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Methylphenidate sensitization is modulated by valproate
Life Sciences 69 (2001) 47–57
Methylphenidate sensitization is modulated by valproate Kary Eckermanna, Anitra Beasleya, Pamela Yanga,b, Osvaldo Gaytana,b, Alan Swannb, Nachum Dafnya,* a
Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77225, USA b Department of Psychiatry and Behavioral Sciences, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77225, USA Received 22 September 2000; accepted 14 December 2000
Abstract Repeated administration of the stimulant methylphenidate (MPD) produces sensitization to its own effects. Glutamate, dopamine, and GABA have been implicated in the underlying mechanism of sensitization to stimulants such as amphetamine and cocaine. We have investigated effects of the GABAergic agent sodium valproate (VAL) on the locomotor response to MPD. Activities of male Sprague-Dawley rats were continuously recorded by a computerized activity monitoring system for 15 days. We studied the dose effect of valproate 1) at 50, 100, and 200 mg/kg (i.p.) on motor activities, 2) on the acute response of motor activities to 2.5 mg/kg MPD, and 3) on behavioral sensitization to subsequent repeated injections of MPD. Valproate alone did not significantly affect motor activities. All three doses of valproate attenuated the acute locomotor effects of MPD, while only the 50mg/kg dose blocked the development of sensitization to subsequent administration. Possible mechanisms involving substrates for the effect of GABA agonists on sensitization are discussed. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Sensitization; Locomotor activity; Methylphenidate; Valproate; GABA
Introduction Repeated administration of stimulants can result in an enhancement of drug-induced locomotor activities with each successive administration. This enhancement in locomotion is referred to as behavioral sensitization [1, 2]. Behavioral sensitization to the locomotor effects of stimulants has been demonstrated to involve the mesolimbic dopamine system [3, 4] and is thought to have properties in common with other forms of neural plasticity such as long-term potentiation [5]. Dopamine (DA) has a pronounced role in the locomotor stimulation such as * Corresponding author. Tel.: (713)-500-5616; fax: (713)-500-0621. E-mail address: [email protected] (N. Dafny) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 9 5 -5
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sensitization, which is produced by repetitive use of psychostimulants (Review by 4). The GABAergic system can alter the activity of the dopamine neurons (Review by 6). Systemic administration of GABA agonist reduces DA turnover in the mesolimbic system [7], and blocks the enhanced locomotor activity and stereotypic behavior produced by dopaminergic stimulation [8,9,10]. Sodium valproate, an anticonvulsant drug used in treating mania and schizophrenia [11], enhances GABA activity in the CNS by inhibiting its degradation, stimulating its synthesis and release, and directly enhancing its postsynaptic effects [11]. Although a growing body of evidence suggests that excitatory amino acid N-methyl-D-aspartate (NMDA) receptors are involved in the development of a sensitized response, less attention has been given to the role of inhibitory amino acids such as GABA or other GABA receptor agonists in this phenomenon. GABAergic agents have been shown to modify the expression of behavioral sensitization produced by methamphetamine, amphetamine, and cocaine. Systemic injection of a benzodiazepine (Clonazepam) prevented the development of sensitization to methamphetamine [12], while baclofen, a GABAb agonist injected into the A10 dopamine region, was able to prevent the development of sensitization to systemic cocaine administration [13]. Moreover, the GABAa agonist THIP, when injected into the anterior cortex 5 minutes prior to systemically administered 12 mg/kg amphetamine, blocked sensitization to an intraperitoneal injection of 8 mg/kg amphetamine 48 hours later [14]. Recently, the NMDA receptor antagonist, MK-801 was shown to block the development of sensitization to the locomotor effects of 2.5 mg/kg MPD [15]. Since both neurotransmitter systems are modulators of the mesolimbic dopamine system and since sensitization is thought to be expressed by changes in the function of these neurons [16], GABA’s role in sensitization warrants investigation. In the present study, we have investigated the effects of sodium valproate, a GABA agonist used in the treatment of bipolar disorder [11], on motor activities and on the acute and chronic responses to MPD. Other GABA agonists have been studied with amphetamine, cocaine, and dopamine [13, 16–19]. However, there are no studies of GABAergic agonist interactions with MPD. Consequently, this study examined: 1) motor effects of 50, 100, and 200 mg/kg of valproate, 2) effects of valproate at the same doses on the acute response to MPD, and 3) effects of valproate pretreatment on response to subsequent MPD administration.
Methods Animals Male Sprague-Dawley rats (N 5 36) weighing from 180–190 g were housed in the experimental room in groups of four at an ambient temperature of 21 6 2 8C and a relative humidity of 37–42%. Animals were maintained on a 12:12 light/dark schedule (light on at 07:00– 19:00 h). Water and food pellets were supplied ad libitum throughout the experiments. After 5–7 days of acclimation in the experimental room, rats were weighed and individually housed in the test cages (i.e., the test cages became their home cages), and allowed a minimum of 24 hours of habituation to the test cages before commencement of continuous recording of locomotor activities.
