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European Journal of Neuroscience, Vol. 15, pp. 514±520, 2002

ã Federation of European Neuroscience Societies

Intravenous cocaine induced-activity and behavioural
sensitization in norepinephrine-, but not dopaminetransporter knockout mice
Andy N. Mead,1,² Beatriz A. Rocha1,4* David M. Donovan3 and Jonathan L. Katz2
1

Behavioural Neuroscience Branch and
Medications Discovery Research Branch, National Institute of Drug Abuse-Intramural Research Program, 5500 Nathan Shock
Drive, Baltimore, MD 21224, USA
3
Gerontology Research Center, IRP, NIA, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224±6825, USA
4
Department of Psychiatry, University of Maryland, Maryland Psychiatric Research Center, PO Box 21247, Baltimore, MD 21228, USA
2

Keywords: DAT, KO mice, NET, nucleus accumbens

Abstract
Previously, it was reported that both norepinephrine transporter (NET) and dopamine transporter (DAT) knockout (KO) mice were
sensitive to the reinforcing effects of cocaine. However, assessing the locomotor-stimulant effects of cocaine in these subjects
has proven dif®cult due to signi®cant differences in their baseline activity compared to wild-type controls. The present studies
were designed to clarify the role of NET and DAT in the stimulant effects of acute and repeated cocaine utilizing these knockout
mice, and thereby assess the role of these substrates in the locomotor stimulant effects of cocaine. Mice were habituated to the
test environment for suf®cient time to ensure equal baselines at the time of cocaine administration. Mice then received cocaine
(3±25 mg/kg) intravenously according to a within-session cumulative dose±response design. Cocaine dosing was repeated at 48h intervals for four sessions to assess behavioural sensitization. NET-KO mice exhibited a reduced response to acute cocaine
administration compared to wild-type (WT) controls. However, comparable sensitization developed in NET-KO and WT mice. The
DAT-KO and DAT-heterozygote (HT) mice displayed no locomotor activation following either acute or repeated cocaine
administration. These data suggest a role for the NET in the acute response to cocaine, but no involvement in sensitization to
cocaine. In contrast, DAT appears to be necessary for both the acute locomotor response to cocaine and the subsequent
development of sensitization. In addition to existing data concerning the reinforcing effects of cocaine in DAT-KO mice, these
data suggest a dissociation between the reinforcing and locomotor stimulant effects of cocaine.

Introduction
The generation of mice lacking either the norepinephrine transporter
(NET) or the dopamine transporter (DAT) has provided an additional
tool in attempts to understand the role of these neurotransmitters in
drug-induced or maintained behaviours. Initial studies of these
knockout (KO) mice have provided surprising, and in some cases
con¯icting data. For example, Rocha et al. (1998a) reported that
DAT-KO mice would self-administer cocaine intravenously, despite
the fact that the DAT was considered to be the primary site for the
reinforcing effects of cocaine. Further, Sora et al. (1998) demonstrated that cocaine established a conditioned place preference in
DAT-KO mice. While these studies do not rule out the possibility that
the DAT is a critical substrate for the reinforcing effects of cocaine in
humans, they do suggest that other substrates may also be suf®cient

Correspondence: Dr A. N. Mead, at ²present address below.
E-Mail: andym@biols.susx.ac.uk
²
Laboratory of Experimental Psychology, School of Biological Sciences,
University of Sussex, Brighton BN1 9QG, UK

*Present address: Department of Pharmacology, Merck Research
Laboratories, R80Y-140, PO Box 2000, Rahway, NJ 07065, USA
Received 8 March 2002, revised 30 May 2002, accepted 31 May 2002
doi:10.1046/j.1460-9568.2002.02104.x

