glycine-site-mediated dopaminergic dysregulation and dopamine transporter function

Neurochemistry International 52 (2008) 119–129 www.elsevier.com/locate/neuint

Characterization of interactions between phencyclidine and amphetamine in rodent prefrontal cortex and striatum: Implications in NMDA/glycine-site-mediated dopaminergic dysregulation and dopamine transporter function§ Henry Sershen a,b, Andrea Balla a,b, John M. Aspromonte a,b, Shan Xie a,b, Thomas B. Cooper a,b, Daniel C. Javitt a,b,* a

Nathan S. Kline Institute for Psychiatric Research, 140 Old Orangeburg Rd., Orangeburg, NY 10962, United States b New York University School of Medicine, Department of Psychiatry, New York, NY, United States Received 27 February 2007; received in revised form 12 July 2007; accepted 13 July 2007 Available online 20 July 2007 In honor of Prof. E.S. Vizi’s 70th birthday.

Abstract N-Methyl-D-aspartate (NMDA) antagonists induced behavioral and neurochemical changes in rodents that serve as animal models of schizophrenia. Chronic phencyclidine (PCP, 15 mg/(kg day) for 3 weeks via Alzet osmotic pump) administration enhances the amphetamine (AMPH)-induced dopamine (DA) efflux in prefrontal cortex (PFC), similar to that observed in schizophrenia. NMDA/glycine-site agonists, such as glycine (GLY), administered via dietary supplementation, reverse the enhanced effect. The present study investigated mechanisms of glycineinduced reversal of PCP-induced stimulation of AMPH-induced DA release, using simultaneous measurement of DA and AMPH in brain microdialysate, as well as peripheral and tissue AMPH levels. PCP treatment, by itself, increased peripheral and central AMPH levels, presumably via interaction with hepatic enzymes (e.g. cytochrome P450 CYP2C11). GLY (16% diet) had no effect on peripheral AMPH levels in the presence of PCP. Nevertheless, GLY significantly reduced extracellular/tissue AMPH ratios in both PFC and striatum (STR), especially following PCP administration, suggesting a feedback mediated effect on the dopamine transporter. GLYalso inhibited acute AMPH (5 mg/kg)-induced DA release in PFC, but not STR. These findings suggest that GLY may modulate DA release in brain by producing feedback regulation of dopamine transporter function, possibly via potentiation of NMDA-stimulated GABA release and presynaptic GABAB receptor activation. The present studies also demonstrate pharmacokinetic interaction between AMPH and PCP, which may be of both clinical and research relevance. # 2007 Published by Elsevier Ltd. Keywords: Phencyclidine; Amphetamine; Dopamine transporter; Glycine; NMDA; GABA; Schizophrenia; Prefrontal cortex; Striatum; Dopamine; NMDA/glycine site

1. Introduction Phencyclidine (PCP) induces schizophrenia-like cognitive dysfunction and psychosis by blocking N-methyl-D-aspartate

§ This work was supported in part by NIDA grant DA03383. Appeared in part at the Society for Neuroscience Meeting, 2003. Trans. Soc. Neurosci. Abstract 461.8. * Corresponding author at: Nathan S. Kline Institute for Psychiatric Research, 140 Old Orangeburg Rd., Orangeburg, NY 10962, United States. E-mail address: [email protected] (D.C. Javitt).

0197-0186/$ – see front matter # 2007 Published by Elsevier Ltd. doi:10.1016/j.neuint.2007.07.011

(NMDA) receptor-mediated neurotransmission (Jentsch et al., 1999; Javitt and Zukin, 1991; Coyle, 1996). Its administrations to rodents has been used frequently to model aspects of schizophrenia, in particular, the noted enhancement of amphetamine-induced dopamine release (Abi-Dargham et al., 1998; Abi-Dargham, 2004; Breier et al., 1998; Laruelle et al., 1996, 1999, 2000; Laruelle, 1998; Laruelle and AbiDargham, 1999). It has been suggested that NMDA receptor dysfunction seen following acute, repeated, and continuous PCP administration to rodents may underlie the dopaminergic hypersensitivity as seen in schizophrenia (Javitt and Zukin, 1991; Javitt et al., 1997, 2004).

