Development of sensitization to methamphetamine in offspring prenatally exposed to
morphine, methadone, and buprenorphine
Yao-Chang Chiang1,2, Tsai-Wei Hung4, Ing-Kang Ho1,3,4
1
Center for Drug Abuse and Addiction, China Medical University Hospital, Taichung,
Taiwan
2
China Medical University, Taichung, Taiwan
3
Graduate Institute of Clinical Medical Science, China Medical University, Taichung,
Taiwan
4
National Health Research Institutes, Zhunan, Miaoli County, Taiwan
Correspondence to: Dr. Ing-Kang Ho, Center for Drug Abuse and Addiction, China
Medical University Hospital, 2 Yude Road, North District, Taichung 404, Taiwan
(ROC)
Tel.: +886-4-2205-2121 ext. 7528; fax: +886-4-2205-2121 ext. 7527
E-mail: iho@mail.cmuh.org.tw
Abstract
Heroin use among young women of reproductive age has drawn much attention
around the world. However, there is lack of information on the long-term effects of
prenatal exposure to opioids on their offspring. Our previous study demonstrated that
prenatally buprenorphine-exposed offspring showed a marked change in the crosstolerance to morphine compared with other groups. In the current study, this animal
model was used to study effects of methamphetamine (METH)-induced behavioral
sensitization in the offspring at their adulthood. The results showed no differences in
either basal or acute METH-induced locomotor activity in any of the groups of
animals tested. When male offspring received METH injections of 2 mg/kg, i.p., once
a day for 5 days, behavioral sensitization was induced, as determined by motor
activity. Furthermore, the distance and rate of development (slope) of locomotor
activity and CPP induced by METH were significantly increased in the prenatally
buprenorphine-exposed animals compared with those in other groups. The dopamine
D1R in the NAc of the prenatally buprenorphine-exposed offspring had lower mRNA
expression; but no significant changes in the µ-, κ-opioid, NOP, D2R, and D3R
receptors were noted. Furthermore, significant alterations were observed in the basal
level of cAMP and the D1R agonist enhanced adenylyl cyclase activity in the
prenatally buprenorphine-exposed group. Overall, the study demonstrates that D1R
and its down-regulated cAMP signals are involved in enhancing METH-induced
behavioral sensitization in prenatally buprenorphine-exposed offspring. The study
reveals that prenatal exposure to buprenorphine caused long-term effects on offspring
and affected the dopaminergic system-related reward mechanism.
Keywords: Behavioral sensitization, buprenorphine, conditioned place preference,
methamphetamine, opioids, prenatal exposure
2
Introduction
According to the U.S. National Survey on Drug Use and Health, 4.4% of
pregnant women ages 15–44 used illicit drugs in 2009–2010 (SAMHSA, 2011). This
report also showed that the rate of current illicit drug use in the combined 2009–2010
data was 16.2% among pregnant women ages 15–17, 7.4% for ages 18–25, and 1.9%
for ages 26–44. The U.S. study indicates that illicit drug use during pregnancy and
among younger women are urgent and important issues.
Continual use of opioids results in addiction and the development of tolerance
to and dependence on these drugs. In the case of opioid abuse, children born from
heroin- or morphine-addicted mothers have been known to suffer from higher
mortality and problems with the central nervous system (Ostrea et al., 1997; Yanai et
al., 2003), such as dysfunction in intellectual ability or in emotional control (Ornoy,
2003; Wilson et al., 1979). These findings suggest that investigating effects of
prenatal opioid exposure upon offspring is important.
Methadone and buprenorphine are two maintenance agents for heroin addicts.
Methadone is a synthetic mu- (µ-) opioid receptor agonist and used for heroin patients
(including pregnant women) on maintenance for over 30 years. It has been shown to
be effective in reducing not only withdrawal symptoms but also the impulsive
continuing injection of heroin (Joseph et al., 2000). However, chronic use of
methadone has also led to addictive liability and respiratory depression in some
subjects (Lobmaier et al., 2010). Studies by Dashe et al. (2002) have shown that
maternal methadone dosage is associated with neonatal abstinence syndrome (NAS)
score on the percentage of treatment for withdrawal and the duration of neonatal
hospitalization. These findings indicate that methadone in high doses would not be a
good therapeutic agent for pregnant women.
3
Recently, high doses (2–32 mg) of buprenorphine have also been used to treat
opioid dependence in human. Buprenorphine is an opioid mixed agonist and
antagonist; it can act on several opioid receptors as a partial agonist for the µ-opioid
receptor, an antagonist for the kappa- (κ-) opioid receptor, and a full/partial agonist for
the nociceptin (NOP) receptor (Bloms-Funke et al., 2000; Lutfy and Cowan, 2004).
