JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 JPETThis Fast article Forward. has not been Published copyedited andon formatted. May 20, The 2008 final asversion DOI:10.1124/jpet.108.138842 may differ from this version.

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Title page

In vivo characterization of a novel phenylisothiocyanate analog at

monoamine transporters in rat brain

Vishakantha Murthy, Thomas J. Martin, Susy Kim, Huw M.L. Davies, and Steven R. Childers Downloaded from

Departments of Physiology/Pharmacology (VM, SRC) and Anesthesiology (TJM, SK), and

Center for the Neurobiological Investigation of Drug Abuse, jpet.aspetjournals.org

Wake Forest University Health Sciences, Winston-Salem, NC 27157;

Department of Chemistry (HMLD), State University of New York at Buffalo, at ASPET Journals on September 26, 2021

1

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version.

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Running Title: Irreversible blockade of monoamine transporters

Corresponding author: Dr. Steven R. Childers

Department of Physiology/Pharmacology

Wake Forest University Health Sciences

Medical Center Blvd.

Winston-Salem, NC 27157

Ph 336-716-3791, Fax 336-716-0237 Downloaded from

[email protected]

jpet.aspetjournals.org Text pages: 35

Tables: 4

Figures: 6 at ASPET Journals on September 26, 2021

References: 42

Abstract: 245 words

Introduction: 745 words

Discussion: 1567 words

Abbreviations: DAT, transporter; SERT, ; GTPγS, guanosine 5’

-O-(3-[35S]thiotriphosphate; DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; DPCPX, 8- cyclopentyl-1,3-dihydroxylxanthine; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin; NPA,

R(-)-propylnorapomorphine.

JPET section assignment: Neuropharmacology

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Abstract

Previous studies have shown that the phenylisothiocyanate tropane analog HD-205 binds covalently to dopamine and serotonin transporters (DAT and SERT) in rat brain membranes

(Murthy et al., 2007). The present studies evaluated the irreversible effects of HD-205 in vivo in rats after intracranial injection. Rats were implanted with unilateral cannulae in rat striatum, and

HD-205 (0.001 to 3 nmol) was administered by intrastriatal injection. In vitro autoradiography of

DAT binding with [125I]RTI-55 on brain sections obtained 24 hr after injection showed a highly Downloaded from localized blockade of binding in striatum, with maximal blockade of binding by 1-3 nmol HD-

205. Similar blockade of SERT binding (using [3H]) was observed in the same area. jpet.aspetjournals.org

No blockade of DAT or SERT binding was observed after intrastriatal injections of the reversible analog HD-206, and HD-205 treatment had no effect on D2- and mu -stimulated

γ [35S]GTP S binding in sections from the same animals. In a time course study, rats administered at ASPET Journals on September 26, 2021 with 1 nmol HD-205 showed recovery of 50% of DAT binding after 3-4 days post-injection, and full recovery after 6 weeks. Rats implanted with bilateral cannulae were tested for - induced locomotor activity. Two days after intrastriatal injection of 1 nmol HD-205, systemic

(20 mg/kg i.p.) cocaine-induced locomotor activity was not affected; however, locomotor activity induced by intrastriatal administration of cocaine (6 nmol) was eliminated. This strategy of site-specific chemical blockade of transporters could serve as a valuable tool to evaluate the neuroanatomical basis of DAT-mediated cocaine effects.

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Introduction

Although cocaine binds to dopamine, serotonin and transporters (DAT,

SERT, and NET, respectively) with approximately equal affinities, it is hypothesized that an increase in extracellular dopamine levels due to blockade of DAT is responsible for its psychostimulant properties (Ritz et al., 1987). The role of DAT in these effects has been studied in DAT knockout mice, which exhibit both a higher level of baseline locomotor activity, as well as a blockade of cocaine-induced locomotor activity (Rocha et al., 1998; Carboni et al., 2001). Downloaded from

Recent behavioral studies using a mutant DAT knock-in mouse strategy eliminated cocaine effects of locomotor activity and conditioned place preference (Chen et al., 2006). Therefore, jpet.aspetjournals.org

DAT is a primary target to study the mechanisms involved in the psychostimulant properties of cocaine, and serves as a target for development of therapeutic agents to treat cocaine addiction.

Various structure-activity studies have resulted in the synthesis of cocaine analogs with at ASPET Journals on September 26, 2021 differing affinities and selectivities at monoamine transporters (Boja et al., 1990a; Davies et al.,

1993). These analogs have made important contributions to understanding the role of DAT in mediating cocaine actions. For example, some tropane analogs have been used as radioligands at monoamine transporters, both in vitro (Boja et al., 1991; Letchworth et al., 2000) and in vivo

(Spencer et al., 2007). Other studies have focused on acute (Daunais et al., 1998) and chronic

(Porrino et al., 1994; Hemby et al., 1995; O'Connor et al., 2004; O’Connor et al., 2005) effects of tropane analogs as psychostimulants, and several analogs have been reported to attenuate cocaine’s effects in animal models of psychostimulant abuse (Desai et al., 2005; Carroll et al.,

2006). Synthesis of using novel schemes involving rhodium catalysts (Davies et al.,

1994) has resulted in a particularly diverse set of analogs, one of which, the 2-napthyl analog

WF-23, is one of the most potent ligands at DAT and SERT (Bennett et al., 1995).

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One class of tropane analogs is designed as irreversible ligands at monoamine transporters. These compounds include azido-based photoaffinity ligands such as [125I]DEEP,

[125I]AD-96-129 (GBR-based derivatives) and [125I]GA-II-34 (a benztropine derivative),

[125I]RTI-82, [125I]MFZ-2-24 (tropane derivatives) (Vaughan et al., 2001; Parnas et al., 2008) as well as isothiocyanate and bromoacetamide analogs such as and tropane derivatives

(Boja et al., 1990b; Husbands et al., 1997; Lever et al., 2005). These compounds provide an

opportunity to precisely map the location of the binding site of cocaine on DAT. For example, Downloaded from recent studies with photoaffinity ligands [125I]RTI-82 and [125I]MFZ-2-24 have identified TM6 and TM1 as binding sites respectively on DAT (Vaughan et al., 2007; Parnas et al., 2008), while jpet.aspetjournals.org similar studies with [125I]DEEP, and [125I]GA-II-34, have implicated TM1 and TM2 for cocaine binding sites on DAT (Vaughan et al., 2005).

