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Neuroscience Vol. 115, No. 1, pp. 153^161, 2002
Published by Elsevier Science Ltd on behalf of IBRO
Printed in Great Britain
0306-4522 / 02 $22.00+0.00
COCAINE MECHANISMS: ENHANCED COCAINE, FLUOXETINE AND
NISOXETINE PLACE PREFERENCES FOLLOWING MONOAMINE
F. S. HALL,a X. F. LI,a I. SORA,a1 F. XU,a M. CARON,b K. P. LESCH,c D. L. MURPHYd and
G. R. UHLa
Molecular Neurobiology Branch, NIDA-IRP, NIH, Box 5180, Baltimore, MD 21224, USA
Deptartments of Cell Biology and Medicine and HHMI, Duke University, Durham, NC 27710, USA
Department of Psychiatry, University of Wu«rzburg, 97080 Wu«rzburg, Germany
Laboratory of Clinical Science, NIMH-IRP, NIH, Bethesda, MD 20892, USA
Abstract;Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine, serotonin and norepinephrine, producing subjective e¡ects in humans that are both euphoric/rewarding and also fearful, jittery and aversive. Mice
with gene knockouts of each of these transporters display cocaine reward, manifest by cocaine place preferences that are
at least as great as wildtype values. Norepinephrine and serotonin receptor knockouts even display enhanced cocaine
reward. One explanation for these observations could be that cocaine produces aversive or anhedonic e¡ects by serotonin
or norepinephrine receptor blockade in wildtype mice that are removed in serotonin or norepinephrine receptor knockouts, increasing net cocaine reward. Adaptations to removing one transporter could also change the rewarding valence of
blocking the remaining transporters. To test these ideas, drugs that block serotonin transporter (£uoxetine), norepinephrine transporter (nisoxetine) or all three transporters (cocaine) were examined in single- or multiple-transporter knockout
mice. Fluoxetine and nisoxetine acquire rewarding properties in several knockouts that are not observed in wildtype mice.
Adding serotonin transporter knockout to norepinephrine transporter knockouts dramatically potentiates cocaine
These and previous data provide evidence that serotonin and norepinephrine transporter blockade can contribute to
the net rewarding valence of cocaine. They identify neuroadaptations that may help to explain the retention of cocaine
reward by dopamine and serotonin transporter knockout mice. They are consistent with emerging hypotheses that actions
at the three primary brain molecular targets for cocaine each provide distinct contributions to cocaine reward and cocaine
aversion in wildtype mice, and that this balance changes in mice that develop without dopamine, norepinephrine or
Published by Elsevier Science Ltd on behalf of IBRO.
Key words: cocaine, dopamine transporter, serotonin transporter, norepinephrine transporter, knockout mice, reward.
alter cocaine self-administration in experimental animals
(Brebner et al., 2000; De Wit and Wise, 1977; Ettenberg
et al., 1982; Kushner et al., 1999; Roberts et al., 1996;
Wilson and Schuster, 1974). Improved understanding of
the molecular underpinnings of cocaine reward and aversion could thus aid understanding of brain reward mechanisms and conceivably even contribute to improved
anticocaine medication development strategies.
Molecular explanations for cocaine reward focused
initially on DAT, and more recently on DAT and
SERT, based on studies of dopamine antagonist e¡ects
(De Wit and Wise, 1977; Yokel and Wise, 1975, 1976),
structure^activity relationships of DAT- and SERTblocking drugs (Kuhar et al., 1991; Spealman et al.,
1989), e¡ects of dopamine lesions (Di Chiara and
Imperato, 1988; Pettit et al., 1984; Roberts et al.,
1977), dopamine release after cocaine administration
(Di Chiara and Imperato, 1988) and modulation of
cocaine reward by serotonin manipulations (Fletcher
and Korth, 1999; Fletcher et al., 1995; Harrison et al.,
1999; Miliaressis, 1977; Poschel, 1974; Redgrave, 1978;
Cocaine blocks uptake by neuronal plasma membrane
transporters for dopamine (DAT), serotonin (SERT)
and norepinephrine (NET) (Kuhar et al., 1991). However, the relationships between these molecular actions
and cocaine reward and aversion remain only partially
understood (Gawin, 1991; Satel et al., 1991; Sora et al.,
1998; Woolverton and Johnson, 1992; Xu et al., 2000).
