THE EFFECTS OF METHYLENEDIOXYMETHAMPHETAMINE … · The mesocorticolimbic dopamine pathway 5.2....

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Progressin NeurobiologyVol.49, pp. 455 to 479, 1996Copyright~ 1996ElsevierScienceLtd. All rights reserved

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THE EFFECTS OF METHYLENEDIOXYMETHAMPHETAMINE(MDMA, “ECSTASY”) ON MONOAMINERGIC

NEUROTRANSMISSION IN THE CENTRAL NERVOUS SYSTEM

S. R. WHITE*, T. OBRADOVIC, K. M. IMEL and M. J. WHEATONDepartment of Veterinary and Comparative Analomy, Pharmacology and Physiology,

Washington State University, Pullman, WA 99164, U.S.A.

(Received 20 March 1996)

Abstract—Methylenedioxymethamphetamine (MDMA, Ecstasy) is a popular recreationally used drugamong young people in Europe and North America. The recent surge in use of MDMA and increasingconcerns about possible toxic effects of the drug have inspired a great deal of research into the mechanismsby which the drug may affect the central nervous system. This paper reviews studies on the neurochemical,behavioral and neurophysiological effects of MDMA, with emphasis on MDMA effects in regions of thebrain that have been implicated in reward.

Experiments in awake, behaving laboratory animals have demonstrated that single injections of MDMAincrease extracelhdar levels of the neurotransmitters dopamine (DA) and serotonin (5HT) in the nucleusaccumbens and in several other brain regions that are important for reward. Most of the behavioral andelectrophysiological changes that have been reported to date for single doses of MDMA appear to bemediated by this MDMA-induced increase in extracelhdar DA and 5HT. As an example, MDMA-inducedhyperthermia and locomotor hyperactivity in laboratory animals can be blocked by administering drugsthat prevent MDMA-induced 5HT release and can be attenuated by administering 5HT receptorantagonists, whereas effects of MDMA on delayed reinforcement tasks appear to be mediated byMDMA-induced increases in extracellular DA. Similarly, the effects of MDMA on neuronal excitabilityin the nucleus accumbens and in several other brain regions can be prevented by administering drugs thatblock MDMA-induced 5HT release and can be attenuated by depleting brain DA levels or byadministering either DA D, receptor antagonists or 5HT receptor antagonists.

In addition to the acute effects of MDMA, it is now well established that repeated or high-doseadministration of MDMA is neurotoxic to a subpopulation of 5HT-containing axons that project to theforebrain in laboratory animals. Recent studies have shown that this neurotoxic effect of MDMA isassociated with long-duration changes in both DA and 5HT neurotransmission in the nucleus accumbens.Whether these long-duration changes in neurotransmission might be related to reports of depression andother psychopathologies by some frequent users of MDMA remains to be determined. Methylene-dioxymethamphetamine has been found to increase extracellular levels of norepinephrine and to alterbrain levels of several neuropeptides as well as altering levels of DA and 5HT. Much additional researchis required to understand the multiple ways in which this complex drug may alter neurotransmission inthe brain, both acutely and in the long term. Copyright KO1996 Elsevier Science Ltd.

CONTENTS1. Introduction2. Acute MDMA, neurochemical changes

2.1. Monoamine release and re-uptake in uitro2.2. Extracellular monoamine levels in uiuo

3. Repeated MDMA, neurochemical changes4. Behavioral effects of MDMA5. Acute MDMA effects on neuronal excitability in brain regions implicated in the rewarding properties

of abused drugs5.1. The mesocorticolimbic dopamine pathway5.2. MDMA effects in the medial prefrontal cortex5.3. MDMA effects in the hippocampus5.4. MDMA effects on 5HT neurons in the dorsal raphe nucleus5.5. MDMA effects on DA neurons in the ventral tegmental area5.6. MDMA effects in the nucleus accumbens

5.6. I. Nucleus accumbens core5.6.2. Nucleus accumbens shell

5.7. MDMA effects on motoneurons in the hypoglossal nucleus6. Repeated MDMA exposure, effects on neuronal firing in the nucleus accumbens7. Concluding remarks

AcknowledgementsReferences

456457457457458459

460460461461461462464464468470472473474474

*Author for correspondence. E-mail: swhite(dvetmed.wsu.edu; Tel: 509 335 1587; Fax: 509 3354650.

455

456 S. R. White et al

AMPTCCKCPCPBGCuZnSODDADOI

EPSPSFLXGABAHPLC60HDA8-OHDPAT5H1AA

ABBREWx-Methyl-p -tyrosineCholecystokininCaudate/putamenI-(m-Chlorophenyl) -biguanideCopper–zinc superoxide dismutaseDopaminel-(2,5-Dimethoxy-4-iodopheny 1)-2-ami nopropaneExcitatory postsynaptic potentialsFluoxetine;-Aminobutyric acidHigh-pressure liquid chromatography6-Hydroxydopamine8-Hydroxy-(2-di-n-propy lamine)tetral in5-Hydroxyindolacetic acid

1. INTRODUCTION

Methylenedioxymethamphetamine (MDMA, “Ec-stasy”) is a legally restricted amphetamine deriva ~ive(Fig. 1) that has become increasingly popular inEurope and North America over the last 10 yearsbecause of its euphoria-inducing and mild stimuimtproperties (Cregg and Tracey, 1993; Cuomo etal,1994; Green et cd., 1995; McKenna and Perou[ka,1990; Steele et al., 1994). MDMA (Ecstasy) is medprimarily by young people in large dance and mllsicsettings, but sometimes it is used in small socialgatherings (Green etal., 1995; Steele et al., 191~4).Although MDMA often is described as a relati. elyharmless drug (for example, note the entries on theMDMA home page on the Internet), acute toxicreactions including malignant hyperthermia, con. ul-sions, hepatitis and death have been attributed toMDMA use by young people (Dykhuizen et al., I’J95;Green etal., 1995; Khakoo et al., 1995; O’Connor,1994; Randall, 1992).

Adding to concerns about possible dangers tohumans, repeated injections of low doses of MDMAproduce long-lasting damage to a selective popu-lation of serotonin-containing axons that project tothe forebrain in laboratory rats and mon~.eys(Battaglia et al., 1991; Mamounas etal.,1’)91;O’Hearn e[ al., 1988; Ricaurte et a/., 1985; Rica lrteet al., 1988). The doses that produce this damag: in

+3,4 -fvlethylenedioxy methamphetamin e

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m“”’Fig. 1. Chemicalstructures for MDMA and amphetaniine.

Popular names for MDMA are Ecstasy. Adam and X.

ATIONS5HTIPSPSKETmPFCMDMA2M5HTMTSGNaClNENMDAPCPATHVTAWAY

5-HydroxytryptamineInhibitory postsynaptic potentialsKetanserinMedial prefrontal cortexMethylenedioxymethamphetamine2-Methyl-5 -hydroxytryptamineMethysergideSodium chlorideNorepinephrineN-methy[-D-aspartatep-ChlorophenylalanineTyrosine hydroxylaseVentral tegmental areaWAY1OO135

monkeys are close to those that are used by humansand there is suggestive evidence that people who haverepeatedly used MDMA also may suffer damage toserotonin neurons in the central nervous system(McCann et al., 1994; Ricaurte et al., 1990; Ricaurteand McCann, 1992). High doses of MDMA causedamage to forebrain dopamine-containing axons aswell as serotonin-containing axons in laboratory rats,and appear to damage selectively only dopamine-con-taining axons in mice (Cadet et al., 1995;Logan et al.,1988; Stone et al., 1987), raising the possibility thatdopaminergic neurons may also be at risk in humansthat repeatedly ingest MDMA. Clinical reports ofpsychopathology including panic attacks, depression,flashbacks and psychosis that occur days to monthsfollowing the last MDMA ingestion suggest thatMDMA may, indeed, produce long-term changes i~neurotransmission in humans as well as laboratoryanimals (Creighton et al., 1991; McCann andRicaurte, 1991; McGuire er al., 1994; Pallanti andMazzi, 1992; Schifano and Magni, 1994).

The recent surge in popularity of illicit use ofMDMA and the increasing reports of toxic reactionsin humans have inspired a great deal of research onthe mechanisms by which MDMA might affectcentral nervous system functioning. It is now wellestablished that administration of single doses ofMDMA to laboratory animals induces acuteincreases in extracellular levels of the monoamineserotonin (5HT), dopamine (DA) and norepinephrine(NE) in several brain regions (Berger et al., 1992;Fitzgerald and Reid, 1990; Gough ef al., 1991;Johnson et al., 1986; Nash and Brodkin, 1991;Nichols et al., 1982; Schmidt er al., 1987; Yamamotoand Spanos, 1988).This observation and the evidencethat multiple doses of MDMA may be neurotoxic toserotonin-containing and perhaps dopamine-contain-ing axons suggest that MDMA may produce complexand widespread changes in neurotransmission in thebrain that may vary depending upon dose andfrequency of exposure to MDMA. The purpose ofthis paper is to review recent laboratory findingsabout the neurochemical, behavioral and neurophys-iological effects of MDMA and to discuss the extentto which the mood-altering effects of MDMA mightbe attributable to alterations in monoaminergic

The Effects of “Ecstasy” on the Central Nervous System 457

neurotransmission in brain regions that are con-sidered to be part of the neural circuitry mediatingthe rewarding properties of abused drugs.

2. ACUTE MDMA, NEUROCHEMICALCHANGES

2.1. Monoamine Release and Re-Uptake In Vitro

Nichols et al. (1982) first demonstrated that bathapplication of MDMA induced release of [3H]5HTfrom synaptosomes prepared from whole rat brain.This observation was confirmed subsequently byseveral laboratories and extended to DA release(Schmidt et al., 1987; McKenna et al., 1991),although MDMA is a more potent releaser of 5HTthan DA in vitro. MDMA released 5HT from striataldices at concentrations about 10-fold lower thanthose that were required to stimulate DA release(Schmidt et al., 1987). MDMA appears to interactwith the 5HT transporter to cause 5HT releasebecause MDMA-induced 5HT release from brainsynaptosomes, hippocampal and brainstem slices,membrane vesicles and fetal 5HT neurons in culturecan be blocked by fluoxetine or imipramine which areinhibitors of the 5HT transporter (Berger et al., 1992;Fitzgerald and Reid, 1990; Gu and Azmitia, 1993;Hekmatpanah and Peroutka, 1990; McKenna et al.,1991; Rudnick and Wall, 1992; Sprouse et al., 1989).The carrier-mediated release of 5HT and DA frombrain slices by MDMA is Ca+ + independent becauserelease of 5HT is not inhibited by removal of Ca + +from the superfusion solution (Schmidt et al., 1987).In addition to stimulating 5HT and DA release,MDMA has been shown to induce NE release fromhippocampal slices (Fitzgerald and Reid, 1990).Presumably MDMA interacts with the NE trans-porter to release NE because NE release was blockedby desmethylimipramine, a specific inhibitor of theNE transporter (Fitzgerald and Reid, 1990). SinceMDMA does not appear to accumulate withinsynaptosomes (Wang et al., 1987), it is thought toinduce monoamine release by interacting with themonoamine carriers to reverse the direction ofneurotransmitter flow (Hekmatpanah and Peroutka,1990).

