Effects of Methyiphenidate on Extracellular Dopamine, Serotonin, and Norepinephrine: Comparison with Amphetamine

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1 Journal ofneurochemistry Lippincott Raven Publishers, Philadelphia 1997 International Society for Neurochemistry Effects of Methyiphenidate on Extracellular Dopamine, Serotonin, and Norepinephrine: Comparison with Amphetamine Ronald Kuczenski and David S. Segal Department of Psychiatry, School of Medicine, University of California San Diego, La Jolla, California, U.S.A. Abstract: Methylphenidate promotes a dose-dependent behavioral profile that is very comparable to that of amphetamine. Amphetamine increases extracellular norepinephrine and serotonin, in addition to its effects on dopamine, and these latter effects may play arole in the behavioral effects of amphetamine-like stimulants. To examine further the relative roles of dopamine, norepinephrine, and serotonin in the behavioral response to amphetamine-like stimulants, we assessed extracellular dopamine and serotonin in caudate putamen and norepinephrifle in hippocampus in response to various doses of methylphenidate (10, 20, and 30 mg/kg) that produce stereotyped behaviors, and compared the results with those of a dose of amphetamine (2.5 mg/kg) that produces a level of stereotypies comparable to the intermediate dose of methylphenidate. The methylphenidate-induced changes in dopamine and its metabolites were consistent with changes induced by other uptake blockers, and the magnitude of the dopamine response for a behaviorally comparable dose was considerably less than that with amphetamine. Likewise, the dose-dependent increase in norepinephrine in response to methylphenidate was also significantly less than that with amphetamine. However, in contrast to amphetamine, methylphenidate had no effect on extracellularserotonin. These results do not support the hypothesis that a stimulant-induced increase in serotonin is necessary for the appearance of stereotyped behaviors. Key Words: Methylphenidate Amphetamine Microdialysis Dopamine Serotonin Norepinephrine Caudate putamen Hippocampus. J. Neurochem. 68, (1997). The ability of amphetamine (AMPH) -like stimulants to enhance dopaminergic transmission in the brain plays a crucial role in the behavioral effects of these drugs. Consistent with this idea, AMPH and methamphetamine, which interact with the dopamine (DA) transporter to release DA, and cocaine (COC), which interacts with the DA transporter to block DA uptake, have all been shown to increase caudate putamen (CP) and nucleus accumbens extracellular DA in a dose-dependent manner. However, the quantitative features of the drug-induced behavioral profile and the regional extracellular DA response can be dissociated under a variety of conditions (for review, see Segal and Kuczenski, 1994). These observations suggest that other neurotransmitter systems likely contribute significantly to psychostimulant-induced behaviors. In this regard, converging evidence suggests that noradrenergic (Geyer et al., 1986; Holman, 1994; Florin et al., 1995; Antelman and Caggiula, 1996) and serotonergic (Brodie and Shore, 1957; Swonger and Rech, 1972; Mabry and Campbell, 1973; Breese et al., 1974; Geyer et al., 1976; Segal, 1976; Gately et al., 1985) systems may modulate the stimulant behavioral response, and AMPH-like stimulants have been shown to increase extracellular norepinephrine (NE) and Serotonin (5-HT) (Kuczenski and Segal, 1989, 1992a; Parsons and Justice, 1993; Florin et al., 1994; Kuczenski et al., 1995). Methylphenidate (MP), which binds to the DA transporter and inhibits DA uptake with a potency similar to that of COC (Ferris et al., 1972; Schweri et al., 1985; Wallet al., 1995; Gatley et al, 1996), promotes a dosedependent behavioral profile that is very comparable to that of AMPH (Browne and Sega!, 1977). Consistent with its actions as a DA-uptake blocker and like the other AMPH-like stimulants, MP has been shown to increase extracellular DA in the CP when administered systemically (Hurd and Ungerstedt, 1989; Butcher et al., 1991; Woods and Meyer, 1991). MP has also been shown to bind to the NE transporter and to be an effective in vitro inhibitor of NE uptake (Ferris et al., 1972; Ross, 1979; Ritz et al., 1987; Andersen, 1989; Wall et Received November 25, 1996; revised manuscript received January 17, 1997; accepted January 17, Address correspondence and reprint requests to Dr. R. Kuczenski at Department of Psychiatry (0603), UCSD School of Medicine, La Jolla, CA 93093, U.S.A. Abbreviations used: AMPH, amphetamine; COC, cocaine; CP, caudate putamen; DA, dopamine; DOPAC, 3,4-dihydroxyphenylacetic acid; HP, hippocampus; 5-HT, serotonin; HVA, homovanillic acid; MP, methyiphenidate; NE, norepinephrine. 2032

