Department of Neurobiology, Pharmacology, and Physiology,
The University of Chicago, Chicago, Illinois (A.H., N.B., R.L., L.W.);
and Department of Applied Mathematics, Illinois Institute of
Technology, Chicago, Illinois (B.H.)
Methamphetamine use by females of child-bearing age has become a major
public health concern in terms of the long-term risk to the exposed
fetus. We examined the possibility of enhanced adult neurotoxic
potential of the drug in offspring that had been exposed to
methamphetamine in utero during gestational days 7 to 18. While basal
levels of monoamines were not affected by prenatal exposure to
methamphetamine, we observed an enhanced neurotoxicity in adult male
offspring following drug challenge with effects localized primarily to
the dopaminergic nigrostriatal projection. This was evidenced by
greater methamphetamine-induced reductions of dopaminergic markers in
the striatum [dopamine (DA), dihydroxyphenylacetic acid, homovanillic
acid (HVA), and 3-methoxytyramine (3-MT)] and ventral brainstem (DA)
of prenatal methamphetamine-treated males compared with saline-treated
animals. Some effects of prenatal methamphetamine exposure were
observed in female offspring, but these were limited to striatal levels
of 3-MT and HVA. Differential gender sensitivity to the neurotoxic
effect of methamphetamine was shown to be correlated with hyperthermic
response. Hyperthermic effects, however, do not account for the
increased susceptibility of prenatal methamphetamine-treated males to
drug-induced striatal DA neurotoxicity since methamphetamine challenge
did not evoke a significantly greater hyperthermic response in these
animals compared with prenatal saline-treated males. The findings raise the concern that male methamphetamine abusers may be at risk for an
enhanced neurotoxic risk if they were exposed to the drug in utero.
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Introduction |
The
psychomotor stimulant methamphetamine has become an increasingly
commonly used drug of abuse. Almost a decade ago, it was estimated that
approximately one-third of methamphetamine users were females of
child-bearing age (Burchfield et al., 1991
). Given the expanded use of
this psychomotor stimulant over the past 10 years in the United States,
particularly in some midwestern states (Marwick, 2000), there is
reasonable concern, but little concrete information, regarding the
long-term health consequences for such exposed individuals.
Methamphetamine is a well established neurotoxic agent producing
degeneration of monoaminergic nerve endings (Seiden et al., 1993).
These effects are quite persistent and the use of the drug is
associated with a number of serious health problems, including
cardiovascular and pulmonary complications and possible brain damage
manifested as psychotic episodes (for review, see Heller et al.,
2000b).
Experimental studies examining the effect of prenatal exposure to
methamphetamine are limited and have produced variable results. When
methamphetamine is administered throughout gestation, elevations in
serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA),
and norepinephrine are observed in several forebrain regions of
9-month-old rat offspring following drug treatment (Tonge, 1973a,b).
Others, however, have reported a reduction in rat cortical DA
concentration as well as 5-HT receptor binding in 5-week postnatal offspring (Sato and Fujiwara, 1986). To add to the complexity, studies
of other neurochemical markers have indicated that low doses (2-5
mg/kg) of methamphetamine, administered throughout pregnancy, result in
a reduction in DA and 5-HT uptake sites in young adult progeny, while
higher doses (10 mg/kg) of the drug, in contrast, produce an increase
in DA and 5-HT uptake sites (Weissman and Caldecott-Hazard, 1993). When
pregnant dams are treated with methamphetamine for a more limited
period, from gestational day 13 to 20, their adult offspring exhibit an
attenuated elevation in plasma renin following exposure to
parachloroamphetamine despite having normal levels of brain 5-HT uptake
sites and 5-HT receptors (Cabrera et al., 1993). The differences
observed in these studies may well be related to the use of different
dosage regimens, periods of gestational exposure to this psychomotor
stimulant, strain of rats used, or postnatal ages at which offspring
are assayed.
It is apparent that the experimental results of prenatal
methamphetamine exposure on basal neurochemical indices in the
offspring can vary. Nevertheless, it is essential that studies on
neurotransmitter function be expanded, given the long-term public
health consequences of prenatal exposure to the drug. We have recently
demonstrated that gestational exposure to methamphetamine results in an
enhanced methamphetamine-evoked release of DA from striatal slices of
adult offspring (Heller et al., 2000a). Since the release of DA is
involved in methamphetamine neurotoxicity (for review, see Seiden and
Sabol, 1996), this suggested that adult animals exposed to the drug in utero might, in fact, be more susceptible to the neurotoxic effects of
the drug. The present study was undertaken to test that possibility and
the results suggest that, at least for male methamphetamine abusers,
there may be enhanced neurotoxic risk to the dopaminergic nigrostriatal
projection from in utero exposure to the drug.
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Materials and Methods |
Animal Housing and Administration of Methamphetamine
Pregnant female C57BL/6 mice, approximately 11 weeks old, were
injected subcutaneously twice daily (8:00 AM and 4:00 PM) with 40 mg/kg
of (+)-methamphetamine hydrochloride (obtained from the National
Institute on Drug Abuse, Rockville, MD) or saline from gestational day
7 through 18 (presence of vaginal plug = gestational day 1). The
dams were housed individually and maintained in an environment with
ambient temperature between 22-24.5°C with a 12:12-h light/dark
cycle and ad libitum access to food and water. Methamphetamine- and
saline-treated dams were killed by cervical dislocation 21 days
postparturition, after weaning. The brains were removed and the
striatum was dissected free-hand for analysis of monoamine levels.