No current medication robustly blocks cocaine reward or
substantially relieves cocaine dependence in humans
(Carroll and Lewis, 1994; Carroll et al., 1999; Kreek,
1997; Lavori et al., 1999), although several agents can
Present address: Psychopharmacology Laboratory, Tokyo Metropolitan Institute of Psychiatry, Tokyo, Japan.
*Corresponding author. Tel. : +1-410-550-1589; fax: +1-410-5501535.
E-mail address: firstname.lastname@example.org (G. R. Uhl).
Abbreviations : ANOVA, analysis of variance ; CPP, conditioned
place preference; DAT, dopamine transporter; KO, knockout;
NET, norepinephrine transporter; PCR, polymerase chain reaction; SERT, serotonin transporter; WT, wildtype.
NSC 1803 14-10-02
F. S. Hall et al.
Robinson et al., 1992; Rocha et al., 1998a,b). The focus
on DAT mechanisms of cocaine action is such that
cocaine binding has been considered almost synonymous
with DAT binding in positron emission tomography
studies (Logan et al., 1997; Volkow et al., 1997).
Studies in transporter knockout mice enrich this picture. Both reported strains of DAT knockouts display
robust cocaine reward despite complete elimination of
cocaine-induced locomotion (Rocha et al., 1998a,b;
Sora et al., 1998). Although pharmacological studies
have supported the idea of a homologous relationship
between cocaine-induced locomotion and cocaineinduced reward (see Wise and Bozarth, 1987) transporter
knockout studies ¢nding complete dissociations of these
argue against homologous mechanisms. Cocaine reward
can even be enhanced in SERT or NET knockout mice
(Sora et al., 1998; Xu et al., 2000). However, combined
DAT and SERT knockouts display no cocaine reward
(Sora et al., 2001b). Current explanations for these ¢ndings combine several perspectives. First, the enhanced
cocaine reward found in NET and SERT knockouts suggests that NET or SERT blockade by cocaine may normally contribute to aversive cocaine e¡ects. Secondly,
mice adapt behaviorally and biochemically to the loss
of single or multiple transporters (Bengel et al., 1998;
Benoit-Marand et al., 2000; Gainetdinov et al., 1999;
Giros et al., 1996; Li et al., 1999, 2000; Rocha et al.,
1998a,b; Sora et al., 1998; Xu et al., 2000). These adaptations could play roles in altering the balance between
rewarding and aversive e¡ects of cocaine and other drug
classes that act via these transporters.
To examine these hypotheses, the rewarding e¡ects of
drugs selective for speci¢c monoaminergic transporters
were tested in single- and multiple-transporter knockout
mice. These data provide support for both emerging
multi-site models for cocaine’s rewarding and aversive
properties and for ideas that adaptations in knockout
mice can alter cocaine’s rewarding properties. These
observations provide tentative explanations for the
rewarding and aversive properties of several drug classes
that act at these transporters but display substantially
distinct abuse liability pro¢les.
