Methamphetamine-induced neurotoxicity alters locomotor activity, stereotypic behavior, and stimulated dopamine release in the rat.
ABSTRACT The neurochemical evidence of methamphetamine (MA)-induced toxicity to dopaminergic nerve terminals is well documented; 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 accumbens 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 accumbens core of either group. Interestingly, the acute administration of higher doses of MA elicited a significantly augmented stereotypic 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 behaviors, as well as a brain region-specific impairment in stimulated dopamine release.
- SourceAvailable from: Baoying Zheng[Show abstract] [Hide abstract]
ABSTRACT: Our previous studies have shown that neonatal exposure to lipopolysaccharide (LPS) resulted in motor dysfunction and dopaminergic neuronal injury in the juvenile rat brain. To further examine whether neonatal LPS exposure has persisting effects in adult rats, motor behaviors were examined from postnatal day 7 (P7) to P70 and brain injury was determined in P70 rats following an intracerebral injection of LPS (1 mg/kg) in P5 Sprague-Dawley male rats. Although neonatal LPS exposure resulted in hyperactivity in locomotion and stereotyped tasks, and other disturbances of motor behaviors, the impaired motor functions were spontaneously recovered by P70. On the other hand, neonatal LPS-induced injury to the dopaminergic system such as the loss of dendrites and reduced tyrosine hydroxylase immunoreactivity in the substantia nigra persisted in P70 rats. Neonatal LPS exposure also resulted in sustained inflammatory responses in the P70 rat brain, as indicated by an increased number of activated microglia and elevation of interleukin-1β and interleukin-6 content in the rat brain. In addition, when challenged with methamphetamine (METH, 0.5 mg/kg) subcutaneously, rats with neonatal LPS exposure had significantly increased responses in METH-induced locomotion and stereotypy behaviors as compared to those without LPS exposure. These results indicate that although neonatal LPS-induced neurobehavioral impairment is spontaneously recoverable, the LPS exposure-induced persistent injury to the dopaminergic system and the chronic inflammation may represent the existence of silent neurotoxicity. Our data further suggest that the compromised dendritic mitochondrial function might contribute, at least partially, to the silent neurotoxicity.Brain Behavior and Immunity 09/2010; 25(2):286-97. · 5.61 Impact Factor
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ABSTRACT: Chronic intake of methamphetamine (METH) causes tolerance to its behavioral and subjective effects. To better mimic human patterns of drug abuse, the present study used a rodent model that took into account various facets of human drug administration and measured METH-induced effects on brain monoamine levels. Adult male Sprague-Dawley rats were injected with METH or saline according to an escalating dose schedule for 2 weeks. This was followed by a challenge regimen of either saline or one of two doses of METH (3 x 10 mg/kg every 2 h or 6 x 5 mg/kg given every hour, both given within a single day). Both challenge doses of METH caused significant degrees of depletion of dopamine in the striatum and norepinephrine and serotonin in the striatum, cortex, and hippocampus. Animals pre-treated with METH showed significant attenuation of METH-induced striatal dopamine depletion but not consistent attenuation of norepinephrine and serotonin depletion. Unexpectedly, METH pre-treated animals that received the 3 x 10 mg/kg challenge showed less increases in tympanic temperatures than saline pre-treated rats whereas METH pre-treated animals that received the 6 x 5 mg/kg METH challenge showed comparable increases in temperatures to saline pre-treated rats. Therefore, pre-treatment-induced partial protection against monoamine depletion is probably not because of attenuated METH-induced hyperthermia in those rats.Journal of Neurochemistry 07/2008; 105(5):1873-85. · 3.97 Impact Factor
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ABSTRACT: Methamphetamine-induced neurotoxicity results in long-lasting depletions of monoamines and changes in basal ganglia function. We previously reported that rats with methamphetamine-induced neurotoxicity no longer engage dorsomedial striatum during a response reversal-learning task, as their performance is insensitive to acute disruption of dorsomedial striatal function by local infusion of an N-methyl-D-aspartate receptor antagonist or an antisense oligonucleotide against the activity-regulated cytoskeleton-associated (Arc) gene. However, methamphetamine-pretreated rats perform the task as well as controls. Therefore, we hypothesized that the neural circuitry involved in the learning had changed in methamphetamine-pretreated rats. To test this hypothesis, rats were pretreated with a neurotoxic regimen of methamphetamine or with saline. Three to five weeks later, rats were trained on the reversal-learning task and in situ hybridization for Arc was performed. A significant correlation between Arc expression and performance on the task was found in nucleus accumbens shell of methamphetamine-, but not saline-, pretreated rats. Consistent with the idea that the correlation between Arc expression in a brain region and behavioral performance implicates that brain region in the learning, infusion of an antisense oligonucleotide against Arc into the shell impaired consolidation of reversal learning in methamphetamine-, but not saline-, pretreated rats. These findings provide novel evidence suggesting that methamphetamine-induced neurotoxicity leads to a shift from dorsal to ventral striatal involvement in the reversal-learning task. Such reorganization of neural circuitry underlying learning and memory processes may contribute to impaired cognitive function in individuals with methamphetamine-induced neurotoxicity or others with striatal dopamine loss, such as patients with Parkinson's disease.Neuropsychopharmacology accepted article preview online, 22 October 2013. doi:10.1038/npp.2013.296.Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 10/2013; · 8.68 Impact Factor
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
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