Methamphetamine-Induced Neurotoxicity Alters Locomotor
Activity, Stereotypic Behavior, and Stimulated Dopamine Release
in the Rat
Tanya L. Wallace,1Gary A. Gudelsky,2and Charles V. Vorhees3
1Neuroscience Graduate Program,2College of Pharmacy, and3The Children’s Hospital Research Foundation, University
of Cincinnati, Cincinnati, Ohio 45267
The neurochemical evidence of methamphetamine (MA)-
induced toxicity to dopaminergic nerve terminals is well docu-
mented; however, the functional consequences are not clearly
defined. The present study was designed to investigate
whether MA-induced dopamine depletions affect locomotor
activity, stereotypic behavior, and/or extracellular dopamine
concentrations in the neostriatum. Male rats were treated with
a neurotoxic regimen of MA (10 mg/kg, i.p., every 2 hr for four
injections) or vehicle and tested for functional effects 1 week
later. Animals that had received the neurotoxic regimen of MA
showed a reduction in both caudate nucleus and nucleus ac-
cumbens dopamine contents of 56 and 30%, respectively.
Furthermore, MA-treated rats exhibited a significant attenuation
in spontaneous activity, as well as a significant diminution in MA
(low dose)-stimulated locomotor activity as compared to
vehicle-treated rats. However, there were no differences in the
MA (low dose)-induced increases in extracellular dopamine
concentrations in the caudate nucleus or the nucleus accum-
bens core of either group. Interestingly, the acute administration
of higher doses of MA elicited a significantly augmented ste-
reotypic response and a significantly attenuated increase in the
extracellular concentration of dopamine in the caudate nucleus
of rats treated with a neurotoxic regimen of MA as compared to
vehicle-treated animals. These data indicate that MA-induced
neurotoxicity results in abnormal dopamine-mediated behav-
iors, as well as a brain region-specific impairment in stimulated
Key words: methamphetamine; neurotoxicity; dopamine; be-
havior; in vivo microdialysis; stereotypy; locomotor activity; sen-
Methamphetamine (MA) is a psychomotor stimulant that in-
creases locomotor activity when administered at low doses and
elicits stereotypic behavior when administered at higher doses
(Kelly et al., 1975; Segal and Kuczenski, 1994). It is generally
believed that dopaminergic transmission in the nucleus accum-
bens and the caudate nucleus mediates MA-induced hyperloco-
motion and stereotypy, respectively (Creese and Iversen, 1974;
Kelly et al., 1975; Kelly and Iversen, 1976; Lucot et al., 1980).
Consistent with this idea, it is well documented that MA in-
creases the extracellular concentration of dopamine in these
brain regions, in part, by reversing the dopamine transporter and
facilitating cytoplasmic dopamine release, as well as by releasing
vesicular stores of dopamine (Liang and Rutledge, 1982; Schmidt
and Gibb, 1985; O’Dell et al., 1991; Seiden et al., 1993; Cubells et
In addition to the acute neurochemical and behavioral effects of
MA, repeated, high-dose administration of this stimulant pro-
duces long-term neurotoxicity to dopaminergic and serotonergic
nerve terminals within the neostriata, as well as to serotonergic
terminals in multiple forebrain regions, of rats, mice, monkeys,
and guinea pigs (Seiden et al., 1975; Wagner et al., 1979; Morgan
and Gibb, 1980; Ricaurte et al., 1980; O’Callaghan, 1991; O’Dell
et al., 1991). The evidence for axon terminal damage includes
long-term decreases in dopamine and serotonin contents, deple-
tion of dopamine uptake sites, and decreases in both tyrosine and
tryptophan hydroxylase activity (Hotchkiss and Gibb, 1980;
Ricaurte et al., 1980; Wagner et al., 1980; Seiden et al., 1988).
Furthermore, there is histochemical evidence of nerve terminal
damage (i.e., reactive gliosis) (Pu and Vorhees, 1993; Broening et
Although the neurochemical consequences of MA-induced
toxicity are well documented, less is known about whether func-
tional effects accompany the long-term depletion of dopamine.
Lucot et al. (1980) have reported that the administration of large
doses (i.e., 100 mg/kg) of MA over several days results in an
attenuation of subsequent MA-stimulated locomotor activity in
rats (stereotypy was not assessed). Consistent with these results,
Cass et al. (1997, 1998) have reported deficits in evoked dopa-
mine release in the caudate nucleus of MA-treated rats. However,
reduced dopamine release is not a consistent finding (Robinson
et al., 1990). More recently, Walsh and Wagner (1992) have
reported impairments in active-avoidance, and Kita et al. (1998)
have shown nocturnal hyperactivity in MA-treated rats.
It has been suggested that dopaminergic systems have substan-
tial reserve capacity and that severe nigrostriatal dopamine re-
ductions are required to reveal Parkinsonian-like symptoms
(Stricker and Zigmond, 1976). However, the aforementioned
studies (i.e., Lucot et al., 1980; Walsh and Wagner, 1992; Kita et
al., 1998) suggest that functional deficits occur at more moderate
levels of dopamine depletion. Therefore, the present study was
designed to determine whether a neurotoxic dosing regimen of
MA, followed by a 1 week recovery interval, results in changes in
spontaneous locomotor activity, as well as in stimulated hyper-
Received June 14, 1999; revised Aug. 5, 1999; accepted Aug. 9, 1999.
This work was supported by National Institute on Drug Abuse Grants DA07427
(G.A.G.) and DA06733 (C.V.V).
