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Research Report

Environmental enrichment increases amphetamine-inducedglutamate neurotransmission in the nucleus accumbens:A neurochemical study

Shafiqur Rahman1, Michael T. Bardo⁎

Center for Drug Abuse Research Translation, University of Kentucky, BBSRB, Room 447, 741 S. Limestone, Lexington, KY 40536-0509, USA

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 859 257 5750.E-mail addresses: Shafiqur.Rahman@sdst

0006-8993/$ – see front matter © 2008 Elsevidoi:10.1016/j.brainres.2007.12.052

A B S T R A C T

Article history:Accepted 26 December 2007Available online 4 January 2008

In addition to dopamine (DA), evidence indicates that glutamatergic regulation of themesolimbic reward pathway is involved in mediating the abuse-related effects ofpsychostimulants, including amphetamine. Since rats raised in an enrichment condition(EC) during development are more sensitive to the locomotor stimulant effects of acuteamphetamine compared to rats raised in an impoverished condition (IC), the present studyexamined amphetamine-induced extracellullar glutamate and aspartate levels in thenucleus accumbens (NAcc) of EC and IC rats using in vivo microdialysis coupled withHPLC-electrochemical detection. Basal extracellular levels of glutamate or aspartate werenot significantly different between EC and IC rats. Acute systemic amphetamine (0.5 or2.0 mg/kg, sc) increased extracellular glutamate levels in NAcc of EC rats (137% or 305% ofbasal) and IC rats (120% or 187% of basal). Similarly, acute systemic amphetamine (0.5 or2.0 mg/kg, sc) elevated aspartate levels in NAcc of EC rats (148% or 237% of basal) and IC rats(115% or 170% of basal). Glutamate levels were elevated by amphetamine to a greater extentin EC rats than in IC rats. Pretreatment with systemic MK-801 (0.25 mg/kg, ip), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, prevented the acuteamphetamine-induced increase in extracellular glutamate and aspartate levels in NAcc.Overall, these results suggest that alterations in glutamate in the NAcc may be involved inthe environment-dependent effects of amphetamine.

© 2008 Elsevier B.V. All rights reserved.

Keywords:Drug abuseEnvironmental enrichmentAmphetamineGlutamateNucleus accumbensIn vivo microdialysis

1. Introduction

Evidence indicates that drug abuse is the result of an interactionof genotype and environmental factors (Commings, 1996;McGue et al., 1996). Previous research has shown that animalsreared in an enriched condition (EC) relative to an impoverishedcondition (IC) exhibit greater sensitivity to the acute effects ofpsychostimulants on mesolimbic dopamine (DA) function andlocomotor activity (Bowling et al., 1993; Bowling and Bardo 1994;Bardo et al., 1999). Results suggest that environmental enrich-

ate.edu (S. Rahman), mba

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ment produces profound neuroanatomical and neurochemicalchanges in various parts of the brain, including neocortex andsub-cortical structures (Jones and Greenough, 1996; Kemper-mann et al., 1997; Van Praag et al., 2000). Enrichment has beenshown to increase neurogenesis (Nilsson et al., 1999; Segoviaet al., 2006), increase dendritic spines on medium size spinyneurons (Comery et al., 1996), increase synaptic density (Ram-pon et al., 2000) and increase the density of astrocytes (Phamet al., 1999). It is possible that these enrichment-induced modi-fications are associatedwith synaptic plasticitywithin the brain

rdo@uky.edu (M.T. Bardo).

.

Fig. 1 – Typical locations of microdialysis probe placementsin NAcc as indicated by black lines. Numbers to the rightindicate distance in mm from bregma according to Paxinosand Watson (1986). The active probe protruded 2 mm belowthe distal end of the guide cannulae for NAcc as described inExperimental procedure.

Fig. 2 – Time course effect of acute amphetamine (AMPH)given at either 0.5 mg/kg (upper panel) or 2 mg/kg (lowerpanel) on extracellular glutamate level in NAcc of EC and ICrats. After collection of basal samples, rats were given saline(SAL) injection, as indicated by left arrow (↑) and thensamples were collected for 60 min before AMPH injection,right arrow (↑). Data represent themean±SEM of 6–7 animals.*pb0.05, significant difference from IC rats at same samplingtime interval.

