AIBREVlATlON&@PCA,p-chloroamphetamlne;MDMA,3,4-methylenedloxymethamphetamine;@-drnuoro-PCA,fi,@-dmuoro-p-chloroamphetamlne;H75/12, 4-methyl-a-ethyl-meta-tyramlne;5-I-I1AA,5-hydrox@1ndoleacetIcacid; DOPAC,3,4-dihydroxyphenylaceticacid; HVA,homovanhlllcacid; SKF525A,proadlfen;MAO,monoamineonidase;5-lIT,5@ydroxytryptan@ne.

0022-3565/93/2671-0417$03.00/ONa Jouarw@or Piiaius@cox.ooyANDExnananmi. Tum@s'su'ricaCopyrightC 1993by Tb. AmericanSocietyfor Pbarmaco1o@and ExperimentalTherapeutics

PossibleInvolvementof Dopamineinthe LongTermSerotoninDepletionbyp-Chloroamphetamineand @3j3-Difluoro-p-ChioroamphetamineinRats

MARKG. HENDERSON,1KENNETHW. PERRYand RAY W. FULLERL@IllyResearch Laboratories,LillyCorporateCenter@EllLillyand Company, Indianapolis,IndianaAccepted for publicationJune 16, 1993

Vol. 267, No. 1Printed in U.S.A.

ABSTRACTThe role of dopamine in the long-term depletion of serotonin inrat brain by p-chloroamphetamine(PCA) and related compoundswas Inves@gatedby comparing effects of @-difluoro-p-chIoroamphetamlne ($ft-dlfluoro-PCA) and 4-methyl-a-ethyl-meta-tyramine (H75/12), reported to cause only short-term serotonindepletion, with those of PCA. A single dose of fi,$-difluoro-PCAhad no long-term effects on serotonin in whole rat brain, evenafter pretreatmentwith proadilenwh@hdecreased the rate atwhich @-difluoro-PCAdisappeared from brain. The possibilitythat proadtfen might antagonize serotonin deple@onwas ruledout; proadifen did not prevent kng-term serotonin depletion byPCA. Long-term depletkn of brain serotonin was found afterrepeated Injections of @9,fi-drnuoro-PCA(fiveinjections 4 hr apart)and was prevented by fluoxetine pretreatment. @-DmUOrO-PCA

given after the monoamine oxidase inhibitor pargyline or aftercarbidopa/L-dopa also caused long-term serotonin depletion,although H75/12 did not. At early times after single dosesproducing the same initial depletion of serotonin, PCA caused alarge increase in dopamine and a largedecrease in the metaboifte3,4-dihydroxyphenylacetic acid inwhole brain,thereby increasingthe ratio dopamine/3,4-dihydroxyphenylacetic, and the other twodrugs caused smaller effects. Extracellular dopamine was increased markedly by PCA, less by @-dmuoro-PCA,and not atall by H75/1 2. These resufts suggest an association betweendopamine release and long-term depletion of serotonin and addto evidencethat dopaminereleaseby PCA may be essentialtoits neurotoxic actions on brain serotonin neurons.

PCA and related chlorinated amphetamines were shown todeplete brain serotonin in rats nearly three decades ago(Pletacher et at., 1963, 1964; Fuller et aL, 1965). Later it wasrecognized that these compounds, even after a single dose,caused long-term depletion of brain serotonin persisting forweeks or months in rats (Frey, 1970; Sanders-Bush et at., 1972).The depletionofbrain serotoninby PCAoccursin two distinctphases. The acute depletion of serotonin during the first fewhours after PCA administration is reversible by subsequentadministration of an inhibitor of the serotonin transporter(Fuller et aL, 1975). The later phase of serotonin depletion isvery long-lasting and is not reversible by uptake inhibition.The long-term depletion of brain serotonin appears to resultfrom a neurotoxic effect on serotonin neurons. Histologic cxamination has shown degeneration of serotonin projections(Mamounas and Molliver, 1988;Berger et aL, 1989) and loss ofretrogradely labeled serotonin neurons in the raphe (Fritschy

Received for publication February 8,1993.1 Current address.- A/M Group, A Unit of Merck & Co., Inc., 4400 Park Road,

Suite 120, Charlotte, NC 28209.

et aL, 1988) after PCA administration. Not only is serotonindepleted at long times after PCA injection, but so are otherparameters associated with brain serotonin neurons, includingtryptophan hydroxylase (Sanders-Bush et aL, 1975), serotoninuptake by synaptosomes (Sanders-Bush and Steranka, 1978),serotonin uptake sites labeled with radioligands (D'Amato etal., 1987) and serotonin turnover measured by several methods(Fuller and Snoddy, 1974). Both the acute, reversible and thelong-term, irreversible phases of serotonin depletion are prevented by pretreatment with an uptake inhibitor before PCAinjection (Meek et aL, 1971; Fuller et at., 1975; Fuller, 1992).

The mechanism underlying the long-term effects of PCA onbrain serotonin neurons continues to be speculative. A comparable long-term, neurotoxic depletion of striatal dopaminehas been reported to occur after high doses of amphetamine inrats (for references, see Fuller, 1985). In that case, autoxidationproducts of dopamine have been postulated to play a role inthe neurotoxicity (Fuller and Hemrick-Luecke, 1982). Dopamine has also been implicated in the neurotoxicity towardserotonin neurons produced by MDMA and methamphetamine

417

418 Hendersonet al. Vol.267

(Schmidt and Gibb, 1985; Schmidt et at., 1990). We investigatedthe possibility that dopamine might be involved in the neurotoxic effects of PCA toward serotonin neurons by doing corn

parative studies on two PCA analogs which resemble PCA inreleasing serotonin acutely but differ markedly from PCA intheir propensity to cause neurotoxicity to serotonin neurons.@3,fi-Difluoro-PCAhas been reported to cause acute depletionof brain serotonin in rats when higher doses are given toproduce brain levels equivalent to those of PCA, but not tocause long-term depletion of brain serotonin (Fuller et at.,1973b). H75/12 is another amphetamine analog that depletesbrain serotonin acutely without causing long-term effects likethose causedby PCA (Carlsson et at., 1969;Fuller et al., 1976;Henderson and Fuller, 1992).