2 BIOGENIC AMINE RESPONSES TO METHYLPHENIDATE 2033 al., 1995; Gatley et al., 1996) and, therefore, it might be expected to increase extracellular NE. However, in contrast to the other AMPH-like stimulants, MP appears to have very weak potency both in binding to the 5-HT transporter and as an in vitro inhibitor of 5-HT uptake (Andersen, 1989; Wallet al., 1995; Gatley et al., 1996). Based on these criteria, MP would not be expected to increase extracellular 5-HT. Disruption of 5-HT function appears to enhance the locomotor effects of AMPH while attenuating the appearance of focused stereotypies (Neill et al., 1972; Sega!, 1976). Based on these and other observations, we suggested that the CP 5-HT response to AMPH may play an important role in the dose-dependent transition from locomotion to stereotypy and, therefore, that enhanced 5-HT function would be a feature common to stereotypy-producing doses of all AMPH-like psychostimulants (Segal, 1976, 1977; Kuczenski and Sega!, 1989). To examine this possible relationship further, we evaluated the CP DA and 5-HT responses across a range of MP doses and compared the responses with a stereotypy-inducing dose of AMPH. Also, to obtain additional information about the possible role of AMPH-induced increases in NE in the behavioral response, we examined the extracellular NE response in the hippocampus (HP) of the same animals. Our results indicate that MP, like AMPH and COC, promotes a dose-dependent increase in extracellular DA in the CP and extracellular NE in the HP, but has no effect on CP dialysate 5-HT. MATERIALS AND METHODS Rats obtained from Simonsen Laboratories (Gilroy, CA, U.S.A.; g) were maintained four per cage on a 14- h/ 10-h light/dark cycle (lights on at 5:00 a.m.) under standard laboratory conditions, with ad libitum access to food and water for at least 1 week prior to surgery. Animals used in this study were maintained in facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care, and all experimentation was conducted in accordance with the guidelines of the Animal Subjects Committee of the University ofcalifornia, San Diego School of Medicine, and the Guidefor Care and Use of Laboratory Animals of the Institute of Laboratory Animals Resources, National Research Council, Department ofhealth, Education and Welfare, Publication (NIH) 85-23, revised Animals were implanted stereotaxically with guide cannulae using procedures previously described in detail (Kuczenski and Segal, 1989). Guide cannulae extended 2.6 mm below the surface of the skull and were aimed at the CP (1.0 mm anterior to bregma, 2.8 mm lateral, and 6.2 mm below dura) and the HP (5.8 mm anterior, 4.8 mm lateral, and 7.5 mm below dura) according to the atlas of Paxinos and Watson (1986). Following surgery, animals were housed individually and allowed at least 1 week to recover before receiving any treatment. Each experimental group included six to eight subjects. Each rat was placed in an experimental chamber, and the dialysis probes were inserted on the day prior to treatment (between 3:00 and 4:00 p.m.) to allow for acclimation to the test environment and for adequate equilibration of the dialysis probes. Concentric microdialysis probes were constructed of Spectra/Por hollow fiber (MW cutoff 6,000, o.d. 250 ~.tm)as previously described (Kuczenski and Segal, 1989). The length of the active probe membranewas 3 mm. Probes were perfused with artificial cerebrospinal fluid (147 mm NaC1, 1.2 mm CaCl 2, 0.9 mm MgC12, 4.0 mm KC1) delivered by a microinfusion pump (1.5 pllmin) via 50 cm of Micro-line ethyl vinyl acetate tubing connected to a fluid swivel. Dailysate was collected through glass capillary tubing into vials containing 20 jil of 25% methanol, 0.2 M sodium citrate, ph 3.8. Under these conditions, levels of dialysate DA, 5-HT, NE, andmetabolites were stable throughout the collection and analysis interval. Samples were collected outside the experimental chamber to avoid disturbing the animal. Individual probe recoveries, whichranged from 7to 10%, were estimated by sampling a standard DA solution in vitro. Preliminary studies indicated that individual