Perinatal Methadone Exposure Affects Dopamine, Norepinephrine, and Serotonin in the Weanling Rat

Neurotoxicology and Teratology, Vol. 19, No. 4, pp. 295–303, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0892-0362/97 $17.00 1 .00

PII S0892-0362(97)00018-4

Perinatal Methadone Exposure Affects Dopamine, Norepinephrine, and Serotonin in the Weanling Rat SUSAN E. ROBINSON, JENNIFER R. MAHER, MELISA J. WALLACE AND PAUL M. KUNKO1 Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0613 Received 10 September 1996; Accepted 31 January 1997 ROBINSON, S. E., J. R. MAHER, M. J. WALLACE AND P. M. KUNKO. Perinatal methadone exposure affects dopamine, norepinephrine, and serotonin in the weanling rat. NEUROTOXICOL TERATOL 19(4) 295–303, 1997.—On gestational day 7 pregnant rats were implanted with osmotic minipumps containing either methadone hydrochloride (initial dose, 9 mg/kg/day) or sterile water. Their offspring were cross-fostered so that they were exposed to methadone prenatally and/or postnatally. On postnatal day 21, dopamine (DA), norepinephrine (NE), serotonin (5-HT), and their metabolites were analyzed. Perinatal methadone exposure disrupted dopaminergic, noradrenergic, and serotonergic activity in a brain region- and gender-specific fashion. The ratio of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) to DA was reduced in the frontal cortex of males exposed to methadone postnatally. No effects of perinatal methadone exposure were observed on DA and DOPAC in the striatum. The ratio of 3-methoxy-4-hydroxyphenylglycol (MOPEG) to NE in the hippocampus was increased significantly in males exposed to methadone prenatally. Striatal and parietal cortical 5-hydroxyindoleacetic acid (5-HIAA), but not its ratio to 5-HT, was increased slightly in rats exposed to methadone postnatally. Although parietal cortical 5-HT, 5-HIAA, and 5-hydroxytryptophan were all affected by perinatal methadone exposure, the ratios of metabolite and precursor to 5-HT were not affected. Effects of methadone exposure appeared to depend upon the developmental stage at which exposure occurred and did not appear to result from the phenomenon of neonatal withdrawal. Changes in activity of these three neurotransmitter systems may contribute to the effects of perinatal methadone on the activity of other neurons, such as cholinergic neurons. © 1997 Elsevier Science Inc. Methadone

Perinatal exposure

Dopamine

Norepinephrine

Serotonin

In addition to disrupting behavior, perinatal exposure to opioids has been demonstrated to disrupt the development of several neurotransmitter systems (10,13,37,43,46). Catecholaminergic and serotonergic neurons develop early, with differentiation beginning as early as gestational days 10–15 (21). Thus, the possibility exists that prenatal methadone exposure can affect these neurons during their development. Indeed, this has been found to be the case in animal studies (6,45). However, it is unclear whether these changes can be attributed to a direct action of the drug itself or to withdrawal from the drug. The few studies including both pre- and postnatal exposures to methadone utilized dosing regimens insufficient

ALTHOUGH it is generally accepted that the use of methadone maintenance in pregnant narcotic addicts is preferable to subjecting the fetus to erratic drug levels and repeated intrauterine withdrawal (17), this prenatal exposure is known to produce both short- and long-term effects in their children (5,44). It is well accepted that infants born to mothers maintained on methadone have low birth weights and undergo an abstinence syndrome, characterized by increased CNS arousal and sleep disturbances. Animal studies have also reported behavioral changes after perinatal opioid exposure (16,47). Considerable controversy remains concerning the causes and extent of behavioral deficits in those exposed to opioids in utero (14,15,22).

Requests for reprints should be addressed to Susan E. Robinson, Ph.D., P.O. Box 980613, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0613. Tel:(804) 828-8396; Fax:(804) 828-2117; E-mail: [email protected] 1Present address: Addiction Research Center, NIDA, Baltimore, MD 21224.

