Address correspondence and reprint requests to Linda P. Dwoskin, PhD, College of Pharmacy, University of Kentucky, 725 Rose Street, Lexington, KY 40536–0082, USA. E-mail: firstname.lastname@example.org
Rats raised in an enriched environmental condition (EC) exhibit a decreased (35%) maximal velocity (Vmax) of [3H]dopamine (DA) uptake in medial prefrontal cortex (mPFC) compared with rats raised in an impoverished condition (IC); however, no differences between EC and IC groups in Vmax for [3H]DA uptake were found in nucleus accumbens and striatum. Using biotinylation and immunoblotting techniques, the present study examined whether the brain region-specific decrease in DA transporter (DAT) function is the result of a reduction in DAT cell surface expression. In mPFC, nucleus accumbens and striatum, total DAT immunoreactivity was not different between EC and IC groups. Whereas no differences in cell surface expression of DAT were found in nucleus accumbens and striatum, DAT immunoreactivity in the biotinylated cell surface fraction of mPFC was decreased (39%) in EC compared with IC rats, consistent with the magnitude of the previously observed decrease in Vmax for [3H]DA uptake in mPFC in EC rats. These results suggest that the decrease in DAT cell surface expression in the mPFC may be responsible for decreased DAT function in the mPFC of EC compared with IC rats, and that there is plasticity in the regulatory mechanisms mediating DAT trafficking and function.
Environmental enrichment during development alters DAT function in a brain-region specific manner (Zhu et al. 2004). The Vmax for [3H]DA uptake in medial prefrontal cortex (mPFC) from EC rats was decreased by 35% compared with that in IC rats, with no differences between EC and IC rats for nucleus accumbens and striatum. Furthermore, no differences between EC and IC rats were found in [3H]GBR12935 binding density. Thus, decreases in mPFC DAT function were observed in environmentally enriched rats compared with impoverished rats, whereas no difference in the total number of DAT sites was found. The latter findings may be explained by a reduction in cell surface expression of DAT protein in EC compared with IC rats.
The present study employed cell surface biotinylation followed by western blot analysis to determine total DAT immunoreactivity, intracellular DAT and cell surface expression of DAT in the mPFC, nucleus accumbens and striatum from EC and IC rats. An antibody recognizing the C-terminal of DAT was used to detect DAT protein in mPFC, as this antibody was used previously to detect the transporter in plasma membrane and intracellular fractions from accumbens and striatum (Salvatore et al. 2003). To validate the use of this DAT antibody in mPFC, preliminary experiments were also conducted in which 6-hydroxydopamine (6-OHDA) was used to lesion mPFC dopaminergic innervation in rats raised in standard housing conditions.
Materials and methods
Antibodies recognizing rat DAT (goat polyclonal antibody), calnexin (rabbit polyclonal antibody) and protein phosphatase 2A (PP2A; mouse monoclonal antibody) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-syntaxin 1A antibody (HPC-1; mouse monoclonal antibody) was purchased from Sigma Chemical Co. (St Louis, MO, USA). Horseradish peroxidase-conjugated goat anti-rabbit antibody was purchased from Bio-Rad (Hercules, CA, USA). Sulfosuccinimidobiotin (sulfo-NHS-biotin) and immunoPure immobilized monomeric avidin gel were purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). Desipramine hydrochloride, 6-OHDA and nomifensine maleate were purchased from Sigma-Aldrich Co. Paroxetine hydrochloride was a generous gift from Beecham Pharmaceuticals (Epsom, Surrey, UK). [3H]DA (3,4-ethyl-2 [N-3H] dihydroxyphenylethylamine; specific activity 31 Ci/mmol) was purchased from New England Nuclear (Boston, MA, USA). d-Glucose was purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI, USA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA).