Correspondence should be addressed to Dr. Charles V. Vorhees, Division of
Developmental Biology, The Children’s Hospital Research Foundation, 3333 Burnet
Avenue, Cincinnati, OH 45229-3039.
Copyright © 1999 Society for Neuroscience 0270-6474/99/199141-08$05.00/0
The Journal of Neuroscience, October 15, 1999, 19(20):9141–9148
locomotion and stereotypic behavior. Furthermore, extracellular
dopamine concentrations in the nucleus accumbens and the cau-
date nucleus were measured to determine whether MA-induced
neurotoxicity results in neurochemical changes that parallel be-
MATERIALS AND METHODS
Male Sprague Dawley CD rats (225–250 gm) were obtained from Charles
Rivers Laboratories (Portage, MI) in groups of 64 and were housed two
or three per cage with food and water available ad libitum. Animals were
maintained on a 12 hr light/dark cycle for 1 week before experimental
treatment began. Rats were housed in a vivarium fully accredited by the
Association for the Assessment and Accreditation for Laboratory Ani-
mal Care. The protocol for this research was approved by the Institu-
tional Animal Care and Use Committee.
Rats were treated in their home cages and randomly assigned to receive
injections of either (?)-methamphetamine hydrochloride (Sigma, St.
Louis, MO) 10 mg/kg, intraperitoneally (expressed as the salt) every 2 hr
for a total of four injections (i.e., neurotoxic regimen of MA) or 0.9%
NaCl (vehicle). Body temperatures were monitored using a Thermistor
thermometer (Cole-Parmer Instruments, Vernon Hills, IL) throughout
the drug treatment regimen. Animals reaching a temperature of ?41.5°C
were wetted and placed in ventilated cages until temperatures dropped to
?40.0°C. These procedures were based on the known role of MA-
induced hyperthermia in the induction of neurotoxicity (Bowyer et al.,
1992, 1994), and the cooling intervention on the known lethal effects of
hyperthermic responses exceeding 42°C.
Behavioral testing. One week after the initial treatment, rats were trans-
ported to the activity testing room in sets of 12 and tested in sets of four.
Animals were placed in one of the four monitors for a 1 hr habituation
period (activity was recorded every 10 min). After this period, each rat
was removed and administered either vehicle or a dose of MA (0.5, 1.0,
2.0, 4.0, or 7.5 mg/kg, i.p.) and placed back in the monitors for another
2 hr (i.e., rats treated previously with a neurotoxic regimen of MA were
administered a subsequent injection of MA or vehicle, similarly, rats
treated previously with the vehicle were administered a subsequent
injection of the vehicle or MA; n ? 16/group). Each set of four animals
tested had one animal from each treatment and each challenge condition
represented (i.e., MA/MA, MA/vehicle, vehicle/MA, and vehicle/vehi-
cle), such that groups were balanced by group for time of day. For the
higher challenge dose experiments (i.e., 4.0 and 7.5 mg/kg MA) only the
vehicle/MA and MA/MA groups were included, because data on 48
MA/vehicle and 48 vehicle/vehicle animals from the 0.5, 1.0, and 2.0
mg/kg MA experiments had already shown no differential response of
MA treatment to a subsequent vehicle injection.
Locomotor activity. Activity chambers (40.6 ? 40.6 cm) (model rxy2z;
Accuscan, Columbus, OH) were equipped with 16-photodetector-LED
pairs in each dimension (i.e., x, y, and two z planes) spaced 2.5 cm apart
and located 2.2 cm above the floor. In addition, the floor contained four
holes (3.2 cm in diameter) located close to each corner (i.e., 6 cm from
each side wall). A set of photodetectors located 1.7 cm below the floor
and another set located 15.9 cm above the floor measured hole pokes and
rearings, respectively. Total distance was measured in centimeters trav-
eled and is defined as sequential photobeam interruptions.
Stereotypic behavior. Video cameras were mounted above each cham-
ber for scoring of stereotypic behavior. After review of the videotape,
behavior was considered stereotypic if the animal remained in a station-
ary position and exhibited repetitive movements, such as sniffing, head-
weaving, licking, or biting. The duration of stereotypic behavior in a 30
sec period was timed at 10 min intervals for 2 hr after the administration
of the subsequent injection of MA or vehicle. Results were reported
based on the mean percent of time each treatment group (i.e., MA or
vehicle) spent during each 10 min interval exhibiting stereotypic behav-
ior, as described by Segal and Kuczenski (1987).
Tissue analysis. Eight days after receiving the initial drug regimen (i.e.,
1 d after behavioral assessment), rats were killed by decapitation, and the
brains were rapidly removed. The nucleus accumbens and the caudate
nucleus were dissected from 1.0 mm coronal sections, frozen rapidly on
dry ice, and stored at ?70°C until assayed. Tissue samples were homog-
enized in 0.2 N perchloric acid. After centrifugation (16,000 ? g for 7
min.), the supernatant was injected onto a C18 reverse-phase column
(Phenomenex, Torrance, CA) connected to a Coulochem II detector
(ESA, Bedford, MA) or an LC-4B detector (BAS, West Lafayette, IN).
