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1 JOURNAL OF NEUROCHEMISTRY doi: /j x Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, USA Abstract Methylphenidate (MPD) is a psychostimulant widely used to treat behavioral problems such as attention deficit hyperactivity disorder. MPD competitively inhibits the dopamine (DA) transporter. Previous studies demonstrated that stimulants of abuse, such as cocaine (COC) and methamphetamine differentially alter rat brain neurotensin (NT) systems through DA mechanisms. As NT is a neuropeptide primarily associated with the regulation of the nigrostriatal and mesolimbic DA systems, the effect of MPD on NT-like immunoreactivity (NTLI) content in several basal ganglia regions was assessed. MPD, at doses of 2.0 or 10.0 mg/kg, s.c., significantly increased the NTLI contents in dorsal striatum, substantia nigra and globus pallidus; similar increases in NTLI were observed in these areas after administration of COC (30.0 mg/kg, i.p.). No changes in NTLI occurred within the nucleus accumbens, frontal cortex and ventral tegmental area following MPD treatment. In addition, the NTLI changes in basal ganglia regions induced by MPD were prevented when D 1 (SCH 23390) or D 2 (eticlopride) receptor antagonists were coadministered with MPD. MPD treatment also increased dynorphin (DYN) levels in basal ganglia structures. These findings provide evidence that basal ganglia, but not limbic, NT systems are significantly affected by MPD through D 1 and D 2 receptor mechanisms, and these NTLI changes are similar, but not identical to those which occurred with COC administration. In addition, the MPD effects on NT systems are mechanistically distinct from the effects of methamphetamine. Keywords: cocaine, dopamine antagonists, dynorphin, methamphetamine, methylphenidate, neurotensin. J. Neurochem. (2011) 117, Attention deficit hyperactivity disorder (ADHD) is a behavioral disturbance diagnosed in 6% of school-age children in the United States (American Psychiatric Association 2000). Methylphenidate (MPD), commonly known as Ritalin is used as an effective treatment for ADHD and other psychiatric conditions (see Wilens 2003 and Collins et al. 2006; for review). There has been an increase in the illicit use of this stimulant presumably attributable to its pharmacological similarity to other drugs of abuse, such as cocaine (COC) (Svetlov et al. 2007; Bogle and Smith 2009). The neurochemical mechanism whereby MPD produces its psychopharmacology effects is still uncertain; however, there is evidence that its effects may be mediated in part by increasing the synaptic content of dopamine (DA) as a result of blocking the DA transporter (Schweri et al. 1985; Kuczenski and Segal 1997; Nestler 2001). The suggestion that MPD produces behavioral and neurochemical alterations similar to other psychostimulants is supported by several observations. First, MPD binds and blocks DA and norepinephrine (NE) transporters similar to COC, but unlike COC and amphetamine, MPD has low affinity for the serotonin transporter (Schweri et al. 1985). Second, MPD, cocaine and amphetamines increase extracellular DA in the CNS (Hurd and Ungerstedt 1989; During et al. 1992; Volkow et al. 1995; Kuczenski and Segal 1997). Like COC, MPD Received October 27, 2010; revised manuscript received February 9, 2011; accepted February 9, Address correspondence and reprint requests to Dr Mario E. Alburges, Department of Pharmacology and Toxicology, University of Utah, 30 South 2000 East, RM 201, Salt Lake City, Utah 84112, USA. Mario.alburges@utah.edu Abbreviations used: ADHD, attention deficit hyperactivity disorder; COC, cocaine; DA, dopamine; METH, methamphetamine; MPD, methylphenidate; NT, neurotensin; NTLI, NT-like immunoreactivity. 470