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The exact nature of this NMDA-mediated PCP-induced activation of cortical dopamine metabolism is still unclear, although it probably involves also inhibitory interneurons (GABAergic) on dopaminergic terminals; blockade of NMDA receptors located on the GABAergic interneurons reduce its inhibitory action, resulting in enhanced dopaminergic transmission (Carlsson, 1988; Carlsson et al., 1999). Similarly, the exact mechanism of the inhibition of the PCP-mediated enhanced AMPH-induced DA release by GLY remains to be determined (Javitt et al., 2004). Recent in vitro studies suggest that GLY may stimulate local GABA release in STR, leading to potentiation of GABAB neurotransmission at presynaptic terminals and subsequent inhibition of DA release, possibly by voltage-dependent regulation of presynaptic dopamine transporters (DAT) (Hoffman et al., 1999). Effects of GLY were inhibited by both NMDA/glycine site antagonists (e.g., HA966) and GABAB (e.g., phaclofen), but not GABAA (e.g., bicuculline) modulators, and were mimicked by novel glycine transport inhibitors (e.g., ALX5407), confirming the critical role of NMDA and GABAB receptors in the circuit (Javitt et al., 2005). The inhibition of PCP-induced behavioral activation and NMDA-mediated or amphetamine-induced dopamine release in vitro or in vivo, by glycine and selective glycine transport inhibitors is consistent with findings that NMDA receptors within the striatum act primarily to inhibit dopamine release via stimulation of inhibitory interneurons (Javitt and Frusciante, 1997; Javitt et al., 1999, 2004). In the course of these studies, it was considered important to take into account additional mechanisms that may be implicated in this PCP-induced hyperdopaminergic model. Potential mechanisms of this hyperdopaminergic response to PCP have also suggested direct effects through the dopamine transporter (DAT). Brain imaging studies have suggested that PCP is a potent releaser of striatal dopamine and amphetamine, although microdialysis studies find little effect of NMDA antagonists on striatal dopamine release (Adams et al., 2002). Others have suggested that PCP is a more potent blocker of the DAT than previously thought (Tsukada et al., 2000, 2001; Schiffer et al., 2001, 2003). In addition, it is also known that PCP is a metabolism-dependent inhibitor of cytochrome P450 enzymes (Hiratsuka et al., 1995; Jushchyshyn et al., 2003; Sharma et al., 1997) and that cytochrome P450 catalyzes ring hydroxylation, a major route of metabolism of amphetamine (Law et al., 2000; Shiiyama et al., 1997; Yamada et al., 1989). There has been no clear study to characterize this metabolic interaction as it could possibly relate to the current PCP-rodent model of schizophrenia, where regionally selective effects on amphetamine-induced dopamine release were observed (Balla et al., 2001a), which would give further information as to the mechanism(s) involved in reversal of PCP-induced dopaminergic dysregulation by NMDA receptor/glycine-site agonists. However, in prior studies no explicit measurements of AMPH levels in either tissue or extracellular fluid were performed, preventing explicit evaluation of transporter function. In the present study, both peripheral and central AMPH levels were monitored during DA microdialysis experiments. Central extracellular/tissue AMPH levels were used as an index

of DAT function. It was examined whether possible drug–drug interactions are involved in the hyperdopaminergic response to amphetamine seen in PCP-treated rats, and the reversal by NMDA/glycine site agonists. In vivo brain microdialysis of prefrontal cortex and striatum was performed to examine the effect of PCP on brain extracellular amphetamine levels, and peripheral tissue fluid and brain tissue concentrations of amphetamine measured during PCP and concurrent glycine treatment. 2. Materials and methods 2.1. Animals Studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Male Sprague–Dawley rats (160–200 and 280–320 g) bred in our animal colony were used. The rats were maintained under a 10 h/14 h dark/ light cycle, and were allowed food and water ad libitum during the microdialysis and ultrafiltration procedures.

2.2. Drug administration Phencyclidine hydrochloride (obtained from the National Institute of Drug Abuse) was dissolved in sterile physiological saline and was given through an osmotic pump model 2ML4 (ALZA Corporation) implanted under the skin. The pumps were filled based on the animal weight at the start of the experiment to deliver 15 mg/(kg day) for 3 weeks. Osmotic pumps filled with saline were used in control animals. The implantation was carried under anesthesia with ketamine hydrochloride and acepromazine maleate 1:1 mixture (1 ml/g i.m.). In addition some animals received a 16% glycine diet (made by Dyets Inc.) in parallel with or without PCP. Amphetamine sulfate (RBI) was solved in physiological saline and was given subcutaneously (0.5, 1, 2 and 5 mg/kg).

2.3. In vivo microdialysis Microdialysis was performed 16–21 days after pump implantation in control, PCP, PCP plus glycine, and glycine treatment groups. Animals were anesthetized with chloral hydrate (400 mg/kg i.p.) and mounted in a stereotaxic frame (David Kopf Instruments). CMA 12 guide cannulas were implanted relative to bregma into medial PFC (AP: +4.1, L: +1.0, V: 1.2 at 158 angle) and into striatum (AP: +1.0, L:+2.5, V: 4.0) according to the rat brain atlas of Paxinos and Watson, 1998, 48 h before microdialysis measurement. The cannulas and two stainlesssteel screws embedded in the skull were cemented with dental acrylic. Buprenorphine (0.5 mg/kg) was given post-op. CMA 12 probes (0.5 mm  2.0 mm or 4.0 mm membrane length with a molecular cut-off 20,000 Da; probe recovery were approximately 18% and 28% for amphetamine, respectively) were used for the striatum and PFC, respectively. The dialysis probes was continually perfused at a flow rate of 1.0 ml/min using a syringe pump CMA/100 (Carnegie Medicine) with Mg2+-free Ringer solution containing (NaCl 147 mM; KCl 4 mM; CaCl2 1.2 mM; degassed). Two hours were allowed to establish the basal level of the extracellular catecholamines. Thirty minutes dialysate samples were collected with a fraction collector (Bioanalytical Systems). After three baseline samples, the rats were challenged with an amphetamine injection. Dialysis samples were collected for an additional 210 min.