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Drugs Methylphenidate hydrochloride (MPD) and sodium valproic acid (VAL) were obtained from Sigma Chemicals (St. Louis, MO) and Research Biochemicals International (Natick, MA), respectively. Valproate (50, 100, and 200 mg/kg) was dissolved in saline and injected intraperitoneally (i.p.), while MPD (2.5 mg/kg) was dissolved in saline and injected subcutaneously (s.c.). All injections were of equal volume (0.8 cc) and given between 13:00 h and 14:00 h. Apparatus The computerized animal activity monitoring (CAAM; AccuScan Instruments, Inc., Columbus, OH) system is the system used to measure the locomotor activity of several indices. The activity chambers consist of clear acrylic open field boxes (40.5340.5331.5 cm) with two levels of infrared motion sensors. The first and second levels of sensors are 6 and 12.5 cm from the cage floor, respectively. The activity monitoring system checks each of the beams at a frequency of 100 Hz to determine whether the beams are interrupted. Interruptions of two or more consecutive beams separated by at least one second are recorded as a movement score. Cumulative counts are compiled and downloaded every 10 minutes into the OASIS data collection program and organized into motor indices [20–22]. The following four locomotor indices were analyzed. The horizontal activity index (HA) measures the overall motor activity in the lowest tier of the testing cages. Total distance (TD) and vertical activity (VA) measure the specific motor behaviors of forward ambulation and rearing, respectively. Number of stereotypic movements (NOS) measures the number of different episodes of stereotypic activity with at least a one-second interval before the beginning of another episode. Procedure Motor activities were continuously recorded for a total of 14 to 15 days as follows: Day 1 – baseline; Day 2 – 0.9% saline injection at 13:00 h (i.p.) and at 14:00 h (s.c.); Day 3 – rats randomly assigned into Groups I, II, III, or IV. Group I received only saline (s.c.) at 13:00 h and 14:00 h, while Groups II, III, and IV received 50 mg/kg, 100 mg/kg, or 200 mg/kg valproate (i.p.), respectively, at 13:00 h and 2.5 mg/kg MPD (s.c.) at 14:00 h. Group I then received 2.5 mg/kg MPD (s.c.) at 14:00 h on Days 4–9, no drug treatment (washout phase) on Days 10–14, and at 13:00 h saline injection followed by a re-challenge dose of 2.5mg/kg MPD (s.c.) at 14:00 h on Day 15. Groups II, III, and IV received 2.5 mg/kg MPD (s.c.) at 14:00 h on Days 4–8, no drug treatment (washout phase) on Days 9–13, followed by saline injection at 13:00 h and a re-challenge dose of 2.5 mg/kg MPD (s.c.) at 14:00 h on Day 14. Data analysis Previous studies indicated that repeated injection of saline did not produce a significant increase in activity for all four motor indices investigated. Thus, the present study used saline as a comparison to the effects of the drugs since activity levels at saline did not fluctuate with time [20–22]. The effect of MPD for each motor index was determined by subtracting the activity score of the 2 hours post-saline injection (Day 2) from that of the 2 hours post-MPD injection (Group I: Days 4 to 9 and Day 15; Groups II-IV: Days 4 to 8 and Day 14). For the
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effect of valproate injection on MPD (Day 3 and Day 15), the activity score for the 2 hours post-saline injection (Day 2) was subtracted from that of the 2 hours post-valproate injection (Day 3 or Day 15). The presence of sensitization for Groups II, III, and IV was determined by comparing the results of all other days (Days 5 to 8 and 14) to that of MPD treatment on Day 4. For Group I, the presence of sensitization was determined by comparing results of all other days (Days 5 to 9 and 15) to that of the initial MPD challenge on Day 4. Values are presented as the mean 6 S.E.M. for each 10 minute or 2 hour sample. Since locomotor baseline activity varies from animal to animal and from group to group, each animal baseline following saline injection was used as its own control, and the difference between the saline control and the subsequent treatment, i.e. the delta, was used for the statistical analysis. The deltas of the groups were represented as the mean 6 S.E.M. for each 10 min sample in the figures. These results were analyzed using repeated measures of ANOVA (two levels: treatment day and 10 minute sample) and post-hoc Fischer’s (LSD) method. Significance for all of the above comparisons was set at * p , 0.05. Results Repeated administration of methylphenidate The effects of repeated MPD (2.5 mg/kg) treatment (Group I) on TD and HA are summarized in Figure 1. The bar graph shows locomotor activity of the first 2 hours after MPD injection for Days 4 to 15 of the experiment. Activity following saline injection was arbitrarily set at zero. Single MPD (2.5 mg/kg) administration on Day 4 elicited an increase in locomotor activity compared to saline, while the same MPD dose given daily for five additional days (Days 5 to 9) caused a further increase in the locomotor activity in all four motor indices (HA, TD, VA, and NOS) studied. There was a significant increase (p , 0.05) in activity after the second MPD injection for HA (Figure 1) and NOS (not shown), and a significant increase (p , 0.05) in activity after the fourth MPD injection for TD (Figure 1, Day 7) and VA (not shown). This significant enhancement in motor activity after multiple MPD administration is interpreted as sensitization to the locomotor effects of the stimulant. Moreover, when MPD was given again (Day 15) after 5 days of drug abstinence (Day 10 to 14), the locomotor activity compared to that on Day 4 was still significantly high (at least p , 0.05) in all four motor indices. Thus, sensitization was observed to develop and persist even in Day 15 for rats given 2.5 mg/kg MPD. Dose-response of valproate Gross observation of the effects of valproate (25, 50, 100, and 200 mg/kg) on locomotion during the initial 60 minutes after valproate administration showed that the 25 and 50 mg/kg dose exerted no behavioral effects, while the 100 and 200 mg/kg doses produced wet dog shakes during the first 20 minutes after valproate administration. Using the CAAM in a pilot study, it was observed that the locomotion activities following the dose of 25 mg/kg valproate were similar to those of saline; consequently, this dosage was removed from use. The 50-mg/kg dose produced a biphasic effect (sigmoidal curve) in locomotor activities compared to saline. Immediately after valproate administration, activities elicited by the 50-mg/kg dose were less
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Fig. 1. The average (N 5 12) total distance (TD) and horizontal activity (HA) traveled in the first 2 hours after saline and MPD administration for the length of the experiment. Sensitization is present by Day 6 and still measurable by Day 15. Values are presented as the mean 6 S.E.M. * p , 0.05.
