HPA axis dysregulation following prenatal opiate exposure and postnatal withdrawal

Neurotoxicology and Teratology 27 (2005) 95 – 103 www.elsevier.com/locate/neutera

HPA axis dysregulation following prenatal opiate exposure and postnatal withdrawal Kathryn L. Hamiltona, Andrew C. Harrisa, Jonathan C. Gewirtza,b,c, Sheldon B. Sparberc,d, Lisa M. Schrotte,* a Department of Psychology, University of Minnesota, Minneapolis, MN 55455, United States Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455, United States c Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN 55455, United States d Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455, United States e Department of Pharmacology and Therapeutics, Louisiana State University Health Sciences Center, 1501 Kings Highway, PO Box 33932, Shreveport, LA 71130-3932, United States b

Received 11 June 2004; received in revised form 20 September 2004; accepted 21 September 2004

Abstract We examined the effects of prenatal exposure to the long acting opiate l-alpha-acetylmethadol (LAAM) followed by postnatal withdrawal on hypothalamic–pituitary–adrenal (HPA) axis reactivity in neonatal and adult rats and anxiety-like behavior in adult rats. Female rats were treated with LAAM (0, 0.2, or 1.0 mg/kg/day) via oral gavage for 28 days prior to and continuing throughout pregnancy. Pups were fostered at birth to nontreated, lactating dams and underwent opiate withdrawal. On postnatal day (PND) 18, prenatal opiate-exposed male and female rat pups displayed a decreased corticosterone response 2 h after the application of an immunological stressor and 15 min following a social stressor compared to controls. In contrast, in adulthood, prenatal opiate-treated rats showed a heightened corticosterone response compared to prenatal water-treated controls at 3 h, but not 8 h, following an immunological stressor. Males prenatally treated with 1.0 mg/kg LAAM displayed elevated startle responding compared to the other prenatally treated male groups, but there was no effect of prenatal treatment in females. There were no effects of prenatal treatment in the open field test in either sex. These results suggest that prenatal opiate exposure followed by postnatal withdrawal dysregulated the HPA axis response to stressors in the neonate and adult and differentially affected adult anxiety-like behavior in males and females. D 2004 Elsevier Inc. All rights reserved. Keywords: HPA axis; Anxiety; Prenatal opiate; Acoustic startle; LAAM

1. Introduction According to the National Survey on Drug Use and Health, 3% of pregnant women had used drugs in 2002 [30], and 19% of pregnant women in rehabilitation programs were using opiates in 1999 [28]. Currently, buprenorphine, l-alpha-acetylmethadol (LAAM), and methadone are all approved for use in the treatment of opiate dependence [29], but only methadone is approved for use during pregnancy in the United States [17]. Fetal exposure to methadone, * Corresponding author. Tel.: +1 318 6757184; fax: +1 318 6757857. E-mail address: [email protected] (L.M. Schrott). 0892-0362/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2004.09.004

however, results in a neonatal abstinence syndrome that can be prolonged and require pharmacological management. Withdrawal signs include irritability, high shrill crying, poor suckling, and inability to sleep (reviewed in Ref. [1]). Prenatal methadone exposure also results in long-term consequences, including fewer goal-directed behaviors, more speech and cognitive deficits, and poorer social skills [2,12]. Children exposed to methadone also show greater anxiety and aggression and poorer fine and gross motor coordination than other children [3,10]. However, human studies may be confounded by environmental factors such as socioeconomic status and maternal care, making it difficult to determine direct effects of prenatal opiate

