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Peri, pre and postnatal morphine exposure: exposure-induced effects and sex differences in the behavioural consequences in rat offspring

Timár, Juliaa; Sobor, Melindaa b; Király, Kornél P.a; Gyarmati, Susannaa; Riba, Pála; Al-Khrasani, Mahmouda; Fürst1, Susannaa c

doi: 10.1097/FBP.0b013e3283359f39
ORIGINAL ARTICLES

This study investigated the behavioural consequences of peri, pre and postnatal morphine (MO) exposure in rats. From gestational day 1 dams were treated with either saline or MO subcutaneously once a day (5 mg/kg on the first 2 days, 10 mg/kg subsequently). Spontaneous locomotor activity in a new environment (habituation) and antinociceptive effects of MO were measured separately in male and female pups after weaning and also in late adolescence or adulthood. The rewarding effect of MO was assessed by conditioned place preference in adult animals. Both exposure-induced and sex differences were observed. A significant delay in habituation to a new environment and decreased sensitivity to the antinociceptive effect of MO were found in male offspring of MO-treated dams. In contrast, the place preference induced by MO was enhanced in the MO-exposed adult animals and this effect was more marked in females. Prenatal exposure to MO resulted in more marked changes than the postnatal exposure through maternal milk. The results indicate that a medium MO dose administered once-daily results in long-term consequences in offspring and may make them more vulnerable to MO abuse in adulthood.

aDepartment of Pharmacology and Pharmacotherapy, Semmelweis University

bNational Institute of Pharmacy

cHungarian Academy of Sciences, Neuropsychopharmacology Research Group, Budapest, Hungary

Correspondence to Dr Julia Timár, PhD, Semmelweis University, Department of Pharmacology and Pharmacotherapy, Budapest, 1085 Nagyvárad tér 4. Hungary

E-mail: timjul@pharma.sote.hu

The authors Julia Timár and Melinda Sobor contributed equally to this study

Received 2 July 2009 Accepted as revised 9 November 2009

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Introduction

Illicit drug use by pregnant women results in substantial medical and social problems. Abused opioids such as heroin, methadone or morphine (MO) cross the placenta and induce dependence in the foetus. Despite the fact that the condition of neonatal abstinence syndrome is well described, reports on longer-term outcome are relatively sparse. Though it is difficult to perform well designed follow-up studies, available results received show that infants exposed to opiates in utero are at risk of neurodevelopmental impairment (Hunt et al., 2008).

Although numerous data on the consequences of the pre, post or perinatal opioid exposure in rats have been published, there are few studies of the long-term consequences of these exposures. Offspring exposed to MO (10 mg/kg/day) during gestational days (GD) 11–18 showed alterations in neurobehavioural development (Slamberova et al., 2005) and in sexual behaviour in adulthood (Vathy and Katay, 1992). Naloxone-precipitated withdrawal syndrome was observed in 5-day-old pups exposed to MO in utero and then through maternal milk (Wu et al., 2005). Similar results were obtained after prenatal methadone or buprenorphine exposure (Robinson and Wallace, 2001) and after postnatal methadone exposure through maternal milk (Kunko et al., 1996).

Perinatal methadone or buprenorphine exposure resulted in reduced analgesia after MO challenge on postnatal day (PD) 4 (Robinson and Wallace, 2001). The fact that continuous opioid administration results in tolerance to the antinociceptive action is well known. The data published on opioid tolerance in neonatal rats are, however, inconsistent. Tolerance was observed in 7–9-day-old pups following either twice-daily MO injection for 4–7 days (Van Praag and Frenk, 1991; Barr and Wang, 1992) or 3 days after fentanyl or MO minipump infusion (Thornton and Smith, 1997; Thornton et al., 1997; Stoller et al., 2002). Others observed tolerance only when the pups were 10 or 15 days old at the beginning of repeated MO administration (Fanselow and Cramer, 1988; Windh et al., 1995).

Prenatal MO exposure also results in long-term effects through adolescence or adulthood. Daily administration of 0.8 mg/kg oxycodone to the dams from GD 8 until PD 5 increased pituitary response to corticotropine releasing hormone in late adolescent (PD 45) male offspring (Sithisarn et al., 2008). Alteration in the hypothalamo-pituitary-adrenal axis regulated stress response was also observed in adulthood following prenatal MO exposure (Slamberova et al., 2004). Decreased proenkephalin and increased prodynorphin mRNA levels were reported in the hippocampus of MO-exposed male offspring on PD 60-70, suggesting that prenatal MO exposure may affect the endogenous opioid system (Schindler et al., 2004). As the endogenous opioid system plays an important role in drug reward, alterations in its function may affect the rewarding properties of opioids. Consistent with this observation, prenatal MO exposure was found to enhance the reinforcing effects of heroin and cocaine measured by self-administration in males at adulthood (Ramsey et al., 1993) and to induce a greater preference for saccharin (Gagin et al., 1996) or higher place preference for MO (Gagin et al., 1997). Riley and Vathy (2006), however, reported no change in the rewarding effect of MO following MO exposure from GD 11 to 18.

Most of these effects were observed following prenatal MO exposure during the gestational period. However, no data can be found in the literature demonstrating long-term behavioural consequence of complete perinatal MO exposure. The purpose of this study was to investigate the short-term and/or long-term behavioural consequences of MO exposure during the whole perinatal period (‘experiment I’) and to compare them to those obtained by prenatal or postnatal exposure (‘experiment II’).