There were 16 litters born to methamphetamine-treated dams and 14 litters born to saline-treated dams. The average litter size was
similar for both treatment groups (n = 7 ± 2 pups
per litter) and the pups remained with the dam until weaning. Although offspring were not cross-fostered to noninjected dams, the
administration of methamphetamine daily to the pregnant dams (2 × 40 mg/kg) from gestational day 7 to 18 had no apparent effect on
nutritional status of the pups as estimated by body weight measured at
postnatal day 14. The mean ± S.D. of the body weights of
methamphetamine-exposed male (6.9 ± 0.6 g, n = 50) and female (6.8 ± 0.7 g, n = 53)
progeny were not significantly different from saline-exposed male
(7.2 ± 0.8 g, n = 65) and female (6.7 ± 0.7 g, n = 50) pups. At 21 days of postnatal
age, male and female offspring were group housed separately at up to
five animals per cage and had free access to food and water. During
methamphetamine exposure, individual postnatal male and female
offspring were housed separately and maintained at an ambient
temperature of 22.2 ± 0.5°C.
All procedures were approved by the University of Chicago's
Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals.
Postnatal Experimental Procedures
Methamphetamine-Induced Hyperthermia and Neurotoxicity.
At
11 weeks of age, offspring from methamphetamine- and saline-treated
dams were evaluated for susceptibility to methamphetamine-induced neurotoxicity over a range of drug doses. For each experimental treatment, four male and four female pups were selected from different litters of the prenatal methamphetamine and prenatal saline groups. The
offspring received two subcutaneous injections (spaced 2 h apart)
of either saline or 5, 10, 15, or 20 mg/kg of (+)-methamphetamine hydrochloride. Rectal temperatures were measured hourly using a small
probe (TMP1530; Kent Scientific, Litchfield, CT) and Barnant thermocouple digital thermometer (TMP1021; Kent Scientific) beginning just before the first injection of methamphetamine or saline and continuing for at least 4 h after the second injection.
All mice were subsequently killed by cervical dislocation 7 days after
drug injection and their brains dissected free-hand on an ice-chilled,
foil-covered metal sheet into five regions: the striatum, cortex,
ventral brainstem, dorsal brainstem, and cerebellum. Briefly, a
transverse cut was made at the posterior edge of the olfactory bulbs.
The brain was then placed on its dorsal surface and a transverse cut
was made just posterior to the optic chiasm. The striatum was obtained
from the piece of brain tissue anterior to the optic chiasm
transection. The tissue was placed on its anterior aspect and the
dorsal cortex removed. The tissue piece was repositioned on its ventral
surface and a cut was made through the lateral ventricles to remove the
septum. The cortex on the ventral and lateral aspects were trimmed from the remaining piece of tissue to obtain the striatum. The remaining cortex tissue lying dorsal to the posterior piece of brainstem was
dissected from the brainstem and added to the anterior cortical areas
obtained in the dissection of striatum. The cerebellum was removed from
the brainstem. For the areas defined as the dorsal and ventral
brainstem, a transverse cut was made at the level of the mammillary
bodies, removing the hypothalamus from the remaining brainstem. Another
transverse cut was made posterior to the inferior colliculus to obtain
a subsection of brainstem. This brainstem piece was bisected coronally
at its midpoint to obtain two slabs of tissue, which were then divided
into dorsal and ventral portions by horizontal cuts just below the
third ventricle in the anterior piece of tissue and at the medial
longitudinal fasciculus in the posterior piece of tissue. The dorsal
and ventral pieces of brainstem were separately pooled. The tissue
samples were flash frozen in liquid nitrogen and stored at 
80°C
until monoamine levels were analyzed by high-performance liquid
chromatography (HPLC).
In a separate experiment on the correlation between absolute body
temperature and the neurotoxic effect of methamphetamine, normal
11-week-old C57BL/6 male mice that did not undergo prenatal treatment
were given two subcutaneous injections (2 h apart) of 20 mg/kg
(+)-methamphetamine hydrochloride or saline. Rectal temperatures were
measured hourly as described above. These animals were killed 7 days
following drug injection and their striata analyzed for DA content.
Comparison of Methamphetamine-Induced Neurotoxicity and Dopamine
Transporter (DAT) Binding.
Fifteen-week-old male offspring from
saline-treated dams were used for examination of
methamphetamine-induced depletion of DA and DAT binding. Five males
selected from four different litters were treated with saline and eight
males from seven litters were treated with methamphetamine. In those
two cases where two animals were selected from a single litter, the
values for DA level and DAT density and affinity were averaged and
treated as a single determination. Mice received two subcutaneous
injections (spaced 2 h apart) of saline or 10 mg/kg of
(+)-methamphetamine hydrochloride. The animals were killed 7 days
postinjection and their brains removed and the striatum dissected. The
tissue was frozen in liquid nitrogen and stored at 80°C. The right
striatum of each brain was used for monoamine analysis by HPLC and the
left striatum was used for measurement of DAT density and affinity.
Biochemical Procedures
Monoamine Analysis.