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dependence, as well as the contribution of the stress of postnatal withdrawal [10,26]. Animal studies have also found behavioral effects of prenatal methadone. Acoustic startle responding is elevated in the juvenile rat after prenatal methadone exposure [16]. Prenatal methadone also alters motor development, pain sensitivity, and morphine self-administration in adult rats [7,15]. However, these studies have some limitations due to the short half-life of methadone in the rat (i.e., less than 3 h) [25], which may have resulted in unintended episodes of withdrawal during prenatal opiate administration [23]. In contrast, because LAAM has active metabolites that prolong its duration of action [14], a single daily dose of LAAM results in continuous dependence in rats (e.g. [22]), making it a good choice for rodent studies. Doses of LAAM during pregnancy that do not induce toxicity [42] can alter neural and behavioral parameters, such as decreasing overall protein levels in the neonatal brain, increasing adult pain sensitivity to noxious stimuli, and reducing the effects of amphetamine on adult operant behavior [21–24]. The aim of the present study was to further determine the consequences of prenatal LAAM dependence followed by neonatal withdrawal. Our focus was on stress responding and anxiety, as these are measures relevant to clinical and behavioral manifestations reported following developmental perturbations, including prenatal drug and prenatal stress exposure. The literature from human studies suggests that there are differences in the hypothalamic–pituitary–adrenal (HPA) axis response to stressors in juveniles and adults following early stress ([6,13], reviewed in [9]). While the effects of prenatal and/or neonatal stress on altered adult stress responding have been well documented in rodents (e.g. [27,40]), there has been less investigation of these effects on juvenile rodents. Thus, our first goal was to measure the stress response in juvenile, as well as in mature rats, following postnatal withdrawal from prenatal LAAM exposure. Our second goal was to measure anxiety-like behaviors in rats exposed prenatally to LAAM followed by withdrawal postnatally since children exposed to methadone prenatally have increased levels of anxiety [3]. We used two well-established measures of rodent anxiety-like behavior: acoustic startle responding and the open field test (reviewed in [34]). Like alterations in HPA axis function, elevations in acoustic startle have been found after other developmental insults, such as exposure to infectious agents or cocaine [8,39]. Furthermore, acoustic startle is elevated in juvenile animals following neonatal methadone withdrawal [16], but to date it has not been examined in adult animals exposed to opiates prenatally. Thus, the current study investigated whether the elevation in startle following neonatal opiate withdrawal persists into adulthood, which would suggest that prenatal exposure and/or postnatal withdrawal produces a long-lasting increase in anxiety-like behavior, similar to what is seen in humans [3]. In addition, we included an analysis of gender effects in these experiments because

there has not been a systematic examination of interactions between gender and prenatal opiate treatment on these measures. This is important, especially in light of recent reports that prenatal opiate exposure can differentially affect adult mu opioid receptor density in male and female rats [38].

2. Method 2.1. Animals Group-housed nulliparous female Sprague–Dawley rats (7–8 weeks of age; Harlan, Madison, WI) weighing approximately 200 g at the start of the experiment were used as subjects. The animals were habituated to an oral gavage procedure using water before initiation of treatment. Treatments began on day 0 and the rats were gradually made chronically opiate dependent via treatments on days 3, 6, and 8, followed by daily treatments from days 10 to 28. After 28 days of treatment, females were harem-bred, with one male placed in each cage of three females. Treatment continued through the 10 days of breeding. Pregnancy was confirmed by a sperm-positive vaginal smear and/or appropriate weight gain. Drug treatment of the females continued during pregnancy until parturition. Females were weighed daily, and throughout gestation they were monitored for signs of opiate withdrawal, such as weight loss, diarrhea, piloerection, and irritability. Pregnant females were placed in individual nesting cages on approximately gestation day (GD) 14. From GD 20–23 the pregnant dams were checked daily at 0700 and 1400 h for births. Litters were handled with gloved hands only after pups had been cleaned and fed. The latter was evidenced by visual inspection of milk in the pups’ stomachs. At this time, the pups were sexed and weighed. Within 12 to 36 h of parturition, all pups were fostered, such that pups from both Water- and LAAM-treated dams were fostered to nontreated dams. The foster mothers had been handled and weighed weekly, but were otherwise undisturbed during pregnancy. In two instances (one Water and one 1.0 mg/kg LAAM), pups were combined from like-treated litters because of small litter size. Pups were culled to 12 per litter at fostering, balancing by sex when possible (6 males and 6 females). Pups were weighed daily for the first 8 days, then at postnatal day 14 (PND 14) and PND 21. Changes in body weight were used to index post-parturition opiate withdrawal since this is a highly sensitive measure of neonatal withdrawal [21,22]. Eye opening was assessed from PND 14–16. A subset of subjects was assessed for HPA axis responsivity on PND 18. The remaining pups were weaned at PND 21, with 3–5 same-sex littermates housed per cage. The offspring were weighed weekly until approximately 15 weeks of age, when non-littermate subjects from each treatment were randomly selected for testing. Separate