In a recent study (Murthy et al., 2007), we reported on the synthesis and pharmacological at ASPET Journals on September 26, 2021 properties of HD-205, a phenylisothiocyanate analog of WF-23 (Fig. 1). Consistent with the actions of an irreversible ligand, HD-205 produced wash-resistant blockade of radioligand binding at DAT, SERT and NET in rat brain membranes. Moreover, its ability to covalently label DAT was confirmed by synthesis of a [125I] derivative of HD-205 that labeled DAT protein after SDS gel electrophoresis of rat striatal membranes (Murthy et al., 2007).

While irreversible analogs have been extensively studied to understand the binding sites of cocaine on DAT, little is known about the in vivo properties of these analogs. Intracranial injection of such compounds could produce a long-lasting (but not permanent) blockade of monoamine transporters and thus provide an opportunity to examine the role of specific transporters in the behavioral effects of cocaine. Such studies would not be practical for photoaffinity agents, but could be feasible for alkylating agents like phenylisothiocyanates and

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JPET #138842 bromoacetamides. In one study, the phenylisothiocyanate tropane analog RTI-76, was administered by intrastriatal injection to produce a prolonged blockade of DAT binding in striatal membranes, with a half life of 6 days (Fleckenstein et al., 1996b). Similar results, with somewhat shorter time course of action, were obtained with intracerebroventricular (i.c.v.) administration on RTI-76, which also produced increases in locomotor activity in rats (Kimmel et al., 2000). However, no detailed anatomical studies have been performed to show that

irreversible tropanes could be used to trace neuroanatomical loci of cocaine actions in brain. Downloaded from

In the present studies, we examine in vivo effects of the phenylisothiocyanate analog HD-

205 after direct injection into rat striatum. Using autoradiography, these results show the jpet.aspetjournals.org localization of the effects of an irreversible tropane analog on transporter binding in striatum.

Moreover, these results also demonstrate that intrastriatal administration of an irreversible drug blocks the locomotor effects of cocaine when injected into the same site, thus providing the at ASPET Journals on September 26, 2021 potential for using this compound to explore the neuroanatomical localization of the role of DAT in mediating behavioral effects of cocaine.

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Materials and Methods

Materials

HD-205 and HD-206 were synthesized as previously described (Murthy et al., 2007).

[35S]GTPγS (1150 Ci/mmol), [125I]RTI-55 (2200 Ci/mmol), [3H]citalopram (81.2 Ci/mmol),

Kodak BioMax MS Films, Kodak BioMax HE TranScreens, and TR tritium sensitive phosphor screens were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Downloaded from Pentobarbital (Nembutal) was purchased form Abbott laboratories (North Chicago, IL), and penicillin G procaine was purchased from Butler Vet (Colombus, OH). Intracranial stainless steel guide cannulae were purchased from Plastics One (Roanoke, VA). Citalopram hydrobromide, jpet.aspetjournals.org

DAMGO, 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), , GDP, R(-)- propylnorapomorphine (NPA), and 8-OH-DPAT were obtained from Sigma-Aldrich (St. Louis,

MO). X-ray films were obtained from Phenix Research (Hayward, CA). Other chemicals used at ASPET Journals on September 26, 2021 were reagent grade chemicals from Sigma and Fisher.

Animals

Male Fisher F-344 rats (Harlan, Indianapolis, IN), 270-350 g, were used in all studies.

Animals were pair-housed before surgery and singly housed post surgery in a climate-controlled room with a 12-h light/dark cycle. Food and water were available ad libitum except for the locomotor chamber sessions. All animals were adapted to vivarium conditions for seven days before surgery. Injections and testing occurred during the dark phase of the cycle (5:00 AM –

5:00 PM). All procedures were carried out in accordance with established practices as described in the National Institutes of Health Guide for Care and Use of Laboratory Animals, reviewed and approved by the Animal Care and Use Committee of Wake Forest University.

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Intracranial surgery and drug treatment

Rats were anesthetized with a combination of atropine methyl nitrate (10.0 mg/kg; i.p.) and pentobarbital (50.0 mg/kg; i.p.) and placed into a stereotaxic frame (Stoelting, Wood Dale,

IL). Cannulae (unilateral cannula for autoradiography studies, and bilateral cannulae in locomotor studies) were implanted into striatum (stereotaxic coordinates: lambda, + 7.5 mm, midline ± 3.0 mm, depth - 4.5 mm from skull). Cannulae were secured to the skull with stainless

steel screws and dental acrylic and the skin incision was closed with 4.0 chromic gut suture. Downloaded from

Animals were administered 75,000 U of penicillin G procaine i.m. and allowed to recover from surgery for at least 7-10 days prior to their involvement in any experiments. Placement of jpet.aspetjournals.org cannulae was verified by initial set of autoradiography experiments with [125I]RTI-55 binding

(see below).

In all experiments involving intrastriatal administration of tropanes, drugs (HD-205 and at ASPET Journals on September 26, 2021

HD-206) were dissolved in DMSO as vehicle. Cocaine and citalopram hydrobromide were dissolved in 0.9% saline. For autoradiography experiments, animals received a single unilateral administration of 1 µl of either HD-205 or HD-206 (at the doses indicated in Results), or vehicle.