probe recoveries for DA, 5- HT, and NE were similar. The behavioral chambers, described in detail elsewhere (Segal and Kuczenski, 1987; Kuczenski and Segal, 1989), were soundproofed, and the animals were maintained on a 14-h/b-h light/dark cycle (lights on at 5:00 a.m.), with constant temperature and humidity. Animals had continuous access to food and water. Each animal was videotaped for observational rating as previously described (Segal andkuczenski, 1987). At the end of the experiment, each animal was perfused with formalin for histological verification of probe placements. All drugs were dissolved in saline and administered in a volume of 1 ml/kg of body weight at about 10:00 a.m. Doses referto the free base. AMPH (NIDA, Rockville, MD, U.S.A.) was administered subcutaneously, and MP (CIBA Geigy, Ardsley, NY, U.S.A.) was administered intravenously. Dialysate samples (30 ~.tl) were collected every 20 mm and were assayed for DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine, 5-hydroxyindoleacetic acid, and 5-HT in the CP samples or for NE in the HP by HPLC with electrochemical detection. In all experiments, solutions of standards revealed a clean separation between 3-methoxytyramine and 5-HT. Each assay by HPLC with electrochemical detection used a 100- mm X 4.6-mm ODS-C18 3 p~mcolumn (Regis) maintained at 40 Cfor DA/5-HT assays or at 30 Cfor NE assays. The mobile phase (0.05 M citric acid, 7% methanol, 0.1 mm Na2EDTA, and 0.2 mm octanesulfonate adjusted to ph , for DA/5-HT; 4% methanol and 1.5 mm octanesulfonate, for NE) was delivered at 0.6 ml/min by awaters model 510 pump. Amines were detected with Waters 460 detectors with glassy carbon electrodes maintained at V relative to a Ag/AgCl reference electrode. The quantification limits for DA, 5-HT, and NE were near 2 3 fmol. Concentrations were estimated from peak areas using a Waters Maxima 820 data station. Substances in the dialysates were corrected for individual probe recoveries to account for this source of variability; although the exact relationship between dialysate concentration and actual extracellular transmitter content is not clear (Wages et a!., 1986; Church and Justice, 1987; Benveniste et al., 1989; Stahle et al., 1991), values are presented as dialysate concentration to allow for meaningful comparisons with other data in the literature. Drug-induced neurochemical and behavioral effects were evaluated statistically using one- or two-way repeated-measures ANOVA. Group/time comparisons were made using t tests with Bonferroni corrections. J. Neurochem., vol. 68, No. 5, 1997

3 2034 R. KUCZENSKI AND D. S. SEGAL FIG. 1. Stereotyped behaviors in response to MP and AMPH. The data represent the percentage oftime animals were engaged in nonoral (focused sniffing, repetitive head and limb movements) and oral (continuous licking, biting) stereotypies during the mm interval following drug injection. RESULTS Consistent with our past results (for review, see Sega! and Kuczenski, 1994), the acute administration of 2.5 mg/kg AMPH produced a typical multiphasic locomotor response profile, which included an initial brief period of locomotion, a prolonged period of focused stereotypies in the absence of locomotion, and a poststereotypy phase of enhanced locomotor activity (data not shown). Stereotypies consisted primarily of repetitive head movements with occasional episodes of oral behaviors (Fig. 1). MPpromoted a dose-dependent pattern of locomotion and stereotypy similar to our previous results with AMPH (Segal and Kuczenski, 1994) and MP (Browne and Segal, 1977). Focused sniffing, the predominant stereotyped behavior at the lowest dose, was replaced by repetitive head movements, then oral behaviors, as the MP dose was increased progressively (Fig. 1). Of particular relevance to the present studies, the behavioral response to 20 mg/kg MP closely paralleled the response to 2.5 mg/kg AMPH, both in terms of the qualitative features of the stereotyped behaviors (Fig. 1) and, as we have previously shown (Browne and Segal, 1977), in the overall pattern and duration of the multiphasic locomotor response (data not shown). The regional extracellular neurotransmitter responses to AMPH and MP are summarized in Figs AMPH (2.5 mg/kg) increased CP DA and 5-HT and HP NE, and the magnitude and duration of the increases were consistent with our past results (Kuczenski and Segal, 1989; Florin et al., 1994; Kuczenski et al., 1995). Also as previously