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reward circuitry where glutamate neurotransmission plays amodulatory role (Wolf, 1998;Wolf and Xue, 1999; Giorgetti et al.,2001; Melendez et al., 2004).

The nucleus accumbens (NAcc) and its associated limbicstructures integrate various neurotransmitter systems, in-cluding DA and glutamate inputs (McGinty, 1999; Mogensonet al., 1980; Pennartz et al., 1994). Acute psychostimulants,including amphetamine, have been shown to increase drug-induced extracellular brain monoamines and excitatoryamino acids in the NAcc and other limbic structures (Giorgettiet al., 2001; Reid et al., 1997; Shoblock et al., 2003; Xue et al.,1996). Both DA and glutamate are important neurotransmittersystems which could be modulated by environmental manip-ulations (Pryce et al., 2002).

Environmental enrichment has been found to alter DAfunction in NAcc in response to psychostimulants (Bardo et al.,1999; Zhu et al., 2004), as well as to alter glutamate functionin hippocampus andmedial prefrontal cortex (Melendez et al.,2004; Naka et al., 2005). Compared to IC rats, EC rats havereduced subunit expression for N-methyl-D-aspartate (NMDA)receptors, but not α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (Wood et al., 2005), perhapsreflecting a subtype-specific down-regulation in response toan enhanced glutamate signal. However, the role of glutamaterelease and its regulation in response to psychostimulants in

EC and IC rats is not known. Therefore, using in vivo micro-dialysis, the present study determined basal glutamate andaspartate levels in NAcc from EC and IC rats, as well as acuteamphetamine-induced extracellular glutamate and aspartatelevels in NAcc. Since NMDA subunit expression is decreased inEC rats (Wood et al., 2005), the effect of the NMDA antagonistMK-801 on amphetamine-induced changes in glutamate lev-els in EC rats was also determined.

2. Results

Representative probe placements for the rats used in thisstudy are shown in Fig. 1. Overlapping probe placements arenot shown in the figure for clarity of presentation.

2.1. Basal extracellular levels of glutamate and aspartatein EC and IC rats

Basal extracellular levels (mean±SEM) of glutamatewere 220±29 pg/μl and 176±44 pg/μl in EC and IC rats, respectively. Basalextracellular levels (mean±SEM) of aspartate were 17±3 pg/μland 16±2 pg/μl in EC and IC rats, respectively. There were no

Fig. 4 – Time course effect of acute amphetamine (AMPH,2 mg/kg, sc) given in combination with systemic MK-801(0.25 mg/kg) on extracellular glutamate or aspartate levels inNAcc of EC rats. After collection of basal samples, rats weregiven saline (SAL) injection as indicated by left arrow (↑) andsamples were collected for 40 min, followed by MK-801, asindicated by middle arrow (↑), and 20 min later, followed by

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significant basal differences between EC and IC rats in eitherglutamate or aspartate.

2.2. Acute amphetamine-induced increase in extracellularlevels of glutamate in EC and IC rats

As shown in Fig. 2, systemic saline injection did not producesignificant changes in basal extracellular glutamate levels for60 min (Fig. 2, top and bottom panels). However, acute sys-temic amphetamine (0.5 or 2 mg/kg) increased extracellularglutamate levels to a peak of ~140% and 318% of basal in ECrats (Fig. 2, top and bottom panels). Glutamate levels returnedto baseline over time during the post-injection collectionperiod. Separate ANOVAs revealed a significant environ-mental condition X sample time interaction [F (19, 110)=4.84,pb0.05 and F (19, 100)=6.36, pb0.05] for amphetamine dosesof 0.5 and 2mg/kg, respectively. Subsequent Tukey's HSD testsrevealed that the difference between EC and IC rats for bothdoses was significant during the first two 20-min post-injection sampling time intervals ( pb0.05), but not duringthe later sampling time intervals.