Rats were euthanized at various time points after drug administration (1 day to 6 weeks), and brains were removed and frozen for autoradiography as described below. For studies of cocaine- induced locomotor activity, animals received one injection of systemic cocaine administration

(20 mg/kg i.p.), then 2-5 days later received bilateral intrastriatal injections of HD-205 (1 nmol in 1 µl) or vehicle (1 µl). 2 days later, rats received another systemic injection of cocaine (20 mg/kg i.p.), and then 5 days later received bilateral intrastriatal injections of cocaine (2 µg, or 6 nmol). All the intrastriatal injections were administered via internal cannulae connected to a

Hamilton syringe through polyethylene tubing. All injections were administered at a flow rate of

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0.2 µl/min and the injector was left in place 5 min after the injection to allow for pressure equilibration.

Radioligand autoradiography

For autoradiography experiments, rats were euthanized by rapid decapitation; brains were removed immediately and immersed slowly in isopentane at -40º to -50º. Coronal sections (20

µm thickness) were made at the level of striatum using a cryostat at -20º and thaw-mounted onto Downloaded from gelatin-coated slides. The sections were stored in -80º until use. In autoradiography experiments, triplicate sections of brain from at least 5 animals were used for each assay. DAT binding was jpet.aspetjournals.org performed using [125I]RTI-55 (Boja et al., 1991), with fluoxetine to block binding of [125I]RTI-55 at SERT (Boja et al., 1992; Tatsumi et al., 1997). Initial results of [125I]RTI-55 binding without fluoxetine showed binding outside the striatum in cortical regions; this binding was eliminated at ASPET Journals on September 26, 2021 by addition of fluoxetine (data not shown). Sections were preincubated for 10 min in TME buffer

(50 mM Tris-HCl, 3 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, pH 7.4), then 0.01 nM

[125I]RTI-55 and 30 nM fluoxetine were added, and the sections were incubated at 25º for 2 hr.

Nonspecific binding was assessed with 1 µM WF-23. After incubation, sections were washed and dried for exposure on phosphoimaging screens and X-ray film.

SERT binding was determined with [3H]citalopram autoradiography on frozen sections

(D'Amato et al., 1987). Sections were preincubated for 10 min in TME buffer, then

[3H]citalopram (0.4 nM) was added and the sections were incubated at 25º for 2 hr. Nonspecific binding was assessed with 1 µM WF-23. Sections were dried and exposed to TR tritium sensitive phosphor screens for 3 weeks.

To determine potential effects of HD-205 treatment on receptor-coupled G-proteins in

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JPET #138842 striatum, agonist-stimulated [35S]GTPγS autoradiography was performed in rat brain sections

(Sim et al., 1995). Sections were preincubated for 10 min in TME buffer, then 15 min with 1 mM

GDP and 1 M 8-cyclopentyl-1,3-diproxylxanthine (DPCPX) at 25º. Agonists were then added, which included 10 µM N-propylapomorphine (NPA) for D2 receptors, 10 µM DAMGO for mu opioid receptors, and 3 µM 8-hydroxy-DPAT for 5-HT1A receptors. Sections were incubated for

90 min at 30º for D2 (Rinken et al., 1999) and 120 min at 25º for mu and 5-HT1A (Sim et al.,

1997). The sections were then washed, exposed to phosphoimaging screens or X-ray film, and Downloaded from analyzed as described previously (Sim et al., 1995).

In all autoradiography experiments, slides were exposed to a phosphoimaging screen, jpet.aspetjournals.org scanned after 72 hrs using Cyclone Storage Phosphor System with OptiQuant image analysis software (version 03.10). Film cassettes contained [14C] microscales for densitometric analysis.

Images were then imported and analyzed in NIH Image J (version 1.30 for MacIntosh). at ASPET Journals on September 26, 2021

Quantification of autoradiograms from unilateral implants allowed each animal to serve as its own control. The effect of drug on the injected side of the striatum was measured by enclosing the area affected using visual inspection, and area was calculated as number of square pixels.

Optical density (OD) in the affected area was calculated by measuring OD values in identical areas of the injected and contralateral sides, subtracting background OD values, and expressing the resulting values as per cent OD of contralateral side in each autoradiogram.

Locomotor Activity

Exploratory behavior was assessed using commercially available equipment and software

(Med Associates In., St. Albans, VT) as previously described (Martin et al., 2004). Briefly, animals were placed in activity chambers equipped with duplicate banks of 16 infrared

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JPET #138842 transmitters spaced 2.5 cm apart with aligned detectors on the opposing sides of the chamber.

Data (locomotor activity) were collected in 6 min bins for 1 hr, and included total distance traveled in the X-Y plane, total beam breaks in the X-Y plane (ambulatory counts/horizontal activity). Locomotor chambers were located within sound- and light-attenuating chambers that contained a ventilator fan to mask extraneous room noise. To acclimate rats to locomotor chambers, hourly sessions were conducted daily (at least 10-15 days) until baseline activity did

not vary by more than 15% of the mean across 5 successive days before any experimentation Downloaded from began.

jpet.aspetjournals.org Statistical Analysis

All densitometric data from autoradiograms are expressed as mean results of triplicate sections from the same animal, with 5-9 animals per groups as described in Results. All at ASPET Journals on September 26, 2021 behavioral data are expressed as mean values of activity, expressed as per cent of average baseline values in each animal, with 5-6 rats per group. Significant differences between groups were determined with a p < 0.05 by a repeated measures one-way ANOVA, with post-hoc analysis using Tukey-Kramer HSD, calculated by JMP statistical software (Cary, NC).