Male Sprague–Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN, USA), and were housed with free access to food and water in a colony room in the Division of Laboratory Animal Resources at the University of Kentucky. For 6-OHDA lesion experiments, rats (200–250 g bodyweight) were housed two rats per cage in standard polycarbonate cages (47 cm long × 26 cm wide × 20 cm high). To determine the effect of environmental enrichment, rats at 21 days of age were housed in one of two rearing conditions as described below. All rats were maintained on a 12-h light–dark cycle with lights on at 07.00 hours. Animal handling procedures were approved by the Institutional Animal Care and Use Committee at the University of Kentucky and were performed in accordance with the 1996 version of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Upon arrival, rats were randomly assigned to either the EC or IC group. EC rats were group housed (8–12 rats per cage) in a large metal cage (120 cm long × 60 cm wide × 45 cm high), with 14 hard non-chewable plastic objects placed randomly in the cage. Each day, these rats were handled while one-half of the objects were replaced with new plastic objects, and the remaining plastic objects were rearranged to enhance novelty. In contrast, IC rats were housed individually in wire mesh hanging cages (25 cm long × 18 cm wide × 17 cm high) with solid metal sides and walls, and were not handled. Rats were maintained in their respective home environments until 53–55 days of age when the neurochemical assays were conducted.
6-OHDA lesion of mPFC DA innervation
To validate the selectivity of DAT immunoreactivity in mPFC, a dopaminergic terminal innervation lesion was made by bilateral infusion of 6-OHDA into mPFC using a method described previously (King and Finlay 1995). Rats housed under standard conditions were assigned randomly to either 6-OHDA or vehicle treatment groups. To prevent uptake of 6-OHDA into noradrenergic nerve terminals in mPFC, rats were pretreated with desipramine hydrochloride (free base, 25 mg/kg i.p.) 30 min before 6-OHDA or vehicle injection. Immediately after injection of desipramine, rats were anesthetized with chloral hydrate (400 mg/kg i.p.) and placed into a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). Body temperature was maintained at 37°C by a heating pad coupled to a rectal thermometer (Harvard Apparatus, Holliston, MA, USA). An injection needle (26 G) attached to a 5-µL syringe (Hamilton Co., Reno, NV, USA) was filled with either 6-OHDA or vehicle and positioned in the mPFC (coordinates: Anteroposterior + 2.9 mm, Mediolateral ± 1.0 mm from bregma, Dorsoventral – 3.3 mm from dura; Paxinos and Watson 1996). The injection needle remained in position for 5 min before infusion of 6-OHDA (1 µg per 2 µL) or vehicle (2 µL of 0.9% NaCl containing 0.03%l-ascorbic acid), which was infused over a 5-min period using a minipump (Model-310; Stoelting Co., Wood Dale, IL, USA). The injection needle remained in position for an additional 5 min after infusion to allow for diffusion into the surrounding tissue. Doses of desipramine and 6-OHDA were chosen based on published results showing a significant 79% DA depletion in mPFC, without a significant loss of norepinephrine content (King and Finlay 1995). After recovery from anesthesia, rats were returned to their home cages in the colony room where they remained for 10 days. Subsequently, rats from 6-OHDA- and vehicle-treated groups were rapidly decapitated, and brains quickly dissected to obtain mPFC, nucleus accumbens and striatum. Synaptosomes for each brain region were prepared from the same groups of rats to determine [3H]DA uptake and amount of DAT immunoreactivity in parallel assays.
Synaptosomal [3H]DA uptake
To determine the extent of the 6-OHDA lesion, [3H]DA uptake assays were conducted using a method published previously (Zhu et al. 2004). The mPFC consisted of anterior cingulate cortex and prelimbic/infralimbic cortex (Deutch 1993). Tissue for each brain region (mPFC, striatum and nucleus accumbens) was pooled from a group of five rats (constituting a single sample for each region), which was used to conduct each independent experiment (n = 1). Thus, ‘n’ refers to the number of independent experiments conducted, rather than the number of rats used.