2.4. Determination of the probe placement The following day after the microdialysis study, the rats were anesthetized with ketamine hydrochloride and acepromazine maleate 1:1 mixture (1 ml/g i.m.). A blood sample was taken from a heart-puncture and plasma obtained after centrifugation for PCP analysis. The rat brain was fixed with 4% formaldehyde solution perfused through the heart; the brain was stored in 30% glucose solution. The placement of the probes was determined histologically.

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2.5. High-pressure liquid chromatography The dialysate samples (30 ml) – collected in 15 mM NaEDTA and 10% ethyl alcohol mixture – were injected with an autosampler. For a determination of catecholamines, a reverse-phase high-pressure liquid chromatography (BAS480 system) with electrochemical detection was used. A microbore C18 100 mm  2 mm millibore column, Classic glassy carbon electrodes versus Ag/AgCl reference electrode at 0.60 and 0.75 V was used (BAS). A filtered, degassed mobile phase (NaH2PO4 25 mM; sodium citrate 50 mM; disodium-EDTA 27 mM; diethylamine–HCl 10 mM; 1-octanesulfonic acid, sodium salt; methanol 3%, v/v; dimethylacetamide 2.1%, v/v; pH 3.5) with a flow rate 0.4 ml/min gave the following retention times in minutes: DOPAC 3.8; DA 4.9; HIAA 6.7; HVA 7.7. The working standard solutions were stored at 80 8C and 10 ml of the standard solution was injected between biological samples.

2.6. In vivo ultrafiltration Animals were anesthetized with chloral hydrate (400 mg/kg i.p.) and ultrafiltration probes (BAS) were implanted under the skin on the back 24 h before ultrafiltration measurement. After a 1 h collection period, 30-min samples were collected using a peristaltic pump (Rainin Inst.); collected sample volumes ranged from 20 to 60 ml/sample. After three baseline samples, the rats were challenged with an amphetamine injection. Ultrafiltration dialysis samples were collected for an additional 210 min; the same as with the microdialysis procedure.

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0.75 and 7.5 ng of amphetamine with each batch of samples, giving the coefficients of variation of 1.7%, 1.8% and 5.1%, respectively. The accuracy of the method, measured as the percentage difference between the mean concentrations found and the amounts added, ranged from 98 to 104.

2.11. Data analysis and statistical testing Data were analyzed to determine significant increases above baseline sample using repeated measures ANOVA with post hoc t-test, with withinsubject measure of time following amphetamine injection (i.e. fraction number) and between-subject factor of drug (PCP or saline). Two-tailed statistics with a significance of p < 0.05 were used. Data are the means  S.E.M.

3. Results 3.1. In vivo microdialysis: effect of amphetamine on dopamine levels in prefrontal cortex and striatum measured by microdialysis Fig. 1 shows that amphetamine (0.5–5 mg/kg s.c.) dosedependently increased extracellular dopamine (expressed as percent baseline dopamine) in prefrontal cortex and striatal

2.7. Brain dissection For measurement of tissue concentration of AMPH, rats (treated with or without PCP and/or GLY) were injected with amphetamine and killed at two points (after an hour or 2.5 h). The animals were decapitated and prefrontal cortex and striatum was rapidly dissected out on an ice-cold Petri dish. The tissue was frozen immediately on dry ice and stored at 80 8C until assayed.

2.8. Amphetamine extraction and derivatization Ampetamine in microdialysis samples were assayed as recently described (Xie et al., 2004). To 25 ml of a microdialysis sample in 0.5 ml of 0.01 N HCl were added 0.5 ng of deuterated ()-amphetamine as an internal standard, 0.5 ml of 0.6 M carbonate buffer (pH 9.0) and 35 ml of 2,3,4,5,6-pentafluorobenzoyl chloride in toluene (30 ml/10 ml). The sample was mixed for 30 min at room temperature. The upper layer was transferred to a vial, and 2 ml was injected into the GC-MS.

2.9. Instrumentation and data acquisition A HP ChemStation data system was used to control a HP 5988B GC-MS system and to collect and quantitate the data. The GC-MS with a HP-1 column (12 m  0.2 mm I.D., 0.33 mm) was operated in a negative chemical ionization (NCI) mode using methane:ammonia (95:5) as the reagent gas. The column was programmed from 80 8C (holding for 1 min) to 280 8C at increasing rate of 30 8C/min. The ion-source temperature was 200 8C, and the temperatures of injector and the interface between the chromatograph and the spectrometer were set at 280 8C.

