1. 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;SKF
525A,proadlfen;MAO,monoamineonidase;5-lIT,5@ydroxytryptan@ne.
0022-3565/93/2671-0417$03.00/O
Na Jouarw@or Piiaius@cox.ooyANDExnananmi. Tum@s'su'rica
CopyrightC 1993byTb. AmericanSocietyfor Pbarmaco1o@and ExperimentalTherapeutics
PossibleInvolvementof DopamineintheLongTermSerotonin
Depletionbyp-Chloroamphetamineand @3j3-Difluoro-p-
ChioroamphetamineinRats
MARKG. HENDERSON,1KENNETHW. PERRYand RAY W. FULLER
L@IllyResearch Laboratories,LillyCorporateCenter@EllLillyand Company, Indianapolis,Indiana
Accepted for publicationJune 16, 1993
Vol. 267, No. 1
Printed in U.S.A.
ABSTRACT
The role of dopamine in the long-term depletion of serotonin in
rat brain by p-chloroamphetamine(PCA) and related compounds
was Inves@gatedby comparing effects of @-difluoro-p-chIo
roamphetamlne ($ft-dlfluoro-PCA) and 4-methyl-a-ethyl-meta-ty
ramine (H75/12), reported to cause only short-term serotonin
depletion, with those of PCA. A single dose of fi,$-difluoro-PCA
had no long-term effects on serotonin in whole rat brain, even
after pretreatmentwith proadilenwh@hdecreased the rate at
which @-difluoro-PCAdisappeared from brain. The possibility
that proadtfen might antagonize serotonin deple@onwas ruled
out; proadifen did not prevent kng-term serotonin depletion by
PCA. Long-term depletkn of brain serotonin was found after
repeated Injectionsof @9,fi-drnuoro-PCA(fiveinjections 4 hr apart)
and was prevented by fluoxetine pretreatment. @-DmUOrO-PCA
given after the monoamine oxidase inhibitor pargyline or after
carbidopa/L-dopa also caused long-term serotonin depletion,
although H75/12 did not. At early times after single doses
producing the same initialdepletion of serotonin, PCA caused a
large increasein dopamineand a largedecrease in the metaboifte
3,4-dihydroxyphenylacetic acid inwhole brain,thereby increasing
the ratio dopamine/3,4-dihydroxyphenylacetic, and the other two
drugs caused smaller effects. Extracellular dopamine was in
creased markedly by PCA, less by @-dmuoro-PCA,and not at
all by H75/1 2. These resufts suggest an association between
dopamine release and long-term depletion of serotonin and add
to evidencethat dopaminereleaseby PCA may be essentialto
its neurotoxic actions on brain serotonin neurons.
PCA and related chlorinated amphetamines were shown to
deplete brain serotonin in rats nearly three decades ago
(Pletacher et at., 1963, 1964; Fuller et aL, 1965). Later it was
recognized that these compounds, even after a single dose,
caused long-term depletion of brain serotonin persisting for
weeks or months in rats (Frey, 1970; Sanders-Bush et at., 1972).
The depletionofbrain serotoninby PCAoccursin two distinct
phases. The acute depletion of serotonin during the first few
hours after PCA administration is reversible by subsequent
administration of an inhibitor of the serotonin transporter
(Fuller et aL, 1975). The later phase of serotonin depletion is
very long-lasting and is not reversible by uptake inhibition.
The long-term depletion of brain serotonin appears to result
from a neurotoxic effect on serotonin neurons. Histologic cx
amination has shown degeneration of serotonin projections
(Mamounas and Molliver, 1988;Berger et aL, 1989) and loss of
retrogradely 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 serotonin
depleted at long times after PCA injection, but so are other
parameters associated with brain serotonin neurons, including
tryptophan hydroxylase (Sanders-Bush et aL, 1975), serotonin
uptake by synaptosomes (Sanders-Bush and Steranka, 1978),
serotonin uptake sites labeled with radioligands (D'Amato et
al., 1987) and serotonin turnover measured by several methods
(Fuller and Snoddy, 1974). Both the acute, reversible and the
long-term, irreversible phases of serotonin depletion are pre
vented by pretreatment with an uptake inhibitor before PCA
injection (Meek et aL, 1971; Fuller et at., 1975; Fuller, 1992).
