Journal of Neuroscience Methods 137 (2004) 161–165

Characterization of dopamine transport in crude synaptosomesprepared from rat medial prefrontal cortex

Jason M. Williams∗, Jeffery D. Steketee

Department of Pharmacology, University of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN 38163, USA

Received 25 August 2003; received in revised form 27 January 2004; accepted 13 February 2004

Abstract

Accumulating evidence suggests that dopamine (DA) uptake into mesocortical neurons may be regulated through mechanisms that aremarkedly different from those observed in nigrostriatal or mesoaccumbens systems. The current studies were conducted to develop a rapidand sensitive DA uptake assay in crude synaptosomes prepared from the medial prefrontal cortex (mPFC) of a single animal. Uptake of DAinto the mPFC was saturable, linear with respect to protein concentration, time dependent, and sensitive to the effects of monoamine transportinhibitors. Saturation analysis revealed theKm andVmax values for DA transport in the mPFC were approximately 60 nM and 6.5 pmol/min mgprotein, respectively. A significant amount of DA uptake in the mPFC was more sensitive to inhibition by nisoxetine compared to GBR12909,fluoxetine (FLX), and cocaine (COC), suggesting the norepinephrine transporter (NET) plays an important role in the clearance of DA withinthis region. The described assay conditions would be useful in examining DA uptake within specific brain regions obtained from a singleanimal.© 2004 Elsevier B.V. All rights reserved.

Keywords: Medial prefrontal cortex; Crude synaptosomes; Dopamine uptake; GBR12909; Nisoxetine; Fluoxetine; Cocaine

1. Introduction

The reinforcing effects of cocaine and other psychomo-tor stimulants are closely associated with their ability toenhance dopamine (DA) transmission within the mesocor-ticolimbic DA system, which extends from cell bodies inthe ventral tegmental area (VTA) to terminal regions in thenucleus accumbens (NAcc) and the medial prefrontal cortex(mPFC) (Kuhar et al., 1991; Roberts et al., 1977). Trans-port into the presynaptic terminal is the primary mechanismfor terminating the effects of released DA (Giros et al.,1996). Cocaine acts at the DA transporter (DAT) to inhibitthe reuptake of DA. While cocaine also blocks serotonin(SERT) and norepinephrine (NET) transporters, the affin-ity with which cocaine and other psychomotor stimulantsbind to DAT and inhibit DA transport is directly correlatedto their potency in animal models of reinforcement (Ritzet al., 1987). Identifying the mechanisms that govern the

∗ Corresponding author. Tel.:+1-901-448-4583;fax: +1-901-448-7206.

E-mail address: jmwilliams@utmem.edu (J.M. Williams).

regulation of DA uptake, as well as how these mechanismsare altered by exposure to cocaine, may provide informationthat is crucial for understanding the development of cocainedependence.

The majority of biochemical and pharmacological studiescharacterizing DA transport in rat brain have utilized synap-tosomes obtained primarily from the dorsal striatum, and,to a lesser extent, the NAcc. In contrast, relatively little re-search effort has focused on the mechanisms of DA clearancewithin regions with relatively low DAT levels, such as themPFC. The need for additional investigation into DA uptakemechanisms within the mPFC is underscored by previousstudies suggesting that DA uptake in this region is regulateddifferently compared to the striatum or the Nacc (Cass andGerhardt, 1995; Elsworth et al., 1993; Garris and Wightman,1994; Izenwasser et al., 1990; Moron et al., 2002).

When studying the mechanisms of drug dependence, itis often helpful to pair behavioral and neurochemical dataobtained from the same subject. For extensive pharmaco-logical analysis with brain tissue preparations, considerableworking volumes may be required for assay. Thus, it issometimes necessary for P2 synaptosomal fractions to be

0165-0270/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.jneumeth.2004.02.022

162 J.M. Williams, J.D. Steketee / Journal of Neuroscience Methods 137 (2004) 161–165

prepared from pooled tissues dissected from several dif-ferent animals. Pooling of tissue is not uncommon forstructures such as the striatum that contain dense DA in-nervation, and may be considered necessary in the mPFC,which contain relatively fewer DA uptake sites. Due to thepaucity of information regarding the role of DA clearancemechanisms within the mPFC in the actions of psychomo-tor stimulants, the objective of the current studies was todevelop an assay for examining DA transport within themPFC obtained from a single animal, which would per-mit the pairing of individual behavioral and neurochemicalfindings.

