cycloheximide 4 h after methamphetamine (O). These injections were repeated 5 times at intervals of 3 and 4 days; 7 days after the last sensitization test the same dose of methamphetamine was given. Each bar represents S.E.M. Abbreviation: MAP, methamphetamine; CXM, cycloheximide.
1st
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Fig. 2. Spontaneous locomotion of mice during the initial 20-min acclimatization period of the sessions in mice. Animals were treated as described in the legend of fig. 1, and are represented by various designs of columns as follows: (m), MAP+saline; (1~) M A P + C X M 0 h; (1~) M A P + C X M 4 h; ([]) saline+CXM; (O) saline. Other details are described in the legend of fig. 1.
m g / k g ) on locomotor activity during the sensitization test were examined, and the results are illustrated in fig. 3. The statistical analysis revealed that there were significant effects of treatment (F(3,32)= 65.616, P < 0.0001), session (F(2,32) = 15.647, P < 0.0001) and time (F(5,32) = 16.884, P < 0.0001). This significance was because the locomotor activity of mice simultaneously injected with methamphetamine and cycloheximide was higher than that of any other group of mice, and because the locomotor activity of mice injected with saline and cycloheximide was lower than that of any other group of mice. The analysis revealed a significant interactive effect of treatment × time (F(15,32)= 16.494, P < 0.0001). This significant interaction reflects a difference among the groups in the time course for for locomotor stimulation across the three sessions. Concomitant administration of methamphetamine with saline produced a stimulation of locomotion, with a maximal effect at 40 min (fig. 3a). Thereafter the effect gradually decreased, locomotion reaching baseline levels within 180 min. In subsequent sessions, the stimulation of locomotion increased progressively (F(2,8) = 15.363, P < 0.001). In contrast, no such stimulation was observed after repeated simultaneous administration of the stimulant with cycloheximide (F(2,8) = 2.807, P > 0.05) (fig. 3b). At the peak level, however, this regimen produced a greater stimulation than co-administration of the stimulant with saline, or administration of the stimulant followed by treatment with the inhibitor 4 h later (F(2,24) = 6.491, P < 0.01). When mice were injected with methamphetamine repeatedly, and then treated with cycloheximide 4 h later, the stimulation of locomotion progressively in-
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creased in much the same manner as that seen after the stimulant and saline (F(2,8) = 8.376, P < 0.01) (fig. 3c). The time course for the stimulation of locomotion was significantly different among the three groups of mice injected with methamphetamine and either saline or the inhibitor (F(10,24) = 10.226, P < 0.0001). Co-administration of the stimulant with cycloheximide resuited in a much more rapid increase in locomotor activity than in the group treated with stimulant and saline. However, the increased activity was of shorter duration than that produced by the stimulant with saline, with activity returning to baseline levels within 120 min. Cycloheximide alone had some effect on locomotion in the 1st and 3rd sessions (fig. 3d). When the inhibitor was administered concomitantly with saline, there was slight hyperlocomotion, which reached a maximum at 10 min and then declined sharply to the basal level within 50 min. However, this hyperlocomotion was not observed after the 5th injection of the inhibitor and saline. The administration of saline alone did not stimulate locomotion. The time course of the locomotor stimulation of mice injected with saline alone was very similar to that observed after the 5th injection with
cycloheximide plus saline (data not shown; see also fig. 6a). 3.3. Challenge test Mice in each group were challenged with 2 m g / k g of methamphetamine 7 days after the last sensitization test; the results are shown in fig. 4. The statistical analysis revealed a significant difference in treatment effect among the groups (F(4,25)= 8.244, P < 0.001). In the two groups of mice that had repeatedly received methamphetamine together with either concomitant saline or cycloheximide 4 h later, a significant increase in locomotion was noted as compared to that of the saline-treated control mice (P < 0.05; Scheff6's S). On the other hand, in mice that had been repeatedly injected with the stimulant and the inhibitor, locomotor activity was not significantly different from that of the saline-treated control mice (P > 0.05; Scheff6's S). In addition, statistical analysis of data obtained from the interval 70-120 rain, indicated a significant decrease in locomotion in the saline + cycloheximideinjected mice when compared to that of mice receiving the stimulant and then the inhibitor (P < 0.05; Scheff6's S), but not when compared to any other groups.
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Fig. 3. Time course of changes in the locomotor stimulating effect of methamphetamine (2 mg/kg) after the 1st (o), 3rd ( / , ) and 5th ( o ) injection of the drugs in mice. Animals received i.p. either MAP + saline (a), MAP + CXM (120 mg/kg) (b), or saline + CXM (d). Another group of mice was injected i.p. with methampbetamine and then treated with cycloheximide 4 h after methamphetamine (c). Other details are described in the legend of fig. 1.
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3.4. Dose-dependent nature of the effect of cycloheximide To characterize the effect of cycloheximide further, we examined the effects of different doses of cycloheximide on the stimulation of locomotion produced by low (1.5 m g / k g ) and high (3 m g / k g ) doses of methamphetamine. Injections of methamphetamine and various doses of cycloheximide (60, 120 or 240 m g / k g ) were repeated 3 times at 3- and 4-day intervals. The challenge dose of methamphetamine was injected 7 days later. The statistical analyses demonstrated a significant stimulation of locomotion after repeated injection of 1.5 m g / k g methamphetamine together with concomitant treatment with either saline or 60 m g / k g of cycloheximide (F(1,9)= 173.267 and 9.761, P < 0.0001 and 0.05 for saline and 60 m g / k g of the inhibitor, respectively) (data not shown). On the other hand, although a substantial difference in locomotion between the 1st and 3rd sessions was noted in mice treated with 120 m g / k g of the inhibitor (F(1,9)= 5.259, P = 0.0475), there was no difference in locomotor activity between the sessions for mice treated with 240 m g / k g of the inhibitor (F(1,5)= 4.634, P > 0.05) (data not shown). These mice were challenged with 1.5 m g / k g of the stimulant and the time course of the stimulation of locomotion was examined (fig. 5a). There was a significant difference in the time course of the locomotor stimulation among the groups (F(4,40)= 4.190, P < 0.01). When compared to the saline-treated control mice, mice treated with saline or 60 m g / k g of cycloheximide showed a stimulation Of locomotion (P < 0.05 and P = 0.11 for treatments with saline and 60 m g / k g
400"
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Fig. 4. Time course of changes in the locomotor-stimulating effect of
the challenge dose of methamphetamine (2 mg/kg) in mice. The challenge session was 7 days after the last sensitization test. Animals are represented as follows: (o) saline alone; ( o ) M A P + saline; (zx) M A P + C X M 0 h; ([3) M A P + C X M 4 h; (11) s a l i n e + C X M . Other details are described in the legend of fig. 1.
