Effects of Repeated Cocaine on the Releaseand Clearance of Dopamine Within the Rat

Medial Prefrontal CortexJASON M. WILLIAMS* AND JEFFERY D. STEKETEE

Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee

KEY WORDS behavioral sensitization; microdialysis; monoamine uptake; psycho-stimulant; withdrawal

ABSTRACT Previous data suggest that cocaine-induced dopamine (DA) transmis-sion within the medial prefrontal cortex (mPFC) undergoes time-dependent changesduring withdrawal from repeated cocaine administration. The current studies assessedtwo potential mechanisms that may underlie this neuroadaptation. One set of experi-ments examined alterations in DA clearance in the mPFC of rats that had beenpretreated with four administrations of cocaine (15 mg/kg, i.p.; once per day for 4 days)and were withdrawn 1, 7, or 30 days. No significant changes in mPFC DA uptake intocrude mPFC synaptosomes or in mPFC DA transporter levels were observed at any ofthe time points examined. Uptake assay and Western blotting sensitivity was confirmedwith prefrontal 6-hydroxydopamine lesions, which significantly reduced [3H]DA uptakeand DA transporter immunoreactivity in mPFC synaptosomes. To evaluate temporalchanges in DA release resulting from repeated cocaine, additional experiments utilizedin vivo microdialysis to locally infuse KCl (10, 30, or 100 mM) into the mPFC over thesame withdrawal time course used in the uptake studies. After 1–7 days of withdrawal,KCl-stimulated DA release was significantly reduced in the mPFC of cocaine-pretreatedanimals. However, after 30 days of withdrawal the evoked release of DA in the mPFC ofsaline- and cocaine-pretreated animals was similar. These data suggest that previouslyreported modulation of cocaine-induced mPFC DA transmission occurring upon with-drawal from repeated cocaine might arise from transient changes in DA releasabilityrather than clearance. The relevance of these findings is discussed in relation to mPFCinvolvement in psychostimulant sensitization. Synapse 55:98–109, 2005.© 2004 Wiley-Liss, Inc.

INTRODUCTION

Behavioral sensitization is the enduring and pro-gressively augmented motor stimulant response thatoccurs with repeated administration of psychomotorstimulants such as cocaine and amphetamine. The in-duction and expression of sensitization model certainaspects of drug dependence and develop through time-dependent and structurally distinct neuroadaptationswithin the mesocorticolimbic dopamine (DA) system(Robinson and Berridge, 1993; White, 1996; Kalivas etal., 1998; Everitt and Wolf, 2002). This system consistsof mesolimbic and mesocortical dopaminergic neuronswithin the ventral tegmental area (VTA) that inner-vate several regions of the limbic forebrain involved incognition, reward, and locomotion, including the nu-cleus accumbens (NAc) and the medial prefrontal cor-tex (mPFC) (Oades and Halliday, 1987). Additionally,glutamatergic neurons of the mPFC project to the VTA

and NAc and serve to regulate the neuronal activationof these regions (Sesack and Pickel, 1992; Karremanand Moghaddam, 1996). In the context of this neuro-anatomical arrangement, it has been postulated thatcocaine-induced sensitization arises in part from a lossof the inhibitory influence of DA in the mPFC, whichmay in turn lead to an enhancement in excitatorytransmission from this region to these other limbic andsubcortical sites that mediate locomotor output (Steke-tee, 2003).

Contract grant sponsor: National Institute on Drug Abuse; Contract grantnumbers: DA13470; DA15965 (to J.D.S.).

*Correspondence to: Jason M. Williams, Department of Pharmacology, Uni-versity of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN38163. E-mail: jmwilliams@utmem.edu

Received 23 July 2004; Accepted 26 September 2004

DOI 10.1002/syn.20093

Published onlinein Wiley InterScience (www.interscience.wiley.com).

SYNAPSE 55:98–109 (2005)

© 2004 WILEY-LISS, INC.

While repeated exposure to cocaine has generallybeen associated with an increase in cocaine-inducedDA transmission within the NAc (Kalivas and Duffy,1990, 1993; Pettit et al., 1990; Parsons and Justice,1993), the mPFC DA response to cocaine challenge isattenuated upon early withdrawal from repeated ad-ministration (Sorg et al., 1997; Chefer et al., 2000;Williams and Steketee, 2004c) and enhanced followinglonger withdrawal periods (Wu et al., 2003; Williamsand Steketee, 2004c). This differential responsivenessto cocaine may be related to functional distinctionsbetween mesocortical and mesolimbic neurons, includ-ing the autoregulatory influences on these systems(Bannon et al., 1981; Chiondo et al., 1984; Wolf andRoth, 1990), their basal and cocaine-stimulated neuro-physiological characteristics (Bannon et al., 1983; Gar-ris and Wightman, 1994; Trantham et al., 2002), ortheir distribution and regulation of DA uptake sites(Elsworth et al., 1993; Garris and Wightman, 1994;Moron et al., 2002).

Given the clear involvement of the mPFC in thedevelopment of sensitization to psychomotor stimu-lants (Schenk and Snow, 1994; Banks and Gratton,1995; Wolf et al., 1995; Beyer and Steketee, 1999; Li etal., 1999; Tzschentke and Schmidt, 2000), as well asthe reinforcing properties of these drugs (Goeders andSmith, 1983; Schenk et al., 1991; Weissenborn et al.,1997; McFarland et al., 2003), the current studies in-vestigated two potential mechanisms that may in partunderlie alterations in cocaine-induced DA transmis-sion within the mPFC that occur with repeated cocaineadministration. In the first set of experiments, cocaine-induced changes in DA uptake and dopamine trans-porter (DAT) immunoreactivity within mPFC synapto-somes were assessed in rats withdrawn for 1 day,1 week, or 1 month from four consecutive injections ofcocaine (15 mg/kg, i.p.) administered once daily.Changes in mPFC DA clearance and DAT levels werealso examined in animals that had undergone 6-hydroxydopamine (6-OHDA) lesioning of the mPFC inan effort to validate the sensitivity of the methodologyused to test these parameters. The second series ofexperiments implemented in vivo microdialysis tostudy whether this time course of withdrawal from thesame repeated cocaine regimen affects depolarization-evoked release of DA in the mPFC.