Buprenorphine is better than methadone for maintenance purposes because it is less
addictive and less likely to produce respiratory depression (Raisch et al., 2002). In
neonatal studies, buprenorphine has been shown to have more beneficial effects on
mortality rate and NAS than methadone in human (Jones et al., 2010) or animals
(Hutchings et al., 1995). However, some clinical and animal studies have provided
different results (Chiang et al., 2010; Kahila et al., 2007; Robinson and Wallace,
2001). A higher dose (3 mg/kg) of buprenorphine increased the number of stillbirths
and raised the mortality index in the animal model (Chiang et al., 2010; Robinson and
Wallace, 2001). A clinical study also showed that 57% of newborns of mothers who
received buprenorphine during pregnancy presented severe NAS and needed therapy;
this was also associated with a higher number of sudden infant deaths (Kahila et al.,
2007). These findings suggest that higher doses of buprenorphine may induce
complex effects or serious systemic toxicities in the offspring, and that more extensive
investigations are needed.
Other evidence also indicates interactions between opioidergic and
dopaminergic systems (Tien and Ho, 2011). Microinjection of µ-opioid receptor
agonist-DAMGO caused an increase in the extracellular level of dopamine (DA) and
its metabolites in the ventral striatum of rats (Devine et al., 1993). Acute morphine
administration also increased the level of extracellular DA in the nucleus accumbens
(NAc) and the striatum of mice (Fadda et al., 2005). Blockade of the μ-opioid receptor
4
attenuated the development of METH-induced sensitization (Chiu et al., 2005; Shen et
al., 2010), which is associated with changes in the extracellular levels of DA and its
metabolites (Lan et al., 2008). However, the prenatal effects of opioids on
dopaminergic system are unclear and need further investigation.
Methamphetamine (METH) is a psychostimulant that increases DA levels in
the mesolimbocortical dopaminergic pathways from the ventral tegmental area (VTA)
to the NAc, and is well known to associate with rewarding circuitry and sensitization
(Segal and Kuczenski, 1997). Chronic exposure to METH induces behavioral
sensitization, which refers to the progressive enhancement of species-specific
behavioral responses upon repetitive drug exposure, and provides a useful model for
investigating sensitization of drug craving (Wolf, 2002).
In our previous study, cross-tolerance to morphine was observed in prenatally
opioid-exposed offspring. This finding may indicate that exposure to opioid during the
prenatal stage causes long-term cellular changes in the central nervous system of the
offspring. Behaviors of animals were also changed by re-exposure to morphine at
their adulthood. In this study, prenatal effects of opioids on the dopaminergic systemrelated behaviors and cellular signals were evaluated in the prenatally opioid-exposed
offspring using METH to perturb dopaminergic systems. For this purpose, we aimed
to investigate if prenatal administration of opioids altered behavioral and cellular
events induced by postnatal systemic METH administration.
5
Materials and Methods
Animals
Pregnant Sprague-Dawley (SD) rats (BioLASCO Taiwan Co.) and their male
offspring were used in the experiments. The pregnant female rats (at E2) were shipped
from animal breading company. After arrival, the dams were acclimatized to a room
with controlled temperature (25°C), humidity (50±10%) and a 12-h day-night cycle
(light on 07:00–19:00) for 24 hours before experimentation. Pregnant rats were kept
individually in separate cages, and their offspring were housed 2–3 per cage after
weaning. All animals were provided with food (Western Lab 7001, Orange, CA,
USA) and water ad libitum. The ethical guidelines provided by Laboratory Animal
Center of the National Health Research Institutes were followed throughout the study.
Drugs
Morphine (NBCD, Taiwan), methadone (USP, USA), and buprenorphine (Sigma
Aldrich, USA) were dissolved in distilled water and were administrated
subcutaneously (s.c.) in a volume of 1.0 ml/kg body weight. METH (NBCD, Taiwan)
was dissolved in saline and was administrated intraperitoneally (i.p.) in a volume of
1.0 ml/kg body weight. Heroin is a major drug of abuse by addicts; however, it is
rapidly converted to morphine after crossing the blood-brain barrier into the central
nervous system. Accordingly, we used morphine directly as a test agent in this study.
Prenatal treatments
Pregnant SD female rats, 10–12 weeks old and weighing 200–250 g, were randomly
assigned to different groups and were s.c. injected with opioids or vehicle during the
gestational period (E3 to E20). The experiments were conducted on four groups of
6
animals. Group 1 (vehicle control) rats received distilled water 1 ml/kg, s.c., twice a
day. Group 2 (morphine) rats received morphine, 2–4 mg/kg (initial to final dose), s.c.,
twice a day; the dose was increased by 1 mg/kg every week. Group 3 (methadone)
rats received methadone, 7 mg/kg, s.c., twice a day (5 mg/kg began at E3). Group 4
(buprenorphine) rats received buprenorphine, 3 mg/kg, s.c., once a day. The offspring
were weaned at postnatal day 28; experiments were performed upon them at 8–12
weeks old with body weight between 250 and 350 g. The number of offspring per
litter and the weight of offspring at birth and 8-12 week were not different among all
the groups tested. The male offspring from the same dam were randomly assigned to
different experiments to avoid the litter effect (the female offspring were not used in
this study).
