EuropeanJournalof Pharmacology200, (1991)53-58 53 ©1991ElsevierSciencePublishersB.V.0014-2999/91/$03.50 ADONIS001429999100478Q F_.JP51949 Microdialysis studies on 3,4-methylenedioxyamphetamine and structurally related analogues J. Frank Nash 1 and David E. Nichols 2 i Departmentsof Psychiatrandy Neuroscience,CaseWesternReserveUniversitySchool, ofMedicine2040,, 4bingtonRoad,ClevelandOH, 44106, U.SA.and: DepartmentofMedicinalChemistrandy PharmacognosSchooly, of PharrnacyandPharmacalSciences,PurdueUnicersity,West LafayetteIN, 47907,U.S.A. Received17April1991,accepted1 May1991 I The acute effect of 3,4-methylenedioxyamphetamine (MDA) and three structural analogues on the extracellular concentra- ti0nsof dopamine (DA) and its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the striatumwas studied using in vivo microdialysis in awake, freely moving rats. MDA significantly (P < 0.001) increased and decreased the extraceUular concentrations of DA and its metabolites, respectively, following J.p. administration. Similarly, acute administration of the N-methyl (MDMA) and N-ethyl (MDE) derivatives of MDA significantly (P < 0.05) increased the concentrationof DA in the striatum. The a-ethyl homologue of MDMA, MBDB, increased the extracellular concentration of DAbut significantly (P < 0.05) !ess than MDA, MDMA or MDE. The rank order of potency for these amphetamine derivatives to increase the extracellular concentration of DA was: MDA > MDMA > MDE > MBDB > vehicle. The increase in extracellu- lar DA concentration following a single administration of these compounds was negatively correlated with the level of serotonin (5-HT)and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in the contralateral striatum measured 7 days following drug administration. Thus, extending the alkyl group on the nitrogen or a-carbon of MDA reduces the ability of these compoundsboth to increase acutely the extracellular concentration of DA and to produce long-lasting depletions of 5-HT in the brain. Dopamine; 5-HT (5-hydroxytryptamine, serotonin); Microdialysis (in vivo); MDA (methylenedioxyamphetamine); MDMA (3,4-methylenedioxymethamphetamine); Neurotoxicity I, Introduction et al., 1990).Moreover,the decreasesin these seroton- ergic parameters have been linked to 5-HT axon termi- The report by Ricaurte et al. (1985) offered the first nal degeneration (O'Hearn et al., 1988). evidencethat repeated administration of high doses of It has been suggested that excessive release of 3,4-methylenedioxyamphetamihe (MDA) produced a dopamine (DA) may play a role in the MDMA-induced 10ng-lastingdecrease in the concentrations of serotonin 5-HT neurotoxicity (Stone et al., 1988; Nash et al., (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in 1990). In this regard, MDA and its N-methyl homo- selectforebrain regions in the rat. That initial study logue, MDMA, have been reported to release DA wasfollowed by numerous reports which have unequiv- from brain slice preparations (Johnson et al., 1986) as 0callyestablished that single or repeated administra- well as to inhibit the uptake of DA into striatal synap- i tionof MDA and structural analogues (i.e. 3,4-methyl- tosomes (Steele et al., 1987). Lengthening the methyl cnedioxymethamphetamine HCI (MDMA), N-methyl- substitution on the nitrogen to ethyl (MDE), decreases ' 141,3-benzodioxol-5-yl)-2-butanamine HCI (MBDB), its potency with respect to DA release from striatal N-ethyl-3,4-methylenedioxyamphetamine HCI (MDE)) brain slices (Schmidt, 1987). Similarly, increasing the i decrease tryptophan hydroxylase activity as well as the length of the alk3'l substitution on the a-carbon of concentrations of 5-HT and 5-HIAA in the brain of these 3,4-methylenedioxy-phenethylamines diminishes rodents and non-human primates (Stone et al., 1986; their effect on DA release and uptake (Johnson et al., Ricaurte et al., 1988; Johnson and Nichols, 1989; Gibb 1986; Steele et al., 1987). Thus, alkyl substituents longer than methyl on either the nitrogen or a-carbon de- crease the ability of these compounds to release neu- -----._..._._ Corres_ndenceto: J.F. Nash, Department of Psychiatry,Hanna ronal stores of DA as measured in vitro. Pavilion,University Hospitals, Cleveland, OH 44106-5000U, .S.A. MDMA has been reported to increase the extracel- 54 · t lular concentration of DA in the striatum, as measured O__--,,_NHR' _ (Nash, 1990; andby inmicrodialysisvivo voltammetry(Hiramatsu(Yamamoandto andCho,Spanos,1990;Nash,1988) (O_ R_ nomenex,(50 tzl)was_Ra 1990). Moreover, Hiramatsu and Cho (1990) found that LC-4B ampe the S-(+) isomer of MDMA increased the concentra- MDA R = CH3, R'= H W. Lafayette tion of DA to a greater extent than the R-(-) isomer. MDMA R = CH3, R' = CH3 trode set at These in vivo studies are in agreement with the afore- MDE R = CH3, R'= C2H5 } Ag/AgC1 ref mentioned in vitro studies and are suggestive that the