In addition to stimulating 5HT, DA and NErelease, MDMA can also increase extracelhrlar levelsof all three monoamine by inhibiting re-uptake(Berger et al., 1992; Johnson et al., 1991a; Steeleet al., 1987) and by delaying metabolism throughinhibition of monoamine oxidase (Gu and Azmitia,1993; Kokotos Leonardi and Azmitia, 1994). Ecstasyalso causes an acute stimulation of striatal dopaminesynthesis that is mediated by 5HTZ receptors(Schmidt et al., 1991). These observations suggestthat a major mechanism by which MDMA mayacutely affect neuronal excitability in the brain is byincreasing extracellular levels of serotonin andcatecholamines and thereby indirectly activating5HT, DA and NE receptors. However, changes inneuropeptide neurotransmission also may mediatesome of the effects of MDMA. Single injections oflow doses of MDMA have been shown to increaseboth neurotensin-like immunoreactivity and dynor-

phin-like immunoreactivity measured 18 hr post-in-jection in homogenates of neostriatum, nucleusaccumbens and substantial nigra (Johnson et al.,1991b). These changes appeared to be mediated byboth dopaminergic and glutaminergic systems be-cause they could be blocked by the dopamine D,receptor antagonist SCH 23390 and by the N-methyl-D-aspartate (NMDA) antagonist MK-801 (Johnsonet al., 1991b).

2.2. Extracelhdar Monoamine Levels Zn Viuo

Advances in microdialysis and in in uivo voltamme-try techniques have allowed investigators to examinedirectly the effects of MDMA administration onextracelhdar levels of monoamine in awake,behaving animals. These studies have revealed thatMDMA produces a marked increase in extracellularDA in regions of the brain that are innervated richlyby DA-containing axon terminals. Most dialysisstudies of MDMA to date have been conducted inlaboratory rats with the dialysis probe inserted in thestriatum (caudate nucleus and putamen), a structurethat is part of the classical “extrapyramidal” motorsystem, but that also may play a role in cognitive andmotivated behaviors. Single systemic injections ofMDMA increased extracehdar levels of DA in thestriatum of awake, behaving rats as measured byhigh-pressure liquid chromatography (HPLC) analy-sis of dialysate samples (Gough et al., 1991; Schmidtet al., 1994; Yamamoto et al., 1995). Systemicinjection of MDMA also increased extracellularlevels of 5HT in the striatum, but the 5HT increasewas much less than that of DA (Gough et al., 1991).Pretreatment of the rats with specific DA uptakeblockers prevented the increase in extracellular DAthat was otherwise produced by infusion of MDMAdirectly into the striatum via the dialysis probe (Nashand Brodkin, 1991). This suggests that MDMAincreases DA in part via a DA transporter-mediatedmechanism which is independent of its effects on 5HTrelease (Nash and Brodkin, 1991). However, systemicinjections of 5HTl receptor antagonists or infusion ofthe antagonists directly into the striatum through thedialysis probe attenuated MDMA-induced DArelease (Nash, 1990; Schmidt et al., 1994;Yamamotoet al., 1995), indicating that DA efflux produced byMDMA is regulated partially by 5HT receptors.

The nucleus accumbens of the forebrain is a brainregion that has been particularly implicated in therewarding properties of abused drugs (Koob, 1992;Wise and Bozarth, 1984). Drugs including opiates,alcohol, amphetamine and cocaine have the commonproperty of increasing extracelhrlar concentrations ofDA (and also of 5HT) in this nucleus in freely movingrats (Di Chiara and Imperato, 1988;Hernandez et al.,1987; Yoshimoto et al., 1991). Similarly to the otherabused drugs, systemic injection of MDMA increasedextracelhtlar DA in the nucleus accumbens measuredby in vivo voltammetry (Yamamoto and Spanos,1988). A recent study from our laboratory confirmedand extended this finding by demonstrating thatMDMA infused directly into the nucleus accumbensthrough the dialysis probe significantly increasedextracellular levels of both DA and 5HT in awake,behaving rats (White et al., 1994). Thus, MDMA can

458 S. R. White et al.

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Fig. 2, MDMA infused through a dialysis probe into thenucleus accumbens of awake rats produced a dose-depen-dent increase in levels of 5HT and DA in dialysate samlles.The histograms indicate the mean monoamine con@n-tration in 20 min dialysis samples taken 60–80 min foliov ingthe onset of MDMA infusion and the bars indicate theSEM. The numbers of animals tested with each dose rangedfrom 6 to 14.The 10and 100PM doses of MDMA prod( cedstatistically significant increases in 5HT and DA kielsabove the basal (Odose) level. Note that the y-axis is a logscale. The maximal 5HT increase produced by MDMA wasabout double the pre-MDMA basal level, whereas themaximal DA increase was about 10-fold higher than thebasal level. “Significant increase (p < 0.01) from kallevels, paired r-test, two-tailed with Bonferonni adjustmentsfor multiple comparisons (modified from White er al., 19’)4).

act directly upon terminals within the nucleusaccumbens to increase extracellular DA and 5HT Aswas reported previously for the striatum (Go~gh~t al., 1991), the magnitude of increase was muchgreater for DA than for 5HT (Fig. 2). However. theDA release may have been mediated at least partiidlyby MDMA-induced 5HT release because it is wellestablished that infusion of 5HT or 5HT agol~istsdirectly into the nucleus accumbens or the stria. umthrough the dialysis probe increases extracellillarlevels of DA measured in dialysate samples(Benloucif and Galloway, 1991; Blandina etal., l’~89;Galloway et al., 1993; Jacocks and Cox, l’~92;Parsons and Justice, 1993a).

3. REPEATED MDMA, NEUROCHEMICALCHANGES

Repeated systemic administration of MDMA tolaboratory rats, guinea-pigs or monkeys produceslong-lasting decreases in neurochemical and histo-logical indices of serotonin function in the forebi ain.MDMA reduces forebrain tissue levels of 5HT and itsmetabolize 5-hydroxyindolacetic acid (5HIAA) anddepresses the activity of tryptophan hydroxylase. thesynthetic enzyme for 5HT (Schmidt et al., 1987;Stone ef al., 1988). These deficits can persist tromweeks to more than a year following multiple dosesof MDMA depending upon the brain area. thespecies studied and the doses of MDMA used(Commins et al., 1987; Fischer et al., 1995; Ric;iurteand McCann, 1992; Scanzello et al., 1993). Uptakesites for 5HT in the cerebral cortex, caudate nucleus,

hippocampus, nucleus accumbens, olfactory tubercleand many thalamic nuclei also are reduced markedlyas measured by quantitative in uitro autoradiographyof [3H]-paroxetine binding in rats after repeatedsystemic injections of MDMA (Battaglia et al., 1991).The MDMA-induced decrease in 5HT uptake sitesoccurred in most brain regions within 24 hr of the lastdose and persisted for at least 2 weeks. The decreasein uptake sites appeared to be specific for theserotonin system in the rat because [qH] mazindolbinding (which labels catecholamine uptake sites) wasunaffected by multiple injections of MDMA at doseslower than 20 mg/kg (Battaglia et a/., 1991).However, repeated injections of MDMA at doses of40 mg/kg or higher decrease NE and DA levels inguinea-pigs (Commins e{ a/., 1987). Single microin-jections of MDMA directly into the dorsal or medianraphe nuclei which contain the 5HT cell bodies thatsend projections to the forebrain do not changeserotonin or catecholamine levels in the hippocampusor striatum of rats measured 2 weeks post-injection(Paris and Cunningham, 1992). This suggests thatMDMA may act at the axon terminals to producedamage. However, effects of repeated microinjectionsof MDMA into the raphe nuclei have not beenexamined.

Neurotoxic effects of MDMA in primates are morepronounced than in rats (Ricaurte and McCann,1992). Serotonin depletion and decreases in 5HTuptake sites in the cerebral cortex of monkeys occurat lower doses of repeated MDMA than for rats, themaximal effect of MDMA on 5HT depletion isgreater in monkey than rat, and the depletion of 5HTlevels and 5HT uptake sites persists for longer periodsof time post-injection in monkeys. In most rats, 5HTand 5HIAA levels and paroxetine-labeled 5HTuptake sites showed nearly complete recovery by 32weeks (Scanzello et al., 1993), but decreases in these5HT markers persisted at 18 months in neocortex,hippocampus and striatum of monkeys and may bepermanent (Ricaurte and McCann, 1992; Fischeret al., 1995). Some neuronal cell bodies in the dorsalraphe nucleus appear to be damaged in squirrelmonkeys following repeated MDMA administration,whereas the cell bodies are spared in rats, perhapsallowing for more successful axonal sprouting andreinnervation (Ricaurte and McCann, 1992). In bothrats and monkeys, the hypothalamus becomesabnormally hyperinnervated by serotonin terminalsby 52–72 weeks following repeated injections ofMDMA (Fischer et al., 1995).

lmmunohistochemical studies indicate that re-peated injections of MDMA appear to cause selectivedegeneration of fine-diameter serotoninergic axonswith small varicosities that arise from the dorsalraphe nucleus and project to the forebrain. Forebrainserotonin-containing axons with large round varicosi-ties and small intervaricose segments (“beaded”fibers) that arise predominantly from the medianraphe nuclei appear to be spared by MDMA(0’ Hearn etal., 1988; Mamounas et al., 1991;Wilsonet al., 1989). Histological evidence of serotonin nerveterminal destruction includes extreme swelling andabnormally shaped and fragmented 5HT-immuno-reactive axon terminals at 1–3 days after multipleMDMA injections followed by a persistent reduction


in the density of fine-diameter 5HT-containing fibersin the forebrain that lasts for many weeks in rats andmay be permanent in some brain regions in monkeys(Wilson et al., 1993; Fischer et al., 1995).

Although the precise mechanisms by whichMDMA causes damage to 5HT axons are not known,DA appears to play a major role in the neurotoxiceffects of MDMA. Acute depletion of DA bypretreatment with ct-methyl-p-tyrosine or reserpineattenuated MDMA-induced DA efflux in thestriatum and prevented subsequent decreases of 5HTuptake sites and tryptophan hydroxylase activity(Brodkin et al., 1993; Stone et al., 1988). 5HT2receptor antagonists also prevent MDMA-inducedacute stimulation of DA synthesis and preventlong-lasting deficits in forebrain 5HT concentrations(Schmidt et al., 1991). It has been proposed bySchmidt et al. (1991) that in the absence of5HT2-mediated enhanced synthesis of DA, the DAneuron cannot sustain the carrier-mediated DArelease which is essential for the development ofMDMA-induced neurotoxicity. Similarly, 5HTZ re-ceptor agonists administered together with MDMA,potentate the MDMA-induced DA release andincrease the toxic effect of MDMA on 5HT levelsmeasured 7 days post-administration (Gudelskyetal., 1994).