(a) MAP 1.5
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Fig. 5. Time course of changes in the locomotor-stimulating effect of the challenge dose of methamphetamine during the challenge session 7 days after the last sensitization test. The panels (a) and (b) represent the results obtained after injection of the challenge doses of 1.5 and 3 mg/kg, respectively, in mice that had been given saline alone (e) or 1.5 m g / k g methamphetamine with either saline (o), 60 (A), 120 ([]) or 240 (m) m g / k g of cycloheximide. Other details are described in the legend of fig. 1.
of the inhibitor, respectively; Scheff6's S). However, no stimulation of locomotion was observed in mice treated with 120 or 240 m g / k g of cycloheximide. Repeated measures A N O V A analysis of data for the sensitization test following repeated injection of 3 m g / k g of methamphetamine revealed significant differences in locomotion between the 1st and 3rd session in all groups of mice (F(1,9) = 27.555, 9.969, 7.556 and 11.591, P < 0.001, 0.05, 0.05 and 0.01 for the treatment with saline, 60, 120 and 240 m g / k g of the inhibitor, respectively) (data not shown). The mice were challenged with 3 m g / k g methamphetamine 7 days later; the results are shown in fig. 5b. There was a significant difference in the time course of the stimulation of locomotion among the groups (F(4,43)= 3.425, P < 0.05). Compared to the saline-treated control mice, mice treated with 60 m g / k g of the inhibitor showed a clear stimulation of locomotion (P < 0.05; Scheff6's S), whereas mice treated with saline showed a slight simulation (P = 0.12; Scheff6's S). Mice treated with 120 and 240 m g / k g of cycloheximide also showed a higher locomotor activity than the saline-treated control mice.
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Statistical significance could be demonstrated only by using a two-way A N O V A (P < 0.0001; Scheff6's S), but not by using repeated measures ANOVA.
3.5. Time-dependent nature of cycloheximide effects The effect of cycloheximide treatment at various times was studied by administration of 1.5 m g / k g methamphetamine followed by treatment with 120 m g / k g of cycloheximide 0, 2 or 4 h later. The injections were repeated 3 times; the results are shown in fig. 6. As described previously, saline injection alone resulted in a slight stimulation of locomotion only in the first 10-min interval, and thereafter locomotor activity returned to baseline levels (fig. 6a). There was no difference in the time course of the stimulation of locomotion between the 1st and 3rd sessions (F(1,9)= 0.097, P > 0.05). Co-administration of methamphetamine with cycloheximide produced a stimulation of locomotion, with a peak at 20 or 30 min (fig. 6b). As shown in fig. 3b, this peak was earlier than that (at 50 min) for mice administered the stimulant followed by
treatment with cycloheximide 2 or 4 h later. There was no significant difference in the time course of locomotion between the 1st and 3rd sessions in these mice (F(1,9) = 0.967, P > 0.05). Mice administered methamphetamine followed by cycloheximide 2 h later showed two peaks of increased locomotion at 50 and 130 min (fig. 6c). The latter peak seemed to result from an effect of cycloheximide injection. The analysis with A N O V A demonstrated no difference in the stimulation of locomotion between the 1st and 3rd sessions in the mice (F(1,9) = 0.019, P > 0.05). In contrast to these results, there was a large stimulation of locomotor activity in mice administered the stimulant followed by cycloheximide 4 h later (F(1,7) = 10.585; P < 0.05) (fig. 6d). This stimulation was again verified in the challenge test session (fig. 7). The statistical analyses revealed a significant difference in the time course of locomotion across the treatments (F(3,32) = 3.970, P < 0.05). Locomotion was significantly stimulated only in mice that had been given cycloheximide 4 h later after the methamphetamine treatment (P < 0.05; Scheff6's S), while in the other two groups there was no difference
(a) Saline Control
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Fig. 6. Time course of changes in the locomotor-stimulating effect of methamphetamine (1.5 mg/kg) after the 1st (©) and 3rd ([:3) injection of the drugs in mice. One group of mice was injected with saline alone (a). The other groups were injected with methamphetamine, and then treated with cycloheximide (120 mg/kg) either at 0 h (b), 2 h (c) or 4 h (d) after methamphetamine. Other details are described in the legend of fig. 1.
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200"
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Time after Inject (mln) Fig. 7. Time course of changes in the locomotor stimulating effect of the challenge dose of m e t h a m p h e t a m i n e (1.5 m g / k g ) during the challenge session 7 days after the last sensitization test. Animals are represented as follows: (e) saline alone; (©) M A P + C X M 0 h; (zx) M A P + C X M 2 h; ([]) M A P + C X M 4 h. Other details are described in the legend of fig. 1.
compared to the saline-treated control mice (P > 0.05; Scheff6's S).
4. Discussion
The present study documents the differential effects of cycloheximide on the locomotor-stimulating effect of methamphetamine depending on the treatment regimen. Repeated administration of methamphetamine resulted in a progressive increase in its locomotorstimulating effect during the sensitization tests and resulted in a significantly higher locomotor activity after the challenge dose as compared to the locomotor activity of saline-treated control mice. This phenomenon has been recognized as behavioral sensitization (Tilson and Rech, 1973; Hirabayashi and Alam, 1981). The locomotor sensitization was remarkably attenuated by simultaneous treatment with high doses of cycloheximide. However, treatment with cycloheximide 4 h later failed to suppress the locomotor sensitization. Several points need to be considered when determining the possible mechanism underlying these paradoxical results. In the present study cycloheximide was injected in a dose of 120 m g / k g , a dose that has been commonly used in the study of long-term memory in mice (Barondes and Cohen, 1967, 1968a,b; Squire and Barondes, 1976). Large doses of protein synthesis inhibitors are known to have toxic effects such as diarrhea, decreased food and water intake, and weight loss (Davis and Squire, 1984). However, the doses of cycloheximide used in this study did not cause significant signs of toxicity. There were no differences in weight gain and spontaneous locomotion among the groups. In addition, mice repeatedly administered methamphetamine and then treated with cycloheximide 4 h
later showed behavioral sensitization in the same manner as mice co-administered the stimulant with saline. The reason we were unable to observe any toxic effects may lie in our protocol, in which the cycioheximide treatments were repeated every 3 or 4 days. Thus it does not seem that toxic side effects of cycloheximide, if any, contributed to the suppression of behavioral sensitization observed in this study. Cycloheximide has been reported to stimulate locomotor activity for 30 min and then to depress it for several hours in mice (Segal et al., 1971). Indeed, the present data revealed a transient stimulation of locomotion by cycloheximide itself. In mice co-administered low doses of the stimulant and cycloheximide, locomotor activity reached a higher maximum faster, and declined more rapidly than in mice co-administered the stimulant with saline. Furthermore, cycloheximide alone caused some hyperlocomotion at 10 min. Based on these findings, the lack in a progressive stimulation of locomotion in the sensitization test in mice co-administered low doses of the stimulant and the inhibitor could possibly be due to the fact that the animals were already displaying a ceiling effect during the 1st session. However, the present study also provides evidence against this interpretation. Co-administered cycloheximide could not prevent the locomotor stimulation observed after repeated administration of a high dose (3 m g / k g ) of methamphetamine, which produced a greater stimulation than low doses of the stimulant co-administered with the inhibitor. In addition, the locomotor sensitization was severely suppressed by the 2-h treatment with cycloheximide, which never interacted with the stimulant at 50 and 60 min. It seems, therefore, that an interaction between these drugs cannot explain the results. It has been shown that protein synthesis inhibitors markedly reduce the rate of synthesis of a rapid turning over pool of dopamine and norepinephrine, the primary source for catecholaminergic neurotransmission, for at least 6.5 h after treatment (Bloom et al., 1977; Lundgren and Carr, 1978). It is thus conceivable that the reduced rate of catecholamine synthesis could account for the suppression of locomotor sensitization observed in this study. If this speculation were true, we could expect a lower rate of synthesis of catecholamines in mice treated with amphetamine and cycloheximide than in mice treated with amphetamine and saline. However, when measured at 1 and 1.5 h after treatment, there is no difference in the rate of dopamine and norepinephrine synthesis between animals treated with the stimulant plus either saline or the inhibitor (Bloom et al., 1977). Therefore, these results suggest that co-administered cycloheximide is unlikely to prevent the development of locomotor sensitization by inhibiting the synthesis of norepinephrine or dopamine.