Endogenous monoamine and metabolite
content was analyzed using HPLC with electrochemical detection. The
HPLC system consisted of a Milton Roy minipump (model 396), an LC4C
amperometric detector with a glassy carbon electrode (Bioanalytical
Systems, Inc., West Lafayette, IN) maintained at a potential of
0.775 V versus an Ag/AgCl reference electrode with a sensitivity of 5 nA/V, and a 5-µm Primesphere C18 column
(Phenomenex, Torrance, CA). The HPLC mobile phase for analysis
of monoamines consisted of 0.04 M sodium acetate, 0.1 mM EDTA (disodium
salt, dihydrate), 0.2 mM octyl sodium sulfate, 1.0% methanol, and 6.0 to 7.5% acetonitrile (depending on brain region) at a pH of 3.74 to
3.75 adjusted with glacial acetic acid. The mobile phase was filtered
and degassed before use. Brain tissues were sonicated in 0.5 N
perchloric acid and centrifuged at 25,000g. The supernatant
was filtered through a 0.2-µm nylon filter and an aliquot was
injected onto the HPLC column. Protein content of the pellet was
determined spectrophotometrically using bicinchoninic acid (Smith et
al., 1985).
DAT Binding.
Measurement of the density
(Bmax) of DATs in striatum was
performed by saturation studies using
125I-RTI-121 (PerkinElmer Life Science Products,
Boston, MA). Assays were performed according to a modification
of the procedure of Boja et al. (1995). Briefly, striata from saline-
and drug-treated animals were suspended in ice-cold incubation buffer
(consisting of 136.9 mM NaCl, 2.7 mM KCl, 12 mM
Na2HPO4, 1.7 mM
KH2PO4, pH 7.4) and
homogenized using a Brinkman Polytron (setting 5 for 20 s) and
then centrifuged twice at 40,000g for 10 min. Striatal homogenates were suspended in incubation buffer at a final tissue concentration of 5.0 mg of original wet weight per milliliter and added
to incubation buffer consisting of labeled
125I-RTI-121 (20 pM final concentration) and
unlabeled RTI-121 (0.1 nM-30 nM) to make a total volume of 1 ml.
Incubation continued for 60 min and was terminated by filtration
through GF/B filters previously soaked in 0.05% polyethylenimine.
Filters were washed with 3 × 5 ml of ice-cold incubation buffer
and radioactivity was measured using an ICN 4/880 Plus gamma counter
(78% efficiency). Nonspecific binding was defined using (±)-cocaine
(30 µM final concentration). Determination of affinity
(Kd) and density of uptake sites
(Bmax) were determined by Scatchard
analysis using the nonlinear iterative curve-fitting program LIGAND
(Munson and Rodbard, 1980).
Statistical Analysis
To determine whether methamphetamine challenge had an effect
with respect to dose, regression analyses of monoamine and metabolite levels were conducted over the entire drug dose range for all brain areas.
To determine whether there was an effect of prenatal methamphetamine
treatment with respect to drug challenge, an analysis of covariance was
conducted. The analysis of covariance is used to compare groups when
measured values depend linearly upon the value of some other variate,
the so-called covariate (Brownlee, 1960). In this case, measured DA and
metabolite values depend upon the covariate that is the effective dose
of challenge methamphetamine.
The aim is to compare DA values and various metabolite values between
two groups, prenatally methamphetamine-treated animals versus
prenatally saline-treated animals in the cases of striatum, cortex,
ventral brainstem, and dorsal brainstem. At methamphetamine challenge
doses of 10, 15, and 20 mg/kg, the responses of these quantities appear
to be linear with respect to dose.
The statistical method of comparison consists of obtaining a regression
line of response variate (DA or metabolite) with respect to
methamphetamine challenge dose for each of the two groups. Then a
statistical test is performed to see whether the two lines can be
considered to be parallel. If so, a second statistical test is
performed to see whether the two lines can be considered to be
coincident. If that hypothesis is rejected (p < 0.05),
we infer that there is a difference in variate value (DA or metabolite) between the two groups. In other words, that the prenatal
methamphetamine treatment had an effect on the neurotoxic response to
methamphetamine in that subdivision of the brain.
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Results |
Our primary objective was to assess the possibility that prenatal
exposure to methamphetamine could result in an increased adult response
to the toxic effects of the drug. To that end, we established that the
doses of methamphetamine used were indeed neurotoxic as assessed by a
correlation between transmitter and transporter loss, which is a
function of anatomic disruption of the nerve endings. The known gender
and temperature dependence of the neurotoxicity was examined by a
comparison of neurotoxicity in females and males and correlated with
hyperthermia. Dose-effect regression analysis of regional depletion of
monoamines and metabolites was used to identify specific neurotoxic
effects and analysis of covariance at the effective depletion doses
used to determine whether there was a significant effect of prenatal
methamphetamine treatment.
Effect of Methamphetamine Administration on Striatal Monoamine
Levels of Dams.
Examination of striatal monoamine and metabolite
levels in the treated dams was conducted to assess the neurotoxic
effectiveness of the administered drug. It was found that
administration of 40 mg/kg methamphetamine twice daily to pregnant mice
during gestational days 7 to 13 produces the expected long-lasting
depletion of striatal dopaminergic markers in the brains of the dams
(Table 1). Such animals demonstrate
significant reductions in striatal DA (66.7%), dihydroxyphenylacetic
acid (DOPAC, 38.4%), and homovanillic acid (HVA, 27.1%) at 3 weeks postinjection. No effects were observed on striatal
3-methoxytyramine (3-MT), 5-HT, or 5-HIAA.
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TABLE 1
Effect of methamphetamine administration during pregnancy on striatal
monoamine and metabolite levels of dams
Pregnant dams were treated twice daily with (+)-methamphetamine (40 mg/kg) or saline from gestational day 7 to 18 and killed 3 weeks
postinjection. Data represent the mean ± S.E.M. striatal
monoamine and metabolite levels (ng/mg of protein).