than those after saline injection, followed by a gradual increase in activity and then activity leveled off at 30–40 minutes after drug injection. The 100 and 200 mg/kg doses elicited a more variable motor response pattern than the 50 mg/kg valproate. The 100 mg/kg dose elicited some increase in TD for 20 minutes after drug injection, while a decrease in activities in
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HA, VA, and NOS occurred during this time. The 200 mg/kg dose elicited a slight decrease in HA and VA for the first 20 minutes after drug injection, while activities fluctuated in TD and NOS during this time. Another study was conducted in which 50 mg/kg valproate was given repeatedly for 6 consecutive days and followed by 5 days of washout and a re-challenge dose of 50 mg/kg valproate. It was found that the 50 mg/kg valproate did not produce any sensitized response to itself. In conclusion, the three valproate doses produced minor changes in locomotor activities compared to saline. Effects of single valproate injection on the acute response to methylphenidate Figure 2 summarizes the acute temporal response of total distance (TD) on Day 3 following administration of 2.5 mg/kg MPD in the four groups, each of which received either 0.9% saline, or 50, 100, or 200 mg/kg valproate 1 hour prior to MPD injection. Values are represented as the mean 6 S.E.M. of the delta for each 10 minute sample. The group pretreated with saline showed a general increase in total distance following MPD injection as expected, while the other three groups pretreated with valproate did not share this similar elevation. Post-hoc analysis revealed that 50 mg/kg and 200 mg/kg valproate given 1 hour prior to MPD administration significantly lowered the MPD-elicited motor response (p , 0.05), particularly during the initial 60 minutes after MPD injection. The 100-mg/kg valproate elicited similar general effects like the 50 and 200-mg/kg valproate, but the effect was not significant. The effects of the three valproate doses on horizontal activities, vertical activities, and number of stereotypic movements given 1 hour prior to MPD were similar to those effects on TD. Effect of single valproate pretreatment on repeated MPD administration Figure 3 summarizes the acute temporal (line graph) and the 2-hour total response (bar graph) of total distance following a challenge dose of 2.5 mg/kg MPD and a re-challenge dose of 2.5 mg/kg MPD on Day 15 for Group I and on Day 14 for Groups II, III, and IV. Group I was given saline pretreatment 1 hour prior to MPD. Groups II, III and IV received 50, 100 and 200 mg/kg valproate 1 hour prior MPD, respectively. Pretreatment with saline, followed by 100 and 200 mg/kg valproate 1 hour prior to MPD failed to modulate the induction and the expression of behavioral sensitization produced by repetitive MPD administration. However, 50 mg/kg valproate pretreatment blocked the MPD elicited sensitization (Figure 3 bar graph: Group II). Vertical activity, horizontal activity, and number of stereotypic movements also showed a similar response as total distance.
Discussion Behavioral response to valproate alone A moderate dose of valproate (50 mg/kg) did not significantly alter motor activity at any dose studied. In higher doses (100 mg/kg and 200 mg/kg), some wet dog shakes occurred transiently. These results confirmed previous reports on the behavioral effects of valproate [23], as well as findings with other GABAergic agonists such as muscimol and baclofen [23].
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Fig. 2. Acute temporal response for total distance (TD) following administration of 2.5 mg/kg MPD in four groups of rats (each N 5 8), each of which received either saline, 50, 100, or 200 mg/kg valproate 1 hour prior to MPD. All three doses of valproate attenuated the behavioral response to MPD. Values are presented as the mean 6 S.E.M. for each 10 minute sample minus the corresponding values following saline injection for each treatment group.
However, one study [24] reported that valproate, along with other GABA agonists, such as chlordiazepoxide, 3- APS, Pentobarbitone, and Clonazepam, produced a dose-related decrease in locomotor activity. This study used a gross measure of locomotor activity rather than quantitative measurement. Variation among results of pertinent studies may arise from
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Fig. 3. Acute temporal response (line graph) and 2-hour total (bar graph) for total distance (TD) following administration of 2.5 mg/kg MPD in four groups of rats. Group I: saline pretreatment 1 MPD. Group II: 50 mg/kg valproate 1 MPD. Group III: 100 mg/kg valproate 1 MPD. Group IV: 200 mg/kg valproate 1 MPD. The 50-mg/kg dose blocked the sensitized response elicited by repetitive MPD administration, while the 100 mg/kg and 200 mg/kg valproate did not block this sensitization. Values are presented as the mean 6 S.E.M. for each 10 minute sample for the line graph and 2 hours for the bar graph. * p , 0.05.
procedural differences or from differences in mechanism of action and effect on motor behavior between all the drugs studied. For example, valproate raises synaptic concentration of GABA by slowing its metabolic degradation. Pentobarbitone acts directly on the chloride ionophore, while 3-APS is a structural analogue of GABA [8]. Nevertheless, valproate’s ma-