Sprague and Nichols (1995) suggest that delamin-ationof excessive DA that has been taken up by 5HTterminals generates hydrogen peroxide that may leadto membrane lipid peroxidation and perhaps otheroxidative insults, resulting in 5HT terminal degener-ation after MDMA administration. Deprenyl, amonoamine oxidase-B inhibitor protects againstMDMA-induced lipid peroxidation and Iong-termdecreases in 5HT, 5HIAA levels and 5HT uptakesites. The MDMA-induced depletion of 5HT fromthe terminals may increase vulnerability to excess DAaccumulation after DA has been released by MDMA.5HT release and depletion alone do not lead toserotonin terminal degeneration. Instead, repeatedexposure to MDMA may produce the followingsequence of events as proposed by Sprague andNichols (1995): MDMA depletes 5HT from serotoninneurons which renders them vulnerable to the toxicprocess; MDMA dramatically increases DA synthesisand release; the DA from the increased extracelluIarpool is transported into depleted 5HT terminals bythe 5HT uptake carrier where it is delaminated byMAO-B to generate hydrogen peroxide; this hydro-gen peroxide then leads to lipid peroxidation andperhaps other oxidative insults and selective 5HTaxonal degeneration. Recent studies in transgenicmice suggest that oxidative insults may, indeed,mediate MDMA-induced neuronal toxicity (Cadetet d., 1995). In mice, MDMA is neurotoxic to DArather than 5HT axons (Logan et al., 1988; Stoneet al., 1987), and Cadet et al. (1995) confirmed thisobservation by showing that in non-transgenic mice,a single high dose of MDMA produced DA depletionin the striatum at 24 days and 2 weeks. However, inhomozygous transgenic mice that carried thecomplete sequence of the human copper–zincsuperoxide dismutase (CuZnSOD) gene, MDMAproduced no DA depletion at either time point, Theincreased cytosolic levels of the antioxidant CuZn-

SOD appeared to protect the DA axons of thetransgenic mice against damage by oxygen-based freeradicals (Cadet et al., 1995).

Excitatory amino acids also have been implicatedin MDMA-induced neurotoxicity because systemicinjections of dizocilpine and other excitatory aminoacid antagonists protect against the neurotoxic effectsof systemic injection of MDMA on 5HT terminals inthe neocortex and hippocampus (Colado et al,, 1993;Farfel et al., 1992). However, systemic injections ofMDMA, unlike injections of methamphetamine, donot increase glutamate in the striatum of awake,freely moving rats as measured by in uivo microdialy-sis (Nash and Yamamoto, 1992). Therefore, fore-brain depletion of 5HT content is probably not dueto an excitotoxic effect of excess extracellularglutamate released by MDMA. The excitatory aminoacid antagonists may prevent MDMA-inducedneurotoxicity by preventing MDMA-induced hyper-thermia (Farfel and Seiden, 1995). Dizocilpine, whichordinarily protects against both MDMA-inducedhyperthermia and MDMA-induced 5HT neurotoxic-ity, does not protect 5HT terminals in the forebrainfrom damage by MDMA when the temperature ofthe rats is artificially maintained above 38.4”C,(Farfel and Seiden, 1995). If MDMA-inducedhyperthermia is prevented by maintaining animals ata reduced ambient temperature, the decrease in 5HTand 5HIAA is prevented (Schmidt er a/., 1990).However, 5HT uptake blockers prevent the MDMA-induced toxicity without preventing the hyperther-mia. Therefore, the hyperthermia produced byMDMA and the 5HT release produced by MDMAappear to contribute independently to the neurotoxi-city (Schmidt et al., 1990). A recent study of MDMAeffects on cuhured glial cells suggests that MDMA-induced neurodegeneration may also be a conse-quence of MDMA-induced depletion of energy storesin the glial cells that surround the 5HT-containingaxon terminals (Poblete and Azmitia, 1995).

In addition to neurotoxic actions on 5HT axons(and DA axons in mice), repeated injections ofMDMA have been reported to have several othereffects including a transient down-regulation of 5HTZreceptors in the forebrain (Scheffel et al., 1992); adecrease in glucocorticoid and mineralocorticoidreceptor mRNA expression and an increase in 5HTZCreceptor mRNA expression in the hippocampus (Yauet al., 1994); a significant increase in glucoseutilization in subregions of the hippocampus despiteprofound reductions in 5HT uptake sites in thehippocampus (Sharkey et al., 1991); and a significantdecrease in preprocholecystokinin mRNA levels inthe substantial nigra (Wotherspoon et al., 1994).Whether any or all of these changes may besecondary to the neurotoxic effects of MDMA onserotonin neurotransmission is not yet known.

4. BEHAVIORAL EFFECTS OF MDMA

Ecstasy is rewarding to laboratory animals as wellas humans; monkeys will press levers to self-adminis-ter MDMA (Beardsley et al., 1986; Lamb andGriffiths, 1987). In addition to its rewardingproperties, MDMA produces locomotor hyperactiv-

460 S. R, White et al.

ity, hyperthermia, head-weaving and other c(Jm-ponents of the “serotonin syndrome” in laboratoryanimals (Green etal., 1995). Systemic injection ofsingle or multiple doses of MDMA incre:meslocomotor activity in laboratory rats (Gold et al.,1988;Matthews eta/., 1989; McNamara et al., 1995).Hyperactivity also can be induced by infusingMDMA directly into the nucleus accumbens(Callaway and Geyer, 1992). The hyperactivityproduced by systemic injection of MDMA appeals tobe mediated at least partially by MDMA-induced5HT release because it is attenuated by pretreatn]entwith the 5HT uptake blocker fluoxetine wi~ichprevents the uptake carrier-mediated release of 5HTby MDMA (Callaway et al., 1990;Hekmatpanah andPeroutka, 1990). The 5HT,, receptor agonist RU24969, like MDMA, produces locomotor hypera,:tiv-ity in rats, whereas agonists for 5HTI,Aand 5H~ ?~,~~receptors decrease locomotion (Rempel et al., I(W3).These observations suggest that MDMA stimulateslocomotor behavior by releasing 5HT which then ~ctson 5HT)~ receptors to produce the locom~torhyperactivity, The hyperthermia that followsMDMA injection can be blocked by the 5HI ,.lCreceptor antagonist ketanserin (Nash et d.,1988).Otherbehavioral effects that have heenreported for MDMA are similar to those prodllcedby amphetamine and appear to be mediated by DArelease. For example, MDMA administration de-creased reinforcement rate and increased resp,)nserate of rats performing on a differential-reinfc ~rce-ment-of-low-rate task in a manner that was typic.d ofamphetamine and other psychomotor stimulants (Liet al., 1989).After repeated injections of MDMA thatresulted in forebrain depletion of 5HT, subsequentinjections of MDMA produced even higher ratus ofresponding (and therefore decreased reinforcementrates) than did the first dose of MDMA. Thus,although MDMA can increase extracellular levels of5HT as well as DA, the high rate of responding thatoccurred during this behavioral task appeared to bemediated by DA (Li et al., 1989). Sympatheticstimulation effects of MDMA (increased heart rateand blood pressure) may be mediated by MDM.A-in-duced monoamine release in both the central anti theperipheral nervous system, and perhaps also by directeffects of MDMA on cq-adrenergic receptors (Greenet al., 1995). Although the exact mechanisms bywhich MDMA produces its many behavioral el~ectsare not known, it is likely that they are mediated byMDMA-induced increases in extracellular levels of5HT, DA and NE in the nervous system.

5. ACUTE MDMA EFFECTS ON NEURONALEXCITABILITY IN BRAIN REGIONSIMPLICATED IN THE REWARDINGPROPERTIES OF ABUSED DRUGS

There is some evidence to suggest that MI.JMAmight have direct postsynaptic effects on neuronalexcitability as well as indirect effects that aremediated by monoamine release. MDMA binds withsimilar, relatively high affinities to 5HTZ receptors,ctz-adrenoreceptors and M-1 muscarinic choliriergicreceptors as to 5HT uptake sites in homogenates of

frontal cortex and striatum (Battaglia et al., 1988).Since 5HT2 receptors, a,-adrenoreceptors and M-1muscarinic receptors are located postsynaptically aswell as presynaptically in several brain regions, thepotential exists for MDMA to have direct postsyn-aptic actions on neurons that contain these receptors.As evidence for direct effects of MDMA on 5HT,receptors, MDMA has been shown to weaklystimulate (compared to 5HT) [3H] inositol mono-phosphate accumulation in cultured fibroblasts thatexpress the cloned 5HTj~ receptor and to stimulatemore strongly accumulation in fibroblasts expressingthe cloned 5HTt,. receptor (Nash eta/,, 1994). Incontrast to the serotonergic system, MDMA hasrelatively low affinity for dopamine D, and Dzreceptors, suggesting that MDMA-induced effectsthat are mediated by DA are indirect (Battaglia etal.,1988). Indeed, most electrophysiological studies todate indicate that MDMA alters central nervoussystem neuronal excitability primarily by increasingextracellular levels of monoamine that are releasedfrom presynaptic terminals. [ntercstingly, MDMAhas been demonstrated to affect neuronal firing byincreasing extracellular levels of monoamine inseveral brain regions that are implicated in mood andin the rewarding effects of abused drugs.

5.1. The Mesocorticolimbic Dopamine Patbway

The mesocorticolimbic dopamine pathway hasbeen implicated widely in drug reinforcement (Dackisand Gold, 1985; Di Chiara and Imperato, 1988;Koob, 1992; Wise and Bozarth, 1984). TheDA-containing cells in this pathway originate mainlyin the ventral tegmental area (VTA) of the midbrainand project to the nucleus accumbens. olfactorytubercle, frontal cortex, amygdala and septal area(Bjorklund and Lindvall, 1984; Oades and Halliday,1987). However, some of the dopaminergic projec-tions to the lateral regions of the nucleus accumbensarise from the medial region of the substantial nigraas well (Brog et a/., 1993). The DA projections fromthe VTA to the nucleus accumbens appear to beessential for the rewarding effects of abused drugs.Destruction of DA terminals in the nucleusaccumbens or of DA cell bodies in the VTA byinjecting the neurotoxin 6-hydroxydopaminemarkedly attenuates intravenous self-administrationof cocaine and amphetamine in laboratory animals(Gawin, 1991; Roberts et al., 1980), and kainic acidlesions of the nucleus accumbens disrupt bothcocaine and heroin self-administration (Zito et al.,1985). Like cocaine, amphetamine, opiates andseveral other abused drugs (Di Chiara and Jmperato,1988; Yoshimoto er al., 1991), MDMA increasesextracellular levels of DA in the nucleus accumbens(White et al., 1994; Yamamoto and Spanos, 1988)and it is very likely that the DA projection to thenucleus accumbens is as essential for the rewardingproperties of MDMA as it is for cocaine andamphetamine.

The role that 5HT terminals in the nucleusaccumbens might play in the rewarding effects ofabused drugs is less clear than that of DA. However,many abused drugs increase extracellular levels of5HT in the nucleus accumbens (White et al., 1994;


Yoshimoto et al., 1991) and 5HT itself increasesrelease of DA in the nucleus accumbens (Benloucifand Galloway, 1991; Parsons and Justice, 1993a).The serotonergic input to the nucleus accumbens is ofmoderate density, with the shell region being moredensely innervated than the core (Steinbusch, 1981).Most of the 5HT terminals in the nucleus accumbensarise from cell bodies in the dorsal raphe nucleus ofthe midbrain, but some innervation arises from themedian raphe nucleus as well (Azmitia and Segal,1978; Lorens and Guldberg, 1974; Vertes, 1991).