2 Effects of methylphenidate on basal ganglia neurotensin 471 increases extracellular DA by blocking DA transporters (Volkow et al. 1995; Nestler 2001) while amphetamines, like methamphetamine (METH), increase DA content by releasing DA from nerve terminals (Jones et al. 1998). Third, MPD, COC, and METH are also potent modulators of the vesicular monoamine transporter-2 (Brown et al. 2000; Riddle et al. 2002; Sandoval et al. 2002) which is responsible for the sequestration of cytoplasmic DA and is an important regulator of DA neurotransmission; however, MPD and COC increase vesicular uptake of DA (Sandoval et al. 2002), whereas METH rapidly decreases vesicular DA uptake (Brown et al. 2000; Riddle et al. 2002). Fourth, MPD affects molecules involved in DA and glutamate cellular signaling cascade by increasing DARPP-32 Thr34 phosphorylation in neostriatal slices from adult mice similar to COC (Fukui et al. 2003). Finally, MPD affects transcription factors (and other genes) like COC and amphetamines by causing DA overflow and overstimulation of DA receptors (for review, see Yano and Steiner 2007). As many of the behavioral effects of MPD appear to be DA-related, it is suggested that MPD alters the dopaminergic activity in the basal ganglia and/or limbic structures (Hurd and Ungerstedt 1989; Gerasimov et al. 2000; Volkow et al. 2001). It has been shown that MPD also alters immediateearly genes (c-fos and zif268) and neuropeptides (dynorphin and substance P) mrna expressions in a similar manner to those produced by other psychostimulants (Brandon and Steiner 2003; Yano and Steiner 2005). However, there are only two studies that have examined interactions between MPD and the neuropeptide neurotensin (NT): Nemeroff et al. (1983) observed that intracisternal injection of NT attenuated MPD-induced locomotor activity and During et al. (1992) reported that MPD treatment stimulated release of both NT and DA from the prefrontal cortex. There are no studies published that examined how MPD treatment influences NT systems in the basal ganglia and the nucleus accumbens. Neurotensin (NT) is a neuropeptide associated with the nigrostriatal and mesolimbic dopaminergic systems (Uhl and Snyder 1976). It has been shown to antagonize the behavioral effects of commonly abused CNS psychostimulants, such as COC and METH (Nemeroff et al. 1983; Wagstaff et al. 1994). There is also evidence that altered activity of DA pathways caused by these psychostimulants substantially impact NT systems as manifested by increases in NT levels in both basal ganglia and limbic structures (Letter et al. 1987; Hanson et al. 1989; Gygi et al. 1994; Castel et al. 1994a,b). Thus, its apparent interaction with dopaminergic systems suggests that NT might influence the addictive properties of these psychostimulants and similar psychostimulants. To understand better the impact of MPD on NT systems, we examined its effects on NT-like immunoreactivity (NTLI) contents in several basal ganglia and limbic structures and compared the NT responses to those caused by comparable COC treatments. Additionally, the impact of D 1 and D 2 receptor antagonists pre-treatment on MPD-induced changes in NT concentration was assessed. The present study is the first report that MPD treatment significantly elevates NT levels in the dorsal striatum, substantia nigra and globus pallidus. Furthermore, this MPD-effect on NT systems was prevented by the pre-treatment with either D 1 or D 2 receptor antagonists. In contrast to other psychostimulants, no changes in NTLI concentrations occurred in limbic regions examined after MPD treatments. Materials and methods Animals Male Sprague Dawley rats (Charles River Laboratories, Raleigh, NC, USA) weighing g were maintained in a temperaturecontrolled environment and cared for according to NIH guidelines. Animals were kept on a 12-h light/dark cycle with food and water available ad libitum. Animals were allowed to acclimate for at least 2 weeks before their use. All the experiments were performed according to the guidelines of the University of Utah Institutional Animal Care and Use Committee. Drug treatment and tissue dissection Following the acclimatization period, two experimental protocols were used in this study. First, animals were injected with four administrations (2-h intervals) of either saline (1.0 ml/kg, s.c.), (dl) MPD (2.0 or 10.0 mg/kg, dose, s.c.) or ())cocaine (30.0 mg/kg, i.p.) and were killed by decapitation 18 h after treatment. This 18 h killing time was used because of results that demonstrate a maximal increase in NTLI content in basal ganglia and limbic systems from 12 to 18 h after drug administration (Letter et al. 1987; Merchant et al. 1988; Gygi et al. 1994; Alburges and Hanson 1999). Second, animals were pre-treated with a D 1 (SCH 23390; 0.5 mg/kg/ injection, i.p.) or a D 2 (eticlopride; 0.5 