The mechanism underlying the long-term effects of PCA on
brain serotonin neurons continues to be speculative. A com
parable long-term, neurotoxic depletion of striatal dopamine
has been reported to occur after high doses of amphetamine in
rats (for references, see Fuller, 1985). In that case, autoxidation
products of dopamine have been postulated to play a role in
the neurotoxicity (Fuller and Hemrick-Luecke, 1982). Dopa
mine has also been implicated in the neurotoxicity toward
serotonin neurons produced by MDMA and methamphetamine
417

2. 418 Hendersonet al. Vol.267
(Schmidt and Gibb, 1985; Schmidt et at., 1990). We investigated
the possibility that dopamine might be involved in the neuro
toxic effects of PCA toward serotonin neurons by doing corn
parative studies on two PCA analogs which resemble PCA in
releasing serotonin acutely but differ markedly from PCA in
their propensity to cause neurotoxicity to serotonin neurons.
@3,fi-Difluoro-PCAhas been reported to cause acute depletion
of brain serotonin in rats when higher doses are given to
produce brain levels equivalent to those of PCA, but not to
cause long-term depletion of brain serotonin (Fuller et at.,
1973b). H75/12 is another amphetamine analog that depletes
brain serotonin acutely without causing long-term effects like
those causedby PCA (Carlsson et at., 1969;Fuller et al., 1976;
Henderson and Fuller, 1992).
Materialsand Methods
Animals. Male Sprague-Dawley rats (Charles River Breeding Lab
oratories, Portage, MI) weighing between 160 to 180 g were group
housed in suspended wire cages under controlled environmental con
ditions with a 12-hr light/dark cycle and ad libitum access to food and
water. In all cases, rats were killed by guillotine. Whole brains were
quickly removedand were rapidly frozen on dry ice.Brains were stored
in a —¿6O@Cfreezer until assayed.
Monoamine analysis. Serotoninand its metabolite,5-HIAAand
dopamine and its metabolites, DOPAC and HVA, were assayed using
high performance liquid chromatography with electrochemical detec
tion as described earlier (Fuller and Perry, 1989).
In uivo microdialysis. Studies using in vivo microdialysis in con
scious freely moving rats were performed as described earlier (Perry
and Fuller,1992),with the exceptionof the constituentsand flowrate
of the artificial cerebrospinal fluid. The artificial cerebrospinal fluid
contained 150 mM NaCl, 3 mM KC1,1.7 mM CaCl2,0.9 mM MgCl2
and Na2HPO4 buffer pH 7.4 pumped through the dialysis probe at 2
@tl/min.
Drug level assay. f3j3-Difluoro-PCAwas measuredin whole rat
brain 1, 2, 3 and 4 hr after injection of 0.4 mmol/kg (96.8 mg/kg of the
hydrochloride salt) i.p. of the compound with or without a 2-hr pre
treatment with 50 mg/kg i.p. SKF-525A. The assay method used
fluorescamine, a reagent for detecting primary amines (Udenfriend et
Fluoxetine
Fig. 1. Fluoxetine pretreatment prevents the acute depletion of brain
serotonin by fl,$-difluoro-PCAin rats. @9j3-Difluoro-PCAhydrochloride
was injectedat a dose of 98.6 mg/kg (0.4 mmol/kg p.) 1 hr after
fluoxetinehydrochloride(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 from
grouptreatedwithflj9-difiuoro-PCAalone(P< .05).
Control@ 343-difluoro-PCA
0 Proadifen •¿@34l-difluoro-PCA+Proadifen
Fig. 2. Inabilityof 13,@9-difiuoro-PCAto causelong-termdepletionof brain
5-hydroxyindoles in rats pretreated with proadifen (SKF-525A). fi,@
Difluoro-PCA hydrochloride was injected at a dose of 98.6 mg/kg (0.4
mmol/kg i.p.) 2 hr after proadifen hydrochloride (50 mg/kg i.p.) and 1
week before rats were killed.Mean values ±standard errors for six rats
per group are shown.
—¿U—-@34@-difIuoro-PCA
—¿ . —¿Proadlfen + f343-dlfluoro-PCA
I
5HT 5HIAA
200
@ 100
0 1 2 3 4
Hours
Fig. 3. Influence of proadifen pretreatment on the rate of disappearance
of fljl-difluoro-PCAfrom rat brain. fljl-Difluoro-PCAhydrochloridewas
injected at a dose of 98.6 mg/kg (0.4 mmol/kg i.p.) alone or 2 hr after
proadifenhydrochloride(50 mg/kg i.p.).Mean values ±standard errors
for six rats per group are shown.
r@ Control@ PCA
Q Proadifen •¿Proadlfen+PCA
5.
4.
I
FIg. 4. Proadifenpretreatmentdoesnotpreventthe long-termdepletion
of 5-hydroxyindolesby PCA. PCA hydrochloride(20.6 mg/kg i.p.)was
injected2 hr after proadifen(50 mg/kg i.p.)and 1 week before rats were
killed.Mean values ±standard errors for six rats per group are shown.