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Correlation between Methamphetamine-Induced Striatal DA Depletion
and DAT Binding Sites.
To establish that the reductions in DA were
a function of dopaminergic neuronal neurotoxicity, 15-week-old male
mice from the prenatal saline group were given two subcutaneous
injections of 10 mg/kg methamphetamine (2 h apart). The animals were
killed 1 week later and their striata obtained for analysis of DA and DAT binding site density and affinity. As can be seen in Table 2, this dose of methamphetamine resulted
in a 35.4% reduction in striatal DAT without any change in affinity of
the transporter. A corresponding 30.0% decrease in striatal DA was
observed. This correspondence provides evidence that the loss of DA
observed at 1 week was, in fact, secondary to degeneration of
dopaminergic axonal endings.
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TABLE 2
Effect of methamphetamine on striatal dopamine and DAT
Fifteen-week-old male offspring exposed to saline in utero were given
two injections of 10 mg/kg methamphetamine 2 h apart and killed 7 days later. Data represent the mean ± S.E.M.
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Comparison of Methamphetamine-Induced Neurotransmitter Depletion in
Male and Female Postnatal Offspring as a Function of Prenatal
Treatment.
To examine the question of whether exposure to
methamphetamine in utero results in an enhanced vulnerability to
methamphetamine neurotoxicity, 11-week-old male and female offspring
exposed to methamphetamine or saline in utero were given two
subcutaneous injections (2 h apart) of 5, 10, 15, or 20 mg/kg
methamphetamine. The animals were killed 7 days later for analysis of
monoamine and metabolite levels in the striatum, ventral and dorsal
brainstem, cortex, and cerebellum.
One critical issue in these studies was the question of whether adult
basal levels of monoamines and metabolites were affected by prenatal
exposure to methamphetamine. It is important to note that basal levels
of monoamines and their metabolites in 3-month-old male and female
offspring were not affected by prenatal drug exposure for any of the
brain regions examined (Tables
3-7).
Despite this lack of effect of prenatal methamphetamine treatment,
there was a clear gender difference in both prenatal methamphetamine
and prenatal saline groups in terms of 5-HIAA. In each region examined, males exhibited significantly lower levels of 5-HIAA than females (striatum: 23%, p < 0.001; ventral brainstem:
26%, p < 0.001; dorsal brainstem: 23%,
p < 0.01; cortex: 24%, p < 0.05;
cerebellum: 28%, p < 0.05).
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TABLE 3
Effect of methamphetamine on striatal monoamine and metabolite levels
of adult mice exposed to the drug in utero
Three-month-old postnatal offspring, exposed in utero to saline or
methamphetamine, were challenged with two doses of saline or
methamphetamine (5, 10, 15, or 20 mg/kg/dose) and killed 7 days later.
Data represent the mean ± S.E.M. of striatal monoamine and
metabolite levels (ng/mg of protein) of individual animals from three
to four litters (n) per treatment group.
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TABLE 4
Effect of methamphetamine on cortical monoamine and metabolite levels
of adult mice exposed to the drug in utero
Three-month-old postnatal offspring, exposed in utero to saline or
methamphetamine, were challenged with two doses of saline or
methamphetamine (5, 10, 15, or 20 mg/kg/dose) and killed 7 days later.
Data represent the mean ± S.E.M. of cortical monoamine and
metabolite levels (ng/mg of protein) of individual animals from three
to four litters (n) per treatment group.
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TABLE 5
Effect of methamphetamine on ventral brainstem monoamine and metabolite
levels of adult mice exposed to the drug in utero
Three-month-old postnatal offspring, exposed in utero to saline or
methamphetamine, were challenged with two doses of saline or
methamphetamine (5, 10, 15, or 20 mg/kg/dose) and killed 7 days later.
Data represent the mean ± S.E.M. of ventral brainstem monoamine
and metabolite levels (ng/mg of protein) of individual animals from
three to four litters (n) per treatment group.
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TABLE 6
Effect of methamphetamine on dorsal brainstem monoamine and metabolite
levels of adult mice exposed to the drug in utero
Three-month-old postnatal offspring, exposed in utero to saline or
methamphetamine, were challenged with two doses of saline or
methamphetamine (5, 10, 15, or 20 mg/kg/dose) and killed 7 days later.
Data represent the mean ± S.E.M. of dorsal brainstem monoamine
and metabolite levels (ng/mg of protein) of individual animals from
three to four litters (n) per treatment group.
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TABLE 7
Effect of methamphetamine on cerebellar monoamine and metabolite levels
of adult mice exposed to the drug in utero
Three-month-old postnatal offspring, exposed in utero to saline or
methamphetamine, were challenged with two doses of saline or
methamphetamine (5, 10, 15, or 20 mg/kg/dose) and killed 7 days later.
Data represent the mean ± S.E.M. of cerebellar monoamine and
metabolite levels (ng/mg of protein) of individual animals from three
to four litters (n) per treatment group.