Consistent with data in the literature (Siddiqui et al., 1997) our preliminary experiments showed that high doses of MO during gestation resulted in premature delivery, still-birth or death of pups after birth. Therefore, in the present series of experiments a fixed medium dose of MO (10 mg/kg) once a day was used.

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Methods

Subjects and treatments

Nulliparous female Wistar rats, weighing 200–220 g (Charles River, Budapest, Hungary) were mated with males at a ratio of 3 : 2. The presence of sperm in the vaginal smear was checked every morning and the sperm-positive females were then separated and housed individually. Females where no sperm was found were excluded after 5 days. From the day sperm was detected (GD 1) the dams were treated either with saline (SAL) or with MO subcutaneously (s.c.) once a day (5 mg/kg on the first 2 days, 10 mg/kg subsequently) always at 08.00 h during the whole period of gestation and lactation. After parturition (PD 1) the pups were sexed, weighed and remained with their mother in the home cage until weaning (PD 21). They were then separated according to sex and housed five per cage. The animals were kept at constant temperature (20–21°C) and humidity (55±5%) under a standard 12–12 h-light/dark cycle (light on at 06.00 h). Water and chow were freely available.

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Experiment I

The dams were divided into two groups: the first group was treated with SAL, the second group with MO during the whole gestation and lactation period. The pups were grouped according to the treatment of their mothers: group I/1 (s/s)-offspring with SAL exposure; group I/2 (m/m)-offspring with MO exposure.

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Experiment II

The dams were divided into three groups: the first group was treated with SAL during both periods, the second group was treated with SAL during gestation and with MO during lactation and the third group was treated with MO during gestation and with SAL during lactation. The pups were grouped according to the treatment of their mothers: group II/3 (s/s)-offspring with SAL exposure; group II/4 (s/m)-offspring with postnatal MO exposure; group II/5 (m/s) -offspring with prenatal MO exposure.

The number of dams in each group was 15–16. All the offspring were used in one experiment only; only one male or one female pup from each litter was put in the same group in order to avoid litter effects. Morphine hydrochloride (ICN, Tiszavasvári, Hungary) was dissolved in physiological SAL and given s.c. in a volume of 0.1 ml/100 g body weight.

Experiments were performed in accordance with the Declaration of Helsinki and the guidelines on the use of experimental animals. The experimental protocols were approved by the Animal Care Ethical Committee at the Semmelweis University.

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Body weight

The pups were weighed on the day of birth (PD 1) than weekly (PD 7, 14) and on the day of weaning (PD 21). The body weight of pups in each litter was averaged (males and females separately) and the analysis was done on litter means.

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Locomotor activity

Locomotor activity was measured by ‘CONDUCTA System for behavioural and activity studies’ (Experimetria Ltd., Budapest, Hungary). The apparatus consisted of three black-painted testing boxes (40×50×50 cm each) set in an isolated room; the movements of rats were detected by high-density arrays of infrared diodes. One animal was placed in each box: the apparatus was able to test three animals at the same time and there was no connection between them. The time spent in ambulation (walking, running) was recorded individually for each box.

Animals were treated with physiological SAL and 30 min later placed into the testing boxes individually. The observation started immediately without any habituation and lasted 40 min, divided into 10 min intervals. The first 10 min of the observation was considered as the exploration of the novel environment (open field activity) and was calculated separately. The locomotor activity of the animals in the following 30 min was considered as habituation to novel surroundings. As locomotor activity normally decreases with time, habituation was examined by comparing activity levels of the time intervals. Delay in habituation is indicated by sustained activity level across the observation.

In experiment I the open field activity and the locomotor activity in the subsequent 30 min (habituation) of group I/1 (s/s) and group I/2 (m/m) were measured 2 days after weaning (PD 23) and at the age of late adolescent (PD 42). In experiment II the open field activity and the locomotor activity in the subsequent 30 min (habituation) of group II/3 (s/s), group II/4 (s/m) and group II/5 (m/s) were measured 2 days after weaning (PD 23).

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Antinociceptive effect

The tail flick test was performed as described earlier (Furst et al., 1993). At the beginning of the experiment the basal latency time of the rats was measured. Animals with latencies over 4 s were excluded from further testing. Three different dose-sets of MO (0.75, 1.5 and 3, or 1.5, 3 and 6 mg/kg) were given to generate dose-response curves. The latencies were tested 30 min after s.c. MO administration. To avoid tissue damage cutoff time was set to 8 s. The antinociceptive effect was calculated as maximum possible effect:

On the semilogarithmic (log2) dose/MPE% relation a linear curve was fitted and the ED50 value was calculated with nonlinear regression (0–100, standard slope). The curves were compared with the 95% confidence limit (CL) of the ED50 values. Groups of five animals were used for each dose of MO (15 animals for one curve).

In experiment I group I/1 (s/s) and group I/2 (m/m) the antinociceptive actions were tested three days after weaning (PD 24) and in adulthood (PD 90). In experiment II group II/3 (s/s), group II/4 (s/m) and group II/5 (m/s) the antinociceptive actions were tested 3 days after weaning (PD 24).

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Conditioned place preference

Conditioned place preference (CPP) was performed according to the method described by Carboni et al. (1989). The apparatus consisted of two identical compartments (38×32×30 cm each), one with white and the other with grey walls, separated by a guillotine door. It was placed in an isolated dark room and the white compartment was illuminated by a standard laboratory lamp (40 W).