for these behavioural effects related to the abuse liability of cocaine.
Furthermore, Carboni et al. (2001) reported that both cocaine and
amphetamine increase extracellular levels of dopamine (DA) in the
nucleus accumbens (NAcc) of DAT-KO mice. In the absence of
DAT, this effect was attributed to the ability of cocaine and
amphetamine to prevent dopamine (DA) uptake via the NET.
Studies of psychostimulant-induced activity in DAT-KO mice have
provided con¯icting results. Giros et al. (1996) and Sora et al. (1998)
reported that cocaine and amphetamine had no effect on activity levels
in the DAT-KO mouse, while Gainetdinov et al. (1999) and Spielewoy
et al. (2001) demonstrated that cocaine and amphetamine suppressed
activity in these mutants. However, interpretation of these results is
complicated due to the basal hyperactivity of DAT-KO mice, as the
effects of stimulants were studied from different baseline levels of
activity; a variable that is known to in¯uence the effects of stimulants
on locomotor activity (Dews & Wenger, 1977; Robbins, 1977).
Presently, very little data exists concerning the response of NETKO mice to psychostimulant administration. With the discovery that
the actions of cocaine in the DAT-KO mouse may be due to its ability
to prevent DA uptake via NET (Carboni et al., 2001), the NET-KO
mouse has the potential to provide valuable insights into the actions
of cocaine. Xu et al. (2000) examined the effects of cocaine and
amphetamine in the NET-KO, and found that these mice showed a

Cocaine effects in NET and DAT knockout mice
TABLE 1. Infusion summary for cocaine sessions
Time into session (min)

515

Apparatus

Experiment
1 (NET)

Experiment
2 (DAT)

Infusion
rate
(ml/min)

Actual
cocaine
dose
(mg/kg)

60
75
90
105

720
735
750
765

30
50
70
100

3
5
7
10

Cumulative
cocaine
dose
(mg/kg)
3
8
15
25

greater locomotor stimulation and conditioned place preference
induced by cocaine. Furthermore, repeated cocaine administration
only resulted in sensitization to the stimulant effects of cocaine in
wild-type (WT) mice, leading Xu and colleagues to conclude that the
NET-KO mouse was presensitized to cocaine. These ®ndings suggest
a potentially important role for the NET in psychostimulant drug
actions.
The purpose of the present study was to investigate the stimulant
effects of repeated cocaine administration in both DAT- and NETKO mice using intravenous (i.v.) drug administration. Adequate
habituation prior to drug administration and administration of cocaine
via a chronically indwelling i.v. catheter allowed baseline levels of
activity to be normalized prior to drug administration, and minimized
the effects on activity of handling and injection procedures.

Materials and methods
Subjects
Experiment 1 was performed with NET-WT (n = 9) and homozygous
NET-KO (n = 12) male mice, originally generated at Howard Hughes
Medical Institute Laboratories, Durham, NC (Wang et al., 1999).
Experiment 2 was performed with DAT-WT (n = 8), DAT-HT
(n = 4) and DAT-KO (n = 6) male mice (Cord et al., 2002). All mice
were obtained from heterozygous breeding pairs, and genotypes of
offspring were con®rmed using PCR. Upon arrival into the animal
colony, all mice were housed individually and allowed a minimum of
14 days habituation prior to any experimental testing. Mice were
housed under a 12-h light : 12-h dark schedule, with lights on at
07.00 h. Throughout the course of the experiment, mice had ad
libitum access to standard lab chow and water (including test-sessions
for experiment 2), in a temperature (70 6 5°F) and humidity
(50 6 15%) controlled room. Testing for experiment 1 took place
during the light-phase between 08.00 h and 16.00 h. Testing for
experiment 2 occurred during the dark-phase between 18.00 h and
07.00 h. Mice were transferred to the separate testing room approximately 30 min prior to test sessions. All experiments were conducted
in strict accordance with the Principles for Care and Use of
Laboratory Animals provided by the NIH, and received prior
approval from the local Animal Care and Use Committee.
Drugs
Ketamine-xylazine anaesthesia (65 mg/kg ketamine and 18 mg/kg
xylazine diluted in 0.9% saline) was administered intraperitoneally
(i.p.) at a volume of 13 mL/kg. Cocaine hydrochloride was dissolved
in 0.9% saline for a ®nal concentration of 2.3±4.5 mg/mL (100 mg/
kg/mL).