DAT (Sora et al., 1998), SERT (Bengel et al., 1998), NET
(Xu et al., 2000) and NET/SERT knockout mice were bred
by heterozygote crosses as previously described. The background strain of the three knockout strains was a chimera of
C57BL/6J with 129/Sv or 129Svj, which was the result of the
original knockout derivation. Mice were genotyped using polymerase chain reaction (PCR) ampli¢cations using oligonucleotide primers targeted at neomycin genomic sequences found in
the knockout constructions, wildtype genomic sequences interrupted in the knockouts, and sequences lying outside the homologous recombinant zones. DAT knockout genotyping used
DAT 9G (5P-GAC TTG CTG TGA CCT GGC TAG GAA3P), DAT 12S (5P-GCC TTG AGC TCT TAT CGG GCC TCC3P) and NEO22B (5P-GCC TCT GTC CGC AGT TCA TTC
AG-3P) at 1.6, 0.6 and 0.6 WM ¢nal concentrations. These reactions produced 640-bp and 900-bp products from wildtype and
knockouts, respectively. NET genotyping used NET1 (5P-CTG
CCA TGA GAG AAA GAA AGT-3P). NET2 (5P-TTG CTT
GAG GAA AGG AAA GGC-3P) and NEO24 (5P-CGG ACA
GGT CGG TCT TGA C-3P) at 1.6, 0.8 and 0.8 WM concentrations. These reactions produced 846-bp and 1000-bp products
from wildtype and knockout alleles. SERT knockouts were
genotyped using primers 5HT11 (5P-GAT GAA GCG CCA
CAC TCT ACG CC-3P), 5HT22 (5P-CAG CGA CAG CTA
CCT TAG GC-3P) and 5HTNEO (5P-YCG ACG TTG TCA
CTG AAG CGG-3P) at 1.6, 0.8 and 0.8 WM concentrations.
Wildtypes produced 340-bp and knockouts 1000-bp ampli¢cation products. Two PCR products of di¡erent sizes that were
identi¢ed after gel electrophoresis thus identi¢ed the wildtype
(WT) and knockout (KO) alleles for each gene. Combined
SERT KO/NET KO mice were generated by crosses of these
lines producing F1 double heterozygote knockout mice and F2
combined double homozygote knockout mice. No gross abnormalities or behavioral phenotypes were found in these combined
knockouts beyond those already reported for the single knockouts. Mice of each genotype were half male and half female and
were tested at 12^20 weeks of age.
Conditioned place preference
Conditioned place preferences (CPP) were assessed as
described using a place preference procedure that has consistently produced signi¢cant place preferences for cocaine and
other drugs of abuse in our laboratory (Hall et al., 2001; Sora
et al., 1997, 1998, 2001a,b; Takahashi et al., 1997). In brief, this
procedure uses a two-compartment Plexiglas chamber, one with
a wire mesh £oor and one with corncob bedding on a smooth
Plexiglas £oor. This test provides a technically tractable and
robust measure of drug reward in mice (Bardo and Bevins,
2000; Tzschentke, 1998). It has been validated by its ability to
detect the rewarding properties of virtually every class of substance abused by humans, and it displays high congruence with
self-administration data (Bardo, 1998). Cocaine produces a
robust place preference such that after conditioning mice spent
more than half their time on the previously non-preferred side of
the apparatus. In all cases there was a slight preference for the
side of the chamber with bedding £ooring, producing a test that
has been termed ‘biased’ place preference. However, this initial
preference did not di¡er signi¢cantly between genotypes (data
Initial preference was assessed by measuring the fraction of a
30-min test period that mice spent on each side of the apparatus.
For conditioning, mice were restricted to single compartments
for 30 min after i.p. injection of either drug (on the initially nonpreferred side) or saline (on the initially preferred side). Mice
were returned to home cages for 4 h, and then provided conditioning while con¢ned to the opposite sides of the cage. Saline
and drug conditioning sessions were counterbalanced. Separate
groups of mice from the DAT KO, NET KO and SERT KO
strains of each genotype (+/+, +/3 and 3/3; n = 8^12 mice/genotype) received cocaine, nisoxetine HCl (RBI) or £uoxetine
HCl (RBI). DAT KO mice received nisoxetine (10 or 30 mg/
kg i.p.) or £uoxetine (5 or 20 mg/kg i.p.). NET mice received 30
mg/kg i.p. nisoxetine or 20 mg/kg £uoxetine i.p. SERT mice
received 30 mg/kg i.p. nisoxetine or 20 mg/kg £uoxetine i.p.
Cocaine conditioning was assessed in the NET/SERT double
knockouts using 10 mg/kg cocaine HCl (Sora et al., 1998). In
this paradigm there were four drug pairings and four saline
pairings for £uoxetine and nisoxetine conditioning, and two
drug pairings and two saline pairings for cocaine.