Fig. 3 – Time course effect of acute amphetamine (AMPH)given at either 0.5 mg/kg (upper panel) or 2 mg/kg (lowerpanel) on extracellular aspartate level in NAcc of EC and ICrats. After collection of basal samples, rats were given saline(SAL) injection, as indicated by left arrow (↑) and thensamples were collected for 60 min before AMPH injection,right arrow (↑). Data represent themean±SEM of 6–7 animals.*pb0.05, significant difference from IC rats at same samplingtime interval.

AMPH as indicated by right arrow (↑). Data represent themean±SEM of 6 animals.

2.3. Acute amphetamine-induced increase in extracellularlevels of aspartate in EC and IC rats

Systemic saline injection did not produce significant changes inbasal extracellular aspartate levels for 60 min (Fig. 3, top andbottom panels). However, acute systemic amphetamine (0.5 or2 mg/kg) increased extracellular aspartate levels to a peak of~148% and 236% of basal in EC rats (Fig. 3, top and bottompanels). Aspartate levels returned to baseline over time duringthe post-injection collection period. Separate ANOVAs revealeda significant environmental condition X sample time interac-tion [F (19, 110)=4.24, pb0.05 and F (19, 100)=3.81, pb0.05] foramphetamine doses of 0.5 and 2 mg/kg, respectively. Subse-quent Tukey's HSD tests revealed that the difference betweenEC and IC rats for both doseswas significant during the first two20-min post-injection sampling time intervals (pb0.05), but notduring the later sampling time intervals.

2.4. Effect of MK-801 on acute amphetamine-inducedincrease in extracellular levels of glutamate and aspartate inEC rats

Pretreatment withMK-801 (0.25mg/kg) prevented the increasein glutamate and in aspartate (Fig. 4) levels induced by acuteamphetamine (2 mg/kg) in EC rats. ANOVA revealed nosignificant effect of sample time for either glutamate oraspartate levels following the combination of MK-801 andamphetamine in EC rats.

3. Discussion

The present study demonstrates that environmental enrich-ment augments the elevation in glutamate and aspartate

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levels in NAcc induced by acute amphetamine. Further, therise in extracellular glutamate and aspartate levels was pre-vented by pretreatment with MK-801, indicating that NMDAreceptors are involved. Our results with MK-801 in blockingthe acute amphetamine-induced increase in glutamate andasparate levels in EC rats are in accord with previous neuro-chemical studies using standard housed rats (Reid et al., 1997;Wolf and Xue, 1999), as well as with behavioral results show-ing that MK-801 blocks amphetamine locomotor sensitizationin standard housed rats (Karler et al., 1989). The enrichment-induced augmentation in the neurochemical effects observedhere is likely related to pharmacodynamic changes in limbicfunction, rather than to an alteration in amphetamine phar-macokinetics, as previous work indicates that treatment with3H-amphetamine produces similar brain uptake of radiolabelin EC and IC rats (Bardo et al., 1999). It is possible that theenrichment-induced augmentation of glutamate levels in res-ponse to amphetamine may explain, at least in part, theexaggerated hyperactivity shown previously in EC rats follow-ing an acute dose of amphetamine (Bowling et al., 1993;Bowling and Bardo 1994).

While the source of glutamate release described in thecurrent report was not determined, it is unlikely that extra-cellular levels reflected primarily synaptic events. Using invivo microdialysis, basal glutamate levels have been shownpreviously to be relatively insensitive to calcium and to thesodium channel blocker tetrodotoxin (Timmerman and Wes-terink, 1997; Xue et al., 1996). In addition, evidence suggeststhat amphetamine-induced glutamate efflux may involve theformation of reactive oxygen species resulting frommetabolicor oxidative stress independent of synaptic activity (Wolfet al., 2000). Future work in this area of investigation mayincorporate microsensor technology, which may be more sen-sitive than microdialysis to neuronally-derived glutamate(Oldenziel et al., 2006).