2. Materials and methods

2.1. Subjects

Male Sprague-Dawley rats (250–300 g) were grouphoused in a temperature and humidity controlled facilityand kept on a 12 h light–dark cycle with food and waterfreely available. All animal protocols were carried out inadherence to the National Institutes of Health Guide for theCare and Use of Laboratory Animals and were approvedby the University of Tennessee Animal Care and UseCommittee.

2.2. Tissue preparation

For each experiment, DA uptake was assessed in crudesynaptosomal suspensions from mPFC tissue pooled fromsix to eight rats. Subjects were sacrificed by decapitation,and their brains were quickly removed, rinsed in ice-coldphosphate-buffered saline and placed upside down on anice-cold glass plate. The mPFC was gross dissected as pre-viously described (Steketee et al., 1998). The average wetweight of this dissection obtained from a single rat was44.3± 1.1 mg. Tissue samples were homogenized on ice in20 vol.% ice-cold phosphate-buffered sucrose (0.32 M) in aglass-Teflon homogenizer (20 up-and-down passes). The ho-mogenate was centrifuged at 800×g (3,200 rpm, Eppendorf5415) for 12 min, 4◦C. The supernatant was centrifuged at23,000×g (15,500 rpm, Sorvall Super T21) for 20 min, 4◦C.The resulting P2 synaptosomal pellet was resuspended inice-cold Kreb’s phosphate buffer, which contained the fol-lowing: (in mM) Na2PO4 16, NaCl 126, KCl 4.8, CaCl21.3, MgSO4 1.4, and ascorbic acid 1.0 (pH 7.4). A prelimi-nary study showed that uptake of 1 nM [3H] DA into crudemPFC synaptosomes was dose-dependently increased in thepresence of 1 nM–1�M pargyline, while higher concentra-tions (10�M–10 mM) dose-dependently attenuated uptake(data not shown). Thus, subsequent experiments included1�M pargyline in the assay buffer to prevent dopaminedegradation by monoamine oxidases. Protein concentrationsfrom synaptosomal preparations were determined using themethod ofBradford (1976).

2.3. [3H] DA uptake assay

Each experiment, run in triplicate, was repeated three orfour times using fresh tissue. Assays were carried out in afinal volume of 1 ml Kreb’s phosphate buffer. Nonspecifictransport and adsorption of DA were determined by run-ning parallel assays at 0◦C (i.e. ice bath), a temperature atwhich monoamine transporters are thought to be inactive.Specific DA uptake was indirectly derived by subtractingthe nonspecific uptake from the total uptake determined at37◦C. Thus, total [3H] DA transport was comprised of spe-cific and nonspecific components. P2 synaptosomes wereresuspended in Kreb’s phosphate buffer at a final tissue con-centration of 2 mg/ml wet weight, except in tissue-responseexperiments in which the final tissue concentration variedfrom 0.5 to 5 mg/ml wet weight. Samples underwent a10 min preincubation either at 37◦C in a shaking water bath(100 rpm) or on ice. For functional assessment of DA uptakein the mPFC, GBR12909, nisoxetine, fluoxetine (FLX), andcocaine (COC) (Sigma Chemical Co. St. Louis, MO, USA)were included in the preincubation step (final concentration10−13 to 10−4 M). Uptake was initiated by the additionof 7,8-[3H] DA (45 Ci/mmol; Amersham, Piscataway, NJ,USA). The final concentration of DA was 1 nM except forsaturation analysis in which the final concentration variedfrom 0.1 to 100 nM in half-log unit increments. Incubationsproceeded at 37◦C for 5 min, except in time-course experi-ments in which incubation times ranged from 2 to 30 min in2 min intervals. Uptake was terminated by immediate place-ment of assay tubes into an ice bath, followed by additionof 5 ml ice-cold 0.32 M sucrose phosphate. Assay contentswere rapidly poured over Whatman GF/B filters presoakedin 0.1% (v/v) polyethyleneimine to reduce nonspecificbinding, which were placed in a vacuum filtration manifold(Millipore, Bedford, MA, USA). Filters were washed twice(5 ml each) in ice-cold 0.32 M sucrose phosphate. Boundradioactivity was measured using a Beckman LS5000TDliquid scintillation counter after addition of 6 ml scintillationcocktail (ScintiSafe Plus, Fisher Scientific, Pittsburgh, PA,USA).

