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SYNAFSE 12:27-36 (1992) High-Affinity Binding of [ 125 IIRTI-55 to Dopamine and Serotonin Transporters in Rat Brain J.W. BOJA, W.M. MITCHELL, A. PATEL, T.A. KOPAJTLC, F.I. CARROLL, A.H. LEWIN, P. ABRAHAM, AND M.J. KUHAR Neuroscience Branch, Addiction Research Center, National Institute on Drug Abuse, Baltimore, Maryland 21224 (J.W.B., W.M.M.,A.P., T.A.K., M.J.K.); Research Triangle Institute, Research Triangle Park, North Carolina 27709 (F.I.C., A.H.L., P.A.) KEY WORDS ABSTRACT RTI-55 (3~-(4-iodophenyl)tropan-2~-carboxylic acid methyl ester), one of the most potent inhibitors of dopamine uptake reported to date, was radioiodinated and tested as a probe for the cocaine receptor in Sprague-Dawley rat brain. Saturation and kinetic studies in the striatum revealed that [1251]RTI-55 bound to both a high- and low-affinity site. The I(d for the high-affinity site was 0.2 nM, while the I(d for the low-affinity site was 5.8 nM. The corresponding number of binding sites in the striatum was 37 and 415 pmol/g protein. The pharmacological profile of specific [1251]RTI-55 binding in the striatum was consistent with that of the dopamine transporter. Addition- ally, [12511RTI-55 was found to bind with high affinity to the cerebral cortex. Scatchard analysis revealed a single high-affinity component of 0.2 nM with a density of 2.5 pmoVg protein. The pharmacological profile demonstrated by [12511RTI-55 in the cerebral cortex matched that of the serotonin transporter. Autoradiographic analysis of sagittal brain sections with [lz5I1RTI-55 binding was consistent with these findings. Specific binding of [12511RTI-55 was blocked by dopamine uptake inhibitors in areas rich in dopaminergic nerve terminals. Conversely, serotonin uptake inhibitors blocked the binding of [1251]RTI-55 in brain areas rich in serotonergic neurons. These results demonstrate that [1251]RTI-55 may be a very useful ligand for the dopamine and serotonin trans- porters. Published 1992 Wiley-Liss, Inc Dopamine uptake, Binding sites, Serotonin uptake, Cocaine INTRODUCTION Much recent research has focused upon the molecular mechanism of action of cocaine. While it has been known for some time that cocaine inhibits the reuptake of dopamine, the exact mechanism by which cocaine produces its inhibition is still unknown. Cocaine has high-affinity binding sites in rodent, nonhuman pri- mate, and human brain (Calligaro and Eldefrawi, 1987; Kennedy and Hanbauer, 1983; Madras et al., 1989a; Reith et al., 1980; Schoemaker et al., 1985). These binding sites show several properties that are associated with biologically relevant receptors. These properties include a significant correlation between the potency for maintaining drug self-administration of various cocaine-related drugs and their potency at the striatal cocaine binding site (Bergman et al., 1989; Ritz et al., 1987). A correlation of potencies also holds true for inhibiting [3H]dopamine uptake and inhibition of [3H]cocaine binding (Kennedy and Hanbauer, 1983; Madras et al., 1998a). The cocaine binding site demon- strates stereoselectivity; (-)cocaine has seven possible PUBLISHED 1992 WILEY-LISS, INC. stereoisomers and these stereoisomers were 60-600 times less potent than (-)cocaine itself (Carroll et al., 1992). Further support for a biological receptor is dem- onstrated by the fact that L3H1cocaine is bound satura- bly at concentrations comparable to that found in the brain following peripheral administration of cocaine (Misra et al., 1977). Thus it appears that cocaine may initiate its mechanism of action via a biologically rele- vant receptor. In order to study the mechanism of action for cocaine, several probes for the cocaine receptor have been devel- oped. However, some of these probes demonstrate tech- nical problems that limit their utility. The use of [3H]~o- caine itself as a probe is limited by the relatively low affinity and rapid dissociation rate (Calligaro and Elde- frawi, 1988; Madras et al., 1989a). Other ligands, such as L3H]GBR 12935 (Andersen, 1987; Janosky et al., 1986), [3H]mazindol (Javitch et al., 19841, L3Hlmeth- Received September 16,1991; accepted in revised form January 27,1992
10

High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

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Page 1: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

SYNAFSE 12:27-36 (1992)

High-Affinity Binding of [ 125 IIRTI-55 to Dopamine and Serotonin Transporters in

Rat Brain J.W. BOJA, W.M. MITCHELL, A. PATEL, T.A. KOPAJTLC, F.I. CARROLL, A.H. LEWIN,

P. ABRAHAM, AND M.J. KUHAR Neuroscience Branch, Addiction Research Center, National Institute on Drug Abuse, Baltimore, Maryland 21224 (J.W.B., W.M.M., A.P., T.A.K., M.J.K.); Research Triangle Institute, Research Triangle Park, North

Carolina 27709 (F.I.C., A.H.L., P.A.)

KEY WORDS

ABSTRACT RTI-55 (3~-(4-iodophenyl)tropan-2~-carboxylic acid methyl ester), one of the most potent inhibitors of dopamine uptake reported to date, was radioiodinated and tested as a probe for the cocaine receptor in Sprague-Dawley rat brain. Saturation and kinetic studies in the striatum revealed that [1251]RTI-55 bound to both a high- and low-affinity site. The I(d for the high-affinity site was 0.2 nM, while the I(d for the low-affinity site was 5.8 nM. The corresponding number of binding sites in the striatum was 37 and 415 pmol/g protein. The pharmacological profile of specific [1251]RTI-55 binding in the striatum was consistent with that of the dopamine transporter. Addition- ally, [12511RTI-55 was found to bind with high affinity to the cerebral cortex. Scatchard analysis revealed a single high-affinity component of 0.2 nM with a density of 2.5 pmoVg protein. The pharmacological profile demonstrated by [12511RTI-55 in the cerebral cortex matched that of the serotonin transporter. Autoradiographic analysis of sagittal brain sections with [lz5I1RTI-55 binding was consistent with these findings. Specific binding of [12511RTI-55 was blocked by dopamine uptake inhibitors in areas rich in dopaminergic nerve terminals. Conversely, serotonin uptake inhibitors blocked the binding of [1251]RTI-55 in brain areas rich in serotonergic neurons. These results demonstrate that [1251]RTI-55 may be a very useful ligand for the dopamine and serotonin trans- porters. Published 1992 Wiley-Liss, Inc

Dopamine uptake, Binding sites, Serotonin uptake, Cocaine

INTRODUCTION Much recent research has focused upon the molecular

mechanism of action of cocaine. While it has been known for some time that cocaine inhibits the reuptake of dopamine, the exact mechanism by which cocaine produces its inhibition is still unknown. Cocaine has high-affinity binding sites in rodent, nonhuman pri- mate, and human brain (Calligaro and Eldefrawi, 1987; Kennedy and Hanbauer, 1983; Madras et al., 1989a; Reith et al., 1980; Schoemaker et al., 1985).