Brain regions were immediately homogenized in 20 mL ice-cold sucrose solution (0.32 m sucrose and 5 mm sodium bicarbonate, pH 7.4) with 16 passes of a Teflon pestle homogenizer (clearance 0.015 in). Homogenates were centrifuged at 2000 g for 10 min at 4°C and resulting supernatants were centrifuged at 20 000 g for 15 min at 4°C. Resulting pellets were resuspended in 2.4 mL ice-cold assay buffer (125 mm NaCl, 5 mm KCl, 1.5 mm MgSO4, 1.25 mm CaCl2, 1.5 mm KH2PO4, 10 mm glucose, 25 mm HEPES, 0.1 mm EDTA, 0.1 mm pargyline and 0.1 mm l-ascorbic acid, saturated with 95% O2/5% CO2, pH 7.4) to obtain synaptosomal suspensions. For assays using mPFC, [3H]DA uptake was determined using buffer that also contained desipramine (1 µm) and paroxetine (5 nm) to prevent [3H]DA uptake into norepinephrine- and serotonin-containing nerve terminals respectively (Masserano et al. 1994). Synaptosomal suspensions of mPFC contained ∼200 µg protein per 100 µL. Striatal and accumbal synaptosomal suspensions contained ∼20 µg protein per 30 µL. Synaptosomes were incubated for 5 min at 34°C. Subsequently, 0.1 µm[3H]DA (final concentration) was added to each assay tube. The total assay volume was 250 µL for mPFC, and 500 µL for nucleus accumbens and striatum. Non-specific [3H]DA uptake was determined in the presence of 10 µm nomifensine. Incubation continued for 10 min at 34°C and was terminated by the addition of 3 mL ice-cold assay buffer containing pyrocatechol (1 mm), followed by immediate filtration through Whatman GF/B glass fiber filters (presoaked with 1 mm pyrocatechol for 3 h). Filters were washed three times with 3 mL ice-cold buffer containing pyrocatechol using a Brandel cell harvester (Model MP-43RS; Biochemical Research and Development Laboratories Inc., Gaithersburg, MD, USA). Radioactivity was determined by liquid scintillation spectrometry (Model B1600TR; Perkin-Elmer Life Sciences, Downers Grove, IL, USA). Protein concentrations were determined using the Bradford protein assay, with bovine serum albumin as the standard (Bradford 1976).
Cell surface expression of DAT
Synaptosomal preparations of mPFC, nucleus accumbens and striatum were obtained as described above to determine total DAT immunoreactivity, intracellular DAT protein and cell surface DAT expression. The use of the impermeant biotinylation reagent sulfo-NHS-biotin for isolation of plasma membrane-associated proteins in brain synaptosomes is well established (Vaughan et al. 1997; Taubenblatt et al. 1999; Salvatore et al. 2003). In the present study, sulfo-NHS-biotin was used to isolate plasma membrane-associated DAT protein prepared from mPFC, nucleus accumbens and striatal synaptosomes; DAT protein was subsequently identified by immunoreactivity using human DAT antibody.
Owing to the lower density of DAT in the mPFC compared with the nucleus accumbens and striatum, larger amounts of mPFC protein (2500 µg) than of accumbal (1000 µg) and striatal (500 µg) protein were used to determine NHS-biotin-labeled cell surface DAT. Samples of synaptosomes containing the respective amounts of total protein were incubated for 1 h at 4°C with continual shaking in 500 µL 1.5 mg/mL sulfo-NHS-biotin in PBS/Ca/Mg buffer (138 mm NaCl, 2.7 mm KCl, 1.5 mm KH2PO4, 9.6 mm Na2HPO4, 1 mm MgCl2, 0.1 mm CaCl2, pH 7.3). After incubation, samples were centrifuged at 8000 g for 4 min at 4°C. To remove the free biotinylation reagent, the resulting pellet was resuspended in 1 mL ice-cold 100 mm glycine in PBS/Ca/Mg buffer, and centrifuged at 8000 g for 4 min at 4°C. The resuspension and centrifugation steps were repeated. Final pellets were resuspended in 1 mL ice-cold 100 mm glycine in PBS/Ca/Mg buffer and incubated with continuous shaking for 30 min at 4°C. Subsequently, the samples were centrifuged at 8000 g for 4 min at 4°C, and the resulting pellets were resuspended in 1 mL ice-cold PBS/Ca/Mg buffer and centrifuged again. The resuspension and centrifugation steps were repeated twice more. The final pellet was lysed by sonication for 2–4 s in 300 µL Triton X-100 buffer (10 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1.0% Triton X-100, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µm pepstatin, 250 µm phenylmethylsulfonyl fluoride) followed by incubation and continuous shaking for 20 min at 4°C. Lysates (300 µL) were centrifuged at 21 000 g for 20 min at 4°C. The pellet was discarded, and 100 µL of the supernatant was stored at − 20°C for determination of total immunoreactive DAT.