Materialsand Methods

Animals. Male Sprague-Dawley rats (Charles River Breeding Laboratories, Portage, MI) weighing between 160 to 180 g were grouphoused in suspended wire cages under controlled environmental conditions with a 12-hr light/dark cycle and ad libitum access to food andwater. In all cases, rats were killed by guillotine. Whole brains werequickly removed and were rapidly frozen on dry ice. Brains were storedin a —¿�6O@Cfreezer until assayed.

Monoamine analysis. Serotoninand its metabolite,5-HIAAanddopamine and its metabolites, DOPAC and HVA, were assayed usinghigh performance liquid chromatography with electrochemical detection as described earlier (Fuller and Perry, 1989).

In uivo microdialysis. Studies using in vivo microdialysis in conscious freely moving rats were performed as described earlier (Perryand Fuller,1992),with the exceptionof the constituentsand flow rateof the artificial cerebrospinal fluid. The artificial cerebrospinal fluid

contained 150 mM NaCl, 3 mM KC1, 1.7 mM CaCl2,0.9 mM MgCl2and Na2HPO4 buffer pH 7.4 pumped through the dialysis probe at 2@tl/min.

Drug level assay. f3j3-Difluoro-PCAwas measuredin whole ratbrain 1, 2, 3 and 4 hr after injection of 0.4 mmol/kg (96.8 mg/kg of thehydrochloride salt) i.p. of the compound with or without a 2-hr pretreatment with 50 mg/kg i.p. SKF-525A. The assay method usedfluorescamine, a reagent for detecting primary amines (Udenfriend et

Fluoxetine

Fig. 1. Fluoxetine pretreatment prevents the acute depletion of brainserotonin by fl,$-difluoro-PCAin rats. @9j3-Difluoro-PCAhydrochloridewas injectedat a dose of 98.6 mg/kg (0.4 mmol/kg p.) 1 hr afterfluoxetinehydrochloride(10 mg/kg p.) and 4 hr before rats were killed.Mean values ±standard errors for six rats per group are shown.*@jgflffi@@difference from control group; ‘¿�significantdifference fromgrouptreatedwithflj9-difiuoro-PCAalone(P< .05).

Control@ 343-difluoro-PCA

0 Proadifen •¿�@34l-difluoro-PCA+Proadifen

Fig. 2. Inabilityof 13,@9-difiuoro-PCAto causelong-termdepletionof brain5-hydroxyindoles in rats pretreated with proadifen (SKF-525A). fi,@Difluoro-PCA hydrochloride was injected at a dose of 98.6 mg/kg (0.4mmol/kg i.p.) 2 hr after proadifen hydrochloride (50 mg/kg i.p.) and 1week before rats were killed.Mean values ±standard errors for six ratsper group are shown.

—¿�U—-@34@-difIuoro-PCA—¿�. —¿�Proadlfen + f343-dlfluoro-PCA

I5HT 5HIAA

200

@ 100

0 1 2 3 4Hours

Fig. 3. Influence of proadifen pretreatment on the rate of disappearanceof fljl-difluoro-PCAfrom rat brain. fljl-Difluoro-PCAhydrochloridewasinjected at a dose of 98.6 mg/kg (0.4 mmol/kg i.p.) alone or 2 hr afterproadifen hydrochloride(50 mg/kg i.p.).Mean values ±standard errorsfor six rats per group are shown.

r@ Control@ PCA

Q Proadifen •¿�Proadlfen+PCA5.

4.

I

FIg. 4. Proadifenpretreatmentdoesnotpreventthe long-termdepletionof 5-hydroxyindolesby PCA. PCA hydrochloride(20.6 mg/kg i.p.) wasinjected2 hr after proadifen(50 mg/kg i.p.)and 1 week before rats werekilled. Mean values ±standard errors for six rats per group are shown.‘¿�Signifi@tdifference from control group (P < .05).

a

+

5HT 5HIAA

1993

U,a,0EC

419

(Philadelphia, PA), L-Dopa was provided by Monsanto (St. Louis,MO), levo-a-(3,4-dihydroxybenzyl)-a-hydrazinopropionic acid monohydrate (carbidopa) was provided by Merck Sharp & Dohme ResearchLaboratories (Rahway, NJ), H75/12 was purchased from Labkemi AB(Goteborg,Sweden) and PCA hydrochloride was purchased from RegisChemical Company (Morton Grove, IL). flj3-Difluoro-PCA, fluoxetine,H75/12 and PCA were dissolved in water, whereas L-dopa and carbidopa were injected in a 5% acacia suspension. All drugs were administered i.p.

Statistical analysis. Comparisons between multiple treatmentgroups were performed using a one-way analysis of variance followedby a Tukey post-hoc test. Comparisons between only two treatmentgroups were analyzed using a two-tailed t test. In all cases P < .05 wasconsidered a statistically significant difference.

Results

The ability of @3j3-difluoro-PCA (0.4 mmol/kg or 96.8 mgIkg) to cause acute depletion of serotonin in whole brain 4 hrafter injection is shown in figure 1. This dose of $,fl-difluoro

PCA is 4 times our usual dose of PCA so that drug concentrations in the brain are similar for the two drugs (Fuller et at.,1973b). The depletion of brain serotonin by flj3-difluoro-PCAwas carrier-dependent in that it was blocked completely bypretreatment with fluoxetine at a dose of 10 mg/kg.