These binding sites show several properties that are associated with biologically relevant receptors. These properties include a significant correlation between the potency for maintaining drug self-administration of various cocaine-related drugs and their potency at the striatal cocaine binding site (Bergman et al., 1989; Ritz et al., 1987). A correlation of potencies also holds true for inhibiting [3H]dopamine uptake and inhibition of [3H]cocaine binding (Kennedy and Hanbauer, 1983; Madras et al., 1998a). The cocaine binding site demon- strates stereoselectivity; (-)cocaine has seven possible PUBLISHED 1992 WILEY-LISS, INC.

stereoisomers and these stereoisomers were 60-600 times less potent than (-)cocaine itself (Carroll et al., 1992). Further support for a biological receptor is dem- onstrated by the fact that L3H1cocaine is bound satura- bly at concentrations comparable to that found in the brain following peripheral administration of cocaine (Misra et al., 1977). Thus it appears that cocaine may initiate its mechanism of action via a biologically rele- vant receptor.

In order to study the mechanism of action for cocaine, several probes for the cocaine receptor have been devel- oped. However, some of these probes demonstrate tech- nical problems that limit their utility. The use of [3H]~o- caine itself as a probe is limited by the relatively low affinity and rapid dissociation rate (Calligaro and Elde- frawi, 1988; Madras et al., 1989a). Other ligands, such as L3H]GBR 12935 (Andersen, 1987; Janosky et al., 1986), [3H]mazindol (Javitch et al., 19841, L3Hlmeth-

Received September 16,1991; accepted in revised form January 27,1992

Page 2: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

28 J.W. BOJA ET AL.

ylphenidate (Janosky et al., 1986), and L3H1nomifen- sine (Dubocovich and Zahniser, 1985), solve the prob- lem of low affinity since they all demonstrate a higher affinity for the cocaine receptor than cocaine itself. However, these four ligands bind to only one high-affin- ity binding site, unlike cocaine, which has both a high- and low-affinity binding site (Calligaro and Eldefrawi, 1988; Madras et al., 1989a). Furthermore, neither GBR 12909 nor mazindol fully displaces r3H1cocaine from its binding site (Kennedy and Hanbauer, 1983; Madras et al., 1989a). The lack of correspondence of the binding properties of these ligands compared to that of [3H]~o- caine may prevent the full characterization of the co- caine receptors with these ligands.

Several cocaine analogs have been developed for use as ligands for the cocaine receptors based on the work of Clarke et al. (1973). These include L3H]WIN 35,428 (Madras et al., 198913) and [3HlWIN 35,065-2 (Ritz et al., 1990). We have developed several compounds that are approximately 100 times more potent than cocaine in inhibiting specifically bound [3H]WIN 35,428 (Boja et al., 1990, 1991) and 20-50 times more potent than cocaine as a psychomotor stimulant (Cline et al., 1992a). The present study describes the in vitro binding and autoradiographic visualization of [12511RTI-55 binding sites. The results of this study suggest that RTI-55 will be a useful ligand for both the in vitro char- acterization and visualization of cocaine receptors.

MATERIALS AND METHODS Tissue sources and preparation

Brains from male Sprague-Dawley rats weighing 200-250 g (Harlan Labs, Indianapolis, IN) were re- moved following decapitation and were washed in ice- cold saline. Specific regions were rapidly dissected and the resulting tissues were frozen and stored at -80°C. Membranes were prepared by homogenizing tissues in 20 volumes (w/v) of assay buffer using a Brinkman Polytron (setting 8, 20 sec) and were centrifuged at 50,000 x g for 10 min. The resulting pellet was resus- pended in 20 volumes of buffer, recentrifuged, and re- suspended in buffer to the desired final concentration.

Ligand binding studies All ligand binding experiments were conducted in

assay tubes containing 2 ml buffer (10 mM sodium phosphate containing 0.32 M sucrose, pH 7.40) at 22- 24°C. Each assay tube contained 15 pM [12511RTI-55 and 0.1 mg tissue (original wet weight) of striatal tissue or 0.3 mg of cortical tissue, except in tissue linearity experiments. Tissue linearity experiments were con- ducted as noted above, except the tissue content of the assay tube varied from 0.006 to 2.0 mg for striatal tis- sue, or in the case of cortical tissue, 0.05-2.0 mg. Non- specific binding of [lz5I1RTI-55 was defined by the addi- tion of 30 p M (-)cocaine. Association experiments were conducted at 22-24°C; tubes were incubated for various

times ranging from 2 to 80 min. Incubation was ter- minated by rapid filtration through Whatman GFA3 filters previously soaked in 0.05% polyethylenimine, using a Brandel R48 filtering manifold (Brandel Instru- ments, Gaithersburg, MD). The filters were washed twice with 7 ml of buffer and counted using a LKB- Wallac CliniGamma 1272 gamma counter (LKB-Wal- lac, Finland). The dissociation rate was determined af- ter allowing equilibrium to be reached (50-60 min at 22-24T) with the addition of 1 pM unlabeled RTI-55 and filtering the samples at various times. Specifically bound [lZ5I]RTI-55 at equilibrium (time 0) was used to determine the percentage of specifically bound [lz5IIRTI-55 at the various time points. Saturation and competition experiments were conducted by using a fixed concentration of [1251]RTI-55 (15 pM) and increas- ing the concentration of unlabeled RTI-55 or another competitor. Total binding was determined in the ab- sence of any inhibitor, while nonspecific binding was determined as previously described.

Data were analyzed by the KINETIC, EBDA, and LIGAND computer radioligand data analysis software (BIOSOFT, UK). Kinetic rate constants were calculated with the KINETIC program from association and disso- ciation data obtained from experiments performed with either striatal or cortical tissue. Mono- or biexponential models were tested for statistical significance using an F test. Competition data were analyzed with EBDA, which provided final estimates of ICbo. The dissociation constant (&) and density of binding sites (Bma) were determined using LIGAND. Statistical analyses were performed using an F test to determine whether those data could be best described by a single- or multiple-site model.