Locomotor activity test
To examine the locomotor activity of exploratory stage and behavioral sensitization
induced by METH, all groups of rats were removed from their home cage and placed
into a locomotor testing box (45 × 45 × 30 cm). Basal locomotor activities were
measured for 30 min; then the rats received 2 mg/kg, i.p., of METH to induce
hyperlocomotor activities for 2 hours. The locomotor activities were monitored and
recorded (500 ms for tracing time intervals) in an acoustically insulated room by
video tracer software (Trace Mouse II, SINGA, Taiwan). Water was used to clean the
inner surface of the apparatus between every test. All experiments were performed
during the light phase (07:00–19:00 h).
Conditioned place preference (CPP) test
7
The CPP apparatus used was a three-compartment acrylic plastic box. A narrower
compartment (10 × 25 × 25 cm) set in the center to separate two equal-size
compartments (30 × 25 × 25 cm), one with black on the four walls and floor as a
visual cue, the other all in white. Subjects were first placed into the central
compartment of the apparatus and given free access to the entire box for 15 min to
measure the pre-drug preference. During the conditioning, METH (0.5 mg/kg, i.p.)
was paired with the non-preferred white compartment, while the vehicle (saline) was
paired with the black compartment. Animals were kept for 1 hour in the
corresponding compartment with the connection doors closed. There were two drugpaired and two vehicle-paired conditioning trials before the post-drug test. The postdrug place preference was examined at day 6 and conducted for 15 min. Behaviors of
the animal were recorded by video tracer software.
Isolation of total RNA and real-time RT-PCR
The NAc region was dissected from a fresh brain and frozen in liquid nitrogen. Total
RNA was isolated from the NAc using the Trisure reagent (Bioline, USA) according
to the manufacturer’s protocol. The mRNA levels of opioid and dopamine receptors
were measured by real-time quantitative RT-PCR using the Bio-Rad iQ5 sequence
detection instrument. The fluorogenic probe of SYBR Green (ABI, USA) was used to
determine the threshold cycle (Ct), which correlated inversely with the target mRNA
level (cycle 6–10). The mRNA levels of the µ-, κ-opioid, NOP, and dopamine
receptors (D1R, D2R, D3R) were normalized with GAPDH mRNA. The sequences of
the forward and reverse primers were designed by Primer Express 3 software (ABI,
USA).
8
Adenylyl cyclase activity assay
For adenylyl cyclase assay, tissues of the NAc were homogenized in a Tris-HCl
buffer (in mM: Tris-HCl 50, NaCl 120, KCl 5, CaCl2 2, and MgCl2 1 containing
proteinase inhibitors PMSF 0.5, p-tosyl-arginine methyl ester 1; pH 7.6). The
homogenate was first centrifuged at 1,000 × g in 4°C for 5 min. The supernatant was
centrifuged again at 34,000 × g in 4°C for 30 min. Afterwards, the resulting pellets
were re-suspended in TE buffer (50 mM Tris–HCl and 1 mM CaCl2, pH 7.4). The
assay was carried out in a volume of 100 µL at 30°C for 10 min. Duplicate membrane
samples (5 µg protein) were incubated in the reaction buffer (in mM: Tris-HCl 50,
MgCl2 5, ATP 2, creatine phosphate 20, 3-isobutyl-1-methylxanthine 0.5, 50 IU/mL
creatine kinase, and 2 mg/mL bovine serum albumin) in the presence or absence of
the dopamine D1R agonist-SKF 38393 (10, 100 nM). After incubation, the reaction
was stopped by adding 200 µL of ice-cold stop buffer (50 mM Tris-HCl and 4 mM
EDTA; pH 7.4). The assay mixture was boiled for 3 min and centrifuged at 13,750 ×
g for 10 min at 4°C. The supernatants were diluted 1:20 to apply to a Cyclic AMP
EIA kit (Cayman, USA) for determining the amount of cAMP produced, details as
described in the manufacturer’s instructions.
Data analyses and statistics
All data were analyzed using GraphPad Prism software. Results were expressed as
mean ± SEM. The results were tested by ANOVA with the post-hoc Bonferroni’s
correction. A P value < 0.05 was considered significant.