This acute depletion of serotonin by j.@,$-difluoro-PCAis notlong lasting as is the depletion of serotonin by PCA (Fuller etat., 1973b), and An experiment designed to see whether pretreatment with an inhibitor of drug metabolism (proadifen orSKF-525A) would allow @3j3-thfluoro-PCAto cause a longlasting depletion of brain serotonin is shown in figure 2. Proadifen had been shown to inhibit the metabolism of flj3-difluoroamphetamine and to increase its brain levels in rats (Fuller etat., 1973a). Accelerated metabolism of @3,fl-difluoro-PCA in ratshad been shown to prevent its serotonin-depletingeffects(Fuller et at., 1974a), so diminished metabolism might be expected to enhance the serotonin-depleting effects. Rats givenproadifen at 50 mg/kg 2 hr before $j3-thfluoro-PCA for thepurpose of inhibiting the metabolism of f3j3-difluoro-PCA bythe cytochrome P-450-linked mixed function oxidase systemwere found not to have depleted levels of serotonin and 5-HIAAat 1 week in whole rat brain (fig. 2). Brain levels of fl,fl-difluoroPCA in rats at 1 to 4 hr after injection of flj3-&fluoro-PCAalone or after proadifen are shown in figure 3. Drug levels werehigher in the proadifen-pretreated group, and the estimatedhalf-life was increased from 2.6 hr in the rats receiving $j3-difluoro-PCA alone to 3.6 hr in the proadifen-pretreated group.

Because f3j@-thfluoro-PCAdid not cause long-term depletioneven though its brain half-life was increased somewhat by

proadifen, the possibility that proadifen might block the longterm depletion of serotonin as uptake inhibitors do was inves

tigated by determining whether the long-term depletion of brainserotonin by PCA occurred in proadifen-pretreated rats. Brainserotonin was depleted at 1 week in rats given 0.1 mmol/kg(20.6 mg/kg) of PCA with or without proadifen pretreatmentis shown in figure 4.

As an alternative means of maintaining brain concentrationsof fl,fl-difluoro-PCA for a longer period, multiple injections ofthe compound were given. In rats receiving an injection of f3j3-difluoro-PCA (0.4 mmol/kg i.p.) every 4 hr for a total of fiveinjections, brain serotonin and 5-HIAA were depleted 1 weeklater (fig. 5). Pretreatment with fluoxetine (a 10-mg/kg i.p.single dose given 1 hr before the first dose of flj3-difluoro-PCA)

Dopamineand SerotoninDepletion

U ControlD @-dlfluoro-PCA

@ Fluoxetlne + @343-dlfluoro-PCA

Flg 5. Depletion of serotonin and 5-HIAA in rat brain 1 week afterrepeatedinjectionsof $,@-difiuoro-PCA. @-Dffluoro-PCAhydrochloridewas injectedevery4 hrat a dose of 98.6 mg/kg (0.4 mmol/kgi.p.),andrats were killed1 week later. Some rats were given fluoxetine hydrochloride (10 mg/kg i.p.) 1 hr before the first dose of fl,fi-difluoro-PCA.Meanvalues±standarderrorsfor fiveto six rats per groupare shown.

@ difference from control group; #5jgnifi@t difference from

group treated with @-difiuoro-PCAalone (P < .05).

0@5HT 5HIAA

. H75/12—¿�. —¿� @-dlfluoro-PCA

—¿�0-—PCA

4

3

2

*

1

0

Fig. 6. Time course of the acute depletion of brain serotonin by H75/12,@3,$-difiuoro-PCAor PCA. H75/12 was injected at a dose of 70 mg/kg(0.3 mmol/kgi.p.). @,$-Difiuoro-PCAhydrochloridewas injectedat a doseof 96.8 mg/kg (0.4 mmol/kgi.p.).PCAhydrochloridewas injectedat adose of 20.6 mg/kg (0.1 mrnol/kgi.p.).Allinjectionswere at zero time.Mean values ±standard errors for five rats per group are shown.Slgnlficantdifferencefrom zero time group (P < .05).

aL, 1972) and was based on a method described earlier for measuringPCA (Fulleret aL,1974b).Brains werehomogenizedin 4 vol of 0.1 NHC1,then 1 vol of 30%HC1O4was addedandthe reactionvessels weremixed and centrifuged for 5 mm. One milliliter of supernatant fluid

was removed and added to tubes containing 0.4 ml of 5 N NaOH and4 ml of toluene; the tube contents were mixed and centrifuged. Threeand one-half milliliters of the toluene was removed and added to a 2-ml wash with 0.2 M borate buffer (pH 10.0) and again mixed andcentrifuged.Three milliliters of the toluenewas then removedandadded to tubes containing 1 ml of 0.1 N HC1mixed and centrifuged.The toluene was then aspirated, and 0.5 ml of the acid layer was addedto tubes containing 1.5 ml of 0.2 M borate buffer (pH 10.0)and mixedwell.Whilethe samplewasvortexed,0.5 ml of a fluorescaminesolution(15 mg of fluorescamine/50 ml of acetone) was added. Fluorescenceintensity was determined in an Aniinco-Bowman spectrophotofluorometer with activation/fluorescence set at 390/490 nm.

Drugs. @,fi-Difluoro-PCA hydrochloride and fluoxetine hydrochloride were synthesized at Lilly Research Laboratories (Indianapolis,IN), SKF.525A was providedby Smith Kline & French Laboratories

2 4 6Hours

420 Hindsison st al Vol. 267

. H75/12—¿�. - @343-dlfluoro-PCA

—¿�0— PCA

0 2 4 6

Fig.7. Effectof H75/12,fi,@9-dlfluoro-PCAand PCAon brainconcentrationsof dopamineandDOPACandon theconcentration ratiodOpamlne/DOPACIn rats. H75/12was Injectedat adose of 70 mg/kg (0.3 mmol/kg i.p.). fi,$-Dmuoro-PCAhydrochloridewas InjeCtedat a dose of 96.8 mg/kg (0.4 mmol/kgi.p.).PCAhydrochloridewas Injectedat a doseof 20.6mg/kg(0.1 mmol/kgi.p.).i@Jlinjectionswere at zero time. Mean values±standard errors for fIve rats per group are shown. Slgnfficantdifferencefrom zero time group; ‘¿�signIfIcantdifferencefromgrouptreatedwith $,@-dIfiUOrO-PCAand 1-175/12groups(P <.05).

a,0EC

2C000

Ca,2a,a.