2.10. Quantitation and validation Quantitation was achieved by the peak area of amphetamine to the calibration standards at the concentration range of 0.025–10.0 ng in 25 ml of saline solution. The within-day precision was determined by using five replicates of each level of the standard curve. The coefficients of variation (CVs) for the back-calculated concentrations of the calibration standards using the regression parameters ranged from 0.6% for the highest standard to 14.9% for the lowest standard. The between-day precision of the method was determined by analyzing quality control samples at the concentration levels of 0.075,

Fig. 1. Dopamine levels, expressed as percent of baseline, from microdialysis samples collected from prefrontal cortex and striatum of rats treated with an acute administration of amphetamine (0.5–5 mg/kg s.c.). After a 2-h period of microdialysis collection, three 30-min baseline samples were collected followed by administration of amphetamine (start of sample four) at indicated doses, and samples collected for an additional 210 min. Results are means  S.E.M. (n = 4–8), expressed as percent baseline dopamine release seen in microdialysis samples taken every 30 min.

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microdialysate samples, dopamine levels peaking at 1 h (fraction 6) after administration. Responses were approximately twofold lower in the prefrontal cortex (700% versus 1300% of baseline for prefrontal cortex and striatum, respectively) with a slower return (clearance) to baseline as compared to striatum. Baseline dopamine values were 1.6  0.15 and 13.48  0.84 pg/10 ml, and when corrected for probe recovery, 6.47  0.60 and 89.51  5.79 pg dopamine/10 ml for prefrontal cortex and striatum. The lower levels of extracellular dopamine in the prefrontal cortex likely reflects the paucity of dopamine transporters (DAT) in this region, and is also reflected in the greater extracellular diffusion and slower clearance in the prefrontal cortex versus striatum (Sesack et al., 1998). Since amphetamine releases dopamine by reversal of the DAT (exchange diffusion) (Connor and Kuczenski, 1986; Khoshbouei et al., 2003), or more correctly by efflux through a dopamine transporter channel (Kahlig et al., 2005), the reduced number of DATs would result in less dopamine release. 3.2. In vivo microdialysis: amphetamine levels in prefrontal cortex and striatum measured by microdialysis Fig. 2 shows also the dose-dependent changes in the extracellular levels of amphetamine in the prefrontal cortex and striatum after acute amphetamine (0.5–5 mg/kg s.c.) administration; higher extracelluar amphetamine is reached with the higher doses. Peak amphetamine levels were reached within 60 min after administration. Clearance rates were similar in both regions. Values shown were not corrected for probe recovery (also see below). Corrected values would indicate similar extracellular amphetamine levels in both regions, and unlike the regional differences observed in the dose-dependent amphetamine-induced increase in extracellular dopamine levels that are DAT-dependent (Fig. 1), the extracellular amphetamine measurements reflects the attainment of equilibrium after peripheral subcutaneous amphetamine administration and correlates more to plasma (extracellular) than intracellular concentrations. Most likely, intracellular amphetamine levels would show a brain regional DAT-dependence to account for the difference in regional dopamine response seen in Fig. 1. 3.3. Effect of PCP and glycine on amphetamine levels in prefrontal cortex and striatum measured by microdialysis Fig. 3 shows the changes in the extracellular levels of amphetamine in the prefrontal cortex and striatum after amphetamine (1 mg/kg s.c.) administration in PCP-treated rats. In controls, amphetamine levels peaked at 60 min (fraction 3) after amphetamine injection and returned near to baseline by 210 min (sample 8) in control animals (prefrontal cortex and striatum). Although peak amphetamine levels appear approximately twofold higher in prefrontal cortex versus striatum in microdialysis samples of controls (0.47  0.04 versus 0.23  0.03 mmol/l), these results are not corrected for probe recovery (25% and 15% for prefrontal cortex (4-mm probe) and striatum (2-mm probe)). If corrected, extracellular levels for amphetamine are similar between regions, for example under

Fig. 2. Amphetamine levels, expressed as mmol/l, in microdialysis samples taken from prefrontal cortex and striatum. Not corrected for probe recovery (15% and 25% for the 2- and 4-mm probes used in striatum and prefrontal cortex, respectively). Rats were treated with given an acute administration of amphetamine (0.5–5 mg/kg, s.c.) at the start of sample number 4 and microdialysis samples (30-min samples) collected for and additional 180 min. Values are means  S.E.M. (n = 3–10) expressed as mmol/l.