9
Results
Effects of METH on locomotor activity and behavioral sensitization in prenatally
opioid-exposed offspring
There was no significant difference observed in locomotor activities of basal
or acute METH administration among prenatally vehicle-, morphine-, methadone-,
and buprenorphine-exposed offspring at their adult age (data not shown). Effects of
chronic METH administration on behavioral sensitization in prenatally opioid
exposed offspring were then assessed. As shown in Figure 1, daily systemic METH
injections induced behavioral sensitization in all groups of animals as compared with
the saline control [(F(4,210)=38.86, p<0.0001), Vehicle-Meth (F(1,70)=103.04, p<0.0001),
M-Meth (F(1,83)=98.83, p<0.0001), Me-Meth (F(1,95)=241.7, p<0.0001), Bu-Meth
(F(1,82)=150.38, p<0.0001) as compared with Vehicle-Sa group]. The locomotor
activity induced by METH showed significant differences in the prenatally
buprenorphine-exposed group when compared with other groups tested (F(3,170)=5.4,
p<0.001). Moreover, the buprenorphine prenatally exposed animals had a significantly
higher degree of sensitization to METH than the saline, morphine and methadone
prenatally treated animals [Vehicle-Meth (F(1,72)=8.65, p<0.01), M-Meth (F(1,85)=5.42,
p<0.05), Me-Meth (F(1,97)=16.48, p<0.0001)]. As calculated by linear regression, the
slope of behavioral sensitization development to METH in prenatally vehicle-,
morphine-, methadone-, and buprenorphine-exposed offspring showed a significant
difference (F(3,
12)=5.42,
p<0.01) in METH-induced behavioral sensitization. A
significant difference was also observed in prenatally buprenorphine-exposed rats as
compared with prenatally vehicle- (F(1,
6)=6.03,
p<0.05), morphine- (F(1,
6)=5.9,
p<0.05), and methadone- (F(1, 6)=7.67, p<0.05) exposed rats. These results showed that
although basal and acute METH-induced locomotor activity was not significantly
10
different among all groups tested, prenatally buprenorphine-exposed animals
developed behavioral sensitization to chronic systemic METH administration was
more quickly than those in other groups.
Effects of low dose of METH on CPP in prenatally opioid-exposed offspring
Prenatal exposure to opioids did not change the basal place preference of rats
(saline/saline) (Figure 2). However, in the case of two conditioned pairs with a lower
dose (0.5 mg/kg) of METH, prenatally buprenorphine-exposed offspring showed
significant changes in METH-induced CPP as compared with the no-drug (saline)
treated prenatally buprenorphine-exposed group and the METH treated prenatally
vehicle-, morphine-, and methadone-exposed groups. These results indicate that
prenatally buprenorphine-exposed offspring exhibited more sensitivity to a lower dose
of METH in the CPP test.
Effects of prenatal exposure to opioids on the mRNA levels of opioid and dopamine
receptors in the NAc
To determine if the transcription levels of opioid receptors were affected by
prenatal exposure to opioids, the mRNA levels of the µ-, κ-opioid and NOP receptors
in the NAc were measured in all groups of animals tested. Results from real-time
quantitative PCR analysis showed no significant difference in the mRNA levels of the
µ-, κ-opioid and NOP receptors in the NAc of prenatally opioid-exposed rats as
compared with the control (data not shown). However, results showed a significant
23% decrease (p<0.05) in mRNA levels of D1R in the NAc of prenatally
buprenorphine-exposed rats as compared with the control group (Figure 3A); but no
difference in D2R (Figure 3B) or D3R (Figure 3C) was observed in any group of
11
animals tested. These results demonstrate that the mRNA expression of the µ-, κopioid and NOP receptors in the NAc was not significantly changed by prenatal
exposure to morphine, methadone, or buprenorphine. However, the dopaminergic
system, especially the mRNA levels of D1R, was affected by prenatal exposure to a
higher dose of buprenorphine.
Effects of prenatal exposure to opioids on the cAMP levels in the NAc
Because of the finding that D1R receptor mRNA was altered in the NAc of rats
prenatally exposed to buprenorphine, the basal levels of cAMP in the NAc were
examined. Results obtained showed that adenylyl cyclase activity was decreased by
23.9% (p<0.05) in prenatally buprenorphine-exposed rats; but no difference was
observed in the prenatally morphine- or methadone-exposed groups (Figure 4). This
result implicates that prenatal exposure to a higher dose of buprenorphine not only
decreased dopamine D1R mRNA expression but also reduced the downstream cAMP
production at the adulthood of the offspring.
Effects of D1R agonist-SKF 38393 on the cAMP levels in the NAc of prenatally
opioid-exposed offspring
In order to test the hypothesis that METH-induced behavioral sensitization is
due to altered sensitivity of D1R-mediated signaling in the NAc, effects of D1R
agonist-SKF 38393 on the level of cAMP was conducted. As shown in Figure 5, SKF
38393 (10 and 100 nM) significantly enhanced adenylyl cyclase activity in the
prenatally buprenorphine-exposed group as compared with the groups prenatally
exposed to vehicle, morphine, and methadone. This suggests that D1R-mediated
cAMP production was more sensitive in the prenatally buprenorphine-exposed group
12
when the D1R was activated.