H75/120 @34@-dIfluoro-PCAI PCA

2 4 6Hours

prevented the depletion of both 5-hydroxyindoles, indicatingthe long-lastingdepletionhadoccurredvia a carrier-dependentmechanism. The repeated injection of @9,$-difluoro-PCAcausedselective depletion of serotonin as PCA itself does in that brainconcentrations of dopamine, DOPAC and HVA were notchanged (data not shown).

The acute depletion of whole brain serotoninproducedbyH75/12 (0.3 mmol or 70 mg/kg), @,fi-difluoro-PCA(04 mmol/kg) and PCA (0.1 mmol/kg) is shown in figure 6. The effectsof the three drugs were virtually identical at 2 hr postinjection,

but at 4 and 6 hr the effectsof PCA were slightlygreaterthanthose of H75/12 or fl,$-difluoro-PCA, whose effects had beenshown earlier not to persist at 24 hr (Fuller et aL, 1973b, 1976).That dopamineconcentration in whole brain was increasedduring this time period by PCA but not by $,@-difluoro-PCA isshown in figure 7. H75/12 significantly decreased dopamine,revealing its depletion of serotonin to be less specific than thatof the chlorinatedcompounds.All three compoundscausedadecrease in DOPAC concentration in brain, possibly via inhibition of monoamine oxidase; the order of effectiveness indecreasing DOPAC was PCA > fi,@-difluoro-PCA > H75/12.HVA concentrations (data not shown) were decreased to alesserextent,andmostby H75/12,the decreasesafter @-difluoro-PCA and PCA being not statisticaily significant. Because DOPAC is the direct product of MAO action on dopamine, the ratio dopamine/DOPAC can be an indication of MAO

inhibition. That the concentration ratio dopamine/DOPACwas increased nearly 4-fold after PCA is shown in figure 8(bottom panel). In contrast, the two non-neurotoxic analogscaused only modest (less than 2-fold) increases in the dopamine/DOPAC ratio.

Experiments with pargyline and L-dopa were done to seewhether treatments that increase dopamine would allow neurotoxicity to be manifest after administration of @-thfluoroPCA or H75/12. The effect of pretreatment with pargyline, aninhibitor ofMAO, on thelong-term depletion ofbrain serotoninby /i,fi-difluoro-PCA or H75/12 was investigated (fig. 8). Pargyline (100 mg/kg) was injected 1 hr before either @-difluoroPCA (0.4 mmol/kg) or H75/12 (0.3 mmol/kg) and 5-hydroxyindole concentrations in whole brain were measured 1 weeklater.Again,the administrationof a singledoseof @,fi-difluoroPCA or H75/12 did not result in long-term serotonin depletion.However,in rats pretreatedwith pargylineto inhibit MAO, asingle dose of fi,@-difluoro-PCA, but not of H75/12, depletedserotoninand 5-HIAA concentrationsat 1 week.The administration of a carbidopa/L-dopa(25:100mg/kg) combinationalso resulted in long-term depletion of serotornn and 5-HIAAby a single dose of fi,@-difluoro-PCA but not H75/12 (fig. 9).

The effectof H75/12 (0.15mmolor 30mg/kg), @-difluoroPCA (0.2 mmol or 48.4 mg/kg) and PCA (0.05 mmol or 10 mgIkg) on extracellular serotonin levels measured by in vivo brainmicrodialysis in conscious, freely moving rats is shown in figure

421

_ Carbidope+L-dopa+ @-dmuoro-PCA

Fig. 9. Brain 5-hydroxyindoleconcentrations 1 week after administrationof @-drnUOrO-PCAorH75/12to ratstreatedwithL-dopacombinedwithcarbidopa. H75/12 was injected at a dose of 70 mg/kg (0.3 mmol/kgi.p.). @3,fi-Dlfiuoro-PCAhydrochloridewas injected at a dose of 96.8 mg/kg (0.4 mmol/kgi.p.). Carbidopa(25 mg/kq i.p.) was injected30 mmbeforeL-dopa(100mg/kgi.p.),wh@hwask@JeCtedat the sametimeas

@,$-drnuoro-PCAand H75/12. Mean values ±standard errors for four tosix rats per group are shown. *#&@j@njf@@differences from controlgroup,grouptreatedwith carbidopa+ L-dopaand grouptreatedwithfi,fi-difiuoro-PCAalone, respectively(P < .05).

20

CU

I!Ua.

Fig. 10. ExtracellularcOncentratiOnSof serotonin in rat striatum after theinjectionof H75/12, $,@-difiUOrO-PCAor PCA.H75/12 was injeCtedat adose of 35 mg/kg (0.15mmol/kgi.p.).fi,ll-Difluoro-PCAhydrochloridewas Injectedat a dose of 48.4 mg/kg(0.2 mmol/kgl.p.).PCAhydrochloride was injectedat a dose of 10.3 mg/kg (0.05 mmol/kg i.p.). Allinjections were at zero time. Mean values ±Standard errors for five tosix rats per group are shown.

in the brain for a substantial time (Fuller et at., 1972). Forexample, m-chloroamphetamine does not cause long-term depletion of brain serotonin unless its usually rapid metabolism(via hydroxylation in the unoccupied pare position of thephenyl ring) is blocked by pretreatment with iprindole or somesimilar agent (Fuller et at., 1972; Fuller and Baker, 1974). Inthis study we have compared two compounds, $,$-difiuoro-PCAand H75/12, which cause a similar acute depletion of brainserotoninas PCA does(fig. 6), but which do not causelongterm depletion of brain serotonin after a single dose as PCAdoes.