2.4. Data analysis

The mean of triplicate determinations (obtained as dpm)was considered a single data point, and each experimentwas replicated three-to-four times. Dpm were convertedto rates of total, specific, and nonspecific DA uptake,which were analyzed using GraphPad Prism (v3.0; Graph-Pad, San Diego, CA, USA). Linear regression was usedto analyze tissue response data, early portions of timecourse data and lower portions of DA concentration re-sponse data. Non-linear least-squares curve fitting withPrism was used to estimateKm and Vmax values fromthe full DA concentration response data and IC50 valuesand Hill coefficients from inhibitor concentration responsedata.

J.M. Williams, J.D. Steketee / Journal of Neuroscience Methods 137 (2004) 161–165 163

3. Results

Assay conditions for DA uptake into crude synaptosomesprepared from the mPFC were optimized for the amount oftissue (i.e. protein) present, incubation time, the final con-centration of [3H] DA and effects of uptake inhibitors onmPFC uptake processes. As shown inFig. 1, incubation ofmPFC synaptosomes for 5 min in the presence of 1 nM [3H]DA produced uptake rates that were linear with respect totissue level from 0.5 to 5 mg/ml wet weight (r2 = 0.8689),which corresponded to approximately 5–50�g of protein.

Fig. 2 shows the time course of [3H] DA uptake intocrude mPFC synaptosomes. The complete time course of up-take (2–30 min) of 1 nM [3H] DA into mPFC synaptosomes(2 mg/ml wet weight) was best fit by a rectangular hyper-bolic function (R2 = 0.8168) that approached asymptote at3.19 ± 0.54 pmol/mg protein. Uptake of DA at early timepoints (2–8 min, inset) increased linearly with time (r2 =0.8953). At later time points uptake of 1 nM [3H] DA be-gan to plateau, reaching a half-saturation time of approxi-mately 26 min. A 5 min incubation, which was in the lin-ear portion of the curve, was utilized in all subsequent DAuptake assays. A second set of experiments conducted toassess the time course of uptake of 100 nM [3H] DA intomPFC synaptosomes revealed that the uptake of this higherconcentration was similarly hyperbolic (R2 = 0.8455) andapproached asymptote at 62.1 ± 4.6 pmol/mg protein (datanot shown). Analyses of both time course studies showedthat nonspecific interactions of the radiolabel within the

Fig. 1. Tissue response for [3H] DA uptake in the rat mPFC. Aftera 10 min preincubation period, crude synaptosomes (0.5–5 mg/ml wetweight) were incubated in the presence of 1 nM [3H] DA for 5 min.Specific transporter-mediated uptake (closed circles) of [3H] DA wasderived indirectly by subtracting nonspecific values (open circles), whichwere determined by incubation of parallel sets of tubes on ice for eachtissue concentration, from the total uptake (closed triangles) measuredat 37◦C. As shown in the figure, specific uptake of [3H] DA increasedlinearly over this range of tissue concentrations. Data represent the meanof three independent experiments each conducted in triplicate.

Fig. 2. Time course of [3H] DA uptake into crude synaptosomes preparedfrom rat mPFC. After a 10 min preincubation period, P2 synaptosomes(2 mg/ml wet weight) were incubated in the presence of 1 nM [3H] DA for2–30 min at 2 min intervals. Specific transporter-mediated uptake (closedcircles) of [3H] DA was derived indirectly by subtracting nonspecificvalues (open circles), which were determined by incubation of parallel setsof tubes on ice for each incubation interval, from the total uptake (closedtriangles) measured at 37◦C. As shown in the figure, specific uptakeof [3H] DA increased in a nearly linear fashion within the first 10 min(inset), but began to plateau when incubated for longer time periods. Datarepresent the mean of three independent experiments each conducted intriplicate.

assay constituted approximately 32 and 80% of the observedtotal transport of 1 and 100 nM [3H] DA, respectively.