74 The sensitization produced by 1.5 m g / k g methamphetamine was markedly attenuated by concurrent treatment with 120 or 240 m g / k g of cycloheximide, but not by treatment with 60 m g / k g of cycloheximide. This result is similar to the finding that mice showed impairment of long-term memory when treated with 150 or 200 m g / k g of cycloheximide at a training test, but not when treated with less than 100 m g / k g of the inhibitor (Kameyama et al., 1986). On the other hand, the behavioral sensitization produced by 3 m g / k g methamphetamine was not affected by treatment with even 240 m g / k g of cycloheximide. It has been demonstrated that the retention of long-term memory is dependent on the voltage of the electric shock delivered in the training test (Kameyama et al., 1986). It has also been suggested that the degree of impairment of long-term memory is related to the extent of inhibition of cerebral protein synthesis during or shortly after training (Davis and Squire, 1984). In addition, the action of cycloheximide has been revealed to be relatively short (Barondes and Cohen, 1968a; Squire and Barondes, 1976), and the locomotor stimulating effects of 3 m g / k g m e t h a m p h e t a m i n e lasted relatively longer. Thus the effects of 3 m g / k g m e t h a m p h e t a m i n e on neurons are too strong to be extinguished by cycloheximide at the dose used. This study also demonstrated the time-dependent nature of the effect of cycloheximide on the locomotor sensitization. The sensitization produced by 1.5 m g / k g m e t h a m p h e t a m i n e was suppressed by the treatment given at either 0 h or 2 h after the stimulant injection, but not by the treatment given 4 h later. It has been shown that when inhibition of cerebral protein synthesis is initiated 1 h or longer after training, long-term memory is not affected (Barondes and Cohen, 1968b; K a m e y a m a et al., 1986). Cycloheximide at a dose of 120 m g / k g has been reported to inhibit by more than 90% cerebral protein synthesis for up to 1.5 h after injection in mice. Thereafter, the inhibition decreases to less than 80% at 2 h, and to 20% at 3 h (Barondes and Cohen, 1968a; Squire and Barondes, 1976). Additionally, it has been suggested that at least 90% inhibition of protein synthesis during training is necessary to impair long-term memory in mice (Barondes and Cohen, 1967). In the present study we have no data about the time course of the inhibition of cerebral protein synthesis. However, based on these findings, the present results suggest that the development of behavioral sensitization depends, at least in part, upon cerebral proteins synthesized 2 h after the injection of the stimulant. The present results have great analogy to the finding that protein synthesis inhibitors, injected just before or shortly after training, impair long-term memory in animals (Barondes and Cohen, 1967; Cohen and Barondes, 1968; Davis and Squire, 1984). It has been re-
ported that behavioral sensitization may result from conditioned drug effects which are, in part, mediated by the mechanism underlying memory or learning of stimuli attending drug injection and the experimental environment (Tilson and Rech, 1973; Hirabayashi and Alam, 1981). It has been reported that conditioned circling behavior is suppressed by treatment with cycloheximide in rats with unilateral 6-hydroxydopamine lesion of substantia nigra (Silverman et al., 1989). Other findings similar to the present results have been described in a number of p h e n o m e n a related to neural plasticity. It has been observed that co-administration of cycloheximide with morphine prevents the development of tolerance to and physical dependence on morphine in mice (Way et al., 1968; Loh et al., 1969). It has also been reported that sensitization to the morphineproduced stimulation of locomotion is blocked by coadministration of cycloheximide in mice (Shuster et al., 1975). Treatment with anisomycin, another protein synthesis inhibitor, during amygdaloid kindling has been revealed to suppress the development of generalized seizures in mice (Cain et al., 1980). These findings strongly suggest that plastic changes in the central neurons involved in p h e n o m e n a such as long-term memory, drug conditioned effects, drug tolerance and dependence, and kindling require de novo protein synthesis in the brain (Davis and Squire, 1984; Goelet et al., 1986). Moreover, the present results also suggest that the development of behavioral sensitization depends upon cerebral protein synthesis within 2 h after m e t h a m p h e t a m i n e administration.
Acknowledgements This research was supported in part by a grant from the Project Research Foundation of the Kawasaki Medical School, Japan (No. 2-413). The authors are grateful to R.J. Marley, Ph.D. and N. Goodman, M.S., NIDA-Addiction Research Center, U.S.A., for helpful comments and suggestions on the manuscript.
References Barondes, S.H. and H.D. Cohen, 1967, Delayed and sustained effect of acetoxycycloheximideon memory in mice, Proc. Natl. Acad. Sci. U.S.A. 58, 157. Barondes, S.H. and H.D. Cohen, 1968a, Arousal and the conversion of 'short-term' to 'long-term' memory, Proc. Natl. Acad. Sci. U.S.A. 61, 923. Barondes, S.H. and It.D. Cohen, 1968b, Memory impairment after subcutaneous injection of acetoxycycloheximide,Science 160, 556. Bloom, A.S., E.E. Quinton and L.A. Carr, 1977, Effects of cycloheximide, diethyldithiocarbamate, and d-amphetamine on protein and catecholamine biosynthesis in mouse brain, Neuropharmacology 16, 411. Browne, R.G. and D.S. Segal, 1977, Metabolic and experiential factors in the behavioral response to repeated amphetamine, Pharmacol. Biochem. Behav. 6, 545.