Discussion
The goal of this study is to evaluate effects of prenatal exposure to morphine,
methadone, and buprenorphine on the offspring when they were exposed to METH at
adulthood. Prenatal exposure to opioids did not affect the basal locomotor activities in
offspring at their adulthood. This suggests that the neuroadaption occurred after
chronic prenatal exposure to opioids, therefore, behavioral outcomes appeared to be
similar to controls. However, a significant increase in METH-induced behavioral
sensitization (the distance and rate of development), as assessed by motor activity,
was observed in the prenatally buprenorphine-exposed offspring as compared with
other groups. A marked and significant effect on the CPP, which was induced by low
dose (0.5 mg/kg) and short-term (2 times) METH administration, was also observed
in the prenatally buprenorphine-exposed offspring. The expression of dopamine D1R
and its downstream cAMP signaling were reduced by prenatal exposure to
buprenorphine. The significant increase in D1R-mediated cAMP production was also
observed in the prenatally buprenorphine-exposed group. These studies demonstrate
that prenatal administration of buprenorphine brought about a significant cellular
change in the dopaminergic system and altered the METH-induced behavioral
sensitization and CPP. The possible mechanisms may be due to changes in the
expression and sensitivity of dopamine D1R and its downstream cAMP signaling in
the NAc.
Early study has shown that rats prenatally exposed to morphine (5–10
mg/kg/twice a day) during embryonic 11–18 period (E11–18), the levels and turnover
of norepinephrine (NE) were decreased and increased, respectively, dependent on the
13
brain regions and gender. However, no changes were observed in basal levels or in the
turnover of dopamine (DA) in any brain regions (hypothalamus, frontal cortex,
striatum, and cerebellum) tested (Vathy et al., 1994). In both adult male and female
rats receiving morphine 5–10 mg/kg prenatally, the lever-pressing behavior for
cocaine reward was not altered (Vathy et al., 2007). The METH-induced behavioral
sensitization and CPP were also not changed in male rats prenatally exposed to
morphine (2–4 mg/kg) in this study. These findings suggest that dopaminergic-related
signals, which were activated by postnatal treatment with cocaine or METH, did not
differ in rats prenatally exposed to morphine or vehicle at their adult age.
It has been reported that the levels of DA and NE in the forebrain significantly
decreased in prenatally methadone-exposed (1–6 mg/kg) offspring, when they were
compared with other tested groups at postnatal days 1 and 20 (McGinty and Ford,
1980). However, the level of NE had returned to normal on day 40, while the contents
of DA remained significantly lower in the stratum. These data demonstrate that
prenatal methadone exposure may retard catecholaminergic axonal growth in the
forebrain of rats. However, in prenatally methadone-exposed offspring, no basal or
METH-induced behavioral differences with their control mates were observed in this
study. The possible explanations may be due to that the level of DA was not difference
from that of the control at 8-12 weeks; because of the whole striatum buffered the
changes in the NAc or by an unknown compensative mechanism.
The present study showed that notable changes in METH-induced behaviors
and the dopaminergic system were observed only in the prenatally buprenorphineexposed group, but not in morphine or methadone prenatally exposed offspring.
Results obtained from biochemical studies also indicate that there are no differences
in the expression of the dopamine receptor, its activity, or its downstream cAMP
14
signaling in morphine or methadone prenatally treated animals. A possible explanation
for these findings is the pharmacological difference of buprenorphine from morphine
and methadone. Both morphine and methadone are pure µ-opioid receptor agonists,
while buprenorphine is a partial µ-opioid and full/partial NOP receptor agonist, as
well as a κ-opioid receptor antagonist. More complex receptor-receptor interactions
may occur during prenatal exposure to buprenorphine than to expose to morphine or
methadone. Prenatal exposure to buprenorphine has been reported to transient change
in the expression of opioid receptors. Prenatal exposure to buprenorphine (0.5–1
mg/kg) caused a reduction in the expression of µ-opioid receptors and an increase in
the expression of κ-opioid receptors in the brain of postnatal day 1 (P1) offspring, but
returned to normal at P7 (Belcheva et al., 1994). Our study also showed that the
mRNA levels of the µ-, κ-opioid and NOP receptors did not differ in the NAc of adult
offspring in any of the groups measured. However, activation of the NOP receptor has
been reported to alter the dopaminergic system. The in vivo study has shown that
activation of the NOP receptor, by treating it with N/OFQ, inhibited the release of
dopamine in the striatum (Flau et al., 2002). N/OFQ also inhibited tyrosine
hydroxylase, dopamine synthesis, D1R-mediated cAMP formation, and NMDA and
AMPA receptor phosphorylation in the NAc slices and primary cells by the activation
of pre- and post-synaptic NOP receptor (Olianas et al., 2008). Additionally,
intracerebral ventricular (i.c.v.) administration of N/OFQ-induced locomotor activity
could be reversed by dopamine D1R antagonist (Florin et al., 1996). The NOP
receptor has also been reported to be expressed early at embryonic 12 (E12) in the
cortical plate, basal forebrain, brainstem, and spinal cord (Neal et al., 2001). These
findings may imply that the NOP receptor is involved in regulating the dopaminergic
system in prenatal stages. However, there still lacks the study of effects of
15
buprenorphine on the release of catecholamines and glutamate in the NAc of the
offspring prenatally exposed to buprenorphine. According to the literatures described
above, prenatal exposure to morphine or methadone reduced the level of DA in the
striatum and NAc, and activation of NOP receptor also decreased the level of DA.