MATERIALS AND METHODSMaterials

Cocaine HCl, GBR12909 2HCl, nisoxetine HCl, flu-oxetine HCl, pargyline HCl, desmethylimipramine(DMI) HCl, 6-OHDA HBr, dihydroxybenzylamine(DHBA) HBr, and monoclonal �-actin antibodies(MW � 42 kDa) were purchased from Sigma Chemical(St. Louis, MO). Monoclonal dopamine transporter(DAT) antibodies were generated against theN-terminal amino acids 42–59 (peptide 16) of the rat

DAT (rDAT; MW � 80 kDa) and were generously pro-vided by Dr. Roxanne Vaughan, University of NorthDakota School of Medicine, Grand Forks, ND. Horse-radish peroxidase (HRP)-conjugated horse antimouseIgG was purchased from Vector Laboratories (Burlin-game, CA).

Animals

One hundred thirty-five male Sprague-Dawley rats(Harlan Sprague-Dawley, Indianapolis, IN) served assubjects (mean weight �300 g at the beginning of ex-periments), which were housed within a temperature-and humidity-controlled, American Association forAccreditation of Laboratory Animal Care (AAALAC)-accredited facility and maintained on a 12:12 h light:dark cycle (lights on at 06:00) with access to food andwater at all times. All experiments were conducted inaccordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals andwere authorized by the University of Tennessee Ani-mal Care and Use Committee.

Treatment schedule

For both the DA clearance and KCl microdialysisstudies, four once-daily injections of isotonic saline(1 ml/kg, i.p) or cocaine HCl dissolved in saline(15 mg/kg, i.p.) were administered between 11:00 and13:00 while animals were in their homecages. The cur-rent studies were designed to limit the potential influ-ence of environmental context on the development ofbehavioral sensitization, an important determinant forthe behavioral effects of psychostimulants (Post et al.,1992). For the DA uptake studies, 1, 7, or 30 daysfollowing the last of the daily injections approximatelyone-half of the saline-pretreated animals (AcuteCocaine group) and the cocaine-pretreated animals(Repeated Cocaine group) received cocaine challengeinjections, while the remaining saline-pretreated ani-mals (Saline group) received challenge saline injec-tions. On test days, the motor activity of these animalswas recorded for 2 h using a computer-assisted MicroAnimal Activity Monitor (AccuScan, Columbus, OH) aspreviously described (Beyer and Steketee, 1999). Forthe DA release experiments, in vivo microdialysis (seebelow) was conducted 1, 7, or 30 days after the last ofthe daily injections. Animals did not receive systemicinjections on microdialysis days, but 24 h afterwardsall animals were challenged with cocaine to verify thatthe treatment regimen produced locomotor sensitiza-tion.

DA uptake assay

Sixty-seven animals were used in DA uptake assays:n � 23 for the 1-day withdrawal time point (Saline, 8;Acute Cocaine, 7; Repeated Cocaine, 8); n � 22 for the7-day period (Saline, 7; Acute Cocaine, 8; Repeated

mPFC DOPAMINE AND COCAINE SENSITIZATION 99

Cocaine, 7); n � 22 for the 30-day period (Saline, 8;Acute Cocaine, 7; Repeated Cocaine, 7). Immediatelyafter collection of locomotor activity data, the mPFCwas dissected on ice according to previously describedmethods (Steketee et al., 1998). This dissection in-cluded prelimbic and infralimbic cortices and was me-dial to the forceps minor of the corpus callosum (fmi)and dorsal to the anterior commissure (aca) at the levelof the rhinal fissure (RF). Dissected tissues were ho-mogenized in ice-cold phosphate-buffered sucrose(320 mM) in a glass-Teflon homogenizer, and crudemPFC synaptosomes were prepared as previously de-scribed (Fleckenstein et al., 1997; Williams and Steke-tee, 2004a). After centrifugation of the homogenate(800g, 12 min, 4°C), the supernatant was centrifuged(23,000g, 20 min, 4°C) and the resulting pellet wasresuspended (2 mg/ml wet tissue wt) in ice-cold Kreb’sphosphate buffer containing (in mM): Na2PO4 16, NaCl126, KCl 4.8, CaCl2 1.3, MgSO4 1.4, ascorbic acid 1.0(pH 7.4). Pargyline (1 �M) was included with thisbuffer to prevent the breakdown of DA by monoamineoxidases.

Crude synaptosomes were divided for use in both theDA uptake and in Western blotting assays (see below).Uptake assays were conducted in triplicate tubes hav-ing a final volume of 1 ml Kreb’s phosphate buffer.Nonspecific uptake was determined by incubating syn-aptosomes on ice; total uptake was determined at 37°Cand normalized to protein concentration (Bradford,1976). Because of the considerable degree of functionaland neurochemical heterogeneity of the mPFC, it wasof interest whether withdrawal from repeated cocainealtered the relative potency of transporters for DA,norepinephrine, or serotonin (DAT, NET, or SERT,respectively) for the clearance of DA in this region.Thus, aliquots of crude mPFC synaptosomes were pre-incubated for 10 min on ice or at 37°C in the absence orpresence of the following inhibitors of monoaminetransport: the DAT-selective inhibitor GBR12909(460 nM); the NET-selective inhibitor nisoxetine(2 nM); the SERT-selective inhibitor fluoxetine(560 nM); and the nonselective reuptake inhibitor co-caine (100 nM). These concentrations correspond topreviously determined apparent IC50 values for DAtransport into mPFC synaptosomes (Williams andSteketee, 2004a). After preincubation, uptake was ini-tiated by the addition of 7,8-[3H]DA (45 Ci/mmol, 1 nMfinal concentration; Amersham, Piscataway, NJ) andincubation continued for 5 min. Uptake was termi-nated by placement of assay tubes in an ice bath fol-lowed by addition of ice-cold 320 mM sucrose phos-phate (5 ml). Tubes were decanted over WhatmanGF/B filters presoaked in 0.1% polyethyleneimine andplaced in a vacuum filtration manifold (Millipore, Bed-ford, MA). After additional washes in ice-cold 320 mMsucrose phosphate (2 � 5 ml) radioactivity was mea-sured by liquid scintillation spectrometry.