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Results

Effects of HD-205 on DAT and SERT binding in rat striatum

Our previous studies (Murthy et al., 2007) showed that the phenylisothiocyanate tropane analog HD-205 binds covalently to DAT in rat brain membranes. To determine whether this compound produced irreversible effects on DAT in vivo, we administered HD-205 (as well as the reversible control analog HD-206) directly into rat striatum using unilateral indwelling cannulae, and examined the resulting blockade of DAT binding by in vitro autoradiography at several time Downloaded from points after injection. Fig. 2A shows the results of injection of 1 nmol HD-205 or HD-206 compared to vehicle (DMSO) on [125I]RTI-55 binding to DAT, with sections prepared from rats jpet.aspetjournals.org

24 hrs after injection. Administration of HD-205 produced a highly localized blockade of

[125I]RTI-55 binding in striatum, while injection of HD-206 or vehicle had no discernible effect

125 on [ I]RTI-55 binding measured 24 hr after injection. The rostral-caudal spread of the effects of at ASPET Journals on September 26, 2021

HD-205 was examined in multiple adjacent sections on either side of the site of injection (data not shown), which showed decreasing effect of HD-205 until it was virtually absent 600 µm away from the injection site.

To confirm that the effect of HD-205 administration was restricted to binding, adjacent sections from HD-205-treated animals were assayed for agonist-

γ stimulated [35S]GTP S binding. Fig. 2B shows typical autoradiograms for both D2- and mu opioid-stimulated [35S]GTPγS binding in sections taken from rats 24 hr after treatment with 1 nmol HD-205. These results show that although HD-205 administration produced highly localized blockade of DAT binding with [125I]RTI-55, there was no effect of HD-205

γ administration on either D2- or mu opioid-stimulated [35S]GTP S binding in sections from the same animals. These data confirmed that blockade of DAT binding by in vivo administration of

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HD-205 was not the result of non-specific tissue damage.

Effects of HD-205 on [125I]RTI-55 binding were quantified using densitometric analysis of autoradiograms of brain sections prepared from rats 24 hr after administration of various doses

(0.001 to 3 nmol in 1 µl DMSO) of HD-205. Since these were all unilateral administrations, each animal served as its own control, and results were expressed as per cent of [125I]RTI-55 binding on the contralateral side. Results (Fig. 3, top) showed that increasing doses of HD-205 increased

125 the area of blockade of [ I]RTI-55 binding in striatum, with 1-3 nmol of HD-205 providing Downloaded from maximal area of blockade. In contrast, there was no dose-related effect of HD-205 on optical density of binding in the affected area of striatum (Fig. 3, bottom). Representative sections are jpet.aspetjournals.org shown on the right (Fig. 3). These results are consistent with the effects of an irreversible ligand, which at sufficient doses would be predicted to block virtually all of specific [125I]RTI-55 binding within the immediate area of injection. at ASPET Journals on September 26, 2021

Our previous results (Murthy et al., 2007) showed that HD-205 produced irreversible blockade of radioligand binding to both DAT and SERT in brain membranes. To determine whether in vivo administration of HD-205 blocked binding to SERT as well as DAT, SERT binding was assayed with [3H]citalopram in brain sections obtained from rats 24 hr after administration of 1 nmol HD-205. Results (Fig. 4) showed highly localized blockade of

[3H]citalopram binding in the area of injection with HD-205, similar to blockade of DAT binding observed in the same sections. These results confirm blockade of both SERT and DAT binding by in vivo treatment of HD-205, as predicted from in vitro effects of HD-205 on DAT and SERT

(Murthy et al., 2007). Because HD-205 had very weak irreversible effects on NET binding in vitro (Murthy et al., 2007), NET binding was not performed in the present study.

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Time course effects of HD-205 in rat striatum

Irreversible ligands would be expected to produce a prolonged blockade of transporters, but this blockade should not be permanent. To determine the recovery of DAT binding after intrastriatal injection of HD-205, rats were sacrificed at different time points (one day to six weeks) after unilateral injection of 1 nmol HD-205. The results (Fig. 5) are plotted as area of striatum affected by HD-205 administration. The results show that DAT binding does recover in

proportion to the time after HD-205 injection, with approximately 50% recovery of DAT binding Downloaded from

3-4 days after injection, approximately 80% recovery after 14 days, and full recovery 42 days (6 weeks) after injection. These results confirm that blockade of DAT binding by HD-205 treatment jpet.aspetjournals.org was not permanent, with an estimated half-life of recovery of 3-4 days.

Long-term blockade of DAT by intrastriatal injection of HD-205 would, in theory, lead to chronic elevated dopamine levels that could potentially produce long-term changes in dopamine at ASPET Journals on September 26, 2021 receptor response, including desensitization. To test this possibility, brain sections were assayed

γ for D2-stimulated [35S]GTP S binding taken from rats 7 and 14 days following HD-205

γ injection. There were no effects on levels of D2-stimulated [35S]GTP S binding in sections prepared 7 days after HD-205 treatment (data not shown), but there was a small but significant

γ decrease (20%) in D2-stimulated [35S]GTP S binding in sections prepared 14 days after HD-205 treatment, measured in striatum on the side ipsilateral to HD-205 injection (Table 1). To determine whether this effect was specific to D2 receptor system, adjacent sections from the same animals were assayed for mu opioid-stimulated [35S]GTPγS binding; results (Table 1) showed no significant effect of HD-205 treatment on this system. Although in theory, HD-205 would also produce desensitization of other monoamine receptor systems, including 5-HT1A and

γ α2-adrenergic (O’Connor et al., 2005), the signals for agonist-stimulated [35S]GTP S binding in

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JPET #138842 striatal sections were too low to be measured accurately for these receptors (data not shown).