75 Cain, D.P., M.E. Corcoran and W.A. Ataines, 1980, Effects of protein synthesis inhibition on kindling in the mouse, Exp. Neurol. 68, 409. Cohen, H.D. and S.H. Barondes, 1968, Effect of acetoxycycloheximide on learning and memory of a light-dark discrimination, Nature 218, 271. Davis, H.P. and L.R. Squire, 1984, Protein synthesis and memory: a review, Psychol. Bull. 96, 518. Goelet, P., V.F. Castellucci, S. Schacher and E.R. Kandel, 1986, The long and the short of long-term memory - a molecular framework, Nature 322, 419. Hirabayashi, M. and M.R. Alam, 1981, Enhancing effect of methamphetamine on ambulatory activity produced by repeated administration in mice, Pharmacol. Biochem. Behav. 15, 925. Ichikawa, J., 1988, Change of behavior and central monoaminergic systems in the rat after repeated methamphetamine pretreatment: presynaptic regulatory mechanism, Jap. J. Psychopharmacol. 8, 389. Kameyama, T., T. Nabeshima and T. Kozawa, 1986, Step-down type passive avoidance and escape learning method. Suitability for experimental methods, J. Pharmacol. Meth. 16, 39. Kilbey, A.E. and E.H. Ellinwood, 1977, Reverse tolerance to stimulant-induced abnormal behavior, Life Sci. 20, 1063. Loh, H.H., F.-H. Shen and E.L. Way, 1969, Inhibition of morphine tolerance and physical dependence development and brain serotonin synthesis by cycloheximide, Biochem. Pharmacol. 18, 2711. Lundgren, P. and L.A. Carr, 1978, Effects of anisomycin and CNS stimulants on brain catecholamine synthesis, Pharmacol. Biochem. Behav. 9, 559. Nishikawa, T., N. Nagata, M. Takashima and M. Toru, 1983, Behavioral sensitization and relative hyperresponsiveness of striatal and limbic dopaminergic neurons after repeated methamphetamine treatment, Eur. J. Pharmacol. 88, 195. Robinson, T.E. and J.B. Becker, 1986, Enduring changes in brain and behavior produced by chronic amphetamine administration:
A review and evaluation of animal models of amphetamine psychosis, Brain Res. Rev. 11, 157. Robinson, T.E., P.A. Jurson, J.A. Bennett and K.M. Bentgen, 1988, Persistent sensitization of dopamine neurotransmission in ventral striatum (nucleus accumbens) produced by prior experience with (+)-amphetamine: a microdialysis study in freely moving rats, Brain Res. 462, 211. Roy, S.N., A.K. Battacharyya, S. Pradhan and S.N. Pradhan, 1978, Behavioral and neurochemical effects of repeated administration of cocaine in rats, Neuropharmacology 17, 559. Segal, D.S. and A.J. Mandell, 1974, Long-term administration of d-amphetamine: progressive augmentation of motor activity and stereotypy, Pharmacol. Biochem. Behav. 2, 249. Segal, D.S., L.R. Squire and S.H. Barondes, 1971, Cycloheximide: its effects on activity are dissociable from its effects on memory, Science 172, 82. Shimosato, K. and S. Watanabe, 1989, Modification of behavioral responses to methamphetamine evoked by the stimulant's metabolite p-hydroxynorephedrine in rats, Pharmacol. Biochem. Behav. 33, 423. Shuster, L., G.W. Webster and G. Yu, 1975, Perinatal narcotic addiction in mice: sensitization to morphine stimulation, Addict. Dis. 2, 277. Silverman, P.B., N.P. Baruch and K.A. Schulz, 1989, One trial conditioning with apomorphine is blocked by cycloheximide, Pharmacol. Biochem. Behav. 34, 663. Squire, L.R. and S.H. Barondes, 1976, Amnetic effect of cycloheximide not due to depletion of a constitutive brain protein with short half-life, Brain Res. 103, 183. Tilson, H.A. and R.H. Rech, 1973, Conditioned drug effects and absence of tolerance to d-amphetamine induced motor activity, Pharmacol. Biochem. Behav. 1, 149. Way, E.L., H.H. Loh and F.-H. Shen, 1968, Morphine tolerance, physical dependence, and synthesis of brain 5-hydroxytryptamine, Science 162, 1290.
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© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 52973
Suppressive effect of cycloheximide on behavioral sensitization to methamphetamine in mice K a z u a k i S h i m o s a t o ~,b a n d Taiichi Saito a a
Department of Pharmacology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-01, Japan and b Behat~ioral Pharmacology and Genetics Laboratory, Preclinical Pharmacology Branch, NIDA Addiction Research Center, P.O. Box 5180, Baltimore, MD 21224, USA Received 11 November 1992, revised MS received 22 December 1992, accepted 5 January 1993
The effect of a protein synthesis inhibitor, cycloheximide, on behavioral sensitization to methamphetamine was investigated in mice. As indicated by the sensitization tests, repeated injection of methamphetamine (2 mg/kg i.p.) at intervals of 3 and 4 days resulted in a progressive augmentation of the locomotor-stimulating effect of methamphetamine. This phenomenon, called locomotor sensitization, was attenuated by simultaneous treatment with cycloheximide (120 mg/kg i.p.) at the time of stimulant injection. In contrast, when mice were treated with cycloheximide 4 h after stimulant injection, locomotor activity was progressively augmented in the same way as observed in mice receiving repeated injections of methamphetamine alone. On challenge, it was noted that locomotor activity was significantly higher in mice injected repeatedly with the stimulant alone and in those mice treated with the inhibitor 4 h after the stimulant injection compared to the saline-treated control mice. However, mice that had been simultaneously treated with cycloheximide and methamphetamine showed almost the same locomotor activity as the saline-treated control mice. These observations indicated that the locomotor sensitization to methamphetamine was possibly suppressed by simultaneous treatment with cycloheximide. We then examined the dose- and time-dependent nature of the effect of cycloheximide on locomotor sensitization. The stimulation of locomotion observed after repeated injection of the stimulant at a dose of 1.5 mg/kg was significantly attenuated by simultaneous treatment with 120 or 240 mg/kg of cycloheximide, but not by treatment with 60 mg/kg of the inhibitor. However, all the treatments failed to suppress the development of locomotor sensitization elicited by 3 mg/kg of methamphetamine. The locomotor stimulation observed after repeated injection of 1.5 mg/kg methamphetamine was effectively suppressed by treatment with 120 mg/kg of cycloheximide given either simultaneously or 2 h after the stimulant injection. Treatment 4 h after methamphetamine did not suppress the stimulation of locomotion. These results suggest that the protein synthesis inhibitor has wsuppressive effect on the development of behavioral sensitization to repeated injections of stimulant drugs in a dose- and time-dependent manner. Locomotion; Sensitization; Methamphetamine; Cycloheximide; (Mouse)
I. Introduction
Repeated administration of stimulant drugs such as methamphetamine, amphetamine or cocaine results in a progressive increase in the locomotor and stereotyped responses to the drugs in animals (Tilson and Rech, 1973; Roy et al., 1978; Hirabayashi and Alam, 1981). This phenomenon, termed behavioral sensitization, has been used as an animal model of stimulant-induced psychosis in humans (Robinson and Becker, 1986). Many different mechanisms have been proposed to explain this phenomenon. Some studies have presented evidence for the involvement of stimulants or
Correspondence to: K. Shimosato, Behavioral Pharmacology and Genetics Laboratory, Preclinical Pharmacology Branch, NIDA Addiction Research Center, P.O. Box 5180, Baltimore, MD 21224, USA.