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jor behavioral effect appears to be a transient increase in wet dog shakes that resolves 20–30 minutes after injection. Effects of valproate on the acute response to methylphenidate All three doses of valproate (50, 100, and 200 mg/kg) administered one hour prior to MPD injection acutely attenuated the locomotor response to MPD. GABAergic drugs have been shown to attenuate the locomotor response to amphetamine [8, 12]. Studies using valproate (200 mg/kg), baclofen (2.5 mg/kg), and THIP (10 mg/kg) found that the administration of high doses, but not lower doses, blocked the acute effects of a locomotor activating dose of amphetamine (1.5 mg/kg) (9). However, all three drugs were administered at the same time as amphetamine, making it difficult to correlate with the present study in which valproate and MPD were given one hour apart. Agmo et al [8] also reported that both low and high doses of valproate and baclofen did not block the motor effect of 3.0 mg/kg amphetamine, suggesting that a normal tonic inhibitory effect of GABA systems on dopamine may be overcome at higher levels of dopaminergic activity. This would account for the finding that all 3 doses used in our study attenuated the motor response to a low dose of MPD [20–22], suggesting that valproate would not alter the effects of a higher dose of MPD. Klitnetick [25] also found that the GABAb agonist baclofen inhibits dopamine transmission in the nucleus accumbens by hyperpolarizing dopaminergic cells in the ventral tegmental area. This provides an alternative explanation for the effect of valproate, which does not act specifically on GABAa or GABAb receptors. Effect of valproate pretreatment on repeated MPD administration Earlier studies in our laboratory showed that a single injection of a noncompetitive NMDA antagonist, MK-801, blocked sensitization to the locomotor effects of MPD. In the present study, only the lowest dose of valproate used, 50 mg/kg, blocked the development of sensitization to methylphenidate. In other studies, Clonazepam [12] and THIP [14] were given repeatedly prior to stimulant administration, whereas our study gave a single injection of valproate 1 hour prior to the first of six repeated MPD injections. This difference in drug administration schedules may account for differences obtained in the results. Other reports have described the ability of the GABAergic agonist THIP to prevent the induction of sensitization. Karler [14] reported that three doses of THIP injected into the anterior cortex but not the posterior cortex, 5 minutes prior to systemically administered 12 mg/kg amphetamine blocked the induction of sensitization produced by 8 mg/kg amphetamine given 48 hours later [14]. THIP injected systemically also blocked the development of sensitization to amphetamine [14]. THIP, whether given systemically or in the cortex prior to the stimulant, blocked the induction of sensitization. These findings and our results are consistent with an inhibitory role of GABA in the induction of sensitization to motor effects of stimulants. Because the projection from the nucleus accumbens to the ventral palladium is GABAergic, this projection is involved in locomotor activity elicited by dopamine release in the nucleus accumbens [26]. A GABAergic influence on dopamine neurons could modulate the effects of stimulants on locomotor activity [13], and could be one interpretation of the effects of val-
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proate on the induction of locomotor sensitization. However, a more likely explanation for the variable effects of GABA agonists on sensitization is that some compounds stimulate GABA receptors directly while others indirectly cause an activation of GABA receptors by several different mechanisms [27]. GABAa agonists regulate dopamine indirectly, while GABAb agonists act directly on dopamine neurons [13]. In response to sensitizing treatment, a change may be observed in the dopamine neurons themselves or a change in GABA regulation of dopamine release. While there are numerous differences, which may account for different GABAergic drug effects on sensitization, benzodiazepines and barbiturates act only on the GABAa receptor complex, while THIP and baclofen act on GABAb and valproate acts on both [27]. It has been well documented that the neural substrates necessary for the induction and expression of sensitization to psychomotor stimulants are not the same. In this regard, an appropriate step in exploring further connections between the GABA-dopamine interaction and the mechanism of sensitization would be to examine whether valproate would block induction and expression of this phenomenon. In summary, a single dose of valproate produced minimal locomotor effects and when given prior to any MPD administration appeared to attenuate the locomotor response to MPD sensitization. Acknowledgment This study was supported in part by the Pat Rutherford Chair in Psychiatry. References 1. Robinson TE, Becker JB. Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychodis. Brain Res. 1986; 396:157–198. 2. Kuczenski R, Segal D. Concomitant characterization of behavioral and striatal neurotransmitter response to amphetamine using in vivo microdialysis. J. Neurosci. 1989; 9: 2051–2065. 3. Robinso TE, Berridge KC. The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res. Rev. 1993; 18: 247–291. 4. Wolf ME. The role of excitatory amino acids in behavioral sensitization to psychomotor stimulants. Prog. In Neurobiol. 1998; 54: 679–720. 5. Karler R, Calder LD, Bedingfield JB. Cocaine behavioral sensitization and the excitatory amino acids. Psychopharmacol. 1994; 115: 305–310. 6. Pierce RC, Kalivas PW. A circuitry model of the expression of behavioral sensitization to amphetamine-like psychostimulants. Brain Res. Rev. 1997; 25:192–216. 7. Waldmeier PC, Maitre L. Effects of baclofen on dopamine metabolism and interaction with neuroleptic effects. Eur. J. Pharmacol. 1978; 47:191–200. 8. Agmo A, Belzung C, Giordano M. Interactions between dopamine and GABA in the control of ambulatory activity. J. Neural Transm. 1996; 103: 925–934. 9. Cott J, Engel J. Suppression by GABAergic drugs of the locomotor stimulation induced by morphine, amphetamine, and apomorphine: evidence for both pre- and post-synaptic inhibition of catecholamine systems. J. Neural Transmission 1997: 40: 253–268. 10. Sandoval MR, Palermo-Neto J. Effect of manipulation of the GABA system on dopamine-related behaviors. Braz. J. Med. Bio. Res. 1995; 28: 88–99. 11. McElroy SL, Keck PE, Pope HG, Hudson JI. Valproate in psychiatric disorders: literature review and clinical guidelines. J. Clin. Psychiatry 1989; 50: 23–29.
