Brain Research 862 (2000) 242–246www.elsevier.com/ locate /bres

Short communication

The 5-HT antagonist WAY 100635 can block the low-dose1A

locomotor stimulant effects of cocaine*Robert Carey , Ernest Damianopoulos, Gail DePalma

Research and Development (151), VA Medical Center and SUNY Health Science Center, 800 Irving Avenue, Syracuse, NY 13210, USA

Accepted 25 January 2000

Abstract

Cocaine treatments of 2.5, 5.0, 10.0 and 15.0 mg/kg induced dose-dependent increases in locomotor behavior. This cocaine-inducedincrease in locomotion was blocked if the animals were administered the selective 5-HT antagonist, WAY 100635 (0.4 mg/kg) prior to1A

the cocaine treatment. The 0.4-mg/kg dose of WAY 100635 did not affect locomotor behavior or alter cocaine availability in brain. 2000 Elsevier Science B.V. All rights reserved.

Themes: Neural basis of behavior

Topics: Drugs of abuse: cocaine

Keywords: Cocaine; Locomotion; 5-HT receptors; WAY 100635; Dopamine1A

Cocaine is a potent stimulant drug with reward prop- sufficient for cocaine-induced reward effects but apparent-erties and a substantial addictive potential [9,11]. It is now ly are not necessary. Another line of evidence whichwell established that cocaine binds with a high affinity to argues against cocaine binding to the dopamine transportercatecholamine and indoleamine transporters and, as a as being sufficient for the elicitation of cocaine stimulantconsequence, cocaine decreases the rate of re-uptake of effects is data provided by pharmaco-behavioral studiesneurotransmitters into presynaptic neurons so that neuro- using tropane analogs which have a high selectivity fortransmitter release is amplified [14]. While cocaine in- binding to the dopamine transporter. Critically, these drugscreases the synaptic availability of several neurotrans- do not elicit cocaine-like locomotor stimulant effects [12].mitters, most experimental attention has been directed at Apparently then, cocaine effects upon the dopamine trans-cocaine-induced increases in extracellular dopamine (DA). porter may be neither necessary nor sufficient for im-The emphasis upon dopamine is not surprising in view of portant behavioral effects of cocaine.the well established involvement of dopaminergic systems In view of the established importance of serotonergicin motoric and reward processes [8,19]. Although a systems to behavior, it is not surprising that cocaine effectsconsiderable body of evidence is compatible with cocaine upon the serotonergic transporter have been considered toinhibition of dopamine transport as the basis for cocaine be an important contributor to the behavioral effects ofstimulant and reward effects [8,13,15,20], there are several cocaine [5,7]. An important receptor site for the mediationfindings inconsistent with the dopaminergic transporter as of 5-hydroxytryptamine (5-HT) effects is the 5-HT1A

being the critical site for the mediation of cocaine effects. receptor [4]. In support of an important role for the 5-HT1A

Perhaps the most direct inconsistency is the observation receptor in the mediation of cocaine locomotor stimulantthat knockout mice which lack the dopamine transporter effects, we report that the selective 5-HT receptor1A

will self-administer cocaine [16]. This finding indicates antagonist [6], 2[2-[4-(2-methoxyphenyl)-1-piperazinyl]-that effects of cocaine at the dopamine transporter may be ethyl]-N-2-pyridinyl-cyclohexanecaroxamide maleate

(WAY 100635) can block the low-dose locomotor stimul-ant effects of cocaine.*Corresponding author. Tel.: 11-315-476-7461; fax: 11-315-476-

Naive male Sprague–Dawley rats from Taconic Farms5348.E-mail address: careyr@cnyrc.org (R. Carey) (Germantown, NY), 4 months old and weighing approxi-

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02084-9

R. Carey et al. / Brain Research 862 (2000) 242 –246 243

mately 400 g at the start of the experiments were used. All Louis, MO) and WAY 100635 (RBI/Sigma, Natick, MA)of the behavioral tests were conducted in square open-field were dissolved in sterile H O. All injections were i.p. (for2

compartments which were 60360345 cm. Closed-circuit the two groups which received cocaine, the cocainevideo cameras (RCA TC7011U) were mounted 50 cm treatments varied and were: 2.5, 5.0, 10.0 and 15.0 mg/kgabove the open-field enclosures. All signals were analyzed cocaine). The saline–saline and WAY 100635 (0.4 mg/by a video tracking system, the Videomex-V from Colum- kg)–saline groups continued to receive the same treatmentbus Instruments (Columbus, OH), and the data imported protocol. The cocaine treatments were given in ascendinginto a PC compatible computer. The walls of the chamber order. All groups also received two interspersed saline–were white and the floor of the open-field was covered by saline tests in order to monitor the behavioral baseline.plain white paper which was changed after each animal. One week after the completion of these tests, the animalsAmbient white noise (80 dB) was provided by a white were administered their drug treatment protocols (thenoise generator (San Diego Instruments, San Diego, CA) cocaine dose was 10 mg/kg) and then sacrificed byand was turned on immediately prior to placement of the guillotine decapitation to validate the effect of the WAYanimal in the test chamber and turned off upon removal 100635 upon cocaine-induced neurochemical effects.from the test chamber. Testing was conducted under Trunk blood was collected in tubes containing 200 ml ofconditions of red light illumination to avoid the aversive 0.5% sodium fluoride and centrifuged for 15 min at 2500quality of white light and to enhance the contrast between rpm. The plasma was frozen at 2708C and subsequentlythe subject and background as well as to reduce the assayed for cocaine. The brain was rapidly removed andanimal’s shadow. The animal’s head was blackened by a dissected on a chilled plate. Under magnification, twomarker pen and the camera only tracked this feature of the brain samples were collected: bilateral striatal and arat’s body. bilateral limbic sample. The striatal sample included the