295

296

ROBINSON ET AL.

to produce sustained exposure to significant levels of the drug (31,41). The production of physical dependence in rats, including pregnant rats, requires a continuous infusion of methadone (4). This requirement is a result of the short plasma halflife of methadone in the rat, which is on the order of 2–4 h (28). To determine the effects of prenatal exposure to and subsequent withdrawal from methadone on the catecholamines dopamine (DA) and norepinephrine (NE) and on the indoleamine serotonin (5-hydroxytryptamine, 5-HT), the following study was performed, using osmotic minipumps to infuse pregnant rats or foster dams with methadone. Pups derived from methadone-treated dams were maintained on methadone, via maternal milk from foster dams treated with methadone, or allowed to withdraw, by placing them with water-treated foster dams. Postnatal methadone exposure by this procedure has been found to produce similar brain concentrations of methadone whether the pups are exposed to methadone both pre- and postnatally or only postnatally, whereas little or no methadone remains in the brains of pups exposed to the drug only prenatally (20). METHOD

Animals Rats were kept on a 12-h light/dark cycle in a temperatureand humidity-controlled room and provided food (Purina Lab Chow) and water ad lib. Nulliparous female “CD” Sprague– Dawley rats (Harlan Labs, Indianapolis, IN) 80–90 days old were placed with male rats of the same strain. The day of detection of a seminal plug was denoted as day 0 of gestation. Beginning on day 7 of gestation, two groups of pregnant rats were exposed to sterile water or methadone hydrochloride (initial dose rate, 9 mg/kg/day) via 28-day osmotic minipumps (Alza, Palo Alta, CA) that had been implanted SC with the rats under methoxyflurane anesthesia. Methadone hydrochloride was obtained from the National Institute on Drug Abuse (NIDA). The above dosing regimen has previously been demonstrated to produce maternal dependence without significant maternal or fetal mortality (8,20,46). Within 24 h of delivery, pups were weighed, sexed, and the litter size reduced to 10, maintaining, if possible, equal numbers of males and females (litters of 8–10 pups were fostered intact). At that time, pups were fostered to dams implanted with minipumps containing either water or methadone, such that the following prenatal/postnatal exposure groups were obtained: water/water (ww), methadone/water (mw), methadone/methadone (mm), and water/methadone (wm). On postnatal day (PD) 10, the dams were briefly anesthetized with methoxyflurane and their minipumps replaced with freshly filled and primed 14-day minipumps. The ww treatment group served as the procedural control. The mw group was allowed to spontaneously withdraw postpartum. The mm group consisted of pups exposed to methadone prenatally, but not allowed to withdraw neonatally. The wm group was included as a control for the effects of early postnatal methadone exposure. On PD 21, rats were killed by focussed microwave radiation (6.5 kW, 0.65–0.95 s, Cober Electronics, Stamford, CT), their brains rapidly removed and dissected on ice into the following regions: frontal cortex, parietal cortex, striatum, and hippocampus. The tissue was stored at 280°C until analyzed for neurotransmitter content. Neurotransmitter Analysis Each brain sample was analyzed for DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), NE and its metabo-

lite 3-methoxy-4-hydroxyphenylglycol (MOPEG), and 5-HT, its precursor 5-hydroxytryptophan (5-HTP), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), by HPLC with electrochemical detection (36). The samples were homogenized in 0.4 N perchloric acid, proteins precipitated, and the pH of the supernatant adjusted to 4.4 by the addition of 7.5 N potassium acetate. Twenty-microliter aliquots were injected onto a reverse-phase (Rainin Microsorb C18, 4.6 3 15 cm, 5 m spherical particle size column) HPLC system with an LC-4C amperometric controller and CC-5 flowcell (BAS, West Lafayette, IN). The mobile phase consisted of 0.1 M potassium phosphate, 0.1 M EDTA, 5.0 mM heptane sulfonic acid, and 13.3% methanol. Neurotransmitter and metabolite contents were expressed per milligram protein, as determined by the Lowry protein assay (25). In addition, the ratios of metabolites to neurotransmitters were examined, as the ratio of metabolite to neurotransmitter can give a good approximation of neuronal activity (40). Data Analysis Data from no more than one male and one female rat from each litter were included in the data analysis. Data were analyzed separately by three-way (sex 3 prenatal treatment 3 postnatal treatment) analysis of variance (ANOVA) with a level of p , 0.05 accepted as statistically significant. Significant interactions in the ANOVAs were followed by tests for simple main effects, for which the p value accepted as statistically significant was adjusted downward based on Dunn’s procedure and the Bonferroni inequality (19). RESULTS