The remaining supernatant was incubated with continuous shaking in the presence of monomeric avidin beads in Triton X-100 buffer (100 µL/tube) for 1 h at room temperature (22 ± 1°C). Samples were then centrifuged at 17 000 g for 4 min at 4°C and supernatants (containing non-biotinylated, intracellular protein) were stored at − 20°C. The efficiency of avidin in isolating the biotinylated and non-biotinylated proteins across the protein range in the tissue extract was verified using avidin-conjugated antibody, which showed that biotinylated proteins were completely absorbed by the avidin and were not present in the supernatant. The resulting pellets containing the avidin-absorbed biotinylated (cell surface) proteins were resuspended in 1 mL 1.0% Triton X-100 buffer and centrifuged at 17 000 g for 4 min at 4°C. The pellet was resuspended and centrifuged twice more. The final pellet consisted of the biotinylated proteins adsorbed to monomeric avidin beads. The biotinylated proteins were eluted by incubating with 50 µL Laemmli buffer (62.5 mm Tris-HCl, 20% glycerol, 2% sodium dodecyl sulfate (SDS), 0.05%β-mercaptoethanol and 0.05% bromophenol blue, pH 6.8) for 20 min at room temperature. The samples were stored at − 20°C.
To obtain the immunoreactive DAT protein in the total, intracellular and cell surface fractions, samples were thawed and subjected to gel electrophoresis and western blotting as described previously (Melikian et al. 1994; Salvatore et al. 2003). Briefly, proteins were separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) for 90 min at 150 V, and transferred to Immobilon-P transfer membranes (0.45 µm pore size; Millipore Co., Bedford, MA, USA) in transfer buffer (50 mm Tris, 250 mm glycine, 3.5 mm SDS) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) for 110 min at 72 V. The membranes were incubated with blocking buffer (5% milk powder in PBS containing 0.5% Tween 20) for 1 h at room temperature, followed by incubation with goat polyclonal DAT antibody (1 µg/mL in blocking buffer) overnight at 4°C. Transfer membranes were washed five times with wash buffer (PBS containing 0.5% Tween 20) at room temperature and then incubated with rabbit anti-goat antibody (1 : 2500 dilution in blocking buffer) for 1 h at 22°C. Membranes were then washed and incubated with peroxidase-conjugated goat anti-rabbit antibody (diluted 1 : 5000) for 1 h at 22°C. Immunoreactive proteins on the transfer membranes were detected using enhanced chemiluminescence and developed on Hyperfilm (ECL-plus; Amersham Biosciences UK Ltd, Little Chalfont, UK). After detection and quantification of DAT protein, each blot was stripped using Tris buffer (62.5 mm Tris-HCl with 2% SDS and 100 mmβ-mercaptoethanol, pH 6.8) and reprobed for detection of syntaxin 1A, calnexin and PP2A. Amounts of syntaxin 1A and of calnexin, a plasma membrane resident protein and an endoplasmic reticular protein respectively, were determined using mouse monoclonal syntaxin 1A antibody (1 : 5000) and rabbit polyclonal calnexin antibody (1 : 5000) to monitor protein loading either between the 6-OHDA- and vehicle-treated groups, or between EC and IC groups. PP2A, an intracellular protein, served as a control to monitor the efficiency of the biotinylation of cell surface proteins and was determined using mouse monoclonal PP2A antibody (1 : 500). Multiple autoradiographs were obtained using different exposure times, and immunoreactive bands within the linear range of detection were quantified by densitometric scanning using Scion image software (Scion Corp., Frederick, MD, USA). Band density measurements, expressed as relative optical density, were used to calculate levels of DAT in total, non-biotinylated and biotinylated fractions. Specifically, total DAT levels were calculated based on the density of DAT-immunoreactive bands in an aliquot of synaptosomal extract multiplied by the total volume of extract and divided by the total volume of synaptosomal extract subjected to SDS–PAGE. DAT levels in the non-biotinylated fractions were calculated as the density of DAT-immunoreactive bands in an aliquot of supernatant after avidin incubation multiplied by the total volume of the extract and divided by the volume of supernatant subjected to SDS–PAGE. Finally, DAT levels in biotinylated fractions were calculated as total DAT levels minus DAT levels in non-biotinylated fractions.