Receptor autoradiography Tissue for visualization of [12513RTI-55 were obtain

from male Sprague-Dawley rats (Harlan Labs) accord- ing to the general method of Kuhar and Unnerstall (1990). The animals were anesthetized with sodium pentobarbital (25 mgkg) and perfused intraaortically with ice-cold, 0.25-M phosphate buffered saline con- taining 0.16 M sucrose (pH 7.2) at a rate of 25 mumin. The brains were immediately removed, embedded in cow brain paste, and frozen in isopentane (-50°C). The frozen brain were sectioned to 10 pM thickness, thaw- mounted onto gelatin-coated slides, dried, and stored desiccated at - 70°C until processed for autoradiog- raphy.

Sections were equilibrated to room temperature and then incubated for 120 min at 23°C with 50 pM [12511RTI-55 in the above assay buffer (in quadrupli- cate). The tissue sections were then washed twice (20 min each) in 2 consecutive assay buffer washes (0°C) followed by a dip in deionized water, also at 0°C. Non- specific binding was defined using 50 pM (-)cocaine. The slide-mounted sections were then either wiped off

Page 3: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

t'2sIlRTI-55 DA AND SEROTONIN BINDING 29

the slide with Whatman GFB filters and were counted using an LKB gamma counter, or were prepared for autoradiographic analysis. These conditions were de- termined in extensive preliminary experiments that systematically varied incubation and wash times (Ku- har and Unnerstall, 1990). In order to determine the pharmacological profile of [12511RTI-55 autoradiogra- phy, various inhibitors of specific [12511RTI-55 binding were also included in the incubation buffer. For autora- diographic studies the slide-mounted sections were im- mediately dried with a stream of desiccated dry air following the deionized water dip. The dry labeled slides and methylmethacrylate [12511 autoradiographic standards (Amersham, Arlington Heights, IL) were co- exposed to Hyperfilm 3H (Amersham) for 3 days. Brain sections were later stained with cresyl violet. Autora- diograms were quantified using a computerized digital image analysis system (Loats, Westminster, MD). Best- fit standard curves of film optical density generated by radiolabeled polymer standards coexposed with labeled slides resulted when a 3rd-order polynomial fit was used to describe the relationship between radioactivity and optical density. Results are expressed as femto- moles per milligram of tissue equivalent.

Drugs The following drugs were used in these studies: am-

fonelic acid, Research Biochemicals Inc. (RBI), Natick MA; atropine sulfate, Sigma Chemical Co., St. Louis, MO; benztropine mesylate, RBI; bupropion hydrochlo- ride, RBI; chloripramine, RBI; cinanserin, Squibb In- stitute for Medical Research, Princeton NJ; citalopram, Pfizer Inc., Groton, CT; (-)cocaine, (+)cocaine, Na- tional Institute on Drug Abuse (NIDA), Rockville, MD; desipramine hydrochloride, Sigma; dimethocaine, Hoff- mann-LaRoche Inc., Research Triangle Park, NC; flu- oxetine hydrochloride, Eli Lilly and Co., Indianapolis, IN; 1-[2-[bis~4-fluorophenyl)methoxylethyll-4-[3-phe- nylpropyllpiperazine dihydrochloride (GBR 12909), RBI; haloperidol, RBI; imipramine hydrochloride, RBI; ketanserin tartrate, RBI; mazindol, Sandoz Pharma- ceuticals, East Hanover, NJ; metergoline, Farmitalia, Milan, Italy; methylphenidate hydrochloride, NIDA; methysergide maleate, Sandoz Pharmaceuticals; nisox- etine hydrochloride, Eli Lilly and Co.; nomifensine maleate, Hoechst-Roussel Pharmaceuticals Inc., Som- erville, NJ: paroxetine hydrochloride, Beecham Phar- maceuticals Research Division, Philadelphia, PA; ser- taline hydrochloride, Pfizer Inc.; R-Sp-(phenyl)tropan- 2p-carboxylic acid methyl ester (WIN 35,065-21, S-3p-(phenyl)tropan-2p-carboxylic acid methyl ester (WIN 35,065-31, 3~-(4-fluorophenyl)tropan-2~-carbox- ylic acid methyl ester (WIN 35,428, CFT), N-nor-3p- (phenyl)tropan-2p-carboxylic acid methyl ester (WIN 35,9811, NIDA. Unlabeled RTI-55 and the precursor to [1251]RTI-55 was synthesized by F.I. Carroll (Carroll

STRIATUM 60000 -

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 TISSUE (mg/assay tube)

CEREBRAL CORTEX LY""",

l r l

g 20000 u

a 16000 Z A Y

I m u 8000 CI

W a Ln 400::

c.0 0.2 0 .4 0.6 0.8 1.0 1.2 TISSUE (mg/assay tube)

Fig. 1. Tissue linearity in rat striatal and cortical membranes. The specific binding of ['251]RTI-55 in striatal membranes was determined as the tissue content in the assay tube was increased from 0.006 to 1.0 mg per assay tube. Nonspecific binding was determined using 30 pM (-)cocaine. The tissue linearity curve for the cerebral cortex was de- termined identically, except the range of tissue content per assay tube varied from 0.05 to 3.0 mg. Each point is the mean of 2 experiments, with each performed in triplicate.

et al., 19911, and radiolabeled RTI-55 was generously supplied by New England Nuclear.

RESULTS Striatal binding

The specific binding of [12511RTI-55 was linear in the striatum as the tissue content in the assay tube was increased from 0.006 to 0.25 mg/2 ml (Figure 1). Fur- ther increases in tissue content produced a nonlinear relationship. The tissue linearity curve for the cerebral cortex (Figure 1) was linear from 0.125 mg/assay tube to 1.2 mg/assay tube; there was less specific binding per milligram of tissue in the cerebral cortex than in the striatum.