It is known that the NAcc and related neural pathways areinvolved in the psychostimulant effects of amphetamine(Wolf, 1998; Rockhold, 1998; Wolf et al., 2004; Kalivas andVolkow, 2005). Neuroanatomical studies have indicated thatDAneurons in theNAcc receive significant glutamatergic inputdirectly from the limbic cortex and sub-cortical structures(Groenewegen et al., 1980; Kelley et al., 1982; Kita and Kitai,1990; Parent and Hazrati, 1995; Sesack et al., 1989; Tzschentkeand Schmidt, 2000). Since activation of direct and indirectglutamate pathways increases glutamate release in NAccfollowing acute amphetamine injection (Robinson and Beart,1988; Parent and Hazrati, 1995; McGinty, 1999; Vanderschurenand Kalivas, 2000), a potential role for glutamate-mediatedmechanisms is implicated in the environment-dependentalteration in amphetamine-induced effects in NAcc.

Regardless of rearing condition, the acute effect of amphe-tamine on extracellular glutamate and aspartate levels in thecurrent study is consistent with results from previous micro-dialysis studiesusing rats raised in standardhousing conditions(Reid et al., 1997; Shoblock et al., 2003; Wolf and Xue, 1999; Xueet al., 1996). However, the underlying mechanism for theenrichment-induced enhancement in amphetamine-inducedglutamate and aspartate levels remains unclear. Previousreports indicate that environmental enrichment can producestructural, functional and biochemicalmodifications in neocor-

tex and hippocampus (Jones and Greenough, 1996; Kemper-mann et al., 1997). Environmental enrichment also has beenfound to increase neurogenesis, synaptic numbers, spinedensity, and growth factor expression in cortex and striatum(Comery et al., 1996; Jones et al., 1997; Pham et al., 1999; Segoviaet al., 2006). Which one of these factors, if any, underline theenriched-induced increase in amphetamine-induced extracel-lular glutamate and aspartate levels in NAcc remains to bedetermined. However, given that the amphetamine-inducedincrease in glutamate was blocked completely by MK-801 in ECrats, an involvement of NMDA receptors is implicated. Con-sistent with this, compared to IC rats, EC rats have increasedglutamate transmission in cortex and hippocampus (Nicholset al., 2007; Segovia et al., 2006) and decreased expression ofNMDA receptor subunits in NAcc (Wood et al., 2005). Thedecrease inNMDA receptor expression inNAccmay represent acompensatory down-regulation in response to an enhancedglutamate signal. Although the basal levels of glutamate andaspartate did not differ significantly in NAcc in the currentreport, it is important to note that amino acid transmitter levelswere measured when both EC and IC rats were removed fromthe home cage environments. Thus, it is possible that repeatedexposure to novelty during development may have producedphasic increases in glutamate andaspartate in EC rats thatwerenot apparent under the current measurement conditions.

Previous work has shown a difference between NAcc coreand shell sub-regions with regard to basal DA levels (Leccaet al., 2006; Rahman et al., 2007). In the present study, ourprobe locations for measurement of glutamate and aspartatewere primarily in NAcc shell. It is not known if there is adifference between NAcc shell and core sub-regions in basalextracellular glutamate and aspartate levels, although NMDAreceptor subunit expression does not differ between core andshell (Wood et al., 2005). Further studies are needed to deter-mine if there is any core-shell regional difference in glutamateand aspartate levels using the enrichment paradigm.

Glutamate transmission in NAcc has been implicated insensitization following repeated stimulant use, which mayunderlie addiction and relapse (Cornish and Kalivas, 2000; DiCiano et al., 2001; Kalivas and McFarland, 2003; Szumlinskiet al., 2000; Wolf, 1998). Although we did not study locomotoractivity or behavioral sensitization in relation to glutamaterelease in NAcc in this report, it is likely that glutamate releasehas a modulatory role in locomotion via activation of NMDAreceptors (Shoblock et al., 2003), as well as via activation ofother glutamate receptor subtypes (Giorgetti et al., 2001; Kimand Vezina, 2002). In the current report, the augmented re-lease of glutamate following acute amphetamine in EC rats,which was blocked completely by MK-801, may explain theexaggerated hyperactivity in response to acute amphetaminenoted previously in EC rats (Bowling et al., 1993; Bowling andBardo 1994). However, since EC rats show less sensitization torepeated amphetamine compared to IC counterparts (Bardoet al., 1995), it is possible that environmental enrichment mayalso blunt the glutamate-mediated neuroadapations thatoccur as a consequence of repeated drug administration,thus protecting against psychostimulant abuse. Consistentwith this possibility, EC rats self-administer less ampheta-mine than IC rats when tested across repeated self-adminis-tration sessions (Bardo et al., 2001).