Each experiment was performed on 8 consecutive days. On the first 3 days (preconditioning period) the animals were allowed to cross the open gate between the two compartments freely for 15 min. On the third day (preconditioning session) the time spent in the white compartment was recorded. Preliminary experiments showed the white compartment to be the less-preferred one for more than 95% of the animals. In the course of the next 4 days (conditioning period) the movement of the animals was limited to one of the compartments by closing the door. All the animals were placed for 60 min into the grey compartment 30 min after SAL administration and then 4 h later for another 60 min into the white compartment 30 min after administration of MO (1 or 3 mg/kg). On the 8th day (postconditioning session) neither MO nor SAL was administered. The animals were allowed to move freely between the two compartments for 15 min and the time spent in the white (drug-paired) compartment was recorded. CPP was measured by calculating the difference in time (Δt) spent in the white (drug-paired) compartment during the post and preconditioning sessions.

In experiment I the sensitivity of animals to MO in the CPP paradigm was studied in perinatally MO exposed (group I/2, m/m) adult animals (PD 90-120) and compared to adult ones exposed perinatally to SAL (group I/1, s/s). In experiment II the sensitivity of adult animals to MO in CPP paradigm in group II/4 (exposed to MO during lactation, s/m) and in group II/5 (exposed to MO during gestation, m/s) was compared.

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Statistical analysis

The data presented text are means with standard errors (±SEM). Statistical significance of difference between mean values was evaluated by unpaired t-test or one-way analysis of variance (ANOVA) for CPP, one-way ANOVA for body weight and open field activity and two-way ANOVA for habituation, with time as a repeated measure. When ANOVA analysis revealed a significant change, Newman-Keuls or Bonferroni post-hoc tests were applied. Tail flick results were analyzed by nonlinear regression and later the data from a selected dose were analysed by one-way ANOVA.

The analyses were performed using GraphPad Prism version 3.00 for Windows, (GraphPad Software, San Diego California USA, www.graphpad.com Copyright©1994–1999 by GraphPad Software). A P value of less than 0.05 was considered to be significant.

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Results

General conditions

There were no difference in the weight gain of dams, the number or size of litters, the sex ratio in litters and in the pup mortality between the MO or SAL treated groups (data not shown).

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Body weight

Experiment I

The results are summarized in Table 1. The birth weight of MO-exposed pups (group I/2, m/m) was significantly lower than SAL-exposed (group I/1, s/s) [F(3,56)=5.93, P<0.01]; Newman–Keuls post-hoc tests revealed significance both for females and males (P<0.05). During the first postnatal week, however, these animals caught up the SAL-exposed pups in body weight, and in the following two weeks the weight gain of MO-exposed offspring was higher than that of the SAL-exposed ones. One-way ANOVA revealed significant differences in the body weight both on PD 14 [F(3,56)=17.04, P<0.001] and PD 21 [F(3,56)=9.10, P<0.01]. According to post hoc tests, significance was shown on PD 14 and 21 both for males and females. Significant sex differences were detected on PD 14 and PD 21 in the SAL-exposed groups (P<0.05).

Table 1

Table 1

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Experiment II

The results are summarized in Table 2. One-way ANOVA showed a significant difference on PD 1 [F(5,84)=4.18, P<0.01]. According to the Newman–Keuls post-hoc test, as expected, there was no difference in birth weight between group II/3 (s/s) and group II/4 (s/m), while the birth weight of pups in group II/5 (m/s, prenatal MO-exposure) was significantly lower both in males and females. Significant differences were obtained by one-way ANOVA on PD 7 [F(5,84)=7.03, P<0.001] and on PD 21 [F(5,84)=19.16, P<0.001]. Post-hoc analysis showed significantly lower body weight both in males and females following MO withdrawal after parturition (group II/5, m/s) on PD 7 and for females also on PD 21, compared to SAL-exposed animals (group II/3, s/s). Body weight of those pups whose MO exposure started after parturition (group II/4, s/m) was significantly lower in females on PD 7 and PD 21 and in males on PD 21. Significant sex difference was observed on PD 21 when the body weight of females was significantly lower in all the three groups.

Table 2

Table 2

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Locomotor activity

Experiment I

There was no difference in the open field locomotor activity between the SAL-exposed (group I/1, s/s) and the MO-exposed (group I/2, m/m) pups on PD 23 [F(3,32)=1.04; Fig. 1, panel a]. In the subsequent 30 min (habituation) the locomotor activity of MO-exposed offspring (Group I/2, m/m) was higher on PD 23, than that of the SAL-exposed ones (group I/1, s/s). Two-way ANOVA analysis revealed a significant exposure difference in males [F(1,48)=8.38, P<0.01], but not in females. Analysis of each session separately by unpaired t-test, however, showed a significant difference at the 30 min interval both in males and females [t=2.63 for males and 2.40 for females, (d.f.=16), P<0.05 in both cases; Fig. 2, panel a].

Fig. 1

Fig. 1

Fig. 2

Fig. 2

On PD 42 ANOVA analysis of the open field activity revealed significance [F(3,32)=10.2, P<0.001]; however, the post-hoc test showed sex, but no exposure difference. There were neither exposure nor sex differences in locomotor activity during habituation at PD 42. The results show that habituation to novel surroundings was slower in perinatally MO exposed pups 2 days after weaning (data not shown).