The association of specific [ 1251]RTI-55 binding in the striatum reached equilibrium at 45-50 min (Figure 2, top) at 20-24°C and was stable for at least 80 min. Analysis of the data with KINETIC revealed that the association occurred in 2 phases with corresponding rate constants kobsl of 2.075 * 0.825 min and kobs2 of 0.074 f 0.009 min (n = 2). Likewise, the dissociation of specifically bound [1251]RTI-55 (Figure 2, bottom) oc-

Page 4: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

J.W. BOJA ET AL. 30

(12511 ~ ~ 1 - 5 5 ASSOCIATION

W z lOOJ{

40

20 U

0 a 0 20 40 60 80

ASSOCIATION TIME (min)

"2511 RTI-55 DISSOCIATION

0 20 40 60 DISSOCIATION TIME (min)

Fig. 2. Association (top) and dissociation (bottom) of t1261]RTI-55 to rat striatal membranes. Association of 15 pM [12611RTI-55 was deter- mined by incubating striatal membranes (0.05 mg/mU at 22-24°C for various times. The dissociation rate was determined (after allowing equilibrium to be reached 50-60 min at 22-24°C) with the addition of 1 FM unlabeled RTI-55 and filtering the samples at various times. Spe- cifically bound [1251]RTI-55 at equilibrium (time 0) was used to deter- mine the percentage of specifically bound [lZ5IJRTI-55 a t the various time points. Data from both association and dissociation experiments were fitted by the KINETIC program to a biexponential model. The results of a typical experiment are presented; the points are the means of triplicate determinations.

curred in 2 phases. The corresponding dissociation rate constants, k-, and kP2, were 0.109 -C 0.016 min-' and 0.029 L 0.004 min-l, respectively. A biexponential model of association and dissociation of specific [1251]RTI-55 binding in the striatum was statistically preferred over a single exponential model (P < 0.001). These data yield a high-affinity Kd of 0.1 nM and a low-affinity I(d of 10.3 nM.

Saturation analysis of specific [ '"IIRTI-55 binding in the striatum was performed using a fixed concentra- tion of [lZ5I]RTI-55 (15 pM) and increasing concentra- tions of unlabeled RTI-55 (1 pM to 100 nM) (Figure 3). Scatchard transformation of the resulting data (Figure 3 insert) revealed a curvilinear relationship, suggesting a 2-binding-component model of the data. The 2-site model was statistically (F test) preferred to a single-site model (P < 0.001). Analysis of the data revealed a high-

100 35, 1

80

60

-

-

40 -

Fig. 3. Saturation of specific ['2511RTI-55 binding in rat striatum. Striatal membranes were incubated with a fixed concentration of [lz5I1RTI-55 (15 pM) and increasing concentrations of unlabeled RTI-55 (1 pM to 100 nM). Nonspecific binding was determined by the addition of 30 p M (-)cocaine. The results of a typical experiment are presented; the points are the means of triplicate determinations. The fit of this plot was determined using EBDA. Insert: Scatchard trans- formation of the resulting data.

and low-affinity site with corresponding &s of 0.2 and 5.8 nM. The density of these binding sites were 37 and 415 pmol/g protein.

The pharmacological profile of specific [1251]RTI-55 binding in the rat striatum was studied using a variety of drugs known to act at the DA, NE, and 5-HT trans- porters, as well as drugs that act at other sites. Overall the profile demonstrated by the inhibition of specific [1251]RTI-55 binding was consistent with that of the dopamine transporter (Table I). Both cocaine and WIN 35,065-2 competed for specifically bound [1251]RTI-55 stereospecifically, with the (-)isomer being approxi- mately 130 and 50 times more potent than their respec- tive (+)isomers. The rank order of potency for cocaine congeners in the striatum was RTI-55 > WIN 35,428 > WIN 35,065-2 > WIN 35,981 > (-)cocaine > (+)co- caine. Noncocaine analogs that act upon the DA trans- porter also competed specific [1251]RTI-55 binding in a rank order consistent with the DA transporter (Table I), with a rank order of GBR 12909 > mazindol > benz- tropine > nomifensine > methylphenidate > amfonelic acid > buproprion > dimethocaine. Representative dis- placement curves of some of the compounds are pre- sented in Figure 4. The total binding of [12511RTI-55 was displaced approximately 95% by (-)cocaine. Other compounds that inhibit dopamine uptake, such as benz- tropine, WIN 35,428, mazindol, and GBR 12909, also inhibited the total binding of [12511RTI-55 to a similar degree. Compounds that act upon the NE or 5-HT transporter, as well as those that act upon postsynaptic DA receptors or cholinergic receptors, were not potent inhibitors of specifically bound ['2511RTI-55 in the stri- atum. The log of the binding IC,,, relative to (-)cocaine [where (-)cocaine = 11, correlated highly (corr. coeff. =

Page 5: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

['251]RTI-55 DA AND SEROTONIN BINDING 31

TABLE I . Inhibition of ['2511RTI-55 binding in rat striatum

DA reuptake inhibitors Cocaine congeners

(-1 Cocaine ( + ) Cocaine WIN 35,065-2 WIN 35,065-3 WIN 35,981 WIN 35,428 RTI-55

Noncocaine analogs Dimethocaine Bupropion Amfonelic acid Methylphenidate Nomifensine Benztropine Mazindol GBR 12909

NE reuptake inhibitors Desipramine Nisoxetine

Citalopram Imipramine Chloripramine Fluoxetine Paroxetine

Atropine Haloperidol

5-HT reuptake inhibitors

Miscellaneous drugs

IC,, (nM) 62.50 2 5.01

7987.72 t 576.56 44.77 ? 4.77

2328.19 2 300.54 25.16 2 3.64 12.49 t 1.69 0.60 t 0.05

460.74 t- 18.41 178.49 f 18.14 160.08 t- 17.79

52.26 2 13.47 37.88 2 2.91 8.70 f 0.86

91.26 t- 3.66

0.79 t- 0.01

1972.39 t- 438.91 156.26 5 32.29

10481.15 f 2692.36

1322.35 t- 191.08 767.10 % 6.38 199.60 f 10.42

3792.83 5 406.68

7011.82 & 1547.80 828.31 t- 39.73

Inhibition of 15 pM ['2511RTI-55 binding in rat striatum by various dopamine, norepi- nephrine, and serotonin reuptake inhibitors and nonrelated drugs. Data are the mean of

trials 2 SE.

1 .oo

80

60

40

20

0 10-12 10-11 10-10 10-3 10-8 10-7 10-6 10-5

COMPETITOR (M)

Fig. 4. Competition of total ['251]RTI-55 binding in rat striatum. Striatal membranes were incubated with 15 pM ['251]RTI-55 and in- creasing concentrations of various inhibitors. The results of a typical experiment are presented; the points are the means of triplicate deter- minations. The fit of this plot was determined using EBDA.

0.99) and significantly (P < 0.0001) t o the log of the IC50 [relative to (-)cocaine, where (-)cocaine = 11 for [3H]DA reuptake (Figure 5). There was no significant correlation with either r3H1NE or L3H15-HT reuptake (corr coeff. = -0.33 and -0.23, respectively).