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Following methamphetamine challenge, adult male and female offspring
exhibited a notable gender difference with respect to drug-induced
reductions in transmitter and metabolite levels. To determine whether
methamphetamine produced a significant reduction in monoamines and
metabolites, regression analyses were conducted using data from all
methamphetamine doses. Significant reductions in female offspring were
seen only for the striatum (Table 3). Both prenatal saline-
(r = 0.49, p < 0.05) and
methamphetamine- (r = 0.69, p < 0.001) treated females demonstrated depletions of striatal DA. This
effect was considerably less than that seen in male offspring. For
example, a challenge dose of 20 mg/kg methamphetamine produced 55 to
70% reductions in striatal DA in males compared with 14 to 25% in
females (Table 3). The only other effect observed in females was a
decrease in striatal HVA in the prenatal methamphetamine group
(r = 0.51, p < 0.05), which was
still less robust than that observed for prenatal methamphetamine- or
saline-treated males.
In the males, dopamine was markedly reduced in the striatum of both
prenatal saline- (r = 0.69, p < 0.01) and methamphetamine- (r = 0.87,
p < 0.001) exposed animals. Effects on other striatal dopaminergic markers were seen in prenatal methamphetamine-treated males (DOPAC: r = 0.71, p < 0.001 and 3-MT: r = 0.57, p < 0.01). Striatal HVA was reduced in both prenatal methamphetamine-
(r = 0.75, p < 0.001) and prenatal
saline-treated males (r = 0.61, p < 0.05). Only three other brain regions in male offspring demonstrated significant methamphetamine-induced depletions. In the case of cortex,
significant regressions were observed for DA (r = 0.68, p < 0.001) and HVA (r = 0.58, p < 0.01) in prenatal methamphetamine males
and for DA (r = 0.62, p < 0.05),
DOPAC (r = 0.47, p < 0.05), and HVA
(r = 0.63, p < 0.01) in prenatal
saline males (Table 4). In the brainstem, regression analyses showed
that methamphetamine challenge only affected prenatal
methamphetamine-exposed males. Significant reductions of DA
(r = 0.72, p < 0.001) were observed
in ventral brainstem and of HVA in both ventral (r = 0.71, p < 0.001) and dorsal brainstem
(r = 0.52, p < 0.05) of these
animals (Tables 5 and 6).
The primary objective of this study was, however, to examine
predisposition to methamphetamine neurotoxicity following fetal exposure to the drug. To carry out a comparison of the extent of
methamphetamine-induced transmitter depletion as a function of prenatal
treatment, an analysis of covariance was carried out over the effective
doses of 10, 15, and 20 mg/kg. Initially, regression lines of response
variates versus the covariate, effective methamphetamine dose, were
constructed for each of the two groups of animals prenatally exposed to
methamphetamine or saline. If the slopes of the two lines were not
statistically different (i.e., the lines were parallel), a subsequent
test was performed to determine whether the two lines were
statistically different or coincident. If the two lines were statistically different (p < 0.05), not coincident,
the inference is that there was a difference in response to
methamphetamine challenge in the effective dose range between
prenatally methamphetamine-exposed animals compared with saline. The
results for males and females were analyzed in separate statistical
analyses. No interactions between males and females were examined.
The effect of methamphetamine challenge was shown to be enhanced in a
number of dopaminergic parameters in the striatum of male offspring
exposed to methamphetamine in utero, including DA (Fig.
1, p < 0.05), DOPAC
(Fig. 2, p < 0.05), and HVA (Fig. 3, p < 0.05). While the
response to methamphetamine challenge for striatal 3-MT was the same
for all the effective doses (10, 15, and 20 mg/kg) for both prenatal
methamphetamine- and saline-treated males, the analysis of covariance
showed that the lines were significantly different (p < 0.01, Fig. 4). Examination of DA
levels in the ventral brainstem of males revealed a similar
exacerbation of methamphetamine neurotoxicity in this subdivision of
brain by fetal exposure to the drug (Fig.
5, p < 0.01). Despite
the fact that methamphetamine reduced cortical DA levels, DA
neurotoxicity in this brain region did not differ as a function of
prenatal exposure in male offspring (Fig.
6). In female offspring, prenatal methamphetamine exposure was only observed to enhance drug evoked striatal 3-MT (Fig. 4, p < 0.05) and HVA (Fig. 3,
p < 0.05) depletions.
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Fig. 1.
Effect of methamphetamine challenge on striatal DA
levels of 11-week-old male and female offspring prenatally exposed to
saline or methamphetamine. Mice were challenged with two doses of
methamphetamine (10, 15, or 20 mg/kg) given 2 h apart and were
killed 1 week following drug injection. Striatal DA depletion is
enhanced in males pretreated with methamphetamine compared with saline.
The regression line for prenatal methamphetamine-exposed males is
significantly lower than that of the prenatal saline group
(p < 0.05) by analysis of covariance. Each value
represents the mean striatal DA level of three to four mice. The error
bars represent the standard deviation of the DA regression at the given
dose. Top line, regression of DA for prenatal methamphetamine and
prenatal saline females; middle line, regression of DA for prenatal
saline males; and bottom line, regression of DA for prenatal
methamphetamine males.
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Fig. 2.
Effect of methamphetamine challenge on striatal DOPAC
levels of 11-week-old male and female offspring prenatally exposed to
saline or methamphetamine. Mice were challenged with two doses of
methamphetamine (10, 15, or 20 mg/kg) given 2 h apart and were
killed 1 week following drug injection. Striatal DOPAC depletion is
enhanced in males pretreated with methamphetamine compared with saline.