This indicates that prenatal buprenorphine may cause a synergistic reducing effect on
the release of DA by both activation of µ-opioid and NOP receptor. In the study of
Parkinson’s disease, MPTP-treated mice lacked pre-synaptic DA, but increased postsynaptic DA receptor sensitivity to apomorphine (Spooren et al., 1998). This finding
may explain the mechanism of the development of D1R super-sensitivity in the
prenatally buprenorphine-exposed offspring, which may due to prolonged presynaptic loss of the neurotransmitter. More detailed study on effects of signals
mediated by the activation of µ-opioid and NOP receptors on the dopaminergic and
glutamatergic systems, especially the level of neurotransmitters, during prenatal
exposure to buprenorphine is warranted.
Another possible mechanism for the METH-induced alterations in the
prenatally buprenorphine-exposed offspring may be the dose effect of opioids on
pregnant rats. In our study, the final doses of opioids used were 4 mg/kg-morphine, 7
mg/kg-methadone, and 3 mg/kg-buprenorphine, respectively. The limitation of this
study was to use a single higher dose of buprenorphine, which almost the highest dose
for human maintenance (~32 mg for 70 kg human, converted based on body surface
areas). The higher dose of buprenorphine would activate both µ-opioid and NOP
receptors. Whether low dose of buprenorphine, which only activates µ-opioid receptor,
causes similar effects like morphine or methadone require further studies. Although,
the dose of buprenorphine used was higher than the other drugs tested, high dose of
buprenorphine may not cause more µ-opioid receptor-induced actions due to its partial
16
agonistic properties, but the NOP-mediated signalings were also activated at this time.
According to the pharmacologic profile and antinociceptive studies, both morphine
and methadone are full µ-opioid receptor agonists. Additionally, compared with
morphine, methadone showed 1–2.5 fold greater efficacy of antinociception (s.c.
injection) in rats (He et al., 2009; Taracha et al., 2009). Most recently, a study showed
a negative genetic correlation between METH consumption and sensitivity to the
opioid-induced effects in the selectively bred mouse lines with different level of
METH intake (Eastwood and Phillips, 2012). This study indicates that the µ-opioid
receptor system is involved in the intake of METH. Although the dose of methadone
used in this study was also higher than that of morphine, there was no significant
dosing effect on the level of µ-opioid receptor and METH-induced behavioral changes
between these two groups. This may imply that significant effects of METH on
behaviors and cellular signaling in prenatally buprenorphine-exposed offspring than
other groups may not only due to the direct agonistic effects on µ-opioid receptor in
pregnant rats, a NOP receptor-mediated mechanism may also be involved.
Previous studies showed that rats prenatally exposed to buprenorphine
exhibited more antinociceptive resistance and tolerance development to morphine in
4-day postnatal pups (Robinson and Wallace, 2001) and adult pups (Chiang et al.,
2010), but failed to obtain changes in the threshold of pain sensitive and acute
morphine-induced antinociception. Robinson and Wallace (2001) also found that
morphine ED25 values were significantly increased in pups prenatally exposed to
buprenorphine, as compared with prenatal methadone after morphine challenge. Pups
exposed to buprenorphine either prenatally, postnatally, or both pre- and post-natally
were more resistant to the antinociceptive response to morphine. These findings
suggest that buprenorphine appears to have a greater ability than methadone to induce
17
tolerance to morphine. Additionally, our previous findings provided direct evidence to
indicate that prenatal exposure to buprenorphine caused faster development of
tolerance to morphine than prenatally saline-, morphine-, and methadone-exposed rats
at adulthood (Chiang et al., 2010). Given the results of previous findings and the
current study, it is suggested that prenatal exposure to buprenorphine does not alter
the basal behavioral outcomes of offspring, which may be due to the adaption of
homeostasis. However, the balances are disrupted after re-exposure to the opioid or
METH at the offspring’s adulthood.