MBDB R = C2H5, R'--CH 3 sisted of 35 active isomer of MDMA resembles amphetamine in its Fig. 1. Molecularstructuresof MDA,MDMA,MDE and MBDB. 0.67 mM disc ability to increase DA concentrations, acid, 0.1% v/ Extension of the nitrogen or a-carbon alkyl reduces pH 4.25. the ability of these 3,4-methylenedioxy-phenethyla- 2. Z Animals Post-morte mines to produce long-lasting decreases in the concen- HVA, 5-HT a trations of 5-HT and 5-HIAA in the brain. For exam- Male Sprague-Dawley rats weighing between 220- ously reporte pie, a single administration of MDE had no long-term 260 g were purchased from Zivic Miller (Allison Park, each sample effect on the concentration of 5-HT in the cortex PA) and used in all experiments. Animals were housed chloric acid/{ (Schmidt, 1987). In contrast, a single administration of two per cage in a temperature controlled room (23 ° C) 20 min. The s MDA or MDMA has been reported to decrease signif- with a 12/12 h lighting schedule (lights on at 06:00 h). assayed for icantly 5-HT and 5-HIAA content, the number of Food and water were available ad libitum, t the HPLC m_ 5-HT uptake sites and tryptophan hydroxylase activity dissolved witl in several forebrain regions (Stone et al., 1986; Gibb et 2.3. In viuo microdialysis determined a al., 1990; Nash et al., 1991). Thus, increasing the length _ (1951). of the alkyl substitution on the nitrogen decreases the Under chloral hydrate (400 rog/kg i.v.) anesthesia, long-term 5-HT depleting effect of MDA. each rat was placed in a stereotaxic frame. A U-shaped _ 2.5. Statistical The objective of the present study was to determine dialysis probe (Nash, 1990) was inserted into the yen- the effect of nitrogen or a-carbon alkyl substitution in trolateral striatum using the following coordinates: A: f The effect MDA on the drug-induced changes in extracellular + 1.0, L: +3.5, V: -6.5, from bregma, incisor bar vehicle on th concentrations of DA and its major metabolites in the -3.3, according to the atlas of Paxinos and Watson HVA in the n ventrolateral striatum, as measured by in vivo micro- (1982). The dialysis probe was secured using dental repeated mca: dialysis. In addition, the ability of a single dose of cement and a skull screw. The dialysis probe was effect of thes_ MDA, MDMA, MDE and MBDB to decrease the flushed with a modified Ringer solution (mM: KC1 1.7, centrations of concentration of 5-HT and 5-HIAA in the striatum 7 KH2PO 4 1.0, CaC12 1.2, NaCI 136.0, NaRHPO 4 6.0 at , one-way AN r days following administration was determined, pH 7.4) and the animals were placed in clear plastic treatment gro cages. On the following day, the dialysis probe was The area um connected to a Harvard infusion pump (Harvard In- using Simpso: struments, South Natick, MA) set to deliver Ringer used to detern 2. Materials and methods solution at a rate of 1.8 tzl/min. Dialysate samples or 5-HIAA o were collected every 30 min. Baseline samples were , dialysis studk collected for 2-3 h, after which an equimolar dose (87 was set at P < 2.1. Materials tzM/kg; approximately 20 mg/kg) of either MDA, MDMA, MDE, MBDB or vehicle was administered Racemic 3,4-methylenedioxyamphetamine HCI and samples were collected over a 3 h time period. At 3. Results (MDA), 3,4-methylenedioxymethamphetamine HCI the end of each study, the rats were returned to their (MDMA) and N-ethyl-3,4-methylenedioxyampheta- home cage. Each rat was killed 7 days following drug , The acute' mine HCI (MDE) were generously provided by the administration and the brain was rapidly removed from MBDB (87 ALI_ National Institute on Drug Abuse. The racemic mix- the skull. The location of the dialysis probe was con- tions of DA, ture of N-methyl-l(1,3-benzodioxol-5-yl)-2-butanamine firmed in each rat and the contralateral striatum ,sas illustrated in HCI (MBDB) was synthesized by procedures estab- removed and rapidly frozen on dry ice. The tissue (P <0.05)inc: lished in one of our laboratories (Nichols et al., 1986). samples were stored at - 80 ° C until the time of assay. DA as comp: The structureofthese amphetaminederivativesisillus- The maximal trated in fig. 1. Chloral hydrate and the HPLC start- 2.4. Biochemical measurements 60 min folio dardswere purchasedfrom SigmaChemicalCo. (St. MDMAand Louis, MO). All drugs were dissolved in saline and The concentrations of DA and its major metabolites significantly ( administered J.p. in a volume of 1 mi/kg. The dialysis 3,4-dihydroxyphenylacetic acid (DOPAC) and ho- time period (t studies were begun at 09:00 h and drugs were adminis- movanillic acid (HVA) were determined in dialysate creased the e,' tered between 12:00-13:00 h. samples using previously established HPLC methods , 90 rain follov 5 t 55 (Nash, 1990; Johnson et al., 1990). Briefly, each sample A. OA (50/zl) was injected onto a 3 /_m C18 column (Phc- ;000 nomenex, Rancho Palos Verdes, CA) connected to an tT_ W.