Effects of HD-205 treatment on intrastriatal cocaine-induced locomotor activity

One of the important goals of this study was to determine whether a highly localized irreversible blockade of DAT by HD-205 could affect cocaine’s behavioral effects. To explore this question, rats were tested for cocaine-induced locomotor activity following intrastriatal

injection of HD-205 or vehicle (DMSO). The results of these experiments are shown in Table 2. Downloaded from

As a control for cocaine-induced behavioral effects, rats in both groups were tested for locomotor activity induced by i.p. injection of 20 mg/kg cocaine before treatment with HD-205 jpet.aspetjournals.org or DMSO. Results (Table 2: i.p. cocaine PRE column) showed that cocaine produced large increases in activity in both groups. Then, 2-3 days after these cocaine pretreatments (a period in which all rats were tested to make sure that the locomotor activity was completely back to at ASPET Journals on September 26, 2021 baseline), 1 µl of either DMSO or HD-205 (1 nmol) was administered bilaterally directly into striatum. Both groups were tested for locomotor activity immediately after the intrastriatal injections to test the direct effects of HD-205 on locomotor activity. Results (Table 2:

DMSO/HD-205 DIRECT column) showed that while DMSO had no effect on activity, the direct effect of HD-205 was not to stimulate activity, but surprisingly to produce a significant inhibitory effect. Subsequently, activity returned to normal in less than one hr after injection, and remained at baseline for two days following intrastriatal injections (data not shown). Two days following injections, both groups were administered i.p. cocaine at 20 mg/kg (Table 2: cocaine i.p. POST column). This cocaine post-treatment produced the same activity in both groups, which was not different from activity observed in the cocaine pretreatment groups. Both groups were allowed to recover for two days (during which time their activities were at baseline; data

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JPET #138842 not shown), then 6 nmol of cocaine was injected intrastriatally through the same cannulae in which HD-205 or DMSO had been injected five days earlier, and rats were tested for locomotor activity immediately. Results (Table 2: cocaine intrastriatal column) showed that intrastriatal cocaine produced significant increase in activity (190% of baseline) in the vehicle group; however, in the HD-205 treated rats, intrastriatal cocaine not only produced no increase in activity, but actually produced a small but significant decrease in activity compared to baseline.

Therefore, the highly localized irreversible blockade of DAT by HD-205 did not affect cocaine- Downloaded from induced activity when cocaine was administered systemically, but did produce a total block of activity when cocaine was administered in the same location in striatum as HD-205. jpet.aspetjournals.org Since HD-205 irreversibly blocks both DAT and SERT in striatum (Fig. 4), it was important to confirm whether effects of HD-205 on cocaine-induced locomotor activity were mediated specifically by DAT. One method to restrict irreversible actions of HD-205 to DAT in at ASPET Journals on September 26, 2021 vivo, and remove its irreversible actions at SERT, is to pre-treat rats with a SERT-specific blocker before injection with HD-205, thus protecting SERT from reaction with HD-205. To accomplish this, rats were injected with citalopram (7.5 mg/kg i.p.), a highly potent SERT blocker (Tatsumi et al., 1997; David et al., 2003). This set of experiments was conducted similar to the previous locomotor studies (Table 2), except that both groups of rats (i.e., DMSO- and

HD-205-injected) were pre-treated with citalopram (7.5 mg/kg i.p.) 20 min prior to the intrastriatal injections of HD-205 and DMSO. Table 3 shows the results of locomotor activity in these citalopram-treated rats. These results are essentially the same as those obtained in normal

(i.e., not administered citalopram) rats (Table 2). Once again, i.p. cocaine pretreatment demonstrated that all animals responded to systemic cocaine administration with the same increase in activity, while bilateral intrastriatal injection of HD-205, two days later, had a small

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JPET #138842 inhibitory effect. Furthermore, as observed previously (Table 2), the effects of i.p. cocaine (20 mg/kg) were not affected two days after HD-205 injection, while the increase in activity observed by intrastriatal injection of cocaine (6 nmol / 2 µg) in DMSO-treated rats (331% of baseline) was completely blocked (104% of baseline) in rats injected with HD-205. Therefore, pretreatment of rats with citalopram had no effect on the ability of HD-205 to block intrastriatal effects of cocaine.

To confirm that this pretreatment with citalopram was effective in blocking irreversible Downloaded from effects of HD-205 at SERT, both DAT and SERT autoradiography was performed in brain sections from these rats immediately after behavioral testing was completed, five days after HD- jpet.aspetjournals.org 205 or DMSO injection (Fig. 7). Typical autoradiograms of SERT binding (Fig. 7A) shows no effect on binding in DMSO-injected rats, and a significant localized area of blockade of SERT binding in rats injected with HD-205 alone. In contrast, rats treated with citalopram before at ASPET Journals on September 26, 2021 injection of HD-205 showed little effect on SERT binding in striatum (Fig. 7A). DAT binding confirmed the specificity of citalopram pretreatment (Fig. 7B): the localized blockade of DAT binding after injection of HD-205 alone was not affected by pretreatment with citalopram before

HD-205. Densitometric analysis (Table 4) of autoradiograms of DAT and SERT binding showed that citalopram pretreatment protected approximately 90% of SERT binding sites from alkylation by HD-205, while blockade of DAT sites was slightly (but not significantly) reduced by citalopram pretreatment. These results confirm that pretreatment with a specific SERT blocker prevents irreversible effects of HD-205 at SERT.

17 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version.

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Discussion

Several irreversible tropanes have been synthesized as covalent ligands for monoamine transporters (Lever et al., 2005), using both photoaffinity and alkylating strategies. While these compounds have been used for mapping cocaine binding sites on DAT (Vaughan et al., 2005;

Vaughan et al., 2007; Parnas et al., 2008), few studies have shown in vivo effects of irreversible tropanes (Fleckenstein et al., 1996b; Vicentic et al., 1999; Kimmel et al., 2000; Kimmel et al.,

2001). The phenylisothiocyanate 2-napthyl tropane analog HD-205 (Murthy et al., 2007) is an Downloaded from ideal compound for this purpose for several reasons. First, HD-205 is one of the most potent irreversible inhibitors of monoamine transporters, with irreversible IC50 values of 4.1 nM and 14 jpet.aspetjournals.org nM at DAT and SERT, respectively. Second, since it uses phenylisothiocyanate as an alkylating reagent, it can be administered directly into brain for irreversible blockade of DAT and SERT,

unlike photoaffinity analogs where such administration is not practical. This is the first study to at ASPET Journals on September 26, 2021 report the in vivo effects of an irreversible isothiocyanate at DAT using autoradiography, and to correlate the effects of this blockade on cocaine-induced locomotor activity.