The regression line for prenatal methamphetamine exposed males is
significantly lower than that of the prenatal saline group
(p < 0.05) by analysis of covariance. Each value
represents the mean striatal DOPAC level of three to four mice. The
error bars represent the standard deviation of the DOPAC regression at
the given dose. Top line, regression of DOPAC for prenatal
methamphetamine and prenatal saline females; middle line, regression of
DOPAC for prenatal saline males; and bottom line, regression of DOPAC
for prenatal methamphetamine males.
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Fig. 3.
Effect of methamphetamine challenge on striatal HVA
levels of 11-week-old male and female offspring prenatally exposed to
saline or methamphetamine. Mice were challenged with two doses of
methamphetamine (10, 15, or 20 mg/kg) given 2 h apart and were
killed 1 week following drug injection. Striatal HVA depletion is
enhanced in males and females pretreated with methamphetamine compared
with saline. The regression line for prenatal methamphetamine-exposed
animals is significantly lower (males, p < 0.05;
females, p < 0.05) than that of the prenatal
saline group by analysis of covariance. Each value represents the mean
striatal HVA level of three to four mice. The error bars represent the
standard deviation of the HVA regression at the given dose. Top line,
regression of HVA for prenatal saline females; second line, regression
of HVA for prenatal methamphetamine females; third line, regression of
HVA for prenatal saline males; and bottom line, regression of HVA for
prenatal methamphetamine males.
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Fig. 4.
Effect of methamphetamine challenge on striatal 3-MT
levels of 11-week-old male and female offspring prenatally exposed to
saline or methamphetamine. Mice were challenged with two doses of
methamphetamine (10, 15, or 20 mg/kg) given 2 h apart and were
killed 1 week following drug injection. Striatal 3-MT depletion is
enhanced in males and females pretreated with methamphetamine compared
with saline. Each value represents the mean striatal 3-MT level of
three to four mice. The error bars represent the standard deviation of
the 3-MT regression at the given dose. Top line, regression of 3-MT for
prenatal saline females; second line, regression of 3-MT for prenatal
methamphetamine females; third line, regression of 3-MT for prenatal
saline males; and bottom line, regression of 3-MT for prenatal
methamphetamine males.
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Fig. 5.
Effect of methamphetamine challenge on ventral
brainstem DA levels of 11-week-old male and female offspring prenatally
exposed to saline or methamphetamine. Mice were challenged with two
doses of methamphetamine (10, 15, or 20 mg/kg) given 2 h apart and
were killed 1 week following drug injection. Ventral brainstem DA
depletion is enhanced in males pretreated with methamphetamine compared
with saline. The regression line for prenatal methamphetamine-exposed
males is significantly lower than that of the prenatal saline group
(p < 0.01) by analysis of covariance. Each value
represents the mean ventral brainstem DA level of three to four mice.
The error bars represent the standard deviation of the DA regression at
the given dose. Top line, regression of DA for prenatal methamphetamine
and prenatal saline females; middle line, regression of DA for prenatal
saline males; and bottom line, regression of DA for prenatal
methamphetamine males.
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Fig. 6.
Effect of methamphetamine challenge on cortical DA
levels of 11-week-old male and female offspring prenatally exposed to
saline or methamphetamine. Mice were challenged with two doses of
methamphetamine (10, 15, or 20 mg/kg) given 2 h apart and were
killed 1 week following drug injection. Cortical DA depletion is
similar in males regardless of prenatal treatment. Each value
represents the mean cortical DA level of three to four mice. The error
bars represent the standard deviation of the DA regression at the given
dose. Top line, regression of DA for prenatal methamphetamine and
prenatal saline females; and bottom line, regression of DA for prenatal
methamphetamine and prenatal saline males.
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Methamphetamine-Induced Temperature Response in Postnatal
Offspring.
Given the temperature dependence of
methamphetamine-evoked neurotoxicity, the rectal temperatures of both
male and female offspring were measured during drug administration and
over a 5- to 6-h period following the last injection. Since the bulk of
the methamphetamine-induced increase in core temperature occurs within
2 h post drug injection, core temperatures obtained from the 4-h
period after the first injection were subtracted from the initial
preinjection temperature (T0) and averaged as a
measure of hyperthermic response (dT). There was a clear
gender difference in hyperthermic response to methamphetamine with no
significant rise in core temperature evident in females regardless of
prenatal treatment (Table 8). In the case
of male mice, a marked temperature response was observed with fairly
similar temperature increases seen in both prenatal treatment groups
following 10, 15, or 20 mg/kg methamphetamine.
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TABLE 8
Effect of methamphetamine challenge on body temperature of adult mice
exposed to the drug in utero
Three-month-old postnatal offspring, exposed in utero to saline or
methamphetamine, were challenged with two doses of saline or
methamphetamine (5, 10, 15, or 20 mg/kg/dose) given 2 h apart.
Data represent the mean ± S.E.M. of initial core body
temperatures (T0) prior to drug challenge and dT is the average
change from initial core temperature over the 4-h period following the
first injection. Body temperatures were obtained from individual
animals of three to four litters (n) per treatment group.
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Relationship between Methamphetamine-Induced Hyperthermic Response
and Striatal DA Level.
While methamphetamine-induced neurotoxicity
was clearly temperature-dependent, it was not possible to determine
across doses whether there was a significant correlation between the
degree of core temperature increase and the reduction in striatal DA due to the low sample number in each experimental group. For this reason, a separate experiment was conducted in which normal 11-week-old male mice received two doses of 20 mg/kg methamphetamine or saline and
were killed 7 days later for analysis of striatal DA levels. Rectal
temperatures were measured in these animals in a similar manner to that
above for prenatal exposed offspring. As seen in Fig.