mg/kg/injection, i.p.) antagonist 15 min prior to MPD high-dose treatment or saline and killed by decapitation 18 h after treatment. The selection of the MPD doses (2.0 or 10.0 mg/kg) was based on previous studies that indicate these MPD doses are therapeutically relevant ( mg/ kg, i.p. or s.c.); (McNamara et al. 1993; Gaytan et al. 1997; Gerasimov et al. 2000; Thanos et al. 2007). In addition, similar MPD treatments increase vesicular [ 3 H]DA uptake and binding of the vesicular monoamine transporter-2 ligand [ 3 H]dihydrotetrabenazine (Sandoval et al. 2002) and also alter the expression of immediate-early genes of neuropeptides (Brandon and Steiner 2003; Yano and Steiner 2005; Adriani et al. 2006; Cotterly et al. 2007). The choice of the COC dose was based on previous studies by Hanson et al. (1989) and Alburges and Hanson (1999), which demonstrated that a 30.0 mg/kg/injection elevates extrapyramidal and limbic NT levels. Brains were removed rapidly, frozen immediately on dry ice and stored at )80 C until dissected and analyzed. For regional studies, consecutive 1-mm thick frozen coronal slices according to the atlas of Paxinos and Watson (1986), were used to dissect dorsal striatum, nucleus accumbens, ventral tegmental area, frontal cortex, globus pallidus, and substantia nigra. All dissected tissue samples were subsequently stored at )80 C until assayed for neuropeptides.

3 472 M. E. Alburges et al. Drugs Eticlopride hydrochloride [S())-3-Chloro-5-ethyl-N-[(1-ethyl-2- pyrrolidinyl)methyl]-6-hydroxy-2-methoxy-benzamide hydrochloride] and SCH hydrochloride [R(+)-7-Chloro-8-hydroxy-3- methyl-1-phenyl-2, 3, 4, 5-tetrahydro-1H-3-benzazepine hydrochloride] were acquired from Sigma-Aldrich (St Louis, MO, USA). (dl) Methylphenidate hydrochloride (MPD) and ()) cocaine hydrochloride were generously supplied by the National Institute on Drug Abuse. All doses were calculated as freebase of the drug and were freshly prepared in physiological saline solution (0.9% wt/vol NaCl, ph 7.4). Radioimmunoassay The solid-phase radioimmunoassay used to analyze the neuropeptide concentrations in this study was adapted from the methods previously described (Maidment et al. 1991). Tissue samples were homogenized in 300 ul 0.01 mol/l HCl. The resulting homogenate was then placed in boiling water for 10 min to inactivate peptidases (these are standard analytical procedures for measuring protein in solution or after precipitation with acid or other agents, see Bradford 1976). Homogenates were centrifuged ( g) for 30 min. Supernatant was then collected and an aliquot was used to determine the total protein for each tissue sample by the method of Bradford (1976). The remaining sample was lyophilized overnight and stored at )80 C until the radioimmunoassay was performed. The concentrations of neuropeptides were determined with a modified solidphase radioimmunoassay technique described for NT by Maidment et al. (1991). Lyophilized samples were reconstituted in 300 ul phosphate-buffered saline (ph 7.4) containing 0.1% (wt/vol) gelatin and 0.1% (vol/vol) Triton X-100. Nunc-Immunoplates (ISC Bio- Express, Kaysville, UT, USA) were incubated overnight at 4 C with 50 ll of protein G solution (50 ng/100 ml in 0.1 mol/l sodium bicarbonate; ph 9.0). After washing the wells three times with wash buffer [0.15 mol/l K 2 HPO 4, 0.02 mol/l NaH 2 PO 4, 0.2 mmol/l ascorbic acid, 0.2% (vol/vol) Tween-20 and 0.1% (wt/vol) sodium azide; ph 7.5], 25 ll of a highly selective antiserum for neurotensin (diluted to 1 : ) or dynorphin (diluted 1 : ) was diluted in assay buffer [same as wash buffer containing 0.1% (wt/vol) gelatin]. Following addition of dynorphin antisera, wells were incubated overnight at 25 C. Wells for NT assays were incubated with the antisera for 2 h at 25 C in order to allow the attachment of antibody to the protein G-coated surface. After incubation, wells were washed three times and 25 ll of sample or standard(s) were added to each well and incubated for 2 h at 25 C. After incubation, 25 ll of the labeled NT ([ 125 I]NT) diluted with assay buffer to approximately 6500 dpm per 25 ll, were added to the wells and incubated for 2 h at 25 C. In studies used for Fig. 4, [ 125 I]dynorphin was added immediately after the samples. After incubation, wells were washed, separated and placed