The mobile phase used for the analysis of dopamine and serotonin
consisted of 35 mM citric acid; 54 mM sodium acetate; 50 mg/l disodium
ethylenediamine tetraacetate, 70 mg/l octanesulfonic acid sodium salt,
100 ?l/l triethylamine, 6% acetonitrile, and 3% methanol, pH 4.2, set at
a flow rate of 0.4 ml/min. Peak heights were quantified using a Hewlett-
In vivo microdialysis. Three to five days after receiving a neurotoxic
regimen of MA or vehicle, rats (separate groups of rats than were used
for behavioral measurements) were anesthetized with an injection of a
ketamine and xylazine (87/13 mg/kg, i.m.), and a guide cannula was
implanted on the cortical surface above the nucleus accumbens core or
the caudate nucleus for in vivo microdialysis. These regions were chosen
specifically because they demonstrate the greatest MA-induced depletion
of dopamine (Morgan and Gibb, 1980; Ricaurte et al., 1980; Broening et
al., 1997). On the morning of the experiment, a concentric style dialysis
probe was inserted through the guide cannula into the nucleus accum-
bens core (anterior (A), 1.7 mm; lateral (L), ?1.4 mm; ventral (V), ?7.5
mm) or the caudate nucleus (A, 1.2 mm; L, 3.0 mm; V, ?7.0 mm)
according to the atlas of Paxinos and Watson (1986). The active portion
of the membrane was 2.0 mm for the nucleus accumbens core and 4.5 mm
for the caudate nucleus. Dulbecco’s PBS containing 1.2 mM CaCl2and 10
mM glucose was perfused through the probe at a constant rate of 2.2
?l/min via an infusion pump. After an equilibration period of 1.5 hr,
dialysis samples were collected every 30 min. At least three baseline
samples were obtained before subsequent MA administration.
HPLC analysis of dopamine. The concentration of dopamine in the
dialysate samples was determined via HPLC with electrochemical detec-
tion using the same procedure used for the analysis of tissue dopamine.
For dialysis experiments, a two-way repeated measure ANOVA was used
followed by post hoc analysis with Duncan’s test. For spontaneous loco-
motor activity, a two-treatment ? six-interval (repeated measure) split-
plot ANOVA was used to analyze the habituation phase, whereas a
two-treatment ? three-challenge ? twelve-interval (repeated measure)
split-plot ANOVA was used to analyze MA-stimulated activity. For
stereotypy, a two-treatment ? two-challenge ? twelve-interval (repeated
measure) split-plot ANOVA was used. Interactions were further ana-
lyzed by simple-effect analysis of variance if significant, after correction
for nonsphericity using the Greenhouse-Geisser epsilon factors. Individ-
ual post hoc group comparisons were made using Duncan’s multiple
MA-induced dopamine and serotonin neurotoxicity
Rats that received the neurotoxic regimen of MA (i.e., 10 mg/kg
every 2 hr for a total of four injections) showed a 56 and 30%
reduction in dopamine concentrations in the caudate nucleus and
the nucleus accumbens, respectively, 8 d after initial treatment
(Table 1). In addition, the serotonin concentrations in the caudate
nucleus and the nucleus accumbens of MA-treated rats were
reduced by 50 and 63%, respectively, as compared to vehicle-
A neurotoxic regimen of MA reduces spontaneous
locomotor activity during the habituation period
To determine whether the MA-induced loss of dopamine was
accompanied by changes in dopamine-mediated behaviors, loco-
motor activity was monitored. Analysis of the total distance
traveled during the habituation period included the rats that
received subsequent low-dose (i.e., 0.5, 1.0, 2.0 mg/kg) injections
of MA or vehicle after the habituation period. The main effect of
the treatment (F(1,228)? 30.82; p ? 0.00001) and the treatment ?
interval interaction (F(5,1140)? 9.44; p ? 0.00001) were significant
9142 J. Neurosci., October 15, 1999, 19(20):9141–9148 Wallace et al. • Functional Effects of Methamphetamine Neurotoxicity
and indicated that the spontaneous activity of rats treated with a
neurotoxic regimen of MA was significantly attenuated as com-
pared to vehicle-treated controls (Fig. 1). A posteriori group com-
parisons indicated that MA-treated animals exhibited lower ac-
tivity during the initial exploratory phases (i.e., the first 30 min)
of the habituation period than vehicle-treated rats; however, the
locomotor activity of both groups reached comparable levels
during the last 30 min of the habituation period. The fact that
both MA- and vehicle-treated animals showed similar levels of
activity at the end of the habituation period (i.e., before receiving
a subsequent injection of MA) suggests that any subsequent
MA-stimulated differences observed between the treatment
groups cannot be attributed to pre-existing activity differences.
A neurotoxic regimen of MA results in the attenuation
of subsequent MA (low dose)-induced
To determine whether receiving a neurotoxic regimen of MA
produced any changes in stimulated locomotor activity, animals
were administered a subsequent low dose of MA (i.e., 0.5, 1.0, or
2.0 mg/kg). The main effects of treatment (i.e., 10 mg/kg ? 4 of
MA or vehicle) (F(1,164)? 4.26; p ? 0.05), challenge (i.e., MA or
vehicle) (F(2,164)? 178.34; p ? 0.00001), dose (i.e., 0.5, 1.0, or 2.0
mg/kg MA) (F(2,164)? 17.89; p ? 0.00001), and interval (i.e.,
10–120 min) (F(11,1804)? 49.24; p ? 0.00001) were significant. In
addition, the challenge ? dose (F(2,164)? 18.37; p ? 0.00001),
interval ? challenge (F(11,1804)? 44.45; p ? 0.00001), interval ?
dose (F(22,1804)? 2.67; p ? 0.05), and interval ? challenge ? dose
(F(22,1804)? 3.32; p ? 0.01) interactions also were significant. No
differences were identified between MA- or vehicle-treated
groups given a subsequent injection of the vehicle.