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12. Ito K, Ohmori T, Abekawa T, Koyama T. Clonazepam prevents the development of sensitization to methamphetamine. Pharmacol., Biochem., Behav. 1997; 58: 875–879. 13. Kalivas PW, Stewart J. Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Res. Rev. 1991;16: 223–244. 14. Karler R, Bedingfield JB, Thai DK, Karler R, Calder LD. The role of frontal cortex in the mouse in behavioral sensitization to amphetamine. Brain Res. 1997; 757: 228–235. 15. Sripada S, Gaytan O, Al-Rahim S, Swann A, Dafny N. Dose-related effects of MK-801 on acute and chronic methylphenidate administration. Brain Res. 1998; 814: 78–85. 16. Karler R, Calder LD, Thai LH, Bedingfield JB. The dopaminergic, glutamatergic, GABAergic bases for the action of amphetamine and cocaine. Brain Res. 1995; 671: 100–104. 17. Antelman SM, Chiodo LA. Dopamine autoreceptor subsensitivity: a mechanism common to the treatment of depression and the induction of amphetamine psychosis. Biological Psychiatry 1981; 16: 717–727. 18. Invernizzi R, Pozzi L, Samanin R. Release of dopamine is reduced by diazepam more in the nucleus accumbens than in the caudate nucleus of conscious rats. Neuropharmacol. 1991; 30: 575–578. 19. Peris J. Repeated cocaine injections decrease the function of striatal gamm-aminobutyric acid (A) receptors. J. Pharmacol. Exp. Ther. 1996; 276: 1002–1008. 20. Gaytan O, Ghelani D, Martin S, Swann A, Dafny N. Dose response characteristics of methylphenidate on different indices of rats’ locomotor activity at the beginning of the dark cycle. Brain Res. 1996; 727: 13–21. 21. Gaytan O, Swann A, Dafny N. Time-dependent differences in the rats’ motor response to amphetamine. Pharmacol. Biochem. Behav. 1998; 59: 459–467. 22. O. Gaytan, R. Nason, R. Alagugurusam, A. Swann and Gaytan O, Nason R, Lalagugurusamy R, Swann A, Dafny N. MK-801 blocks the development of sensitization to the locomotor effects of methylphenidate. Brain Res. Bull 2000; 51: 485–492. 23. Dalvi A, Rodgers RJ. GABAergic influences on plus-maze behavior in mice. Psychopharmacol. 1996; 128: 380–397. 24. Herberg LJ, Williams SF. Anti-conflict and depressant effects by GABA agonists and antagonists, benzodiazepines and non-gabergic anticonvulsants on self-stimulation and locomotor activity. Pharmacol. Biochem. Behav. 1983; 19: 625–633. 25. Klitenick MA, Dewitte P, Kalivas PW. Regulation of somatodendritic dopamine release in the ventral tegmental area by opioids and GABA: an in vivo microdialysis study. J. Neurosci. 1992; 12: 2623–2632. 26. Mogenson GJ, Nielsen MA. Evidence that an accumbens to subpallidal GABAergic projection contributes to locomotor activity. Brain Res. Bull. 1983; 11: 309–314. 27. Cooper JR, Bloom FE and Roth, RH. The Biochemical Basis of Neuropharmacology. Oxford University New York, 1996. pp.126–193.
Methylphenidate sensitization is modulated by valproate Kary Eckermanna, Anitra Beasleya, Pamela Yanga,b, Osvaldo Gaytana,b, Alan Swannb, Nachum Dafnya,* a
Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77225, USA b Department of Psychiatry and Behavioral Sciences, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77225, USA Received 22 September 2000; accepted 14 December 2000
Abstract Repeated administration of the stimulant methylphenidate (MPD) produces sensitization to its own effects. Glutamate, dopamine, and GABA have been implicated in the underlying mechanism of sensitization to stimulants such as amphetamine and cocaine. We have investigated effects of the GABAergic agent sodium valproate (VAL) on the locomotor response to MPD. Activities of male Sprague-Dawley rats were continuously recorded by a computerized activity monitoring system for 15 days. We studied the dose effect of valproate 1) at 50, 100, and 200 mg/kg (i.p.) on motor activities, 2) on the acute response of motor activities to 2.5 mg/kg MPD, and 3) on behavioral sensitization to subsequent repeated injections of MPD. Valproate alone did not significantly affect motor activities. All three doses of valproate attenuated the acute locomotor effects of MPD, while only the 50mg/kg dose blocked the development of sensitization to subsequent administration. Possible mechanisms involving substrates for the effect of GABA agonists on sensitization are discussed. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Sensitization; Locomotor activity; Methylphenidate; Valproate; GABA
Introduction Repeated administration of stimulants can result in an enhancement of drug-induced locomotor activities with each successive administration. This enhancement in locomotion is referred to as behavioral sensitization [1, 2]. Behavioral sensitization to the locomotor effects of stimulants has been demonstrated to involve the mesolimbic dopamine system [3, 4] and is thought to have properties in common with other forms of neural plasticity such as long-term potentiation [5]. Dopamine (DA) has a pronounced role in the locomotor stimulation such as * Corresponding author. Tel.: (713)-500-5616; fax: (713)-500-0621. E-mail address: [email protected] (N. Dafny) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 9 5 -5
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sensitization, which is produced by repetitive use of psychostimulants (Review by 4). The GABAergic system can alter the activity of the dopamine neurons (Review by 6). Systemic administration of GABA agonist reduces DA turnover in the mesolimbic system [7], and blocks the enhanced locomotor activity and stereotypic behavior produced by dopaminergic stimulation [8,9,10]. Sodium valproate, an anticonvulsant drug used in treating mania and schizophrenia [11], enhances GABA activity in the CNS by inhibiting its degradation, stimulating its synthesis and release, and directly enhancing its postsynaptic effects [11]. Although a growing body of evidence suggests that excitatory amino acid N-methyl-D-aspartate (NMDA) receptors are involved in the development of a sensitized response, less attention has been given to the role of inhibitory amino acids such as GABA or other GABA receptor agonists in this phenomenon. GABAergic agents have been shown to modify the expression of behavioral sensitization produced by methamphetamine, amphetamine, and cocaine. Systemic injection of a benzodiazepine (Clonazepam) prevented the development of sensitization to methamphetamine [12], while baclofen, a GABAb agonist injected into the A10 dopamine region, was able to prevent the development of sensitization to systemic cocaine administration [13]. Moreover, the GABAa agonist THIP, when injected into the anterior cortex 5 minutes prior to systemically administered 12 mg/kg amphetamine, blocked sensitization to an intraperitoneal injection of 8 mg/kg amphetamine 48 hours later [14]. Recently, the NMDA receptor antagonist, MK-801 was shown to block the development of sensitization to the locomotor effects of 2.5 mg/kg MPD [15]. Since both neurotransmitter systems are modulators of the mesolimbic dopamine system and since sensitization is thought to be expressed by changes in the function of these neurons [16], GABA’s role in sensitization warrants investigation. In the present study, we have investigated the effects of sodium valproate, a GABA agonist used in the treatment of bipolar disorder [11], on motor activities and on the acute and chronic responses to MPD. Other GABA agonists have been studied with amphetamine, cocaine, and dopamine [13, 16–19]. However, there are no studies of GABAergic agonist interactions with MPD. Consequently, this study examined: 1) motor effects of 50, 100, and 200 mg/kg of valproate, 2) effects of valproate at the same doses on the acute response to MPD, and 3) effects of valproate pretreatment on response to subsequent MPD administration.