Four activity monitors (Digiscan system, AccuScan instruments Inc.
Ohio, USA) were divided into quadrants by two perpendicular
Plexiglas divisions crossing the mid-point of the monitor. This
resulted in four testing regions (20 cm 3 20 cm 3 31 cm) per
monitor, of which only two were used concurrently for activity
monitoring. Floors and walls were made of smooth clear Plexiglas.
Sixteen regularly spaced photocells (eight along each axis) located
2 cm above the ¯oor detected horizontal movements (total distance
travelled). Each monitor was enclosed in a sound and lightattenuating chamber, and was connected to a PC running Digipro
software (AccuScan instruments Inc. Ohio, USA) for data acquisition.
For i.v. infusions, a multisyringe Harvard Pump 22 (Harvard
Apparatus, MA, USA) was connected to the same PC, and controlled
using a program written in Q-Basic software. Syringes were
connected to single-channel ¯uid swivels (Instech Laboratories Inc.,
Plymouth Meeting, PA, USA) via Tygon tubing (0.06-inch o.d.;
Tygon Microbore Tubing, Norton Performance Plastics, Akron, OH,
USA). The swivel was mounted on a counterbalanced arm (Instech
Laboratories Inc., Plymouth Meeting, PA, USA), and a Tygon line
exited the swivel and entered the testing region of the activity
monitor.
Procedures
Catheter Implantation
Under anaesthesia (ketamine/xylazine solution i.p.), a silastic catheter
(0.009-inch i.d., 0.028-inch o.d.; Speciality Manufacturing Inc.,
Saginaw, MI, USA) was inserted into the right external jugular and its
tip advanced towards the right atrium, before being secured by
sutures, following procedures described by Rocha et al. (1997). The
distal end of the catheter was connected to a modi®ed cannula
assembly (C313G-5UP; Plastics One, Roanoke, VA, USA). The
entire unit consisting of catheter and guide cannula was then
embedded in dental acrylic cement (100±1673; Henry Schein, Port
Washington, NY, USA) and ®xed to the skull using dental cement
(GlasIonomer cement, Shofu Inc., Henry Schein, Port Washington,
NY, USA). The open end of the cannula assembly was sealed with a
silicone cap. Mice were allowed 36 h to recover following surgery.
The total dead space of the cannula assembly was approximately
5 mL and the catheter was ¯ushed daily with 0.02 mL of 30-U/mL
heparin in 0.9% saline.
Behavioural Testing
The general methods have been described previously (Mead et al.,
2002). Mice were placed into the activity monitors for six sessions.
Sessions 1±3 occurred on consecutive days (days 1±3), while sessions
4±6 were performed at 2-day intervals (on days 5, 7 and 9). During
the ®rst 60 min (Experiment 1) or 720 min (Experiment 2) of each
session, no infusions were administered (habituation phase). At 60,
75, 90 and 105 min (Experiment 1) or at 720, 735, 750 and 765 min
(Experiment 2), mice received i.v. infusions of either saline or
cocaine. During sessions 1 (novel), and 2 (saline), mice received
saline infusions, while during sessions 3±6, mice received cocaine
infusions in an ascending cumulative dose sequence. All infusions
occurred over 60 s and the rate of infusion was varied to control
the dose. For cocaine sessions, doses of 3, 5, 7 and 10 mg/kg
cocaine were administered. Therefore, the maximum cumulative
dose reached at each infusion time was 3, 8, 15 and 25 mg/kg
cocaine, respectively. Table 1 provides a summary of the infusion
details.

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 15, 514±520

516 Andy N. Mead, et al.

FIG. 1. Response to novelty in NET- and DAT-KO mice. Data show the mean square-root total cm travelled (6 SEM) following exposure to a novel
environment for NET (A and B) and DAT (C and D)-KO mice. Panels A and C indicate total distance travelled over the ®rst 60 min, and B and D show the
time course of habituation to the environment. NET-KO mice displayed a signi®cant reduction in activity over the ®rst 60 min compared to WT controls (A),
due to an increased rate of habituation (B). DAT-KO mice displayed an increased response to the novel environment as indicated by a reduced rate of
habituation, while DAT-HT mice did not differ from WT controls (C and D). *P < 0.05 compared to WT.

Statistical analysis
The dependent variable measured was total distance travelled, and
all data were square root transformed prior to analysis in order to
produce homogeneity of variance and allow parametric analysis.
Responses to novelty were analysed using two-way mixed factor
ANOVA with genotype and time as factors. Data for analysis of the
novelty response was taken from the ®rst hour of session 1. For
analysis of dose±response functions following acute administration
of cocaine, data from the 15-minute period following each
infusion was pooled, with data from the saline session (session 2)
being compared to data from the ®rst cocaine session (session 3).
Dose±response data were analysed using three-way ANOVA with
dose (3, 8, 15 or 25 mg/kg or saline control infusions) and drug
(saline or cocaine) as within-subjects factors, and genotype as a
between subjects factor. Data from repeated cocaine treatments
were analysed using two-way repeated measures ANOVA with dose
and session (cocaine sessions 1±4) as factors. Signi®cant maineffects of session were further investigated using one-way
repeated measures ANOVA followed by post hoc comparisons of
each session with cocaine session 1 (Dunnett's test).