their metabolites accumulated in the cerebrum (Segal and Mandell, 1974; Shimosato and Watanabe, 1989), whereas other research has provided evidence against this interpretation (Browne and Segal, 1977). There have also been disputes concerning the neurochemical changes in the brain. Although it has been suggested that chronic injection with amphetamine or methamphetamine causes up-regulation of postsynaptic dopamine receptors (Kilbey and Ellinwood, 1977; Nishikawa et al., 1983), many studies have shown no evidence for increased postsynaptic receptor sensitivity after chronic injections (Robinson and Becker, 1986). Furthermore, it has been proposed that changes in presynaptic dopaminergic function account for the sensitization (Robinson et al., 1988; Ichikawa, 1988). In addition, some findings have indicated that conditioning to the cues associated with drug administration and behavioral testing may contribute to the phenomenon (Tilson
68 and Rech, 1973; Hirabayashi and Alam, 1981), while other results have suggested that this is not so (Browne and Segal, 1977; Robinson and Becker, 1986). Thus, the mechanisms underlying the phenomenon have not yet been elucidated. Conditioned drug effects have been defined as the effects which are conditioned to neutral stimuli attending drug administration and behavioral testing (Tilson and Rech, 1973). A possible explanation for conditioned drug effects is that optimal conditioning for learning is presented during behavioral testing, and the learned response is maintained by the reinforcing properties of the drugs. It has been proposed that many phenomena, such as learning or long-term memory, drug tolerance, kindling, and long-term potentiation, may be dependent upon de novo protein synthesis in the brain (Davis and Squire, 1984; Goelet et al., 1986). With regard to conditioned drug effects, it has also been reported that conditioned circling behavior is completely suppressed by treatment with cycloheximide, a protein synthesis inhibitor, in rats with a unilateral 6-hydroxydopamine lesion of the substantia nigra (Silverman et al., 1989). If the behavioral sensitization following repeated injection of stimulant drugs results from conditioned drug effects which are mediated by a process similar to learning or long-term memory, the development of behavioral sensitization may be prevented by treatment with protein synthesis inhibitors. In the present study, we investigated the effect of cycloheximide on the behavioral sensitization to the locomotor-stimulating effect of repeated administration of methamphetamine to mice. The results indicate that treatment with cycloheximide, when given either simultaneously or shortly after the stimulant injection, probably prevents the development of behavioral sensitization to methamphetamine in a dose- and time-dependent manner.
2. Materials and methods
2. l. Animals and drugs
Male mice of the ICR strain (Charles River Japan, Inc.), weighing 25-32 g at the start of experiments, were used (N = 6-10 in each group). The animals were kept four per cage under our standard animal facility conditions on a constant light/dark cycle (illuminated at 7 : 00-21 : 00) and at 23 _+ 1°C. Food and water were freely available except during the behavioral test. d-Methamphetamine hydrochloride (Dainippon Pharmaceutical Co., Japan) was dissolved in physiological saline and injected i.p. at a dose of either 1.5, 2 or 3 mg/kg (0.005 m l / g body weight). Cycloheximide (Sigma Chemical Co., U.S.A.) was dissolved in saline
and administered i.p. in a dose of either 60, 120 or 240 mg/kg (0.01 m l / g body weight for 60 and 120 mg/kg, or 0.02 m l / g for 240 mg/kg). Control animals were injected with an equivalent volume of saline. 2.2. Behauioral assessment
The experiment consisted of three or five sensitization tests and a challenge test. Locomotor activity was measured with sensitivity-matched activity meters (Animex; Muromachi Kikai Co., Japan and LKB Farad, Sweden) in a quiet room (20-25°C). Measurements were made during the 1st, 3rd and 5th sensitization sessions and the challenge session. In the 2nd and 4th sessions, mice received drug injections as during the odd-numbered sessions but locomotor activity was not measured. By measuring locomotor activity only in the odd-numbered sessions and by staggering sets of mice, twice as many animals could be tested in the same period. For each session, at least 3 h before drug injection, mice were placed in individual transparent test boxes (30 × 40 × 30 cm) in order to become adjusted to the experimental surroundings. During this period, spontaneous locomotion was assessed for 20 min in each group. In the sensitization test of one experiment, each mouse was assigned to one of four groups and was injected i.p. with one of the following combinations of drugs: methamphetamine (2 mg/kg) + saline, methamphetamine + cycloheximide (120 mg/kg), saline + cycloheximide or saline alone. After administration, locomotor activity was measured at 10-min intervals over 180 min. Another group of mice was injected i.p. with methamphetamine (2 mg/kg) and then locomotor activity was measured for 180 min. Four hours after the injection, these mice were treated with cycloheximide (120 mg/kg). These administrations were repeated 5 times at intervals of 3 and 4 days. In the challenge test, mice from each group were challenged with the same dose of methamphetamine 7 days after the last sensitization test. In the dose-effect experiment, we examined the effect of 60, 120 and 240 mg/kg of cycloheximide on the locomotor sensitization elicited by 1.5 or 3 mg/kg of methamphetamine. Based on these results, we studied the time course of the suppression of locomotor sensitization using a treatment combination of 120 mg/kg of cycloheximide and 1.5 mg/kg of methamphetamine. The effect of various times of cycloheximide treatment were examined after its administration simultaneously with, or 2 or 4 h after injection with methamphetamine. 2.3. Statistics
The data were analyzed with repeated measures analysis of variance (ANOVA) techniques except when
69
indicated otherwise. To evaluate the significance of difference among the factors, a post-hoe analysis was done with Scheff6's S-test. For the locomotion data, the analyses were carried out with data obtained during the initial 60-min and 120-min intervals. As there was no difference between the results for these two periods, only the statistics computed using the data for the initial 60-min interval are presented in the text.
300"
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3. Results
3.1. Toxicity o
The general toxicity of cycloheximide was examined by evaluating its effects on weight gain and spontaneous locomotion during the acclimatization period. Administration of cycloheximide (120 m g / k g ) with either methamphetamine (2 m g / k g ) or saline produced no obvious signs of toxicity during behavioral assessment. There was no overall significant effect of the various drug injections on weight gain (F(4,28) = 1.217, P > 0.05) (fig. 1). In addition, the injections did not have a significant effect on spontaneous locomotion during the initial 20-min acclimatization period in each session (F(3,32)= 0.064, P > 0.05 for the sensitization test, repeated measures A_NOVA; F(4,40) = 0.329, P > 0.05 for the challenge test, one-way A.NOVA) (fig. 2). 3.2. Sensitization test
The effects of repeated administration of methamphetamine (2 m g / k g ) a n d / o r cycloheximide (120
40-
A
36"
7: O
32'
28
1st
J
i
i
3rd
5th
Challenge
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Fig. 1. Weight gain in mice. Animals were injected i.p. with either saline alone (o), MAP (2 m g / k g ) + s a l i n e (©), M A P + C X M (120 mg/kg) (zx), or saline + CXM ( • ) . Another group of mice were injected i.p. with methamphetamine and then treated with cycloheximide 4 h after methamphetamine (O). These injections were repeated 5 times at intervals of 3 and 4 days; 7 days after the last sensitization test the same dose of methamphetamine was given. Each bar represents S.E.M. Abbreviation: MAP, methamphetamine; CXM, cycloheximide.
