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The significance of central noradrenergic, dop-aminergic and serotonergic neural systems for the locomotor stimulant effects of methylphenidate was investigated in the rat. In order to study the role of brain catecholamines, rats were pretreated with reserpine (2.5 mg/kg) followed 24 hrs later by treatment with α-methyltyrosine (25 mg/kg) or U-14,624 (75 mg/kg), a dopamine-β-hydroxylase inhibitor. In these experiments, methylphenidate stimulated motor activity was antagonized by α-methyltyrosine and enhanced after treatment with U-14,624, suggesting that release of newly synthesized dopamine is important to a locomotor stimulant action of methylphenidate. Evidence implicating brain serotonin in the actions of methylphenidate was obtained in rats pretreated with pargyline or p-chlorophenylalanine (PCPA). Administration of pargyline 1 hr prior to methylphenidate was found to reduce the locomotor activity induced by methylphenidate and this was antagonized by pretreatment with low doses of PCPA. Higher doses of PCPA caused a significant elevation of methylphenidate induced activity which could be reduced by 5-hydroxytryptophan. Destruction of serotonergic neurons with 5,7-dihydroxytryptamine also potentiated methylphenidate induced locomotion. These latter findings suggest that serotonergic fibers have an inhibitory function in brain. These results are discussed in relation to the possible mechanism by which methylphenidate may act in hyperkinesis.
Much attention has been directed toward understanding the actions of those stimulant drugs found to be of value in the treatment of hyperkinetic syndrome (MBD) (Snyder and Meyerhoff, 1973). Of these drugs, d-amphetamine has been the most widely studied. From animal experiments, the stimulant effects of d-amphetamine have been suggested to be mediated through the indirect release of catecholamines (Weissman et al., 1966; Hanson, 1966; Fibiger et al., 1973; Von Voigtlander and Moore, 1973; Hollister et al., 1974). Based upon such findings, Snyder and Meyerhoff (1973) have proposed that the therapeutic effectiveness of d-amphetamine in MBD was related to this action. These workers, however, did not provide an explanation for the paradoxical quieting effects of d-amphetamine in this syndrome.
Recently, several studies in animals have provided evidence that the neurochemical effects of d-amphetamine to induce motor activity (Neill et al., 1972; Mabry and Campbell, 1973; Breese et al., 1974b) and to increase rates of responding on a variable-interval schedule of reinforcement (Green and Harvey, 1974) are also influenced by central serotonergic fibers. In these latter studies, reduction of brain serotonin caused a marked enhancement of the stimulant properties of d-amphetamine, suggesting an inhibitory influence of serotonergic pathways. These findings subsequently led to the proposal that this serotonergic inhibitory action produced by d-amphetamine may have relevance to its therapeutic use in hyperkinesis (Breese et al., 1974a, b).
Although methylphenidate is widely used in MBD (Millichap, 1968; Seger and Hallum, 1974), the basic neurochemical actions of methylphenidate have been less widely studied than those of d-amphetamine. The stimulatory properties of this drug have been proposed to be related to indirect release of catecholamines (Franklin and Herberg, 1974; Costall and Naylor, 1974) as well as direct actions on receptors (Thornburg and Moore, 1973; Costall and Naylor, 1974). In addition, a high dose of methylphenidate (100 mg/kg) has been reported to increase 5-HIAA content in rat brain without changing content of 5-hydroxytryptamine (Scheel-Krüger and Hasselager, 1974). The purpose of the present study was to examine the role of endogenous catecholamines in the action of methylphenidate and to determine if alterations in 5-hydroxytryptamine content in brain would influence methylphenidate-induced motor activity.
Male Sprague-Dawley rats (250–400 g) were housed in rooms with controlled lighting (10 hr light and 14 hr dark). Motor activity was recorded from activity cages with six photocell sensors mounted on the outer wall of a circular runway in a dark, soundproof room away from recording equipment (Breese et al., 1974b). Counts were automatically recorded at 15 min intervals over a 2.5 or a 3 hr period. Animals were “habituated” to the chamber for 1 hr before receiving methylphenidate intraperitoneally.
Some rats were injected with 2.5 mg/kg reserpine (s.c.) 24 hrs before receiving 75 mg/kg U-14,624,. 25 mg/kg α-methyltyrosine, or vehicle. These animals were then tested with 5 mg/kg methylphenidate 1 hr later. In addition, methylphenidate was also administered to rats pretreated with various monoamine oxidase inhibitors, p-chlorophenylalanine (PCPA), or 5,7-dihydroxytryptamine (5,7-DHT). The PCPA (200 mg/kg) was suspended in a 0.5% methylcellulose solution and administered orally for 1 day or 2 successive days to reduce serotonin (Koe and Weissman, 1966). The methylphenidate (10 mg/kg) was administered 24 hrs after the last dose of PCPA. Some rats that received PCPA were given 5-hydroxytryptophan (75 mg/kg) 1 hr before methylphenidate. The 5,7-DHT (200 µg) was administered intracisternally in 25 µl of saline containing 0.5 % ascorbic acid 30 min after pargyline (50 mg/kg, i.p.) to destroy serotonergic fibers (Baumgarten et al., 1973; Breese and Cooper, 1975). Animals were examined 10 days after treatment. Rats were given methylphenidate. 1 hr after receiving pargyline, trancylcypromine, nialamide, clorgyline, iproniazid, or pheniprazine.