Further analyses of the individual low doses of MA indicated
that the treatment and the treatment ? interval interactions were
significant for the 1.0 mg/kg (both p ? 0.00001) and 2.0 mg/kg
doses of MA ( p ? 0.0001 and p ? 0.05, respectively) (Fig. 2A,B).
Neither factor was significant for the 0.5 mg/kg MA dose. Group
comparisons demonstrated that the animals treated previously
with a neurotoxic regimen of MA and administered a subsequent
low dose injection of MA showed an increase in locomotor
activity, however, the increase was significantly attenuated as
compared to vehicle-treated animals administered the same low
dose of MA.
In addition to locomotor activity, horizontal activity, rearing,
and hole-poking measures also were analyzed. MA-treated ani-
mals administered a subsequent low-dose injection of MA exhib-
ited a suppressed response for all three behaviors (i.e., horizontal
activity, rearing, and hole-poking) as compared to control ani-
mals (data not shown).
A neurotoxic regimen of MA results in the
augmentation of subsequent MA (high dose)-induced
To determine whether the MA-induced depletion of dopamine
produced changes in stereotypic behavior, animals were admin-
istered a subsequent dose of MA (i.e., 4.0 or 7.5 mg/kg) that was
known to elicit stereotypy as the dominant behavior (i.e., loco-
motor activity is suppressed). In contrast to the previous results
indicating locomotor activity induced by lower doses of MA was
attenuated in rats treated with a neurotoxic regimen of MA,
stereotyped behavior elicited by higher doses of MA was signif-
icantly enhanced in these rats. At the 4.0 mg/kg dose of MA,
treatment (F(1,28)? 6.71; p ? 0.05) and treatment ? interval
(F(11,308)? 4.49; p ? 0.001) effects were significant (Fig. 3A). The
same factors were significant at the 7.5 mg/kg dose, i.e., treatment
(F(1,28)? 15.11; p ? 0.001) and treatment ? interval (F(11,308)?
3.27; p ? 0.01) (Fig. 3B). Group comparisons performed at each
interval to further analyze the interactions showed that at both
the 4.0 and 7.5 mg/kg MA doses, the rats treated previously with
a neurotoxic regimen of MA exhibited significantly augmented
stereotypy at multiple early and middle test intervals as compared
to their respective vehicle-treated controls.
A neurotoxic regimen of MA produces alterations in
the subsequent MA-induced increase of extracellular
dopamine that are dose-dependent and brain
In vivo microdialysis was used to determine whether the MA-
induced depletion of dopamine content in both the caudate
nucleus and the nucleus accumbens would result in deficits in the
extracellular concentrations of dopamine. There was no signifi-
cant difference in the basal extracellular concentration of dopa-
mine in rats treated previously with the neurotoxic regimen of
MA or vehicle in either the caudate nucleus (MA, 6.0 ? 0.8 pg/20
?l; vehicle, 6.8 ? 0.4 pg/20 ?l) or the nucleus accumbens core
(MA, 1.9 ? 0.3 pg/20 ?l; vehicle, 1.3 ? 0.2 pg/20 ?l). Extracel-
lular concentrations of dopamine increased ?400% in both the
Table 1. MA-induced reduction of dopamine and serotonin contents in
the nucleus accumbens and caudate nucleus
Vehicle-treated (n ? 16)
MA-treated (n ? 12)
Vehicle-treated (n ? 14)
MA-treated (n ? 9)
74.5 ? 4.0
52.3 ? 6.3*
10.3 ? 0.4
4.5 ? 0.9*
9.1 ? 0.4
3.4 ? 0.3*
0.44 ? 0.03
0.22 ? 0.04*
Rats were killed 8 d after treatment with MA (10 mg/kg every 2 hr for four
injections) or with the vehicle. Tissue was homogenized in 0.2 N PCA and analyzed
by HPLC-EC. Data are expressed as mean ? SEM.
*p ? 0.05 compared with appropriate vehicle-treated control (Student’s t test).
regimen of MA. MA (10 mg/kg, i.p.) or the vehicle (VEH) was admin-
istered every 2 hr for a total of four injections. After 7 d, rats were placed
in activity chambers and monitored for a 1 hr habituation period. *Indi-
cates values that differ significantly from those of the vehicle-treated
controls ( p ? 0.05).
Reduced spontaneous activity in rats treated with a neurotoxic
Wallace et al. • Functional Effects of Methamphetamine Neurotoxicity J. Neurosci., October 15, 1999, 19(20):9141–9148 9143
caudate nucleus and the nucleus accumbens core after the acute
injection of a low dose of MA (1.0 mg/kg) (Fig. 4A,B). However,
there was no significant difference in the magnitude of the in-
crease in the extracellular concentration of dopamine in either
brain region of MA- or vehicle-treated rats.
In addition, the extracellular concentration of dopamine in
both brain regions was determined after the administration of a
higher dose of MA. In rats that had previously received vehicle
injections, the administration of MA (7.5 mg/kg) produced an
increase in extracellular dopamine to ?2200% of baseline values
in both the caudate nucleus and the nucleus accumbens core (Fig.
5A,B). In rats previously treated with a neurotoxic regimen of
MA, the magnitude of the increase of extracellular dopamine in
the caudate nucleus elicited by this dose of MA was significantly
diminished (?45%) (treatment ? interval, F(8, 128)? 4.33; p ?
0.0001). However, there was no difference in the MA-induced
increase of extracellular dopamine in the nucleus accumbens core
of MA or vehicle-treated rats.