the same treatment conditions; that is, there were no significant regional differences between prefrontal cortex and striatum (1.89  0.12 versus 1.54  0.19 mmol/l for controls) in extracellular amphetamine. PCP treatment (15 mg/(kg day) for 3 weeks) induced a significant enhancement in the prefrontal cortex and striatum levels of amphetamine between 30 and 210 min post-injection versus control rats. In contrast, GLY treatment by itself had no effect on extracellular amphetamine levels (control versus glycine), but significantly attenuated the PCP-induced increase in amphetamine in prefrontal cortex and striatum of PCP-treated rats (PCP versus PCP + GLY). The level of amphetamine in the PCP plus GLY treatment group was reduced versus PCP-treated rats and similar to control rats throughout the 60–210 min in prefrontal cortex and the 30–210 min in the striatum. 3.4. Effect of PCP and glycine on peripheral ultrafiltrate levels of amphetamine Fig. 4 shows the peripheral body-ultrafiltrate fluid amphetamine levels in control, PCP- and PCP + glycine-treated rats.

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Fig. 4. Amphetamine levels measured every 30 min after an acute administration (1 mg/kg s.c.), collected from peripheral fluid using an ultrafiltration probe implanted subcutaneously in PCP-treated rats (15 mg/(kg day) for 2 weeks) with or without dietary glycine (16%). Values are means  S.E.M. (n = 6–9) expressed as mmol/l. *P < 0.05; **P < 0.001 control vs. PCP or + P < 0.001 control vs. PCP + gly, samples 5–8.

increased dopamine release in the prefrontal cortex, but not in the striatum. The regional effect of PCP on dopamine release is in agreement with the studies by Nishijima et al., showing a dramatic increase in DA in the dialysates from medial prefrontal cortex and much lower augmentation of DA

Fig. 3. Amphetamine levels, expressed as mmol/l, in microdialysis samples taken from prefrontal cortex and striatum. Not corrected for probe recovery (15% and 25% for the 2- and 4-mm probes used in striatum and prefrontal cortex, respectively). Rats were treated with PCP (15 mg/(kg day) for 2 weeks) with or without dietary glycine (16%) or glycine alone, and then given an acute administration of amphetamine (1 mg/kg, s.c.) at the start of sample number 2 and microdialysis samples (30-min samples) collected for and additional 180 min. Values are means  S.E.M. (n = 9–11) expressed as mmol/l. *P < 0.05; **P < 0.01 control vs. PCP or PCP + gly; #P < 0.01 PCP vs. PCP + gly.

Amphetamine levels were elevated approximately twofold at 90–180 min after amphetamine (1 mg/kg s.c.) administration in peripheral ultrafiltrate samples of PCP-treated (15 mg/ (kg day) for 3 weeks) rats versus controls, reflecting at least in part a presumed effect of PCP on P450 metabolism of amphetamine (Laurenzana and Owens, 1997; Yamada et al., 1989). Unlike brain tissue content or brain microdialysate levels, glycine did not attenuate the PCP-induced increase in amphetamine ultrafiltrate levels, indicating that glycine effects cannot be attributed to alterations in peripheral amphetamine metabolism. 3.5. Effect of acute PCP on amphetamine-induced dopamine levels in prefrontal cortex and striatum measured by microdialysis Fig. 5 shows the dopamine response (microdialysis samples) in prefrontal cortex and striatum to an acute PCP (5 mg/kg i.p) and amphetamine (1 mg/kg s.c.) administration. PCP alone

Fig. 5. Effect of acute PCP (5 mg/kg i.p.) on amphetamine (1 mg/kg s.c.)induced dopamine release in prefrontal cortex and striatum. Rats were injected with PCP followed by amphetamine 30 min later. Results are means  S.E.M. (n = 3–8), expressed as percent baseline dopamine release seen in microdialysis samples taken every 30 min.

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release in the striatum that may relate to the regional variation of the combination between NMDA receptor blocking and DA reuptake inhibition by PCP (Nishijima et al., 1996), and the findings that ketamine and PCP are not effective dopamine releasers and that the effects are smaller compared to amphetamine (Adams et al., 2002; Zetterstrom et al., 1986). Amphetamine increased dopamine release in both regions, but dopamine levels were not significantly increased above controls if given after the acute PCP administration in prefrontal cortex and striatum. The absence of additive effect may relate to the relatively high dose of PCP used and its ability to block the DAT (Schiffer et al., 2001, 2003), preventing amphetamine exchange efflux of dopamine.

3.6. Effect of glycine (16% in diet) on acute amphetamineinduced dopamine release in prefrontal cortex and striatum measured by microdialysis Rats were treated with high-dose amphetamine (5 mg/kg) (Fig. 6) to mimic the dopamine response seen in PCP-treated rats given 1 mg/kg amphetamine (Javitt et al., 2004) and the elevated amphetamine levels reached in the present study after chronic PCP (Fig. 3). In the absence of PCP treatment, GLY treatment (16%—3 weeks) attenuated the amphetamine-induced dopamine release only in prefrontal cortex (Fig. 6) and only at 5 mg/kg amphetamine; when tested against 2 mg/kg amphetamine there was no effect. The effect was more noted during the later fractions (8–10) than the peak response (fraction 6). There was

Fig. 6. Effect of glycine on acute amphetamine (2 or 5 mg/kg s.c.)-induced dopamine release and AMPH levels in microdialysis samples from prefrontal cortex and striatum. Rats were treated with glycine (16% diet) for 2 weeks and given an acute amphetamine administration and extracellular dopamine (expressed as pg/10 ml, n = 9–10) and AMPH (expressed as mmol/l, n = 5–7, not corrected for probe recovery) (lower graphs-after 5 mg/kg AMPH) measured; means  S.E.M. *P < 0.05 glycine vs. control.