Methylphenidate HCl and reserpine were furnished by Ciba-Geigy Corp., Summit, N.J. The 5,7-dihydroxytryptamine and α-methyltyrosine were purchased from Regis Chemical Company (Chicago, Ill.). P-Chlorophenylalanine was kindly supplied by Pfizer Inc. (Groton, Conn.). The pargyline, (Abbott Laboratories, North Chicago, Ill.), pheniprazine (Lakeside Laboratories, Milwaukee, Wis.), tranylcypromine (Smith, Kline and French Lab., Philadelphia, Pa.), iproniazid (Hoffmann LaRoche Laboratories, Nutley, N.J.), nialamide (Pfizer Inc., Groton, Conn.) and clorgyline (May and Baker, LTD, Dagenham) were all gifts. The L-5-hydroxytryptophan was purchased from H & H Bulk Biochemical (Los Angeles, California). The U-14,624 (1-phenyl-3-[2-thiazolyl]-2-thiourea) was purchased from Aldrich Chemical Co. (Milwaukee, Wis.).
Determinations of brain norepinephrine, dopamine, and serotonin were carried out as previously described (Breese and Traylor, 1970).
Statistical comparisons between groups were made with either Students’s or Dunnett’s t test (Two tailed probability values are reported). Correlational analyses were performed by multiple regression and partial correlation.
While the effects of d-amphetamine-induced motor activity have been intensively studied (Weissman et al., 1966; Creese and Iversen, 1973; Hollister et al., 1974), less information concerning the neurochemical mediation of methylphenidate-induced locomotor activity was available. For that reason, the effects of methylphenidate were tested in reserpinized rats treated with either α-methyltyrosine to inhibit synthesis of both catecholamines or U-14,624 to inhibit synthesis of norepinephrine (Fig. 1). Following treatment with α-methyltyrosine, methylphenidate-induced locomotion was drastically reduced, suggesting that the response elicited after reserpine treatment was related to release of newly synthesized catecholamines. Treatment with the dopamine-β-hydroxylase inhibitor, U-14,624, did not antagonize the increased locomotion caused by methylphenidate, but rather produced a significant elevation in locomotor activity. Although Thornburg and Moore (1973) did not observe a significant increase in methylphenidate-stimulated activity in mice after U-14,624, this latter observation deserves further investigation with regard to a possible inhibitory role for norepinephrine in rats. Regardless, these findings seemed to implicate dopaminergic pathways in the mediation of the increased activity induced by methylphenidate.
In order to search for possible involvement of serotonergic fibers in the action of methylphenidate, methylphenidate was administered to rats following treatment with either PCPA or 5,7-DHT. The increase in motor activity induced by this drug was found to be markedly enhanced following these treatments (Fig. 2). This potentiation of methylphenidate-induced stimulation produced by PCPA was subsequently found to be significantly antagonized by 5-hydroxytryptophan (P < 0.05; Table 1). The effect of these various treatments on brain monoamines is shown in Table 1.
In accord with experiments with d-amphetamine (Breese etal., 1974b), administration of pargyline (50 mg/kg) also significantly reduced the locomotor response induced by methylphenidate (Fig. 3). This reduced response included a decrease in peak activity as well as in total activity during the 3 hr period following drug administration. At a higher dose of methylphenidate (10 mg/kg), pargyline pretreatment did not produce a significant reduction in the locomotor response to methylphenidate (Table 2). The degree of inhibition of methylphenidate-induced locomotor activity could also be altered if the dose of pargyline were varied (Table 3, top).
If the inhibition of methylphenidate-induced motor activity by pargyline were due to brain serotonin, pretreatment with PCPA should reverse this inhibition. Fig. 4 indicates that 24 hrs after a single dose of PCPA the inhibitory effects of pargyline on activity stimulated by 5 mg/kg methylphenidate were antagonized. In this case, PCPA pretreatment reduced brain serotonin content by approximately 40% and antagonized the rise in serotonin due to pargyline by the same degree.