$,fi-Difiuoro-PCA, like PCA, releases serotonin by a carrierdependent mechanism; pretreatment with fluoxetine, a selective inhibitor of the serotoninuptake carrier, prevents serotonindepletionby @-difiuoro-PCA(fig.1)justasitprevents

1993

2

DopamineandSerotonlnDepletion

rz@c@t@i@ Carbidopa+L-dopa+E Carbidopa+L-dopa0 @-dffluoro-PCAElIllH75/12

Control

Pargyllne treated

Fig.8. BraIn5-hydrox@indoleconcentrations1weekafteradministrationof @-dIfIUOrO-PCAor H75/12to ratstreatedwithpargyline,an inhibftorof MAO.H75/12 was Injectedat a dose of 70 mg/kg (0.3 mmol/kg i.p.).fi,fl-DIfIUOrO-PCAhydrochloridewas InjeCtedat a dose of 96.8 mg/kg(0.4 mrnol/kgi.p.).PargyIk@ehydrochloridewas Injectedat a dose of 100mg/kg i.p. 1 tw before @-dIflUOrO-PCAor H75/12. Mean values ±standarderrorsfor threeto six rats per groupare shown. Significantdifferencefromgrouptreatedwithpargylinealone(P< .05).

10. All three compounds caused a rapid, marked increase inextracellular serotonin, the effect of PCA being slightly greaterthan that off3,fi-difiuoro-PCA or H75/12. Despite the similarityof their effects on extracellular serotonin, the three drugs hadstrikingly different effects on extracellular dopamine (fig. 11).PCA caused about a 4-fold increase in extracellular dopamine,

@,fl-difiuoro-PCAcaused a slightly smaller increase and H75/12 had virtually no effect on extraceilular dopamine. Theselower dosesof all three drugswereusedin the microdiatysisexperiments becausethe higher doseswere not well toleratedin rats with implanted microdialysis probes.

Discussion

The mechanism(s) for the long-term serotonin depletion andneurotoxicity produced by PCA has never been firmly established. Some investigators have suggested the involvement ofexcitatory amino acid receptors (Finnegan et aL, 1991), theformation of autoxidationproductssuchas5,6-dihydroxytryptaxnine (Commins et aL, 1987)and the involvement of dopamine(Schmidt et aL, 1991a). One requirement for the long-termdepletion of brain serotonin seems to be that the drug persists

r@ Control0 @l,@-dlf1uoro-PCA

@ H75/12 5.

4

5HT 5HIAA

5HT 5HIAA

F@Pargyllnep Pargyllne+@343—dIfluoro-PCA

@ Pargyline+ H75/12

—¿�0•-PCA- U - @-dffiuoro-PCA

—¿�S.—H75/12

5HT 5HIAA

150 180 210 240 270

Minutes

422 Hendersonet al Vol.267

—¿�•0--PCA- U - @d1fIuoro-PCA

—¿�a,.-- H75/12

Evidence for a role of dopamine in the serotonergic neurotoxicity of substituted amphetamines has been reported byother investigators who used a variety of paradigms. For example, 5-HT2 receptor antagonists were found to attenuate theserotonin neurotoxicity measuredafter MDMA administrationto rats (Schmidtet aL, 1990, 1991b).Thesefindingsimplicateddopamine because 5-HT2 receptors facilitate dopamine synthesis and 5-HT2 receptor antagonists can block the release ofdopamine by amphetamine and MDMA (Sorensen et aL, 1992;Nash, 1990). Perhaps more direct evidence for dopamine'sinvolvement in serotonin neurotoxicity came from a study inwhich L-dopa, the precursor to dopamine, was found to potentiate serotonergic deficits produced by MDMA, methamphetamine and PCA in rats (Schmidt et at., 1991a). Johnson and

Nichols (1991) demonstrated that a non-neurotoxic analog ofMDMA, 5-methoxy-6-methyl-2-aminoindan, could be made todecreasespecificserotonergicparameterslong-termbycombining it with a nonvesiculardopaminereleaserS-(+)-amphetamine. Axt and Seiden (1990) showed that a-methyl-p-tyrosine,an inhibitor of dopamine synthesis, attenuated PCA-inducedserotonin depletion in rat brain, although they also pointed outthe a-methyl-p-tyrosine pretreatment did not completely prevent the formation of 5,6-dihydroxytryptamine, a serotoninneurotoxin, in response to methamphetamine administration.

Our investigation of dopamine involvement in PCA-inducedserotonergic neurotoxicity centered on comparing the dopamine-releasing effects of two non-neurotoxic amphetamine analogs, fl,fi-difluoro-PCA and H75/12, to those of PCA. Thedoses of @1j9-difluoro-PCA(0.4 mmol/kg) and H75/12 (0.3mmol/kg) were chosen to produce the same initial (2 hr)serotonin depletion in whole rat brain. These three drugs had

markedly different effects on tissue and extracellular concentrations of dopamine at these early times. PCA caused a largeincrease in the dopamine/DOPAC concentration ratio, suggestive of marked inhibition of dopamine oxidation by MAO(Fuller, 1966), and a large increase in extracellular dopamineconcentration,suggestingit releaseddopaminefrom intraneuronal stores. In contrast, @-difluoro-PCAhad much less effectthan PCA on the dopamine/DOPAC concentration ratio, suggesting it was a weaker MAO inhibitor. Nonetheless, fi,fidifluoro-PCA did increase extracellular dopamine, suggestingit released dopamine perhaps not as effectively as PCA. H75/12 had only a small effect on the dopamine/DOPAC concentration ratio and essentially no effect on extracellular dopamine. This order of efficacy—PCA> @,fi-difluoro-PCA> H75/12—parallels the order of efficacy of the drugs in causingserotonin neurotoxicity. PCA is the most neurotoxic, H75/12

is not neurotoxic and $,fi-difluoro-PCA is not neurotoxic exceptwhen given by repeated injections, in combination with anMAO inhibitor, or in combination with carbidopa/L-dopa toincrease dopamine stores available for release.