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In summary, results from the present study suggest thatalterations in glutamate function in NAcc may be involved inthe enrichment-induced augmentation in the stimulanteffects of acute amphetamine, an effect that depends onactivation of brain NMDA receptors. Further work is needed todetermine if this enrichment-induced augmentation in exci-tatory amino acid release following acute amphetaminegeneralizes to other drug and non-drug reinforcers.

4. Experimental procedure.

4.1. Animals

Male Sprague–Dawley rats were obtained from Harlan Labora-tories (Indianapolis, IN) at 21 days of age and were housedwith free access to food and water in a colony room in theDivision of Laboratory Animal Resources at the University ofKentucky. Rats were maintained on a 12:12 h light/dark cycle(lights on at 0700 h). All animal handling procedures wereapproved by the Institutional Animal Care and Use Committee(IACUC) at the University of Kentucky and were performed inaccordance with the 1996 version of the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals.

4.2. Environmental conditions

Upon arrival, rats were assigned randomly to an EC or IC homecage for the duration of the study. EC rats were housed in alarge metal cage (60×120×45 cm) with cohorts (8–10 per cage).The environment contained novel hard plastic objects (i.e.,commercially available children's toys, PVC pipe, plasticcontainers, etc.; 14 objects per cage). Rats were removedbriefly each day so that 7 of the objects could be replaced withnew objects; the remaining objects were rearranged into anovel configuration. IC rats were housed individually in ahanging cage with wire mesh floor and front panel (17×24×20 cm), and solid metal sides, back and top. IC rats werenot handled during this rearing period (21–53 days of age). Allexperiments commencedwhen rats were 53 days of age underboth conditions.

4.3. Drugs

D-Amphetamine sulfate and MK-801 were obtained fromSigma-Aldrich (St. Louis, MO). Drugs were dissolved in 0.9%NaCl (saline) and were injected subcutaneously (sc). Dosesrepresented the salt weight. The doses of amphetamine andMK-801 used in the present studywere similar to doses used inprevious studies (Shoblock et al., 2003; Xue et al., 1996).

4.4. Surgery

Surgeries for microdialysis were performed as describedpreviously (Rahman et al., 2007) in aseptic conditions underanesthesia induced by xylazine (8 mg/kg, ip) and ketaminehydrochloride (60 mg/kg, ip). Rats were anesthetized and plac-ed in a stereotaxic apparatus (Stoelting, Wood Dale, IL).Microdialysis guide cannulae (18 ga; Plastics One, Roanoke,VA) were implanted unilaterally in the NAcc according to the

atlas of Paxinos and Watson (1986). The cannula wasimplanted at a 10° angle from the midline using the followingcoordinates with the incisor bar set at −3.3 mm: AP+1.7 mmfrom bregma, L+2.2 mm, and DV −6.2 mm. To minimize dis-comfort and pain, rats were given carpofen (5 mg/kg, sc), anon-opioid analgesic, for 3 days after the surgical procedure.Rats were allowed to recover for 3–4 days in their home cagesfollowing surgery.

4.5. Microdialysis probe

Loop-style probes were made as described previously (Rah-man and McBride, 2002; Rahman et al., 2007) with dialysismembrane (Spectra/Por regenerated cellulose dialysis mem-brane, molecular weight cutoff of 13,000; Medical Industries,Los Angeles, CA). The outside diameter of the dialysis mem-brane was 220 µm. The length of the probe was 2 mm activedialysis membrane. Loop-style probes were used instead ofconcentric probes because they provide consistent, high basallevels of glutamate, and they sample a large portion of thetarget area (Rahman and McBride, 2002). All microdialysisprobes were inserted 18–24 h before the microdialysis pro-cedure (see below).