Methods Animals Male Sprague-Dawley rats (N 5 36) weighing from 180–190 g were housed in the experimental room in groups of four at an ambient temperature of 21 6 2 8C and a relative humidity of 37–42%. Animals were maintained on a 12:12 light/dark schedule (light on at 07:00– 19:00 h). Water and food pellets were supplied ad libitum throughout the experiments. After 5–7 days of acclimation in the experimental room, rats were weighed and individually housed in the test cages (i.e., the test cages became their home cages), and allowed a minimum of 24 hours of habituation to the test cages before commencement of continuous recording of locomotor activities.
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Drugs Methylphenidate hydrochloride (MPD) and sodium valproic acid (VAL) were obtained from Sigma Chemicals (St. Louis, MO) and Research Biochemicals International (Natick, MA), respectively. Valproate (50, 100, and 200 mg/kg) was dissolved in saline and injected intraperitoneally (i.p.), while MPD (2.5 mg/kg) was dissolved in saline and injected subcutaneously (s.c.). All injections were of equal volume (0.8 cc) and given between 13:00 h and 14:00 h. Apparatus The computerized animal activity monitoring (CAAM; AccuScan Instruments, Inc., Columbus, OH) system is the system used to measure the locomotor activity of several indices. The activity chambers consist of clear acrylic open field boxes (40.5340.5331.5 cm) with two levels of infrared motion sensors. The first and second levels of sensors are 6 and 12.5 cm from the cage floor, respectively. The activity monitoring system checks each of the beams at a frequency of 100 Hz to determine whether the beams are interrupted. Interruptions of two or more consecutive beams separated by at least one second are recorded as a movement score. Cumulative counts are compiled and downloaded every 10 minutes into the OASIS data collection program and organized into motor indices [20–22]. The following four locomotor indices were analyzed. The horizontal activity index (HA) measures the overall motor activity in the lowest tier of the testing cages. Total distance (TD) and vertical activity (VA) measure the specific motor behaviors of forward ambulation and rearing, respectively. Number of stereotypic movements (NOS) measures the number of different episodes of stereotypic activity with at least a one-second interval before the beginning of another episode. Procedure Motor activities were continuously recorded for a total of 14 to 15 days as follows: Day 1 – baseline; Day 2 – 0.9% saline injection at 13:00 h (i.p.) and at 14:00 h (s.c.); Day 3 – rats randomly assigned into Groups I, II, III, or IV. Group I received only saline (s.c.) at 13:00 h and 14:00 h, while Groups II, III, and IV received 50 mg/kg, 100 mg/kg, or 200 mg/kg valproate (i.p.), respectively, at 13:00 h and 2.5 mg/kg MPD (s.c.) at 14:00 h. Group I then received 2.5 mg/kg MPD (s.c.) at 14:00 h on Days 4–9, no drug treatment (washout phase) on Days 10–14, and at 13:00 h saline injection followed by a re-challenge dose of 2.5mg/kg MPD (s.c.) at 14:00 h on Day 15. Groups II, III, and IV received 2.5 mg/kg MPD (s.c.) at 14:00 h on Days 4–8, no drug treatment (washout phase) on Days 9–13, followed by saline injection at 13:00 h and a re-challenge dose of 2.5 mg/kg MPD (s.c.) at 14:00 h on Day 14. Data analysis Previous studies indicated that repeated injection of saline did not produce a significant increase in activity for all four motor indices investigated. Thus, the present study used saline as a comparison to the effects of the drugs since activity levels at saline did not fluctuate with time [20–22]. The effect of MPD for each motor index was determined by subtracting the activity score of the 2 hours post-saline injection (Day 2) from that of the 2 hours post-MPD injection (Group I: Days 4 to 9 and Day 15; Groups II-IV: Days 4 to 8 and Day 14). For the
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effect of valproate injection on MPD (Day 3 and Day 15), the activity score for the 2 hours post-saline injection (Day 2) was subtracted from that of the 2 hours post-valproate injection (Day 3 or Day 15). The presence of sensitization for Groups II, III, and IV was determined by comparing the results of all other days (Days 5 to 8 and 14) to that of MPD treatment on Day 4. For Group I, the presence of sensitization was determined by comparing results of all other days (Days 5 to 9 and 15) to that of the initial MPD challenge on Day 4. Values are presented as the mean 6 S.E.M. for each 10 minute or 2 hour sample. Since locomotor baseline activity varies from animal to animal and from group to group, each animal baseline following saline injection was used as its own control, and the difference between the saline control and the subsequent treatment, i.e. the delta, was used for the statistical analysis. The deltas of the groups were represented as the mean 6 S.E.M. for each 10 min sample in the figures. These results were analyzed using repeated measures of ANOVA (two levels: treatment day and 10 minute sample) and post-hoc Fischer’s (LSD) method. Significance for all of the above comparisons was set at * p , 0.05. Results Repeated administration of methylphenidate The effects of repeated MPD (2.5 mg/kg) treatment (Group I) on TD and HA are summarized in Figure 1. The bar graph shows locomotor activity of the first 2 hours after MPD injection for Days 4 to 15 of the experiment. Activity following saline injection was arbitrarily set at zero. Single MPD (2.5 mg/kg) administration on Day 4 elicited an increase in locomotor activity compared to saline, while the same MPD dose given daily for five additional days (Days 5 to 9) caused a further increase in the locomotor activity in all four motor indices (HA, TD, VA, and NOS) studied. There was a significant increase (p , 0.05) in activity after the second MPD injection for HA (Figure 1) and NOS (not shown), and a significant increase (p , 0.05) in activity after the fourth MPD injection for TD (Figure 1, Day 7) and VA (not shown). This significant enhancement in motor activity after multiple MPD administration is interpreted as sensitization to the locomotor effects of the stimulant. Moreover, when MPD was given again (Day 15) after 5 days of drug abstinence (Day 10 to 14), the locomotor activity compared to that on Day 4 was still significantly high (at least p , 0.05) in all four motor indices. Thus, sensitization was observed to develop and persist even in Day 15 for rats given 2.5 mg/kg MPD. Dose-response of valproate Gross observation of the effects of valproate (25, 50, 100, and 200 mg/kg) on locomotion during the initial 60 minutes after valproate administration showed that the 25 and 50 mg/kg dose exerted no behavioral effects, while the 100 and 200 mg/kg doses produced wet dog shakes during the first 20 minutes after valproate administration. Using the CAAM in a pilot study, it was observed that the locomotion activities following the dose of 25 mg/kg valproate were similar to those of saline; consequently, this dosage was removed from use. The 50-mg/kg dose produced a biphasic effect (sigmoidal curve) in locomotor activities compared to saline. Immediately after valproate administration, activities elicited by the 50-mg/kg dose were less
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Fig. 1. The average (N 5 12) total distance (TD) and horizontal activity (HA) traveled in the first 2 hours after saline and MPD administration for the length of the experiment. Sensitization is present by Day 6 and still measurable by Day 15. Values are presented as the mean 6 S.E.M. * p , 0.05.