Western blotting

An aliquot of crude synaptosomes obtained from eachsubject that participated in the DA uptake studies un-derwent a subcellular fractionation procedure (Sando-val et al., 2002) in which they were centrifuged(23,000g, 20 min, 4°C) and the resulting P3 pelletswere lysed by resuspension in ice-cold deionized water(50 mg/ml wet tissue wt). An aliquot of P3 was retainedas the whole synaptosomal fraction; the remainder wasagain centrifuged (23,000g, 20 min, 4°C). The resultingP4 pellets containing plasma membranes were resus-pended in 2% SDS. Both fractions were probe sonicatedon ice and stored at –80°C until Western blotting couldbe performed. Protein concentrations were determinedusing the Micro BCA kit (Pierce, Rockford, IL) withbovine serum albumin (BSA) as the standard.

Samples (50 �g total protein) were diluted in electro-phoresis sample buffer (final concentration � 60 mMTris HCl (pH 6.8), 10% glycerol, 5 mM dithiothreitol,2% SDS, and 0.01% bromophenol blue) and loaded onto10% polyacrylamide gels. After separation by SDS-PAGE proteins were electroblotted to polyvinylidenefluoride (PVDF) membranes and blocked in 3% BSA �5% nonfat dried milk (NFDM; Carnation) in Tris-buff-ered saline with 0.1% Tween-20 (TBST). After rinsingin TBST, blots were incubated in anti-rDAT (1:1,000)or anti-�-actin (1:5,000), rinsed in TBST again andincubated in HRP-conjugated antimouse IgG (1:5,000).All antibodies were diluted in 0.3% BSA � 0.5% NFDMin TBST; blocking and antibody incubations were doneat room temperature for 1 h with mild agitation. Im-munoblots of whole synaptosomal fractions obtainedfrom animals withdrawn from repeated cocaine for 1and 7 days underwent a stripping procedure for 30 min(55°C) in a buffer containing Tris (pH 6.7), 2% SDS and0.7% �-mercaptoethanol; membranes were then re-probed with DAT antibodies. After several rinses inTBST target proteins were visualized by enhancedchemiluminescence (SuperSignal�, Pierce) and ana-lyzed by densitometry using a computer-assisted geldocumentation system (ChemiDoc™, Bio-Rad Labora-tories, Hercules, CA).

6-OHDA lesions

In a separate group of animals, the sensitivity of thetransporter assays described above was determined byinjecting the catecholaminergic neurotoxin 6-hydroxy-dopamine (6-OHDA) into the mPFC. In order to pre-vent lesion-mediated destruction of cortical noradren-ergic terminals, animals were pretreated with thenorepinephrine reuptake inhibitor desmethylimipra-mine (DMI) (25 mg/kg, i.p.) or saline (vehicle; 1 ml/kg,i.p.) 30 min prior to being anesthetized with 3.3 ml/kg(i.p.) Equithesin (9.72 mg/ml sodium pentobarbital and42.5 mg/ml chloral hydrate) according to previouslydescribed methods (Beyer and Steketee, 1999). Approx-imately 5 min after Equithesin administration, ani-

100 J.M. WILLIAMS AND J.D. STEKETEE

mals were placed in a stereotaxic apparatus (DavidKopf, Tujunga, CA) and two sets of bilateral infusions(0.2 �l/min; 1 �l/injection) of 6-OHDA (4 �g free base/�l;prepared fresh immediately prior to surgery) or 20%ascorbic acid in saline (sham control) were injectedwithin the mPFC via 30-gauge injectors connected to aSage syringe pump (Orion Research, Boston, MA). Thecoordinates for the first set of infusions were as follows:A/P, �2.7 and M/L, �0.7 from bregma and D/V, –2.5from dura with a flat skull. Five minutes later thesecond set of infusions were made at the followingcoordinates: A/P, �3.2 and M/L, �0.6 from bregma andD/V, –5.0 from dura with a flat skull (Paxinos andWatson, 1986). Injectors remained in place 5 min aftereach set of bilateral infusions; animals were then su-tured, returned to their homecages, and allowed 7 daysto recover from surgery. Afterwards, animals were sac-rificed and mPFC tissues were rapidly dissected andprepared into crude synaptosomes, which were assayedfor lesion-induced changes in DA uptake and DAT im-munoreactivity as described above. A total of 19 ani-mals [vehicle (veh)/sham, n � 4; DMI/sham, n � 5;veh/6-OHDA, n � 6; DMI/6-OHDA, n � 4] were used inthese studies.

In vivo microdialysis of the mPFC

Forty-nine animals were used in microdialysis exper-iments: n � 17 for the 1-day withdrawal time point(Saline, 7; Cocaine, 10); n � 15 for the 7-day period(Saline, 8; Cocaine, 7); n � 17 for the 30-day period(Saline, 8; Cocaine, 9). Seven days prior to daily cocainepretreatment animals were anesthetized with Equith-esin (3.3 ml/kg, i.p.) and placed in a stereotaxic frame(David Kopf). Guide cannulae (20-gauge, 14 mm) wereimplanted 3 mm above the mPFC at the followingcoordinates: A/P, �3.2 mm and M/L, �0.6 mm frombregma, D/V, –1.5 mm from dura with a flat skull(Paxinos and Watson, 1986). Cannulae were secured tothe skull with three screws and cemented with dentalacrylic. Cannula patency was maintained by insertionof 25-gauge (14 mm) obturators.