Saturation analysis for [3H] DA transport in the mPFCis shown inFig. 3. Specific transport of DA (0.1–100 nM)into crude mPFC synaptosomes (2 mg/ml wet weight, 5 minincubation) occurred via a concentration-dependent processaccording to Michaelis Menton kinetics assuming a singleuptake site (R2 = 0.9283). TheKm andVmax values were58.46± 3.37 nM and 6.53± 1.34 pmol/min mg protein, re-spectively. Uptake of DA into P2 synaptosomes was linearfrom 0.1 to 10 nM (r2 = 0.9228, inset); in subsequent ex-periments a final concentration of 1 nM [3H] DA was used.

Fig. 4demonstrates the effects of inhibitors of monoaminetransport on total specific DA uptake into the mPFC.GBR12909, nisoxetine, fluoxetine or cocaine (10−13 to10−4 M) was preincubated for 10 min at 37◦C in the pres-ence of crude synaptosomes of the mPFC (2 mg/ml wetweight). Uptake was assessed by 5 min incubation with[3H] DA (1 nM final concentration) in order to determinethe relative contribution of DAT, NET, and SERT to totalDA uptake in the mPFC. Hill coefficients for the inhi-bition of [3H] DA uptake by GBR12909 or nisoxetinewere −0.275± 0.034 (R2 = 0.9964) or−0.421± 0.055(R2 = 0.9984), respectively. Interestingly, the lowest con-centration of GBR12909 (0.1 pM) inhibited DA uptake intomPFC synaptosomes by approximately 16%. Fluoxetine-or cocaine-mediated inhibition of [3H] DA uptake yielded

164 J.M. Williams, J.D. Steketee / Journal of Neuroscience Methods 137 (2004) 161–165

Fig. 3. Concentration response analyses for [3H] DA uptake in the ratmPFC. After a 10 min preincubation period, crude synaptosomes (2 mg/mlwet weight) were incubated in the presence of 0.1–100 nM [3H] DA for5 min. Specific transporter-mediated uptake (closed circles) of [3H] DAwas derived indirectly by subtracting nonspecific values (open circles),which were determined by incubation of parallel sets of tubes on ice foreach concentration of [3H] DA, from the total uptake (closed triangles)measured at 37◦C. As shown in the figure, the concentration-dependentincrease in specific uptake of [3H] DA (0.1–10 nM) into crude mPFCsynaptosomes was nearly linear (inset), while higher concentrations beganto saturate DA uptake sites. Data represent the mean of three independentexperiments each conducted in triplicate.

Fig. 4. Concentration-dependent effects of DAT, NET, and SERT inhibitorson [3H] DA uptake in the rat mPFC. Transport of 1 nM [3H] DA intomPFC P2 synaptosomes (2 mg/ml wet weight) was determined for 5 minat 37◦C in the presence of 10−13 to 10−4 M GBR12909, nisoxetine,fluoxetine or cocaine. For each inhibitor curve, nonspecific uptake (averageof triplicate on ice determinations) was subtracted from total uptake(measured at 37◦C) to indirectly yield specific rates of [3H] DA uptakeat each inhibitor concentration. Data are expressed as percent-of-control,which was defined as the specific uptake of [3H] DA in the absenceof any inhibitors, and represent the mean of three-to-four independentexperiments each conducted in triplicate.

Hill coefficients of −0.478 ± 0.069 (R2 = 0.9951) or−0.641± 0.173 (R2 = 0.9937), respectively. Estimated ap-parent IC50 values for each of the uptake inhibitors were asfollows: GBR12909= 467.7 nM, nisoxetine= 2.072 nM,fluoxetine= 567.5 nM, and cocaine= 96.06 nM.

4. Discussion

The purpose of the present studies was to develop an assayto analyze DA uptake within brain regions containing lowlevels of DAT, specifically the mPFC, in a single subject exvivo. The current assay was modified from previous methodsdeveloped for the striatum (Fleckenstein et al., 1997) withseveral issues under consideration, including abundance ofuptake sites, time course of uptake, concentration of sub-strate, and maintenance of function. Uptake of DA in themPFC was sufficiently optimized when P2 synaptosomes(2 mg/ml wet weight) were incubated at 37◦C for 5 min inthe presence of 1 nM [3H] DA.