Numerous main effects of sex were observed on the neurotransmitters and/or ratios of metabolite to neurotransmitter in the various brain regions. For the sake of brevity, significant sex differences are mentioned only in cases where there are interactions between sex and pre and/or postnatal treatments. Dopamine The only brain region exhibiting effects of perinatal methadone exposure on DA and its metabolite DOPAC was the frontal cortex (Fig. 1). Significant main effects for postnatal treatment, F(1, 60) 5 6.10, p , 0.05, and sex, F(1, 60) 5 15.70, p , 0.05, were observed on DA, as well as an interaction between sex and postnatal treatment, F(1, 60) 5 4.34, p , 0.05. Postnatal exposure to methadone increased frontal cortical DA content in the females (Fig. 1A), F(1, 33) 5 6.15, p , 0.0375, but had no effect on DA content in the males. In the case of the DA metabolite DOPAC, a significant postnatal treatment effect, F(1, 51) 5 8.23, p , 0.05, as well as significant prenatal treatment by postnatal treatment, F(1, 51) 5 8.5, p , 0.05, sex by postnatal treatment, F(1, 51) 5 14.08, p , 0.05, and sex by prenatal treatment by postnatal treatment, F(1, 51) 5 10.50, p , 0.05, interactions were observed. Postnatal exposure to methadone increased frontal cortical DOPAC when the data are collapsed across sex (Fig. 1B), F(1, 58) 5 5.33, p , 0.05. Inspection of the data indicated that this effect was restricted to females (Table 1). Furthermore, the increase in frontal cortical DOPAC can be largely attributed to the females exposed to methadone both pre- and postnatally, which had significantly more DOPAC in the frontal cortex compared to females exposed to methadone either only prenatally, F(1, 12) 5 12.86, p , 0.05, or only postnatally, F(1, 15) 5 8.10, p , 0.05 (Table 1). Not surprisingly, the ratio DOPAC/DA was affected by perina-

PERINATAL METHADONE AND NEUROTRANSMITTERS

297

FIG. 1. Effect of perinatal methadone exposure on frontal cortical dopaminergic neurons in female and male 21day-old rats. (A) Effect of postnatal methadone on frontal cortical DA in females; (B) effect of postnatal methadone on frontal cortical DOPAC, collapsed across sex; and (C) effect of postnatal methadone on frontal cortical DOPAC/DA in males. Data are expressed as mean 6 SEM. *p , 0.05.

tal methadone exposure. The effects of perinatal methadone exposure on DOPAC/DA vary according to gender: significant sex by postnatal treatment, F(1, 46) 5 9.04, p , 0.05, and significant sex by prenatal treatment by postnatal treatment, F(1, 46) 5 4.30, p , 0.05, interactions were observed. In this case, males exposed to methadone postnatally exhibited a smaller DOPAC/DA ratio than males exposed to water postnatally (Fig. 1C), F(1, 26) 5 5.60, p , 0.0375. On the other hand, postnatal methadone exposure did not significantly alter the ratio DOPAC/DA in females. In females exposed to

methadone prenatally only (mw), however, the ratio DOPAC/DA is significantly less than in females in the ww control group, F(1, 11) 5 7.71, p , 0.05, or in the mm exposure group, F(1, 11) 5 6.03, p , 0.05 (Table 1). Norepinephrine Several effects were observed on NE and its metabolite MOPEG in the hippocampi of weanling rats (Fig. 2). A significant interaction was observed between prenatal and postnatal

TABLE 1 EFFECT OF PERINATAL METHADONE EXPOSURE ON NEUROTRANSMITTERS IN 21-DAY-OLD FEMALE RATS Treatment

Striatum w/w m/w w/m m/m Hippocampus w/w m/w w/m m/m Frontal Cortex w/w m/w w/m m/m Parietal Cortex w/w m/w w/m m/m

DA

DOPAC

DOPAC/DA

218 6 14 221 6 11 200 6 20 244 6 26

36 6 3 35 6 2 35 6 3 43 6 6

0.17 6 0.11 0.16 6 0.10 0.18 6 0.01 0.18 6 0.02

2.1 6 0.1 1.2 6 0.1 3.0 6 0.1 29 6 28

36 6 4 36 6 2 35 6 4 41 6 6

40 6 30 43 6 26 10 6 2 49 6 33

14 6 3 14 6 3 25 6 5 35 6 12

7.2 6 2.3 2.0 6 0.5 7.8 6 1.6 20 6 4‡

7.2 6 1.6 6.3 6 1.2 6.2 6 1.3 5.7 6 1.0

7.4 6 2.5 8.2 6 0.2 6.7 6 1.5 5.0 6 2.8

NE

3.3 6 1 3.6 6 1 5.2 6 1.1 4.4 6 1.6

MOPEG

20 6 2 21 6 3 28 6 8 25 6 6

5.7 6 0.9 6.6 6 1.6 6.8 6 1.9 16 6 3.5 7.9 6 1.1 37 6 19 4.6 6 1.3 5.9 6 2.1

0.45 6 0.08 8.7 6 1.1 20 6 4 0.15 6 0.04† 6.4 6 1.8 14 6 3 0.38 6 0.07 9.7 6 1.8 62 6 40 0.62 6 0.16 19 6 1.3 9.9 6 2.5 1.1 6 0.3 1.1 6 0.2 1.0 6 0.3 0.6 6 0.3