‘¿Signifi@tdifference from control group (P < .05).
a
+
5HT 5HIAA

3. 1993
U,
a,
0
E
C
419
(Philadelphia, PA), L-Dopa was provided by Monsanto (St. Louis,
MO), levo-a-(3,4-dihydroxybenzyl)-a-hydrazinopropionic acid mono
hydrate (carbidopa) was provided by Merck Sharp & Dohme Research
Laboratories (Rahway, NJ), H75/12 was purchased from Labkemi AB
(Goteborg,Sweden)and PCA hydrochloridewas purchased from Regis
Chemical Company (Morton Grove, IL). flj3-Difluoro-PCA, fluoxetine,
H75/12 and PCA were dissolved in water, whereas L-dopa and carbi
dopa were injected in a 5% acacia suspension. All drugs were adminis
tered i.p.
Statistical analysis. Comparisons between multiple treatment
groups were performed using a one-way analysis of variance followed
by a Tukey post-hoc test. Comparisons between only two treatment
groups were analyzed using a two-tailed t test. In all cases P < .05 was
considered a statistically significant difference.
Results
The ability of @3j3-difluoro-PCA (0.4 mmol/kg or 96.8 mgI
kg) to cause acute depletion of serotonin in whole brain 4 hr
after injection is shown in figure 1. This dose of $,fl-difluoro
PCA is 4 times our usual dose of PCA so that drug concentra
tions in the brain are similar for the two drugs (Fuller et at.,
1973b). The depletion of brain serotonin by flj3-difluoro-PCA
was carrier-dependent in that it was blocked completely by
pretreatment with fluoxetine at a dose of 10 mg/kg.
This acute depletion of serotonin by j.@,$-difluoro-PCAis not
long lasting as is the depletion of serotonin by PCA (Fuller et
at., 1973b), and An experiment designed to see whether pre
treatment with an inhibitor of drug metabolism (proadifen or
SKF-525A) would allow @3j3-thfluoro-PCAto cause a long
lasting depletion of brain serotonin is shown in figure 2. Proad
ifen had been shown to inhibit the metabolism of flj3-difluoro
amphetamine and to increase its brain levels in rats (Fuller et
at., 1973a). Accelerated metabolism of @3,fl-difluoro-PCA in rats
had been shown to prevent its serotonin-depletingeffects
(Fuller et at., 1974a), so diminished metabolism might be ex
pected to enhance the serotonin-depleting effects. Rats given
proadifen at 50 mg/kg 2 hr before $j3-thfluoro-PCA for the
purpose of inhibiting the metabolism of f3j3-difluoro-PCA by
the cytochrome P-450-linked mixed function oxidase system
were found not to have depleted levels of serotonin and 5-HIAA
at 1 week in whole rat brain (fig. 2). Brain levels of fl,fl-difluoro
PCA in rats at 1 to 4 hr after injection of flj3-&fluoro-PCA
alone or after proadifen are shown in figure 3. Drug levels were
higher in the proadifen-pretreated group, and the estimated
half-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 depletion
even though its brain half-life was increased somewhat by
proadifen, the possibility that proadifen might block the long
term depletion of serotonin as uptake inhibitors do was inves
tigated by determining whether the long-term depletion of brain
serotonin by PCA occurred in proadifen-pretreated rats. Brain
serotonin was depleted at 1 week in rats given 0.1 mmol/kg
(20.6 mg/kg) of PCA with or without proadifen pretreatment
is shown in figure 4.
As an alternative means of maintaining brain concentrations
of fl,fl-difluoro-PCA for a longer period, multiple injections of
the 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 five
injections, brain serotonin and 5-HIAA were depleted 1 week
later (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 Control
D @-dlfluoro-PCA
@ Fluoxetlne+ @343-dlfluoro-PCA
Flg 5. Depletion of serotonin and 5-HIAA in rat brain 1 week after
repeatedinjectionsof $,@-difiuoro-PCA. @-Dffluoro-PCAhydrochloride
was injectedevery4 hrat a dose of 98.6 mg/kg(0.4 mmol/kgi.p.),and
rats were killed1 week later. Some rats were given fluoxetine hydrochlo
ride (10 mg/kg i.p.)1 hr before the firstdose of fl,fi-difluoro-PCA.Mean
values±standarderrorsfor fiveto six rats per groupare shown.
@ difference from control group; #5jgnifi@t difference from
group treated with @-difiuoro-PCAalone (P < .05).
0@
5HT 5HIAA
. H75/12
—¿. —¿ @-dlfluoro-PCA
—¿0-—PCA
4
3
2
*
1
0
Fig. 6. Time course of the acute depletion of brain serotonin by H75/12,
@3,$-difiuoro-PCAor PCA. H75/12 was injected at a dose of 70 mg/kg
(0.3mmol/kgi.p.). @,$-Difiuoro-PCAhydrochloridewas injectedata dose
of 96.8 mg/kg (0.4 mmol/kgi.p.).PCAhydrochloridewas injectedat a
dose 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.