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Experiment II

There was no difference in the open field locomotor activity of pups exposed to SAL-(group II/3, s/s), to MO postnatally (group II/4, s/m) or to MO prenatally (group II/5, m/s) [F(5,44)=1.04] on PD 23 (Fig. 1, panel b). In the subsequent 30 min (habituation) the locomotor activity of both the postnatally MO-exposed (group II/4, s/m) and the prenatally MO-exposed (group II/5, m/s) male pups was significantly higher than that of the SAL-exposed (group II/3, s/s) ones [F(1,54)=8.96, P<0.01 and F(1,39)=19.96, P<0.001, respectively]. The locomotor activity of male pups exposed to MO during gestation (group II/5, m/s) was significantly higher [F(1,45)=4.17, P<0.05] than those exposed to MO during lactation (group II/4, s/m). The results show that habituation of both MO-exposed male groups was slower; the difference was more marked in the case of prenatal exposure. There was no difference in the habituation between the female groups (Fig. 2, panel b).

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Tail-flick test

Experiment I

Sensitivity of MO-exposed male offspring (group I/2, m/m) to the antinociceptive effect of MO was lower on PD 24 (3 days after weaning) than that of the SAL-exposed ones (group I/1, s/s) and a moderate decrease of sensitivity was observable in the group of MO-exposed females, too. 95% CL values depicted in Table 3 show no overlap in the case of males. As another approach, a test dose of MO causing an MPE of approximately 50% was selected. This common dose was 3 mg/kg. Applying one-way ANOVA to the data sets of MPEs obtained with this dose, a significant difference [F(3,16)=4.95, P<0.05] was shown; Newman–Keuls post-hoc test indicated a P value of 0.05 for males.

Table 3

Table 3

The tendency of difference was observed even on PD 90 in males; however, the difference was already not statistically significant. In the case of females, the moderate decrease absolutely disappeared by PD 90 (Fig. 3). ED50 (95% CL) values are shown in Table 3.

Fig. 3

Fig. 3

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Experiment II

There was no difference in sensitivity to the antinociceptive effect of MO among the offspring exposed to SAL (group II/3, s/s), exposed to MO postnatally (group II/4, s/m) or prenatally (group II/5, m/s), checked on PD 24. However, the prenatally MO-exposed females showed a moderate, albeit not significant reduction (Fig. 4).

Fig. 4

Fig. 4

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Conditioned place preference

Experiment I

MO (1 and 3 mg/kg) failed to induce CPP in SAL-exposed (group I/1, s/s) males; however, it induced a moderate place preference in MO exposed (group I/2, m/m) animals. The difference was statistically significant [F(3,28)=11.70, P<0.001]. Sensitivity of females to the CCP-inducing effect of MO was more marked both in SAL and MO-exposed groups than that of males. In females the difference between the SAL-exposed (group I/1, s/s) and the MO-exposed (group I/2, m/m) animals was highly significant [F(3,28)=49.01, P<0.001]. The Newman–Keuls post-hoc test showed significant exposure differences for both challenge doses of MO (Fig. 5, panel a).

Fig. 5

Fig. 5

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Experiment II

Animals exposed to MO during gestation (group II/5, m/s) displayed significantly higher place preference after MO conditioning (3 mg/kg) than animals exposed to MO during lactation (group II/4, s/m) (t=5.86 for males and 12.06 for females (d.f.=14) P<0.001 in both cases; Fig. 5, panel b).

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Discussion

The aim of the present experiments was to differentiate the behavioural consequences of peri, pre and postnatal MO exposure in rats. As far as we know this is the first comprehensive long-term behavioural study on the consequences of a complete perinatal MO exposure.

In preliminary studies (not shown) a starting dose of 5 mg/kg MO twice a day was used and the dose was increased gradually. The maximal dose (30 mg/kg twice daily) was reached on the 5th day, and then this dose was continued. Although all the dams survived throughout the experiment, there was no normal parturition in the group of MO-treated dams and abortion, premature delivery and death of pups after birth were observed. The average size of litters was less than one. Siddiqui et al. (1997) managed a long MO treatment before and during pregnancy with relatively high doses (the maximal dose during gestation was 30 mg/kg/day) and reported that only 43% of MO-treated dams became pregnant and the number of still-births was significantly higher. That is why in the present series of experiments a constant medium dose of MO (10 mg/kg) was applied once a day. This schedule did not result in differences in the weight gain of dams, the size of litters, or in pup mortality, indicating that the dose of MO did not influence the physical state of dams.

Prenatal MO-exposure significantly decreased the birth weight of offspring measured on PD 1. Similar results have been published following prenatal methadone exposure (Kunko et al., 1996), while Slamberova et al. (2005) and Gagin et al. (1997), did not observe reduced birth weight as a consequence of MO exposure. In their experiments, however, shorter period of MO exposure and other strains (Sprague--Dawley and Fischer 344, respectively) were applied.

The weight gain of pups varied according to the period of MO exposure. When the animals were exposed to MO during the whole perinatal period (experiment I) their weight gain was significantly higher than that of the SAL-exposed peers. The decreased birth weight of MO exposed offspring observed by us at the beginning and followed by a weight gain similar to control peers, are in good correlation with human data. Birth weight of human babies born with neonatal abstinence syndrome was significantly lower compared to control ones, while no difference was shown at the age of 18 months (Hunt et al., 2008). In contrast to our results Siddiqui et al. (1997) found significant slowness in weight gain until PD 60, following a much more marked MO exposure.