In addition to the monoaminergic input from theVTA and the dorsal raphe nucleus, the nucleusaccumbens gets excitatory amino acid (glutamateand/or aspartate) input from such Iimbic systemassociated areas as the medial prefrontal cortex, thehippocampus, the amygdala and the midline thalamicnuclei (Fuller et al., 1987; Groenewegen et al., 1990;see Kalivas, 1993 for review). Over the past decade,several electrophysiological studies have examinedthe effects of MDMA on neuronal excitability insome of the brain regions that are part of themesocorticolimbic system and these studies arereviewed below.

5.2. MDMA Effects in the Medial PrefrontalCortex

The medial prefrontal cortex (mPFC) whichprovides a major excitatory drive to the nucleusaccumbens (Brog e~ al., 1993; Fuller et al., 1987;Walaas, 1981; Wright and Groenewegen, 1995) andto the VTA and adjacent substantial nigra of themidbrain (Carter, 1982; Christie et al., 1989) is richlyinnervated by both 5HT- and DA-containing axonterminals (Steinbusch, 1981; Ungerstedt, 1971). Panand Wang 1991a); Pan and Wang, 1991b) demon-strated that acute systemic injection of MDMAinhibited spontaneous firing recorded extracelhrlarlyfrom cells in the mPFC of anesthetized rats. Theinhibition induced by MDMA appeared to bemediated at least in part by 5HT because it waspartially reversed by systemic injections of the 5HTantagonists granisetron and metergoline. TheMDMA-induced inhibition did not appear to bemediated by DA or other catecholamines because itwas attenuated by pretreating rats with the 5HTsynthesis inhibitor p-chlorophenylalanine (PCPA)but not by pretreatment with the catecholaminesynthesis inhibitor a-methyl-p-tyrosine (AMPT).Furthermore, systemic injection of the 5HT precursor5-hydroxytryptophan but not the DA precursorL-DOPA restored the inhibitory effect of MDMA inPCPA-pretreated rats. The MDMA-induced inhi-bition was also prevented by fluoxetine, an inhibitorof the 5HT transporter which prevents MDMA-in-duced 5HT release, but not by GBR 12909,a specificblocker of DA uptake. These observations by Panand Wang 1991a), 1991b) suggest that the excitatorydrive from the mPFC to the nucleus accumbenswould be reduced following systcmic administrationof MDMA and that this effect of MDMA is mediatedprimarily indirectly by MDMA-induced increases inextracelhdar 5HT. Similarly, the excitatory drivefrom the mPFC to the VTA (the source ofdopaminergic afferents to the nucleus accumbens)

would also presumably be reduced followingadministration of MDMA. However, becauseMDMA and the 5HT antagonists were administeredsystemically by Pan and Wang 1991a), 1991b), it isnot known whether the MDMA was acting onpresynaptic terminals within the mPFC to producethe inhibition or was acting in some other brainregion that projects to the mPFC.

5.3. MDMA Effects in the Hippocampus

The hippocampus also provides an excitatory driveto the nucleus accumbens (Boeijinga et al., 1990;Fuller et al., 1987;Pennartz and Kitai, 1991;Yim andMogenson, 1988). Effects of MDMA on excitabilityof hippocampal cells in vivo or in brain slicepreparations have not yet been studied. However,bath application of MDMA produced a dose-depen-dent increase in inward current recorded fromcultured rat hippocampal neurons (Premkumar andAhern, 1995).The inward current was associated witha decrease in the conductance of a barium-sensitiveresting K + channel and with increased neuronalexcitability. The blockade of the resting potassiumchannel did not appear to be mediated byMDMA-induced 5HT release within the culturepreparation because direct application of 5HT to thecultured hippocampal cells activated K + conduc-tance and decreased the excitability of the cells(Premkumar and Gage, 1994).Whether MDMA mayhave similar effects on hippocampal cells in vivo is notknown. However, a K+ channel conductanceincrease produced by 5HT released from presynapticterminals in vivo by MDMA may override the K+conductance decrease that was observed in thecultured neurons. The MDMA-induced DA releasemay have a predominantly inhibitory effect in thehippocampus. DA application to guinea-pig hippo-campal neurons in brain slices hyperpolarized theresting membrane potential and increased theamplitude and the duration of the spike after-hyper-polarization (Suppes et al., 1985), both of whichwould lead to decreased firing rates of the cells.Furthermore, Hsu (1996) has reported recently thatDA decreased excitatory postsynaptic potentials(EPSPS) that were evoked by stimulation of theSchaffer Collateral-Commissural pathway in hippo-campal slices. The inhibition of the EPSPS wasdose-dependent and appeared to be mediated bypresynaptic DA D, receptors. Studies of MDMAeffects on hippocampal neuronal excitability in brainslices and in vivo would be useful for determiningwhether MDMA is likely to reduce excitatoryafferent input to the nucleus accumbens from thehippocampus as well as from the prefrontal cortex.

5.4, MDMA Effects on 5HT Neurons in the DorsalRaphe Nucleus

Most of the 5HT-containing axons that project tothe forebrain arise from the dorsal raphe nucleus andthe median raphe nucleus of the brainstem (Azmitiaand Segal, 1978;Steinbusch, 1981).The median raphenucleus gives rise to 5HT fibers with large sphericalvaricosities while the 5HT fibers arising from dorsalraphe cells are very fine and have small varicosities

JPN 49/5–-F


(Kosofsky and Molliver, 1987). The fine fibers ~hatproject from cells in the dorsal raphe nucleus are theones that are most vulnerable to damage by repeit tedadministration of MDMA (Wilson et al., 1989). “rhedorsal raphe nucleus is also the source of most of the5HT-containing fibers within the nucleus accumbens(Vertes, 1991). Sprouse et a/. (1989), Sprouse e/ al.(1990)) and Bradberry et al. (1991) examined theeffects of acute bath application of MDMA onextracellularly recorded spontaneous firing of ph} sio-Iogically identified serotonergic cells in the dorsalraphe nucleus in a midbrain slice preparation.Serotonin cells in the raphe nucleus are inhibited by5HT acting at autoreceptors located on thesomatodendritic regions of the cells (Aghajanian andVanderMaelen, 1982). Bath application of MDMAinhibited firing of the cells in a concentra~iondependent manner, presumably by releasing 5HTfrom terminals in the slice. Dialysis studies conductedin parallel with the recording studies indicated thatapplication of MDMA to the bath medium causedthe release of 5HT with a time course Ihatcorresponded to the decrease in dorsal raphe cellfiring rate (Sprouse et al., 1989). The selective 5HTuptake inhibitor fluoxetine which prevented MDMA-induced 5HT release in the midbrain slice (Bradberryet al., 1991) also blocked the MDMA-ind~icedinhibition of 5HT cell firing in the raphe nuclel!s inthe slice preparation. In contrast, the selective NEuptake inhibitor desipramine did not alter theMDMA-induced inhibition of tiring. Furthermore,pretreatment of the slices with the 5HT precursorL-tryptophan lowered the dose of MDMA requiredto inhibit neuronal firing and increased the amt~untof 5HT released from the slice by MDMA (Bradberryet al., 1991; Sprouse etal., 1990). This Sei ofexperiments strongly suggests that MDMA cat actdirectly within the dorsal raphe nucleus to release5HT which in turn, inhibits firing of the serotmincells in the nucleus. Intracellular studies have no{ yetbeen conducted in the raphe nuclei to determinewhether MDMA may have any direct effects OJ!theexcitability of the serotonin cells that are not

mediated by MDMA-induced release of 5HT oranother neurotransmitter.

If MDMA also inhibits the firing of serotonergiccells in the dorsal raphe nucleus in t]iuo,itwould beexpected to decrease impulse-mediated release of5HT from terminals in the nucleus accumbens andfrom terminals in the VTA that are the origin of theDA-containing fibers in the nucleus accumbens.However, direct stimulation of 5HT and DA releasefrom terminals in the nucleus accumbens by MDMAmay override any MDMA-induced inhibitory effectson firing rates of the dorsal raphe nucleus cells.Indeed, in uiuo dialysis results indicate that bothsystemic and local administrations of MDMAincrease extracellular levels of 5HT and DA in thenucleus accumbens and adjacent striatum (Coughet al., 1991;White et al., 1994)despite any effects thatit may have on firing rates of the serotonergic cells.

5.5. MDMA Effects on DA Neurons in the VentralTegmental Area

The dopaminergic cells in the VTA and substantialnigra that project to the nucleus accumbens and thestriatum can be distinguished from non-dopamin-ergic cells in the same brain regions by theircharacteristic slow firing rates, irregular firingpatterns and long-duration biphasic or triphasicaction potential waveforms (Aghajanian and Bunney,1977; Bunney et al., 1973; Grace and Bunney, 1983).Spontaneous firing of these DA-containing cells isinhibited by application of DA which acts directly onD, autoreceptors to hyperpolarize the cells (Aghaja-nian and Bunney, 1977; Grace and Bunney, 1983;Johnson and North, 1992)and indirectly by acting onD, receptors on non-dopaminergic cells to stimulaterelease of the inhibitory neurotransmitter GABA(Cameron and Williams, 1993). Acute systemicinjections of MDMA also partially inhibit spon-taneous firing of DA cells in the VTA and thesubstantial nigra of anesthetized rats (Kelland et a/.,1989; Matthews et al., 1989) and the MDMA-in-duced inhibition is attenuated significantly following

DA DA DA MDMA Mt))A Mt))A Mt))A M133A40 80 60 20——— —— ———

Fig. 3. Effects of microiontophoretically ;tpplied DA and MDMA on spontaneous firing of twophysiologically identified dopaminergic cells i:l the VTA. The oscillograph pen that recorded cumulativespikes was reset every 10 sec. Lines above ~he records indicate periods of drug ejection and numbers

indicate ejection current (nA).


25t MDMA

O 30 60 90 120

Time (sec.)Fig. 4. The time course of the effect of MDMA (60 nA, 60see) on spontaneous tiring of 18 dopaminergic cells in theVTA is plotted (means and SEM). The firing rate duringeach 10sec period during and following MDMA applicationis expressed as a percentage of the baseline firing rateaveraged over a 2 min period just prior to MDMA

application.

depletion of either 5HT or DA (Kelland et al., 1989).Our laboratory recently has extended these obser-vations by demonstrating that MDMA appliedlocally by microiontophoresis also partially inhibitsspontaneous firing of electrophysiologicaliy identifiedDA cells in the VTA of chloral-hydrate anesthetizedrats (Fig. 3). Records from a DA cell that firedspontaneously at about 3 Hz (top) and a cell that firedat about 8 Hz (bottom) are shown. Microion-tophoretic application of MDMA (20–60 nA, 60 see)inhibited the spontaneous firing rate of both ceI1s,asdid microiontophoretic application of DA. Theamount of inhibition produced by MDMA ejectionvaried both within and between cells (Fig. 3). Thetime course for the effect of MDMA (60 nA, 60 see)on 18 dopaminergic cells in the VTA is plotted inFig. 4. The inhibitory effect of MDMA wasstatistically significant (p < 0.01) but failed to

E%#ahclMA m m+auLP

ar@c 1000am8eg 50@bl

t%

%

#“Drug

Fig. 5. The DA D, receptor antagonist sulpiride (SULP)attenuated significantly the inhibitory effect of MDMA onspontaneous firing of dopaminergic cells in the VTA.Meansand SEM from nine cells that were tested with MDMA (60nA, 120 see) and then retested with the same dose ofMDMA applied during ejection of SULP (30 nA, 6 rein).*Significant difference between MDMA and MDMA+

SULP, p <0.05, paired f-test, two-tailed.

suppress entirely the firing of most cells as was foundpreviously with systemic injections of MDMA(Matthews et al., 1989). The inhibition developedslowly following onset of the MDMA ejection periodand the firing rate slowly recovered to pre-injectionbaseline levels within 2–3 min following offset ofMDMA application for all cells. Microiontophoreticapplication of the D2 antagonist sulpiride (30 nA)beginning 2 min before and continuing for 2 min afterthe offset of MDMA ejection (60 nA, 120 see)significantly attenuated the inhibitory effect ofMDMA (Fig. 5).