observed, AMPH decreased CP DOPAC to ~-~20%and HVA to -~60%of predrug baseline values (data not shown). A different neurotransmitter profile was observed with MP. As with AMPH, MP increased CP extracellular DA in a dose-dependent manner with respect to both the peak and the duration of the response (Fig. 2). However, although 2.5 mg/kg AMPH and 20 mg/kg MP promoted similar behavioral responses, and the duration of the DA responses was comparable, the peak DA response to MP was substantially lower. In addition, in contrast to AMPH, MP had no effect on CP DOPAC and increased HVA following the highest MP dose (30 mg/kg) (data not shown). Differences between MP and AMPH were also observed in the HP NE responses (Fig. 3). The duration of the NE response to MP was progressively longer as a function of increasing dose, and the peak NE responses over this MP dose range were all significantly less than the response to 2.5 mg/kg AMPH. The greatest differences between MP and AMPH were observed in the CP 5-HT responses (Fig. 4). In contrast to 2.5 mg/kg AMPH, none of the doses of MP tested significantly altered extracellular 5-HT. DISCUSSION The dialysate neurotransmitter responses to MP are generally consistent with the results of in vitro studies of the interactions of this psychostimulant with biogenie amine transporter systems. MP has been shown to inhibit DA and NE uptake (Ferris et al., 1972; Andersen, 1987, 1989; Wall et al., 1995) and to bind with high affinity to the DA and NE transporters (Schweri et al., 1985; Ritz et al., 1987; Gatley et al., 1996). In contrast, MP is substantially less potent in inhibiting 5-HT uptake compared with the catecholamines (Andersen, 1989; Wall et al., 1995) and has an affinity one to two orders of magnitude lower in binding to the 5-HT transporter (Ritz et al., 1987; Gatley et al., 1996). Thus, the ability of systemic MP to increase extracellular DA and NE at doses that do not affect extracellular 5-HT is consistent with these in vitro data. However, the failure of stereotypy-inducing doses of MP to increase extracellular 5-HT in the CP is not consistent with our hypothesis that a stimulantinduced increase in 5-HT plays an important role in the dose-dependent transition from locomotor activation to focused stereotypies. There have been no reports in the literature of the effects of MP on extracellular 5-HT or NE, and the few characterizations of MP-induced changes in extracellular DA involved anesthetized animals at short intervals following probe implantation (Hurd and Ungerstedt, 1989; Nomikos et al., 1990; Butcher et al., 1991; Woods and Meyer, 1991; During et al., 1992). Each of these latter studies revealed MP-induced increases J. Neurochem., vol. 68, No. 5, 1997

4 BIOGENIC AMINE RESPONSES TO METHYLPHENIDATE 2035 FIG. 2. Temporal profile of the CP DA response to increasing doses of MP compared with AMPH (2.5 mg/ kg). Histograms represent the peak response or the area under the curve (A.U.C.) during the indicated interval. p<0.01, compared with the response to AMPH. ~p < 0.05, ~ < 0.01, compared with the response to 30 mg/kg MP. in extracellular DA, but, because of the limitations of early sampling after probe insertion (Westerink and De Vries, 1988; Santiago and Westerink, 1990), as well as the use of preparations from anesthetized animals, the results do not provide a data base with which the quantitative aspects of the present results in awake animals can be compared. However, the DA and NE responses to MP reported above are generally compatible with the results from our previous studies of other uptake blockers with respect to their efficacy and potency relative to releasers such as AMPH and methamphetamine (Kuczenski et al., 1991, 1995; Kuczenski and Segal, 1992b; Florin et al., 1994). With regard to CP DA, several features of the response to AMPH-like stimulants appear to differentiate AMPH, a DA releaser, from DA-uptake blockers. For one, we have shown previously that the dose-dependent increases in extracellular DA promoted by AMPH are substantially and significantly greater than behaviorally similar doses of the uptake blockers, COC and fencamfamine (Kuczenski et al., 1991; Kuczenski and Segal, 1992b). Consistent with those data and the presumed mechanism of action of MP as a DA-uptake blocker (Ferris et al., 1972; Schwen et al., 1985; Wall et al., 1995; Galley et al., 1996), the maximum CP DA response to 2.5 mg/kg AMPH was about threefold greater FIG. 3. Temporal profile of the HP NE response to increasing doses of MP compared with AMPH (2.5 mg/kg). Histograms represent the peak response or the area under the curve (A.U.C.) during the indicated interval. *p < 0.05, compared with the response to AMPH. +*p < 0.01, compared with the response to 30 mg/kg MP. J. Neurochem., vol. 68, No. 5, 1997