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subjects were tested for acoustic startle reactivity, in the open field, and for adult HPA axis responsivity. All animals were maintained on a 12:12 light/dark schedule, with lights on at 0700 or 0800. Food and water were provided ad libitum throughout the experiment. All animal experimentation was conducted at the University of Minnesota. Experimental procedures conformed to the Guidelines for the Care and Use of Laboratory Animals and were approved by University of Minnesota Animal Care and Use Committee. 2.2. Drugs 2.2.1. LAAM Two doses of LAAM HCl (kindly provided by the National Institute on Drug Abuse through the Research Triangle Institute, Research Triangle Park, NC) were used: 1.0 and 0.2 mg/kg/day. LAAM was dissolved in tap water filtered with a 0.2 Am filter. An 18-gauge, 7.6 cm long gavage tube (Popper and Sons, New Hyde Park, New York) was used for each drug administration. Gavage volumes were 1.0 ml/kg. The 0.2 mg/kg LAAM dose was based on prior studies demonstrating continuous dependence without significant toxicity [21], while the 1.0 mg/kg dose was chosen because prior studies indicated that higher doses (e.g., 2.0 mg/kg) led to significant neonatal morbidity [21,42]. 2.2.2. Lipopolysaccharide (LPS) LPS (serotype 0128:B12; Sigma, St. Louis, MO) was dissolved in 0.9% NaCl solution to form a 100 Ag/kg dose. LPS was injected i.p. in a volume of 1.0 ml/kg. 2.3. HPA axis responsivity HPA axis responsivity was measured in both neonatal (PND 18) and mature offspring (20 weeks of age). On PND 18, basal corticosterone or corticosterone concentrations following social isolation or immune challenge were measured. There was at least one male or female per treatment condition within each litter. Littermate means were used for analysis if there were multiple subjects per treatment. For the adult assessment, male and female rats (no more than one male and female per litter) were injected with LPS to measure the adult responsivity to an immunological stressor. 2.3.1. Procedure for neonatal response Rats in the immune challenge group received LPS and were placed back into the home cage with littermates and dam for 2 h. Animals that received the social isolation treatment were individually placed in a tub with fresh bedding for 15 min in a room outside of the colony room. Non-stress control animals were left undisturbed in the home cage. The assessment times of 2 h for LPS and 15 min for isolation were based on preliminary studies demonstrat-

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ing corticosterone elevations in control animals. Trunk blood was obtained via rapid decapitation of unanesthetized rats. All blood collection was done between 0930 and 1045. Blood samples were collected in tubes containing EDTA as an anti-coagulant and then kept on ice until centrifuged to obtain plasma. Plasma samples were stored at 80 8C until analysis. 2.3.2. Procedure for adult response Rats were injected with LPS and returned to their home cages. At two time points (3 and 8 h) they were rapidly decapitated without anesthesia. Preliminary studies found that corticosterone elevations following LPS were evident by 3 h in control subjects. Blood sampling occurred between 1030 and 1245 for the 3-h time point and at 1530 to 1700 for the 8-h time point. At blood collection, samples were centrifuged to collect serum and frozen at 80 8C until analysis for corticosterone. 2.3.3. Corticosterone assay The plasma or serum samples were diluted 1:250 or 1:500 and heated at 100 8C for 10 min to denature corticosterone-binding globulin. The samples were then incubated with an antibody directed against corticosterone (ICN Biomedical, Costa Mesa, CA) and 3H-corticosterone (New England Nuclear, Boston MA). Charcoal was added and samples were centrifuged to separate bound 3Hcorticosterone. Bound 3H-corticosterone was counted in a liquid scintillation counter to an error of F1.5%. All samples and standards were run in triplicate. Standards were used to generate a curve from which sample values were interpolated. These values were subsequently converted and expressed as ng/ml plasma or sera. Assay sensitivity was between 0.5 and 1 ng/ml, assay range from 1 to 1000 ng/ml, the intra-assay coefficient of variation was 3.1% for the neonatal and 3.5% for the adult subjects, and the inter-assay coefficient of variation was 4.6% for the neonatal and 7.9% for the adult subjects. 2.4. Acoustic startle responsivity At 16.5 weeks of age, animals were tested for their responsiveness to acoustic startle stimuli. Acoustic startle responsivity is a marker of anxiety-like behavior, as greater startle responses have been seen during opiate withdrawal and in response to anxiogenic drugs [11,18,37]. 2.4.1. Apparatus Animals were tested in four identical Stabilimeter devices (81515 cm) made of Plexiglas cages on four compression springs in sound-attenuating chambers. Cage movement resulted in displacement of a Type 338B35 accelerometer (PCB Piezotronics, Depew, NY) attached to the top of the cage. The accelerometer voltage was amplified by a signal processor (Model 482A20; PCB Piezotronics) and was proportional to the velocity of cage