11 6 2 16 6 2 17 6 3 12 6 2

11 6 3 13 6 4 12 6 4 12 6 3

MOPEG/NE

5-HT

5-HIAA

5-HTP

5-HIAA/5-HT 5-HTP/5-HT

12 6 8 3.4 6 0.4 5.1 6 1.3 5.1 6 1.6

22 6 1 37 6 16 22 6 3 29 6 4

29 6 2 29 6 3 30 6 2 34 6 4

4.3 6 1.2 2.5 6 0.5 4.0 6 0.6 4.7 6 1.8

1.4 6 0.1 1.4 6 0.1 1.4 6 0.1 1.3 6 0.1

0.19 6 0.06 0.12 6 0.03 0.20 6 0.03 0.17 6 0.05

1.5 6 0.4 2.5 6 0.5 4.2 6 1.6 1.1 6 0.4*

20 6 2 19 6 2 19 6 2 18 6 2

29 6 5 29 6 3 28 6 3 26 6 4

3.9 6 2.0 6.8 6 1.3 3.6 6 1.8 1.5 6 1.4

1.4 6 0.1 1.5 6 0.2 1.5 6 0.2 1.4 6 0.2

0.20 6 0.10 0.30 6 0.08 0.23 6 0.13 0.09 6 0.09

2.3 6 0.5 2.2 6 0.6 7.8 6 4.7 1.5 6 0.3

21 6 3 21 6 6 23 6 6 29 6 12

18 6 3 16 6 5 25 6 8 27 6 6

1.0 6 0.2 0.78 6 0.07 1.7 6 0.9 0.90 6 0.17 0.7 6 0.4 1.1 6 0.21 0.5 6 0.4 1.1 6 0.19

1.0 6 0.2 0.82 6 0.22 0.78 6 0.18 1.1 6 0.3

15 6 0.9 19 6 2 23 6 3 15 6 1

20 6 2 3.7 6 1.9 28 6 3 11 6 3.8 27 6 2† 7.2 6 1.8 19 6 2 2.9 6 0.4

1.4 6 0.1 1.6 6 0.2 1.3 6 0.2 1.3 6 0.2

0.81 6 0.02 0.20 6 0.13 0.07 6 0.04 0.03 6 0.02 0.24 6 0.12 0.64 6 0.23 0.30 6 0.06 0.20 6 0.02

Values are mean 6 SEM. w/w, prenatal water/postnatal water; m/w, prenatal methadone/postnatal water; w/m, prenatal water/postnatal methadone; m/m, prenatal methadone/postnatal methadone. *p , 0.05, compared to m/w. †p , 0.05, compared to w/w and m/m. ‡ p , 0.05, compared to m/w and w/m.

298

ROBINSON ET AL.

FIG. 2. Effect of perinatal methadone exposure on hippocampal noradrenergic neurons in female and male 21-day-old rats. (A) Effect of perinatal methadone on hippocampal NE content, collapsed across sex; (B) effect of prenatal methadone exposure on hippocampal MOPEG in males; and (C) effect of prenatal methadone exposure on hippocampal MOPEG/NE in males. Abbreviations are as follows: ww, prenatal water/postnatal water; mw, prenatal methadone/postnatal water; wm, prenatal water/postnatal methadone; and mm, prenatal methadone/postnatal methadone. Data are expressed as mean 6 SEM. In (A), *p , 0.05, compared to wm. In (B, C), *p , 0.05.

treatments on NE content in the hippocampus, F(1, 56) 5 5.09, p , 0.05. Further analysis of the data indicated that the hippocampi of rats exposed to methadone both pre- and postnatally had significantly less NE than those of rats that were exposed to the drug in the postnatal period only, when the data are collapsed across sex (Fig. 2A), F(1, 30) 5 7.21, p , 0.0375. Although no main effects were observed on MOPEG content, a significant interaction between sex and prenatal treatment was

observed on hippocampal MOPEG content, F(1, 57) 5 4.78, p , 0.05. Further analysis of the data indicated MOPEG content was significantly increased in the hippocampi of males exposed to methadone prenatally (Fig. 2B), F(1, 31) 5 5.35, p , 0.0375. Similar to the change in MOPEG, the ratio MOPEG/NE also exhibited an interaction between sex and prenatal treatment, F(1,48) 5 7.22, p , 0.05, such that the MOPEG/NE ratio was elevated in the males exposed to methadone prenatally (Fig.