Results
Response to novelty
Experiment 1 revealed that NET-KO mice displayed a reduced
activity response compared to WT controls when placed into a novel

environment. This difference was apparent over the ®rst hour of
exposure to the novel environment in Session 1 (Fig. 1A) as indicated
by a signi®cant difference between genotypes (t1,20 = 2.175,
P < 0.05). In Experiment 2, DAT KO mice showed signi®cantly
greater levels of activity than both WT and DAT-HT mice when
placed into a novel environment (main-effect of genotype,
F2,15 = 5.14, P < 0.05; Student Newman±Keuls, P < 0.05; Fig. 1C).
This increased response was apparent for approximately 10±12 h,
suggesting a reduced rate of habituation, rather than an enhanced
response to novelty, and it was because of this prolonged hyperactivity that infusions were not administered until 12 h after
placement in the apparatus during Experiment 2. During the twelfth
hour, there were no signi®cant differences in activity between
genotypes (mean total distance traveled 6 SEM: WT 321.3 6 106.6;
HT 369.5 6 252.4; KO 328.0 6 243.5, One-way ANOVA,
F2,17 = 0.017, P = 0.98).
Response to acute cocaine administration
In Experiment 1, the ®rst session of cocaine infusions (session 3)
produced a dose-dependent increase in total distance travelled in
WT and NET-KO mice (dose by drug interaction, F3,57 = 21.93,
P < 0.01; Fig. 2A). However, the magnitude of this effect differed
between genotypes, with the response in NET-KO mice being
lower than that seen in WT mice (drug by genotype interaction,
F1,19 = 4.80, P < 0.05). Post hoc analysis revealed that the
activity response was signi®cantly greater in WT mice at a
dose of 8 mg/kg cocaine (P < 0.05). This difference was also

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 15, 514±520

Cocaine effects in NET and DAT knockout mice

517

FIG. 2. Response to acute cocaine administration in NET- and DAT-KO mice. Data show the mean square-root total cm travelled (6 SEM) over a 15-minute
period following acute administration of cocaine or saline for NET- (A) and DAT- (B) KO mice. Doses of cocaine represent cumulative doses, with
individual infusions of 3, 5, 7 and 10 mg/kg administered at 15-min intervals. For saline infusions, infusion rate was matched to rate for cocaine infusions.
NET-KO mice displayed a signi®cantly lower activity response compared to WT mice, but still displayed a signi®cant increase in activity compared to saline.
Both DAT-KO and DAT-HT mice displayed a signi®cantly lower response to cocaine than WT controls, and this response did not differ from saline.
*P < 0.05, **P < 0.01 compared to corresponding saline infusion for respective genotype. #P < 0.05 compared to respective KO response to cocaine at
indicated dose.