Intrastriatal injection of HD-205 resulted in a highly localized blockade of both DAT and

SERT binding in rat striatum, consistent with previous in vitro studies with HD-205 in striatal membranes (Murthy et al., 2007). This highly localized effect is similar to the blockade of mu receptor binding by intrastriatal injection of the mu antagonist 5’- naltrindole-isothiocyanate

(Martin et al., 2006), and may be due to the localized effect of the highly reactive isothiocyanate reacting with other proteins (Murthy et al., 2007). This highly localized effect of HD-205 represents a distinct advantage to provide a neuroanatomical localization of the role of monoamine transporters in mediating cocaine effects in brain.

The effects of intrastriatal injection of HD-205 were not the result of non-specific tissue

18 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #138842 damage, since neither intrastriatal injections of DMSO nor the reversible analog HD-206 had any effects on DAT binding 24 hours after injection (Fig. 2A). The specificity of HD-205 was confirmed by showing that HD-205 administration had no effect on D2- and mu opioid receptor- stimulated [35S]GTPγS binding, using adjacent sections from the same animals 24 hr after treatment with HD-205 (Fig. 2B). Interestingly, a decrease in D2-activated G-proteins was observed in the injected side 14 days, but not seven days, after a single injection of HD-205

(Table 1). This appeared to be homologous desensitization (i.e., specific for monoamine Downloaded from receptors), since no effect was observed on mu-stimulated [35S]GTPγS binding in adjacent sections from the same rats. This is consistent with previous reports of desensitization of D2- jpet.aspetjournals.org activated G-proteins in striatum after chronic i.p. administration of the potent tropane WF-23

(O'Connor et al., 2004; O’Connor et al., 2005); in fact, the time course in observing significant desensitization in the D2 system is consistent with these previous results as well. at ASPET Journals on September 26, 2021

Increasing doses of HD-205 administration produced increasing areas of blockade of

DAT binding. Thus, varying the dose of HD-205 can determine the area affected, and in the future this strategy could be used to vary the portion of brain to be studied in cocaine behavioral studies. It is possible that even larger areas of brain could be affected by intracerebroventricular rather than intrastriatal injections, as shown with RTI-76 (Kimmel et al., 2000); however, the possibility of non-specific effects and toxicity may also increase with such injections.

Time course studies revealed that the blockade of DAT binding by intrastriatal injection of HD-205 was not permanent, with 50% recovery of DAT binding 3-4 days after HD-205 treatment, and total recovery on binding in 6 weeks (Fig. 5). These results are consistent with previous studies with the irreversible analog RTI-76 (Fleckenstein et al., 1996b), which showed a half-life of DAT recovery of 6 days after intrastriatal injection of RTI-76, and a value of 2-3 days

19 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #138842 after i.c.v. administration. These results suggest that optimal studies using HD-205 to examine the role of DAT on cocaine behavioral actions should be accomplished within a week of injection for optimal results. These rates of recovery may reflect the turnover rate of DAT protein in brain; however, this conclusion is complicated by the fact that chronic inhibition of

DAT in vivo by cocaine or other tropanes may produce upregulation of DAT (Tella et al., 1996).

DAT blockers, including cocaine and other tropanes, increase synaptic levels of

dopamine and increase locomotor activity (Porrino et al., 1994; Fleckenstein et al., 1996a; Downloaded from

Daunais et al., 1998). Indeed, i.c.v. administration of the irreversible analog RTI-76 increased locomotor activity at relatively high doses (Kimmel et al., 2001), but no studies have yet reported jpet.aspetjournals.org the effects of irreversible DAT blockade on in vivo cocaine actions. In the present study, we report that intrastriatal injection of HD-205 blocked cocaine-induced locomotor activity when cocaine was injected intrastriatally at the same sites, but not after i.p. administration of cocaine. at ASPET Journals on September 26, 2021

This lack of effect on systemic actions of cocaine can be explained by the highly localized blockade of DAT binding by the irreversible drug effects. With injections of 1 nmol drug in 1 µl volumes, the effect of HD-205 was localized to a small portion of the total volume of striatum, leaving the remaining portions of the basal ganglia unaffected and allowing systemic cocaine to act on the remaining DAT. HD-205 did block activity produced by intrastriatal injection of a relatively small dose of cocaine (6 nmol), but the effects of HD-205 were reduced when the dose of intrastriatal cocaine was raised to 24 nmol (data not shown). This is explained by the fact that larger doses of cocaine diffuse further in striatum and can bind to unaffected DAT to increase locomotor activity. Therefore, these results demonstrate that blockade of cocaine effects by HD-

205 is not only dependent on the dose of HD-205 used, but also on the dose of cocaine as well.

The effect of HD-205 to block cocaine-induced locomotor activity may have important

20 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #138842 implications in understanding of cocaine actions at DAT. Interestingly, intrastriatal HD-205 produced no increase in locomotor activity by itself. This was unexpected, since previous studies showed that a variety of tropanes with different structures inhibit dopamine uptake in synaptosomes with potencies similar to their binding potencies at DAT (Bennett et al., 1995).