Cerebral cortical binding Equilibrium of specific [1251]RTI-55 binding in the

cerebral cortex was achieved after 50-60 min at 22-

Carr C o e f . = 0.99 p < 0.0001

-3 -2 -1 0 1 2 3 Relative 13H]DA Uptake I n h i b i t i o n

Fig. 5. Correlation of the log of relative potencies (where cocaine = 1) of various druga in striatal ['261]RTI-55 binding assay with [3HIIIA uptake assay. Values for inhibition of dopamine uptake are from Hyttel et al. (1982) and Andersen (1989).

24°C (Figure 6, top). Analysis of the data with KI- NETIC revealed that a monoexponential model of asso- ciation for [12511RTI-55 in the cerebral cortex best fit the data with a single rate constant kobsl of 1.109 2 0.013 min (n = 2). The dissociation of specifically bound ['2511RTI-55 in the cerebral cortex (Figure 6, bottorn) again was best fit to a single exponential model with a dissociation rate constant kPl of 0.025 f 0.001 min-' (n = 2). Division of the dissociation rate by the associa- tion rate yielded a I(d of 0.3 nM.

Saturation analysis of specific [ 12511RTI-55 binding sites in the cerebral cortex was again performed using a fixed concentration of labeled RTI-55 (15 pM) and in- creasing concentrations of unlabeled RTI-55 (1 pM - 100 nM) (Figure 7). Scatchard transformation of the resulting data (Figure 7 insert) revealed a linear rela- tionship, suggesting a single binding site model of the data. A 2-site model was not able to be fi t t o the data in 4 of the 5 trials. Analysis of the data revealed a single high-affinity site with a I(d of 0.2 nM and a correspond- ing B,, of 2.5 pmoVg protein.

Inhibition of specific [ 1251]RTI-55 binding in the cere- bral cortex demonstrated a pharmacological profile consistent with that of binding to the 5-HT transporter (Table 11). The rank order of inhibitory potency in the cerebral cortex was sertraline > citalopram > paroxe- tine > chloripramine > GBR 12909 > WIN 35,428 :> mazindol > (-)cocaine > imipramine. Dimethocaine and benztropine compounds very selective for the DA transporter (Ritz et al., 1987) were not potent inhibitors of [1251]RTI-55 binding in the cerebral cortex. Com- pounds that were also not potent inhibitors of specific [1251]RTI-55 binding in the cerebral cortex included all NE reuptake inhibitors and all 5-HT receptor antago- nists. The log of the binding IC,, in the cerebral cortex relative to (-)cocaine [where (-)cocaine = 11 correlated highly (corr. coeff. = 0.81) and significantly (P < 0.02)

Page 6: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

J.W. BOJA ET AL. 32

['2511RTI-55 ASSOCIATION

ASSOCIATION TIME ( m i n )

[ 12511 RTI-55 DISSOCIATION W z 0 z H -

m U I

L L b - a : u - w -

H

m m

C I H

a m * l i j + - z - W U U W a

0 20 40 60 DISSOCIATION TIME ( m i n l

Fig. 6. Association (top) and dissociation (bottom) of [12511RTI-55 to rat cerebral cortical membranes. Association of 15 pM [12511RTI-55 was determined by incubating cortical membranes (0.05 mg/ml) a t 22-24°C for various times. The dissociation rate was determined after allowing equilibrium to be reached (50-60 min at 22-24°C) with the addition of 1 pM unlabeled RTI-55 and filtering the samples at various times. Specifically bound ['2511RTI-55 at equilibrium (time 0) was used to determine the percentage of specifically bound [1z511RTI-55 at the various time points. Data from both association and dissociation ex- periments were fitted by the KINETIC program to a monoexponential model. Presented are the results of a typical experiment; points are the means of triplicate determinations.

to the IC,, [again, relative to (-)cocaine, where (-)co- caine = 11 for l3H15-HT reuptake (Figure 8). There was no significant correlation with either [3H]DA (r = -0.25) or L3H1NE (r = -0.26) reuptake and the rank order of inhibition of specific [ 1251]RTI-55 binding in the cerebral cortex.

Receptor autoradiography In the in vitro labeling autoradiographic experiments

(Figure 9), sections incubated with [12511RTI-55 alone sowed high levels of binding in the striatum, olfactory tubercle, and substantia nigra, and lesser levels in the cerebral cortex, hippocampus, brain stem, and thala- mus. Benztropine (400 nM), a compound relatively se- lective for the dopamine transporter rather than the 5-HT transporter, displaced binding from regions rich in dopamine transporter, i.e., striatum and olfactory tubercle. Paroxetine (10 nM), a compound selective for

j o 0 j 80

l o l " f 0 , -\ , 0 I0 20 30 40

EDUNO llnolestagl

Fig. 7. Saturation of specific [lz5I1RTI-55 binding in rat cerebral cortex. Cortical membranes were incubated with a fixed concentration of ['2511RTI-55 (15 pM) and increasing concentrations of unlabeled RTI-55 (1 pM to 100 nM). Nonspecific binding was determined by the addition of 30 pM (-)cocaine. Presented are the results of a typical experiment; points are the means of triplicate determinations. The fit of this plot was determined using EBDA. Insert: Scatchard transfor- mation of the resulting data.

TABLE II. Inhibition of ['251]RTI-55 binding in rat cerebral cortex

DA reuptake inhibitors Dimethocaine Benztropine Nomifensine (-) Cocaine Mazindol WIN 35,428 GBR 12909

Desipramine Nisoxetine

Imipramine Chloripramine Paroxetine Citalopram Sertaline

Methysergide Cinanserin Metergoline Ketanserin

NE reuptake inhibitors

5-HT reuptake inhibitors

5-HT receptor antagonists

IC,, (nM) 34572.33 ? 150.15

1261.84 i- 28.96 1067.58 ? 146.58

60.61 i- 8.06 45.08 ? 1.12 23.70 i 2.04

3.93 i- 0.52

629.12 -+ 56.43 635.70 i- 105.69

62.69 f 0.70 2.05 i- 0.26

0.49 i- 0.08 0.30 '-t 0.05

34842.37 * 4504.56 1986.74 f 46.09 595.07 i 14.86 307.67 5 30.05

1.23 ? 0.35

Inhibition of 15 pM ['2511RTI-55 binding in rat cerebral cortex by various dopamine, norepinephrine, and serotonin reuptake inhibitors and nonrelated drugs. Data are the mean of .%4 trials f SE.