than those after saline injection, followed by a gradual increase in activity and then activity leveled off at 30–40 minutes after drug injection. The 100 and 200 mg/kg doses elicited a more variable motor response pattern than the 50 mg/kg valproate. The 100 mg/kg dose elicited some increase in TD for 20 minutes after drug injection, while a decrease in activities in
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HA, VA, and NOS occurred during this time. The 200 mg/kg dose elicited a slight decrease in HA and VA for the first 20 minutes after drug injection, while activities fluctuated in TD and NOS during this time. Another study was conducted in which 50 mg/kg valproate was given repeatedly for 6 consecutive days and followed by 5 days of washout and a re-challenge dose of 50 mg/kg valproate. It was found that the 50 mg/kg valproate did not produce any sensitized response to itself. In conclusion, the three valproate doses produced minor changes in locomotor activities compared to saline. Effects of single valproate injection on the acute response to methylphenidate Figure 2 summarizes the acute temporal response of total distance (TD) on Day 3 following administration of 2.5 mg/kg MPD in the four groups, each of which received either 0.9% saline, or 50, 100, or 200 mg/kg valproate 1 hour prior to MPD injection. Values are represented as the mean 6 S.E.M. of the delta for each 10 minute sample. The group pretreated with saline showed a general increase in total distance following MPD injection as expected, while the other three groups pretreated with valproate did not share this similar elevation. Post-hoc analysis revealed that 50 mg/kg and 200 mg/kg valproate given 1 hour prior to MPD administration significantly lowered the MPD-elicited motor response (p , 0.05), particularly during the initial 60 minutes after MPD injection. The 100-mg/kg valproate elicited similar general effects like the 50 and 200-mg/kg valproate, but the effect was not significant. The effects of the three valproate doses on horizontal activities, vertical activities, and number of stereotypic movements given 1 hour prior to MPD were similar to those effects on TD. Effect of single valproate pretreatment on repeated MPD administration Figure 3 summarizes the acute temporal (line graph) and the 2-hour total response (bar graph) of total distance following a challenge dose of 2.5 mg/kg MPD and a re-challenge dose of 2.5 mg/kg MPD on Day 15 for Group I and on Day 14 for Groups II, III, and IV. Group I was given saline pretreatment 1 hour prior to MPD. Groups II, III and IV received 50, 100 and 200 mg/kg valproate 1 hour prior MPD, respectively. Pretreatment with saline, followed by 100 and 200 mg/kg valproate 1 hour prior to MPD failed to modulate the induction and the expression of behavioral sensitization produced by repetitive MPD administration. However, 50 mg/kg valproate pretreatment blocked the MPD elicited sensitization (Figure 3 bar graph: Group II). Vertical activity, horizontal activity, and number of stereotypic movements also showed a similar response as total distance.
Discussion Behavioral response to valproate alone A moderate dose of valproate (50 mg/kg) did not significantly alter motor activity at any dose studied. In higher doses (100 mg/kg and 200 mg/kg), some wet dog shakes occurred transiently. These results confirmed previous reports on the behavioral effects of valproate [23], as well as findings with other GABAergic agonists such as muscimol and baclofen [23].
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Fig. 2. Acute temporal response for total distance (TD) following administration of 2.5 mg/kg MPD in four groups of rats (each N 5 8), each of which received either saline, 50, 100, or 200 mg/kg valproate 1 hour prior to MPD. All three doses of valproate attenuated the behavioral response to MPD. Values are presented as the mean 6 S.E.M. for each 10 minute sample minus the corresponding values following saline injection for each treatment group.
However, one study [24] reported that valproate, along with other GABA agonists, such as chlordiazepoxide, 3- APS, Pentobarbitone, and Clonazepam, produced a dose-related decrease in locomotor activity. This study used a gross measure of locomotor activity rather than quantitative measurement. Variation among results of pertinent studies may arise from
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Fig. 3. Acute temporal response (line graph) and 2-hour total (bar graph) for total distance (TD) following administration of 2.5 mg/kg MPD in four groups of rats. Group I: saline pretreatment 1 MPD. Group II: 50 mg/kg valproate 1 MPD. Group III: 100 mg/kg valproate 1 MPD. Group IV: 200 mg/kg valproate 1 MPD. The 50-mg/kg dose blocked the sensitized response elicited by repetitive MPD administration, while the 100 mg/kg and 200 mg/kg valproate did not block this sensitization. Values are presented as the mean 6 S.E.M. for each 10 minute sample for the line graph and 2 hours for the bar graph. * p , 0.05.