These results strongly suggest that MDMA can actlocally within the VTA to partially inhibit spon-taneous firing of the dopaminergic cells in the VTAby increasing extracellular concentrations of DA.Systemic administration of MDMA is known toincrease extracelhdar levels of DA in the adjacentsubstantial nigra (Yamamoto et al., 1995) and verylikely does so in the VTA as well. The DA-mediatedinhibitory effects of MDMA on dopaminergicneurons in the VTA may be counteracted partially by5HT-mediated excitatory effects. The VTA is richlyinnervated by 5HT-containing terminals, some ofwhich make synaptic contact with the DA cells(Herve et al., 1987)and MDMA probably induces anincrease in extracelhrlar levels of 5HT in the VTA asit does in other brain regions that contain 5HTterminals. Microiontophoresis of 5HT into thesubstantial nigra of anesthetized rats or electricalstimulation of the dorsal raphe nucleus cells that send5HT projections to the substantial nigra inhibit thefiring of dopaminergic cells in that region (Crossmaner al., 1974; Dray ef al., 1976; Kelland et al., 1990).However, there is evidence that 5HT may also haveexcitatory effects on the dopaminergic cells. Microin-fusion of 5HT into the VTA increases DA efflux inthe nucleus accumbens, suggesting that 5HT doesincrease the firing of these dopaminergic cells thatproject to the accumbens (Guan and McBride, 1989).One mechanism by which 5HT appears to increasethe excitability of dopaminergic cells in the VTA is byinteracting with 5HTl~ receptors to inhibit presyn-aptic release of GABA (Johnson et al., 1992).However, 5HT also may stimulate somatodendriticDA release from the dopaminergic cells in the VTA(as it does from DA terminals in the nucleusaccumbens) which could, in turn, inhibit the firing ofthe cells by acting on the Dz autoreceptors. In brainslices, bath application of serotonin was found toenhance DA-induced inhibition of VTA dopamin-ergic neurons whether 5HT increased or decreasedspontaneous firing of the cells when applied alone(Brodie and Bunney, 1994). Although 5HT and DAhave complex effects in the VTA, the predominanteffect of MDMA on spontaneous firing of thedopaminergic cells is inhibitory. The mechanisms bywhich MDMA decreases spontaneous tiring of thedopaminergic cells in the VTA are not known.However, cocaine, which also has a predominantlyinhibitory effect on spontaneous firing of dopamin-ergic cells, has been demonstrated to both hyper-polarize the dopaminergic cells by blocking DAuptake (Lacey et al., 1990) and to inhibit GABArelease from presynaptic terminals by blocking 5HTuptake (Cameron and Williams, 1994). MDMA


probably has effects in the VTA that are at least ascomplex because, like cocaine, it blocks reuptakc of5HT and DA, but in addition, it also stimuktesrelease of DA and 5HT from presynaptic termil ials(see Section 2).

5.6. MDMA Effects in the Nucleus Accumbens

As discussed previously (Section 2), drugs witheuphoric properties increase extracellular DA in thenucleus accumbens whether the drugs are cen[ralnervous system stimulants such as cocaine, ampheta-mine and MDMA (Di Chiara and Imperato, 1088;Hernandez et al., 1987; Kalivas and Duffy, 1’}90;White el al., 1994; Yamamoto and Spanos, 1988i orcentral nervous system depressants such as opiiitesand alcohol (Di Chiara and Imperato, 1‘/88;Yoshimoto et cd., 1991). Food reward also incre<lsesextracelluar DA in the nucleus accumbens (HerT.an-dez and Hoebel, 1988) and some neurons within thenucleus accumbens of awake, behaving animals firespecifically preceding or just after reward deliver orwhen the animals view a stimulus associated ~vithreward (Apicella et al., 1991; Rolls and Willi~lms,1988; Schultz et al., 1992). In addition to firin{l inassociation with food or water reward, some neul onsin the nucleus accumbens alter firing pattern incocaine self-administering rats just prior to the [/mewhen the animal will press a lever to ob~ainintravenous cocaine or for a few minutes just af[~r alever press delivered cocaine (Carelli and Deadw: ler,1994; Chang et al., 1994). These findings combinedwith early observations that lesions in the nucleusaccumbens disrupted cocaine and heroin self-adl min-istration (Zito et al., 1985) indicate that the nucleusaccumbens is an essential component of brainpathways that mediate reward as proposed more {han10 years ago by Roberts et a/. (1980) and Wise .mdBozarth (1984).

No studies of MDMA effects on neuronal act. vityin the nucleus accumbens of awake, behaving animalshave been conducted to date, but our laboratoryrecently has examined the effects of MDMA onneuronal firing in the nucleus accumbens ofanesthetized rats using extracellular unit recor.lingcombined with microiontophoresis (Fig. 6). Thenucleus accumbens neurons were driven by c>cledpulses of glutamate because most nucleus accum oensneurons are silent in anesthetized rats (White andWang, 1986; Woodruff et al., 1976). The effects ofMDMA on glutamate-evoked firing were compmedto the effects of DA and 5HT which are known to bereleased in the nucleus accumbens by MDMA (M’hiteet al., 1994). In addition, DA and 5HT depictingdrugs and receptor antagonists were employed tc testwhether MDMA effects on nucleus accum bensexcitability might be mediated by DA and/or 5HT.Both 5HT and DA previously have been delnon-strated to inhibit glutamate-evoked firing of nestnucleus accumbens cells (White, 1986; White andWang, 1986). Since many neurochemical andanatomical differences have been described for thecore and shell regions of the nucleus accumbens(Brog et al., 1993; Deutsch and Cameron, !992;Groenewegen and Russchen, 1984; Heimer e( al.,1991; Meredith et al., 1992; Pennartz et a[., ‘994;

Fig. 6. Single unit recording from the nucleus accumbenscore combined with microiontophoresis using a multi-barrelglass microelectrode, The hatched area medial to andventral to the nucleus accumbens core indicates the nucleusaccumbens shell. The micropipettes contained NaCl forrecording and for automatic current balance, dye to markthe recording site, glutamate (GLU) to drive the quiescentcells, MDMA, and some combination of 5HT, DA, agonistsand antagonists for specific subtypes of 5HT and DAreceptors or a specific 5HT uptake blocker. The drawing ofthe brain section is modified from Paxinos and Watson

(1986).

Zahm and Heimer, 1993), effects of MDMA in thesetwo regions of the accumbens are discussedseparately.

5.6.1. Nucleus Accumbens Core

Microiontophoretic application of MDMA pro-duced a dose-dependent inhibition of glutamate-evoked firing in nearly every ceil that has been testedin the core region of the nucleus accumbens(Obradovic er al., 1996:White ef al., 1994). Examplesof this inhibition are illustrated in Fig. 7. Theinhibition was not mimicked by ejection of Na + ionsfrom a NaCl control solution, but was similar toinhibition produced by DA and 5HT ejection.Simultaneous application of MDMA potentiated theinhibition of glutamate-evoked tiring that wasproduced by low ejection currents of DA or 5HT(White et al., 1994). Dose (current)-response curvesindicated that the inhibitory effect of MDMA wasstatistically significant compared to the effect ofequivalent currents applied to the saline controlsolution for MDMA doses of 30 nA, 60 sec andhigher (Obradovic et al., 1996). Applications of 5HTor the selective 5HTz~,,C receptor agonist l-(2,5-dimethoxy-4-iodopheny l)-2-aminopropane (DOI)(Martin and Humphrey, 1994; Van Wijngaardenet al., 1990) also significantly inhibited glutamate-evoked firing of the nucleus accumbens cells with adose–response relationship that was not differentfrom MDMA (Fig. 8). A selective agonist for 5HT,,receptors, 8-hydroxy-(2-di-n-propylamine) tetralin(8-OHDPAT) and relatively selective agonists for5HT3 receptors, 2-methyl-5 -hydroxytryptamine


(2M5HT) and l-(m-chlorphenyl)-biguanide (CPBG)also were effective inhibitors of glutamate-evokedfiring. An agonist with high affinity for 5HT, ~receptors, CGS12066B also inhibited glutamate-evoked firing of nucleus accumbens cells (Obradovicet al., 1996), but there is no highly selective agonistfor this receptor subtype currently available (VanWijngaarden et al., 1990). These results suggest that5HT released by MDMA may interact with avariety of 5HT receptor subtypes to depressexcitation of nucleus accumbens cells by excitatoryamino acids. Blockade of MDMA-induced 5HTrelease by application of the selective 5HT uptakeinhibitor fluoxetine (FLX) prevented inhibition ofglutamate-evoked firing by MDMA, as did appli-cation of the 5HT2~,2creceptor antagonist ketanserin(KET) and the selective 5HT,, receptor antagonistWAY1OO135 (Fig. 9), providing further evidencethat MDMA inhibits neuronal firing in the nucleus

.

accumbens at least in part by increasing extracellu-Iar levels of 5HT.

The MDMA-induced inhibition of glutamate-evoked firing in the nucleus accumbens also ispartially dependent upon MDMA-induced increasesin extracellular DA. Pretreatment of rats with the5HT synthesis inhibitor PCPA did not preventMDMA-induced inhibition of glutamate-evokedfiring in the nucleus accumbens. However, pretreat-ment with both PCPA and the catecholaminesynthesis inhibitor AMPT did significantly attenuatethe inhibitory effect of MDMA (White et al., 1994).The selective DA D, receptor antagonist SCH39166(Chipkin et al., 1988), but not the D, receptorantagonist sulpiride, also significantly attenuated theinhibitory effect of MDMA on neuronal firing in thenucleus accumbens (White et al., 1994). Furthermore,depletion of DA by combined pretreatment with theneurotoxin 6-hydroxydopamine (60HDA) and the

MDMAB& Q

MDMA40

D% DAc“&A — —20 20

650MDMA

5HT MDMA20 20

G35 2 min

Fig. 7. Effects of MDMA, 5HT and DA on glutamate-evoked firing of four different nucleus accumbenscells. The cells were driven by cycled pulses of glutamate and the oscillograph pen that recordedcumulative spikes was reset at the onset of each glutamate pulse. The height of each histogram indicatesthe total number of spikes that occurred in each glutamate-application cycle. Lines above each recordindicate periods of drug ejection and numbers indicate ejection currents (nA). The Na in (B) representssodium (and hydrogen) ions ejected from an acidic saline control solution. (A) and (B) MDMA produceda dose-dependent inhibition of glutamate-evoked firing. (C) and (D) MDMA also potentiated the

inhibitory effects of low doses of DA and 5HT (White ef al., 1994).