in mm polypropylene tubes and counted in a five-channel Packard Cobra II Auto-Gamma counter (Packard Instrument Co., Meriden, CT, USA). The total and non-specific binding were defined by adding 25 ll of the labeled peptide to protein G-untreated and -treated wells, respectively. Determinations of the quantities of NT and dynorphin, were made by comparing bound to free [ 125 I]NT and [ 125 I]dynorphin in each sample to a standard curve (from 1 to 125 pg/assay tube). The reproducibility of the assay was evaluated using cerebellum tissue spiked with 62 and 250 pg of each peptide. This technique has been demonstrated to be very reproducible, resulting in less than 10% variability between assays and less than 5% between sample and standard duplicates with an uniform NTLI recovery of 77 93%. Antisera The NT and dynorphin antisera were raised in New Zealand White rabbits as previously described (Letter et al. 1987). These antisera recognize the NT or dynorphin carboxy terminus (respectively) and are highly selective, expressing no cross-reactivity with 1000-fold excess concentrations of other endogenous neuropeptides such as dynorphin (for NT antiserum), metenkephalin, cholecystokinin, substance P (for NT or dynorphin antiserum) or substance K. Data analysis Results from these experiments are expressed as percentages of their respective controls in order to facilitate comparisons between groups (mean value ± SEM). The control values (pg of NT- or dynorphinlike immunoreactivity per mg of protein) for each experiment are indicated in the corresponding figure legend. Differences between means were analyzed using one-way analysis of variance followed by Fisher-protected least significant difference. Differences were considered significant when the probability that they were zero was less than 5%. Results To determine if MPD treatment influences the basal ganglia and/or limbic structures-associated NT systems, the effect of multiple administrations (four injections, 2-h intervals) of varying doses of MPD (2.0 and 10.0 mg/kg/injection, s.c) on NTLI concentrations in these brain regions was determined and compared quantitatively with a similar COC treatment (30.0 mg/kg/injection, i.p.). Eighteen hours after four injections of 2.0 or 10.0 mg/kg/injection of MPD, the NTLI content was significantly increased in dorsal striatum (Fig. 1a), globus pallidus (Fig. 1b) and substantia nigra (Fig. 1c). As previously observed, multiple administrations of 30 mg/kg/injection, i.p., of COC also significantly increased NTLI concentration in the dorsal striatum (Fig. 1a), globus pallidus (Fig. 1b) and substantia nigra (Fig. 1c) in a quantitative manner similar to that caused by the 2.0 mg/kg, s.c. dose of MPD. The effects of multiple administrations of MPD (2.0 and 10.0 mg/kg, s.c.) on NTLI content in areas of the limbic NT systems (nucleus accumbens, ventral tegmental area and frontal cortex) were also examined. No significant changes in the NTLI concentration on these brain structures were observed at 18 h following MPD treatments in contrast to the increases in all three regions after COC treatment (Fig. 2a c). Because the most consistent and robust response to the MPD treatment occurred with the 10.0 mg/kg/injection, this dose was used to study the role of DA receptors in the MPDinduced changes in basal ganglia NT systems. By itself, SCH had no significant effect on NTLI contents in any of the brain regions examined, whereas eticlopride

4 Effects of methylphenidate on basal ganglia neurotensin 473 Fig. 1 Effects of multiple administrations of MPD or COC on NTLI content in the dorsal striatum (a), globus pallidus (b) and substantia nigra (c). Animals were administered four injections (2-h intervals) of either saline (1.0 ml/kg/injection, s.c.), (dl) methylphenidate (2.0 or 10.0 mg/kg, s.c.) or ()) cocaine (30.0 mg/kg/injection, i.p.) and were killed 18 h after treatment. Results are expressed as percentages of control and represent mean values ± SEM (n = 8 control and 11 drugtreated animals per group). The average control values of NTLI concentration for the dorsal striatum, globus pallidus, and substantia nigra were 499 ± 46, 886 ± 135, and 688 ± 74 pg/mg protein, respectively. *p < 0.05, **p < versus saline. pre-treatment elevated NTLI concentration in dorsal striatum (Fig. 