Slgnlficantdifferencefromzero time group (P < .05).
aL, 1972)and was based on a method described earlier for measuring
PCA (Fulleret aL,1974b).Brainswerehomogenizedin 4 vol of 0.1 N
HC1,then 1vol of 30%HC1O4wasaddedandthe reactionvesselswere
mixed and centrifuged for 5 mm. One milliliter of supernatant fluid
was removed and added to tubes containing 0.4 ml of 5 N NaOH and
4 ml of toluene; the tube contents were mixed and centrifuged. Three
and 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 and
centrifuged.Three milliliters of the toluenewasthen removedand
added 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 added
to tubes containing 1.5ml of 0.2 M borate buffer (pH 10.0)and mixed
well.Whilethe samplewasvortexed,0.5 mlof a fluorescaminesolution
(15 mg of fluorescamine/50 ml of acetone) was added. Fluorescence
intensity was determined in an Aniinco-Bowman spectrophotofluo
rometer with activation/fluorescence set at 390/490 nm.
Drugs. @,fi-Difluoro-PCA hydrochloride and fluoxetine hydrochlo
ride were synthesized at Lilly Research Laboratories (Indianapolis,
IN), SKF.525Awas providedby Smith Kline & FrenchLaboratories
2 4 6
Hours

4. 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 brain
concentrationsof dopamineandDOPACandontheconcentra
tion ratiodOpamlne/DOPACIn rats. H75/12was Injectedat a
dose of 70 mg/kg (0.3 mmol/kg i.p.).fi,$-Dmuoro-PCAhydro
chloridewas InjeCtedat a doseof 96.8 mg/kg (0.4 mmol/kg
i.p.).PCAhydrochloridewas Injectedat a doseof 20.6mg/kg
(0.1 mmol/kgi.p.).i@Jlinjectionswere at zero time. Meanvalues
±standard errors for fIve rats per group are shown. Slgnfficant
differencefrom zero time group; ‘¿signIfIcantdifferencefrom
grouptreatedwith $,@-dIfiUOrO-PCAand 1-175/12groups(P <
.05).
a,
0
EC
2
C
0
0
0
C
a,
2a,
a.
H75/12
0 @34@-dIfluoro-PCA
I PCA
2 4 6
Hours
prevented the depletion of both 5-hydroxyindoles, indicating
the long-lastingdepletionhadoccurredvia a carrier-dependent
mechanism. The repeated injection of @9,$-difluoro-PCAcaused
selective depletion of serotonin as PCA itself does in that brain
concentrations of dopamine, DOPAC and HVA were not
changed (data not shown).
The acute depletion of whole brain serotoninproducedby
H75/12 (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 effects
of the three drugs were virtually identical at 2 hr postinjection,
but at 4 and 6 hr the effectsof PCA wereslightlygreaterthan
those of H75/12 or fl,$-difluoro-PCA, whose effects had been
shown earlier not to persist at 24 hr (Fuller et aL, 1973b, 1976).
That dopamineconcentrationin whole brain was increased
during this time period by PCA but not by $,@-difluoro-PCA is
shown in figure 7. H75/12 significantly decreased dopamine,
revealing its depletion of serotonin to be less specific than that
of the chlorinatedcompounds.All three compoundscauseda
decrease in DOPAC concentration in brain, possibly via inhi
bition of monoamine oxidase; the order of effectiveness in
decreasing DOPAC was PCA > fi,@-difluoro-PCA > H75/12.
HVA concentrations (data not shown) were decreased to a
lesserextent,andmostby H75/12,the decreasesafter @-
difluoro-PCA and PCA being not statisticaily significant. Be
cause DOPAC is the direct product of MAO action on dopa
mine, the ratio dopamine/DOPAC can bean indication of MAO
inhibition. That the concentration ratio dopamine/DOPAC
was increased nearly 4-fold after PCA is shown in figure 8
(bottom panel). In contrast, the two non-neurotoxic analogs
caused only modest (less than 2-fold) increases in the dopa
mine/DOPAC ratio.
Experiments with pargyline and L-dopa were done to see
whether treatments that increase dopamine would allow neu
rotoxicity to be manifest after administration of @-thfluoro
PCA or H75/12. The effect of pretreatment with pargyline, an
inhibitor ofMAO, on thelong-term depletion ofbrain serotonin
by /i,fi-difluoro-PCA or H75/12 was investigated (fig. 8). Par
gyline (100 mg/kg) was injected 1 hr before either @-difluoro
PCA (0.4 mmol/kg) or H75/12 (0.3 mmol/kg) and 5-hydroxy
indole concentrations in whole brain were measured 1 week
later.Again,the administrationof asingledoseof @,fi-difluoro
PCA or H75/12 did not result in long-term serotonin depletion.