Data are presented as mean ± SEM, and n represents the number of independent experiments for each group. Data from [3H]DA uptake experiments were analyzed using unpaired Student's t-test. Data from immunoblotting experiments were analyzed using separate between-subject one-way anovas for each of the fractions (total, non-biotinylated and biotinylated); DAT levels between 6-OHDA- and vehicle-treated groups or between EC and IC groups were compared. Simple effect comparisons were made for post hoc analysis as appropriate. The accepted level of significance was p < 0.05.
6-OHDA infusion into mPFC decreased synaptosomal [3H]DA uptake and DAT expression in mPFC, but not in nucleus accumbens and striatum
In the vehicle control group, [3H]DA uptake in mPFC, nucleus accumbens and striatum was 2.2 ± 0.6, 35 ± 1.5 and 39 ± 1.2 pmol per min per mg respectively. Infusion of 6-OHDA into mPFC significantly reduced [3H]DA uptake in mPFC, by 93 ± 0.6% of that in the vehicle control group. However, there was no effect of 6-OHDA infusion on [3H]DA uptake in the nucleus accumbens or striatum (Fig. 1).
Synaptosomes from 6-OHDA-treated and vehicle control rats were evaluated in immunoblotting assays to determine DAT levels in total, non-biotinylated (intracellular) and biotinylated (cell surface) fractions in mPFC (Fig. 2). In vehicle control rats, DAT-immunoreactive bands (∼ 75 kDa) were detectable in all three fractions. Levels of DAT in the biotinylated fraction were 55.5 ± 7.9% of total DAT in the mPFC. Infusion of 6-OHDA into the mPFC markedly reduced (89.2 ± 5.6%) total DAT levels compared with vehicle control levels. Equivalent reductions in DAT levels were observed in non-biotinylated and biotinylated fractions. Infusion of 6-OHDA did not alter levels of the control protein, syntaxin 1A, in mPFC subcellular fractions. DAT-immunoreactive bands in the biotinylated fraction from nucleus accumbens (Fig. 3a) and striatum (Fig. 3b) from vehicle control rats were 77.4 ± 4.7% and 69.3 ± 2.0% of total DAT respectively. Infusion of 6-OHDA into mPFC did not alter DAT levels in total and subcellular fractions from nucleus accumbens and striatum (Fig. 3). The decrease in DAT levels in the mPFC, which correlated with the decrease in [3H]DA uptake in this brain region following lesion with 6-OHDA, validated the use of the DAT antibody for investigating transporter levels in mPFC from EC and IC rats.