In the present study, the repeated high dose administration of
MA to rats resulted in alterations in both spontaneous and
response to a subsequent low-dose injection of MA. MA (10 mg/kg, i.p.)
or the vehicle (VEH) was administered every 2 hr for a total of four
injections. After 7 d, rats were placed in activity chambers and monitored
for a 1 hr habituation period, after which they received an injection of
MA, 1.0 mg/kg (A) or 2.0 mg/kg (B), or the vehicle. *Significantly
different from VEH/MA-treated rats ( p ? 0.05).
Reduced stimulated locomotor activity in MA-treated rats in
sponse to a subsequent high-dose injection of MA. MA (10 mg/kg, i.p.)
or the vehicle (VEH) was administered every 2 hr for a total of four
injections. After 7 d, rats were placed in activity chambers and monitored
for a 1 hr habituation period, after which they received an injection of
MA, 4.0 mg/kg (A) or 7.5 mg/kg (B), or the vehicle. *Significantly
different from VEH/MA-treated rats ( p ? 0.05).
Augmented stereotypic behavior in MA-treated rats in re-
9144 J. Neurosci., October 15, 1999, 19(20):9141–9148Wallace et al. • Functional Effects of Methamphetamine Neurotoxicity
stimulated behavior, as well as in evoked dopamine release. The
MA-induced depletion of dopamine content may be responsible,
in part, for these observations. Joyce et al. (1983) have reported
that bilateral injections of 6-hydroxydopamine into the nucleus
accumbens result in a marked decrease in spontaneous activity.
Although Joyce et al. (1983) obtained almost a 95% depletion of
dopamine in the nucleus accumbens with the 6-hydroxydopamine-
induced lesions, our results with a 30% decrease in dopamine
content in this same brain region of MA-treated rats suggest that
a nearly complete loss does not have to be obtained in order to
affect the exploratory phase of spontaneous activity.
Alternatively, the suppressed spontaneous activity of MA-
treated animals observed in the present study could be caused by
the dosing regimen of MA (i.e., 10 mg/kg of MA administered
every 2 hr for a total of four injections within a single day) that
was used rather than the depletion of dopamine. Robinson and
Camp (1987) have reported a decrease in basal locomotor activity
in rats that have received repeated, daily doses of D-amphetamine.
Whereas the dosing paradigm of Robinson and Camp (1987) was
not neurotoxic to dopamine neurons, the deficit in basal activity
that they reported for D-amphetamine-treated rats was similar to
that reported in the present study for rats that received a neuro-
toxic regimen of MA. Thus, it is not clear whether the deficit in
i.p.) or the vehicle (VEH) was administered every 2 hr for a total of four
injections. Seven days later, rats were administered a subsequent injection
of MA (1.0 mg/kg, i.p.) at time 0, and extracellular dopamine concentra-
tions were measured in either the caudate nucleus (A) or the nucleus
accumbens core (B).
MA (low dose)-stimulated dopamine release. MA (10 mg/kg,
mg/kg, i.p.) or the vehicle (VEH) was administered every 2 hr for a
total of four injections. Seven days later, rats were administered a
subsequent injection of MA (7.5 mg/kg, i.p.) at time 0, and extracel-
lular dopamine concentrations were measured in either the caudate
nucleus (A) or the nucleus accumbens core (B). *Significantly different
from VEH/MA rats ( p ? 0.05).
MA (high dose)-stimulated dopamine release. MA (10
Wallace et al. • Functional Effects of Methamphetamine NeurotoxicityJ. Neurosci., October 15, 1999, 19(20):9141–9148 9145
spontaneous activity of MA-treated rats observed presently is the
result of dopamine depletion or is a compensatory response to
repeated dopaminergic stimulation.
The administration of a low dose of MA (1.0 or 2.0 mg/kg, i.p.)
resulted in significant deficits in stimulated locomotor activity in
MA-treated rats as compared to vehicle-treated controls. The
attenuated locomotor response seen in MA-treated rats was not
caused by the emergence of stereotypic behavior as determined
by videotape scoring. Our data are consistent with results ob-
tained by Lucot et al. (1980) for the 1.0 mg/kg dose of MA,
although these investigators reported no difference in the loco-
motor activity of MA- and vehicle-treated rats in response to a 2.0
mg/kg dose of MA. However, Lucot et al. (1980) did not monitor
stereotypic behavior even though locomotion was highest at the
1.0 mg/kg dose, reduced at 2.0 mg/kg, and absent at 4.0 mg/kg.
Thus, it is possible in the Lucot et al. (1980) study that the
animals became less hyperactive and more stereotypic at the 2.0
and 4.0 mg/kg doses of MA.
In the present study, it was hypothesized that the observed
deficits in stimulated locomotor activity of rats treated with a
neurotoxic regimen of MA may be caused by the loss of dopa-
mine. However, no significant differences in the extracellular
concentration of dopamine were observed between MA- and
vehicle-treated rats in either the caudate nucleus or the nucleus
accumbens core before or after a subsequent injection of MA (1.0
mg/kg). Thus, there appeared to be no correlation between
MA-induced dopamine release and the observed behavioral ef-
fects. Such a lack of correlation has been reported several times in
animals repeatedly exposed to D-amphetamine and later tested
for functional changes (Callaway et al., 1989; Kuczenski and
Segal, 1989; Kuczenski et al., 1991; Segal and Kuczenski, 1992).