Based on these observations, one might predict that HD-205 would act as a psychostimulant by itself, and would produce locomotor activity additive to that of cocaine. The lack of effect of

HD-205 on activity was not caused by a difference in the doses of cocaine and HD-205 (2 nmol Downloaded from and 13 nmol, respectively); since HD-205 is approximately 40 times more potent than cocaine in binding to DAT (Murthy et al., 2007), there should have been more than sufficient HD-205 jpet.aspetjournals.org present to increase activity. This lack of activity is probably also not associated with any unique effects of the irreversible HD-205 analog on DAT binding: preliminary studies (not shown) revealed that bilateral intrastriatal injection of the reversible analog HD-206 (2 nmol) in the same at ASPET Journals on September 26, 2021 vehicle (DMSO) also had no effect on locomotor activity by itself. At this point, there is no explanation for this lack of effect of HD-205 and HD-206 on locomotor activity, but it could be associated with the fact that these compounds, administered in DMSO, may diffuse through a smaller area in striatum compared to the same volume of cocaine in saline, and thus recruit fewer

DAT molecules to activate behavior. These compounds may also inhibit dopamine uptake somewhat less than other tropanes, and future studies will compare the effects of intrastriatal cocaine and HD-205 on extrasynaptic levels of dopamine by microdialysis.

Not only did HD-205 produce no increase in locomotor activity, but it also produced a small decrease in activity by itself (Tables 2 and 3). Do these data suggest that HD-205 is actually a sedative rather than a psychostimulant? While further studies would have to be performed to explore this question, it is more likely that this decrease in activity is a non-specific

21 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #138842 action of the drug. First, this decrease in activity is short-lived, with activity returning to baseline levels within a couple of hours after drug administration (not shown), despite that fact that both

DAT and SERT are blocked for several days after HD-205 administration. In addition, previous studies with other alkylating analogs also showed non-selective effects of sedation (Keck and

Lakoski, 1997). Thus, intracranial administration of an isothiocyanate in DMSO, reacting with many synaptic proteins in addition to DAT and SERT, could produce a temporary loss in

activity. Downloaded from

One disadvantage of HD-205 as a pharmacological tool is the fact that it binds with high affinity to both DAT and SERT. To specifically examine the role of DAT in cocaine actions, it is jpet.aspetjournals.org critical to pharmacologically block the irreversible effects of HD-205 at SERT without affecting its reaction with DAT. Our studies using citalopram, administered 20 min before injection of

HD-205, showed that alkylation of SERT by HD-205 could be prevented without affecting at ASPET Journals on September 26, 2021 alkylation of DAT binding. In theory, the reverse experiment could be performed, protecting

DAT from alkylation to explore the role of SERT blockade. Therefore, by carefully choosing the site of injection, as well as the dose of HD-205 and cocaine, together with the use of specific blocking agents, these results demonstrate that HD-205 could be used to evaluate the role of discrete populations of DAT and SERT in the psychostimulant and reinforcing actions of cocaine.

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Footnotes

This research was supported by grant DA-06634 from the National Institute on Drug

Abuse.

Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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Figure Legends

Fig. 1. Chemical structures of tropane analogs, including cocaine, WF-23, HD-205 and HD-206

(Murthy et al, 2007).

Fig. 2. Effects of intrastriatal injection of HD-205 on DAT binding (top) and mu opioid- and D2 dopamine-stimulated [35S]GTPγS binding (bottom), in rat brain sections. A. Blockade of DAT binding in striatum 24 hr after intrastriatal injection of HD-205, showing representative coronal Downloaded from sections from rats treated with unilateral injections of 1 µl DMSO, 1 nmol HD-205 (1 µl) and 1 nmol HD-206 (1 µl), with sections obtained 24 hr after injection. DAT binding was performed jpet.aspetjournals.org using [125I]RTI-55 as described in Methods. B. Lack of effect of HD-205 administration on receptor stimulated [35S]GTPγS binding. Shown are the representative autoradiograms of

sections from rat brains obtained 24 hr after injection of HD-205. Coronal sections were assayed at ASPET Journals on September 26, 2021

γ for agonist-stimulated [35S]GTP S binding; D2 (left) using 10 µM NPA and mu opioid (right) using 10 µM DAMGO.

Fig. 3. Dose response effects of HD-205 in vivo. Rats were treated with unilateral intrastriatal injections of various doses (0.001 – 3 nmol) of HD-205 in 1 µl DMSO, and sections were obtained 24 hr later. DAT binding was determined with [125I]RTI-55 as described in Methods, and autoradiograms were calculated densitometrically using two different parameters: top, by area (number of pixels) affected; bottom, optical density (plotted as per cent OD of contralateral side in each autoradiogram). Shown on right are typical autoradiograms from rats administered

0.003, 1.0 and 3.0 nmol HD-205. In all cases, data were obtained from sections at the point of injection, determined by locating the section of maximal area affected in a series of at least 20

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JPET #138842 serial sections around the point of injection. Data are expressed as mean values ± SEM from triplicate sections from the same rat, using 5-6 rats.

Fig. 4. Blockade of SERT binding in rat striatum after HD-205 injection. Rats were injected unilaterally with 1 nmol of either HD-205 or HD-206, coronal sections at the level of striatum were obtained 24 hr later, and SERT binding was determined with [3H]citalopram as described in

Methods. Shown are representative autoradiograms from rats treated with HD-205 (left) and HD- Downloaded from

206 (right).

jpet.aspetjournals.org Fig. 5. Time course of recovery of DAT binding in rat striatum after intrastriatal injection of HD-

205 (1 nmol in 1 µl DMSO). Rats were treated with unilateral injection of HD-205 and coronal sections at the level of striatum were obtained at various time points (1 to 42 days) after at ASPET Journals on September 26, 2021 injection. DAT binding was determined with [125I]RTI-55 as described in Methods, and the area affected by DAT blockade was calculated densitometrically from autoradiograms (see Fig. 3).

Data represent mean values ± SEM of area calculations (in pixels) of triplicate autoradiograms from each animal (n=5-9 rats in each group).