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Table 1 Effect of PCP and PCP plus glycine on brain tissue amphetamine content at 1 and 2.5 h after amphetamine (1 mg/kg s.c.) administration Treatment

Control PCP GLY PCP + GLY

Prefontal cortex (amphetamine (pmol/mg))

Striatum (amphetamine (pmol/mg))

1h

2.5 h

1h

2.5 h

6.2  0.38 10.9  0.58* 5.1  0.66 7.8  1.16#

1.2  0.12 4.2  0.34* 2.3  0.70** 3.9  0.34

5.0  0.61 8.5  0.46* 4.2  0.48 6.4  0.80#

1.2  0.13 4.1  0.30* 2.4  0.55** 3.6  0.26

Rats were treated with PCP (15 mg/(kg day) for 3 weeks) with or without dietary glycine (16%), and then given an acute administration of amphetamine (1 mg/kg, s.c.), killed 1- and 2.5-h later, and brain regions dissected. Values are means  S.E.M. (n = 5–7) expressed as pmol/mg tissue weight. *P < 0.001 control vs. PCP; **P < 0.05 control vs. GLY; #P < 0.005 PCP vs. PCP + GLY.

enhanced effect of glycine on amphetamine (5 mg/kg s.c.)induced dopamine release in the striatum only in fraction 5. Extracelluar amphetamine levels were not affected by GLY in the prefrontal cortex, but were even slightly higher in the striatum of GLY-treated rats.

source (tissue/microdialysis)  glycine interaction in both prefrontal cortex (F = 7.2, d.f. = 1.37, p = .011) and striatum (F = 5.3, d.f. = 1.33, p = .03).

3.7. Comparison of the extracellular and brain tissue levels of amphetamine after chronic PCP and glycine treatment in prefrontal cortex and striatum

Dysregulation of brain DA circuits has been well documented in schizophrenia, based especially upon in vivo neuroimaging evidence (Abi-Dargham et al., 1998; AbiDargham, 2004; Breier et al., 1998; Laruelle et al., 1996, 1999, 2000; Laruelle, 1998; Laruelle and Abi-Dargham, 1999). At present, the basis for these deficits are unknown, but are posited to result from underlying disturbances in NMDA receptor-mediated neurotransmission, leading to failures in GABAergic feedback circuitry (e.g.(Coyle, 2006; Javitt, 2007)). This theory is supported by the observation that the NMDA antagonist ketamine induces in vivo DA release (Breier et al., 1998; Schiffer et al., 2001, 2003) and schizophrenia-like DAergic hyperactivity in human (Kegeles et al., 2000), whereas high affinity NMDA antagonists such as PCP or MK-801 induce similar effects in rodent models (Balla et al., 2001b, 2003; Miller and Abercrombie, 1996). More recently, NMDA/ glycine-site agonists, such as GLY and GYT1 transport inhibitors have been found to antagonize both PCP-induced DA release (Javitt et al., 1999) and PCP-induced enhancement of AMPH-induced DA release (Javitt et al., 2004), consistent with their ability to ameliorate both positive and negative symptoms of schizophrenia in at least some clinical trials (Javitt, 2006). It has been widely hypothesized that effects of NMDA antagonists on brain DA levels are due to their effects on GABAergic feedback circuits in brain (Carlsson, 1988; Carlsson et al., 1999). How such effects might alter DA levels in regions such as PFC or STR, however, is presently unknown. The present study follows our recent proposal that agents such as GLY may inhibit presynaptic DA release by potentiating local GABA release in target brain regions, and that the resultant tonic increase in GABA activates presynaptic GABAB receptors on DA terminals (Javitt et al., 2005). GABAB activation on DA terminals is known to lead to hyperpolarization of the terminals, leading to net increase in inward DA transport via voltage-sensitive DATs and thus reduced extracellular/intracellular ratio (Fleckenstein et al., 2007) (Hoffman et al., 1999; Gulley and Zahniser, 2003). In the present study, equilibrium of AMPH across the DAT is used as a