1st
3rd
5th
Challenge
Session
Fig. 2. Spontaneous locomotion of mice during the initial 20-min acclimatization period of the sessions in mice. Animals were treated as described in the legend of fig. 1, and are represented by various designs of columns as follows: (m), MAP+saline; (1~) M A P + C X M 0 h; (1~) M A P + C X M 4 h; ([]) saline+CXM; (O) saline. Other details are described in the legend of fig. 1.
m g / k g ) on locomotor activity during the sensitization test were examined, and the results are illustrated in fig. 3. The statistical analysis revealed that there were significant effects of treatment (F(3,32)= 65.616, P < 0.0001), session (F(2,32) = 15.647, P < 0.0001) and time (F(5,32) = 16.884, P < 0.0001). This significance was because the locomotor activity of mice simultaneously injected with methamphetamine and cycloheximide was higher than that of any other group of mice, and because the locomotor activity of mice injected with saline and cycloheximide was lower than that of any other group of mice. The analysis revealed a significant interactive effect of treatment × time (F(15,32)= 16.494, P < 0.0001). This significant interaction reflects a difference among the groups in the time course for for locomotor stimulation across the three sessions. Concomitant administration of methamphetamine with saline produced a stimulation of locomotion, with a maximal effect at 40 min (fig. 3a). Thereafter the effect gradually decreased, locomotion reaching baseline levels within 180 min. In subsequent sessions, the stimulation of locomotion increased progressively (F(2,8) = 15.363, P < 0.001). In contrast, no such stimulation was observed after repeated simultaneous administration of the stimulant with cycloheximide (F(2,8) = 2.807, P > 0.05) (fig. 3b). At the peak level, however, this regimen produced a greater stimulation than co-administration of the stimulant with saline, or administration of the stimulant followed by treatment with the inhibitor 4 h later (F(2,24) = 6.491, P < 0.01). When mice were injected with methamphetamine repeatedly, and then treated with cycloheximide 4 h later, the stimulation of locomotion progressively in-
70
creased in much the same manner as that seen after the stimulant and saline (F(2,8) = 8.376, P < 0.01) (fig. 3c). The time course for the stimulation of locomotion was significantly different among the three groups of mice injected with methamphetamine and either saline or the inhibitor (F(10,24) = 10.226, P < 0.0001). Co-administration of the stimulant with cycloheximide resuited in a much more rapid increase in locomotor activity than in the group treated with stimulant and saline. However, the increased activity was of shorter duration than that produced by the stimulant with saline, with activity returning to baseline levels within 120 min. Cycloheximide alone had some effect on locomotion in the 1st and 3rd sessions (fig. 3d). When the inhibitor was administered concomitantly with saline, there was slight hyperlocomotion, which reached a maximum at 10 min and then declined sharply to the basal level within 50 min. However, this hyperlocomotion was not observed after the 5th injection of the inhibitor and saline. The administration of saline alone did not stimulate locomotion. The time course of the locomotor stimulation of mice injected with saline alone was very similar to that observed after the 5th injection with
cycloheximide plus saline (data not shown; see also fig. 6a). 3.3. Challenge test Mice in each group were challenged with 2 m g / k g of methamphetamine 7 days after the last sensitization test; the results are shown in fig. 4. The statistical analysis revealed a significant difference in treatment effect among the groups (F(4,25)= 8.244, P < 0.001). In the two groups of mice that had repeatedly received methamphetamine together with either concomitant saline or cycloheximide 4 h later, a significant increase in locomotion was noted as compared to that of the saline-treated control mice (P < 0.05; Scheff6's S). On the other hand, in mice that had been repeatedly injected with the stimulant and the inhibitor, locomotor activity was not significantly different from that of the saline-treated control mice (P > 0.05; Scheff6's S). In addition, statistical analysis of data obtained from the interval 70-120 rain, indicated a significant decrease in locomotion in the saline + cycloheximideinjected mice when compared to that of mice receiving the stimulant and then the inhibitor (P < 0.05; Scheff6's S), but not when compared to any other groups.
2 + CXM (0 h)
(a) MAP 2 + Saline 400
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200,
200'
0 60
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Inject
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0
60
Time after
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(c) MAP 2 + CXM (4 h)
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(rain)
(d) Saline + CXM (0 h)
400"
400'
200'
200'
o o ._1
0
60
Time
after
120
Inject
180
(rain)
0
60
Time
after
120
Inject
180
(min)
Fig. 3. Time course of changes in the locomotor stimulating effect of methamphetamine (2 mg/kg) after the 1st (o), 3rd ( / , ) and 5th ( o ) injection of the drugs in mice. Animals received i.p. either MAP + saline (a), MAP + CXM (120 mg/kg) (b), or saline + CXM (d). Another group of mice was injected i.p. with methampbetamine and then treated with cycloheximide 4 h after methamphetamine (c). Other details are described in the legend of fig. 1.
71
3.4. Dose-dependent nature of the effect of cycloheximide To characterize the effect of cycloheximide further, we examined the effects of different doses of cycloheximide on the stimulation of locomotion produced by low (1.5 m g / k g ) and high (3 m g / k g ) doses of methamphetamine. Injections of methamphetamine and various doses of cycloheximide (60, 120 or 240 m g / k g ) were repeated 3 times at 3- and 4-day intervals. The challenge dose of methamphetamine was injected 7 days later. The statistical analyses demonstrated a significant stimulation of locomotion after repeated injection of 1.5 m g / k g methamphetamine together with concomitant treatment with either saline or 60 m g / k g of cycloheximide (F(1,9)= 173.267 and 9.761, P < 0.0001 and 0.05 for saline and 60 m g / k g of the inhibitor, respectively) (data not shown). On the other hand, although a substantial difference in locomotion between the 1st and 3rd sessions was noted in mice treated with 120 m g / k g of the inhibitor (F(1,9)= 5.259, P = 0.0475), there was no difference in locomotor activity between the sessions for mice treated with 240 m g / k g of the inhibitor (F(1,5)= 4.634, P > 0.05) (data not shown). These mice were challenged with 1.5 m g / k g of the stimulant and the time course of the stimulation of locomotion was examined (fig. 5a). There was a significant difference in the time course of the locomotor stimulation among the groups (F(4,40)= 4.190, P < 0.01). When compared to the saline-treated control mice, mice treated with saline or 60 m g / k g of cycloheximide showed a stimulation Of locomotion (P < 0.05 and P = 0.11 for treatments with saline and 60 m g / k g
400"
C
_o O
E o
200'
0 ,.1
o 0
60 Time
120 after
Inject
180
(min)
Fig. 4. Time course of changes in the locomotor-stimulating effect of
the challenge dose of methamphetamine (2 mg/kg) in mice. The challenge session was 7 days after the last sensitization test. Animals are represented as follows: (o) saline alone; ( o ) M A P + saline; (zx) M A P + C X M 0 h; ([3) M A P + C X M 4 h; (11) s a l i n e + C X M . Other details are described in the legend of fig. 1.