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displacement. An Instrunet 100b board (GW Instruments, Somerville, MA) interfaced to a Macintosh G3 microcomputer digitized the analog output of the accelerometer on a scale of 0–1000 units. Startle amplitude was defined as the peak accelerometer voltage that occurred during the first 200 ms after the onset of the startle stimulus. High frequency speakers (Radio Shack Supertweeters, range 5– 40 kHz) located 5 cm from the cage delivered the 50-ms (rise-decay: 5-ms) bursts of white noise (low pass, 22 kHz) at 90, 95, 100, and 105 dB. White noise through the high frequency speakers, together with the ventilation system, elevated the background noise to 67 dB. 2.4.2. Procedure Response to the acoustic startle stimuli was assessed across 3 days. The first 2 days served to habituate the rats to the chambers and stimuli, and to establish a stable startle baseline [11]. The data from the third day were used for analysis. Two males and two females were tested at the same time, with two chambers assigned to each gender for the duration of testing to minimize odor distractants. A test session consisted of a 5-min habituation period followed by 20 min of testing. During testing, an acoustic startle stimulus was presented every 30 s, with 10 stimuli of each intensity presented in a semi-random order during the session. After the testing session was over, the chambers were cleaned with 70% ethanol. The startle response was measured as the mean startle amplitude collapsed across all decibel levels [11]. 2.5. Open field test A subset of prenatally opiate-exposed offspring was used for open-field testing. One male and one female per litter were used. The open field test was used to measure emotionality-related behavior and locomotor activity. Testing occurred at approximately 20 weeks of age. 2.5.1. Apparatus The open field (90.7590.75 cm) was divided into 25 equal squares (18.1518.15 cm). The walls were 22.50 cm high. The floor of the open field was gray, and the walls were black; yellow lines demarcated the squares. The open field was placed on the floor in a dark room, and a 25-W white light bulb was attached to the ceiling above the center square, so the illumination was equal in all parts of the apparatus (2 lux). A low light-sensitive 8-mm video camera (Sony, Japan) recorded activity. 2.5.2. Procedure An animal was placed in the center square and allowed to move freely through the open field for 5 min. The open field was cleaned with 70% ethanol between testing sessions. Males and females were tested on different days to prevent odors from the opposite gender from altering behavior. Testing occurred from 1530 to 1900. Videotapes were

scored for latency to leave the center square, inner line crossings (a measure of emotionality-related behavior) and outer line crossings and rearings (as measures of locomotor behavior). Inner line crossings were defined as line crossings into one of the nine inner squares and outer line crossings were defined as line crossings into one of the 16 squares along the walls. Rearings were defined as the snout facing upwards with the front paws off the floor. Scoring was done by an observer blind to treatment. 2.6. Data analysis To determine changes resulting from LAAM administration prior to and during pregnancy, analyses of variance (ANOVAs) were used with Treatment as the betweensubjects measure and Treatment Day, if appropriate, as a within-subjects factor. Postnatal withdrawal measures were analyzed using a mixed-model ANOVA with Gender and Prenatal Treatment as between-subject factors and Day as a within-subjects factor. For the neuroendocrine and behavioral measures two-way ANOVAs with Gender and Prenatal Treatment as factors were used. Time post-injection was included as a between-subjects measure for the adult HPA axis assessment. Fisher’s PLSD planned comparisons or Dunnett’s contrasts were used to determine differences among the prenatal treatment groups. It should be noted that for the adult HPA axis study, there was one outlier (fell greater than 2 standard deviations below the mean) in the 1.0 mg/kg LAAM treatment group at 8 h post-LPS injection and that subject was eliminated from the analyses. Because of concerns about the large variability in the adult HPA axis measures, especially in the prenatal LAAM-treated groups, the data were assessed for heterogeneity of variance using the Brown–Forsyth test. There was no significant effect of Prenatal Treatment, thus the data were analyzed using parametric statistics. Data are depicted as meanFS.E.M. for all measures.

3. Results 3.1. Induction of opiate dependence Body weight was assessed across the initial 28 days of LAAM administration. There was a significant effect of Day ( F 14,28=55.34, pb0.0001) and a TreatmentDay interaction ( F 26,364=7.23, pb0.0001). There was no difference in the pre-treatment body weights (197–201 g across the three groups). But as treatment continued, the LAAM-treated rats gained weight at a slower rate than the water-treated controls. One-way ANOVAs revealed significant treatment effects for body weight on D16–D28, with rats treated with 1.0 mg/kg LAAM significantly lighter than controls ( pb0.05 for all; Fisher’s PLSD). There was no evidence of behavioral or somatic signs of opiate withdrawal during this phase (e.g., piloerection, diarrhea, and irritability),

K.L. Hamilton et al. / Neurotoxicology and Teratology 27 (2005) 95–103 Table 1 Pregnancy and birth statistics Prenatal treatment