TABLE 2 EFFECT OF PERINATAL METHADONE EXPOSURE ON NEUROTRANSMITTERS IN 21-DAY-OLD MALE RATS Treatment

Striatum w/w m/w w/m m/m Hippocampus w/w m/w w/m m/m Frontal Cortex w/w m/w w/m m/m Parietal Cortex w/w m/w w/m m/m

DA

DOPAC

DOPAC/DA

219 6 27 204 6 20 219 6 11 235 6 17

36 6 5 37 6 4 37 6 2 42 6 3

0.16 6 0.01 2.6 6 1 0.18 6 0.01 2.2 6 1 0.17 6 0.01 3.7 6 1 0.18 6 0.01 2.6 6 1

5.7 6 2.1 2.5 6 1.3 4.6 6 1.2 1.3 6 0.1

7.3 6 3.2 1.2 6 0.3 7.3 6 2.2 11 6 3.7 7.4 6 5.7 2.2 6 1.3 9.1 6 2.4 27 6 9 7.9 6 1.8 4.1 6 1.9 11 6 3.1 13 6 2.6 1.7 6 1.7 0.72 6 0.72 4.8 6 1.6 27 6 8

8.4 6 2.4 6.5 6 1.7 8.8 6 2.2 8.9 6 3.5

3.1 6 1.1 2.4 6 0.8 2.4 6 0.7 0.7 6 0.7

3.4 6 0.8 5.9 6 1.7 2.9 6 1.1 3.3 6 1.0

5.3 6 1.7 1.6 6 0.4 5.3 6 1.6 17 6 4 3.5 6 1.7 0.36 6 0.17 8.4 6 1.4 19 6 4 5.3 6 0.9 0.43 6.0 6 1.6 10 6 2 1.8 6 0.4 0.63 4.4 6 1.6 17 6 5