(a) MAP 1.5
300-
E
150
0 o 0 ,-I
0
60 Time after
0
Inject
120 (min)
180
(b) MAP 3 400-
E
o
2OO
0 0 ,J
0
u
,
,
,
0
60
120
180
Time
after
Inject
(mln)
Fig. 5. Time course of changes in the locomotor-stimulating effect of the challenge dose of methamphetamine during the challenge session 7 days after the last sensitization test. The panels (a) and (b) represent the results obtained after injection of the challenge doses of 1.5 and 3 mg/kg, respectively, in mice that had been given saline alone (e) or 1.5 m g / k g methamphetamine with either saline (o), 60 (A), 120 ([]) or 240 (m) m g / k g of cycloheximide. Other details are described in the legend of fig. 1.
of the inhibitor, respectively; Scheff6's S). However, no stimulation of locomotion was observed in mice treated with 120 or 240 m g / k g of cycloheximide. Repeated measures A N O V A analysis of data for the sensitization test following repeated injection of 3 m g / k g of methamphetamine revealed significant differences in locomotion between the 1st and 3rd session in all groups of mice (F(1,9) = 27.555, 9.969, 7.556 and 11.591, P < 0.001, 0.05, 0.05 and 0.01 for the treatment with saline, 60, 120 and 240 m g / k g of the inhibitor, respectively) (data not shown). The mice were challenged with 3 m g / k g methamphetamine 7 days later; the results are shown in fig. 5b. There was a significant difference in the time course of the stimulation of locomotion among the groups (F(4,43)= 3.425, P < 0.05). Compared to the saline-treated control mice, mice treated with 60 m g / k g of the inhibitor showed a clear stimulation of locomotion (P < 0.05; Scheff6's S), whereas mice treated with saline showed a slight simulation (P = 0.12; Scheff6's S). Mice treated with 120 and 240 m g / k g of cycloheximide also showed a higher locomotor activity than the saline-treated control mice.
72
Statistical significance could be demonstrated only by using a two-way A N O V A (P < 0.0001; Scheff6's S), but not by using repeated measures ANOVA.
3.5. Time-dependent nature of cycloheximide effects The effect of cycloheximide treatment at various times was studied by administration of 1.5 m g / k g methamphetamine followed by treatment with 120 m g / k g of cycloheximide 0, 2 or 4 h later. The injections were repeated 3 times; the results are shown in fig. 6. As described previously, saline injection alone resulted in a slight stimulation of locomotion only in the first 10-min interval, and thereafter locomotor activity returned to baseline levels (fig. 6a). There was no difference in the time course of the stimulation of locomotion between the 1st and 3rd sessions (F(1,9)= 0.097, P > 0.05). Co-administration of methamphetamine with cycloheximide produced a stimulation of locomotion, with a peak at 20 or 30 min (fig. 6b). As shown in fig. 3b, this peak was earlier than that (at 50 min) for mice administered the stimulant followed by
treatment with cycloheximide 2 or 4 h later. There was no significant difference in the time course of locomotion between the 1st and 3rd sessions in these mice (F(1,9) = 0.967, P > 0.05). Mice administered methamphetamine followed by cycloheximide 2 h later showed two peaks of increased locomotion at 50 and 130 min (fig. 6c). The latter peak seemed to result from an effect of cycloheximide injection. The analysis with A N O V A demonstrated no difference in the stimulation of locomotion between the 1st and 3rd sessions in the mice (F(1,9) = 0.019, P > 0.05). In contrast to these results, there was a large stimulation of locomotor activity in mice administered the stimulant followed by cycloheximide 4 h later (F(1,7) = 10.585; P < 0.05) (fig. 6d). This stimulation was again verified in the challenge test session (fig. 7). The statistical analyses revealed a significant difference in the time course of locomotion across the treatments (F(3,32) = 3.970, P < 0.05). Locomotion was significantly stimulated only in mice that had been given cycloheximide 4 h later after the methamphetamine treatment (P < 0.05; Scheff6's S), while in the other two groups there was no difference
(a) Saline Control
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Time after Inject (min) (c) CXM, 2 h
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Fig. 6. Time course of changes in the locomotor-stimulating effect of methamphetamine (1.5 mg/kg) after the 1st (©) and 3rd ([:3) injection of the drugs in mice. One group of mice was injected with saline alone (a). The other groups were injected with methamphetamine, and then treated with cycloheximide (120 mg/kg) either at 0 h (b), 2 h (c) or 4 h (d) after methamphetamine. Other details are described in the legend of fig. 1.
73
200"
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Time after Inject (mln) Fig. 7. Time course of changes in the locomotor stimulating effect of the challenge dose of m e t h a m p h e t a m i n e (1.5 m g / k g ) during the challenge session 7 days after the last sensitization test. Animals are represented as follows: (e) saline alone; (©) M A P + C X M 0 h; (zx) M A P + C X M 2 h; ([]) M A P + C X M 4 h. Other details are described in the legend of fig. 1.
compared to the saline-treated control mice (P > 0.05; Scheff6's S).