Microdialysis was conducted within chambersequipped with counterbalanced lever arms and liquidswivels, which were connected to concentric-style mi-crodialysis probes. The active membrane surface ofthese probes (2 mm) consisted of regenerated celluloseand had a MW cut-off of 5,000 (Spectrum Laboratories,Rancho Dominguez, CA). Animals were placed in mi-crodialysis chambers and probes were inserted intoguide cannulae on the night before dialysis to reducethe influence of damage-induced release of neurotrans-mitters (Westerink and De Vries, 1988); the dialysispump (Harvard Apparatus, Holliston, MA) remainedoff overnight. The next morning the pump was turnedon and probes were continuously perfused (2 �l/min)with dialysis buffer containing (in mM): KCl 2.7;NaCl 140; CaCl2 1.2; MgCl2 1.2; phosphate-buffered

saline 0.2, pH � 7.4. After this 1–2 h equilibrationperiod, baseline samples were collected over 20-minintervals into tubes containing 10 �l of internal stan-dard (30 fmol DHBA in 50 mM HCl). Afterwards, per-fusion with modified dialysis buffers containing 10, 30,and 100 mM KCl (NaCl concentration adjusted to132.7, 112.7, and 47.7 mM, respectively) began. Four20-min samples were collected for each concentrationof KCl, which were switched in ascending order bytemporarily disconnecting probes from the liquid swiv-els and purging the perfusion lines with the replace-ment buffer. Probes were reattached to the liquid swiv-els and sample collection continued once consistentperfusion was reestablished; dialysate flow was inter-rupted for 2–3 min for each buffer replacement. Pre-liminary studies from our laboratory indicated thatbaseline DA values remained stable across the experi-ment despite the momentary interruption of dialysisperfusion (unpubl. obs.). Samples were collected andstored at –80°C until HPLC analysis could be con-ducted (�2 weeks). The day after completion of dialy-sis, animals were tested for the expression of locomotorsensitization as described above.

HPLC analysis of DA

Mobile phase containing 50 mM NaH2PO4, 2 mMdecanesulfonic acid, 0.7 mM EDTA, 11% acetonitrile,and 11% methanol (pH 6.0) was pumped through anoctadecasilane column (2 mm � 15 cm, 3 �m; ESA,Chelmsford, MA) at a flow rate of 0.25 ml/min. Samples(50 �l) were injected with an ESA Model 542 autosam-pler and, following separation of monoamines, wereanalyzed by electrochemical detection (ESA Model5041; guard cell, �400 mV; amperometric cell,�150 mV). Dopamine levels were determined by linearregression from external DA and DHBA standardcurves (1–300 fmol). Under these HPLC conditions, thelimit of detection of DA was 1 fmol/sample.

Histology

Immediately after completion of the locomotor sensi-tization tests (i.e., 24 h after completion of dialysis),subjects were anesthetized with sodium pentobarbital(100 mg/kg, i.p.) and intracardially perfused with0.2 mM PBS followed by 4% formaldehyde. Brainswere quickly removed and placed in 4% formaldehydefor at least 3 days until sectioning (100 �m) on avibratome (Technical Products International, St.Louis, MO). Coronal sections were mounted on gelatin-coated slides and stained with cresyl violet for verifi-cation of probe placement by light microscopy.

Statistics

Measures of DA uptake, expressed as fmol/min/mgprotein, were compared across treatment groups foreach withdrawal period (1, 7, or 30 days), for each

mPFC DOPAMINE AND COCAINE SENSITIZATION 101

assay condition (Total, GBR12909, nisoxetine, fluox-etine, or cocaine), using one-way analysis of variance(ANOVA). Western blot data were obtained as back-ground-subtracted optical densities and normalized to�-actin expression. Normalized values were convertedto percent-of-saline for each gel; two gels were run foreach subcellular fraction (whole synaptosomes, plasmamembrane) at each withdrawal time. These data wereaveraged and compared across treatment groups byone-way ANOVA in a similar manner as the uptakedata. Multiple comparisons of DA uptake and westernblotting data were made using a Student Newman-Keuls test. Microdialysis data were converted to per-cent-of-baseline, defined as the average of last threevalues prior to the replacement of the physiologic(2.7 mM KCl) dialysis buffer with one containing 10mM KCl. Locomotor activity (photocell beam breaks)and microdialysis data collected after each withdrawalperiod were analyzed by two-way (treatment and time)ANOVA with one repeated measure (time). Group dif-ferences in treatment-time interaction were consideredsignificant at P � 0.05. Following two-way ANOVA,post hoc comparisons were made with a modified leastsignificant differences test (Milliken and Johnson,1984).

RESULTSWithdrawal from repeated cocaine and

behavioral sensitization

Figure 1 shows the combined locomotor activity datacollected in animals used in both the DA uptake anddialysis experiments. Since Repeated Cocaine animalsin both studies exhibited a significantly enhanced mo-tor-stimulant response to a cocaine challenge injectionat each withdrawal time, data were combined into onerepresentative figure. Compared to daily saline pre-treatment, subjects that received repeated cocaine in-jections displayed an augmented motor stimulant re-sponse to cocaine challenge (15 mg/kg, i.p.) deliveredafter 1–2 days [Fig. 1A; treatment F(2,37) � 3.459, P �0.042; time F(10,370) � 16.356, P � 0.001; interactionF(20,370) � 3.468, P � 0.001], 7–8 days [Fig. 1B;treatment F(2,34) � 5.714, P � 0.007; time F(10,340) �20.254, P � 0.001; interaction F(20,340) � 4.595, P �0.001] or 30–31 days [Fig. 1C; treatment F(2,36) �

Fig. 1. Expression of behavioral sensitization in animals partici-pating in DA uptake studies and KCl-evoked DA release studies.Animals pretreated with daily cocaine displayed a significantly aug-mented motor stimulant response to cocaine challenge (15 mg/kg, i.p.;arrow) administered after 1–2 (A), 7–8 (B), or 30–31 (C) days ofwithdrawal from repeated cocaine (15 mg/kg/d � 4 d, i.p.), indicatingthat these animals were expressing behavioral sensitization. Data arerepresented as photocell counts (mean � SEM) for each withdrawalperiod. Since the cocaine-pretreated groups from both the uptake andrelease studies exhibited sensitization at each withdrawal time, datawere collapsed, reanalyzed, and collectively presented in the figure.The combined number of subjects in each treatment group for bothstudies is indicated in parentheses. *P � 0.05 compared with Saline;#P � 0.05 compared with Acute cocaine.