A single CPP assessment session followed the last conditioning session by 24 h. The proportion of the 30-min session spent
on each side was recorded during this place preference assessment. Time spent on the initially non-preferred side after conditioning was compared to the time spent on that side in prior to
conditioning (Sora et al., 1998).
Statistical comparisons focused on the six groups used in this
study: NET WT/SERT WT, NET KO/SERT WT, NET WT/
SERT KO, NET HET/SERT HOM, NET HOM/SERT HET,
and NET HOM/SERT HOM. These tests compared di¡erences
NSC 1803 14-10-02
Cocaine mechanisms in transporter knockout mice
in place preferences (post-conditioning^preconditioning) using
analyses of variance (ANOVA) followed by Fisher’s PLSD
post-hoc analyses (Statview), or Sche¡e’s post-hoc comparisons,
and are presented as mean R S.E.M. for each experimental
group. Data were analyzed with the between subjects factors
DOSE or GENOTYPE (wild-type, heterozygote, and homozygote) for DAT GENOTYPE, SERT GENOTYPE and NET
GENOTYPE. NET/SERT data were compared using one-factor
ANOVAs, and NET WT/SERT WT, NET KO/SERT WT,
NET WT/SERT KO, and NET HOM/SERT HOM groups subsequently compared using two-factor ANOVAs as NET GENOTYPE vs. SERT GENOTYPE. In some experiments there
appeared to be a signi¢cant place aversion produced by £uoxetine, in some groups, as well as place preferences, in the other
groups. In these cases a separate ANOVA was performed
without the data from the homozygous KO mice to determine
if there was a signi¢cant aversion produced in the other
No gross abnormalities or behavioral phenotypes were
found in these individual or combined knockouts beyond
those already reported for the single knockouts. SERT/
NET combined knockout mice were produced at nearly
expected ratios from double-heterozygote crosses and
were normal in their fertility, baseline locomotor
responses, habituation, screen hang time and rotarod
performance (data not shown).
Fluoxetine CPP in DAT knockout mice
Fluoxetine failed to produce signi¢cant place preferences in either wildtype or heterozygote DAT KO mice.
Indeed, mice of both of these two genotypes manifested
conditioned aversions to the compartments where they
received 20 mg/kg £uoxetine (Fig. 1A; ANOVA of wildtype and heterozygous groups only F[1,28] = 14.1,
P 6 0.001). Sche¡e post-hoc comparisons (P 6 0.05)
revealed that the wildtype 20 mg/kg group had a signi¢cant place aversion compared to the wildtype 5 mg/kg
group. By contrast, £uoxetine produced a substantial
place preference in homozygous DAT KO mice, as demonstrated by a highly signi¢cant main e¡ect of DAT genotype (F[2,39] = 24.8, P 6 0.0001) con¢rmed by Sche¡e
post-hoc comparisons (P 6 0.05) with wildtype and heterozygote preferences.
Nisoxetine CPP in DAT KO mice
Nisoxetine also failed to produce signi¢cant place preferences in wildtype or heterozygous DAT KO mice.
However, nisoxetine produced a substantial place preference in homozygous DAT KO mice as manifested by a
Fig. 1. (A) Fluoxetine establishes CPP in DAT KO mice not shown by wildtype mice. CPP induced by £uoxetine (5 and
20 mg/kg, i.p.) in wildtype (+/+, open bars), heterozygous (+/3, gray bars) and homozygous (3/3, black bars) DAT KO
mice. Homozygous knockout mice treated with this SERT-selective blocker (20 mg/kg dose) displayed signi¢cant preferences
for the place where they received the drug (*P 6 0.01 compared to wildtype controls). Wildtype and heterozygous knockout
mice displayed conditioned place aversions to the high dose of £uoxetine (V P 6 0.05; 20 mg/kg £uoxetine versus 5 mg/kg
£uoxetine ; Sche¡e’s comparison). (B) Nisoxetine establishes CPP in DAT KO mice not shown by wildtype mice. CPP
induced by nisoxetine (10 and 30 mg/kg, i.p.) in wildtype (+/+, open bars), heterozygous (+/3, gray bars) and homozygous
(3/3, black bars) DAT KO mice. Homozygous knockout mice treated with this NET-selective blocker (30 mg/kg dose)
displayed signi¢cant preferences for the place where they received the drug (*P 6 0.05 compared to wildtype controls and
heterozygote knockout mice).