100

75

50

25

0

vMDMA

owl

ADOI

12:=

o 20 40 60 0 20 40 60

Current (nA) Current (nA)

Fig. 8. MDMA, 5HT and several 5HTagon,sts that are relatively selective for specific 5HT receptorsubtypes all significantly inhibited glutamat;-evoked firing of cells in the nucleus accumbens corecompared to the effects of equivalent current, applied to an acidic saline (NaCl) control solution, Foreach ejection current tested (20, 30,40 and 60 :nA),the decrease in firing during the60 sec ejection periodis expressed as a percentage of the baseline Ilring rate that occurred in the 2 min period immediatelypreceding drug ejection, Means and SEM arc plotted and the NaCl results are shown on both the left-and the right-hand sides for ease of comparis{ n with the other drugs. The MDMA was tested on 57 cellsin 28 rats, 5HT on 44 cells in nine rats, DOI on 35 cells in 10 rats, 2M5HT on 13 cells in five rats, CPBGon 27 cells in seven rats and 8-OHDPAT on 1$cells in six rats. *Significant differences (p < 0,01) betweenNaCl and all the other drugs, two-way repeated measures analysis of variance with POSIhoc comparisons

of means (~)bradovic ?( a/., 1996).

synthesis inhibitor AMPT significantly attenuated theinhibitory effect of MDMA applied with all bu[ thehighest ejection currents (Fig. 10). The inhibitoryeffects of 5HT, however, were unaltered in the s:imecells in which the inhibitory effects of MDMA wereattenuated (Fig. 10). Taken together, the ability offluoxetine, 5HT2~,,c and 5HT,,. receptor antagonists,the D, receptor antagonist, and DA depletion toattenuate MDMA-induced inhibition in the nulleusaccumbens strongly suggests that both MDMA-in-duced DA release and an intermediate step ofMDMA-induced 5HT release are the means by whichMDMA alters neuronal activity in this nucleus

Anatomical and electrophysiological studiessuggest that complex mechanisms underlie alterationsin nucleus accumbens neuronal excitability thal areproduced by MDMA-induced release of 5HT andDA. Ultrastructural analysis showed that a I:mgeproportion of 5HT terminals in the nucleusaccumbens make direct synaptic contact withdendrites in the nucleus (Van Bockstaele and Pickel,1993). In addition, many 5HT terminals arc inapposition to tyrosine hydroxylase (TH)-containingterminals (presumably dopaminergic), and man! arein apposition to unlabeled (presumably non-dopa-minergic) terminals. Thus, the anatomical frameworkis present for 5HT to affect directly postsynaptic cellsin the nucleus accumbens and to modulate (or bemodulated by) presynaptic release of DA and otherneurotransmitters. Nearly every known 5HT receptorsubtype is present within the nucleus accumbensincluding high densities of 5HT4 receptors and highlevels of mRNA for 5HT6 receptors (Jakeman <I al.,1994; Ruat et al., 1993);high to intermediate densitiesof 5HT2A(formerly named 5HTj), 5HTjC (fornlerly5HT,C) and 5HT,, receptors (Bruinvels et d., 1994;

Mengod et al., 1990a, Mengod et al., 1990b; Pazosand Palacios, 1985; Pazos et al., 1985; Pompeianoet al., 1994; Voigt e? al., 1991); moderate to lowdensities of 5HT, receptors (Barnes et al., 1990;Kilpatrick et al., 1987)and low or sparse densities of5HTl~ receptors (Khawaja, 1995). Thus, 5HT that isreleased by MDMA has the potential to havemultiple direct and indirect effects on nucleusaccumbens neuronal excitability. For example,GABAergic cells are a major constituent of both theshell and the core of the nucleus accumbens(Mugnaini and Oertel, 1985) and are both theprojection neurons of the nucleus and constitute apopulation of interneurons that synapse within thenucleus. Excitability of these cells may be modified bydirect, postsynaptic effects of 5HT or by indirect 5HTmodulation of presynaptic terminal release of GABAfrom projection neuron axon collaterals or inter-neurons, DA from mesoaccumbens terminals, and/orother neurotransmitters from presynaptic terminalsof interneurons within the accumbens or frompresynaptic terminals of other afferent neurons.

The inhibitory effects of 5HT on glutamate-evokedfiring of nucleus accumbens cells that are illustratedin Fig. 7 and Fig, 8 are compatible with previousobservations that electrical stimulation of the dorsalraphe nucleus produced depression of neuronal firingin the anatomically similar striatum (Miller et d.,1975; Olpe and Koella, 1977). The raphe-evokeddepression was prevented by pharmacological de-pletion of brain 5HT, suggesting that the inhibitionwas mediated by 5HT release. However, it is notknown whether the effects of 5HT released by raphestimulation or 5HT applied by iontophoresis aredirect effects on the post-synaptic cells or aremediated by presynaptic modulation of the release of


M::A MC):A M~t#A M~~A—

F~X.....................................

WOMA

KET‘xA& M MA

5. . . . . . . . . . . . . . . . . . . . . . . . . . .

MOWA&

M~;A M::A M;:A

W;Y —. . . . . . . . . . . . . . . . . . . . . . . . . .

Q% 2MINFig. 9. The inhibitory effect of MDMA on glutamate-evoked firing of cells in the nucleus accumhens corewas markedly attenuated by the 5HT uptake blocker fluoxetine (FLX, top record) which preventsMDMA-induced 5HT release, by the 5HTM,,c receptor antagonist ketanserin (KET, middle record) andby the 5HT,A receptor antagonist WAY1OOI35 (WAY, bottom record). Note that the inhibitory effectof MDMA recovered by 5–10 min after offset of the uptake blocker and the antagonists. Ejection periodsfor FLX and the 5HT receptor antagonists are indicated by dotted lines above the records, ejection periodsfor MDMA are indicated by solid lines and ejection currents (nA) are indicated by the numbers (modified

from Obradovic et al., 1996).

other transmitters. In brain slices, 5HT produced adirect (tetrodotoxin insensitive) depolarization ofaccumbens cells that was blocked by the relativelyselective 5HT2 antagonists ketanserin and mianserin(North and Uchimura, 1989). Selective 5HTI, and5HT, agonists did not affect accumbens cellmembrane potentials in this in uitrw study. Theseobservations suggest that 5HT may have quitedifferent and perhaps opposite direct effects onpostsynaptic membrane potentials and indirect effectson terminals that are presynaptic to the cells. Nicolaet al. (1996) demonstrated recently that bathapplication of 5HT reduced the amplitude ofelectrically evoked excitatory postsynaptic potentials(EPSPS) in nucleus accumbens cells in brain slices.Further studies to determine whether this is apresynaptic effect of 5HT, what neurotransmittermight mediate the effect if it is presynaptic, andwhether 5HT may also alter IPSPS in the accumbenshave not yet been reported.

The potential mechanisms by which MDMA-in-duced DA release might alter neuronal excitability inthe nucleus accumbens are as complex as those of5HT. The GABAergic medium spiny neurons aremost likely the exclusive output route of theaccumbens (Chang and Kitai, 1985) and are likely tobe the neurons that are most often examined in unitrecording studies. The nucleus accumbens also

contains a population of GABAergic interneurons,and GABA-containing neurons are the most numer-ous cells in the nucleus (Mugnaini and Oertel (1985).Ultrastructural studies revealed that terminals in thenucleus accumbens that are positive for TH-likeimmunoreactivity (which are presumably DA-con-taining terminals) make synaptic contacts withGABA-immunoreactive dendrites and somata indi-cating that DA can have direct postsynaptic actionsin the accumbens, perhaps on both GABAergicprojection neurons and GABAergic interneurons(Pickel ej al., 1988).In addition, terminals that werepositive for TH-like immunoreactivity were found inapposition to presynaptic terminals in the accum-bens, some of which were also positive for TH-likeimmunoreactivity and some of which were non-dopa-minergic (Zahm and Brog, 1992). Thus DA isanatomically situated in locations that would allowfor modulation of presynaptic release of transmittersas well as for direct postsynaptic effects. As a furthercomplication, DA routinely escapes from thesynaptic cleft during dopaminergic synaptic trans-mission in the nucleus accumbens and so could act ina paracrine fashion to alter neuronal excitability at adistance from the synaptic terminal (Garris et al.,1994).The presence of multiple DA receptor subtypesin the nucleus accumbens (Bardo and Hammer, 1991;Booze and Wallace, 1995;Mansour e( al., 1992;Yung


100 A B *.100 ‘?

75 %+. 75 R.\ .>

i

o ‘$\-

50 50 \‘\T‘ay

25 0 60 HOA.PreT. 25 0 60 HOA.PreT.oc—_—0 20 40 60

5HT Current

● Vehlc..P,.aT.

o0 20 40 60

MDMA Current

Fig. 10. Depletion of DA by pretreatment with the neurotoxin 6-hydroxydopamine and the synthesisinhibitor AMPT (60 HDA-PreT group) did n, ,t alter inhibition of glutam~te-evoked firing produced by5HT (A), but reduced significantly the inhibition produced by MDMA (B) compared to effects on cellsin vehicle pretreated control animals (Vehic.- PreT group). For the six rats in the 60 HDA-PreT group,MDMA was tested on 23 cells and 5HT was tested on 15 of the 23 cells. For the six rats in the Vehic,-PreTgroup, MDMA was tested on 27 cells and 5H”I’was tested on 25 of these 27 cells. *Significant differences(p < 0.01) between the 60 HDA-PreT and the Vehic.-PreT. groups, two-way repeated measures analysis

of variance with past hoc comparisons of means (modified from Obradovic er al., 1996).

et al., 1995) provides yet more possibilities forcomplex actions of DA in the nucleus.

Woodruff et al. (1976)was the first to demonstratethat microiontophoretic application of DA inhibitsspontaneous and excitatory amino acid-evoked firingof most neurons in the nucleus accumbens in I ivo.This finding has been reproduced many times (i.e.Akaike et a/., 1983; Henry and White, 1991; Whiteand Wang, 1984, White and Wang, 1986; White e al.,1995a, White et al., 1995b) and appears tc bephysiologically relevant because electrical stimulationof the VTA inhibits glutamate-evoked and synapti-cally evoked firing of nucleus accumbens cells, andthe inhibition is blocked by selective D, antagonists(Yim and Mogenson, 1982; Hara e( al., 1989).Incontrast to these inhibitory findings, intracellularrecording from nucleus accumbens cells in Pivoindicated that DA application produced a consistentsmall depolarization of the cells even though it didnot increase spontaneous firing (Yim and Mogenson,1988). However, despite this depolarizing effect, DAstrongly inhibited the postsynaptic depolarization ofaccumbens cells that was evoked by amyg.ialastimulation (Yim and Mogenson, 1988). Recorcilngsfrom accumbens cells in guinea-pig tissue slicesrevealed both hyperpolarizing (Dl receptor-mediated)and depolarizing (Dz receptor-mediated) tetrodot~)xininsensitive effects of DA on membrane potentials,and the majority of cells responded to DA with aninitial hyperpolarization followed by depolarize tion(Uchimura et al., 1986; Higashi et al., 1989).Pennartz et al. (1992) found that DA attenuated hothexcitatory and inhibitory synaptic inputs to nm.Ieusaccumbens shell cells in rat brain slices by acting atpresynaptic D, receptors. However, direct effects ofDA on membrane potential and input resistance werevariable and did not correlate with changes in thepostsynaptic potentials. Whether this discrepancy in

DA direct postsynaptic effects on membrane poten-tials can be attributed to differences in recordinglocations within the nucleus accumbens or to speciesdifferences has not been resolved. Nicola el al. (1996)also found that DA acted at presynaptic D, receptorsto reduce EPSPS elicited in nucleus accumbens cellsby stimulation of cortical afferents in rat brain slices.Similarly to Pennartz ef a/. (1992), DA reduced thesize of the EPSPS without affecting the restingmembrane potential or input resistance. Cocaine andamphetamine also reduced the excitatory synapticresponses elicited by stimulation of cortical afferents,and although the mechanism by which cocaine actedwas not tested, amphetamine appeared to act viastimulation of a presynaptic D1 receptor (Nicolaef al., 1996). The response to amphetamine appearedto be mediated by DA release because it was reducedfollowing depletion of DA by the neurotoxin6-hydroxydopamine. The identity of the neurotrans-mitter that was affected by DA was not established.However, DA did not appear to be activating GABArelease because the EPSPS were depressed by DA tothe same extent whether or not a GABA receptorantagonist was present. Although similar studies havenot been conducted with MDMA, it is very likely thatMDMA reduces firing of nucleus accumbens cellsin vivo by releasing DA and 5HT that act at least inpart on presynaptic terminals to reduce tonicexcitatory synaptic transmission.