102 J.M. WILLIAMS AND J.D. STEKETEE

6.564, P � 0.004; time F(10,360) � 21.920, P � 0.001;interaction F(20,360) � 5.004, P � 0.001] after with-drawal from repeated cocaine injections, indicatingthat behavioral sensitization was expressed in theseanimals. On each of these test days, the sensitizedresponse was statistically significant 45 min after chal-lenge. A potential trend toward a progressive enhance-ment over the time course of withdrawal was apparentin the peak motor-stimulant response to cocaine chal-lenge in animals withdrawn from repeated daily co-caine, which occurred during the first 15 min intervalafter cocaine challenge (Fig. 1). However, subsequentone-way ANOVA of behavioral data from sensitizedanimals revealed no statistically significant increase inbehavioral sensitization occurred over the time courseof withdrawal: F(2,43) � 0.661, P � 0.522 (Fig. 1).Sample sizes for the Saline groups were low, comparedto the other two groups, because this group was notincluded in the KCl-evoked dopamine release studies.

Withdrawal from repeated cocaine andmPFC DA clearance

Measurement of DA uptake within mPFC synapto-somes was done immediately following locomotor datacollection and is displayed in Figure 2. The averagerates of DA transport were not significantly affected byeither acute or repeated cocaine exposure at any of thewithdrawal time points examined. Rates of DA trans-port in saline-pretreated control groups were similarover the time course of withdrawal: 258 � 39 (1 day),194 � 36 (7 days), 328 � 92 (30 days) fmol/min/mgprotein (Fig. 2A–C, respectively). Furthermore, theamount of DA clearance in the presence of previouslydetermined (Williams and Steketee, 2004a) IC50 con-centrations of GBR12909 (GBR), nisoxetine (NSX), flu-oxetine (FLX), or cocaine (COC) did not significantlydiffer among treatment groups at any of the with-drawal time points (Fig. 2A–C). One-way ANOVA ofuptake data revealed that no significant cocaine-in-duced changes in mPFC uptake after 1 day [Fig. 2A;Total F(2,20) � 1.310, P � 0.292; GBR F(2,20) � 0.283,P � 0.757; NSX F(2,20) � 1.452, P � 0.258; FLXF(2,20) � 1.392, P � 0.272; COC F(2,19) � 1.607, P �

Fig. 2. Effects of withdrawal from repeated cocaine (15 mg/kg/d �4 d, i.p.) on DA uptake within crude mPFC synaptosomes. After 1 (A),7 (B), or 30 (C) days of withdrawal from repeated cocaine, and imme-diately following behavioral data collection, animals were sacrificedand DA uptake assays in mPFC synaptosomes were conducted. Spe-cific uptake of [3H]DA was derived by subtracting nonspecific values(determined on ice) from values determined at 37°C without inhibi-tors (Total) or in the presence of 460 nM GBR12909 (GBR), 2 nMnisoxetine (NSX), 560 nM fluoxetine (FLX), and 100 nM cocaine(COC). Assays were conducted in individual subjects (triplicate mea-sures for each inhibitor condition) and averaged; data representmean � SEM rates of DA uptake for each treatment group tested after1, 7, or 30 days withdrawal. The number of subjects in each treatmentgroup is indicated in parentheses. Total specific uptake as well asinhibitor-sensitive uptake in the three treatment groups did not sig-nificantly differ regardless of withdrawal duration.

mPFC DOPAMINE AND COCAINE SENSITIZATION 103

0.227], 7 days [Fig. 2B; Total F(2,19) � 0.102, P �0.904; GBR F(2,19) � 0.141, P � 0.869; NSX F(2,19) �0.082, P � 0.922; FLX F(2,19) � 0.084, P � 0.920; COCF(2,19) � 0.045, P � 0.956] or 30 days [Fig. 2C; TotalF(2,20) � 0.250, P � 0.781; GBR F(2,20) � 0.154, P �0.858; NSX F(2,19) � 0.148, P � 0.863; FLX F(2,20) �0.068, P � 0.935; COC F(2,20) � 0.017, P � 0.983] ofwithdrawal.

The effects of withdrawal from repeated cocaine onmPFC DAT levels as determined by Western blottingare summarized in Table I. Consistent with the overalllack of effects on DA clearance, neither acute nor re-peated cocaine exposure significantly altered DAT lev-els within mPFC whole synaptosomal lysates or plas-malemmal fractions, regardless of withdrawalduration. One-way ANOVA of Western blot data re-vealed no significant cocaine-induced changes in DATimmunoreactivity, regardless of withdrawal duration,in either whole synaptosomal [1 day F(2,17) � 0.725,P � 0.499; 7 days F(2,20) � 0.237, P � 1.550; 30 daysF(2,20) � 0.232, P � 0.795] or plasmalemmal [1 dayF(2,17) � 0.146, P � 0.866; 7 days F(2,17) � 0.239, P �0.790; 30 days F(2,20) � 2.977, P � 0.074] fractions ofthe mPFC (Table I).

Effects of 6-OHDA lesions on DA clearancewithin the mPFC

As shown in Figure 3A, 6-OHDA lesions of the mPFCproduced significant changes in prefrontal cortical DAclearance among the treatment groups [F(3,15) �11.691, P � 0.001]. Post hoc analysis revealed that inlesioned animals that were not protected with the nor-adrenergic reuptake blocker DMI prior to surgery, therate of mPFC DA uptake was significantly attenuatedcompared to vehicle-treated controls [16 � 4 vs. 66 �5 fmol/min/mg protein; P � 0.001]. However, DMI pro-tection in lesioned animals blocked the 6-OHDA-medi-ated attenuation in mPFC DA uptake (Fig. 3A).