In contrast to complete perinatal MO exposure (experiment I), both withdrawal of MO after parturition or starting the MO exposure after parturition only resulted in lower weight gain (experiment II). Why MO-exposure through maternal milk resulted in reduced weight gain in animals exposed only postnatally (experiment II), while just the opposite, higher weight gain was observed in pups exposed already prenatally (experiment I) remains to be elucidated. One possible explanation may be that prenatally MO-exposed pups were already tolerant to the anorexigenic effect of MO. This is supported by the results, that decreased sensitivity to the antinociceptive action of MO in the tail-flick test was also shown.

We can also presume that administration of MO to MO-naïve dams until parturition disturbed the maternal behaviour and/or lactation, while it failed to influence these behavioural patterns in the dams already treated with MO during gestation. Our results that withdrawal of MO after parturition also reduced the weight gain of pups support the role of disturbed maternal behaviour. In this case, however, we cannot exclude the direct effect of MO withdrawal on pups.

In our experiments the complete perinatal MO exposure (experiment I) resulted in delayed habituation to a new environment, decreased sensitivity to the antinociceptive effects of MO and enhanced sensitivity to the reinforcing effect of MO. Sex differences in these behavioural tests were also observed.

The development of tolerance to the antinociceptive effect of MO in newborn rats has been reported earlier. In the majority of these reports, however, the pups were treated with twice-daily high dose (20 mg/kg) MO (Van Praag and Frenk, 1991; Barr andt Wang, 1992), or opioids were administered by infusion (Thornton and Smith, 1997; Thornton et al., 1997). Reduced analgesic response to MO challenge was found in 4-day-old pups exposed perinatally to methadone or buprenorphine through maternally implanted osmotic minipumps (Robinson and Wallace, 2001), or on PD 14 in offspring whose mother was treated s.c. with MO during gestation and lactation (Chiou et al., 2003). None of these papers reported sex differences.

It is necessary to emphasize that, in contrast to the above cited papers, we measured decreased sensitivity to the antinociceptive action of MO 3 days after weaning (i.e. 3 days after cessation of MO exposure) and found this to be significant only in males. In addition, more than 2 months after cessation of MO exposure (PD 90) a tendency to respond only to higher doses of MO was still detectable.

A large body of literature shows sex differences in the sensitivity to the acute effects of opioids in sexually mature adult rats. Males were more sensitive to the antinociceptive action (Cicero et al., 1997; Craft et al., 1999), or to the locomotor suppressant effect of MO (Craft et al., 2006). Both MO-naïve and MO-tolerant neonatal rats, however, were reported to respond sex-independently to the antinociceptive effect of MO or fentanyl (Thornton and Smith, 1997; Thornton et al., 1997) and no sex difference in the reduced analgesic response was observed in 20-day-old rats treated daily with 3 mg/kg MO from PD 1 until PD 9 (Zhang and Sweitzer, 2008). In contrast to these results, we observed a greater decrease in the analgesic effect of MO in males 3 days after weaning; the differences in the results can be explained by the different postnatal ages and/or the different methods to develop tolerance.

The delayed habituation to the new environment measured 2 days after weaning was also more marked in males in our experiments. Our results are in contrast to some recent data which indicate that opioid-naïve periadolescent (PD 29) males are less active in a novel environment than females (White et al., 2008). It is important to emphasize, however, that we observed the delayed extinction of investigatory behaviour in perinatally MO-exposed rats.

However, the role of withdrawal 2 days after weaning must be taken into consideration, and the sex differences found in habituation might be related to this factor. MO-dependent males show higher sensitivity than females to naloxone precipitated withdrawal (Craft et al., 1999). Although naloxone decreases locomotor activity in adult MO-dependent rats, which is considered as a sign of withdrawal (Schulteis et al., 1994; Timár et al., 2005), in neonatal MO-dependent rats naloxone enhanced locomotor activity (Thornton et al., 1997; Stoller et al., 2002). If the delayed habituation that we observed can be considered as a moderate spontaneous withdrawal symptom, the more marked withdrawal in males may explain the slower habituation. The role of withdrawal in delayed habituation is supported by the result that three weeks later, in late adolescence (PD 42) no deficit in habituation was shown. This hypothesis, however, cannot explain the results of experiment II, where higher delay in habituation was observed in animals exposed to MO during gestation than during lactation.

In the present experiments the place preference-inducing effect of MO significantly increased in perinatally MO-exposed adult animals in contrast to a reduced analgesic effect of MO measured in MO-exposed offspring after weaning and partly in adulthood. The reinforcing effect of MO in CPP, however, was significantly higher in the case of prenatal than postnatal MO exposure. While no SAL-exposed ‘control’ animals were studied in experiment II, comparing the results of experiments I and experiment II in the CPP test, it seems that MO exposure through the maternal milk did not influence the MO sensitivity of adult animals.

The enhanced reinforcing capacity of MO in perinatally MO-exposed animals is not surprising, although data in the literature are rather equivocal. While Riley and Vathy (2006) did not show enhanced rewarding properties, either in CPP or in self-administration in adult males exposed to MO prenatally, Ramsey et al. (1993) reported that prenatal MO exposure enhanced heroin and cocaine self-administration in adult males. Gagin et al. (1996, 1997) also reported enhanced sucrose preference and higher place preference for MO, both in males and females exposed to MO from GD 11 to 18, but sex differences were not found. In contrast, in our experiments, the enhanced reinforcing effect of MO was more marked in females. In addition, sex difference was also observed in perinatally SAL-exposed ‘control’ animals. According to Shoaib et al. (1995) 5 mg/kg MO induced a significant CPP in Wistar rats. In our CPP experiments we intended to choose MO doses, which do not induce strong reinforcing effects; therefore, we administered MO at doses of 1 and 3 mg/kg, which failed to induce place preference in perinatally SAL-exposed males. However, these doses of MO did induce a CPP in SAL-exposed females.