Pregnancy rate Weight gained (g) % Weight gained # Live births Sex ratio (M:F)

Water

0.2 mg/kg LAAM

1.0 mg/kg LAAM

7/10 136.9F14.61 150.6F5.42 11.1F1.72 1.17

8/9 140.7F6.50 164.3F2.91 12.1F0.72 1.29

8/10 125.7F9.02 159.8F4.60 10.6F1.65 1.25

which is consistent with the long duration of action of LAAM and previous studies using this dosing paradigm (e.g. [36]). 3.2. Pregnancy and birth statistics Table 1 depicts the pregnancy and birth statistics. Treatment did not affect reproductive success, with all groups displaying similar pregnancy rates. There were differences in the absolute body weight before pregnancy among the groups (Water=234.6F6.54 g; 0.2 mg/kg LAAM=222.20F5.43; and 1 mg/kg LAAM=208.63F3.16). However, the amount of weight gained and the percent change in weight during pregnancy did not differ as a consequence of treatment (Table 1). The birth statistics also indicated a lack of toxicity for both doses of LAAM, as there was no difference in the number of live births or in the sex ratio at birth. 3.3. Post-parturition opiate withdrawal We assessed the presence of opiate withdrawal by examining weight loss after the cessation of the opiate treatment in both the dams and their offspring. For the females treated during pregnancy we determined weight change between parturition day and 2 days post-parturition. There was an overall effect of Prenatal Treatment ( F 2,18=4.56, pb0.03), with the dams treated with 1.0 mg/ kg LAAM losing significantly more weight than the other treated groups (Table 2). We also saw differences in weight gain in the prenatally opiate-treated pups. Although gender differences in body weight emerged as the animals aged, there were no

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interactions between gender and treatment, so for simplicity, males and females were combined for the following analyses and graphs. Repeated measure ANOVAs for body weight across D0–D5 revealed a significant Prenatal Treatment effect ( F 2,18=5.16, pb0.02; Fig. 1). There was no difference in birth weight (D0). However, after the pups were fostered to untreated dams and thus no longer receiving opiates, they underwent withdrawal, as indicated by reduced weight gain. A dose-related pattern was found, with the pups from dams treated prenatally with 1.0 mg/kg LAAM showing the least weight gain and those treated with the 0.2 mg/kg LAAM dose showing intermediate weight gain. The withdrawal-associated body weight reduction was transient, and by D5 there was no difference in body weight among the groups. This pattern continued throughout the neonatal period, with no differences in body weight at D21 (Table 2). There was also no difference in the number of pups per litter that died between birth and fostering or during the first week of life, and the time of eye opening did not differ amongst the groups (Table 2). 3.4. Neonatal HPA axis responsivity Circulating corticosterone was measured under three conditions: basal (non-stress), LPS challenge, and social isolation. There was no effect of Gender, or a Gender Prenatal Treatment interaction, so means were taken for all pups within a litter exposed to the same condition. Oneway ANOVAs were used to analyze the corticosterone concentrations for each manipulation. In the basal (nonstress) condition, there was no effect of prenatal treatment. In the prenatal water-treated controls, corticosterone was increased 4-fold following social isolation and 8-fold 2 h after LPS injection. However, as can be seen in Fig. 2, there was a blunted corticosterone response to LPS challenge and social isolation for both LAAM-treated groups (LPS

Table 2 Characteristics of dams and pups in the post-parturition and neonatal period Prenatal treatment Water Dam weight lost (g) Number of pup deaths Birth to fostering First week post-foster Day of eye opening Pup weight at D21 (g)

0.2 mg/kg LAAM

1.0 mg/kg LAAM

3.80F4.02

3.80F2.12

15.51F3.55*

0.33F0.33 0.00F0.00 15.32F0.11 47.85F2.61

0.00F0.00 0.12F0.12 15.78F0.23 45.23F1.48

1.00F0.58 0.29F0.17 15.61F0.27 47.71F2.70

* pb0.05 versus 0.2 mg/kg LAAM and Water (Fisher’s PLSD).

Fig. 1. Analysis of the meanFS.E.M. body weight (g) during the first five postnatal days revealed less weight gain in the offspring of dams treated with 1.0 mg/kg/day of LAAM (n=8 litters). *pb0.05 versus Water-treated (n=6 litters) and 0.2 mg/kg LAAM-treated (n=8 litters) offspring; **pb0.05 versus Water-treated offspring (Fisher’s PLSD).