However,in rats pretreatedwith pargylineto inhibit MAO, a
single dose of fi,@-difluoro-PCA, but not of H75/12, depleted
serotoninand 5-HIAA concentrationsat 1 week.The admin
istration of a carbidopa/L-dopa(25:100mg/kg) combination
also resulted in long-term depletion of serotornn and 5-HIAA
by a single dose of fi,@-difluoro-PCAbut not H75/12 (fig. 9).
The effectof H75/12 (0.15mmolor 30mg/kg), @-difluoro
PCA (0.2 mmol or 48.4 mg/kg) and PCA (0.05 mmol or 10 mgI
kg) on extracellular serotonin levels measured by in vivo brain
microdialysis in conscious, freely moving rats is shown in figure

5. 421
_ Carbidope+L-dopa+ @-dmuoro-PCA
Fig. 9. Brain 5-hydroxyindoleconcentrations 1 week after administration
of @-drnUOrO-PCAorH75/12to ratstreatedwithL-dopacombinedwith
carbidopa. H75/12 was injected at a dose of 70 mg/kg (0.3 mmol/kg
i.p.). @3,fi-Dlfiuoro-PCAhydrochloridewas injectedat a dose of 96.8 mg/
kg (0.4 mmol/kgi.p.).Carbidopa(25 mg/kq i.p.)was injected30 mm
beforeL-dopa(100mg/kgi.p.),wh@hwask@JeCtedat thesametimeas
@,$-drnuoro-PCAand H75/12. Mean values ±standard errors for four to
six rats per group are shown. *#&@j@njf@@differences from control
group,grouptreatedwith carbidopa+ L-dopaandgrouptreatedwith
fi,fi-difiuoro-PCAalone, respectively(P < .05).
2
0
C
U
I!U
a.
Fig. 10. ExtracellularcOncentratiOnSof serotonin in rat striatum after the
injectionof H75/12,$,@-difiUOrO-PCAor PCA.H75/12was injeCtedat a
doseof 35 mg/kg (0.15mmol/kgi.p.).fi,ll-Difluoro-PCAhydrochloride
was Injectedat a dose of 48.4 mg/kg(0.2 mmol/kgl.p.).PCAhydrochlo
ride was injectedat a dose of 10.3 mg/kg (0.05 mmol/kgi.p.). All
injections were at zero time. Mean values ±Standard errors for five to
six rats per group are shown.
in the brain for a substantial time (Fuller et at., 1972). For
example, m-chloroamphetamine does not cause long-term de
pletion of brain serotonin unless its usually rapid metabolism
(via hydroxylation in the unoccupied pare position of the
phenyl ring) is blocked by pretreatment with iprindole or some
similar agent (Fuller et at., 1972; Fuller and Baker, 1974). In
this study we have compared two compounds, $,$-difiuoro-PCA
and H75/12, which cause a similar acute depletion of brain
serotoninas PCA does(fig. 6), but which do not causelong
term depletion of brain serotonin after a single dose as PCA
does.
$,fi-Difiuoro-PCA, like PCA, releases serotonin by a carrier
dependent mechanism; pretreatment with fluoxetine, a selec
tive inhibitor of the serotoninuptake carrier, prevents sero
tonindepletionby @-difiuoro-PCA(fig.1)justasitprevents
1993
2
DopamineandSerotonlnDepletion
rz@c@t@i@ Carbidopa+L-dopa+
E Carbidopa+L-dopa0 @-dffluoro-PCA
ElIllH75/12
Control
Pargyllne treated
Fig.8. BraIn5-hydrox@indoleconcentrations1weekafteradministration
of @-dIfIUOrO-PCAor H75/12to ratstreatedwithpargyline,aninhibftor
of MAO.H75/12 was Injectedat a dose of 70 mg/kg (0.3 mmol/kgi.p.).
fi,fl-DIfIUOrO-PCAhydrochloridewas InjeCtedat a dose of 96.8 mg/kg
(0.4 mrnol/kgi.p.).PargyIk@ehydrochloridewas Injectedat a dose of 100
mg/kg i.p. 1 tw before @-dIflUOrO-PCAor H75/12. Mean values ±
standarderrorsfor threeto six rats per groupare shown. Significant
differencefromgrouptreatedwithpargylinealone(P< .05).