The ability of pargyline, a nonselective monoamine oxidaseinhibitor, to allow fi,$-difluoro-PCA to cause long-term depletion of serotonin and 5-HIAA does not in itself implicatedopamine specifically, because other monoamines are also affected by pargyline. Those fmdings, and the finding that Ldopa in combination with the peripheral decarboxylase inhibitor carbidopa allowed fij9-difluoro-PCA to cause long-termdepletion of 5-hydroxyindoles, implicate dopamine availabilityas a factor influencing neurotoxicity of substituted amphetamines toward serotonin neurons. H75/12 did not cause longterm depletion of 5-hydroxyindoles atone (Fuller et aL, 1976)

450

400

350

1300

I 250@ 200

C@ 150@ 100

50

40 -so 40 0 30 60 90 120 150 180 210 240 270

Fig. 11. Extracellularconcentrationsof dopaminein rat striatumafterthe injectionof H75/12, @,fi-difiuoro-PCAor PCA. H75/12 was injectedat a dose of 35 mg/kg (0.15 mmol/kg i.p.). @@,$-Dffluoro-PCAhydrochloride was injectedat a dose of 48.4 mg/kg (0.2 mmol/kg i.p.). PCAhydrochloride was injected at a dose of 10.3 mg/kg (0.05 mmol/kg i.p.).All injections were at zero time. Mean values ±standard errors for sixratspergroupare shown.

serotonin depletion by PCA (Fuller et at., 1975). It was important to show the carrier dependence of the serotonin depletionby this compound because it is much more lipid-soluble thanPCA (Fuller et aL, 1973b) and might have acted independentlyof the membrane transporter. @,fi-Difluoro-PCA,which is rapidly and extensively deaminated due to the influence of the twofluorines on the@ carbon, has a much shorter half-life thandoes PCA in rat brain (Fuller et aL, 1973b). A single dose offi,fi-difluoro-PCA does not cause the long-lasting neurotoxicdepletion of brain serotonin as caused by PCA. To prolong thepersistence of fi,fl-difluoro-PCA in rat brain, SKF-525A, aninhibitor of drug-metabolizing enzymes was administered torats to determine whether longer persistence of fi,fi-difluoroPCA could result in long-term serotonin depletion. A singleinjection of $,fi-difluoro-PCA did not deplete brain serotoninat 1 week even in proadifen-pretreated rats. Proadifen pretreatment slightly increased brain concentrations of @,$-difluoroPCA by slowing its rate of disappearance, but the effect wasrelatively small and did not cause the compound to be neurotoxic toward brain serotonin neurons. The possibility thatproadifen might be capable of antagonizing brain serotonindepletion by agents of this sort was considered because proadifen doeshavesomepharmacologiceffectsin additionto inhibiting microsomalenzymes(Viana and Osswatd,1970;Ho et at.,1978; Choo et aL, 1986) and has been shown to compete withmembrane binding of a serotonin uptake inhibitor, alaproclate(Ross, 1987). However, proadifen pretreatment did not alterthe long-term depletion of brain serotonin by PCA, so thefailure of flj3-difluoro-PCA to cause long-term depletion ofbrain serotonin after proadifen pretreatment was apparentlynot due to any protective action of proadifen.

When multiple injections of fi,@9-difluoro-PCAwere given torats (five injections spaced 4 hr or approximately two half-livesapart), brain serotonin and 5-HIAA concentrations were decreased 1 week later. This finding reveals that fi,$-difluoroPCA has the capability of causing PCA-like neurotoxicity andthat it fails to do so after a single dose because the drug isremoved from brain too rapidly. Although proadifen hadslightly prolonged the half-life of fi,fi-difluoro-PCA in brain,the persistence of @-difluoro-PCAin proadifen-pretreatedrats was still too short to produce the neurotoxicity which wasproduced by repeated injections.

Minutes

1993 Dopamine and Serotonin Depletion 423

FINNEGAN, K. T., KERR, J. T. AND LANGSTON, J. W.: Dextromethorphanprotects against the neurotoxic effects of p-chloroamphetainine in rats. BrainRca.558: 109—111,1991.

FREY,H. H.:p-Chloroamphetaniine—Similaritiesanddissimilaritiesto amphetamine. In Amphetamines and Related Compounds: Proceedings of the MarioNegriInstitute for PharmacologicalResearch,ed. by E. Costaand S. Garattini,pp. 343-355, Raven Press, New York, 1970.

FRITScHY,J. M., LYONS,W. E., MowvER, M. E. ANDGRzANNA,R.: Neurotoxiceffects of p-chloroamphetamine on the serotoninergic innervation of the tngeminal motor nucleus: A retrograde transport study. Brain Rae. 473: 261-270,1988.

FULLER,R. W.: Serotonin oxidation by rat brain monoamine oxidase: Inhibitionby 4-chioroaniphetamine. Life Sci. 5: 2247—2252,1966.

FULLER,R. W.: Persistent effects of amphetamine, p-chloroamphetamine, andrelated compounds on central dopamine and serotonin neurons in rodents.PsychopharmacoLBull.21: 528—532,1985.

FULLER,R. W.: Effects of p-chloroamphetamine on brain serotonin neurons.Neurochem. Rae. 17:449-456,1992.

FULLER, R. W. AND BAKER, J. C.: Long-lasting reduction of brain 5-hydroxytryptamine concentration by 3-chloroamphetamine and 4-chioroamphetaminein ipnindole-treated rats. J. Pharm. PharmacoL 26: 912-914, 1974.

FULLER,R. W., BAKER,J. C., PERRY,K. W. AND MOLLOY,B. B.: Effect ofphenobarbital on the metabolism and serotonin-depleting action of 4-chioro$,@9-&fluomaznphetamine in rats. Arch. mt. Pharinacodyn. 208: 274-278,1974a.

FULLER, R. W. AND HEMRICK-LUECKE,S. K.: Further studies on the long-termdepletion of stniatal dopamine in ipnindole-treated rats by amphetamine. Neuropharmacology 21: 433-438, 1982.

FULLER,R. W., HINES, C. W. ANDMILLS, J.: Lowering of brain serotonin levelby chloramphetamines. Biochem. PharmacoL 14: 483-488, 1965.