NSC 1803 14-10-02
F. S. Hall et al.
Fig. 2. (A) Fluoxetine establishes CPP in NET KO mice not shown by wildtype mice. CPP induced by £uoxetine (20 mg/kg,
i.p.) in wildtype (+/+, open bars), heterozygous (+/3, gray bars) and homozygous (3/3, black bars) NET KO mice. Homozygous knockout mice treated with this SERT-selective blocker displayed signi¢cant preferences for the place where they
received the drug (*P 6 0.01 compared to wildtype controls). (B) Nisoxetine does not establish CPP in NET KO mice. CPP
induced by nisoxetine (30 mg/kg, i.p.) in wildtype (+/+, open bars), heterozygous (+/3, gray bars) and homozygous (3/3,
black bars) NET KO mice. None of these genotypes treated with this NET-selective blocker displayed signi¢cant preferences
for the place where they received the drug.
signi¢cant main e¡ect of DAT genotype (Fig. 1B;
F[2,44] = 7.7, P 6 0.002).
Fluoxetine CPP in NET KO mice
Fluoxetine failed to produce a signi¢cant place preference in wildtype or heterozygous NET KO mice. However, it did produce a place preference in homozygous
NET KO mice as manifested by a signi¢cant main e¡ect
of NET genotype (Fig. 2A; F[2,21] = 3.5, P 6 0.05). In
this experiment there was no indication that the £uoxetine produced any aversive e¡ects (e.g. reduced preference for the drug-paired side).
Nisoxetine CPP in NET KO mice
Nisoxetine failed to produce a signi¢cant place preference in NET KO mice, as anticipated, and as indicated
Fig. 3. (A) Fluoxetine does not establish a CPP in SERT KO mice. CPP induced by serotonin (20 mg/kg, i.p.) in wildtype
(+/+, open bars), heterozygous (+/3, gray bars) and homozygous (3/3, black bars) SERT KO mice. None of these genotypes treated with this NET-selective blocker displayed signi¢cant preferences for the place where they received the drug.
(B) Nisoxetine does not establish a CPP in SERT KO mice not shown by wildtype mice. CPP induced by nisoxetine (30 mg/
kg, i.p.) in wildtype (+/+, open bars), heterozygous (+/3, gray bars) and homozygous (3/3, black bars) SERT KO mice.
None of these genotypes treated with this NET-selective blocker displayed signi¢cant preferences for the place where they
received the drug.
NSC 1803 14-10-02
Cocaine mechanisms in transporter knockout mice
by a non-signi¢cant e¡ect of NET genotype (Fig. 2B;
F[2,20] = 0.7, not signi¢cant).
Fluoxetine CPP in SERT KO mice
Fluoxetine failed to produce a signi¢cant place preference in SERT KO mice, as anticipated, and as indicated
by a non-signi¢cant e¡ect of SERT genotype (Fig. 3A;
F[2,34] = 0.0, not signi¢cant). As before for the DAT KO
mice the data indicate that £uoxetine produced a small
conditioned aversion although, in this case, there is no
changes in cocaine reward were observed in mice that
were heterozygous knockouts at one locus and homozygous at the other locus. When the heterozygous groups
were excluded from the analysis so that the only groups
included were NET WT/SERT WT, NET KO/SERT
WT, NET WT/SERT KO and NET HOM/SERT
HOM, a two-way ANOVA (NET GENOTYPEUSERT
GENOTYPE) revealed a signi¢cant NET GENOTYPEUSERT GENOTYPE interaction (F[1,28] = 4.7,
P 6 0.04) as well as a signi¢cant main e¡ect of SERT
GENOTYPE (F[1,28] = 8.1, P 6 0.01) and the e¡ect of
NET GENOTYPE did not reach statistical signi¢cance
(F[1,28] = 3.4, P 6 0.08).