10. All three compounds caused a rapid, marked increase in
extracellular serotonin, the effect of PCA being slightly greater
than that off3,fi-difiuoro-PCA or H75/12. Despite the similarity
of their effects on extracellular serotonin, the three drugs had
strikingly 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. These
lower dosesof all three drugswereusedin the microdiatysis
experiments becausethe higher doseswere not well tolerated
in rats with implanted microdialysis probes.
Discussion
The mechanism(s) for the long-term serotonin depletion and
neurotoxicity produced by PCA has never been firmly estab
lished. Some investigators have suggested the involvement of
excitatory amino acid receptors (Finnegan et aL, 1991), the
formationof autoxidationproductssuchas5,6-dihydroxytryp
taxnine (Commins et aL, 1987)and the involvement of dopamine
(Schmidt et aL, 1991a). One requirement for the long-term
depletion of brain serotonin seems to be that the drug persists
r@ Control
0 @l,@-dlf1uoro-PCA
@ H75/12 5.
4
5HT 5HIAA
5HT 5HIAA
F@Pargyllne
p Pargyllne+@343—dIfluoro-PCA
@ Pargyline+H75/12
—¿0•-PCA
- U - @-dffiuoro-PCA
—¿S.—H75/12
5HT 5HIAA
150 180 210 240 270
Minutes

6. 422 Hendersonet al Vol.267
—¿â€¢0--PCA
- U - @d1fIuoro-PCA
—¿a,.-- H75/12
Evidence for a role of dopamine in the serotonergic neuro
toxicity of substituted amphetamines has been reported by
other investigators who used a variety of paradigms. For ex
ample, 5-HT2 receptor antagonists were found to attenuate the
serotonin neurotoxicity measuredafter MDMA administration
to rats (Schmidtet aL, 1990,1991b).Thesefindingsimplicated
dopamine because 5-HT2 receptors facilitate dopamine synthe
sis and 5-HT2 receptor antagonists can block the release of
dopamine by amphetamine and MDMA (Sorensen et aL, 1992;
Nash, 1990). Perhaps more direct evidence for dopamine's
involvement in serotonin neurotoxicity came from a study in
which L-dopa, the precursor to dopamine, was found to poten
tiate serotonergic deficits produced by MDMA, methampheta
mine and PCA in rats (Schmidt et at., 1991a). Johnson and
Nichols (1991) demonstrated that a non-neurotoxic analog of
MDMA, 5-methoxy-6-methyl-2-aminoindan, could be made to
decreasespecificserotonergicparameterslong-termbycombin
ing it with a nonvesiculardopaminereleaserS-(+)-ampheta
mine. Axt and Seiden (1990) showed that a-methyl-p-tyrosine,
an inhibitor of dopamine synthesis, attenuated PCA-induced
serotonin depletion in rat brain, although they also pointed out
the a-methyl-p-tyrosine pretreatment did not completely pre
vent the formation of 5,6-dihydroxytryptamine, a serotonin
neurotoxin, in response to methamphetamine administration.
Our investigation of dopamine involvement in PCA-induced
serotonergic neurotoxicity centered on comparing the dopa
mine-releasing effects of two non-neurotoxic amphetamine an
alogs, fl,fi-difluoro-PCA and H75/12, to those of PCA. The
doses of @1j9-difluoro-PCA(0.4 mmol/kg) and H75/12 (0.3
mmol/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 concen
trations of dopamine at these early times. PCA caused a large
increase in the dopamine/DOPAC concentration ratio, sugges
tive of marked inhibition of dopamine oxidation by MAO
(Fuller, 1966), and a large increase in extracellular dopamine
concentration,suggestingit releaseddopaminefrom intraneu
ronal stores. In contrast, @-difluoro-PCAhad much less effect
than PCA on the dopamine/DOPAC concentration ratio, sug
gesting it was a weaker MAO inhibitor. Nonetheless, fi,fi
difluoro-PCA did increase extracellular dopamine, suggesting
it released dopamine perhaps not as effectively as PCA. H75/
12 had only a small effect on the dopamine/DOPAC concen
tration ratio and essentially no effect on extracellular dopa
mine. This order of efficacy—PCA> @,fi-difluoro-PCA> H75/
12—parallels the order of efficacy of the drugs in causing
serotonin neurotoxicity. PCA is the most neurotoxic, H75/12
is not neurotoxic and $,fi-difluoro-PCA is not neurotoxic except
when given by repeated injections, in combination with an
MAO inhibitor, or in combination with carbidopa/L-dopa to
increase dopamine stores available for release.