FULLER, R. W., MOLLOY,B. B. AND PARLI, C. J.: The effect of fl,$-difluorosubstitution on the metabolismand pharmacologyof amphetamines. In Paychopharmacology, Sexual Disorders and Drug Abuse, ed. by T. A. Ban, J. R.Boissier, G. J. Geese, H. Heimann, L Hollister, H. E. Lehmann, I. Munkvad,H. Steinberg, F. SWear,A. Sundwall and 0. Vinar, pp. 615-624, North-HollandPublishing Company, Amsterdam, 1973a.

FULLER,R. W., Pmuty, K. W., BAKER,J. C., P@u, C. J., LEE, N., DAY,W. A.AND M0LL0Y, B. B.: Comparison of the oxime and the hydroxylamine denivatives of4-chloroamphetamine as depletors ofbrain 5-hydroxyindoles. Biochem.Pharrnacol. 23: 3267—3272,1974b.

FULLER, R. W., PERRY, K. W. AND M0LL0Y, B. B.: Reversible and irreversiblephases of serotonin depletion by 4-chioroamphetamine. Eur. J. Pharmacol. 33:119—124,1975.

FULLER, R. W., PERRY, K. W. AND B@.KER,J. C.: Duration of the effects ofalpha-ethyl-4-methyl-m-tyramine (H75/12) on brain 5-hydroxyindole concentrationsin rats.J. Pharm.Pharmacol.28: 649—650,1976.

FULLER, R. W. AND PERRY, K. W.: Effects ofbuspirone and its metabolite, 1-(2-pynimidinyl)piperazine, on brain monoamines and their metabolites in rats. J.PharmacoL Exp. Ther. 248:50-56,1989.

FULLER, R. W., SCHAFFER,R. J., RousH, B. W. AND MOLLOY,B. B.: Drugdisposition as a factor in the lowering of brain serotonin by chioroamphetamines in the rat. Biochem. Pharmacol. 21: 1413—1417,1972.

FULLER, R. W., SNODDY,H. D. AND MOLLOY,B. B.: Effect of fl,fl-difluorosubstitution on the disposition and pharmacological effects of 4-chioroamphetamine in rats. J. Pharinacol. Exp. Ther. 184: 273-284, 1973b.

FULLER,R. W. ANDSNODDY,H. D.: Long-term effects of 4-chloroamphetamineon brain 5-hydroxyindolemetabolismin rats. Neuropharmacology13: 85-90,1974.

HENDERSON, M. G. AND FULLER, R. W.: Dextromethorphan antagonizes theacute depletion of brain serotonin by p-chloroaznphetamine and H75/12 inrats. Brain Ens. 594: 323-326, 1992.

Ho, T. K., LABELLA, F. S. AND PINSKY, C.: Opiate properties ofSKF 525A. Can.J. PhysioL PharniacoL 56: 550-554, 1978.

JOHNSON, M. P. AND NICHOLS, D. E.: Combined administration of a nonneurotoxic 3,4-methylenedioxymethamphetamine analogue with amphetamineproduces serotonin neurotoxicity in rats. Neuropharmacology 30: 819-822,1991.

MAMOUNAS,L A. ANDMOLLIVER,M. E.: Evidence for dual serotonergic projections to neocortex: Axons from the dorsal and median raphe nuclei are differentially vulnerable to the neurotoxin p-chloroamphetamine (PCA). Exp. Neurol 102: 23-36, 1988.

Msmc, J. L., FuxE, K. ANDCARLSSON,A.: Blockade of p-chloroamphetamineinduced 5-hydroxytryptamine depletion by chloriinipraznine, chlorpheniramineand mepenidine. Biochem. PharmacoL 20: 707-709, 1971.

NASH, J. F.: Ketansenin pretreatment attenuates MDMA-induced dopaininerelease in the striatuni as measured by in vivo microdialysis. Life Sci. 47:2401—2408,1990.

PERRY, K. W. AND FULLER, R. W.: Effect of fluoxetine on serotonin anddopamine concentration in microdialysis fluid from rat stniatum. Life Sci. 50:1683—1690,1992.

PLETSCHER,A., BURKHARD,W. P., BRUDERER,H. ANDGEY, K. F.: Decrease ofcerebral 5-hydroxytryptamine and 5-hydroxyindoleacetic acid by an arylalkylamine. Life Sci. 11: 828-833, 1963.

PLm@sCHan,A., BARm0LINI, G., BRUDERER,H., BURKHARD,W. P. ANDGEY,K. F.: Chlorinated arylkalkylamines affecting the cerebral metabolism of 5-

or after pretreatment with either pargyline or L-dopa/carbidopa, suggesting it does not have the same propensity as @,$-difluoro-PCA to mimic the neurotoxic effects of PCA.

The data from our studies in which serotonin depletion wasmeasured in whole brain tissue suggestedthat dopamine maybe involved in the serotonergic neurotoxicity of PCA. Presumably, dopamine would have to be released from neurons contaiuiing it and then taken up into the serotonin neuron. Apossible action there would involve autoxidation of dopamineto reactiveproductswhich would damagethe serotoninnerveterminals. To determine the ability of the neurotoxic drugs torelease dopamine, we measured extracellular concentrations ofdopamine after the administration of PCA, @,fi@difluoro-PCAor H75/12 using the technique of in vivo microdialysis (Perryand Fuller, 1992). The drug doses used in the microdiatysisstudy had to be reduced from those used in experiments measwing serotonin depletion becausethe mortality rates after thehigher doses in rats during microdiatysis probe implantationapproached100%.The lower doseswere adequateto releaseserotonin, and extracellular levels of serotonin in rat striatumwere significantly increased over base-line levels to a similarextent by all three drugs. On the other hand, dopamine wasincreased by PCA to a greater extent than by $,@9-difluoroPCA, and H75/12 had essentially no effect.