After completion of DA uptake assays, an aliquot ofP2 synaptosomes from each subject was lysed and sub-sequently analyzed by Western blotting (Fig. 3B). One-way ANOVA revealed significant lesion-induced differ-ence in DAT immunoreactivity among the four

treatment groups [F(3,15) � 21.111, P � 0.001]. Inter-estingly, sham-operated control animals that receiveda single injection of DMI prior to the bilateral intracra-nial infusions demonstrated a significant elevation[162 � 24% of veh/sham, P � 0.01] in DAT proteinlevels within mPFC synaptosomes. Consistent with theresults of the DA uptake assay, lesioned animals thatwere not pretreated with DMI displayed a significantdecrease [17 � 5% of veh/sham, P � 0.01] in mPFCDAT levels. Not surprisingly, DMI pretreatment wasunable to significantly prevent 6-OHDA-induced lossesin mPFC DAT expression [33 � 17% of veh/sham, P �0.01].

Withdrawal from repeated cocaine and evokedDA release within the mPFC

Figure 4 shows the time course of withdrawal fromrepeated cocaine on evoked DA release within themPFC. Notably, there appeared to be no significantdifference between the baseline levels of DA in cocaine-vs. saline-pretreated animals after 1 day [Fig. 4A; Sa-line, 6.8 � 2.9 vs. Cocaine, 10.8 � 1.8; t � 1.251; d.f. �15; P � 0.230], 7 days [Fig. 4B; Saline, 7.8 � 2.3 vs.Cocaine, 15.9 � 4.2; t � 1.762; d.f. � 13; P � 0.102] or30 days [Fig. 4C; Saline, 7.8 � 2.5 vs. Cocaine, 6.7 �2.7; t � 0.303; d.f. � 15; P � 0.766] of withdrawal.Within the first week of withdrawal from repeatedcocaine, the KCl-stimulated release of DA in the mPFCwas markedly attenuated, an effect that reached sta-tistical significance at the 100 mM KCl concentration(Fig. 4A,B). Two-way repeated-measures ANOVA dem-onstrated that, compared to baseline DA concentra-tions, 1 day of withdrawal from repeated saline injec-tion resulted in a significant increase in 100 mM KCl-evoked DA release in the mPFC; cocaine-pretreatedanimals did not display this effect [Fig. 4A; treatmentF(1,15) � 4.776, P � 0.045; time F(14,210) � 1.882, P �0.030; interaction F(14,210) � 1.795, P � 0.041]. Like-wise, in animals withdrawn for 7 days from repeatedinjection, saline-pretreated controls exhibited a signif-icant increase in mPFC extracellular DA levels in re-sponse to KCl infusion; however, in cocaine-pretreatedsubjects no significant effect of KCl infusion on mPFC

TABLE I. Effects of withdrawal from repeated cocaine administration on mPFC DAT levels

Whole synaptosomesDays of withdrawal Saline Acute cocaine Repeated cocaine

1 day 100 � 7.9 (7) 83.1 � 13.5 (6) 85.4 � 11.1 (7)7 days 100 � 8.7 (8) 83.6 � 8.7 (7) 109.5 � 12.7 (8)

30 days 100 � 10.9 (8) 85.3 � 15.0 (7) 97.8 � 20.4 (8)

Plasma membranesDays of withdrawal Saline Acute cocaine Repeated cocaine

1 day 100 � 13.3 (7) 73.1 � 16.2 (6) 83.4 � 11.3 (7)7 days 100 � 12.3 (8) 86.1 � 14.5 (7) 119.4 � 12.7 (8)

30 days 100 � 10.2 (8) 76.8 � 8.7 (7) 80.9 � 13.5 (8)

Data were obtained as background-subtracted optical densities and normalized to �-actin expression. Values representmean � SEM percent-of-saline for each gel (two gels per fraction for each withdrawal time). The number in parenthesesrepresents the number of subjects analyzed from each treatment group.

104 J.M. WILLIAMS AND J.D. STEKETEE

DA release was observed compared to baseline [Fig.4B; treatment F(1,13) � 2.702, P � 0.124; timeF(14,182) � 2.937, P � 0.001; interaction F(14,182) �1.829, P � 0.037]. However, after 30 days of with-drawal animals pretreated with cocaine or saline ex-hibited similar responses to KCl stimulation, as both

groups displayed significant elevations above baselinein evoked mPFC DA release [Fig. 4C; treatmentF(1,15) � 0.069, P � 0.796; time F(14,210) � 3.771, P �0.001; interaction F(14,210) � 0.617, P � 0.850]. Thus,early withdrawal from repeated cocaine (1 or 7 days)was associated with significant losses in KCl-stimu-lated DA release.

Histological verification of dialysisprobe placement

Figure 5 shows a representative photomicrographobtained from an animal that had undergone in vivomicrodialysis. Typically, the 2 mm active membranesurface of the dialysis probe spanned the prelimbic/infralimbic border within the mPFC and was medial tothe forceps minor of the corpus callosum (fmi). Subjectswith improper probe placement in the mPFC were ex-cluded from statistical analysis.

DISCUSSION

The current studies reveal that, compared to saline-pretreated controls, animals withdrawn from a re-peated cocaine regimen (15 mg/kg/d � 4 d, i.p.) dis-played an augmented motor stimulant response to acocaine challenge injection (15 mg/kg, i.p.) deliveredafter 1–2, 7–8, or 30–31 days of withdrawal, indicatingthat repeated cocaine resulted in the expression ofbehavioral sensitization. During the first week of with-drawal from repeated cocaine, the sensitized behaviorswere associated with a significant loss in the ability ofmesocortical neurons to release DA in response tointra-mPFC KCl infusion (10, 30, or 100 mM). After30 days of withdrawal, the KCl-induced release of DAin the mPFC was similar in both cocaine- and saline-pretreated animals. These effects were the result ofcocaine-mediated reductions in the stimulated releaseof DA, since baseline dialysate levels of DA from themPFC were unchanged after each withdrawal period.The current studies also demonstrated that 1, 7, or30 days of withdrawal from repeated cocaine was notassociated with significant changes in DA clearance orDAT protein expression when compared to saline oracute cocaine control subjects. Taken together, thesedata suggest that early withdrawal from repeated co-caine may be characterized by an initial hyporespon-sivity in mesocortical neurons mediated by a transientattenuation in depolarization-induced DA release thatis restored over more prolonged withdrawal periods.

