Age-Related Reductions in [H]WIN 35,428 Binding to the Dopamine Transporter in Nigrostriatal and Mesolimbic Brain Regions of the Fischer 344 Rat

In the present study, we used the potent cocaine analog [H]WIN 35,428 to map and quantify binding to the dopamine transporter (DAT) within the dorsal striatum, nucleus accumbens, substantia nigra, and ventral tegmental area in young (6-month-old), middle-aged (12-month-old), and aged (18and 24-month-old) Fischer 344 rats. Quantitative autoradiographic analysis of indirect [H]WIN 35,428 saturation curves revealed two-site binding for all four brain regions in every age group. The percentage of binding to the highor low-affinity sites did not differ with age or region and was approximately 50%. However, significant age-related decreases in the overall density (Bmax) of [ H]WIN 35,428-binding sites were observed in the striatum, nucleus accumbens, substantia nigra, and ventral tegmental area. The Bmax within all brain regions declined by more than 15% every 6 months, with the Bmax in the aged (24-month-old) group being approximately half that measured in the young adult (6-month-old) group. Competition experiments indicated that nomifensine also exhibited two-site binding to the DAT in Fischer 344 rats. No consistent age-related differences in binding affinities were noted with either [H]WIN 35,428 or nomifensine. Taken together, these results support the hypothesis that functional DATs within the nigrostriatal and mesolimbic systems are down-regulated with age, without changing their affinity for ligands. Dopamine transporters (DATs) are integral neuronal membrane proteins that function to terminate dopaminergic neurotransmission by the rapid reuptake of synaptic dopamine (DA) into dopaminergic neurons. Because DAT is the main mechanism for clearing extracellular DA, it is the primary element that regulates the intensity and duration of dopaminergic neurotransmission (Giros et al., 1996). The uptake process is important for normal brain function because the administration of drugs that block the uptake process have dramatic behavioral and physiological effects (Ritz and Kuhar, 1993). Because it is responsible for the translocation of neurotoxins that can cause parkinsonian symptoms, DAT is also implicated in age-related neurodegenerative disorders, such as Parkinson’s disease (Edwards, 1993). Dramatic age-related deficits in the in vivo function of the DAT have been documented in aged animals. Significant reductions in the clearance rate of endogenous DA were measured using in vivo electrochemical methods in middleaged and aged Fischer 344 (F344) rats and middle-aged nonhuman primates (Friedemann and Gerhardt, 1992; Gerhardt et al., 1995; Hebert and Gerhardt, 1998). Using the same technique, age-related differences in the capacity to clear extracellular DA were observed, with the maximum rate of DA transport being reduced by 50% in the striatum and nucleus accumbens of 24-month-old F344 rats (Hebert and Gerhardt, 1999). It has been speculated that changes in the density of functional DATs may be the basis for observed changes in capacity and rate of DA uptake (Hebert and Gerhardt, 1999). Alternatively, age-related changes in DA uptake may reflect altered affinity of DAT for DA. In vitro localization of DATs in brain tissue has been routinely performed using radioligand binding and hybridization assays. Radiolabeling of uptake inhibitors, which bind to a recognition site associated with the DAT, and of antisense cDNA probes, which hybridize with DAT mRNA, has created the opportunity for quantitative autoradiographic assessment of the density and distribution of DATs (Himi et al., 1995). Radioligand binding to the human DAT has been reported to decrease linearly with age (Volkow et al., 1994). Received for publication July 24, 1998. 1 This work was supported by U.S. Public Health Service Grants NS09199, AG06434, and DA04216 and National Institutes of Health Training Grant HD07408. In addition, this work was supported in part by a Level II Research Scientist Development Award (MH01245) from NIMH (G.A.G.) and RSDA Grant DA00174 from NIDA (N.R.Z.). ABBREVIATIONS: DA, dopamine; DAT, dopamine transporter; F344, Fischer 344. 0022-3565/99/2883-1334$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 288, No. 3 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 288:1334–1339, 1999 1334 at A PE T Jornals on N ovem er 2, 2017 jpet.asjournals.org D ow nladed from Similarly, reports indicate that the expression of human DAT mRNA declines significantly with age (Bannon and Whitty, 1997). Although DAT mRNA was also found to be significantly reduced in aged rats (Himi et al., 1995), there is a lack of consensus regarding age-related changes in radioligand binding to the DAT in laboratory rats. In two studies involving aged rats, significant reductions were seen in [H]mazindol binding density and [H]GBR 12783 binding within the striatum (Araki et al., 1997). In contrast, in another study, a similar number of striatal [H]GBR 12935-binding sites were reported in aged and young rats (Inglefield and Richfield, 1992). Differential binding characteristics of the various radioligands used may account for the observed inconsistencies in DAT-binding densities in aged rats. [H]GBR compounds and [H]mazindol have been reported to vary in specificity for the DAT, leading to differences in reported transporter density in various brain regions (Madras et al., 1989; Izenwasser et al., 1994; Pristupa et al., 1994). Furthermore, [H]GBR 