apparent at doses of 15 and 25 mg/kg cocaine, but failed to reach
statistical signi®cance (compare ®lled and open circles in
Fig. 2A). There were no signi®cant differences between genotypes
following saline infusions.
In Experiment 2, cocaine produced a dose-dependent increase in
activity compared to saline infusions in WT mice, although this effect
was not observed in DAT-HT or DAT-KO mice (dose by genotype
interaction, F6,51 = 3.81, P < 0.01; drug by genotype interaction,
F2,17 = 10.44, P < 0.01; Fig. 2B). While cocaine produced a signi®cant enhancement in activity in WT mice at doses of 8±25 mg/kg, no
enhancement in activity was seen at any dose of cocaine in DAT-HT
or DAT-KO mice, when compared to saline infusions. Although this
difference between genotypes only reached signi®cance at the 25 mg/
kg dose of cocaine (P < 0.05), it was also apparent at the 15 mg/kg
dose (compare ®lled and open circles in Fig. 2B). There were no
signi®cant between-genotype differences in activity following any of
the saline infusions.
Response to repeated cocaine administration
In Experiment 1, repeated cocaine administration produced a
progressive increase in activity, indicative of behavioural sensitization (Fig. 3A±B). This increase was observed in both NET-WT and KO mice (main-effect of session; F3,57 = 18.07, P < 0.01), and the
effect was similar between genotypes (main-effect of genotype;
F1,19 = 0.15, n.s). In WT mice, a signi®cant enhancement in activity
compared to cocaine session 1 was observed at doses of 3±15 mg/kg
(session by dose interaction; F9,72 = 2.24, P < 0.05). In NET-KO
mice, sensitization occurred at all doses (main effect of session;
F3,33 = 13.48, P < 0.01, session by dose interaction; F9,99 = 1.45,
n.s). The lack of sensitization at the 25 mg/kg cocaine dose in WT
may have been due to a ceiling effect, suggesting that this dose
produced an activity response close to maximum acutely, and further
increases could not be observed (Fig. 3A).
In Experiment 2, repeated cocaine administration only produced
sensitization in WT mice (main-effect of session, F3,21 = 11.86,
P < 0.01; dose by session interaction, F9,63 = 2.49, P < 0.05). This
increase across sessions was observed at doses of 3±15 mg/kg
(P < 0.05) (Fig. 3C). In DAT-HT and -KO mice, there was no

signi®cant effect of session (F3,9 = 2.55; F3,15 = 1.03, respectively)
or session by dose interaction (F9,27 = 0.93; F9,45 = 0.43, respectively), indicating that repeated cocaine did not result in an
enhancement in cocaine's stimulant effects (Fig. 3D±E).

Discussion
The results of the present study showed that NET-KO mice displayed
a decreased locomotor response to a novel environment, a decreased
locomotor response to an acute administration of cocaine, but
comparable behavioural sensitization compared to WT controls. In
contrast, DAT-KO mice displayed a reduced rate of habituation to the
novel environment. Cocaine administration had no effect on activity
levels in DAT-KO or DAT-HT mice following either acute or
repeated administration, although DAT-HT mice did not differ from
WT controls in their response to novelty, or rate of habituation.
The observation that NET-KO mice displayed a reduced level of
activity when placed in a novel environment is similar to that reported
by Xu et al. (2000), who observed that NET-KO mice displayed
lower levels of activity than WTs during the ®rst 35 min of exposure
to a novel environment. The observation that NET-KO mice
displayed a reduced activity response following an acute cocaine
administration was unexpected as Xu et al. (2000) reported that these
mice showed an enhanced response to acute cocaine administration. It
is unlikely that this difference is due to differences in the NET-KO's
since the two studies used mice from the same source. In addition,
mice were bred according to the same strategy (i.e., heterozygous
breeding pairs). One obvious difference between the two studies is
the route of drug administration, with Xu and colleagues giving
cocaine via the i.p. route. The route of drug administration may
account for differences in results in a number of ways. One possibility
concerns the kinetics of drug distribution and concentration.
Following i.v. administration, one would expect a more rapid rise
in drug levels and a higher peak drug concentration. However, no
data exists on cocaine concentrations in the brain following systemic
cocaine administration in NET-KO mice, so further testing is needed
to examine this possibility. A second explanation is that NET-KO

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 15, 514±520

518 Andy N. Mead, et al.

FIG. 3. Effects of repeated cocaine administration on activity in NET- and DAT-KO mice. Data show the mean square-root total cm travelled (6 SEM) over
a 15-min period following repeated administration of cocaine for NET- (A and B) and DAT- (C±E) KO mice, over four successive sessions. Doses of cocaine
represent cumulative doses, with individual infusions of 3, 5, 7 and 10 mg/kg administered at 15-min intervals. Cocaine sessions occurred at 48-h intervals.
Both NET-KO and WT mice displayed signi®cant behavioural sensitization following repeated cocaine (A and B). In DAT-KO and DAT-HT mice, no
sensitization occurred following four treatments. In WT mice, sensitization occurred at doses of 3±15 mg/kg *P < 0.05 compared to cocaine session 1 at
respective dose (indicating signi®cant behavioural sensitization). P < 0.05 compared to cocaine session 1 for all doses.