The ability of pargyline, a nonselective monoamine oxidase
inhibitor, to allow fi,$-difluoro-PCA to cause long-term deple
tion of serotonin and 5-HIAA does not in itself implicate
dopamine specifically, because other monoamines are also af
fected by pargyline. Those fmdings, and the finding that L
dopa in combination with the peripheral decarboxylase inhibi
tor carbidopa allowed fij9-difluoro-PCA to cause long-term
depletion of 5-hydroxyindoles, implicate dopamine availability
as a factor influencing neurotoxicity of substituted ampheta
mines toward serotonin neurons. H75/12 did not cause long
term 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 striatumafter
the injectionof H75/12, @,fi-difiuoro-PCAor PCA.H75/12 was injected
at a dose of 35 mg/kg (0.15 mmol/kgi.p.). @@,$-Dffluoro-PCAhydrochlo
ride was injectedat a dose of 48.4 mg/kg (0.2 mmol/kgi.p.). PCA
hydrochloride was injected at a dose of 10.3 mg/kg (0.05 mmol/kg i.p.).
Allinjections were at zero time. Mean values ±standard errors for six
ratspergroupareshown.
serotonin depletion by PCA (Fuller et at., 1975). It was impor
tant to show the carrier dependence of the serotonin depletion
by this compound because it is much more lipid-soluble than
PCA (Fuller et aL, 1973b) and might have acted independently
of the membrane transporter. @,fi-Difluoro-PCA,which is rap
idly and extensively deaminated due to the influence of the two
fluorines on the@ carbon, has a much shorter half-life than
does PCA in rat brain (Fuller et aL, 1973b). A single dose of
fi,fi-difluoro-PCA does not cause the long-lasting neurotoxic
depletion of brain serotonin as caused by PCA. To prolong the
persistence of fi,fl-difluoro-PCA in rat brain, SKF-525A, an
inhibitor of drug-metabolizing enzymes was administered to
rats to determine whether longer persistence of fi,fi-difluoro
PCA could result in long-term serotonin depletion. A single
injection of $,fi-difluoro-PCA did not deplete brain serotonin
at 1 week even in proadifen-pretreated rats. Proadifen pretreat
ment slightly increased brain concentrations of @,$-difluoro
PCA by slowing its rate of disappearance, but the effect was
relatively small and did not cause the compound to be neuro
toxic toward brain serotonin neurons. The possibility that
proadifen might be capable of antagonizing brain serotonin
depletion by agents of this sort was considered because proad
ifen doeshavesomepharmacologiceffectsin additionto inhib
iting microsomalenzymes(Viana and Osswatd,1970;Ho et at.,
1978; Choo et aL, 1986) and has been shown to compete with
membrane binding of a serotonin uptake inhibitor, alaproclate
(Ross, 1987). However, proadifen pretreatment did not alter
the long-term depletion of brain serotonin by PCA, so the
failure of flj3-difluoro-PCA to cause long-term depletion of
brain serotonin after proadifen pretreatment was apparently
not due to any protective action of proadifen.
When multiple injections of fi,@9-difluoro-PCAwere given to
rats (five injections spaced 4 hr or approximately two half-lives
apart), brain serotonin and 5-HIAA concentrations were de
creased 1 week later. This finding reveals that fi,$-difluoro
PCA has the capability of causing PCA-like neurotoxicity and
that it fails to do so after a single dose because the drug is
removed from brain too rapidly. Although proadifen had
slightly prolonged the half-life of fi,fi-difluoro-PCA in brain,
the persistence of @-difluoro-PCAin proadifen-pretreated
rats was still too short to produce the neurotoxicity which was
produced by repeated injections.
Minutes

7. 1993 Dopamine and Serotonin Depletion 423
FINNEGAN, K. T., KERR, J. T. AND LANGSTON, J. W.: Dextromethorphan
protects against the neurotoxic effects of p-chloroamphetainine in rats. Brain
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FREY,H.H.:p-Chloroamphetaniine—Similaritiesanddissimilaritiesto amphet
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pp. 343-355, Raven Press, New York, 1970.
FRITScHY,J. M., LYONS,W. E., MowvER, M. E. ANDGRzANNA,R.: Neurotoxic
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FULLER,R. W.: Persistent effects of amphetamine, p-chloroamphetamine, and
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tryptamine concentration by 3-chloroamphetamine and 4-chioroamphetamine
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FULLER,R. W., BAKER,J. C., PERRY,K. W. ANDMOLLOY,B. B.: Effect of
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FULLER, R. W. AND HEMRICK-LUECKE,S. K.: Further studies on the long-term
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FULLER,R. W., HINES, C. W. ANDMILLS, J.: Lowering of brain serotonin level
by chloramphetamines. Biochem. PharmacoL 14: 483-488, 1965.