Sensitization and DA uptake within the mPFC

During early withdrawal from repeated cocaine,mPFC DA overflow elicited by a cocaine challenge in-jection is attenuated in cocaine-sensitized animals(Sorg et al., 1997; Chefer et al., 2000; Williams andSteketee, 2004c). Previous studies have attempted toexplain this effect by showing that basal DA uptake

Fig. 3. Effects of 6-OHDA lesions on DA uptake and DAT proteinlevels in the mPFC. Thirty minutes after i.p. injection of saline (ve-hicle) or desmethylimipramine (DMI), animals received two sets ofbilateral infusions of vehicle (sham) or 6-OHDA (OHDA). Seven dayslater, DA uptake (A) and Western blotting (B) were conducted usingmPFC synaptosomes from these animals. The number of subjects ineach treatment group is indicated in parentheses. A: 6-OHDA lesionssignificantly reduce specific uptake of [3H]DA in animals that werenot pretreated with DMI. Assays were conducted in individual sub-jects (in triplicate); data represent mean � SEM rates of DA uptakefor each treatment group. B: 6-OHDA lesions significantly attenuateDAT levels within the mPFC in vehicle-pretreated and DMI-pre-treated subjects. DMI pretreatment significantly increased mPFCDAT levels in sham-operated animals. Data were obtained as back-ground-subtracted optical densities, normalized to �-actin expressionand represented as mean � SEM percent-of-veh/sham for each of twogels. Representative blots of DAT and �-actin are included below thefigure. **P � 0.01, ***P � 0.001 compared with veh/sham; ##P � 0.01compared with DMI/sham.

mPFC DOPAMINE AND COCAINE SENSITIZATION 105

within the mPFC was enhanced after animals werewithdrawn from repeated cocaine (Meiergerd et al.,1997; Chefer et al., 2000). These studies assessed DAuptake 3–7 days after withdrawal from the daily treat-ment regimen, and animals did not receive a cocainechallenge injection. However, in the current study acocaine challenge was delivered 1, 7, or 30 days afterwithdrawal from repeated cocaine, and DA uptake wasmeasured 2 h later, i.e., immediately after completionof locomotor activity data collection. Additionally, theprevious studies utilized DA concentrations that were100–1,000-fold higher than that used in the currentstudies; however, use of these higher DA concentra-tions in the present mPFC synaptosomal assay re-sulted in significant amounts of nonspecific [3H]DAbinding. Notably, the concentration of DA used in thepresent study has previously been shown to exhibitdose-dependent sensitivity to the inhibitory effects ofGBR12909, nisoxetine, fluoxetine, and cocaine ontransport into mPFC synaptosomes (Williams andSteketee, 2004a). In that study, the relative potencies(IC50 values) of these drugs to inhibit mPFC DA clear-ance were determined for use in the current uptakeexperiments to demonstrate that withdrawal from re-peated cocaine did not significantly alter the proportionof total mPFC DA transport that was GBR12909-,nisoxetine-, or fluoxetine-sensitive. Finally, while re-cent in vitro studies suggest monoamine transportersmay undergo dynamic cell surface regulation in thepresence of psychostimulants (for review, see Blakelyand Bauman, 2000), the present data suggest with-drawal from repeated cocaine was not associated withsignificant changes of DAT protein expression in eitherwhole synaptosomes or the plasma membranes of themPFC. These data, while in contrast to previous find-ings for the NAc and caudate putamen (e.g., Wilson etal., 1994; Letchworth et al., 2001), are consistent withthe currently observed lack of cocaine-mediated effectson DA uptake into mPFC synaptosomes.

It was of potential concern that the present experi-mental methodology was insufficient to detect cocaine-induced changes in DA transport or DAT protein levelsin mPFC synaptosomes. To address this issue, a previ-ously established protocol (Beyer and Steketee, 1999)was used to lesion the mPFC in order to examine thesensitivity of the current uptake assay and Westernblotting conditions. Drug-naı̈ve animals received6-OHDA lesions of the mPFC, and 7 days later mPFCsynaptosomes were prepared from these animals and

Fig. 4. Effects of 1, 7, or 30 days withdrawal from repeated cocaine(15 mg/kg/d � 4 d, i.p.) on KCl-stimulated DA release within themPFC (A, B, or C, respectively). Intra-mPFC infusion of 10, 30, and100 mM KCl (arrows) induced the local release of DA that was sig-nificantly blunted 1 (A) and 7 (B) days after withdrawal from repeateddaily cocaine injections; by 30 days (C), the KCl-evoked response wassimilar in saline- and cocaine-pretreated subjects. *P � 0.05 com-pared with Saline; �P � 0.05 compared with baseline.

106 J.M. WILLIAMS AND J.D. STEKETEE

assayed for lesion-induced changes in DA clearance.Lesion-mediated depletion of DA resulted in a signifi-cant loss of DA uptake into mPFC synaptosomes whenanimals were not pretreated with the NET-selectiveinhibitor DMI, consistent with previous reports sug-gesting that a significant amount of DA clearance inthe mPFC may occur via noradrenergic nerve termi-nals (Carboni et al., 1990; Pozzi et al., 1994; Yamamotoand Novotney, 1998; Moron et al., 2002). Furthermore,the ability of 6-OHDA to significantly diminish DATimmunoreactivity in mPFC synaptosomes suggeststhat the lesion was effective and that the present trans-porter and Western blotting assays were sensitive toexperimental manipulation.

Sensitization and evoked DA releasewithin the mPFC

To investigate changes in the releasability of DAwithin the mPFC, a modified in vivo microdialysis pro-cedure was used in which KCl (10, 30, and 100 mM)was locally infused via the dialysis probe implanted inrats withdrawn for 1, 7, or 30 days from the samerepeated cocaine regimen that was implemented in thecurrent uptake studies. In animals withdrawn fromrepeated cocaine for up to 1 week, intra-mPFC infusionof KCl was unable to evoke DA release significantlyabove baseline; however, saline-pretreated animalswere sensitive to KCl-mediated stimulation of mPFCDA release. In contrast, after 30 days of withdrawal,cocaine- and saline-pretreated animals displayed sim-ilar KCl-evoked DA responses in the mPFC. This sug-gests that during early withdrawal from repeated co-caine administration stimulus-evoked DA release inthe mPFC may be considerably attenuated, but, aswithdrawal duration is extended, neuroadaptive mech-anisms may take place within mesocortical neuronsthat begin to restore DA releasability by subsequentstimuli.