mice respond differently to the handling and injection procedure
associated with i.p. drug administration. However, neither Xu et al.
(2000) nor Bohn et al. (2000) observed such differences following
vehicle injections. Despite this, the possibility remains that there is an
interaction between the injection procedure and the effects of
cocaine, such that stress only in¯uences activity in the presence of
the drug. In order to test this hypothesis, a full investigation into the
physiological response to the injection procedure in NET-KO is
needed. In support of this possibility is the known involvement of
norepinephrine (NE) in the response to stressors, and in particular, the
ability of the injection procedure to increase extracellular hypothalamic NE levels in rats (Pacak et al., 1995). In the context of the
present study, these ®ndings would suggest that the injection
procedure would have a more pronounced effect in the NET-KO
than in the WT, as clearance of NE would be reduced substantially in
the absence of NET. Furthermore, systemic cocaine administration
has been demonstrated to increase extracellular NE levels in the
NAcc and ventral tegmental area of rats (Chen & Reith, 1994; Reith
et al., 1997; Pepper et al., 2001). Therefore, the combination of
injection procedure and cocaine-induced NE increases may explain
the more pronounced activity in the NET-KO following i.p. cocaine,
while in the present study, the absence of injection procedure-induced
NE increases would have resulted in a lower NE response to cocaine.
Following repeated cocaine administration, both NET-KO and WT
mice displayed a progressive increase in response to cocaine,

indicative of behavioural sensitization. Again, this contrasts with
previous ®ndings, where NET-KO mice failed to show an increased
locomotor response following repeated cocaine administrations (Xu
et al., 2000). One explanation for this difference is that in the study
by Xu and colleagues, the dose of cocaine chosen produced a
response which may well have been at maximum following the ®rst
drug administration, and therefore further increases could not be
observed. The dose of 20 mg/kg chosen by Xu et al. is at or near
maximal effectiveness in several mouse strains (Rocha et al., 1998b;
Chausmer & Katz, 2001). Such a ceiling effect would have obscured
any evidence of behavioural sensitization, at least with the single
dose studied. As we observed that NET-KO mice displayed
sensitization similar to that observed in WT controls, it appears
that the NET does not play a role in the development of sensitization
to cocaine. However, the reduced response of NET-KO mice to an
acute administration of cocaine suggests that the NET does have a
role in mediating the acute effects of cocaine on activity.
The initial results from DAT-KO mice were comparable with
previous reports using these mice, in that they are hyperactive when
exposed to a novel environment, and habituate more slowly than
controls (Gainetdinov et al., 1999; Giros et al., 1996; Sora et al.,
1998). This hyperactivity has been attributed to hyperdopaminergic
tone in the KO mouse. Interestingly, DAT-HT mice do not differ
from WTs in their activity response to novelty despite a signi®cant
reduction in levels of DAT as assessed by [3H]CFT binding (Sora