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Fig. 2. Prenatal LAAM treatment followed by postnatal withdrawal had no effect on basal corticosterone concentrations but resulted in a blunted corticosterone response to both 15-min social isolation and LPS injection (n=5 Water-treated; n=6 0.2 mg/kg LAAM-treated, and n=4 1.0 mg/kg LAAM-treated subjects per PND 18 condition). The meanFS.E.M. corticosterone concentrations (ng/ml) are depicted. *pb0.05 versus Water controls in same postnatal condition (Fisher’s PLSD).

challenge: F 2,13=5.84, pb0.02; social isolation: F 2,13=4.26, pb0.04). The corticosterone response to the stressors was decreased 30–50% as a consequence of prenatal LAAM exposure followed by postnatal withdrawal. 3.5. Adult HPA axis responsivity There was no effect of Gender on the corticosterone response to LPS injection. Therefore, data were collapsed across sex. A two-way ANOVA with Time post-LPS injection and Prenatal Treatment revealed a main effect of Time ( F 1,61=9.83, pb0.003) and a Prenatal Treatment Time interaction ( F 2,61=3.52, pb0.04). One-way ANOVAs were conducted at each time point (3 and 8 h). There was a marginal Prenatal Treatment effect at 3 h ( pb0.09). An a priori comparison using Dunnett’s contrasts revealed a heightened response to LPS at 3 h in adult rats prenatally treated with 1.0 mg/kg LAAM compared to controls. As can be seen in Fig. 3, corticosterone was increased in the high dose LAAM group 2-fold over the prenatal Watertreated controls and nearly 1.5 fold over the 0.2 mg/kg LAAM-treated rats. There was no effect of Prenatal Treatment at 8 h.

Fig. 3. Prenatal exposure to the high dose of LAAM and/or postnatal withdrawal caused a heightened corticosterone response 3 h after LPS injection, but there was no effect 8 h after LPS injection (n=11–12 for Water-treated; n=12–14 for 0.2 mg/kg LAAM-treated, and n=10 for 1.0 mg/kg LAAM-treated subjects per post-injection time). The meanFS.E.M. corticosterone concentrations (ng/ml) are depicted. *pb0.05 versus Water controls (Dunnett’s contrast).

Males in the 1.0 mg/kg LAAM group showed a 1.5-fold and a 2-fold greater mean startle amplitude than the males in the Water group and 0.2 mg/kg LAAM group, respectively ( pb0.05, Fisher’s PLSD). 3.7. Open field test The number of outer line crossings was significantly different between males and females ( F 1,32=5.13, pb0.05), with females crossing 22% more lines than males crossed. There was no prenatal treatment effect or interaction between Gender and Prenatal Treatment. There were no

3.6. Acoustic startle responding There was a significant difference between the genders ( F 1,30=4.26, pb0.05), with males having a greater response to acoustic stimuli than females. This effect was not due to differences in body weight because startle reactivity and body weight were not significantly correlated (r=0.24). There was also a Prenatal Treatment effect ( F 2,30=3.38, pb0.05) and an interaction between Prenatal Treatment and Gender ( F 2,30=3.56, pb0.05; Fig. 4). In females, there was no Prenatal Treatment effect, but for males the magnitude of startle amplitude was influenced by prenatal treatment.

Fig. 4. Treatment with the high dose of LAAM in utero followed by postnatal withdrawal resulted in an elevated acoustic startle response in males. MeanFS.E.M. startle amplitude (arbitrary units) between males and females prenatally treated with Water, 0.2 mg/kg/day of LAAM, or 1.0 mg/ kg/day of LAAM (n=6 per gender per prenatal treatment). *pb0.05 compared to Water-treated controls in same gender (Fisher’s PLSD).

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differences between the treatment groups or genders for the number of inner line crossings. There was a marginal effect of Gender for the number of rearings ( pb0.07), with females having more rearings than males (data not shown).