3a) and globus pallidus (Fig. 3c), as previously reported with other D 2 antagonists (Frey et al. 1986; Letter et al. 1987; Merchant et al. 1988; Hanson et al. 1989; Alburges and Hanson 1999). The pre-treatment with either a D 1 or D 2 antagonist prevented the MPD-induced increases in Fig. 2 Effects of multiple administrations of MPD or COC on NTLI content in the nucleus accumbens (a), ventral tegmental area (b) and frontal cortex (c). Animals were given injections of MPD, COC, or saline (as described for Fig. 1) and killed 18 h following the treatment. Results are expressed as percentages of control and represent mean values ± SEM (n = 8 control and 11 drug-treated animals per group). The average control values of NTLI concentration for the nucleus accumbens, ventral tegmental area, and frontal cortex were 752 ± 88, 538 ± 62, and 58 ± 14 pg/mg protein, respectively. *p < 0.05 versus saline. NTLI in all the basal ganglia-related brain regions examined (Fig. 3a c). The presence of D 2 receptor antagonist caused approximately the same increases in NTLI contents in both dorsal striatum (Fig. 3a) and globus pallidus (Fig. 3c) with or without MPD. Finally, we determined if basal ganglia neuropeptide systems other than NT were also affected by the MPD

5 474 M. E. Alburges et al. treatment (10.0 mg/kg, s.c.; 4 2-h intervals) by measuring dynorphin- and substance P-like immunoreactivity (dynorphin-like immunoreactivity and substance P-like immunoreactivity) in basal ganglia areas. This MPD treatment significantly increased the dynorphin-like immunoreactivity content in dorsal striatum (Fig. 4a), substantia nigra (Fig. 4b), and globus pallidus (Fig. 4c); however, MPD had no significant effect on substance P-like immunoreactivity concentrations in these brain regions (data not shown). Discussion Fig. 3 Effects of pre-treatment with selective dopamine receptor antagonists on MPD-induced changes in NTLI content in dorsal striatum (a), substantia nigra (b) and globus pallidus (c). Animals were given four administrations of MPD (10.0 mg/kg/injection, s.c., 2-h intervals) or saline (S; control), alone or 15 min after administration of SCH (SCH; D 1 receptor antagonist; 0.5 mg/kg/injection, i.p.), or eticlopride (ETI; D 2 receptor antagonist; 0.5 mg/kg/injection, i.p.). Animals were killed 18 h following the last treatment. Values represent the mean ± SEM expressed as percentages of control (n = 8). The control values ± SEM for NTLI concentration for the dorsal striatum, substantia nigra, and globus pallidus were 295 ± 20, 699 ± 46, and 685 ± 99 pg/mg protein, respectively. (a) **p < versus S-S, SCH-S and SCH-MPD groups; # p < 0.05 versus all groups except S-MPD; (b) **p < versus all other groups; (c) **p < versus S-S, SCH-S and SCH-MPD groups. Psychostimulants, such MPD and dextroamphetamine are the treatment of choice for ADHD (Faraone et al. 2004). Furthermore, MPD is becoming more widely used for nonmedical purposes by college students and adolescents (Svetlov et al. 2007; Bogle and Smith 2009). Like with COC, the reinforcing effect of MPD is primarily caused by its blockade of the DA transporter (Kuczenski and Segal 1997; Nestler 2001). Although several studies have elucidated the interaction of MPD with dopaminergic systems in dorsal striatal and limbic structures, relatively little is known about the effect of MPD on other related transmitter systems. For example, neuropeptide pathways, such as NT are also associated with basal ganglia regions and contribute to the regulation of DA efferent pathways and their functional outcomes. In a reciprocal manner, changes in D 1 and D 2 activity alter elements of NT systems such as neuropeptide levels (Letter et al. 1987; Merchant et al. 1988; Hanson et al. 1989; Gygi et al. 1994; Alburges and Hanson 1999), expression of related mrna (Merchant et al. 1992; Kalivas 1993; Hanson and Keefe 1999; Zahm et al. 2001) and NT release (Hernandez et al. 1985; Wagstaff et al. 1994). From previous studies, it appears that increases in striatal NT synthesis correlate with elevated NT tissue levels h later whether caused by D 1 agonists, psychostimulants or D 2 antagonists (Merchant et al. 1991, 1992, 1994; Castel et al. 1993, 1994a), and are likely associated with striatonigral (D 1 -related) or striatopallidal (D 2 -related) neurons (Castel et al. 1994b). Because of these NT/DA interactions, it has been suggested that central NT pathways contribute to the etiology of schizophrenia and may be potential therapeutic targets for neuropsychiatric and substance of abuse disorders (Nemeroff et al. 1977; Richelson et al. 2003). To understand better the role of these basal ganglia and NT pathways, particularly as it relates to MPD effects, we examined the influence of MPD treatment on tissue levels of NT in discrete brain regions. The selection of the MPD dose schedule was based on previous studies (McNamara et al. 1993; Gaytan et al. 1997; Gerasimov et al. 2000; Brandon and Steiner 2003; Thanos et al. 2007) and is considered to be in the upper range of the clinical relevant dose. In response to multiple administrations of MPD, we observed that MPD

6 Effects of methylphenidate on basal ganglia neurotensin 475 Fig. 4 Effects of multiple administrations of MPD on dynorphin-like immunoreactivity content in the dorsal striatum (a), substantia nigra (b), and globus pallidus (c). Animals were given four injections (2-h intervals) of either MPD (10.0 mg/kg/injection, s.c.) or saline (1.0 ml/ kg/injection, s.c.) and killed 18 h following the treatment. Results are expressed as percentages of control and represent mean values ± SEM (n = 8 control and 11 drug-treated animals per group). The average control values of DYNLI concentration for the dorsal striatum, substantia nigra, and globus pallidus were 149 ± 30, 1121 ± 266, and 348 ± 114 pg/mg protein, respectively. *p < 0.05 versus corresponding saline. (2.0 or 10.0 mg/kg, s.c.; 4 2-h intervals) elicited similar effects to those of COC (30.0 mg/kg, i.p.; 4 2-h intervals; Fig. 1a c), and increased NTLI levels in basal ganglia structures (dorsal striatum, globus pallidus and substantia nigra). In contrast to COC, MPD had little or no effect on NTLI content in limbic system structures (i.e. nucleus accumbens, ventral tegmental area and frontal cortex; Fig. 2a c). Our observed effects of COC exposure were consistent with original observations from Hanson et al. (1989) and Alburges and Hanson (1999), that levels of NTLI are elevated in striatum, substantia nigra, as well as nucleus accumbens and frontal cortex after administration of COC. Because MPD treatment affected only dorsal striatum, substantia nigra and globus pallidus and not limbic structures it appears that this drug has some selective action on basal ganglia circuitry systems (Merchant et al. 1991, 1992). These results suggest that the nigrostriatal dopaminergic pathways mediated the effects of MPD on striatal NT pathways. In comparison, METH, a potent DA releaser, like COC increases NTLI concentrations in both limbic and basal ganglia NT pathways (Letter et al. 1987; Merchant et al. 1988; Castel et al. 1993, 1994a; Gygi et al. 1994). Because the limbic NT system is more sensitive to the effects of COC or METH than MPD, this perhaps explains why MPD appears to have less abuse potential than those other stimulants (Svetlov et al. 2007; Thanos et al. 2007; Yano and Steiner 2007) and a distinctive therapeutic value (Levin et al. 1998; Collins et al. 2006; Svetlov et al. 2007). Our results that MPD influences more the basal ganglia NT pathways than the limbic NT pathways are consistent with previous studies (Lin et al. 1996; Brandon and Steiner 2003; Yano and Steiner 2005) which indicate that MPD treatment (2 10 mg/kg, i.p.) also produces dose-dependent increases in the expression of two immediate early-genes (c-fos and zif 268 mrnas) and neuropeptides (dynorphin, substance P) that are more robust in the dorsal sensorimotor striatal regions of the basal ganglia and weakly or absent in the nucleus accumbens (limbic system). We confirmed that these MPD-mediated NT changes were linked to the dopaminergic systems because the NT responses were blocked by either D 1 or D 2 receptor antagonists (Fig. 3a c). These observations were similar to previous reports that SCH also blocks the NTLI increases by COC (Hanson et al. 1989; Alburges and Hanson 1999) and METH (Letter et al. 1987; Merchant et al. 1988). As for D 2 mechanisms, several studies have shown that D 2 blockade alone with haloperidol, sulpiride, or eticlopride elevates the NTLI concentrations in striatum and nucleus accumbens, but not in the substantia nigra (Frey et al. 1986; Letter et al. 1987; Merchant et al. 1988; Hanson et al. 1989; Gygi et al. 1994; Alburges and Hanson 1999), and these increases in NTLI tissue levels are additive with effects of METH, but not COC. The present study showed that like COC the eticlopride and MPD effects on NTLI were not additive in the dorsal striatum and eticlopride blocked the MPD-mediated NTLI changes in the substantia nigra (Fig. 3a c). Thus, like COC, MPD affects the NT systems of the basal ganglia by activation of both D 1 and D 2 receptors. In contrast, METH-induced NT changes in these