Nisoxetine CPP in SERT KO mice
Nisoxetine failed to produce a signi¢cant CPP in
SERT KO mice as indicated by a non-signi¢cant e¡ect
of SERT genotype (Fig. 3B ; F[2,30] = 0.5, not signi¢cant).
Cocaine CPP in combined NET/SERT KO mice
Combined NET KO/SERT KO mice had a much
larger cocaine CPP than wildtype mice or mice of any
other genotypes when all groups where compared in initial one-way ANOVA (Fig. 4; GENOTYPE: F[5,39] =
2.6, P 6 0.05). This was con¢rmed by post-hoc analyses
using Fisher’s PLSD in which the cocaine place preferences of NET KO/SERT KO double knockout mice
were signi¢cantly greater than the place preferences
observed in all other groups (P 6 0.05 Fisher’s PLSD).
The combined NET KO/SERT KO increased the time
spent on the non-preferred side by 735 R 75 s compared
to 251 R 44 in the NET WT/SERT WT group. No
The simplest hypotheses that explain current and previous data are that lifelong deletion of DAT, SERT,
NET or combined transporter deletions each provide a
distinctive pattern of alteration in the reward resulting
from blockade of SERT or NET or of all three transporters. The current results are consistent with the idea
that cocaine may normally work as a dirty drug that
provides both rewarding and aversive properties by distinct actions at these three transporters. The combined
observations document adaptations that occur in knockout mice. They can also be tentatively ¢t with the human
abuse liability of a number of widely used drug classes
whose members act di¡erentially at these three monoamine transporters.
Other than the previously published behavioral di¡erences in these mice, they appear grossly normal aside
from the speci¢c alterations in drug responses noted
Fig. 4. Cocaine produces substantially enhanced CPP in combined NET/SERT KO mice (NET 3/3 SERT 3/3). CPP
induced by cocaine (10 mg/kg, i.p.) in combined NET/SERT KO mice: NET +/+ SERT +/+, NET 3/3 SERT +/+, NET +/
+ SERT 3/3, NET +/3 SERT 3/3, NET 3/3 SERT +/3, NET 3/3 SERT 3/3. Double homozygous knockout mice
(NET 3/3 SERT 3/3) displayed a greatly enhanced CPP for cocaine compared to all other groups (*P 6 0.01, Fisher’s
NSC 1803 14-10-02
F. S. Hall et al.
below and in other work describing these mice (Sora et
al., 1998, 2001a,b). In addition, general tests of motor
function are normal (unpublished data). There may be
more subtle e¡ects on attention, learning, or response
selection, but gross general de¢cits seem unlikely given
the fact that each of these strains exhibits normal
responses, including conditioned responses, to some
drugs, but not others.
Possible contributions of each transporter’s blockade
to cocaine’s rewarding and aversive properties can be
tentatively outlined based on results in wildtype and
knockout mice. DAT appears to be the transporter
most associated with rewarding properties of cocaine in
CPP tests. Compounds with substantial selectivity for
DAT, including GBR 12909 and 12935, can produce
reward, and are self-administered by animals, although
often less avidly than cocaine (for review see Rothman
and Glowa, 1995). However, deletion of DAT alone did
not eliminate cocaine reward in previous studies with
these mice (Sora et al., 1998, 2001a,b). The striking
rewarding e¡ects that £uoxetine and nisoxetine confer
in DAT KOs, but not in wildtype mice, clearly indicate
that blockade of SERT or NET can acquire rewarding
properties in DAT’s lifelong absence. The adaptive
mechanisms that make £uoxetine and nisoxetine rewarding in DAT KOs could well contribute to the reward that
cocaine confers in DAT KO mice, since cocaine retains
its ability to block both SERT and NET in these animals. It should be stressed that DAT KO basal dopamine levels are ¢ve times those found in wildtype mice
and in fact higher than in wildtype animals treated with
cocaine (Jones et al., 1998). Recent reports that NET
blocker can enhance nucleus accumbens dopamine in
DAT KO but not in wildtype mice (Carboni et al.,
2001) provide one biochemical substrate for the nisoxetine reward found in our current studies. The supra-additive e¡ects of SERT/NET combined knockouts in
enhancing cocaine reward, compared to e¡ects of individual knockouts alone, would also be consistent with
the idea that each of these transporter deletions might
change cocaine reward through di¡erent mechanisms.