4. Discussion In the present study, as in past studies [36], daily administration of low to moderate doses of LAAM to pregnant opiate-dependent rats did not interfere with successful pregnancy, but did produce dependent pups that underwent withdrawal postnatally. These offspring had alterations in their response to stressors in the late neonatal period, as well as in adulthood. While basal corticosterone concentrations were similar for all groups in the present study, prenatally exposed juvenile rats showed a blunted corticosterone response compared to controls. Conversely, there was a heightened response to stressors in the adult following prenatal opiate exposure. There was an interaction between gender and prenatal opiate exposure for acoustic startle responding, such that males prenatally treated with a higher dose of LAAM displayed greater startle responding than males in other prenatal treatment groups. These results will be discussed in turn. HPA axis dysregulation was found in both preweanling and mature animals, with the dysregulation in opposite directions depending on age. In young rats treated prenatally with opiates, two stressors—LPS challenge and social isolation—produced a blunted corticosterone response. One explanation for this blunted response in the present study may be that the stress of opiate withdrawal during the first week of life resulted in a short-term desensitization of the HPA axis. Additionally, during the neonatal period, there is a stress hyporesponsive period during which pups exhibit a limited elevation of plasma corticosterone in response to stressors [4]. Since the corticosterone measures were taken soon after the completion of the stress hyporesponsive period, there may have been some residual or compensatory effects in the prenatal LAAM-treated subjects that were not present in the controls. We cannot rule out the possibility that the prenatal opiate exposure shifted the timing of the corticosterone response to the stressors, since we only examined a single time point. However, the present results are similar to our previous finding demonstrating a blunted corticosterone response to LPS in young chicks following embryonic cocaine exposure [31], as well as to the finding of a blunted ACTH response to an immunological stressor following prenatal alcohol exposure in juvenile rats [19]. Together, these studies provide convergent evidence that prenatal exposure to abused substances alters the expression of the stress response in juvenile animals. In contrast to the young rat, mature rats that were prenatally exposed to LAAM showed an elevated corticosterone response to an immunological stressor. These results are similar to those of studies observing the HPA axis

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response following prenatal or neonatal stress [27,40]. Because we did not examine the response to the isolation stressor in adulthood, we do not know if the exaggerated corticosterone response was a consequence of the type of stressor, or the reflection of a more general HPA axis dysregulation in the LAAM-exposed offspring. However, evidence from other studies where adult HPA axis function was assessed following prenatal or neonatal stress (e.g. [27,40]) suggests that our results would extend beyond immunological stimulation. In the adult rats we examined corticosterone at 3 and 8 h following LPS. Elevated corticosterone concentrations were present only at 3 h in the prenatal opiate-treated rats, suggesting that the termination of the corticosterone response was not altered by the prenatal treatment. However, to determine more conclusively the extent to which negative feedback regulation of the HPA axis is altered by prenatal opiate exposure and/or postnatal opiate withdrawal, investigation of additional time points, as well as glucocorticoid receptor concentrations will be necessary. The opposite stress response pattern found in young and mature rats exposed prenatally to LAAM is intriguing, particularly since similar patterns have been reported in humans. For example, early adversity, such as abuse or rearing in an orphanage, can result in a blunted response to stressors and reduced diurnal variation of HPA axis hormones in children (reviewed in [9]). Conversely, following child abuse, adult females display a heightened cortisol response to stressors, particularly if they show other psychopathology, such as depression or post-traumatic stress disorder [6,13]. Since both hypoactive and hyperactive responding to stressors can be maladaptive for the physical and mental health of an organism, examination of the mechanisms that may underlie the HPA axis dysregulation of both young and older animals is warranted. Potential candidates include changes in glucocorticoid receptor number or function, hypothalamic corticotropin releasing hormone (CRH) expression, and sympathetic nervous system regulation of the adrenal gland. Unlike the HPA axis dysregulation, where both males and females exposed prenatally to opiates were affected, only males treated with the highest dose of LAAM showed alterations in acoustic startle responding. This pattern indicates that the elevation in anxiety-like behavior in the males was due to greater prenatal opiate exposure and/or more severe neonatal opiate withdrawal in the 1.0 mg/kg LAAM dose group. Interestingly, a single daily administration of heroin during gestation from GD 8 to GD 20 resulted in an elevation in acoustic startle in 3-week-old females [43]. Similarly, startle was elevated at 3 but not 4 weeks of age in prenatally opiate-exposed male and female rats, an effect that may have been related to a protracted withdrawal from methadone [16,44]. These results suggest that in addition to the drug type and dosing paradigm, the age at testing may be a critical variable. We only examined the startle response in animals at 16.5 weeks of age, so we