5.6.2. Nucleus Accumbens Shell

There are several differences in DA and 5HTdistribution in the shell and core subregions of thenucleus accumbens. Concentrations of both DA and5HT are significantly greater in the shell than in thecore, reflecting differences in the density of terminalsthat contain these monoamine (Deutsch and


Cameron, 1992; Steinbusch, 1981; Voorn ef al.,1989). The morphology of the 5HT-containing fiberterminals also differs in these two subdivisions of thenucleus accumbens. Most of the 5HT-containingfibers in the core region are very fine in diameter withsmall varicosities (Van Bockstaele and Pickel, 1993).In contrast, a large number of fibers in the shellregion have thicker diameter with large varicosities.Many more of the DA-containing terminals in themedial shell than in the core region of the nucleusaccumbens contain co-localized cholecystokinin(CCK); and CCK receptors are more denselyconcentrated in the medial shell than in the core(Hokfelt et al., 1980; Zarbin et al., 1983). White andWang (1984) demonstrated that the anatomicaldifference in CCK terminal and receptor distributionis complimented by a functional difference. Cellslocated in the dorsomedial nucleus accumbens (shellregion) were excited by significantly lower doses ofCCK applied by microiontophoresis than were cellslocated in the lateral (core) region. Part of the effectof CCK on neuronal excitability in the nucleusaccumbens may be mediated by DA release. Bathapplication of CCK inhibits DA release from slices ofthe anterior nucleus accumbens and stimulates DArelease from dices of the posterior nucleus accumbens(Marshall et al., 1991). Whether diverse effects ofCCK on DA release might be observed in shell vscore regions of the nucleus accumbens is not known.Pennartz et al. (1992) found that DA decreasedpostsynaptic responses that were recorded from cellsin the shell region but not the core region of thenucleus accumbens in vitro, further suggesting thatthe neurochemical differences between the core andthe shell might reflect functional differences in theeffectiveness of the neurotransmitters.

Since the effects of MDMA on neuronal excit-ability in the nucleus accumbens appear to be

100

75

50

25

0

0- Shell, MDMA

● ✍ core, MOMA

A- NAG, NsCI

mediated largely by MDMA-induced increases inextracellular DA and 5HT (White et al., 1994;Obradovic e[ al., 1996), MDMA might also beexpected to have differential effects in the twosubregions of the nucleus. This hypothesis was testedrecently in our laboratory (White et al., 1995b).Effects of microiontophoretically applied MDMA onglutamate-evoked firing were compared for cells inthe nucleus accumbens core and shell. Responses ofthe nucleus accumbens cells also were compared toresponses of cells in the caudate/putamen (CP,striatum) because the core region of the nucleusaccumbens has input-output connections and neuro-transmitter distributions that are more analogous tothose of the striatum than to the shell of the nucleusaccumbens (Heimer er al., 1991; Zahm and Heimer,1993). Similarly to nucleus accumbens cells, gluta-mate-evoked firing of most cells in the CP issuppressed by microiontophoretic application of DA,DA D, receptor agonists and 5HT (Hu and Wang,1988). Applications of MDMA (20-60 nA, 60 see)produced a dose-dependent inhibition of glutamate-evoked responding in the core, the shell and the CP(Fig. 11). The inhibition produced by the highest doseof MDMA (60 nA, 60 see) significantly inhibited cellsin all three regions compared to the effect of anequivalent current applied to a NaCl controlsolution. However, ceils in the shell region of thenucleus accumbens were significantly less inhibitedthan cells in the core region by all but the highest dose(60 nA, 60 see) of MDMA tested (Fig. 11, left).Responses to MDMA did not significantly differbetween cells in the core and cells in the CP (Fig. 11,right-hand side).

Microiontophoretic application of DA, the D,agonist SKF 38393, 5HT and the 5HT,.,,C agonistDOI all produced equivalent inhibition of glutamate-evoked firing in the nucleus accumbens core and shell

100

75

50

25

● �

m-1

CP,MDMA

A - core, NaCl. 0’

10 100 10 100

Current (nA) Current (nA)

Fig. 11. The MDMA (applied at 30, 40, and 50 nA for 60 sec. but not at 20 or 60 nA) was significantlyless inhibitory on glutamate-evoked tiring of cells in the shell region of the nucleus accumbens than oncells in the core region of the accumbens or in the striatum (CP). The data from the cells in the nucleusaccumbens core are plotted on both the left- and the right-hand sides for ease of comparison to cells inthe other regions. Effects of the highest ejection current (60 nA, 60 see) applied to an acidic saline controlsolution (NaCl) are indicated by the triangles (filled triangle represents cells from the core and shell regionscombined; open triangle represents cells from the core only). Note that the x-axis is a log scale. *Significantdifferences (p < 0.01) between effects of MDMA on cells in the shell and cells in the core, two-way

repeated measures analysis of variance with pos/ hoc comparisons of means.

470 S. R. White et al,

MDUA MONA20 20

ld:$A

M:#G

ii. .... .. .... .. . ..... . .. ..

imwmvldfffI/iMmluM~~ 120

i ::

zc%

UDMA M$y rll:M30

KET

Q40

Fig. 12. MDMA produced a long-lasting fac Iitation of glutamate-evoked firing of motoneurons in thehypoglossal nucleus that was sometimes pt.xeded by a short-lasting inhibition. The 5HT receptorantagonists methysergide (MTSG) and ketanwrin (KET) attenuated the excitatory effect of MDMA andenhanced the inhibitory effect. The motoneur ms were driven by cycled pulses of glutamate (G) and theoscillograph pen that recorded cumulative act!on potentials was reset at the onset of each glutamate cycle,The height of each histogram represents the Iotal number of spikes that occurred during the glutamatecycle. The dotted lines above the records ind]-ate periods of MTSG and KET application and the solid

lines indicate periods of MDMA application. Numbers indicate ejection currents (nA).

(White etal.,1995b), as has been reported previ,:uslyfor DA (Hu and White, 1994). Therefore, thedifference in shell and core sensitivity to the effec.s ofMDMA cannot be attributed to difference: insensitivity of cells in the two regions to DA and 5HT.The most likely explanation for the differential el~ectof MDMA in the two regions is that MDMA nayincrease extracelltdar levels of 5HT and/or DA LO agreater extent in the core region than in the shell, venthough 5HT and DA terminals are denser in the hellthan in the core. Although there is no direct evidmcein support of this mechanism to date, thel~ issuggestive evidence. MDMA is selectively toxic tc thefine-diameter 5HT-containing fibers that predl lnli-nate in the core region (Mamounas e{ al., 199I; VanBockstaele and Pickel, 1993; Wilson etal., 1989) andthis neurotoxicity depends upon initial 5HT depl(tionfrom the terminals (Sprague etal.,1994). The largerdiameter, beaded fibers in the sheli may be sptredfrom the toxic effects of MDMA because MDMAmay not reiease 5HT as readiiy from these fibels asfrom the fibers in the core. The MDMA-indllcedinhibition of giutamate-evoked firing in the co e isknown to be dependent upon 5HT reiease, beciusefluoxetine, which biocks 5HT reiease, also bl)cksMDMA-induced inhibition (Fig. 9). If MDMJ’i is,indeed, iess effective at reieasing 5HT from terml naisin the sheii, iess 5HT wouid be avaiiabie to stim~,iateDA reiease, and the combination of iess reiease ofboth of these mcmoamines wouid be expected to !eadto iess inhibition of giutamate-evoked firing in thesheii region. As additional evidence that this mayoccur, Pierce and Kaiivas (1995) reported data thatshowed that a high dose of amphetamine (30 .LM)appiied through a diaiysis probe in the mediai rejionof the accumbens sheii of saiine-treated rats incre.wedextraceiiuiar DA iess than an equivalent dos: ofamphetamine infused into the core region of thenucieus accumbens.

The specific ceii types from which recordings vere

made in the studies discussed above are not known.Since the medium spiny GABAergic neurons are themost numerous ceii type in the accumbens, it is iikeiythat many recordings were from those ceiis. However,the acetyichoiine-containing interneurons have iargeceii bodies (Meredith et al., 1989) and so may havebeen sampied disproportionateiy in the recordingstudies even though they are relatively few in numberin the nucieus accumbens. Further studies todetermine whether MDMA has differential effects onactivity of identified projection celis in the sheli andcore would ciarify whether MDMA has a greaterinhibitory effect on the output pathways from thecore that are more ciassicaiiy “basai gangiia-iike”pathways (Zahm and Heimer, 1993) than on outputpathways from the sheii which appear to be more“iimbic-iike”.

5.7. MDMA Effects on Motoneurons in theHypoglossal Nucleus

In addition to euphoric properties, MDMA is asympathetic nervous system stimuiant which aisostimulates somatic motor tone. Foiiowing ingestionof MDMA, human subjects experience jaw clenching,increased deep tendon reflexes and gait instability asweli as increased heart rate and biood pressure (Steeieet al., 1994). It is iikeiy that both the vasomotor andthe sornatomotor effects of MDMA aiso aremediated by increases in extraceiiuiar ieveis ofmonoamine. Our laboratory recentiy examined theeffect of microiontophoretic application of MDMAon giutamate-evoked firing of motoneurons in thehypogiossai nucieus of anesthetized rats. MDMAproduced a siowiy developing, long-lasting increasein giutamate-evoked firing of the motoneurons thatwas preceded in some celis by short-iasting inhibition(Fig. i2), effects that are mimicked by 5HT or NEapplication to faciai or spinai motoneurons (McCaiiand Aghajanian, 1979; White and Neuman, i980).


$ ‘o 60 120 180 240 300

Time (sec.)

Fig. 13. The time course of MDMA (30 nA, 120 see) effectson glutamate-evoked firing of 22 hypoglossal motoneuronsis shown. The dotted rectangle indicates the highest andlowest changes from baseline firing that were produced byapplication Of an equivalent ejection current to an acidicsaline control solution. The MDMA produced a briefinhibition followed by a very long-lasting facilitation ofglutamate-evoked firing. The firing rates did not return topre-MDMA baseline rates until 5–10 min after ejection

offset.