Decreased DAT cell surface expression in mPFC of EC rats
Using the above immunoblotting protocol, DAT levels in the total and subcellular fractions of mPFC from EC and IC rats were determined. There were no differences in DAT levels in either total or non-biotinylated (intracellular) fractions from mPFC between EC and IC rats (Fig. 4). However, the DAT level in the biotinylated fraction (cell surface) from the mPFC was 39% lower in EC rats than in IC rats (F5,54 =2.65, p < 0.05). There were no differences between EC and IC rats in the levels of control proteins, including syntaxin 1A, calnexin and PP2A, in either the total or subcellular fractions obtained from mPFC (Fig. 4). Thus, the difference in DAT mPFC levels between EC and IC rats was not due to differences in protein loading or biotinylation efficiency. Determination of DAT levels in the nucleus accumbens and striatum revealed no significant differences in either total or subcellular fractions between EC and IC rats (Fig. 5). These results indicate that lower amounts of DAT were distributed to the plasma membrane of the mPFC in EC compared with IC rats.
The present study is the first to report the use of biotinylation and western blot analysis to evaluate DAT trafficking in mPFC. After validating these methods for the evaluation of subcellular localization of DAT in the mPFC, the present study assessed subcellular localization of the transporter in the mPFC of EC and IC rats. Exposure to different environmental conditions, enrichment and impoverishment, during development altered the distribution of DAT protein in subcellular fractions of the mPFC. Specifically, the levels of DAT at the cell surface were lower in mPFC from EC rats compared with IC rats. This effect of environmental enrichment was brain-region specific, as differences were observed in the mPFC, but not in the nucleus accumbens or striatum.
DAT localized at the presynaptic membrane reflects functional transporter that is involved in the clearance of extracellular DA. Changes in DAT surface expression have been shown to be induced by changes in the activity of protein kinase C, mitogen-activated protein kinase and phophotidylinositol-3-kinase, as well as exposure to psychostimulants. Such changes correlate with alterations in DA uptake in both brain and cell systems expressing DAT (Copeland et al. 1996; Vaughan et al. 1997; Pristupa et al. 1998; Carvelli et al. 2002; Chi and Reith 2003; Morón et al. 2003).
In the present study, exposure to an enriched environment during development resulted in comparable changes in DAT surface expression and DAT function. The decrease (39%) in cell surface DAT expression in mPFC in EC relative to IC rats was comparable to the magnitude (35%) of the decrease in Vmax for [3H]DA uptake in mPFC in EC compared with IC rats demonstrated in our previous report (Zhu et al. 2004). The present study also showed no differences between EC and IC rats in the levels of total DAT protein in the mPFC. This is consistent with our previous finding that the maximal density of [3H]GBR12935 binding in the mPFC, which reflects both cell surface and internalized DAT, does not differ between EC and IC rats (Zhu et al. 2004). Thus, the difference between EC and IC rats in both cell surface levels of mPFC DAT and in DA uptake into mPFC synaptosomes is not due to different levels of total DAT protein, but results from a change in expression of cell surface DAT.
The decrease (39%) in DAT immunoreactivity at the cell surface in mPFC from EC rats was not accompanied by an increase in intracellular DAT. However, with no change in total DAT levels, a decrease in cell surface DAT levels would be expected to be accompanied by an increase in intracellular DAT levels. In another study using N2A cells, cocaine exposure was shown to significantly increase (28%) DAT cell surface expression, with no change in total DAT, and intracellular DAT levels were also unchanged (Little et al. 2002). Phorbol ester exposure also resulted in decreased DAT levels at the cell surface of PC12 cells, with no significant change in the intracellular pool, although a tendency for an increase in DAT was observed (Melikian and Buckley 1999). The present findings are therefore consistent with those of other studies that used similar cell surface biotinylation techniques, in that changes in cell surface DAT expression were not accompanied by intracellular changes in expression.