Though human data are rather controversial, females appear to be more vulnerable than males to the reinforcing effect of opiates, psychostimulants and nicotine during many phases of the addiction process, for example, acquisition, maintenance, disregulation-escalation, relapse (Lynch et al., 2002). In animal experiments female rats were found to be more vulnerable than males to the acquisition of cocaine and heroin self-administration (Lynch and Carroll, 1999). Mu-opioid receptor agonists showed higher reinforcing effect in females than in males (Cicero et al., 2003). Female rats discriminated MO at lower doses than males and acquired it in significantly fewer sessions (Craft et al., 1996). Sex difference was also reported by Vathy et al. (2007) for cocaine reward in adult animals exposed to MO from GD 11 to 18; however, prenatal MO exposure did not affect cocaine reward. All these data indicate that female rats are more sensitive to the rewarding effect of abused drugs.

The mechanism underlying the sex differences are not yet understood but sex-related alterations in pharmacokinetic characteristics do not seem to provide an explanation. Cicero et al. (1997) did not find sex-related differences in adult rats following single doses of MO in a range from 2.5 to 15 mg/kg, either in elimination half-life or in disappearance from the brain.

The elimination half life of MO alters in line with the age of pups: it was measured to be 2.5 h in neonates and 26 min in weanlings (Windh and Kuhn, 1995). However, no data were found in the literature about MO levels in the brain of pups exposed to MO pre or postnatally. Kunko et al. (1996) measured the methadone level in the brain of pups exposed to methadode either prenatally in utero or postnatally through maternal milk. While no detectable quantity was measured 4 days after parturition in the case of prenatal exposure, a measurable quantity of methadone was detected in the brain of pups exposed to it through maternal milk, but this decreased gradually as the pups aged. Even if the different pharmacokinetic properties of methadone are taken into consideration, we presume that the presence of MO in the brain of pups during prenatal or postnatal exposure may not qualitatively differ from that measured after methadone exposure.

Some studies suggested differences in the density of opioid receptors in the preoptic area in males and females (Hammer, 1984), or showed that adult females possess smaller μ-receptor reserve or less μ-receptor mediated signal transduction, than males (Craft et al., 2001). If perinatal MO exposure decreases the μ-receptor reserve in offspring, we may hypothesize that the same degree of change results in more marked consequences when the reserve is higher, and this may explain the more marked decrease in sensitivity to the antinociceptive effect of MO in males. The finding that the decrease of μ-receptor binding density in the preoptic area was more marked in perinatally MO-exposed males than in females (Hammer et al., 1991), supports this hypothesis. Other data (Windh et al., 1995; Stoller et al., 2002), however, speak against the role of receptor density in sex differences.

The long-term consequences of perinatal opioid exposure on transmitters and hormones, especially female gonadal hormones, are rather equivocal. A three-fold increase in dopamine (DA) content was measured in prenatally methadone-exposed females in the hypothalamus (Robinson et al., 1991) but not in the striatum (Vathy et al., 1994; Robinson et al., 1997) on PD 21. In contrast, Siddiqui et al. (1997) observed no changes in the DA level of the hypothalamus and amygdala in perinatally MO-exposed adult female rats. Sexually dimorphic alteration of norepinephrine level was observed in the hypothalamus of adult prenatally MO-exposed animals. Decreased levels were measured in females (Vathy and Katay, 1992; Siddiqui et al., 1997) but an increase was observed in males (Vathy and Katay, 1992) and similar (but not significant) sex difference was observed in frontal cortex. No change of norepinephrine content was reported in the striatum (Vathy et al., 1994) and in the amygdala (Siddiqui et al., 1997).

The effects of gonadectomy on sensitivity to the antinociceptive effects of morphine are rather inconsistent (Kepler et al., 1989; Candido et al., 1992; Ali et al., 1995). The role of gonadal hormones might be especially important when the reinforcing effect of MO is studied. In animal experiments female gonadal hormones enhance DA release in the striatum and in accumbens (Becker, 1999), the brain regions that mediate the positive reinforcing effects of abused drugs. Data obtained for humans with modern imaging techniques suggest that women have higher synaptic concentration of DA in the striatum, which may be associated with sex differences in vulnerability (Laakso et al., 2002).

As for the endocrinological consequences, Siddiqui et al. (1997) found reduced plasma estradiol, ovarian estradiol and ovarian progesterone levels in perinatally MO-exposed adult females, which indicates that the enhanced rewarding effect of MO cannot be explained simply by the changes in gonadal hormone system.