Fig. 6. Autoradiography of DAT and SERT binding in rats pre-treated with citalopram before injection with HD-205. Rats were either injected i.p. with citalopram (7.5 mg/kg) 20 min before intrastriatal injection of DMSO or 1 nmol HD-205 (Table 3), or injected directly intrastriatal with DMSO or HD-205 without citalopram (Table 2). Rats were tested for cocaine-induced locomotor activity for 5 days (see Tables 2 and 3), then brain sections were obtained for DAT

([125I]RTI-55) and SERT ([3H]citalopram) binding autoradiography. A. Typical autoradiograms

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JPET #138842 of SERT binding. B. Typical autoradiograms of DAT binding. Treatments: DMSO, HD-205: rats injected intrastriatally with DMSO or HD-205, respectively, with no citalopram pretreatment;

CIT/HD-205: rats pre-treated with i.p. citalopram before intrastriatal injection with HD-205.

Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021

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TABLE 1.

Long-term (14 days) effect of HD-205 on receptor-stimulated [35S]GTPγS binding.

Rats were injected intrastriatally with either 1 nmol HD-205 in 1 µl, or 1 µl DMSO, then euthanized after 14 days and brain sections prepared for autoradiography. Sections were assayed

γ for agonist stimulated [35S]GTP S binding, using 10 µM NPA for D2 and 10 µM DAMGO for mu opioid as described in Methods. Data represent nCi/g of [35S] from densitometric analysis of

autoradiograms (with per cent of contralateral side in parentheses) and are mean values of ± Downloaded from

S.E.M of triplicate sections of brains of same rat from five HD-205 treated and five DMSO treated control animals. *, p < 0.05, significantly different from contralateral side, ANOVA. jpet.aspetjournals.org

Contralateral Ipsilateral Treatment Receptor nCi/g nCi/g

at ASPET Journals on September 26, 2021 DMSO D2 37.9 ± 6 41.8 ± 5 (110%)

HD-205 D2 49.4 ± 8 30.8 ± 8 (80%) *

DMSO Mu 138 ± 15 133 ± 15 (95%)

HD-205 Mu 146 ± 9 135 ± 11 (93%)

34 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #138842

TABLE 2.

Effects of intrastriatal administration of HD-205 on cocaine-induced locomotor activity.

Rats were surgically implanted with bilateral cannulae as described in Methods, then after 7-10 days recovery, were pre-tested for locomotor activity with 20 mg/kg i.p. cocaine

(cocaine i.p. PRE column). After two days of baseline sessions, rats were injected either with 1

µl DMSO (n=6) or 1 nmol HD-205 in 1 µl DMSO (n=6), and tested directly for activity

(DMSO/HD-205 Direct column). After two further days of baseline sessions, rats were injected Downloaded from i.p. with 20 mg/kg cocaine (cocaine i.p. POST column). Finally, after another two days of baseline sessions, rats were injected intrastriatally with 6 nmol cocaine through the same jpet.aspetjournals.org cannulae (cocaine intrastriatal column). Data are expressed as percent of baseline activity, and represent mean values ± S.E.M. *, p < 0.05 different from DMSO controls; §, p < 0.05 significantly different from baseline, ANOVA. at ASPET Journals on September 26, 2021

Treatment Cocaine: DMSO or Cocaine: Cocaine: (i.p.) PRE HD-205: (i.p.) POST (Intrastriatal) DIRECT 6 nmol DMSO 980 ± 129% § 100 ± 18% 896 ± 133% § 190 ± 28% §

HD-205 601 ± 115% § 51 ± 9% *§ 811 ± 76% * 85 ± 8% *§

35 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version.

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TABLE 3.

Effects of HD-205 on cocaine-induced locomotor activity after pretreatment with citalopram.

Rats were surgically implanted with bilateral cannulae as described in Methods, then after 7-10 days recovery, were pre-tested for locomotor activity with 20 mg/kg i.p. cocaine

(cocaine i.p. PRE column). After two days of baseline sessions, rats were injected i.p. with

citalopram (7.5 mg/kg), then 20 min later injected intrastriatally with either 1 µl DMSO (n=5) or Downloaded from

1 nmol HD-205 in 1 µl DMSO (n=6). Locomotor activity was tested directly after HD-205 treatment (Direct column), after i.p injection of 20 mg/kg cocaine (cocaine POST column), and jpet.aspetjournals.org after intrastriatal injection of 6 nmol cocaine (cocaine intrastriatal column), as described in Table

2. Data are expressed as percent of baseline activity, and represent mean values ± S.E.M. *, p <

0.05 different from DMSO controls; §, p < 0.05 significantly different from baseline, ANOVA. at ASPET Journals on September 26, 2021

+ Citalopram:

Cocaine: DMSO or Cocaine: Cocaine: Treatment (i.p.) PRE HD-205: (i.p.) POST (Intrastriatal) Direct 6 nmol DMSO 820 ± 125% § 126 ± 35% 983 ± 202% § 331 ± 53% §

HD-205 1062 ± 200% § 72 ± 19% *§ 1433 ± 305% § 104 ± 14% *

36 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version.

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TABLE 4.

Densitometric analysis of DAT and SERT binding autoradiograms from rats pretreated with and without citalopram before injection of HD-205.

Rats tested for intrastriatal cocaine-induced locomotor activity with and without i.p. citalopram treatment (see Tables 2 and 3) were euthanized immediately after the final locomotor activity session. Brain coronal sections at the level of striatum were evaluated for effects of HD-

125 3 205 blockade on both DAT and SERT with [ I]RTI-55 and [ H]citalopram, respectively, as Downloaded from described in Methods. The autoradiograms were quantified by densitometric analysis for the area affected by HD-205 in both the groups. Data are expressed as area of blockade of binding jpet.aspetjournals.org (pixels) and are mean values ± S.E.M; *, p < 0.001 different from HD-205 group, ANOVA.

Treatment: at ASPET Journals on September 26, 2021

Citalopram, Transporter HD-205 HD-205 DAT 991 ± 93 812 ± 94

SERT 871 ± 117 72 ± 61 *

37 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on May 20, 2008 as DOI: 10.1124/jpet.108.138842 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021