4. Discussion
The present study documents the differential effects of cycloheximide on the locomotor-stimulating effect of methamphetamine depending on the treatment regimen. Repeated administration of methamphetamine resulted in a progressive increase in its locomotorstimulating effect during the sensitization tests and resulted in a significantly higher locomotor activity after the challenge dose as compared to the locomotor activity of saline-treated control mice. This phenomenon has been recognized as behavioral sensitization (Tilson and Rech, 1973; Hirabayashi and Alam, 1981). The locomotor sensitization was remarkably attenuated by simultaneous treatment with high doses of cycloheximide. However, treatment with cycloheximide 4 h later failed to suppress the locomotor sensitization. Several points need to be considered when determining the possible mechanism underlying these paradoxical results. In the present study cycloheximide was injected in a dose of 120 m g / k g , a dose that has been commonly used in the study of long-term memory in mice (Barondes and Cohen, 1967, 1968a,b; Squire and Barondes, 1976). Large doses of protein synthesis inhibitors are known to have toxic effects such as diarrhea, decreased food and water intake, and weight loss (Davis and Squire, 1984). However, the doses of cycloheximide used in this study did not cause significant signs of toxicity. There were no differences in weight gain and spontaneous locomotion among the groups. In addition, mice repeatedly administered methamphetamine and then treated with cycloheximide 4 h
later showed behavioral sensitization in the same manner as mice co-administered the stimulant with saline. The reason we were unable to observe any toxic effects may lie in our protocol, in which the cycioheximide treatments were repeated every 3 or 4 days. Thus it does not seem that toxic side effects of cycloheximide, if any, contributed to the suppression of behavioral sensitization observed in this study. Cycloheximide has been reported to stimulate locomotor activity for 30 min and then to depress it for several hours in mice (Segal et al., 1971). Indeed, the present data revealed a transient stimulation of locomotion by cycloheximide itself. In mice co-administered low doses of the stimulant and cycloheximide, locomotor activity reached a higher maximum faster, and declined more rapidly than in mice co-administered the stimulant with saline. Furthermore, cycloheximide alone caused some hyperlocomotion at 10 min. Based on these findings, the lack in a progressive stimulation of locomotion in the sensitization test in mice co-administered low doses of the stimulant and the inhibitor could possibly be due to the fact that the animals were already displaying a ceiling effect during the 1st session. However, the present study also provides evidence against this interpretation. Co-administered cycloheximide could not prevent the locomotor stimulation observed after repeated administration of a high dose (3 m g / k g ) of methamphetamine, which produced a greater stimulation than low doses of the stimulant co-administered with the inhibitor. In addition, the locomotor sensitization was severely suppressed by the 2-h treatment with cycloheximide, which never interacted with the stimulant at 50 and 60 min. It seems, therefore, that an interaction between these drugs cannot explain the results. It has been shown that protein synthesis inhibitors markedly reduce the rate of synthesis of a rapid turning over pool of dopamine and norepinephrine, the primary source for catecholaminergic neurotransmission, for at least 6.5 h after treatment (Bloom et al., 1977; Lundgren and Carr, 1978). It is thus conceivable that the reduced rate of catecholamine synthesis could account for the suppression of locomotor sensitization observed in this study. If this speculation were true, we could expect a lower rate of synthesis of catecholamines in mice treated with amphetamine and cycloheximide than in mice treated with amphetamine and saline. However, when measured at 1 and 1.5 h after treatment, there is no difference in the rate of dopamine and norepinephrine synthesis between animals treated with the stimulant plus either saline or the inhibitor (Bloom et al., 1977). Therefore, these results suggest that co-administered cycloheximide is unlikely to prevent the development of locomotor sensitization by inhibiting the synthesis of norepinephrine or dopamine.
74 The sensitization produced by 1.5 m g / k g methamphetamine was markedly attenuated by concurrent treatment with 120 or 240 m g / k g of cycloheximide, but not by treatment with 60 m g / k g of cycloheximide. This result is similar to the finding that mice showed impairment of long-term memory when treated with 150 or 200 m g / k g of cycloheximide at a training test, but not when treated with less than 100 m g / k g of the inhibitor (Kameyama et al., 1986). On the other hand, the behavioral sensitization produced by 3 m g / k g methamphetamine was not affected by treatment with even 240 m g / k g of cycloheximide. It has been demonstrated that the retention of long-term memory is dependent on the voltage of the electric shock delivered in the training test (Kameyama et al., 1986). It has also been suggested that the degree of impairment of long-term memory is related to the extent of inhibition of cerebral protein synthesis during or shortly after training (Davis and Squire, 1984). In addition, the action of cycloheximide has been revealed to be relatively short (Barondes and Cohen, 1968a; Squire and Barondes, 1976), and the locomotor stimulating effects of 3 m g / k g m e t h a m p h e t a m i n e lasted relatively longer. Thus the effects of 3 m g / k g m e t h a m p h e t a m i n e on neurons are too strong to be extinguished by cycloheximide at the dose used. This study also demonstrated the time-dependent nature of the effect of cycloheximide on the locomotor sensitization. The sensitization produced by 1.5 m g / k g m e t h a m p h e t a m i n e was suppressed by the treatment given at either 0 h or 2 h after the stimulant injection, but not by the treatment given 4 h later. It has been shown that when inhibition of cerebral protein synthesis is initiated 1 h or longer after training, long-term memory is not affected (Barondes and Cohen, 1968b; K a m e y a m a et al., 1986). Cycloheximide at a dose of 120 m g / k g has been reported to inhibit by more than 90% cerebral protein synthesis for up to 1.5 h after injection in mice. Thereafter, the inhibition decreases to less than 80% at 2 h, and to 20% at 3 h (Barondes and Cohen, 1968a; Squire and Barondes, 1976). Additionally, it has been suggested that at least 90% inhibition of protein synthesis during training is necessary to impair long-term memory in mice (Barondes and Cohen, 1967). In the present study we have no data about the time course of the inhibition of cerebral protein synthesis. However, based on these findings, the present results suggest that the development of behavioral sensitization depends, at least in part, upon cerebral proteins synthesized 2 h after the injection of the stimulant. The present results have great analogy to the finding that protein synthesis inhibitors, injected just before or shortly after training, impair long-term memory in animals (Barondes and Cohen, 1967; Cohen and Barondes, 1968; Davis and Squire, 1984). It has been re-
ported that behavioral sensitization may result from conditioned drug effects which are, in part, mediated by the mechanism underlying memory or learning of stimuli attending drug injection and the experimental environment (Tilson and Rech, 1973; Hirabayashi and Alam, 1981). It has been reported that conditioned circling behavior is suppressed by treatment with cycloheximide in rats with unilateral 6-hydroxydopamine lesion of substantia nigra (Silverman et al., 1989). Other findings similar to the present results have been described in a number of p h e n o m e n a related to neural plasticity. It has been observed that co-administration of cycloheximide with morphine prevents the development of tolerance to and physical dependence on morphine in mice (Way et al., 1968; Loh et al., 1969). It has also been reported that sensitization to the morphineproduced stimulation of locomotion is blocked by coadministration of cycloheximide in mice (Shuster et al., 1975). Treatment with anisomycin, another protein synthesis inhibitor, during amygdaloid kindling has been revealed to suppress the development of generalized seizures in mice (Cain et al., 1980). These findings strongly suggest that plastic changes in the central neurons involved in p h e n o m e n a such as long-term memory, drug conditioned effects, drug tolerance and dependence, and kindling require de novo protein synthesis in the brain (Davis and Squire, 1984; Goelet et al., 1986). Moreover, the present results also suggest that the development of behavioral sensitization depends upon cerebral protein synthesis within 2 h after m e t h a m p h e t a m i n e administration.
Acknowledgements This research was supported in part by a grant from the Project Research Foundation of the Kawasaki Medical School, Japan (No. 2-413). The authors are grateful to R.J. Marley, Ph.D. and N. Goodman, M.S., NIDA-Addiction Research Center, U.S.A., for helpful comments and suggestions on the manuscript.
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