FULLER, R. W., MOLLOY,B. B. AND PARLI, C. J.: The effect of fl,$-difluoro
substitution on the metabolismand pharmacologyof amphetamines.In Pay
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H. Steinberg, F. SWear,A. Sundwall and 0. Vinar, pp. 615-624, North-Holland
Publishing 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 deniva
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FULLER, R. W., PERRY, K. W. AND M0LL0Y, B. B.: Reversible and irreversible
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119—124,1975.
FULLER, R. W., PERRY, K. W. AND B@.KER,J. C.: Duration of the effects of
alpha-ethyl-4-methyl-m-tyramine (H75/12) on brain 5-hydroxyindole concen
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substitution on the disposition and pharmacological effects of 4-chioroamphe
tamine in rats. J. Pharinacol. Exp. Ther. 184: 273-284, 1973b.
FULLER,R. W. ANDSNODDY,H. D.: Long-term effects of 4-chloroamphetamine
on brain 5-hydroxyindolemetabolismin rats. Neuropharmacology13: 85-90,
1974.
HENDERSON, M. G. AND FULLER, R. W.: Dextromethorphan antagonizes the
acute depletion of brain serotonin by p-chloroaznphetamine and H75/12 in
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Ho, T. K., LABELLA, F. S. AND PINSKY, C.: Opiate properties ofSKF 525A. Can.
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neurotoxic 3,4-methylenedioxymethamphetamine analogue with amphetamine
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1991.
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Msmc, J. L., FuxE, K. ANDCARLSSON,A.: Blockade of p-chloroamphetamine
induced 5-hydroxytryptamine depletion by chloriinipraznine, chlorpheniramine
and mepenidine. Biochem. PharmacoL 20: 707-709, 1971.
NASH, J. F.: Ketansenin pretreatment attenuates MDMA-induced dopainine
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2401—2408,1990.
PERRY, K. W. AND FULLER, R. W.: Effect of fluoxetine on serotonin and
dopamine concentration in microdialysis fluid from rat stniatum. Life Sci. 50:
1683—1690,1992.
PLETSCHER,A., BURKHARD,W. P., BRUDERER,H. ANDGEY, K. F.: Decrease of
cerebral 5-hydroxytryptamine and 5-hydroxyindoleacetic acid by an arylalkyl
amine. 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/carbi
dopa, 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 was
measured in whole brain tissue suggestedthat dopamine may
be involved in the serotonergic neurotoxicity of PCA. Presum
ably, dopamine would have to be released from neurons con
taiuiing it and then taken up into the serotonin neuron. A
possible action there would involve autoxidation of dopamine
to reactiveproductswhich would damagethe serotoninnerve
terminals. To determine the ability of the neurotoxic drugs to
release dopamine, we measured extracellular concentrations of
dopamine after the administration of PCA, @,fi@difluoro-PCA
or H75/12 using the technique of in vivo microdialysis (Perry
and Fuller, 1992). The drug doses used in the microdiatysis
study had to be reduced from those used in experiments meas
wing serotonin depletion becausethe mortality rates after the
higher doses in rats during microdiatysis probe implantation
approached100%.The lower doseswereadequateto release
serotonin, and extracellular levels of serotonin in rat striatum
were significantly increased over base-line levels to a similar
extent by all three drugs. On the other hand, dopamine was
increased by PCA to a greater extent than by $,@9-difluoro
PCA, and H75/12 had essentially no effect.
Taken together, these data suggest that the non-neurotoxic
serotonin releaserH75/12 (Caisson et aL, 1969; Henderson
and Fuller, 1992) and the less neurotoxic serotonin releaser
$,@-difluoro-PCA (Fuller et aL, 1973b) differ from PCA mainly
in their effects on dopamine neurons. Perhaps there is a re
quired threshold concentration of extracellular dopamine which
is exceeded1) by PCA and 2) by fl,fi-difluoro-PCA when do
paminelevelsareelevatedbyL-dopaorbyinhibitingdopamine
metabolismwith pargyline.The fact that L-dopaandpargyline
did not make H75/12 neurotoxic may result from the lack of
any significant effect of H75/12 on the dopamine system.
Because repeated injections of @-difluoro-PCA appear to be
neurotoxic, this threshold ofextracellular dopamine concentra
tion may include not only an initial surge in extracellular
dopamine,but an increasedareaunderthe curvewith respect
to time for dopamine.The apparentability of PCA but not fi,@
difluoro-PCA to inhibit dopamine oxidation by MAO may also
contribute to the greater neurotoxicity of PCA.
Acknowledgments
We thank Joan Hager and Susan K. Hemrick-Luecke for assistance in pre
paring the manuscript and the illustrations.
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Send reprint requests to: Ray W. Fuller, Lilly Research Laboratories, EliLilly
and Company,LillyCorporateCenter, IndianapOlis,IN 46285.