7, depletion of striatal DA following
methamphetamine treatment was significantly correlated with the average
increase in rectal temperature attained during the first 4 h after
the initial drug injection.
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Fig. 7.
Relationship of average increase in core temperature
(°C) to striatal DA level following administration of two doses of 20 mg/kg methamphetamine to 11-week-old male mice. The increase in core
temperature from preinjection baseline was calculated and averaged for
the 4-h period after the first drug injection. Each point on the graph
represents an individual animal (n = 8). There is a
significant correlation (r = 0.85, p < 0.01) between the average increase in core
temperature at the time of drug injection and striatal DA level
measured 1 week after injection. Mean striatal DA level of
saline-injected males from the same experiment was 76.5 ng/mg protein
(n = 6).
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Discussion |
Prenatal exposure to methamphetamine has been shown to result in
gender-dependent and region-specific long-term alterations in
dopaminergic function. Adult methamphetamine-exposed male offspring demonstrate an enhanced dopaminergic neurotoxic response when challenged with the drug. Greater methamphetamine-induced reductions of
dopaminergic markers in the striatum (DA, DOPAC, HVA, and 3-MT) and
ventral brainstem (DA) were seen in prenatal methamphetamine-treated males compared with saline-treated animals. Effects of prenatal methamphetamine exposure in female offspring were limited to striatal levels of 3-MT and HVA.
The mechanism by which prenatal exposure to methamphetamine potentiates
drug-induced DA neurotoxicity in the striatum and ventral brainstem of
offspring is of considerable interest. The neurotoxic effect of
methamphetamine depends upon the release or redistribution of
intraneuronal DA. Methamphetamine is transported into the cell via DAT
and releases DA into the extracellular space through exchange diffusion
across the plasma membrane (Seiden et al., 1993). Accumulation of high
concentrations of methamphetamine following administration of the drug
also decreases intracellular pH causing displacement of DA from
synaptic vesicles (Sulzer and Rayport, 1990), resulting in increased
cytoplasmic concentrations of DA. The release and/or redistribution of
DA is believed to result in the formation of neurotoxic reactive oxygen
species (Cubells et al., 1994) and DA quinones (LaVoie and Hastings,
1999) involved in axonal destruction. The balance between DAT and the vesicular monoamine transporter-2 (VMAT-2) may determine cell vulnerability to methamphetamine (Fumagalli et al., 1999). DAT plays an
important role in methamphetamine neurotoxicity since the toxicity can
be prevented by neuronal DA uptake blockers (Seiden and Sabol, 1996).
Furthermore, DAT knockout mice are resistant to methamphetamine-induced
DA neurotoxicity (Fumagalli et al., 1998). In contrast, enhanced
drug-induced neurotoxicity is observed in VMAT-2 knockout mice
(Fumagalli et al., 1999).
Given these considerations, it is possible that prenatal exposure to
methamphetamine results in an impaired ability of DAT and/or VMAT-2 to
maintain normal compartmentalization of DA within the axon terminal. We
have demonstrated that in utero exposure to methamphetamine enhances
methamphetamine-induced release of striatal DA in adult offspring
(Heller et al., 2000a). This heightened responsivity occurs despite the
fact that basal levels of striatal DA are similar to prenatal saline
animals. Similar results have been obtained in rabbit progeny with in
utero exposure to cocaine causing an increased amphetamine-induced
release of striatal DA in the adult animal (Du et al., 1999). Given an
enhanced release of neurotransmitter following methamphetamine and the
participation of DA release in producing neurotoxicity, it seemed
reasonable to investigate whether in utero exposed animals might show
an increased neurotoxic response to the drug, the primary finding of
the current study.
Enhancement of psychostimulant-evoked striatal DA release in adult
offspring that were repetitively exposed to methamphetamine in utero
may be analogous to the biochemical sensitization observed in adults
following repetitive exposure to amphetamine (Robinson and Becker,
1982). Prior exposure of adult animals to psychostimulants has also
been shown to produce increased self-administration of these drugs
(Lorrain et al., 2000). Given this sensitized behavioral responding, it
will be interesting to examine whether there is enhanced propensity of
methamphetamine-exposed offspring to self-administer psychostimulants
as has been demonstrated in rats exposed to cocaine in utero (Keller et
al., 1996).
It is important to note that gestational exposure to methamphetamine
did not effect basal concentrations of monoamine or metabolite levels.
Female offspring did, however, exhibit higher levels of 5-HIAA than
males in all brain regions analyzed. Others have reported altered basal
monoamine and/or metabolite concentrations in forebrain and brainstem
of rat progeny following gestational exposure to methamphetamine
(Tonge, 1973a,b; Sato and Fujiwara, 1986). Differences between the
present and previous studies may involve drug dose, route of
administration, period of prenatal treatment, or species. Alterations
in central serotonergic function are also seen following prenatal
exposure to methamphetamine (Cabrera et al., 1993). Serotonin-mediated plasma renin secretion is reduced in the adult despite the fact that
such animals have normal levels of 5-HT uptake sites, and 5-HT1 and 5-HT2 receptors.
Studies of methamphetamine neurotoxicity using male and female mice
have shown marked gender sensitivity to the effects of the drug (Yu and
Wagner, 1994; Yu and Liao, 2000a,b). In the present study,
methamphetamine produced greater depletion of striatal DA levels in
male compared with female animals, irrespective of prenatal treatment.