In order to determine whether the inhibition of methylphenidate by pargyline might be shared by other MAO inhibitors, several compounds in this class were examined for their effect on methylphenidate-induced motor activity. As shown in Table 3, lower doses of tranylcypromine, nialamide, and clorgyline, in addition to pargyline, were found that would significantly inhibit the locomotion induced by methylphenidate. Lower doses of pheniprazine showed a tendency for reduction of methylphenidate-induced motor activity, but the reduction was not great enough to be significant. In most cases, higher doses of the MAO inhibitors produced no change or enhanced the increase in locomotor activity caused by methylphenidate. All doses of iproniazid examined seemed to enhance the effects of methylphenidate. The effect of these MAO inhibitors on brain monoamines at the time methylphenidate was injected is also shown in Table 3. Based on changes in brain monoamines, these latter results did not provide an obvious predictor of how these MAO inhibitors would interact with methylphenidate-induced motor activity. (Correlation coefficients: activity with norepinephrine, r = −0.0159; activity with dopamine, r = 0.217; activity with serotonin, r = −0.0878). However, in those groups of animals which exhibited a significant inhibition of methylphenidate-induced activity, a negative correlation between activity and brain serotonin content was found which approached the 0.05 level of significance (r = −0.865, n = 5, 0.1 > P > 0.05).
In accord with the view that methylphenidate acts by releasing catecholamines (Costall and Naylor, 1974), the locomotor stimulatory actions of this compound were found to be reduced by pretreating reserpinized animals with α-methyltyrosine. Since blockade of norepinephrine synthesis with U-14,624 in the reserpinized rats did not decrease methylphenidate-induced motor activity, it would appear that the locomotor stimulation induced by methylphenidate is dependent upon an intact dopaminergic mechanism. Although a similar conclusion has been reached concerning d-amphetamine-induced locomotor activity (Thornburg and Moore, 1973; Hollister et al., 1974), the mechanism of action of methylphenidate would appear to differ from that of d-amphetamine in that methylphenidate stimulation is not antagonized by α-methyltyrosine unless reserpine is present (see Thornburg and Moore, 1973; Franklin and Herberg, 1974).
This study also provides evidence for an involvement of serotonergic fibers in the action of methylphenidate. Similar to reports that reduction of serotonin in brain will enhance locomotor activity induced by d-amphetamine (Neill et al., 1972; Mabry and Campbell, 1973; Breese et al., 1974a, b), methylphenidate hyperactivity was found to be potentiated following reduction of brain serotonin with PCPA or 5,7-DHT. Although it is possible to argue that PCPA may interfere with methylphenidate metabolism or alter its uptake into brain, this would not explain why the activity response to methylphenidate is enhanced 10 days after treatment with 5,7-DHT or why 5-hydroxytryptophan would antagonize the effect of PCPA. In addition, 50 mg/kg of pargyline administered 1 hr before methylphenidate was found to reduce locomotor activity. Since this action of pargyline was antagonized by PCPA, serotonin fibers were implicated in the inhibition produced by pargyline. However, it was found that one MAO inhibitor, iproniazid, enhanced rather than reduced methylphenidate locomotor activity. Whether this could be related to changes in drug metabolism or to other interactions between these compounds will require additional investigation.
The mechanism for the paradoxical quieting effects of d-amphetamine and methylphenidate in hyper-kinetic children has yet to be resolved. By studying the basic mechanisms responsible for the actions of such drugs, we have attempted to define new therapeutic approaches to alleviate the symptoms of this disease. Since the present data suggest that both d-amphetamine and methylphenidate activate a serotonergic neural system which inhibits locomotor activity, it would not seem unreasonable to believe that this action may have relevance to the mechanism by which these drugs act in hyperkinetic children. Such a proposal need not be contradictory with the view that brain dopamine is involved in the effects of such drugs upon the symptoms of MBD (Snyder and Meyerhoff, 1973), if an intact dopaminergic function is necessary for activation of this inhibitory serotonergic system (Breese et al., 1974b). Obviously, further work will be necessary to define interactions between these monoamine systems. Nevertheless, the observation that serotonergic systems play an inhibitory role in the actions of methylphenidate suggests the possibility that administration of serotonin precursors, such as tryptophan or 5-hydroxytryptophan, may be of value in the symptomatic control of hyperkinesis. Such therapy would be expected to enhance the “tone” of the serotonergic system, allowing its inhibitory function on locomotor activity to predominate. If this approach should be successful, it would not only support the proposal concerning the mechanism of action of d-amphetamine and methylphenidate, but also may provide additional information concerning the neurochemical basis of MBD.
We are grateful to Marcine Kinkead, Susan Hollister, Edna Edwards, and Joseph Farmer for their excellent technical assistance and to Roger Formisano for his expert statistical assistance. This work was supported by USPHS grants MH-16522 and HD-03110. Alan S. Hollister is Predoctoral Fellow in Neurobiology (MH-11107 and the Sloan Foundation) and is supported by a grant from the Pharmaceutical Manufacturers Association, Inc. George R. Breese is a USPHS Career Development Awardee (MH-00013).