5-HT transport, displaced binding in all regions, except those containing high concentrations of dopamine transporter. When benztropine and paroxetine were added together, all binding was reduced to background levels. These data are compatible with the view that RTI-55 binds to the dopamine transporter in striatum and olfactory tubercle, and to the serotonin transporter in cerebral cortex and some other areas.

DISCUSSION The results of this study confirm and expand upon

the earlier report of the high-affinity binding of [12511RTI-55 to the dopamine transporter (Boja et al.,

Page 7: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

PZ5I]RTI-55 DA AND SEROTONIN BINDING 33

(5,

LT ?.I

GEA 12909 0

Carr Caef = 0 81

P ( 0 0 2

Relative f3H1 5-HT Uptake Inhibition

Fig. 8. Correlation of the log of relative potencies (where cocaine = 1) of various drugs in cerebral cortical 112511RTI-55 binding assay with L3H]5-HT uptake assay. Values for inhibition of serotonin uptake are from Hyttel et al. (1982) and Andersen (1989).

1991). [12511RTI-55 demonstrates binding properties and a pharmacological profile in the striatum that are similar to that shown by t3H]cocaine (Calligaro and Eldefrawi, 1988; Kennedy and Hanbauer, 1983; Ma- dras et al., 1989a; Schoemaker et al., 19851, [3HlWIN 35,065-2 (Ritz et al., 19901, and i3H1WIN 35,428 (Ma- dras et al., 1989b). The & ratio for the high-affinity to low-affinity binding site was 1:38 for [1251]RTI-55. This compares to the ratio of 158 for F3H1cocaine (Madras et al., 1989a1, 1:29 for t3H1WIN 35,065-2 (Ritz et al., 19901, and 1:14 for L3H1WIN 35,428 (Madras et al., 1989b). A ratio of the number of high-affinity binding sites to the number of low-affinity binding sites can be calculated as well. The B,, ratio was 1:11 for [12511RTI-55. This compares to the ratio of 1:15 for [3H]cocaine (Madras et al., 1989a), 1:26 for r3H1WIN 35,065-2 (Ritz et al., 19901, and 1:13 for [3HlWIhJ 35,428 (Madras et al., 1989b). These results suggest that since the & and B,,, ratios of [12511RTI-55 are very similar to the ratio shown by L3H1cocaine, [lZ5I]RTI-55 is binding to the same sites that are recog- nized by L3H]cocaine.

The pharmacological profile demonstrated by [lz51]RTI-55 in the rat striatum is comparable to that shown by [3H]cocaine (Calligaro and Eldefrawi, 1988; Madras et al., 1989a), 13HIWIN 35,065-2 (Ritz et al., 19901, and 13H1WIN 35,428 (Madras et al., 198913). Inhi- bition of specific ['2511RTI-55 binding was stereospe- cific; the (-)isomers of cocaine and WIN 35,065 were more potent than either (+)isomer of these compounds. Furthermore, compounds that were very potent inhibi- tors of specific L3H]cocaine, [3H]WIN 35,065-2, or r3H]WIN 35,428 binding were also potent inhibitors of specific [1251]RTI-55 binding. Such compounds include mazindol, GBR 12909, nomifensine, and methylpheni- date. Mazindol and GBR 12909 displaced total ['251]RTI-55 binding to about the same extent as either

(-)cocaine or WIN 35,428. This observation is in con- trast to that observed by Madras et al. (1989b) with i3H1WIN 35,428, where mazindol and GBR 12909 dis- placed striatal L3HlWIN 35,428 at a level of 95% of that found with cocaine. This observation may reflect a dif- ference between the binding of [12511RTI-55 and r3H]WIN 35,428; certainly this deserves further inves- tigation. Compounds such as desipramine, citalopram, and nisoxetine, which were weak inhibitors of specific [3Hlcocaine, i3H1WIN 35,065-2, or i3H1WIN 35,428 binding, were also weak inhibitors of specific [12511RTI- 55 binding in the striatum. Further support that ['2511RTI-55 in the striatum binds to the dopamine transporter is evidenced by the observation that inhibi- tion of specific [ 1251]RTI-55 binding correlates highly (r = 0.99) with the rank order for inhibition of [3H]DA uptake (Figure 7).

In contrast to that seep in the striatum, [1251]RTI-Ei5 demonstrates binding properties and a pharmacologi- cal profile in the cerebral cortex that are similar to that shown for the serotonin transporter. Previous work by Calligaro and Eldefrawi (1987) demonstrated both a high- and low-affinity binding site for cocaine in the frontal (&high = 14 nM and GIOw = 143 a) and occipital cortices (&hlgh = 17 nM and Glow = 68 nM). In contrast, [12511RTI-55 only recognized a high-affinity binding site in the cerebral cortex with a Ifd of 0.2 nM. The number of binding sites in the cerebral cortex rec- ognized by [12511RTI-55 (B,,, = 28.98 fmol/mg tissue) corresponds roughly to the number of serotonin up- take sites (B,,, = 338 fmol/mg protein) recognized by [3Hlparoxetine (Battaglia et al., 1987).

The pharmacological profile for inhibition of specific [1251]RTI-55 binding in the cerebral cortex did not match that of the norepinephrine or dopamine trans- porter, but instead matched that of the serotonin trans- porter (Habert et aL, 1985; D'Amato et al., 1987). Drugs such as imipramine, citalopram, chloripramine, and sertraline, which are potent inhibitors of serotonin re- uptake, were all potent inhibitors of specific [12511RTI- 55 binding. The specific dopaminergic selective drugs, benztropine and dimethocaine (Ritz et al., 19871, while potent inhibitors of specific [12511RTI-55 binding in the striatum, were not potent inhibitors in the cortex. Fur- ther evidence that [lz5I1RTI-55 in the cerebral cortex binds to the serotonin transporter is that the potency of inhibition of specific [ 1251]RTI-55 binding in the cere- bral cortex correlates well (r = 0.81) with the potency for inhibition of [3H15-HT uptake (Figure 8).

In preliminary experiments, the specific binding of [lZ5I]RTI-55 in other brain regions, such as the nucleus accumbens, hypothalamus, and prefrontal cortex, is riot as clear as that in either the striatum or cortex. Since the binding profile of [1251]RTI-55 is much iike that of cocaine, ['2511RTI-55 could bind to several different sites. In preliminary studies, the inhibition curves pro- duced by [1251]RTI-55 in these areas are often biphasic

Page 8: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

Figure 9.