SERT blockade could contribute to both rewarding
and aversive properties of cocaine in wildtype animals.
Shifts in this reward/aversion balance in DAT and in
NET KO mice are some of the most plausible explanations for otherwise apparently contradictory data that
demonstrate (1) enhanced cocaine reward in SERT KO
mice (Sora et al., 1998), (2) enhanced £uoxetine reward
in DAT and in NET KO mice (present data), and (3)
ablated cocaine reward in DAT/SERT combined knockout mice (Sora et al., 2001a,b). SERT blockade could
normally produce a combination of rewarding and aversive features by augmenting serotonin actions at di¡erent
serotonin receptor subtypes expressed di¡erentially in
distinct serotoninergic circuits. If these rewarding and
aversive features normally balanced each other to produce little net reward or aversion with SERT blockade,
the failure of £uoxetine or other selective serotonin reuptake inhibitors to display substantial aversion or abuse
liability in humans would make sense (Frank and
Zubrycki, 1989; Tella, 1995). The striking £uoxetine
reward acquired by DAT and by NET KOs is consistent
with the idea that each of these knockouts shifts the
balance between rewarding and aversive portions of the
serotonin systems. These considerations do not necessarily imply that the reward/aversion balance changes in
identical ways in both strains, however.
NET blockade may well contribute less to rewarding
and more to aversive properties of cocaine in wildtype
animals. These observations are consistent with frequent
human clinical self-reports of ‘jitteriness’ with cocaine
use (Grabowski et al., 1997; Lobl and Carbone, 1992).
They accord with evidence for cocaine aversive features
in some animal models (DeVries et al., 1998; Ettenberg
et al., 1999). They ¢t with the failure of mazindol, a
widely used combined DAT/NET blocker, to exhibit
any striking human clinical abuse liability and with its
not-infrequent discontinuation due to jitteriness and
sleep disturbances (Chait et al., 1987; Wilson and
Schuster, 1976; Yanagita et al., 1982).
Combined NET/SERT KO mice provide a means of
studying possible interactions between the e¡ects of
deleting these two transporters. Our data show no evidence for complementation of these two genetic deletions. In fact, the robust increase in cocaine reward
manifested by mice with combined NET/SERT KOs is
more than supra-additive when compared to the variable
e¡ects of each transporter deletion on cocaine reward
when they are studied separately. This robust synergy
suggests that di¡erent, although possibly interacting,
alterations could be produced by SERT and by NET
deletions so that cocaine reward manifests an even larger
increase in mice with both deletions than would have
been predicted from the results of individual knockouts
of these two transporters. The large increases in cocaine
reward found in NET/SERT double knockouts also provide striking contrasts to the large decreases in cocaine
reward found in DAT/SERT double knockouts (Sora et
The precise brain changes that contribute to the di¡erences in the rewarding consequences of single- and multiple-transporter blockers in single- and multipletransporter knockout mice are unknown. The brains of
mice of each of these knockout strains appear grossly
normal, although the smaller brains of DAT KO mice
are consistent with the smaller overall size of these mice.
Each single transporter knockout exhibits a distinctive
pattern of brain neurochemical rearrangements (Bengel
et al., 1998; Gainetdinov et al., 1999; Sora et al.,
2001a,b; Xu et al., 2000). Microarray studies of mRNA
from the brains of DAT and of SERT KOs also identify
dozens of genes whose expression is consistently altered
more than two-fold (Liu, Hall, Sora and Uhl, unpublished data). None of these initial characterizations provides substantial evidence for supra-additive knockout
in£uences on any brain neurochemical, microdialysis or
gene expression pro¢le when mice with multiple knockouts have been examined to date, however.