It has previously been shown that local KCl-stimu-lated release of DA is calcium-dependent and tetrodo-toxin-insensitive (Westerink et al., 1989). Thus, theKCl-stimulated extracellular DA sampled from themPFC is likely to represent vesicular DA release thatmay be only partially impulse-dependent. Based inpart on this finding, Sorg et al. (1997) suggested thatthe tolerance of the mPFC DA response to systemiccocaine challenge after withdrawal from repeated co-caine is due primarily to actions of the drug at the levelof the DA cell bodies in the VTA. In support of thishypothesis, and in contrast to the effects of systemiccocaine administration, local infusion of cocaine di-rectly into the mPFC of animals withdrawn from re-peated cocaine resulted in a significant increase inmPFC DA in sensitized vs. nonsensitized animals(Sorg et al., 1997). However, in light of the current datait appears possible that other effects of cocaine may betaking place presynaptically in the mPFC at the lev-

el of the dopaminergic nerve terminals, includingcocaine-mediated changes in the DA synthesis, vesicu-lar storage or autoregulation of release, which havebeen shown to be altered in other mesocorticolimbicand mesostriatal brain areas after repeated adminis-tration of psychostimulants (Trulson and Ulissey,1987; Beitner-Johnson and Nestler, 1991; Brown et al.,2002). Collectively, these changes may at least in partaccount for the observed diminution of KCl-evoked DAreleasability following early withdrawal from repeatedcocaine.

Role of mPFC DA transmission inbehavioral sensitization

In general, studies have shown that, in contrast tothe transient neuroadaptations occurring within theVTA (White, 1996), the NAc DA response to cocainechallenge after pretreatment with psychostimulants isdelayed in appearance and can persist over extendedperiods of withdrawal (Pettit et al., 1990; Kalivas andDuffy, 1993; Parsons and Justice, 1993). However, rel-atively few studies have evaluated cocaine-induced DAresponses within the mPFC, which appear to be atten-uated during early withdrawal from repeated cocaine(Sorg et al., 1997; Chefer et al., 2000; Williams andSteketee, 2004c). In light of these reports, it has beenhypothesized that, in addition to the VTA and NAc, theearly recruitment of the mPFC immediately following

Fig. 5. Representative photomicrograph indicating the typicalplacement of dialysis probes within the mPFC. Coronal sections(100 �m) were stained with cresyl violet to visualize the 2 mm probetract (arrow), which typically spanned the border between prelimbicand infralimbic subregions of the mPFC. Animals with inaccurateprobe placement were excluded from analysis. RF, rhinal fissure; fmi,forceps minor of corpus callosum; aca, anterior commissure. Magnifi-cation �20.

mPFC DOPAMINE AND COCAINE SENSITIZATION 107

(or possibly during) repeated psychomotor stimulantadministration is essential for the behavioral and neu-rochemical manifestations associated with sensitiza-tion (Kalivas and Stewart, 1991; Wolf et al., 1995;Steketee, 2003).

Within the mPFC, DA released from mesocorticalneurons generally attenuates the activity of pyramidalglutamatergic neurons—the primary excitatory effer-ents of the mPFC—either by its direct effects on thesecells or indirectly through stimulation of local -amino-butyric acid (GABA) release from local inhibitory inter-neurons (Sesack and Bunney, 1989; Cowan et al., 1994;Grobin and Deutch, 1998). These glutamatergic pyra-midal neurons comprise the corticotegmental and cor-ticoaccumbal circuits have been implicated in the de-velopment of behavioral sensitization to psychomotorstimulants (Wolf et al., 1995; Reid and Berger, 1996;Pierce et al., 1998; Li et al., 1999) and in the reinstate-ment of cocaine self-administration (Cornish and Kali-vas, 2000; McFarland et al., 2003). Additional supportfor the involvement of the mPFC afferents in the de-velopment of behavioral sensitization to psychostimu-lants comes from previous studies showing that meso-cortical DA modulates spontaneous (Tassin et al.,1978) as well as acute cocaine-induced (Beyer andSteketee, 2000) or amphetamine-induced (Vezina etal., 1991) locomotor activity. This hyperactive sensi-tized state, which is thought to model certain aspects ofpsychomotor stimulant addiction (Robinson and Ber-ridge, 1993; Kalivas et al., 1998), may in part resultfrom a disinhibition of mPFC pyramidal cells caused byattenuation in the ability of mesocortical neurons torelease DA. Furthermore, it has recently been shownthat repeated cocaine administration might also beassociated with an enhancement in cocaine-inducedglutamate, the primary excitatory input to cortical py-ramidal neurons, in the mPFC during the first week ofwithdrawal (Williams and Steketee, 2004b). Collec-tively, the dopaminergic effects of the present study,combined with the recently observed effects on cocaine-induced mPFC glutamate transmission, may be pre-dicted to lead to the enhanced excitatory cortical out-put proposed to be critical for the development ofcocaine-induced sensitization (Steketee, 2003).

CONCLUSIONS

The current studies provide additional support forthe involvement of the mPFC in the development ofcocaine-induced behavioral sensitization. These datasuggest that the attenuation in cocaine-induced mPFCDA transmission that occurs through the first week ofwithdrawal from repeated cocaine administration andthat has been associated with the development of be-havioral sensitization to cocaine (Sorg et al., 1997;Chefer et al., 2000; Williams and Steketee, 2004c) mayresult from a decreased DA releasability rather thanan enhanced DA clearance. This diminution in KCl-

evoked DA transmission within the mPFC may haveimportant consequences for subsequent excitatory out-put from this region to other components of the meso-corticolimbic DA system, and thus may play a consid-erable role in the development of psychomotorstimulant sensitization.

ACKNOWLEDGMENT

Preliminary data from these studies were submittedin abstract form for presentation at the 2004 Society forNeuroscience conference.

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