Page 9: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

1lZ5I1RTI-55 DA AND SEROTONIN BINDING 3 5

and therefore are difficult to interpret. Work is cur- rently underway to resolve the binding characteristics of [lZ5I1RTI-55 in these regions.

Recent in vivo binding studies using [1251]RTI-55 con- firm our findings that RTI-55 binds to both dopaminer- gic and serotonergic transporters. The striatum dis- played binding to the dopamine transporter (Cline et al., 1992b) and the cerebral cortex demonstrated binding to the serotonin transporter (Scheffel et al., 1992). SPECT studies utilizing [lZ5I]RTI-55 (Innis et al., 1991) also found significant binding in the striatum, hypothalamus, and cortex.

The pattern of specific [ 1251]RTI-55 binding in the autoradiographic experiments provides additional evi- dence that [1251]RTI-55 binds both to the dopamine and serotonin transporters. Displacement of [ 1251]RTI-55 specific binding by benztropine (400 nM), a relatively specific inhibitor of the dopamine transporter (Ritz et al., 1987), occurred in areas rich in the dopamine transporter, such as the striatum, nucleus accumbens, and olfactory tubercle. Specific binding in areas that have few dopamine transport sites, such as the thala- mus, hippocampus, and cerebral cortex, was relatively unaffected by benztropine. The pattern of [12511RTI-55 binding to areas rich in the dopamine transporter corre- sponds to that seen with L3H1WIN 35,428 or ["FIGBR 13119 (Canfield et al., 1990; Ciliax et al., 1990). Dis- placement of [12511RTI-55 specific binding by paroxe- tine (10 nM) occurred in areas rich in the serotonin transporter, such as the thalamus, hippocampus, and cerebral cortex. Specific binding in areas that have rel- atively fewer serotonin transport sites, such as the stri- atum and olfactory tubercle, was relatively unaffected by paroxetine. However, it must be pointed out that [lZ5I]RTI-55, like cocaine, may bind to other sites in different regions of the brain. Also binding sites other than dopamine and serotonin may be present in small, undetectable quantities in the striatum or the cerebral cortex.

Since [1251]RTI-55 binds to both serotonin and dopamine transporters with high affinity, it is an excel- lent probe for the cocaine binding sites. However, its use as a routine assay tool for either the dopamine o r serotonin transporter could be problematic. However, one site can be pharmacologically blocked by the addi- tion of appropriate drugs. For example, for measuring

Fig. 9. In vitro autoradiographic localization of [1251JRTI-55 binding. Top section is autoradiograms generated by incubation with 50 pM [lZ5I IRTI-55 showing high levels of binding in striatum, olfactory tu- bercle, substantia nigra, and lesser levels in cerebral cortex, hippo- campus, and thalamus. The second section demonstrates selective blockade of the dopamine transporter with 400 nM benztropine, thus revealing apparent serotonergic binding of ['2511RTI-55. The third section shows the selective blockade of the serotonin transporter by 10 nM paroxetine, revealing apparent dopaminergic binding of [12511RTI- 55. The addition of both 400 nM benztropine and 10 nM paroxetine eliminates all 112511RT1-55 binding, bottom section. Bar = 1 mm.

dopamine transporters the addition of 10 nM par0x.e- tine will selectively block potential binding to the sero- tonin transporter. Similarly, for the assay of serotonin transporters, the addition of 5 FM dimethocaine or 400 nM benztropine will preferentially block dopamine transporters.

In summary, these results indicate that [1251]RTI-!S5 labels a high- and low-affinity binding site in the stria- tum that appears to be the cocaine binding site on the dopamine transporter. Furthermore, [1251]RTI-55 binds to a single high-affinity site in the cerebral cortex. The very high affinity and high specific activity suggest that [12511RTI-55 will be a very useful ligand for cocai:ne binding sites.

ACKNOWLEDGMENTS The authors thank Drs. Elizabeth Cline and Bertha

K. Madras for helpful discussions. This work was su.p- ported in part by NIDA grant DA05477 to F.I. Carroll.

REFERENCES Andersen, P.H. (1987) Biochemical and pharmacological characteriza-

tion of L3HJGBR 12935 binding in vitro to rat striatal membranes: Labeling of the dopamine uptake complex. J. Neurochem., 48:1887- 1896.

Andersen, P.H. (1989) The dopamine uptake inhibitor GBR 12909: Selectivity and molecular mechanism of action. Eur. J . Pharmacol., 166:493604.

Battaglia, G., Yeh, S.Y., OHearn, E., Molliver, M.E., Kuhar, M.J., amd De Souza E.B. (1987) 3,4-Methylenedioxymethamphetamine and 3,4-methyIenedioxyamphetamine destroy serotonin terminals in rat brain: Quantification of neurodegeneration by measurement of 13Hlparoxetine-labeled serotonin uptake sites. J. Pharmacol. Exp. Ther., 242:911-916.

Bergman, J., Madras, B.K., Johnson, S.E., and Spealman, R.D. (1989) Effects of cocaine and related drugs in nonhuman primates. III. Self-administration by squirrel monkeys. J . Pharmacol. Exp. Th8er., 219:150-155.

Boja, J.W., Carroll, F.I., Rahman, M.A., Philip, A,, Lewin, A.H., and Kuhar, M.J. (1990) New, potent cocaine analogs: Ligand binding and transport studies in rat striatum. Eur. J. Pharmacol., 184:3%9- 332.

Boja, J.W., Patel, A., Carroll, F.I., Rahman, M.A., Lewin, A.H., and Kuhar, M.J. (1991) ['"I]RTI-55: A potent ligand for dopamine transporters. Eur. J. Pharmacol. 194:133-134.

Calligaro, D.O. and Eldefrawi, M.E. (1987) Central and peripheral cocaine receptors. J . Pharmacol. Exp. Ther., 243:61-67.

Calligaro, D.O. and Eldefrawi, M.E. (1988) High affinity stereospecific binding of L3Hlcocaine in striatum and its relationship to the dopa- mine transporter. Membr. Biochem., 7:87-106.

Canfield, D.R., Spealman, R.D., Kaufman, M.J., and Madras, E:.K. (1990) Autoradiographic localization of cocaine binding sites by I3H1CFT (I3H1WIN 35,428) in the monkey brain. Synapse, 6:189- 195.

Carroll, F.I., Lewin, A.H., Boja, J.W., and Kuhar, M.K. (1992) Coca.ine receptor: Biochemical characterization and structure-activity rela- tionships for the dopamine transporter. J . Med. Chem., 6:969-981.