In contrast, other studies have reported an increase in either intracellular expression of the DAT, the norepinephrine transporter (NET) or the serotonin transporter (SERT) coincident with decreases in cell surface expression of the respective transporters. With respect to DAT cellular localization, Morón et al. (2003) treated HEK293 cells expressing the human DAT with 50 µm PD98059 (a mitogen-activated protein kinase inhibitor), and found a 28% decrease in cell surface expression of DAT and a concomitant, significant increase (< 20%) in intracellular DAT localization using the biotinylation assay. The latter study was more likely to detect small significant differences than the present study because a homogeneous population of cells directly exposed to PD98059 in vitro was likely to show less variability in response than the brain synaptosome preparations from rats exposed to different environments in the present experiments. Furthermore, the density of DAT expression in the HEK293 cell line is several-fold higher than that in rat mPFC synaptosomes, facilitating detection of changes in intracellular DAT expression in the cell line.
With respect to NET cellular localization, Jayanthi et al. (2004) treated rat placental trophoblast cultures with 0.5 µmβ-phorbol-12-myristate-13-acetate (PMA, a protein kinase C activator), which resulted in a 40% decrease in cell surface expression of NET that was accompanied by a 40% increase in intracellular NET levels, measured in a biotinylation assay. The peripheral cells used in this study expressed a high level of NET in comparison with DAT expression in rat mPFC synaptosomes, which probably facilitated detection of differences in NET expression in cellular compartments in placental trophoblasts following drug treatment. Furthermore, the culture was exposed directly to the PMA, providing a homogenous response of the culture samples.
With respect to SERT cellular localization, Samuvel et al. (2005) recently reported a 70% decrease in SERT cell surface expression in rat midbrain synaptosomes after treatment with 10 µm PD169316 (a p38 mitogen-activated protein kinase inhibitor), which was accompanied by a 70% increase in the amount of SERT in the intracellular fraction. Again, in this experimental model, the density of SERT in midbrain was probably higher than that of DAT at the terminals in mPFC in the present study. Furthermore, the midbrain synaptosomes were exposed directly to PD169316, providing a homogenous response.
In the above three studies (Morón et al. 2003; Jayanthi et al. 2004; Samuvel et al. 2005), cell expression systems, cell culture systems or synaptosomes were used. The density of expression of the transporters in the respective tissues was probably higher in these model systems than the level of DAT expression in mPFC synaptosomes in the present study. Furthermore, in each study the tissues were exposed to drug directly, providing conditions with minimal variability in response, which further facilitated the detection of changes in transporter localization in response to direct drug treatment. Thus, decreases in cell surface expression of the respective transporters, with a concomitant increase in intracellular levels using the biotinylation assay were reported. Interestingly, despite the use of optimal conditions (i.e. homogeneous cell culture populations and tissues exposed directly to drug) for detecting changes in transporter expression, other studies did not observe changes in intracellular levels of DAT (Melikian and Buckley 1999; Little et al. 2002). Thus, there is variability reported in the literature with regard to changes in intracellular expression of transporter occurring with changes in surface expression, even when optimal model systems and assay conditions are used. In the present study, differences in cell surface expression of the DAT were observed in mPFC, a heterogeneously innervated brain region with a low density of DAT expression. Environmental enrichment resulted in decreases in DAT expression at the cell surface, but changes in DAT expression were not detected in the intracellular compartment. In contrast to previous studies that employed drug treatment, the environmental enrichment paradigm used in this study may not produce a homogenous response in all dopaminergic terminals in the mPFC, leading to greater variability in response and probably diminishing the likelihood of detecting small differences in DAT expression in the intracellular compartment. The fact that a change in DAT cell surface expression in the mPFC was observed following a physiological manipulation is a novel finding, which may have implications for understanding neural mechanisms involved in addictive disorders and how environmental factors influence addiction.
The mechanism by which environmental enrichment reduces DAT cell surface expression may involve regulation of intracellular signaling cascades. Activation of protein kinase C by phorbol esters and inhibition of mitogen-activated protein kinase or phophotidylinositol-3-kinase have been shown to decrease cell surface DAT expression in cell systems and in brain (Melikian and Buckley 1999; Carvelli et al. 2002; Morón et al. 2003). Moreover, membrane depolarization has also been shown to inhibit DAT function in Xenopus oocytes (Sonders et al. 1997). Taken together, these results suggest that exposure to novelty during development might activate neural systems in the mPFC that may subsequently alter cell signaling cascades leading to a decrease in cell surface DAT expression and function in this brain region.