7 476 M. E. Alburges et al. regions are principally mediated by increased D 1, and not D 2, activity (Letter et al. 1987; Merchant et al. 1988; Castel et al. 1993, 1994a,b). These findings suggest that the MPDmediated changes in basal ganglia NT systems are equally dependent on both D 1 and D 2 receptor activation mediated by MPD. Although the functional relevance of this basal ganglia NT change caused by MPD is not known, it has been demonstrated that NT pathways influence the effects of other psychostimulants. For example, NT contributes to the following effects of COC: sensitization (Rompre and Bauco 2006); reinstatement of COC-seeking behavioral (Lopak and Erb 2005); brain stimulation reward (Rompre et al. 1992); conditioned place preference (Glimcher et al. 1984); and hyperactivity (Boules et al. 2001). Consequently, it is likely that the basal ganglia NT systems contribute to some of the reinforcing and psychiatric effects of MPD as well. Finally, we examined the possibility that other DA-linked neuropeptides systems in the basal ganglia are also altered by this MPD treatment. Previous studies showed that repeated MPD administrations to adolescent rats increase dynorphin and substance P gene expression in the striatum in a manner similar to other psychostimulants that increase DA receptor stimulation (Brandon and Steiner 2003; Yano and Steiner 2005, 2007). The increases in the expression of dynorphin mrna reported previously (Harlan and Garcia 1998; Brandon and Steiner 2003; Willuhn et al. 2003) were highly correlated with the immediate-early gene response in different striatal brain regions, specifically the induction of the expression of c-fos and zif 268 mrnas by MPD treatment was principally in the dorsal part of the middle to caudal striatum and slight or absent in the nucleus accumbens. In the present study, as already mentioned, we observed that multiple MPD administration also increased dynorphin-like immunoreactivity concentrations in the basal ganglia structures of dorsal striatum, substantia nigra and globus pallidus (Fig. 4a c) at 18 h after drug treatment, but the substance P-like immunoreactivity content was not altered (data not shown). The response by dynorphin pathways to MPD treatment was very similar to that observed in the basal ganglia NT systems (Fig. 1a c) and also closely resembled the effects of COC (i.e. increased dynorphin-immunoreactivity concentrations) on extrapyramidal dynorphin systems observed by Smiley et al. (1990). This pattern of changes in dynorphin-immunoreactivity in basal ganglia structures following MPD and COC treatment was in agreement with previous reports (Harlan and Garcia 1998; Brandon and Steiner 2003; Willuhn et al. 2003). These results indicate that other neuropeptides systems associated with the basal ganglia DA pathways also contribute to the psychopharmacological effects of MPD in addition to NT. In conclusion, these findings revealed for the first time that multiple administrations of MPD significantly increase NT tissue levels in the basal ganglia but not in the limbic structures. Consistent with our previous work (Hanson et al. 1989; Alburges and Hanson 1999) similar increases in NTLI basal ganglia contents also occurred after multiple COC administrations. As with COC, these MPD-related NT changes were mediated by both D 1 and D 2 mechanisms. Acknowledgements This work was supported by U.S. Public Health Service Grants DA011389, DA019447, DA09407, and DA The authors declare that they have no biomedical financial interests or other conflicts of interest. The experiments described within this publication are all in compliance with the laws of the USA. 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