4.6. In vivo microdialysis procedure

To assess the changes on extracellular glutamate and aspar-tate levels in NAcc following acute amphetamine, EC and ICrats were assigned randomly to one of 2 groups (n=8–10 pergroup): (1) amphetamine (0.5 mg/kg) or (2) amphetamine(2mg/kg). Rats from each groupwere treatedwith injections ofsaline and amphetamine, separated by 60 min. In a separatestudy designed to evaluate if blockade of NMDA receptorsprevented the effect of amphetamine, EC rats were treatedwith saline and MK-801 (0.25 mg/kg) separated by 40 min,followed 20 min later by amphetamine (2 mg/kg).

Microdialysis experiments were performed in clear Plexig-las chambers (25×44×38 cm). Artificial cerebrospinal fluid(ACSF; inmM: 145NaCl, 2.7 KCl, 1.0MgCl2, 1.2 CaCl2; pHadjustedto 7.3–7.4 with 2mM sodium phosphate buffer; filtered througha 0.2 μm sterile filter) was perfused through the probe at a flowrate of 1 μl/min for 2hprior to collectionof thebaseline samples.After this equilibration period, three baseline samples werecollected into polyethylenemicrofuge tubes every 20min for anadditional 60 min. Five minutes after the last basal samplecollection, rats were given saline, followed 60 min later byamphetamine. For the MK-801 group, EC rats were treated withsaline and MK-801 (0.25 mg/kg) separated by 40 min, followed20 min later by amphetamine (2 mg/kg). Samples continued tobe collected at 20 min intervals for 80 min following theamphetamine injection. Samples were frozen on dry ice andstored at −70 °C for HPLC analysis. At the end of the experi-ments, a 1% bromphenol blue solution was perfused throughthe probes to verify the placements. Animalswere anesthetizeddeeply with pentobarbital. Brains were removed and fixed forsubsequent sectioning to determine the location of the micro-dialysis probe. Probe placements were evaluated according tothe atlas of Paxinos andWatson (1986). Only data from animalswith correct probe placements in the NAcc were used in thestatistical analyses.

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4.7. Quantification of glutamate and aspartate content

Samples were analyzed for glutamate and aspartate levelsusing high performance liquid chromatography coupled withelectrochemical detection (HPLC-ECD, ESA Inc., Chelmsford,MA) as previously described (Melendez et al., 2004). The sys-tem consisted of a solvent delivery unit, a Coulochem IIIelectrochemical detector equippedwith a 5014B analytical celland 5020 guard cell. The guard cell was set at +600 mV,electrode 1 at −150 mV, and electrode 2 at +550 mV withthe gain at 1 µA. Precolumn derivatization of glutamatewith O-Phthalaldehyde (OPA; Pickering Lab Inc, CA) and 2-Mercaptoethanol (Sigma-Aldrich, MO) was performed usingan ESA 542 autosampler. The mobile phase consisted of100 mM Na2HPO4, 22% methanol and 3.5% acetonitrile; pH6.75 adjusted with phosphoric acid, and pumped through thesystem at a flow rate of 0.7 µl/min. Glutamate and aspartatewere separated using an analytical column (50 mm×3 mm,Thermo Hypersil-Keystone, PA). The concentration of gluta-mate and aspartate in the dialysis samples was quantified bycomparing peak heights from samples and external standardsusing an ESA Chromatography data system (EZChrom Elite,Chelmsford, MA).

4.8. Data analysis

Values were not corrected for in vitro probe recovery effi-ciency, which was ~15% and in close agreement withpublished values (Melendez et al., 2004). The baseline wasdefined as the average glutamate or aspartate level in threesamples prior to the saline injection and normalized to 100%.Data were expressed as percent of baseline andwere analyzedby one-wayANOVAusing SPSS (version 12.0; Chicago, IL).Whenappropriate, data were further analyzed using post-hoc tests(Tukey's HSD) for multiple comparisons. The significance levelwas set at pb0.05.

Acknowledgments

This work was supported by USPHS grant DA12964. Weacknowledge Dr. Eric Engleman for his comments on the glu-tamate assay and Emily Geary for her assistance duringsurgery and the dialysis procedure.

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