5 2036 R. KUCZENSKI AND D. S. SEGAL FIG. 4. Temporal profile of the CP 5-HT response to increasing doses of MP compared with AMPH (2.5 mg/kg). Histograms represent the area under the curve (A.U.C.) during the indicated interval. The A.U.C. for 2.5 mg/ kg AMPH was significantly different from those for all doses of MP. **p < than the maximal increase following the behaviorally similar dose of MP (20 mg/kg), although the duration of the responses for the two drugs was not significantly different. In addition, the pattern of DA metabolite changes following MP administration, including both the absence of a drug-induced change in DOPAC and an increase in HVA at the higher MP doses, is similar to the effects we have reported for COC and fencamfamine (Kuczenski et al., 1991; Kuczenski and Segal, 1992b) and in marked contrast to the effects of AMPH. Thus, the features of the DA-response profile that distinguish the DA-uptake blockers from AMPH are reflected in the MP response as well and substantiate the generally held view that MP interacts with DA terminals primarily as an uptake blocker. MP also increased extracellular NE in the HP, as we have reported for both COC and AMPH (Florin et a!., 1994, 1995). The duration of the NE response to MP was dose-dependent, but the maximal NE response was achieved at the lowest dose we tested. In contrast, over a behaviorally similar dose range, both AMPH and COC induce dose-dependent increases in the NE peak response. It is possible that the lowest dose of MP we used in the present study was sufficient to block NE fully but not DA uptake, because some (Ferris et al., 1972; Andersen, 1989), though not all (Pan et al., 1994; Gatley et al., 1996), in vitro studies suggest that MP is more potent in its interactions with the NE transporter than with the DA transporter. However, the maximal NE response to MP was significantly less than the response to AMPH. Likewise, the COC-induced increases in extracellular NE are substantially less than the AMPH-induced increases. Presumably, this differential effect between AMPH compared with COC and MP reflects the ability of AMPH not only to block NE uptake, but also to facilitate NE release (Florin et al., 1994, 1995). In summary, acute MP administration increases extracellular DA and NE, consistent with its presumed mechanism of action as an uptake blocker of these nerve terminal transporters. The absence of an effect of MP on CP extracellular 5-HT at doses that produce intense, focused stereotypies clearly indicates that a stimulant-induced increase in 5-HT is not a prerequisite for the appearance of perseverative behaviors. Furthermore, although 5-HT may play a modulatory role in COC- and AMPH-induced behaviors, to the extent that the behavioral responses to MP are comparable, these data further suggest that a stimulant-induced increase in 5-HT is not critical to the range of behaviors typical of AMPH-like stimulants. Acknowledgment: This work was supported in part by PHS grantsda andda-01568; D.S.S. is the recipient of PHS Career Scientist Award MH The authors thank Molly Roznoski and Joseph Higgins for their excellent technical assistance. REFERENCES Andersen P. H. (1987) 3H]GBRI Biochemical 2935 binding and in pharmacological vitro to rat striatal characterization of membranes: labeling [ of the dopamine uptake complex. J. Neurochem. 48, Andersen P. H. (1989) The dopamine uptake inhibitor GBR 12909: selectivity and molecular mechanism of action. Eur. J. Pharmacol. 166, Antelman S. M. and Caggiula A. R. (1996) Norepinephrine dopamine interactions and behavior. Science 195, Benveniste H., Hansen A. J., and Ottosen N. 5. (1989) Determination of brain interstitial concentrations by microdialysis. J. Neurochem. 52, Breese G. R., Cooper B. R., and Mueller R. A. (1974) Evidence for involvement of 5-hydroxytryptamine in the action of amphetamine. Br. J. Pharmacol. 52, Brodie B. B. and Shore P. A. (1957) A concept for a role of serotonm and norepinephrine as chemical mediators in the brain. Ann. NYAcad. Sci. 66, J. Neurochem., Vol. 68, No. 5, 1997

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