procedural differences or from differences in mechanism of action and effect on motor behavior between all the drugs studied. For example, valproate raises synaptic concentration of GABA by slowing its metabolic degradation. Pentobarbitone acts directly on the chloride ionophore, while 3-APS is a structural analogue of GABA [8]. Nevertheless, valproate’s ma-
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jor behavioral effect appears to be a transient increase in wet dog shakes that resolves 20–30 minutes after injection. Effects of valproate on the acute response to methylphenidate All three doses of valproate (50, 100, and 200 mg/kg) administered one hour prior to MPD injection acutely attenuated the locomotor response to MPD. GABAergic drugs have been shown to attenuate the locomotor response to amphetamine [8, 12]. Studies using valproate (200 mg/kg), baclofen (2.5 mg/kg), and THIP (10 mg/kg) found that the administration of high doses, but not lower doses, blocked the acute effects of a locomotor activating dose of amphetamine (1.5 mg/kg) (9). However, all three drugs were administered at the same time as amphetamine, making it difficult to correlate with the present study in which valproate and MPD were given one hour apart. Agmo et al [8] also reported that both low and high doses of valproate and baclofen did not block the motor effect of 3.0 mg/kg amphetamine, suggesting that a normal tonic inhibitory effect of GABA systems on dopamine may be overcome at higher levels of dopaminergic activity. This would account for the finding that all 3 doses used in our study attenuated the motor response to a low dose of MPD [20–22], suggesting that valproate would not alter the effects of a higher dose of MPD. Klitnetick [25] also found that the GABAb agonist baclofen inhibits dopamine transmission in the nucleus accumbens by hyperpolarizing dopaminergic cells in the ventral tegmental area. This provides an alternative explanation for the effect of valproate, which does not act specifically on GABAa or GABAb receptors. Effect of valproate pretreatment on repeated MPD administration Earlier studies in our laboratory showed that a single injection of a noncompetitive NMDA antagonist, MK-801, blocked sensitization to the locomotor effects of MPD. In the present study, only the lowest dose of valproate used, 50 mg/kg, blocked the development of sensitization to methylphenidate. In other studies, Clonazepam [12] and THIP [14] were given repeatedly prior to stimulant administration, whereas our study gave a single injection of valproate 1 hour prior to the first of six repeated MPD injections. This difference in drug administration schedules may account for differences obtained in the results. Other reports have described the ability of the GABAergic agonist THIP to prevent the induction of sensitization. Karler [14] reported that three doses of THIP injected into the anterior cortex but not the posterior cortex, 5 minutes prior to systemically administered 12 mg/kg amphetamine blocked the induction of sensitization produced by 8 mg/kg amphetamine given 48 hours later [14]. THIP injected systemically also blocked the development of sensitization to amphetamine [14]. THIP, whether given systemically or in the cortex prior to the stimulant, blocked the induction of sensitization. These findings and our results are consistent with an inhibitory role of GABA in the induction of sensitization to motor effects of stimulants. Because the projection from the nucleus accumbens to the ventral palladium is GABAergic, this projection is involved in locomotor activity elicited by dopamine release in the nucleus accumbens [26]. A GABAergic influence on dopamine neurons could modulate the effects of stimulants on locomotor activity [13], and could be one interpretation of the effects of val-
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proate on the induction of locomotor sensitization. However, a more likely explanation for the variable effects of GABA agonists on sensitization is that some compounds stimulate GABA receptors directly while others indirectly cause an activation of GABA receptors by several different mechanisms [27]. GABAa agonists regulate dopamine indirectly, while GABAb agonists act directly on dopamine neurons [13]. In response to sensitizing treatment, a change may be observed in the dopamine neurons themselves or a change in GABA regulation of dopamine release. While there are numerous differences, which may account for different GABAergic drug effects on sensitization, benzodiazepines and barbiturates act only on the GABAa receptor complex, while THIP and baclofen act on GABAb and valproate acts on both [27]. It has been well documented that the neural substrates necessary for the induction and expression of sensitization to psychomotor stimulants are not the same. In this regard, an appropriate step in exploring further connections between the GABA-dopamine interaction and the mechanism of sensitization would be to examine whether valproate would block induction and expression of this phenomenon. In summary, a single dose of valproate produced minimal locomotor effects and when given prior to any MPD administration appeared to attenuate the locomotor response to MPD sensitization. Acknowledgment This study was supported in part by the Pat Rutherford Chair in Psychiatry. References 1. Robinson TE, Becker JB. Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychodis. Brain Res. 1986; 396:157–198. 2. Kuczenski R, Segal D. Concomitant characterization of behavioral and striatal neurotransmitter response to amphetamine using in vivo microdialysis. J. Neurosci. 1989; 9: 2051–2065. 3. Robinso TE, Berridge KC. The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res. Rev. 1993; 18: 247–291. 4. Wolf ME. The role of excitatory amino acids in behavioral sensitization to psychomotor stimulants. Prog. In Neurobiol. 1998; 54: 679–720. 5. Karler R, Calder LD, Bedingfield JB. Cocaine behavioral sensitization and the excitatory amino acids. Psychopharmacol. 1994; 115: 305–310. 6. Pierce RC, Kalivas PW. A circuitry model of the expression of behavioral sensitization to amphetamine-like psychostimulants. Brain Res. Rev. 1997; 25:192–216. 7. Waldmeier PC, Maitre L. Effects of baclofen on dopamine metabolism and interaction with neuroleptic effects. Eur. J. Pharmacol. 1978; 47:191–200. 8. Agmo A, Belzung C, Giordano M. Interactions between dopamine and GABA in the control of ambulatory activity. J. Neural Transm. 1996; 103: 925–934. 9. Cott J, Engel J. Suppression by GABAergic drugs of the locomotor stimulation induced by morphine, amphetamine, and apomorphine: evidence for both pre- and post-synaptic inhibition of catecholamine systems. J. Neural Transmission 1997: 40: 253–268. 10. Sandoval MR, Palermo-Neto J. Effect of manipulation of the GABA system on dopamine-related behaviors. Braz. J. Med. Bio. Res. 1995; 28: 88–99. 11. McElroy SL, Keck PE, Pope HG, Hudson JI. Valproate in psychiatric disorders: literature review and clinical guidelines. J. Clin. Psychiatry 1989; 50: 23–29.
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