A conventional procedure in this type of investigation is caudate nucleus dorsal to the anterior commissure. Theto use several groups with different doses of WAY 100635. limbic tissue sample included nucleus accumbens, olfac-In view of the fact that each dose level treatment given tory tubercle and overlying pyriform cortex. Followingwith cocaine requires non-cocaine /WAY 100635 control dissection, the samples of brain tissue are weighed, placedtreatments, a very large number of animals becomes in tubes containing 0.5 ml of 0.1 M perchloric acid and 4.5necessary to conduct even a basic experiment. In this ml of 10 mg/ml dihydroxybenzylamine (DHBA) as anstudy, we used a different approach. We first conducted an internal standard, and then homogenized and centrifuged.experiment in which we gave several doses of WAY The resulting supernatant is filtered through 0.2-mm pore100635 (0.2–0.8 mg/kg) in combination with cocaine (10 filters and the extracts are stored at 2708C until themg/kg). Using ex vivo measurements of dopamine and HPLC–EC analysis, which is completed within 24–48 h.5-HT turnover, we determined the lowest dose of WAY The tissue samples are analyzed for dopamine, DA (3-100635 which selectively blocked the effects of cocaine on hydroxytyramine), the dopamine metabolite, DOPAC (3,4-5-HT turnover. We found that 0.4 mg/kg WAY 100635 dihydroxyphenyl-acetic acid), serotonin, 5-HT (5-hydroxy-was the lowest dose level which blocked the effect of tryptamine), and the serotonin metabolite, 5-HIAA (5-cocaine (10 mg/kg) 5-HT turnover. We then used this dose hydroxyindole-3-acetic acid). For the catecholamine andlevel of WAY 100635 to assess the effect of blockade of indoleamine analyses in brain tissue, a BAS biophasethe 5-HT receptors on the locomotor stimulant effect of column (C reversed-phase (4.63250 mm 5 mm)) is used.1A 18

cocaine. The buffer used is 0.15 M moncholoroacetic acid, pH 3.1,Initially, all animals underwent 10 days of daily hand- 2 mM EDTA, 0.86 mM SOS (sodium octyl sulfate). This is

ling including 3 days of saline injections to acclimate the added to 35 ml acetonitrile (3.5%) to make 1 liter. Thisanimals to manipulation and injection procedures. Next, all solution is then filtered and degassed and 18 ml (1.8%)animals were given two 10-min tests in the test environ- tetrahydrofuran (THF) are added. The mobile phase flowment in order to form groups which were statistically rate is 1.2 ml /min and a BAS 4B EC detector is set at 0.8equivalent with respect to the dependent variable of V. For cocaine [3], the sample (0.5 ml serum) is preparedlocomotion. Four days later, the four matched groups by precipitating out the protein with 1.5 ml 100% acetoni-received 20-min tests spaced 4 days apart in which trile. 0.3 ml of 0.1 M sodium phosphate buffer (pH 6.0) isspontaneous locomotor behavior was recorded. Each test added to the supernate and the pH is between 4 and 6. Ainvolved two injections (i.p.): one 20 min prior to testing Bond Elut Certify (Varian, Harbor City, CA) solid-phaseand one immediately before testing. Two groups received column (125 mg and 3 ml) is conditioned with methanolsaline 20 min prior to testing and two groups received 0.4 and 0.1 M sodium phosphate buffer. Before the columnmg/kg WAY 100635 20 min before testing. Subsequently, can run dry the prepared sample is passed through theone saline group and one WAY 100635 group received column. The column is then washed with 3 ml HPLC-saline immediately before testing and the remaining saline grade water, 3 ml 0.1 M HCl, and 10 ml methanol. Thegroup and WAY 100635 group received cocaine immedi- cocaine is eluted with 2 ml methylene chlo-ately before testing. Cocaine hydrochloride (Sigma, St. ride:isopropanol:ammonium hydroxide (77:19:4). The