Summarizing, the major findings of the present paper are the followings:

  • (1) We have found differences in the consequences of peri, pre and postnatal MO exposure according to the period of MO exposure. Birth weight of pups exposed to MO in utero was significantly lower. However, the weight gain after parturition was normal or even higher when the exposure was continued through maternal milk. In contrast, when the MO exposure started only after parturition through maternal milk, or when MO was withdrawn after parturition, the weight gain was slower.
  • (2) Reduced sensitivity to the antinociceptive effect of MO was observed in the tail-flick test when the MO exposure occurred during the whole perinatal period.
  • (3) Habituation to a new environment was slower in all the peri, pre or postnatally MO-exposed male pups.
  • (4) Based on the CPP results, the enhanced sensitivity of peri or prenatally MO-exposed offspring to the rewarding effect of MO in adulthood might be considered as a predictive model of ‘vulnerability’.

In conclusion, while the procedure of MO exposure that we have used does not simulate exactly the drug-taking habits of pregnant women, we can state that even this relatively small, constant once-daily administration of MO during gestation and lactation results in long-term consequences in the offspring and may make them more vulnerable to MO abuse in adulthood.

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Acknowledgements

Sponsorship: This study was supported by Hungarian grants OTKA K-60999 and ETT-441/2006.

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References

Ali BH, Sharif SI, Elkadi A 1995. Sex-differences and the effect of gonadectomy on morphine-induced antinociception and dependence in rats and mice. Clin Exp Pharmacol Physiol 22:342–344.
Barr GA, Wang SN 1992. Tolerance and withdrawal to chronic morphine treatment in the week-old rat pup. Eur J Pharmacol 215:35–42.
Becker JB 1999. Gender differences in dopaminergic function in striatum and nucleus accumbens. Pharmacol Biochem Behav 64:803–812.
Candido J, Lutfy K, Billings B, Sierra V, Duttaroy A, Inturrisi CE, Yoburn BC 1992. Effect of adrenal and sex-hormones on opioid analgesia and opioid receptor regulation. Pharmacol Biochem Behav 42:685–692.
Carboni E, Acquas E, Leone P, Dichiara G 1989. 5HT3 receptor antagonists block morphine-induced and nicotine-induced but not amphetamine-induced reward. Psychopharmacology 97:175–178.
Chiou LC, Yeh GC, Fan SH, How CH, Chuang KC, Tao PL 2003. Prenatal morphine exposure decreases analgesia but not K+ channel activation. Neuroreport 14:239–242.
Cicero TJ, Nock B, Meyer ER 1997. Sex-related differences in morphine's antinociceptive activity: relationship to serum and brain morphine concentrations. J Pharmacol Exp Ther 282:939–944.
Cicero TJ, Aylward SC, Meyer ER 2003. Gender differences in the intravenous self-administration of mu opiate agonists. Pharmacol Biochem Behav 74:541–549.
Craft RM, Kalivas PW, Stratmann JA 1996. Sex differences in discriminative stimulus effects of morphine in the rat. Behav Pharmacol 7:764–778.
Craft RM, Stratmann JA, Bartok RE, Walpole TI, King SJ 1999. Sex differences in development of morphine tolerance and dependence in the rat. Psychopharmacology (Berl) 143:1–7.
Craft RM, Tseng AH, McNiel DM, Furness MS, Rice KC 2001. Receptor-selective antagonism of opioid antinociception in female versus male rats. Behav Pharmacol 12:591–602.
Craft RM, Clark JL, Hart SP, Pinckney MK 2006. Sex differences in locomotor effects of morphine in the rat. Pharmacol Biochem Behav 85:850–858.
Fanselow MS, Cramer CP 1988. The ontogeny of opiate tolerance and withdrawal in infant rats. Pharmacol Biochem Behav 31:431–438.
Furst Z, Buzas B, Friedmann T, Schmidhammer H, Borsodi A 1993. Highly potent novel opioid receptor agonist in the 14-alkoxymetopon series. Eur J Pharmacol 236:209–215.
Gagin R, Cohen E, Shavit Y 1996. Prenatal exposure to morphine alters analgesic responses and preference for sweet solutions in adult rats. Pharmacol Biochem Behav 55:629–634.
Gagin R, Kook N, Cohen E, Shavit Y 1997. Prenatal morphine enhances morphine-conditioned place preference in adult rats. Pharmacol Biochem Behav 58:525–528.
Hammer RP Jr 1984. The sexually dimorphic region of the preoptic area in rats contains denser opiate receptor binding sites in females. Brain Res 308:172–176.
Hammer RP Jr, Seatriz JV, Ricalde AR 1991. Regional dependence of morphine-induced mu-opiate receptor down-regulation in perinatal rat brain. Eur J Pharmacol 209:253–256.
Hunt RW, Tzioumi D, Collins E, Jeffery HE 2008. Adverse neurodevelopmental outcome of infants exposed to opiate in-utero. Early Hum Dev 84:29–35.
Kepler KL, Kest B, Kiefel JM, Cooper ML, Bodnar RJ 1989. Roles of gender, gonadectomy and estrous phase in the analgesic effects of intracerebroventricular morphine in rats. Pharmacol Biochem Behav 34:119–127.
Kunko PM, Smith JA, Wallace MJ, Maher JR, Saady JJ, Robinson SE 1996. Perinatal methadone exposure produces physical dependence and altered behavioral development in the rat. J Pharmacol Exp Ther 277:1344–1351.