The excitatory effect of MDMA was inhibitedmarkedly by the non-specific 5HT antagonistmethysergide (MTSG) and by the selective 5HTz~,2Creceptor antagonist ketanserin (KET). Both MTSGand KET (to a greater extent) partially inhibitedglutamate-evoked firing when applied alone (perhapsby blocking the excitatory effects of tonically released

5HT) and increased the initial inhibitory effects ofMDMA (Fig. 12). This 5HT antagonist-inducedinhibition of the excitation produced by MDMA and“unmasking” of the inhibition produced by MDMAmimicked the effects that these antagonists have on5HT-mediated excitation and inhibition of gluta-mate-evoked tiring of spinal motoneurons (Jacksonand White, 1990), suggesting that the MDMA effectswere mediated in part by increasing extracelhdarlevels of 5HT. Effects of MDMA (30 nA, 120 see) on22 hypoglossal motoneurons are shown in Fig. 13.MDMA produced a short-lasting but statisticallysignificant inhibition of glutamate-evoked firing thatwas followed by a statistically significant increase infiring that did not return to baseline until 5–10 minafter MDMA ejection offset.

It is well established that 5HT, NE and DA all haveexcitatory effects on motoneurons in the brainstemand the spinal cord (Bayliss et al., 1995; Katakuraand Chandler, 1990;Kubin et al., 1992;Larkman andKelly, 1992; Parkis et al., 1995; Smith et al., 1995;VanderMaelen and Aghajanian, 1980; Wang andDun, 1990;White and Fung, 1989;White et al., 1991)and neuronal terminals that contain these mono-amine are found in both the ventral horn of thespinal cord and in cranial motor nuclei in thebrainstem. Therefore, MDMA ingestion by humansmay increase deep tendon reflexes and muscle tone byincreasing extracelhrlar levels of all three of theseamines. Effects of MDMA on blood pressure andheart rate may be mediated similarly by monoaminereleased from terminals in the intermediolateralnucleus of the spinal cord (the location of

Saline PreTDA40

DA60

MDMA PreTDA DA

:: 40 60

AG36 2 MIN

Fig. 14. Effects of DA application on glutamate-evoked firing of cells in the nucleus accumbens core ofrats tested 14 days after the last injection of saline (Saline PreT) or MDMA (MDMA PreT). Lines abovethe records indicate periods of DA ejection and numbers indicate current intensity (nA). Cells were drivenby cycled pulses of glutamate (G), and onset of each glutamate application reset the curvilinearoscillograph pen that recorded cumulative spikes. The peak of each histogram indicates the number of

spikes that occurred during each glutamate cycle.


Saline PreT

g*Q

gEa

G30

MDMA PreT6HT 5HT 5HT30 40 60

G :0 7imi--Fig, 15. Effects of 5HT on a nucleus accumbens core cell in a saline-pretreated rat (Saline PreT) and in

an MDMA-pretreated (MDMA PreT) are s’mwn.

sympathetic preganglionic neurons that are exctedby both NE and 5HT), or by catecholamines releasedperipherally. Effects of MDMA on excitability! ofvisceromotor neurons in the spinal cord or brainsiemhave not yet been tested.

6. REPEATED MDMA EXPOSURE, EFFEC rsON NEURONAL FIRING IN THE NUCLEt S

ACCUMBENS

The neurotoxic effects that repeated MDMA hason 5HT-containing axons in the forebrain oflaboratory animals are long-lasting. Levels of f HTare reduced and 5HT immunoreactive fibers are

25

to~

O 20 40 60

DA Current

Fig. 16. Dose (current)–response curves for DA effecls onglutamate-evoked firing of 33 nucleus accumbens core cellsfrom rats in the MDMA-PreT group and 28 cells from ratsin the Saline-PreT group are plotted. *Significant differcrices(p < 0.01) between the cells from the two pretreat)nentgroups, two-way repeated measures analysis of variance

with post hoc comparisons of means.

Lines and numbers are as described for Fig. 14

diminished for several months or longer after the lastadministration of MDMA in some brain regions,including the striatum and nucleus accumbens(Fischer et al., 1995; Scanzello et al., 1993). It ishighly likely, therefore, that toxic doses of MDMAwould produce long-term changes in serotonergic andperhaps dopaminergic neurotransmission in thenucleus accumbens. These long-term changes mayunderlie some of the psychopathologies that arereported by human patients long after the lastingestion of MDMA (Steele e? al., 1994). Ourlaboratory (Obradovic e~ al., 1995) has investigatedwhether responses of cells in the nucleus accumbenscore to microiontophoretic application of DA or5HT are altered 9–15 days after the last adminis-

100 * *

75L@<.Q.\‘.?+50

25

00 20 40 60

- 0- MDMA.Pr.T.

–*– S.li”e-prel.

5HT Current

Fie. 17. Dose (current)–resuonse curves for 5HT effects ongl~tamate-evoked firin~ of !34nucleus accumbens core cellsfrom rats in the MDMA-PreT group and 20 cells from ratsin the Saline-PreT group, Reponses to 5HT for cells in thetwo groups were significantly different only for the 30 and

40 nA doses.


tration of an eight-dose sequence of MDMA that isknown to be selectively neurotoxic to 5HT axons inrats (20 mg/kg, subcutaneous, twice/day for foursuccessive days). Records of DA and 5HT effects onglutamate evoked firing are compared for cells fromsaline pretreated (PreT) and MDMA pretreated ratsin Fig. 14 and Fig. 15. The inhibitory effects of DAand 5HT on glutamate-evoked firing were virtuallyabolished in the two cells from MDMA pretreatedrats that are shown.

Comparisons of the effects of DA on 33 cells fromsix MDMA pretreated rats and 28 cells from sixsaline pretreated rats indicated that the inhibitoryeffect of DA was significantly (p < 0.01) attenuatedin the MDMA pretreated rats for all but the lowestdose of MDMA-tested (Fig. 16, two-way repeatedmeasures analysis of variance with post hoccomparisons of means). The 5HT was tested on 34cells from six MDMA pretreated rats and 20 cellsfrom six saline pretreated rats (Fig. 17). Theinhibitory effect of the 30 and 40 nA (60 see) dosesof 5HT was attenuated significantly by MDMA-pre-treatment, but the highest dose of 5HT (60 nA, 60see) was equally inhibitory on cells from both groupsof animals. The reduced ability of DA and 5HT toinhibit the nucleus accumbens cells in MDMApretreated rats was specific in that microiontophoret-ically applied GABA-produced equivalent dose-de-pendent inhibitions of glutamate-evoked tiring in thenucleus accumbens of both groups of animals.Furthermore, the doses of glutamate that wererequired to tire cells from both groups of animals atequivalent baseline firing rates did not differ. Thus,pretreatment with neurotoxic doses of MDMAselectively reduced the inhibitory effects of DA and5HT on nucleus accumbens neurons tested more tbana week following the last administration of MDMA.

The finding that the inhibitory effects of DA werereduced following pretreatment with multiple dosesof MDMA was opposite to the effect of repeateddoses of cocaine. Multiple doses of cocaine, anabused drug that also increases extracelhdar levels ofDA and 5HT in the nucleus accumbens (Kalivas andDuffy, 1990, Kalivas and Duffy, 1993; Parsons andJustice, 1993b), increase the inhibitory effect of DAon glutamate-evoked firing of nucleus accumbenscells (Henry and White, 1991; White et al., 1995a).Cocaine, however, is not neurotoxic to serotonergicaxons (Kleven et al., 1988)and this might account forthe opposite effects of the two abused drugs.Repeated injections of cocaine might be expected toproduced repeated episodes of increased extracelhdarlevels of both DA and 5HT, whereas repeatedMDMA injections may produce only an initialepisode of increased extracellular 5HT and DAfollowed by repeated episodes of increased extracellu-lar DA only. Whether this is indeed the means bywhich repeated exposure to the two abused drugsproduces opposite effects on nucleus accumbensneuronal responses to DA and 5HT remains to bedetermined. Reports of psychopathologies includingdepression and psychosis following cessation of useare common for frequent users of cocaine and appearto be increasing for frequent users of MDMA, but,interestingly, frequent users of MDMA do not reportcravings for MDMA during abstinence from the drug

(Schifano and Magnij 1994;Steele et al., 1994). Thesedifferential long-term changes in humans may reflectthe fact that MDMA is neurotoxic to 5HT-contain-ing axons in the forebrain and cocaine is not.

7. CONCLUDING REMARKS

A major mechanism by which MDMA produceseuphoric effects is undoubtedly by increasingextracellular levels of DA and 5HT in the nucleusaccumbens, a property that it has in common withmany other abused drugs such as cocaine, alcoholand opiates. However, the potential ways in whichthis increased extracelh.dar DA and 5HT mightinteract with presynaptic and postsynaptic receptorsto alter neurotransmission in the nucleus accumbensand in brain regions that project to the nucleusaccumbens are exceedingly complex. As a furthercomplexity, the projection neurons from the nucleusaccumbens do not seem to act as a functional unit,but rather constitute numerous input/output “neur-onal ensembles” that are functionally distinct(Pennartz et al., 1994). Almost nothing is known todate about whether MDMA may affect differentiallydistinct ensembles within the nucleus accumbens.However, the fact that MDMA is significantly lessinhibitory to glutamate-evoked firing in the shellregion than in the core suggests that particularlocations of the postsynaptic neurons within theaccumbens, and perhaps, the neurotransmittercontent of the postsynaptic neuron may be determi-nants of the response to MDMA.

The ability of MDMA to alter neurotransmissionin the brain is not restricted to brain regions that areimplicated in the rewarding effects of abused drugs.MDMA produces the facilitation of somatic mo-toneuron excitability that would be expected of adrug that increased extracellular levels of 5HT, DAand NE. It is probable that the muscle hypertonusand increased deep tendon reflexes that occur in someMDMA users are attributable to this action of thedrug. It is very likely that MDMA increasesextracellular levels of monoamine in every brainregion that contains substantial numbers ofmonoaminergic terminals, and so can mimic most ofthe neurophysiological effects of monoamine.

It is not yet certain that repeated MDMA ingestionis neurotoxic to forebrain 5HT terminals in humansas it is in laboratory rodents and non-humanprimates. However, it is now clear that the neurotoxiceffects of MDMA in laboratory rats are accompaniedby changes in 5HT and DA neurotransmission in thenucleus accumbens that last for at least 2 weeksfollowing the final exposure to MDMA (the longestpost-drug time period that has been tested to date).The effects of changes in brain levels of neuropeptidessuch as CCK and neurotensin that occur followingMDMA exposure are just beginning to be studied.Whether changes in the neuropeptides may belong-lasting and what the effects of those changesmay be on neurotransmission in the brain are not yetknown. However, the numerous and complex effectsof MDMA on neurotransmission in the braincombined with increasing reports of psychopatholo-gies from frequent users of MDMA suggest that

474 S. R. White et a[.

repeated exposure to this drug may be much moreharmful than seems to be believed by many y<~ungrecreational users.

Ackno}t>[edg<,menf.f-This research was supported in part byN[H grant DA-08116 to SRW. The authors thank N IDA(MDMA), Janssen Pharmaceuticals (Ketanserin), Eli LillyCo. (tluoxetine) and Wyeth Research (WAY100135~ forgifts of drugs.

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