It is well known that the dopamine D1R is involved in the development of
METH-induced behavioral sensitization in the induction and expression stages. Early
studies have shown that systemic or intra-VTA administration of D1R antagonist
during a period of repeated amphetamine (AMPH) treatment blocked the induction of
behavioral sensitization (Bjijou et al., 1996; Vezina, 1996). In addition, repeated intraVTA administration of D1R agonist-SKF 38393 produced behavioral sensitization to
AMPH and cocaine (Pierce et al., 1996). Recently, a study of two-injection protocol
for the induction of sensitization, which is a simple animal model to explore the
sensitization mechanisms, demonstrated that the D1R, NMDA receptor and cAMPdependent pathway were required for induction stage of psychostimulants-induced
sensitization (Valjent et al., 2010). Although, a study showed that locomotor activating
effects and sensitization of AMPH were reduced, but not fully blocked, in D1Rdeficient mice (Xu et al., 2000), sensitized behavioral responses to D1R agonists in
psychostimulant pre-treated animals have not been observed following either systemic
or intra-NAc administration (Vanderschuren and Kalivas, 2000). These findings
suggested that D1R and its-mediated signaling are essential for the induction of abuse
drug-induced sensitization but may not be for the expression stage. Beulter et al.
18
(2011) showed that the NMDA receptor, which was removed by the conditional
knockout method on the D1R-expressed medium spiny neurons, significantly
attenuated AMPH-induced behavioral sensitization in mice. This indicates that
METH-induced behavioral sensitization may do so through altering other relevant
receptors (such as NMDA), enzymes (such as DARPP-32), or genes (such as Fos) in
the NAc (Chen et al., 2009; Nestler, 2004). Exposure to METH, cocaine, or morphine
up-regulated the cAMP-PKA signaling pathway in the NAc, which also contributed to
the behavioral changes induced by cocaine and METH (Chiang and Chen, 2007; Self,
2004). In the current study, the D1R-mediated cAMP signaling showed more sensitive
to D1R agonist in the prenatally buprenorphine-exposed group than other groups
tested. The increased sensitivity to METH in the prenatally buprenorphine-exposed
offspring may be due to the D1R-mediated cAMP pathway.
The balance of the dopamine D1R and D2-like (D2R and D3R) receptors also
plays a critical role in the development of addiction (Chen et al., 2009; Vanderschuren
and Kalivas, 2000). The anatomical and physiological evidence showed that the D1R
and D2R are co-localized in the same neurons of the NAc but influence different
downstream brain areas (Self, 2004). The D2-like receptor is believed to function in a
manner opposite of D1R for development of addiction in behaviors and signaling
(Chen et al., 2009; Self, 2004; Zhang et al., 2012). The level of D1R but not D2-like
(D2R and D3R) receptor was altered by prenatal exposure to buprenorphine in this
study. Park et al. (2001) has shown that the mRNA level of D1R increased more than
that of D2R in the NAc of µ-opioid receptor knockout mice. This may indicate that the
expression of D1R was more sensitive to the changes of opioid system and also
provide a possible mechanism to explain why the expression of D1R, not D2R, was
significantly altered in this study.
19
In summary, we compared three kinds of opioids in METH-induced behavioral
changes to verify effects of prenatal exposure to opioids on the dopaminergic system
of the offspring at their adulthood. Although buprenorphine is considered a newer and
safer therapeutic agent than methadone for treating heroin addicts, the current study
shows that prenatal exposure to buprenorphine can induce faster behavioral
sensitization and CPP development to METH. This study further provides evidence to
show that prenatal effects of buprenorphine in the dopaminergic system exhibit longterm changes in the expression and sensitivity of dopamine D1R and its downstream
cAMP signaling in the NAc, even at the adulthood. This cellular evidence provides a
possible mechanism for the marked changes of METH-induced behaviors in
prenatally buprenorphine-exposed offspring. Several previous studies have suggested
that changes of opioid receptors in prenatal exposure to opioids may be the primary
factor in behaviors. Although differences in the mRNA levels of the µ-, κ-opioid and
NOP receptors were not observed in the NAc in any of the groups tested, details of the
changes in the opioid receptors in other brain areas — especially in the roles of the
NOP receptor for higher dose buprenorphine administration during pregnancy — are
still unclear. For this reason, we intend to investigate the roles of the NOP receptor in
the NAc of rats in prenatal buprenorphine exposure. Finally, prenatal exposure to
buprenorphine caused more notable effects on METH-induced behaviors and the
dopaminergic system than the other opioids tested in the current study. This raises the
question of whether higher buprenorphine is an ideal maintenance medication for
treating pregnant women. This animal study could provide an important reference for
clinical usage of buprenorphine in treating pregnant women who are heroin addicts.