DAT and NET each share relatively high a⁄nities for
dopamine and norepinephrine. Recent authors have even
advocated uptake of dopamine by serotonin terminals. It
is conceivable that these monoamines could become
NSC 1803 14-10-02
Cocaine mechanisms in transporter knockout mice
‘false transmitters’ in monoaminergic neurons adjacent
to terminals in DAT KO mice. Dopamine could become
a transmitter at adjacent norepinephrine or serotonin
terminals in DAT KOs, norepinephrine a false transmitter at adjacent dopaminergic or serotoninergic terminals
in NET KOs, and/or serotonin a false transmitter in
SERT KO dopamine or norepinephrine terminals.
These mechanisms are not consistently prominent in
DAT KOs, since NET and SERT blockade by cocaine
can fail to robustly enhance dopamine e¥ux in DAT KO
striata (Gainetdinov et al., 1999). SERT blockade by
£uoxetine may not enhance dopamine e¥ux in nucleus
accumbens of DAT KOs (Carboni et al., 2001). However, in nucleus accumbens of DAT KO mice, cocaine is
reported to enhance dopamine e¥ux and the NET
blocker reboxetine acquires the ability to enhance dopamine e¥ux in a way not found in wildtype mice (Carboni
et al., 2001). Enhanced accumbens dopamine e¥ux could
thus contribute to the reward that nisoxetine acquires in
our DAT KO studies, although it may not contribute to
the £uoxetine reward in these animals. Further studies
will be necessary to de¢ne how much of the enhanced
reward is due to false transmitter mechanisms in, for
example, accumbens noradrenergic terminals that lie
near dopaminergic terminals, and how much might represent adaptations in circuits such as those that regulate
dopamine cell ¢ring rates and dopamine release after
cocaine administration (Berretta et al., 2000; Bonhomme
et al., 1995; Boulenguez et al., 1996; Bowers et al., 2000;
Di Giovanni et al., 2000; Di Matteo et al., 2000; Lejeune
and Millan, 1998; Shi et al., 2000; West and Galloway,
1996; Yadid et al., 1994).
Even in the absence of exact knowledge about mechanisms, the current striking results provide novel pathways toward thinking about therapies for the treatment
of cocaine addiction based on selectivity for speci¢c
plasma membrane monoamine transporters. Although
cocaine abuse remains a daunting challenge with complications of non-compliance, frequent psychiatric comorbidity, etc., these sorts of information could inform
several pathways of pharmacological approach to this
clinical problem. ‘Replacement’ theories in which a
postulated ‘reward de¢cit’ (Blum et al., 2000) could be
treated analogously to the model used to develop methadone (Dole et al., 1966) need to take into account the
possible balances between rewarding and possibly aversive e¡ects of blocking di¡erent constellations of transporters. Pharmacodynamically based approaches in
which slower-onset replacements might yield fewer
euphoric properties should have similar sets of concerns.
Cocaine ‘antagonist’ strategies that seek compounds that
would block cocaine access to some of the transporters
a¡ected by cocaine in the absence of antagonist, yet
spare these transporters’ abilities to accumulate monoamines, could also be informed by these observations.
Drugs that block cocaine’s uptake-inhibiting actions at
DAT and SERT but allow it to continue to block uptake
by NET could provide one possible therapeutic avenue
to this di⁄cult problem. Such compounds could provide
cocaine antagonism at ‘rewarding’ transporters and spare
its actions at ‘aversive’ sites, dramatically altering the
rewarding valence of cocaine and potential anti-cocaine
therapeutics. It is even conceivable that use of transporter-blocking agents in the depressive disorders, and
other psychiatric disorders often comorbid with substance abuse (Beitchman et al., 2001; Farrell et al.,
1998), could also be informed by the current observations.
Acknowledgements'We acknowledge that ¢nancial support of
the NIDA-IRP; the substantial contributions to this work from
the Charles River animal care sta¡, Triad division, as well as
comments by Drs. Elliot Gardner and Roy Wise.
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(Accepted 18 June 2002)
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