Specific uptake of DA increased linearly at tissue concen-trations from 0.5 to 5 mg/ml wet weight. Subsequent assayswere conducted using 2 mg/ml wet weight to ensure suffi-cient working volume to assess the effects of monoamineuptake inhibitors on uptake. Based on the time course andDA concentration response data, a 5 min incubation of [3H]DA (1 nM final concentration) was utilized for subsequentuptake experiments since these values were well within theobserved linear range of the assay. TheKm for DA transportinto mPFC in the current assay was approximately 60 nM,a value comparable to that previously determined in brainslice preparations of the mPFC (Izenwasser et al., 1990). Inother studies using similar uptake conditions in crude synap-tosomes from rat striata theKm values ranged from 90 nMwhen the DA concentration was 1 nM (Vaughan et al., 1997)to 250 nM at a DA concentration of 10 nM (Peris et al.,1990). The current estimates ofVmax for DA transport in themPFC are also generally consistent with those previouslyreported for this region (Chefer et al., 2000; Elsworth et al.,1993). Thus, the present kinetic measures in mPFC synapto-somes are similar to previously reported values and suggestthat functional changes in DA transport within this regioncan be examined using the current assay.

Once assay conditions were optimized, the functionalityof the mPFC transport complex was assessed by determiningIC50 values for the inhibition of DA uptake using GBR12909(DAT), nisoxetine (NET), fluoxetine (SERT), and cocaine(nonselective). Nisoxetine was the most potent inhibitor ofDA uptake with an IC50 of 2.072 nM, similar to previouslyreported values (Izenwasser et al., 1990), further illustrat-ing the potential importance of NET-mediated DA clear-ance in the mPFC (Carboni et al., 1990; Moron et al., 2002;Yamamoto and Novotney, 1998). Both GBR12909 (IC50 =467.7 nM) and fluoxetine (IC50 = 567.5 nM) showed lowerpotencies for inhibiting DA uptake compared to nisoxe-tine. The effects of fluoxetine were consistent with previous

J.M. Williams, J.D. Steketee / Journal of Neuroscience Methods 137 (2004) 161–165 165

findings, thus suggesting a limited role for serotonergic sys-tems in the clearance of DA within the mPFC (Moron et al.,2002; Pozzi et al., 1999). Previous studies on DA uptake intocrude synaptosomes from the prefrontal cortex suggest thatthe IC50 for GBR12909 was in the low nM range (1.9 nM)(Elsworth et al., 1993). Differences in methodology couldaccount for the discrepancy in IC50 values for GBR12909.First, the previous study used a concentration range of 10−9

to 10−5 M GBR12909 to inhibit the uptake of DA whereasthe present study used a range of 10−13 to 10−4 M (Elsworthet al., 1993). Given that there was some inhibition of uptakeat the lowest concentrations of GBR12909 tested, using anexpanded range of concentrations may have influenced thecalculation of the IC50 value. Second, the previous studiesexamined the effects of GBR12909 in the presence of theNET inhibitor desipramine whereas the present study did notinclude NET inhibitor in the presence of the DAT inhibitor(Elsworth et al., 1993). Inclusion of desipramine in the assayconditions may have influenced the capacity of GBR12909to inhibit DA uptake into synaptosomes. In addition to theselective inhibitors, cocaine was shown to possess a modestpotency for inhibiting DA transport in the mPFC as demon-strated by previous reports (Elsworth et al., 1993; Izenwasseret al., 1990). Taken together the data support previous stud-ies that suggest NET is the primary carrier responsible forclearance of DA in the mPFC.

In summary, a new method was presented for the studyof DA transport in discrete brain regions with low levels ofDAT. Parameters were optimized specifically for use withmPFC tissue obtained from an individual rat, which mayenable direct comparisons between behavioral and neuro-chemical measures within the same subject.

Acknowledgements

The authors wish to extend special thanks to Drs. An-nette E. Fleckenstein and James M. O’Donnell for their con-structive comments and fruitful discussions regarding thismanuscript. This work was supported by a grant from theNational Institute on Drug Abuse (DA13470, J.D.S.).

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