Laakso A, Vilkman H, Bergman J, Haaparanta M, Solin O, Syvalahti E, et al. 2002. Sex differences in striatal presynaptic dopamine synthesis capacity in healthy subjects. Biol Psychiatry 52:759–763.
Lynch WJ, Carroll ME 1999. Sex differences in the acquisition of intravenously self-administered cocaine and heroin in rats. Psychopharmacology (Berl) 144:77–82.
Lynch WJ, Roth ME, Carroll ME 2002. Biological basis of sex differences in drug abuse: preclinical and clinical studies. Psychopharmacology (Berl) 164:121–137.
Ramsey NF, Niesink RJ, Van Ree JM 1993. Prenatal exposure to morphine enhances cocaine and heroin self-administration in drug-naive rats. Drug Alcohol Depend 33:41–51.
Riley MA, Vathy I 2006. Mid to late gestational morphine exposure does not alter the rewarding properties of morphine in adult male rats. Neuropharmacology 51:295–304.
Robinson SE, Wallace MJ 2001. Effect of perinatal buprenorphine exposure on development in the rat. J Pharmacol Exp Ther 298:797–804.
Robinson SE, Guo HZ, McDowell KP, Pascua JR, Enters EK 1991. Prenatal exposure to methadone affects central cholinergic neuronal activity in the weanling rat. Brain Res Dev Brain Res 64:183–188.
Robinson SE, Maher JR, Wallace MJ, Kunko PM 1997. Perinatal methadone exposure affects dopamine, norepinephrine, and serotonin in the weanling rat. Neurotoxicol Teratol 19:295–303.
Schindler CJ, Slamberova R, Rimanoczy A, Hnactzuk OC, Riley MA, Vathy I 2004. Field-specific changes in hippocampal opioid mRNA, peptides, and receptors due to prenatal morphine exposure in adult male rats. Neuroscience 126:355–364.
Schulteis G, Markou A, Gold LH, Stinus L, Koob GF 1994. Relative sensitivity to naloxone of multiple indices of opiate withdrawal: a quantitative dose-response analysis. J Pharmacol Exp Ther 271:1391–1398.
Shoaib M, Spanagel R, Stohr T, Shippenberg TS 1995. Strain differences in the rewarding and dopamine-releasing effects of morphine in rats. Psychopharmacology (Berl) 117:240–247.
Siddiqui A, Haq S, Shah BH 1997. Perinatal exposure to morphine disrupts brain norepinephrine, ovarian cyclicity, and sexual receptivity in rats. Pharmacol Biochem Behav 58:243–248.
Sithisarn T, Bada HS, Dai H, Reinhardt CR, Randall DC, Legan SJ 2008. Effects of perinatal oxycodone exposure on the response to CRH in late adolescent rats. Neurotoxicol Teratol 30:118–124.
Slamberova R, Rimanoczy A, Riley MA, Vathy I 2004. Hypothalamo-pituitary-adrenal axis-regulated stress response and negative feedback sensitivity is altered by prenatal morphine exposure in adult female rats. Neuroendocrinology 80:192–200.
Slamberova R, Riley MA, Vathy I 2005. Cross-generational effect of prenatal morphine exposure on neurobehavioral development of rat pups. Physiol Res 54:655–660.
Stoller DC, Thornton SR, Smith FL 2002. Loss of antinociceptive efficacy in rat pups infused with morphine from osmotic minipumps. Pharmacology 66:11–18.
Thornton SR, Smith FL 1997. Characterization of neonatal rat fentanyl tolerance and dependence. J Pharmacol Exp Ther 281:514–521.
Thornton SR, Wang AF, Smith FL 1997. Characterization of neonatal rat morphine tolerance and dependence. Eur J Pharmacol 340:161–167.
Timár J, Gyarmati Z, Furst Z 2005. The development of tolerance to locomotor effects of morphine and the effect of various opioid receptor antagonists in rats chronically treated with morphine. Brain Research Bulletin 64:417–424.
Van Praag H, Frenk H 1991. Evidence for opiate tolerance in newborn rats. Brain Res Dev Brain Res 60:99–102.
Vathy I, Katay L 1992. Effects of prenatal morphine on adult sexual behavior and brain catecholamines in rats. Brain Res Dev Brain Res 68:125–131.
Vathy I, Rimanoczy A, Eaton RC, Katay L 1994. Modulation of catecholamine turnover rate in brain regions of rats exposed prenatally to morphine. Brain Res 662:209–215.
Vathy I, Slamberova R, Liu X 2007. Foster mother care but not prenatal morphine exposure enhances cocaine self-administration in young adult male and female rats. Dev Psychobiol 49:463–473.
White DA, Michaels CC, Holtzman SG 2008. Periadolescent male but not female rats have higher motor activity in response to morphine than do adult rats. Pharmacol Biochem Behav 89:188–199.
Windh RT, Kuhn CM 1995. Increased sensitivity to mu opiate antinociception in the neonatal rat despite weaker receptor-guanyl nucleotide binding protein coupling. J Pharmacol Exp Ther 273:1353–1360.
Windh RT, Little PJ, Kuhn CM 1995. The ontogeny of mu opiate tolerance and dependence in the rat: antinociceptive and biochemical studies. J Pharmacol Exp Ther 273:1361–1374.
Wu CC, Chen JY, Tao PL, Chen YA, Yeh GC 2005. Serotonin reuptake inhibitors attenuate morphine withdrawal syndrome in neonatal rats passively exposed to morphine. Eur J Pharmacol 512:37–42.
Zhang GH, Sweitzer SM 2008. Neonatal morphine enhances nociception and decreases analgesia in young rats. Brain Research 1199:82–90.
Keywords:

antinociception; conditioned place preference; morphine exposure; offspring; perinatal; rat; sex differences; tolerance

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