Table 1 and Fig. 7 shows that in controls, tissue amphetamine levels were similar in both prefrontal cortex and striatum tissue extracts at 1 and 2.5 h after amphetamine (1 mg/kg s.c.) administration, but were significantly elevated in the PCP-treated (15 mg/(kg day) for 3 weeks) rats at 1 and 2.5 h compared to controls. In the PCP + glycine treatment groups, amphetamine levels were slightly lower (25%) than the PCPtreatment group at 1 h. In addition, in the glycine-alone-treated rats, amphetamine levels were also slightly deceased at 1 h, and increased at 2.5 h, versus controls, suggesting that glycine may affect the DAT membrane potential; that is, the amphetamine gradients indirectly reflect DAT activity. The graphs also show the actual levels (from Fig. 3 corrected for probe recovery) of extracellular/microdialysis amphetamine at 1 and 2.5 h after administration in control, PCP-treated (15 mg/(kg day) for 3 weeks), glycine (16% diet for 3 weeks), and PCP + glycine-treated rats in the two regions. The results show parallel AMPH levels to that seen with the tissue samples; i.e., higher level after PCP with attenuation in the PCP + glycine treatment group. The lower bar graphs express the data as the ratio of extracelluar/tissue amphetamine. Whereas the ratio at 1 and 2.5 h of extracellular/tissue amphetamine were similar at both time points in controls for both regions, glycine reduced the ratio at 2.5 h versus 1 h. In the PCP + glycine treated rats the ratios at both time points were similar, but lower than controls, reflecting an effect of glycine on maintaining higher tissue levels of amphetamine versus extracellular levels. The changes (decrease) in the ratios between 1 and 2.5 h seen in the glycinealone-treated rats, also represents a slower decrease in tissue versus extracellular amphetamine over the time period. Further, glycine significantly reduced microdialysis amphetamine levels (in the PCP-treated group) at all time points (Figs. 3 and 7), disproportionately to its effects on tissue levels in both prefrontal cortex and striatum, as reflected by significant

4. Discussion

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Fig. 7. Summary comparisons of amphetamine tissue levels (pmol/mg tissue)(bottom axes) and extracellular (microdialysis) levels (mmol/l) (left axes) at 1 and 2.5 h after administration in control, PCP-treated (15 mg/(kg day) for 3 weeks), glycine (16% diet for 3 weeks), and PCP + glycine-treated rats in the two regions. Extracellular AMPH levels are corrected for probe recovery. Results are means  S.E.M. (n = 5–12). Lower bar graphs are the ratio of extracellular to tissue AMPH at the two time points.

marker of DAT function. This equilibrium was reduced following GLY treatment in the presence, but not absence, of PCP, suggesting that effects of GLY on local GABA release or presynaptic GABAB receptors may be particularly relevant in the presence of partial NMDA blockade. In contrast, PCP did not significantly alter extracellular/intracellular AMPH distribution, suggesting that PCP-induced increases are not primarily mediated by alteration of DAT equilibrium. The ability of NMDA antagonists, such as PCP, to induce DA release within PFC and STR has been extensively evaluated using a variety of in vivo and in vitro techniques, with no definitive mechanism determined to date. Different theories postulate direct effects of PCP on D2 receptors (Kapur and

Seeman, 2002), DAT transporters (Schiffer et al., 2003), or NMDA receptors located on presynaptic DA terminals or GABAergic feedback neurons (Javitt et al., 2005). The concept that NMDA antagonists function, in part, through modulation of GABAergic tone is consistent with PET findings in primates that PCP effects on 11C-raclopride binding are antagonized by treatment with the GABA-transaminase inhibitor gamma-vinyl GABA (GVG) (Schiffer et al., 2003). Neurochemical effects of GVG are thought to be mediated primarily through its effects at GABAB receptors (Ashby, Jr. et al., 1999). Thus, as suggested in present study, GABAB receptors may represent a critical target at which to modulate subcortical and cortical DA release. Other PET studies also suggest that the glutamatergic system