Taken together, these data suggest that the non-neurotoxicserotonin releaserH75/12 (Caisson et aL, 1969; Hendersonand Fuller, 1992) and the less neurotoxic serotonin releaser$,@-difluoro-PCA (Fuller et aL, 1973b) differ from PCA mainlyin their effects on dopamine neurons. Perhaps there is a re

quired threshold concentration of extracellular dopamine whichis exceeded 1) by PCA and 2) by fl,fi-difluoro-PCA when dopaminelevelsareelevatedbyL-dopaorbyinhibitingdopaminemetabolismwith pargyline.The fact that L-dopaandpargylinedid not make H75/12 neurotoxic may result from the lack ofany significant effect of H75/12 on the dopamine system.Because repeated injections of @-difluoro-PCA appear to beneurotoxic, this threshold ofextracellular dopamine concentration may include not only an initial surge in extracellulardopamine,but an increasedareaunder the curvewith respectto time for dopamine.The apparentability of PCA but not fi,@difluoro-PCA to inhibit dopamine oxidation by MAO may alsocontribute to the greater neurotoxicity of PCA.

Acknowledgments

We thank Joan Hager and Susan K. Hemrick-Luecke for assistance in preparing the manuscript and the illustrations.

Reference.AXT, K. J. AND SnmgN, L S.: a-Methyl-p-tyrosine partially attenuates p

chloroamphetamine-induced 5-hydroxytryptamine depletions in the rat brain.PharmacoLBiochem.Behav.35:995-997,1990.

Basoan, U. V., GRzANNA,R. ANDMowvnn, M. E.: Depletion of serotoninusing p-chlorophenylaianine (PCPA) and reserpine protect. against the neurotoxic effects ofp-chloroamphetamine (PCA) in the brain. Exp. NeuroL 103:111—115,1989.

C@uu.a8oN,A.,CoRRoDI,H.,Fuxa,K.ANDHOKFELT,T.:Effectofantidepressant drugs on the depletion ofintraneuronal brain 5-hydroxytryptamine storescaused by 4-metbyl-alpba.ethyl-meta-tyramine. Eur. J. PhSZmSCOL5: 357—366,1969.

CHoo, L K., MALTA,E. ANDM1TCHEL8ON,F.: Investigation of the antimuscarinic and other actions of proadifen in vitro. J. Pharm. PharmaCOL 38: 898—901,1986.

CoMMiNs,D. L, AXT,K. J., Vos@aEn,G. ANDSnmE@,L S.: Endogenouslyproduced5,6-dihydroxytryptaminemaymediatethe neurotoxiceffectsof parschlorarnphetamine. Brain Re.. 419:253-261,1987.

D'Ma@To,R.J., LaRGaNT,B.L, SNowM,@ti,A.M.ANDSNYDER,S.H.:Selectivelabeling of serotonin uptake sites in rat brain by [‘H]citalopramcontrasted tolabeling of multiple sites by [5Hjixnipramine. J. PharmacoL Exp. Ther. 242:364—371,1987.

424 Hendersonat al. VoL267

hydroxytryptainine.J. PharmacoLExp. Then. 145: 344-350,1964.Rosa, S. B.: Proadifen-sensitive high affinity binding of 3H-alaproclate to liver

membranes@PharmaCOL ToxicoL 61:282-287,1987.SANDERS-BUSH, E., BUsHiNG, J. AND Sut.sna, F.: Long-term effects of p

chioroamphetamineon tryptophan hydroxylaseactivity and on the levelsof 5-hydroxytryptamineand5-hydroxyindoleaceticacidin brain. Eur. J. PhSYInaCOL20: 385—388,1972.

SANDERS-BUSH, E., BUSHING, J. A. AND Sut.snn, F.: Long-term effects of pchloroamphetamineandrelateddrugsoncentralserotonergicmechanisms.J.PhSrISSCOLExp. Then. 192: 33-41, 1975.

SANDana-BusH, E. ANDSTERANKA,L. R.: Immediate and long-term effects ofp-chloroamphetamine on brain amines@Ann. NY AcacL Sd. 305: 208-221,1978.

SCHMIDT, C. J., Ann@Tn, G. M., Bi@Acic,C. K. @t@DTAYLOR, V. L: Selective 5-hydroxytryptamine@ receptor antagonists protect against the neurotoxicity ofznethylenedioxymethaxnphetaminein rats. J. PharmacOL Exp. Then. 255:478-483,1990.

SCHMIDT, C. J., Bi@tcK, C. K. @DTAYLOR, V. L: L-DOPA potentiation of theserotonergic deficits due to a single administration of 3,4-methylenedioxymethamphetamine, p-chloroamphetamine or znethamphetaniine to rats. Eur. J.PhSnInSCOL203: 41-49, 1991a.

SCHMIDT,C. J. ANDGina, J. W.: Role of the dopamine uptake carrier in theneurochemicalresponseto methamphetainine:EffeCtsof amfonelicacid. Eur.J. PharmaCOL109: 73—80,1985.

SCHMIDT,C. J., TAYLOR,V. L, AsnAva, G. M. ANDNIEDUZAK,T. R.: 5-HT,antagonists stereoeelectively prevent the neurotoxicity of 3,4-methylenedioxymethamphetamineby blockingthe acute stimulation of dopamine synthesis:Reversal by L-DOPA. J. PharInacOL Exp. Ther 256: 230-235, 1991b.

SoanNsnI@i,S.M., Huupiranvs,T. M., TAYLOR,V. L @NDSCHMIDT,C.J.: 5-HT2receptorantagonistsreverseamphetamine-inducedslowingof dopaminergic neurons by interfering with StimUlated dopainine synthesia@J. PharmacoL Exp. Then. 260: 872-878, 1992.

UDENFRIEND, S., STEIN, S., BOHLEN, P., DAIRMAN, W., LEIMGRUBER,W. ANDWalonLE,M.:Fluonescamine:areagentfortheassayofaminoacids,peptides,proteins, and primary amine. in the picomole range. Science (Wash. DC) 178:871—872,1972.

VniNA,A. P. ANDOsswALD, W.: Antiarrhythmic effects ofproadifen hydrochloride (SKF 525-A). Arzneim.-Forach. 20: 851-853, 1970.

Send reprint requests to: Ray W. Fuller, Lilly Research Laboratories, EliLillyand Company,LillyCorporateCenter, IndianapOlis,IN 46285.