Gender-specific methamphetamine-induced striatal DA depletions in mice
are strain-dependent (Yu and Liao, 2000b) and may (Yu and Liao,
2000a,b) or may not (Yu and Wagner, 1994) be modulated by circulating
ovarian hormones.
Hyperthermia is clearly a factor contributing to differential gender
sensitivity to methamphetamine-induced neurotoxicity. Systemic
administration of amphetamines to rodents at ambient temperature
results in an increase in core body temperature (Bowyer et al., 1994;
Albers and Sonsalla, 1995; Miller and O'Callaghan, 1995) as well as
brain temperature (Clausing and Bowyer, 1999). The magnitude of
hyperthermia produced by methamphetamine is correlated with the
severity of striatal DA depletion (Bowyer et al., 1994; Fukumura et
al., 1998; Clausing and Bowyer, 1999). There is greater accumulation of
methamphetamine by the DA transporter at higher temperatures associated
with increased DA release and the production of reactive oxygen species
(Xie et al., 2000). Prevention of drug-induced hyperthermia reduces or
blocks the neurotoxicity (Ali et al., 1996). In mice (Miller and
O'Callaghan, 1995) and rats (Fukumura et al., 1998)
methylenedioxymethamphetamine or methamphetamine, respectively,
increases maximum core temperatures more in male than female animals.
We have extended studies on the relation of methamphetamine and
hyperthermia to include a more extensive range of drug doses. Our data
support the contention that differential gender sensitivity to the
neurotoxic effect of methamphetamine is correlated with hyperthermic
response. Hyperthermic effects, however, do not account for the
increased susceptibility of prenatal methamphetamine-treated males to
drug-induced striatal DA neurotoxicity since methamphetamine challenge
did not evoke a significantly greater hyperthermic response in these
animals compared with prenatal saline-treated males.
The neurotoxic effects of methamphetamine in a number of species are
well documented. Methamphetamine produces persistent reductions in
striatal DA in male mice, rats, and monkeys (Seiden et al., 1993) as
well as a loss of rat serotonergic markers (Bakhit et al., 1981).
Central dopaminergic systems apparently do not respond uniformly to
methamphetamine, the striatum and substantia nigra being most sensitive
to neurotransmitter depletion (Seiden and Sabol, 1996). This is
consistent with our results of enhanced methamphetamine-induced
dopaminergic neurotoxicity in the striatum and ventral brainstem of
male offspring prenatally exposed to the drug. Methamphetamine-induced
depletion of transmitter is thought to effect monoaminergic endings
without concomitant loss of neurons as documented both in vivo and in
three-dimensional reaggregate tissue culture in which the nigrostriatal
projection can be reconstructed and allowed to develop to the adult
stage (Kontur et al., 1991). A 45% reduction, however, has been
observed in the number of dopaminergic cell bodies of C57BL adult male mice 5 to 8 days following methamphetamine with a corresponding 92%
decrease in striatal DA levels (Sonsalla et al., 1996). In the present
study, a significant reduction in ventral brainstem levels of DA was
observed for males in the prenatal methamphetamine group challenged
with methamphetamine. Since the ventral brainstem contains the
dopaminergic cell bodies of the midbrain, there is a possibility that
the decrease in transmitter in this area may reflect a loss of cell bodies.
The primary finding of the current study is that there is an
enhancement of methamphetamine neurotoxicity following prenatal exposure to the drug. This effect is selective for the nigrostriatal dopaminergic system of male offspring. The results raise potential concern since maternal administration of 40 mg/kg to pregnant dams, the
dose used in the present study, results in fetal brain levels of
methamphetamine in the range of 10
5 M (Won et
al., 2001). Similar brain drug levels have been reported for
premature infants delivered by women abusing methamphetamine (Bost et
al., 1989).
The functional significance of the enhanced dopaminergic neurotoxic
response to in utero methamphetamine exposure is obviously an important
issue. The relation of loss of nigrostriatal DA to function needs to be
considered in light of the large redundancy in dopaminergic content.
Frank clinical symptoms of Parkinsonism are not seen until there is at
least an 80% loss of the transmitter (Hornykiewicz and Kish, 1987). A
recent study in previous human methamphetamine users, abstinent for at
least 3 years, showed that such individuals had significant reductions
in DAT binding sites in the caudate and putamen (McCann et al., 1998).
Although clinical signs of Parkinsonism were not evident, the authors
speculated that with age, the persistent damage to the dopaminergic
system may predispose these individuals to extrapyramidal or
neuropsychiatric disorders. The enhanced neurotoxicity in response to
methamphetamine in male animals exposed to the drug in utero may be an
additional risk factor in the development of frank signs and symptoms
of Parkinsonism in such individuals.
In conclusion, the present study provides evidence that prenatal
exposure to methamphetamine has long-term effects on dopaminergic function in the offspring, which are region-specific and
gender-dependent. The findings raise serious concerns for male
methamphetamine abusers, of an enhanced neurotoxic risk from in utero
exposure to methamphetamine.
5-HT, 5-hydroxytryptamine;
5-HIAA, 5-hydroxyindoleacetic acid;
DA, dopamine;
HPLC, high-performance liquid
chromatography;
DAT, dopamine transporter;
DOPAC, dihydroxyphenylacetic
acid;
HVA, homovanillic acid;
3-MT, 3-methoxytyramine;
VMAT-2, vesicular monoamine transporter-2.
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Abstract
Introduction