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may exert significant control over DAT function and expression (Kagawa et al., 2002; Nakano et al., 1998), and that DAT transporter availability may be affected by NMDA antagonists such as ketamine (Kahlig and Galli, 2003; Kahlig et al., 2004, 2006; Tsukada et al., 2000, 2001). DA release, in general, is divided into impulse-dependent and impulse-independent. Electrically evoked release is consistent with an exocytotic process involving the vesicular pool. Spontaneous DA release occurs mainly from a cytoplasmic pool of DA that is maintained by ongoing transmitter synthesis. Whereas impulse-dependent release depends upon vesicular fusion unrelated to sustained alterations in membrane potential, impulse-independent release depends upon reverse transport of DA via presynaptic DAT-type DA transporters. The voltagedependence of DAT transport is well established, with more depolarized potentials favoring greater reverse transport and higher extracellular levels, and more negative potentials favoring greater inward transport and lower extracellular levels. To the extent that AMPH gradient across DATs can be used as a marker of the DA gradient, the present finding suggest that NMDA antagonists induce increased DA release primarily via increase in impulse-dependent release, whereas agents that stimulate GABAergic tone, such as GVG or GLY, can reverse this via modulation of impulse-independent release. This hypothesis is consistent as well with prior research demonstrating that GLYT1 inhibitors reverse NMDA-, but not electrically-, induced STR DA release in vitro (Javitt et al., 2000), as well as in vivo studies showing that stimulatory effects of the NMDA antagonist MK801 can be reversed in vivo by local administration of TTX into PFC (Kashiwa et al., 1995). Feedback between regions may also be important, given preferential effects of GVG on cortical over subcortical dopamine release (Schiffer et al., 2001). In addition to stimulating reverse transport of DA due to its own transport via the DAT, AMPH also induces DAT internalization within minutes following acute administration, and, at high concentrations, stimulates reverse transport of DA from synaptic vesicles, increasing the pool available for reverse transport (Fleckenstein et al., 2000, 2007). How such an effect would affect extracellular/intracellular ratios, however, needs to be determined. NMDA receptors may also modulate the activity of the DAT via triggering phosphorylation through activation of protein kinase C and calcium calmodulindependent protein kinase II (inhibition and internalization of the DAT) (Page et al., 2000, 2001). GABAB receptors also couple to intracellular signaling pathways common to dopamine D2 receptors, and can rapidly up-regulate DAT function (Gulley and Zahniser, 2003; Hoffman et al., 1999), again suggesting the possibility for complex interactions. A second finding of the present study is that, chronic treatment with PCP significant alters acute AMPH levels, measured either peripherally or centrally. Although this effect has not been demonstrated previously, it is highly consistent with the fact that PCP inhibits the hepatic cytochrome P450 (CYP2C11) system (Sharma et al., 1997; Jushchyshyn et al., 2003; Hiratsuka et al., 1995), which is a major route of AMPH metabolism (Yamada et al., 1989; Shiiyama et al., 1997; Law et al., 2000), leading to a pharmacokinetic interaction between

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the two compounds. Such an interaction would be relevant clinically during mixed abuse situations, and would predict that chronic PCP abusers may be predisposed to increase AMPHinduced psychosis. In the present study, based upon the dose range of AMPH effects, the PCP effect on AMPH metabolism would explain, in part, the higher PFC and STR DA release observed in animals treated subchronically with PCP. However, pharmacokinetic interaction cannot explain effects of GLY in the presence of PCP, as GLY did not affect peripheral AMPH levels in the presence of PCP. Further, effects of GLY on DA release induced by combined PCP and AMPH (1 mg/kg) treatment were qualitatively similar to those induced by AMPH (5 mg/kg) alone, suggesting that GLY effects are unrelated to metabolic interactions between PCP and AMPH. A third finding of this study is that, as in prior studies, regional differences were observed in rates of DA clearance from the extracellular space in PFC versus STR. The regional differences in the dopaminergic response to NMDA antagonists (Fig. 5) agrees with previous findings (Adams et al., 2002; Balla et al., 2001b). The lack of effect in striatum was suggested not to be related to a more rapid reuptake mechanism in striatum, since nomifensine did not influence the effect of PCP (Adams et al., 2002). However, others have suggested differences in DAT distribution or availability are involved or that PCP exerts direct effects on the DAT as an underlying mechanism of altered dopamine release (Schiffer et al., 2003; Tsukada et al., 2000), and that the absence of effect of nomifensine may be related to a U-shaped dose-response curve for PCP (Schiffer et al., 2003). In our previous study, dopamine efflux responses to local administration of amphetamine or nomifensine in PCP-treated rats were similar to saline controls in prefrontal cortex, and responses were even lower in striatum versus controls, suggesting that neither the DAT nor the D2 autoreceptor were affected by chronic PCP (at least in the prefrontal cortex), so that the enhanced response to systemic AMPH administration in PCP-treated rats reflects predominantly effects rather at the circuit level (Balla et al., 2001a). Other clearance mechanisms for DA in PFC, such as norephinephrine transporters (NET) and monoamine oxidase (MAO), may also be relevant (Wayment et al., 2001), and account in part for differential regional effects. Finally, other potential cross-reactivities must be taken into account. For example, it has been suggested that AMPH has selective influence on the metabolism of PCP, leading to enhanced duration of motor effects of PCP but not MK-801 (Pesic et al., 2003). PCP levels were not measured in the present study so it is unknown whether AMPH treatment altered PCP levels during the course of the study. AMPH may also act, in part, as an antagonist at the NMDA receptor close to or at the PCP site (Yeh et al., 2002), potentially contributing also to the observed AMPH/PCP interactions. Nevertheless, these effects are also unlikely to have contributed to the observed ability of GLY to alter AMPH ratios or AMPH-induced DA release. In summary, the present results support the concept that GLY, and potentially other direct or indirect acting NMDA/ glycine-site agonists, modulate DA release in part through modulation of presynaptic DAT-type transporters in PFC and STR, consistent with effects on GABAergic feedback and

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