One explanation for the lack of a deficit observed in the present
study may be attributable to presynaptic compensatory changes
that occur in the remaining dopamine neurons. For example,
6-hydroxydopamine-induced lesions in the striatum result in an
increase in the amount of dopamine efflux per remaining nerve
terminal (Snyder et al., 1986; Stachowiak et al., 1987; Zigmond et
al., 1989). Therefore, residual dopamine nerve terminals may be
able to compensate for the MA-induced loss of dopamine. Alter-
natively, the MA-induced decrease of high-affinity dopamine
reuptake sites may allow dopamine to remain in the synapse
longer and to diffuse to more distant sites, thereby maintaining
neurochemical function (Wagner et al., 1980; Doucet et al., 1986;
Kelly and Wightman, 1987).
Although the amount of extracellular dopamine measured may
remain unchanged in MA- and vehicle-treated animals, the ob-
served deficit in locomotor activity may still be evident. For
example, a decrease in dopamine receptor binding has been
demonstrated in the caudate putamen after repeated high-dose
injections of MA without any alteration in receptor affinity
(Schmidt et al., 1985; McCabe et al., 1987), although this is not a
consistent finding (Robinson and Becker, 1986). Based on these
data, a decrease in binding sites could result in deficits in
dopamine-mediated functions, e.g., locomotor activity.
Although MA-treated rats demonstrated deficits in locomotor
activity when injected subsequently with a low dose of MA (i.e.,
1.0 or 2.0 mg/kg), these rats exhibited an augmentation in ste-
reotypic behavior when given higher doses of MA (4.0 or 7.5
mg/kg). This illustrates the utility of pharmacological challenges
in unmasking underlying functional changes. Moreover, the en-
hanced behavioral response (i.e., quicker onset and greater inten-
sity of stereotypy) exhibited by rats treated with a neurotoxic
regimen of MA is similar to results observed in animals that have
become sensitized to D-amphetamine (Segal and Kuczenski,
1994) and is noteworthy in light of the concomitant attenuation in
the MA-induced increase of the extracellular dopamine concen-
tration. Although an increase in stimulated dopamine release has
been reported in amphetamine-sensitized rats, there are several
reports indicating that animals exhibiting sensitization have di-
minished dopamine release after stimulation (Kuczenski and
Segal, 1988, 1989; Segal and Kuczenski, 1992). Therefore, these
results suggest that the neurochemical and behavioral responses
are not necessarily correlated.
In addition, whereas MA-treated rats demonstrated a signifi-
cant reduction in the MA (7.5 mg/kg)-induced increase of the
extracellular concentration of dopamine in the caudate nucleus as
compared to vehicle-treated rats, an attenuated response was not
observed in the nucleus accumbens core. This result may be
attributed, in part, to a greater loss of dopamine in the caudate
nucleus that results in the activation of different compensatory
mechanisms (e.g., upregulation of postsynaptic receptors), which
does not occur in the nucleus accumbens. Moreover, the differing
circuitry of the two brain regions also may contribute to the
observed consequential differences of MA-induced neurotoxicity.
The seemingly paradoxical findings observed in MA-treated
rats in the present study [i.e., augmented stereotypy with concom-
itant reduction in the MA (7.5 mg/kg)-stimulated increase in the
extracellular concentration of dopamine in the caudate] suggests
that presynaptic mechanisms do not account for the enhanced
stereotypy. Alternatively, the augmented response may be caused
by a postsynaptic upregulation of dopamine receptors in the
caudate nucleus. Although, it has been suggested that for an
upregulation of receptors to occur within the dopaminergic sys-
tem, damage to caudate dopamine neurons has to be nearly
complete, i.e., ?90% (Mishra et al., 1974; Stricker and Zigmond,
1976; Creese et al., 1977; Graham et al., 1990; Schwarting and
Huston, 1997). However, increases in dopamine receptor sensi-
tivity have been shown to occur in response to the repeated
exposure to D-amphetamine (Robinson and Becker, 1986).
In the present study, it is difficult to determine whether the
augmented behavioral response demonstrated by MA-treated
rats was caused by the repeated exposure to MA (i.e., sensitiza-
tion) or by the depletion of dopamine from nerve terminals.
Certainly the temporal pattern of stimulant administration is an
important factor in the development of behavioral augmentation,
although a great deal of variation occurs in the dosing regimens
with little difference in the production of sensitization (Segal and
Geyer, 1985; Paulson et al., 1991). For example, one or two daily
injections of D-amphetamine for 2 weeks or a single injection of
D-amphetamine have both been shown to elicit a sensitized be-
havioral response (Browne and Segal, 1977; Robinson and
In summary, the treatment of rats with a neurotoxic regimen of
MA results in a depletion of dopamine and serotonin in the
caudate nucleus and the nucleus accumbens that is accompanied
by a reduction in spontaneous locomotor activity, low-dose MA-
induced locomotion, and augmented high-dose MA-induced ste-
reotypy. The MA-induced depletion of dopamine or the repeated
administration of MA may be responsible for the decrease in
stimulated dopamine release and the development of augmented
stereotypic responses. The present results demonstrate that MA-
9146 J. Neurosci., October 15, 1999, 19(20):9141–9148 Wallace et al. • Functional Effects of Methamphetamine Neurotoxicity
induced neurotoxicity alters dopamine-mediated function and
may serve as a useful model of functional consequences of
moderate dopamine depletion.
Bowyer JF, Tank AW, Newport GD, Slikker Jr W, Ali SF, Holson RR
(1992) The influence of environmental temperature on the transient
effects of methamphetamine on dopamine levels and dopamine release
in rat striatum. J Pharmacol Exp Ther 260:817–824.