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do not know if there would be alterations due to the present prenatal opiate paradigm at earlier ages. Since prior studies found that pre-weaning females had enhanced startle after prenatal heroin exposure [16], the lack of an effect of prenatal LAAM exposure on startle responding in the adult females in the present study is interesting and warrants further study. For example, circulating ovarian hormones may be affecting the expression of the startle response, potentially masking the effects that were seen in young animals. It is also possible that the startle response of young animals is qualitatively different that of adults, and that regions of the male brain that underlie adult startle reactivity are more sensitive to developmental perturbation by opiate exposure and withdrawal than those in the female brain. Our findings indicate that processes involved in the modulation of the acoustic startle response are altered as a consequence of prenatal opiate exposure and neonatal withdrawal. Based on the anatomical substrates of acoustic startle and startle modulation, potential candidates include activity differences within the extended amygdala and brainstem [20]. It is important to note that anxiety, as indicated by elevated acoustic startle responding, has been observed in humans and rodents undergoing precipitated withdrawal from opiates [11,18,36]. Thus, the finding that prenatal LAAM resulted in greater startle responding in otherwise untreated adult males may indicate an increased vulnerability to drug abuse, given that both prenatal opiate exposure and anxiety increase drug self-administration ([15], reviewed in [41]). To further explore the effects of prenatal LAAM exposure on affective behavior, the open field test was used. There was no effect of prenatal LAAM treatment for this measure, although females were more active. Similar gender differences on this measure have been reported previously [5,35]. In addition, all rats, regardless of gender or prenatal treatment, crossed more outer than inner lines, revealing that that they had similar levels of emotional behavior. Therefore, prenatal exposure to opiates did not increase these measures of emotional behavior in the open field, which is similar to effects seen following prenatal morphine exposure [35]. Overall, these findings indicate that prenatal opiate exposure and/or neonatal withdrawal alter development such that stress responding and anxiety-like behaviors are changed. The types of measures affected by the prenatal LAAM exposure suggest that responsiveness to adverse circumstances (e.g., stress, anxiogenic stimuli) is sensitive to these developmental manipulations. Gender also appears to interact with exposure and withdrawal on some measures, making males and females differentially susceptible to subsequent anxiogenic challenges. The effects of prenatal drug exposure can be difficult to interpret because multiple interacting factors are involved. For example, in addition to the direct pharmacological effects of the drug on the developing embryo and fetus,

there can be indirect effects on maternal physiology and behavior due to the drug or the method used for drug administration. Superimposed upon this, in the case of opiates, are the direct effects of withdrawal from the opiates on the neonate. For example, we have found activation or sensitization of the HPA axis in chicken embryos and young chickens undergoing withdrawal from embryonic exposure to the active LAAM metabolite nor-LAAM [32,33]. In addition, there are potentially indirect withdrawal effects due to altered maternal physiology and behavior. Differences in the potency, duration of action, and receptor subpopulations targeted amongst various opiates also add complexity to interpreting results in the context of other studies. Some of these factors can be controlled for in the study design, such as the use of foster mothers after birth in the present study, to eliminate altered maternal behavior and milk production while the dam is undergoing withdrawal. It is more difficult to evaluate or control for other factors, such as the effects of the route of drug administration, or altered behavior of the drug-exposed pup affecting maternal behavior. The goal of the present study was not to determine the magnitude of the individual contributions of any these factors, but rather to examine the global effect of prenatal opiate exposure followed by withdrawal on stress and anxiety measures. Once reliable effects are determined, it will be important to determine the relative contributions of the various interacting factors, specifically to address how the stress of postnatal withdrawal may be affecting neonatal and adult stress responsivity. Acknowledgements This work was supported, in part, by USPHS grants T32 DA 07097 (support of KLH and ACH) and K01 DA 00362 (LMS). References [1] C. Archie, Methadone in the management of narcotic addiction in pregnancy, Curr. Opin. Obstet. Gynecol. 10 (1998) 435 – 440. [2] P.S. Bauman, S.A. Levine, The development of children of drug addicts, Int. J. Addict. 21 (1986) 849 – 863. [3] M.M. de Cubas, T. Field, Children of methadone-dependent women: developmental outcomes, Am. J. Orthopsychiatr. 63 (1993) 266 – 276. [4] E.R. De Kloet, P. Rosenfeld, J.A. Van Eekelen, W. Sutanto, S. Levine, Stress, glucocorticoids and development, Prog. Brain Res. 73 (1988) 101 – 120. [5] M. Dubovicky, I. Skultetyova, D. Jezova, Neonatal stress alters habituation of exploratory behavior in adult male but not female rats, Pharmacol. Biochem. Behav. 64 (1999) 681 – 686. [6] B.M. Elzinga, C.G. Schmahl, E. Vermetten, R. van Dyck, J.D. Bremner, Higher cortisol levels following exposure to traumatic reminders in abuse-related PTSD, Neuropsychopharmacology 28 (2003) 1656 – 1665. [7] E.K. Enters, H. Guo, U. Pandey, D. Ko, S.E. Robinson, The effect of prenatal methadone exposure on development and nociception during the early postnatal period of the rat, Neurotoxicol. Teratol. 13 (1991) 161 – 166.

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