Environmental enrichment during development influences both structural and functional maturation of the mPFC in rats. For example, EC rats exhibit a higher density of DA innervation (Winterfeld et al. 1998) and a decreased DA metabolism (decreased dihydroxyphenylacetic acid content) in mPFC relative to IC rats (Zhu et al. 2004). In the present study, EC rats had reduced cell surface DAT, presumably increasing the extracellular DA concentration, which is consistent with previously reported changes in DA metabolism. Moreover, a decrease in cell surface DAT expression was observed only in mPFC, and not in the nucleus accumbens or striatum, suggesting that this effect of environment on DAT function and trafficking is region specific. Numerous studies have suggested that the mPFC plays an important role in mediating drug reward, reinstatement of drug-seeking behavior and drug addiction (Isaac et al. 1989; Karreman and Moghaddam 1996; Tzschentke and Schmidt 2000; Steketee 2003).
Studies using rodent models have suggested that the DAT may have a major role in influencing the behavioral response to a novel environment. DAT-deficient mice exhibit augmented locomotor, rearing and stereotypic behavior in response to a novel environment (Giros et al. 1996; Gainetdinov et al. 1999; Gainetdinov et al. 2002). Recent studies in rats have also shown alterations in DAT function that are correlated with intrinsic response to a novel environment. Specifically, high-responder rats, which exhibit greater locomotor activity in an open field, show a higher Vmax for [3H]DA uptake compared with low responders but no change in total DAT levels in the nucleus accumbens (Chefer et al. 2003). The individual differences in DAT function between high and low responders may be due to differences in surface expression of the DAT in the nucleus accumbens. Similarly, the locomotor stimulant effect of cocaine has been shown to be correlated with differences in DAT function in striatum (Sabeti et al. 2002; Briegleb et al. 2004). In the study by Briegleb et al. (2004), rats with a high locomotor response to cocaine exhibited increased DA clearance in vivo and increased [3H]DA uptake in vitro compared with rats with a low response to cocaine, whereas no between-group difference in [3H]WIN 35428 binding was observed. Although these results indicate that differences in DAT function may contribute to individual differences in the locomotor response to cocaine, the contribution of cell surface DAT expression and DAT trafficking has not been determined.
Differences in DAT subcellular localization in mPFC observed in the present study suggest that this brain structure may be critically involved in the behavioral consequences of environmental manipulations during development. Interestingly, clinical evidence has implicated this region in behavioral inhibitory processes that modulate the excitatory subcortical limbic regions that motivate drug and other reward-related behaviors (Gray 1976; Fillmore 2003; Asahi et al. 2004). Perhaps most relevant to the present work, 6-OHDA-induced DA depletion in the mPFC has been shown to produce impulsive behavioral patterns and enhancement of the locomotor stimulant effects of amphetamine in rats (Sokolowski and Salamone 1994). Although DA levels were not determined in the present study, the reduced expression of DAT at the cell surface in the mPFC of EC rats suggests that these rats have increased extracellular DA, and so would be expected to perform well in tasks measuring behavioral inhibition. Consistent with this EC rats show less impulsivity than IC rats (Ough et al. 1972), less sensitization to repeated injections of stimulant drugs (Bardo et al. 1995; Zhu et al. 2004) and less amphetamine self-administration (Bardo et al. 2001). Because impulsivity and enhanced response to drugs may both be important indicators of risk for drug abuse vulnerability, the present results suggest that DAT function in mPFC may be an important neural mechanism involved in risk for drug abuse among individuals with different environmental histories.
Authors thank Ms. Jackie Huller for technical assistance. This research was supported by National Institutes of Health grants DA00399, DA05312, DA12964, DA14040, RR15592, National Alliance for Research on Schizophrenia and Depression and the University of Kentucky Center on Drug and Alcohol Research.