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 15, 514±520

Cocaine effects in NET and DAT knockout mice
et al., 1998) and reduced DA uptake in striatal synaptosomes (Giros
et al., 1996).
Previous reports of the effects of psychostimulants on activity in
DAT-KO mice have been complicated by this basal hyperactivity of
the KO phenotype. As this hyperactivity is often of a similar
magnitude to the hyperactivity seen in WT mice following stimulant
administration, it is not clear whether the basal level of activity is
masking any effects of drug in DAT-KO mice. However, by using i.v.
administration of cocaine and providing an adequate habituation time
period, this problem was averted in the present study. In order to
ensure that activity levels across genotypes were comparable at time
of drug administration, mice in this study were allowed 12 h
habituation to the testing environment prior to the ®rst drug
administration. As the results of the saline infusions show, this
period was suf®cient to provide equal baselines across genotypes.
Results from the ®rst cocaine session indicated that within the
dose-range tested, cocaine stimulated activity in WT mice, but not in
either the DAT-KO or DAT-HT mice. It is unlikely that the range of
doses chosen was too low to observe a stimulant effect of cocaine in
the DAT-KO or DAT-HT as pilot studies indicated that higher doses
induced seizures in WT mice. While the results for DAT-KO mice are
consistent with previous ®ndings (Giros et al., 1996; Sora et al.,
1998), the lack of effect in DAT-HT mice is surprising, as cocaine
has previously been reported to increase activity in DAT-HT mice
(Giros et al., 1996; Sora et al., 1998). One possibility is that the
presence of stereotyped behaviours interfered with the expression of
ambulatory behaviour in the present study, however, this is unlikely
as no cocaine-induced activity was seen at the lower doses, at which
stereotypy was unlikely to occur. Indeed, this discrepancy does not
appear to be due to the dose of cocaine used, as we tested over a wide
range of doses, and previous studies had utilized doses of 10 mg/kg
(subcutaneous), and 40 mg/kg (i.p). It is possible that the injection
procedure may in¯uence the response of the DAT-HT to cocaine
administration. Although there are no reports of a saline injection
increasing activity in the DAT-HT mice, reconciling the published
results with the present ones suggests that the procedure associated
with the injection is suf®cient to enhance the stimulant effects of an
otherwise inactive dose of cocaine.
The repeated administration of cocaine produced behavioural
sensitization to the drugs stimulant effects in WT, but not in DAT-KO
or DAT-HT mice. These results are consistent with the known role of
DAT in mediating the stimulant effects of acute cocaine. The
observation that DAT-HT mice displayed no cocaine-induced activity
shows that even a reduction of approximately 50% in the level of
DAT is suf®cient to abolish the stimulant effects of cocaine. This
®nding is consistent with observations of in vivo displacement of the
DAT ligand, WIN 35,428, and locomotor stimulation both produced
by the cocaine analogue, RTI-31 (Cline et al., 1992). In that study,
maximal stimulation of horizontal activity was obtained at a dose of
RTI-31 that virtually displaced [3H]WIN 35 428 fully. Doses that
produced less than half occupancy were not particularly effective in
stimulating activity.
In light of recent ®ndings showing that cocaine is still capable of
producing increases in extracellular DA in the NAcc of DAT-KO
mice by acting at NET (Carboni et al., 2001), the lack of stimulant
effect observed in the DAT-KO and DAT-HT mice is maybe
surprising, as stimulant-induced hyperactivity has often been associated with increased extracellular DA levels in the NAcc (Sharp
et al., 1987; Kuczenski et al., 1991). However, there is also evidence
suggesting that locomotor activity levels are not simply a re¯ection of
NAcc DA concentrations. First, it has been reported that DA levels in
the NAcc do not increase proportionately with behavioural activity

519

ratings (Kuczenski et al., 1991; Hemby et al., 1995; Kimmel et al.,
2001), suggesting that NAcc DA is not mediating the locomotor
response per se. Second, while D1 antagonism with SCH-23390 in the
NAcc prevented the reinforcing effects of cocaine (Baker et al.,
1998), it had no effect on the locomotor stimulant effects of cocaine
(Baker et al., 1998; Neisewander et al., 1998), suggesting that areas
other than the NAcc mediate the locomotor response to cocaine.
These observations are compatible with the ®ndings that DAT-KO
mice will self-administer cocaine (Rocha et al., 1998a) and display a
conditioned place preference to cocaine (Sora et al., 1998), but do not
display a locomotor response to cocaine. As there are differences in
regional cocaine-induced extracellular DA levels between DAT-KO
and WT mice (e.g., caudate putamen), further analysis of these
differences may provide insight into the regions ultimately responsible for mediating the locomotor response to cocaine.
In summary, we observed that the stimulant effects of cocaine are
reduced in the NET-KO mouse, while sensitization to cocaine
remains unaltered. Therefore, while there appears to be a minor role
of NET in the acute hyperactivity response to cocaine, the NET is not
important in the development of sensitization to cocaine. In the DATKO and DAT-HT mice, cocaine does not induce hyperactivity
following either acute or repeated administration. These results
con®rm that the normal levels of DAT are crucial for cocaine-induced
hyperactivity and subsequent behavioural sensitization, and in light of
recent ®ndings, provide evidence for a dissociation between cocaine
induced activity and reinforcing effects, at the level of the NAcc.

Acknowledgements
Funding provided by NIDA-IRP [NIDA grant R29 DA 12579 (BR)]. The
authors thank Dr M. Caron for supplying the NET mutant mice.

Abbreviations
DA, dopamine; DAT, dopamine transporter; HT, heterozygote; KO, knockout;
NAcc, nucleus accumbens; NE, norepinephrine; NET, norepinephrine transporter; WT, wild-type.

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