Bowyer JF, Davies DL, Schmued L, Broening HW, Newport GD, Slikker
Jr W, Holson RR (1994) Further studies of the role of hyperthermia in
methamphetamine neurotoxicity. J Pharmacol Exp Ther 268:1571–1580.
Broening HW, Pu C, Vorhees CV (1997) Methamphetamine selectively
damages dopaminergic innervation to the nucleus accumbens core
while sparing the shell. Synapse 27:153–160.
Browne RG, Segal DS (1977) Metabolic and experiential factors in the
behavioral response to repeated amphetamine. Pharmacol Biochem
Callaway CW, Kuczenski R, Segal DS (1989) Reserpine enhances am-
phetamine stereotypies without increasing amphetamine-induced
changes in striatal dialysate dopamine. Brain Res 505:83–90.
Cass WA (1997) Decreases in evoked overflow of dopamine in rat stri-
atum after neurotoxic doses of methamphetamine. J Pharmacol Exp
Cass WA, Manning MW, Dugan MT (1998) Effects of neurotoxic doses
of methamphetamine on potassium and amphetamine evoked overflow
of dopamine in the striatum of awake rats. Neurosci Lett 248:175–178.
Creese I, Iversen SD (1974) A role of forebrain dopamine systems in
amphetamine induced stereotyped behaviour in the rat. Psychophar-
Creese I, Burt DR, Snyder SH (1977) Dopamine receptor binding en-
hancement accompanies lesion-induced behavioral sensitivity. Science
Cubells JF, Rayport S, Rajendran G, Sulzer D (1994) Methamphet-
amine neurotoxicity involves vacuolation of endocytic organelles and
Doucet G, Descarries L, Garcia S (1986) Quantification of the dopamine
innervation in adult rat neostriatum. Neuroscience 19:427–445.
Graham WC, Crossman AR, Woodruff GN (1990) Autoradiographic
studies in animal models of hemi-parkinsonism reveal dopamine D2but
not D1receptor supersensitivity I: 6-OHDA lesions of ascending mes-
encephalic dopaminergic pathways in the rat. Brain Res 514:93–102.
Hotchkiss AJ, Gibb JW (1980) Long-term effects of multiple doses of
methamphetamine on tryptophan hydroxylase and tyrosine hydroxylase
activity in rat brain. J Pharmacol Exp Ther 214:257–262.
Joyce EM, Stinus L, Iversen SD (1983) Effect of injections of 6-OHDA
into either nucleus accumbens septi or frontal cortex on spontaneous
and drug-induced activity. Neuropharmacology 22:1141–1145.
Kelly PH, Iversen SD (1976) Selective 6OHDA-induced destruction of
mesolimbic dopamine neurons: abolition of psychostimulant-induced
locomotor activity in rats. Eur J Pharmacol 40:45–56.
Kelly RS, Wightman RM (1987) Detection of dopamine overflow and
diffusion with voltammetry in slices of rat brain. Brain Res 423:79–87.
Kelly PH, Seviour PW, Iversen SD (1975) Amphetamine and apomor-
phine responses in the rat following 6-OHDA lesions of the nucleus
accumbens septi and corpus striatum. Brain Res 94:507.
Kita T, Takahashi M, Wagner GC, Kubo K, Nakashima T, (1998)
Methamphetamine-induced changes in activity and water intake during
light and dark cycles in rats. Prog Neuropsychopharmacol Biol Psychi-
Kuczenski R, Segal DS (1988) Psychomotor stimulant-induced sensitiza-
tion: behavioral and neurochemical correlates. In: Sensitization in the
nervous system (Kalivas P, Barnes T, eds), pp 175–205. Caldwell, NJ:
Kuczenski R, Segal DS (1989) Concomitant characterization of behav-
ioral and striatal neurotransmitter response to amphetamine using in
vivo microdialysis. J Neurosci 9:2051–2065.
Kuczenski R, Segal DS, Aizenstein ML (1991) Amphetamine, cocaine
and fencamfamine: relationship between locomotor and stereotypy
response profiles and caudate and accumbens dopamine dynamics.
J Neurosci 11:2703–2712.
Liang N, Rutledge C (1982) Evidence for carrier-mediated efflux of
dopamine from corpus striatum. Biochem Pharmacol 302:479–484.
Lucot JB, Wagner GC, Schuster CR, Seiden LS (1980) The effects of
dopaminergic agents on the locomotor activity of rats after high doses
of methylamphetamine. Pharmacol Biochem Behav 13:409–413.
McCabeRT, Hanson GR,Dawson
Methamphetamine-induced reduction in D1and D2dopamine recep-
tors as evidenced by autoradiography: comparison with tyrosine hy-
droxylase activity. Neuroscience 23:253–261.
Mishra RK, Gardner EL, Katzman R, Makman MH (1974) Enhance-
ment of dopamine-stimulated adenylate cyclase activity in rat caudate
after lesions in substantia nigra evidence for denervation supersensi-
tivity. Proc Natl Acad Sci USA 71:2883–2887.
Morgan ME, Gibb JW (1980) Short-term and long-term effects of meth-
amphetamine on biogenic amine metabolism in extrastriatal dopami-
nergic nuclei. Neuropharmacology 19:989–995.
O’Callaghan (1991) Neurotoxicity profiles of substituted amphetamines
in the C57BL/6J mouse. J Pharmacol Exp Ther 270:197–206.