12935, the radioligand used in the study in which no age-related differences in binding density were observed, binds not only to the DAT but also to a piperazine acceptor site (Richfield, 1991). We have chosen to use a highly selective ligand for the DAT, [H]WIN 35,428 ([H]2-b-carbomethoxy-3b-(4-fluorophenyl)tropane), to investigate differences in binding densities within the dopaminergic cell bodies and terminal regions in F344 rat brain. [H]WIN 35,428 has excellent qualities for autoradiographic studies. Its DAT binding characteristics and pharmacokinetic properties have been very well characterized in vitro, ex vivo, and in vivo in rodents (Haaparanta et al., 1996), primates (Kaufman and Madras, 1993), and humans (Laakso et al., 1998). The lack of an ester bond makes it relatively resistant to metabolism. It has good selectivity for DAT over other monoamine transporters (Aloyo et al., 1995), and it shows relatively low nonspecific binding in the brain (Kaufman and Madras, 1993). In membranes and intact sections from rodent brain, [H]WIN 35,428 has been shown to bind with nanomolar affinity in a reversible, saturable, and stereoselective manner (Izenwasser et al., 1994). In the only aging study to use [H]WIN 35,428 to date, in vivo accumulation of this ligand in the striatum of aged monkeys was reduced by more than 50% compared with young adult monkeys (Kaufman and Madras, 1993). The purpose of these experiments was to evaluate agerelated changes in the binding characteristics of DAT proteins in both the terminal and cell body regions of the nigrostriatal and mesolimbic systems in young, middle-aged, and aged F344 rats. Our studies addressed the following questions: 1) Are there age-related changes in the number of DATs? 2) Are there differences in DAT-binding affinities between the age groups? 3) Are the differences region dependent? To approach these questions, two experiments were performed using in vitro radioligand binding with quantitative autoradiographic analysis. In experiment 1, indirect saturation curves for [H]WIN 35,428 binding were generated. In a second experiment, we studied the displacement of [H]WIN 35,428 by the DA uptake inhibitor nomifensine. We have previously reported significant age-related deficits in nomifensine-induced behavior and in vivo uptake inhibition (Hebert and Gerhardt, 1998, 1999) and wanted to investigate further whether age-related alterations in the affinity of DAT for this ligand were related to functional changes. Experimental Procedures Animals. Male F344 young adult (6 months old, n 5 9), middleaged (12 months old, n 5 9), and aged (18 months old, n 5 9; and 24 months old, n 5 9) rats were used for [H]WIN 35,428 indirect saturation experiments. Male F344 young adult (6 months old, n 5 6) and aged (24 months old, n 5 6) rats were used in the nomifensine competition experiments. All rats were obtained from the National Institute on Aging (Harlan Sprague-Dawley, Inc., Indianapolis, IN). Protocols for animal use were approved by the Institutional Animal Care and Use Committee. Animals were housed according to approved guidelines, with food and water available ad libitum. Tissue Preparation. Rats were anesthetized with urethane (1.5 g/kg i.p.) and sacrificed by decapitation. The brains were rapidly removed, frozen in powdered dry ice, and stored at 280°C. For sectioning, the brains were allowed to equilibrate to 220°C for 1 h, mounted on a chuck, serially sectioned with a cryostat in 10-mm coronal sections, and thaw-mounted onto Fisherbrand Superfrost slides (Fisher Scientific, Pittsburgh, PA). The sections were cut at the levels of the striatum/nucleus accumbens and substantia nigra/ ventral tegmental areas based on the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986). The slide-mounted sections were stored at 280°C until assayed. Binding Assays and Quantitative Autoradiography. Optimal binding conditions for [H]WIN 35,428 were established in preliminary studies. For both the indirect saturation analysis and the nomifensine competition experiments, slides were preincubated in 30 mM sodium phosphate buffer, pH 7.4, for 10 min at 4°C. The assays consisted of 30 mM sodium phosphate buffer containing 0.32 M sucrose (Reith and Coffey, 1993), 3.7 nM [H]WIN 35,428, and 8 to 10 concentrations of unlabeled WIN 35,428 (0.3 nM to 1 mM) or nomifensine (0.3 nM to 10 mM). In both experiments, total binding was defined in the absence of added unlabeled drugs, and nonspecific binding was defined in the presence of benztropine (30 mM). The slides were incubated for 90 min at 4°C. Unbound ligand was removed by washing the sections twice for 1 min in 4°C buffer that did not contain sucrose, followed by a quick rinse in 4°C water to remove buffer salts. Excess water was removed immediately from the slide under a stream of cool air. The slides were dried on a slide warmer (55°C) and then stored overnight at room temperature. The next day, the slide-mounted brain sections were apposed to H-labeled Hyperfilm (Amersham Corp., Arlington Heights, IL) along with calibrated H-labeled microscales (Amersham Corp.) in film cassettes. Sections containing the striatum and nucleus accumbens were exposed to film for 12 days, and sections containing the substantia nigra and ventral tegmental area were exposed for 6 weeks. The films were developed for 4 min with Kodak D19 (Rochester, NY), rinsed for 45 s in an acid stop bath (Kodak Quick Stop), fixed for 4 min (Kodak Fixer), and rinsed for 10 min in distilled