Carroll, F.I., Rahman, M.A., Abraham, P., Parham, K., Lewin, A.H., Dannals, R.F., Shaya, E., Scheffel, U., Wong, D.F., Boja, J.W., and Kuhar, M.K. (1991) [12"113~-(4-iodophenyl)tr~pan-2~-carboxylic acid methyl ester (RTI-551, a unique cocaine receptor ligand for imaging the dopamine and serotonin transporters in vivo. NIed. Chem. Res., 1:289-294.

Ciliax, B.J., Kilbourn, M.R., Haka, M.S., and Penney, J.B., Jr . (1990) Imaging the dopaminergic uptake site with ex vivo ['"FIGBR 13119 binding autoradiography in rat brain. J. Neurochem., 55:619-623.

Clarke, R.L., Daum, S.J., Gambino,A.J., Aceto, M.D., Pearl, J., Levitt, M., Cumiskey, W.R., and Bogado, E.F. (1973) Compounds affecting the central nervous system. 4. 3-Beta-phenyltropane-2-carboxylic esters and analogs, J . Med. Chem., 16:1260.

Cline, E.J., Scheffel, U., Boja, J.W., Carroll, F.I., Katz, J.L., and Kuhar, M.J. (1992a) Behavioral effects of novel analogs: A compari-

Page 10: High-affinity binding of [125I]RTI-55 to dopamine and serotonin transporters in rat brain

36 J.W. BOJA ET AL.

son with in vivo receptor binding potency. J . Pharmacol. Exp. Ther., 260:1174-1179.

Cline, E.J., Scheffel, U., Boja, J.W., Mitchell, W.M., Carroll, F.I., Abra- ham, P., Lewin, A.H., and Kuhar, M.J. (1992b) In vivo binding of L'251JRTI-55 to dopamine transporters: Pharmacology and regional distribution with autoradiography. Synapse, 12:37-46.

D'Amato, R.J., Largent, B.L., Snowman, A.M., and Snyder, S.H. (1987) Selective labeling of serotonin uptake sites in rat brain by L"H1citalo- pram contrasted to labeling of multiple sites by [3H]imipramine. J. Pharmacol. Exp. Ther., 242:364-371.

Dubocovich, M.L. and Zahniser, N.R. (1985) Binding characteristics of the dopamine uptake inhibitor L3Hlnomifensine to striatal mem- branes. Biochem. Pharm., 34:1137-1144.

Habert, E., Graham, D., Tahraqui, L., Claustre, Y., and Langer, S.Z. (1985) Characterization of ["Hlparoxetine binding to rat cortical membranes. Eur. J. Pharmacol., 118:107-114.

Hyttel, J . (1982) Citalopram-Pharmacological profile of a specific serotonin uptake inhibitor with antidepressant activity. Prog. Neuro-Psychopharmacol. Biol. Psychiat., 6:277-295.

Innis, R., Baldwin, R., Sybirska, E., Zea, Y., Laruelle, M., Al-Tikriti, Charney, D., Zoghbi, S., Smith, E., Wisniewski, G., Hoffer, P., Wang, S., Milius, R., and Neumeyer, J. (1991) Single photon emission com- puted tomography imaging of monoamine reuptake sites in primate brain with [lz3I]CIT. Eur. J . Pharmacol., 200:369-370.

Janosky, A., Berger, P., Vocci, F., Labarca, R., Skolnick, P., and Paul, S.M. (1986) Characterization of sodium-dependent ["HIGRB-12935 binding in brain: A radioligand for selective labeling of the dopam- ine transport complex. J . Neurochem., 46:1272-1276.

Javitch, J.A., Blaustein, R.O., and Snyder, S.H. (1984) [3H1Mazindol binding associated with neuronal dopamine and norepinephrine up- take sites. Mol. Pharmacol., 26:35-44.

Kennedy, L.T. and Hanbauer, I. (1983) Sodium-sensitive cocaine bind- ing to rat striatal membrane: Possible relationship to dopamine uptake sites. J. Neurochem., 41:172-178.

Kuhar, M.J., and Unnerstall, J.R. (1990) Receptor autoradiography. In: Methods in Neurotransmitter Analysis. H. Yamamura editor, Raven Press, New York, pp. 177-218.

Madras, B.K., Fahey, M.A., Bergman, J., Canfield, D.R., and Speal- man, R.D. (1989a) Effect of cocaine and related drug in nonhuman primates. I. ["Hlcocaine binding sites in caudate-putamen. J . Phar- macol. Exp. Ther., 251:131-141.

Madras, B.K., Spealman, R.D., Fahey, M.A., Neumeyer, J.L., Saha, J.K., and Milius, R.A. (1989b) Cocaine receptors labeled by 13H]2p- carbomethoxy-3~-(4fluorophenyl)tropane. Mol. Pharmacol., 36 518-524.

Misra, A.L., Giri, V.V., Patel, M.N., Alluri, V.R., and Mule, S.J. (1977) Disposition and metabolism of 13Hlcocaine in acutely and chroni- cally treated monkeys. Drug Alcohol Depend., 2:261-272.

Reith, M.E.A., Shershen, H., and Lajtha, A. (1980) Saturable L3H]co- caine binding in central nervous system of mouse. Life Sci., 27:1055-1062.

Ritz, M.C., Lamb, R.J., Goldberg, S.R., and Kuhar, M.J. (1987) Cocaine receptors on dopamine transporters are related to self-administra- tion of cocaine. Science, 237:1219-1223.

Ritz, M.C., Boja, J.W., Grigoriadis, D.E., Zaczek, R., Carroll, F.I., and Kuhar, M.J. (1990) [3HlWIN35,065-2: A ligand for cocaine receptors in striatum. J . Neurochem., 55:1556-1562.

Schoemaker, H., Pimoule, C., Arbilla, S., Scattom, B., Javoy-Agid, F., and Langer, S.Z. (1985) Sodium dependent [3Hlcocaine binding as- sociated with dopamine uptake sites in the rat striatum and human putamen decrease after dopaminergic denervation and in Parkin- son's disease. Naunyn Schmiedebergs Arch. Pharmacol., 329:227- 235.

Scheffel, U., Dannals, R.F., Cline, E.J., Ricaurte, G.A., Carroll, F.I., Abraham, P., Lewin, A.H., and Kuhar, M.J. (1992) ['23/1251]RTI-55, an in vivo label for the serotonin transporter. Synapse, 11:134-139.