O’Dell SJ, Weihmuller FB, Marshall JF (1991) Multiple methamphet-
amine injections induce marked increases in extracellular striatal do-
pamine which correlates with subsequent neurotoxicity. Brain Res
Paulson PE, Camp DM, Robinson TE (1991) Time course of transient
behavioral depression and persistent behavioral sensitization in rela-
tion to regional brain monoamine concentrations during amphetamine
withdrawal in rats. Psychopharmacology 103:480–492.
Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates.
San Diego: Academic.
PuC,Vorhees CV (1993) Developmental
methamphetamine-induced depletion of dopaminergic terminals and
astrocyte reaction in rat striatum. Dev Brain Res 72:325–328.
Ricaurte GA, Schuster CR, Seiden LS (1980) Long-term effects of re-
peated methylamphetamine administration on dopamine and serotonin
neurons in the rat brain: a regional study. Brain Res 193:153–163.
Robinson TE, Becker JB (1986) Enduring changes in brain and behavior
produced by chronic amphetamine administration: a review and eval-
uation of animal models of amphetamine psychosis. Brain Res Rev
Robinson TE, Camp DM (1987) Long-lasting effects of escalating doses
of D-amphetamine on brain monoamines, amphetamine-induced ste-
reotyped behavior and spontaneous nocturnal locomotion. Pharmacol
Biochem Behav 26:821–827.
Robinson TE, Yew J, Paulson PE, Camp DM (1990) The long-term
effects of neurotoxic doses of methamphetamine on the extracellular
concentration of dopamine measured with microdialysis in striatum.
Neurosci Lett 110:193–198.
Schmidt CJ, Gibb JW (1985) Role of dopamine uptake carrier in the
neurochemical response to methamphetamine and effects of amfonelic
acid. Eur J Pharmacol 109:73–80.
Schmidt CJ, Gehlert DR, Peat MA, Sonsalla PK, Hanson GR, Wamsley
JK, Gibb JW (1985) Studies on the mechanism of tolerance to meth-
amphetamine. Brain Res 343:305–313.
Schwarting RKW, Huston JP (1997) Behavioral and neurochemical dy-
namics of neurotoxic meso-striatal dopamine lesions. Neurotoxicology
Segal DS, Geyer MA (1985) Animal models of psychopathology. In:
Psychiatry (Cavenar JO, ed), pp 1–18. Philadelphia: Lippincott.
Segal DS, Kuczenski R (1987) Individual differences in responsiveness
to single and repeated amphetamine administration: behavioral char-
acteristics and neurochemical correlates. J Pharmacol Exp Ther
Segal DS, Kuczenski R (1992) In vivo microdialysis reveals a diminished
amphetamine-induced dopamine response corresponding to behavioral
sensitization produced by repeated amphetamine pretreatment. Brain
Segal DS, Kuczenski R (1994) Behavioral Pharmacology of Amphet-
amine. In: Amphetamine and its analogs: psychopharmacology, toxi-
cology and abuse (Cho AK, Segal DS, eds), pp 115–150. San Diego:
Seiden LS, Fischman MW, Schuster CR (1975) Long-term metham-
phetamine induced changes in brain catecholamines in tolerant rhesus
monkeys. Drug Alcohol Depend 1:215–219.
Seiden LS, Commins D, Vosmer G, Axt L, Marek G (1988) Neurotox-
icity in dopamine and serotonin terminal fields: a regional analysis in
TM, Gibb JW (1987)
Wallace et al. • Functional Effects of Methamphetamine Neurotoxicity J. Neurosci., October 15, 1999, 19(20):9141–9148 9147
Seiden LS, Sabol KE, Ricaurte GA (1993) Amphetamine: effects on
catecholamine systems and behavior. Annu Rev Pharmacol Toxicol
Snyder GL, Stachowiak M, Keller Jr RW, Stricker EM, Zigmond MJ
(1986) Release of endogenous DA and DOPAC from striatal slices
after DA-depleting lesions: effects of stimulation frequency and DA
synthesis inhibition. Soc Neurosci Abstr 12:136.
Stachowiak MK, Keller RW, Jr, Stricker EM, Zigmond MJ (1987) In-
creased dopamine efflux from striatal slices during development and
after nigrostriatal bundle damage. J Neurosci 7:1648–1654.
Stricker ED, Zigmond MJ (1976) Recovery of function following dam-
age to central catecholamine containing neurons; a neurochemical
model of the lateral hypothalamic syndrome. In: Progress in psychobi-
and mesolimbic projections.AnnNY AcadSci ology and physiological psychology (Sprague JM, Epstein AN, eds), pp
121–189. New York: Academic.
Wagner GC, Seiden LS, Schuster CR (1979) Methamphetamine induced
changes in brain catecholamines in rats and guinea pigs. Drug Alcohol
Wagner GC, Ricaurte GA, Seiden LS, Schuster CR, Miller RJ, Westley J
(1980) Long-lasting depletions of striatal dopamine and loss of dopa-
mine uptake sites following repeated administration of methamphet-
amine. Brain Res 181:151–160.
Walsh SL, Wagner GC (1992) Motor impairments after methamphet-
amine-induced neurotoxicity in the rat. J Pharmacol Exp Ther
Zigmond MJ, Berger TW, Grace AA, Stricker ED (1989) Compensa-
tory responses to nigrostriatal bundle injury. Mol Chem Neuropathol
9148 J. Neurosci., October 15, 1999, 19(20):9141–9148Wallace et al. • Functional Effects of Methamphetamine Neurotoxicity