244 R. Carey et al. / Brain Research 862 (2000) 242 –246

sample is then evaporated under a stream of nitrogen and 5-HT ((F )51.07, P.0.05 and (F )50.66, P.0.05),3,24 3,24

reconstituted in 0.2 ml of buffer. For cocaine (plasma and respectively. There were, however, statistically significantbrain samples), the mobile phase is 24% acetonitrile and effects of the WAY 100635 and cocaine treatments upon76% 0.02 M potassium phosphate buffer (pH 3.0). A DOPAC/DA and 5-HIAA/5-HT ratios in the limbic tissueNucleosil C column (10034 mm, 3 mm) is used with a samples. The WAY 100635 treatment blocked the effect of18

flow rate of 0.5 ml /min. Cocaine is detected using the 10 mg/kg cocaine upon 5-HIAA/5-HT but not DOPAC/BAS variable wavelength UV detector at a setting of 235 DA ratios in limbic brain tissue. One-way ANOVAsnm. indicated that there was a significant drug treatment effect

As a consequence of the matching procedure, the mean upon 5-HIAA/5-HT ratios ((F )59.7, P,0.001). Group3,24

locomotion distance scores for the four treatment groups in comparisons using Duncan’s multiple range test showedbaseline saline–saline tests were similar (F )50.62, P. that the cocaine group had a lower ratio than all other3,24

0.05. Fig. 1 presents the effects of the drug treatments groups (P,0.01) and that the WAY–saline group had aupon locomotion. In the four tests in which cocaine was higher ratio than all other groups (P,0.01), but that theadministered there was a statistically significant treatment saline and WAY–cocaine groups did not differ (P.0.05).effect (F )57.1, P,0.01. Importantly, a two-way For DOPAC/DA ratios there were also statistically signifi-3,24

ANOVA indicated that there was a statistically significant cant group differences ((F )57.7, P,0.001). Individual3,24

treatment group by dose level interaction (F )53.4, group comparisons using Duncan’s multiple range test9,24

P,0.01. In order to ascertain at which dose level signifi- indicated that the cocaine and the WAY 100635 pluscant effects occurred, one-way ANOVAs were performed cocaine groups had lower ratios (P,0.01) than the otherat each dose level. Statistically significant F values were two groups, but did not differ statistically from each other.obtained at the 10.0- and 15.0-mg/kg doses (F )57.7, These results are presented in Fig. 2, with the DOPAC/DA3,24

P,0.01, (F )54.9, P,0.01, respectively. Specific group results in the upper panel and the 5-HIAA/5-HT results in3,24

comparisons with Duncan’s multiple range test indicated the middle panel. In order to assess whether the WAYthat the cocaine group differed significantly (P,0.01) from 100635 compound may have altered cocaine availability,all other groups which did not differ from each other. In serum and brain concentrations of cocaine were comparedterms of DA and 5-HT measurements, there were no in the cocaine and the WAY 100635 plus cocaine groups.statistically significant differences among groups in the t-Tests for independent samples were used for the statisti-measurements obtained from striatal tissue samples (DA cal comparison and the results showed that for serum(F )50.89, P.0.05; 5-HT (F )50.98, P.0.05; (t50.37, P.0.05) and for brain (t50.41, P.0.05), the3,24 3,24

DOPAC/DA (F )51.5, P.0.05; 5-HIAA/5-HT group differences were not statistically significant. The3,24

(F )50.43, P.0.05). In the limbic tissue samples, there cocaine concentrations in limbic brain tissue samples is3,24

were also no statistically significant differences in DA and shown in the bottom panel of Fig. 2.In this study, the 5-HT antagonist, WAY 100635 was1A

found to prevent the low-dose locomotor stimulant effectof cocaine. When administered by itself, however, theWAY compound did not modify locomotor activity incomparison to saline-treated animals, indicating that theWAY compound did not induce a non-specific responsesuppression effect. In that WAY 100635 has been exten-sively studied and shown to be a selective 5-HT antago-1A

nist [6], the present findings implicate the 5-HT receptor1A

in the mediation of cocaine stimulant effects. Thesefindings appear to contribute to the growing evidence fornon-dopaminergic systems as important contributors to thebehavioral effects of cocaine [1]. The present resultsindicate that cocaine-induced changes in dopamine aloneare not sufficient to account for cocaine locomotor stimul-ant effects. In prior studies of low-dose cocaine-inducedlocomotor stimulation, we have reported that the be-havioral effects appear to be an increase in spontaneously

Fig. 1. The effects of four doses of cocaine 2.5, 5.0, 10.0 and 15.0 mg/kg occurring exploratory behaviors rather than repetitiouson locomotor activity. The cocaine treatments were given to two groups stereotypical motoric responses [2]. It may be that neuralof rats either with 0.4 mg/kg WAY 100635 or with saline. The saline– systems, such as 5-HT systems, contribute to this type ofsaline and the 0.4-mg/kg WAY–saline groups did not receive cocaine.

behavior, as opposed to the more repetitive stereotypicalThe locomotion scores are presented as mean and S.E.M. for totalbehavior associated with higher doses of cocaine [17].locomotion distance in meters (m) during 20-min test sessions. *P,0.01

versus all other groups. Perhaps it is these latter types of motoric effects which are

R. Carey et al. / Brain Research 862 (2000) 242 –246 245

Acknowledgements

This research was supported by NIDA grantRO1DA05366-12 and a VA Merit Review grant.

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