Authors contribution
20
YCC designed and performed the experiments, analyzed the data, and drafted the
manuscript. TWH co-performed the experiments. YCC and IKH conceived the study
and revised the final manuscript. All authors have critically reviewed the content and
approved the final version for publication.
Acknowledgments
We thank Dr. M. Swofford for English editing before submission of the paper. The
work was supported by the National Health Research Institutes (NHRI-101A1-PDCO1312141 and NHRI-EX102-10224NC) and the China Medical University Hospital
(DMR-101-117) in Taiwan. The authors declare no conflict of interest to report.
21
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Figure legends
Figure 1. Behavioral sensitization development to METH in prenatally vehicle-,
morphine-, methadone-, and buprenorphine-exposed offspring. Animals were injected
METH, 2 mg/kg, i.p., and placed in a locomotor test box to measure locomotor
activities. The X-Y plot graphs illustrate the cumulative distances of locomotor
activity for a 2-hour observation period. All data are expressed as mean ± SEM. (n=712 per group). **p<0.01 as compared with the prenatally vehicle-exposed group,
#p<0.05 as compared with the prenatally morphine-exposed group, and +++p<0.001
as compared with the prenatally methadone-exposed group with methamphetaminetreatment (Two-way ANOVA).
Figure 2. Effects of METH on CPP in prenatally vehicle-, morphine-, methadone-,
and buprenorphine-exposed offspring. Time scores show differences between postconditioning and pre-conditioning time spent in the saline or 0.5 mg/kg METH-paired
environment in the prenatally buprenorphine-exposed group. All data are expressed as
mean ± SEM. (n=8-12 per group). *p<0.05 as compared with the METH-treated
prenatally vehicle-exposed group. #p<0.05 as compared with the METH-treated
prenatally morphine-exposed group, +p<0.05 as compared with the METH-treated
prenatally methadone-exposed group, &p<0.05 as compared with the prenatally
buprenorphine-exposed control group (One-way ANOVA).
Figure 3. The mRNA levels of dopamine receptor expressions in the NAc of rats
prenatally exposed to vehicle, morphine, methadone, or buprenorphine. Amounts of
mRNA were measured by real-time quantitative RT-PCR using SYBR Green as a
probe and adjusted with GAPDH mRNA from the same sample. The mRNA levels of
28
(A) D1R, (B) D2R, and (C) D3R expressed in the NAc. Data are expressed as mean ±
SEM. (n=8-12 per group). *p<0.05 as compared with the prenatally vehicle-exposed
group (One-way ANOVA).
Figure 4. Basal levels of cAMP in the NAc of rats prenatally exposed to vehicle,
morphine, methadone, and buprenorphine. Amounts of cAMP were measured by
cAMP ELISA kit for testing the basal adenylyl cyclase activities of the NAc. The data
were adjusted to the prenatally vehicle-exposed group (control group). Data are
expressed as mean ± SEM. (n=7 per group). *p<0.05 as compared with prenatally
vehicle-exposed group (One-way ANOVA).
Figure 5. Effect of dopamine D1R agonist, SKF 38393, on the levels of cAMP
accumulation in the NAc of rats prenatally exposed to vehicle, morphine, methadone,
and buprenorphine. The data of each group were adjusted with the basal (non-agonist
treated) group. Data are expressed as mean ± SEM. (Numbers of samples in the 10
nM and 100 nM are 6 and 7, respectively). *p<0.05 as compared with the prenatally
vehicle-exposed group, #p<0.05 as compared to the prenatally morphine-exposed
group, +p<0.05 as compared with the prenatally methadone-exposed group (One-way
ANOVA).
29
Distance in 2-hour (cm)
40000
**
#
30000
+++
Vehicle-Sa (n=9)
Vehicle-Meth (n=7)
M-Meth (n=10)
Me-Meth (n=12)
Bu-Meth (n=10)
20000
10000
3000
0
0
1
2
3
4
Time (days)
Figure 1.
30
5
Postconditioning-preconditioning
on less preferred side (sec)
+
#
*&
100
Prenatal Vehicle (n=12)
Prenatal Morphine (n=12)
Prenatal Methadone (n=8)
Prenatal Buprenorphine (n=8)
75
50
25
0
-25
-50
Saline/Saline
Saline/Meth-0.5 mg
Figure 2.
31
Figure 3.
32
cAMP concentration
(% of control)
150
100
*
50
0
Figure 4.
33
Prenatal Vehicle (n=7)
Prenatal Morphine (n=7)
Prenatal Methadone (n=7)
Prenatal Buprenorphine (n=7)
Figure 5.
34