0.37 6 0.14 0.34 6 0.12 0.16 6 0.04 0.06 6 0.06

NE

16 6 2 16 6 1 18 6 2 21 6 4

MOPEG MOPEG/NE

5-HT

5-HIAA

5-HTP

19 6 3 16 6 3 20 6 2 19 6 2

27 6 3 27 6 3 32 6 2 37 6 7

5.6 6 3.4 8.1 6 6.3 2.9 6 1.3 4.1 6 1.2

1.5 6 0.1 1.6 6 0.2 1.9 6 0.3 1.9 6 0.3

0.37 6 0.26 0.51 6 0.38 0.14 6 0.06 0.22 6 0.07

1.3 6 0.4 2.6 6 0.7 1.3 6 0.1 4.1 6 1.4*

20 6 4 20 6 2 19 6 3 16 6 2

29 6 4 26 6 2 31 6 2 27 6 5

5.6 6 2.8 5.0 6 1.3 5.5 6 1.2 6.4 6 2.3

2.1 6 0.8 1.4 6 0.1 3.7 6 1.5 1.7 6 0.2

0.41 6 0.24 0.23 6 0.05 0.74 6 0.33 0.42 6 0.11

16 6 4 0.94 6 0.16 59 6 26 5.1 6 3.0 17 6 4 0.89 6 0.13 19 6 3 1.0 6 0.3

26 6 3 28 6 3 34 6 6 39 6 7

18 6 3 27 6 3 26 6 4 31 6 6

8.8 6 1.6 0.72 6 0.12 9.6 6 1.5 1.0 6 0.1 11 6 3 0.78 6 0.12 13 6 3 0.80 6 0.07

0.37 6 0.08 0.34 6 0.04 0.32 6 0.06 0.32 6 0.04

9.3 6 1.2 13 6 1 9.7 6 1.1 8.8 6 0.9

11 6 1 13 6 2 11 6 2 9.3 6 1.1

1.4 6 0.6 3.4 6 2.7 2.2 6 1.5 0.8 6 0.6

0.22 6 0.13 0.20 6 0.14 0.18 6 0.11 0.07 6 0.05

22 6 6 23 6 5 19 6 5 23 6 5

7.5 6 3.6 13.3 6 7.3 8.6 6 4.4 7.5 6 3.3

3.0 6 1.5 2.3 6 0.6 1.4 6 0.2 6.4 6 3.0

5-HIAA/5-HT 5-HTP/5-HT

1.2 6 0.1 1.0 6 0.1 1.2 6 0.1 1.1 6 0.2

Values are mean 6 SEM. w/w, prenatal water/postnatal water; m/w, parental methadone/postnatal water; w/m, prenatal water/postnatal methadone; mm/, prenatal methadone/postnatal methadone. *p , 0.05, as compared to m/w.

PERINATAL METHADONE AND NEUROTRANSMITTERS

FIG. 3. Effect of perinatal methadone exposure on 5-HT in parietal cortex in female and male 21-day-old rats, collapsed across sex. Abbreviations are the same as in Fig. 2. Data are expressed as mean 6 SEM. *p , 0.05, compared to mm and ww treatment groups.

2C) F(1, 25) 5 9.16, p , 0.0375. However, subsequent analyses following a significant three-way interaction of sex, prenatal treatment, and postnatal treatment, F(1, 48) 5 5.9, p , 0.05, on the MOPEG/NE ratio indicated that males exposed to methadone both pre- and postnatally exhibited a higher MOPEG/NE ratio than males exposed to the drug postnatally only (Table 2), F(1, 10) 5 7.95, p , 0.05. On the other hand, females exposed to methadone both pre- and postnatally exhibited a lower MOPEG/NE ratio than females exposed to the drug prenatally only (Table 1), F(1, 13) 5 5.79, p , 0.05. Noradrenergic neurons appear to be minimally affected by perinatal methadone exposure in the frontal cortex (Tables 1 and 2). NE content was not significantly altered. An interaction between sex and prenatal treatment was observed, F(1, 61) 5 4.88, p , 0.05, on the metabolite MOPEG. However, no significant effect of prenatal treatment was observed in either the females, F(1, 34) 5 2.15, p 5 0.15, or the males, F(1, 33) 5 3.35, p 5 0.077, for MOPEG content. A significant interaction between prenatal and postnatal treatments was observed on NE, F(1, 57) 5 7.09, p , 0.05, and the ratio MOPEG/NE, F(1, 50) 5 4.4, p , 0.05, in the parietal cortex. The prenatal by postnatal treatment interaction reflects a trend for increased NE content in the parietal cortex of animals exposed to methadone prenatally only compared to the controls when the data are collapsed across sex [mw, 11.7 6 1.4 pmol/mg protein, n 5 16, vs. ww, 7.7 6 1.4 pmol/mg protein, n 5 16; F(1, 31) 5 3.931, p 5 0.0566], although no significant differences were found among the groups. The prenatal by postnatal treatment interaction reflects a trend for an increased MOPEG/NE ratio in the rats exposed to methadone pre- and postnatally compared to the ratio in the rats exposed postnatally only when the data are collapsed across sex [mm, 3.57 6 1.54, n 5 13, vs. wm, 1.05 6 0.16, n 5 16; F(1, 28) 5 3.29, p 5 0.0809]. Again, however, differences were not found between any of the treatment groups, suggesting that the significant interaction is attributable to some linear combination of the means. Serotonin The 5-HT neurons appear to be the neurons most greatly affected in the parietal cortex (Tables 1 and 2). Interactions of pre and postnatal treatments were observed on 5-HT, F(1, 57) 5

299

13.62, p , 0.05, and its precursor 5-HTP, F(1, 57) 5 4.84, p , 0.05. 5-HT content in the pre- and postnatal exposure group (mm) was similar to that of the ww control group, whereas the values for the mw and wm groups tended to be higher (Tables 1 and 2). Further analysis of the data found that parietal cortical 5-HT content in rats exposed to methadone prenatally only was significantly greater than that of rats exposed to methadone both pre- and postnatally (Fig. 3), F(1, 30) 5 5.18, p , 0.0375, as well as greater than that of rats exposed to water both pre- and postnatally, F(1, 31) 5 6.69, p , 0.0375, when the data are collapsed across sex. The difference in 5-HT values of the mm group from those of the wm group did not attain statistical significance, F(1, 32) 5 2.89, p 5 0.099. Similarly, the parietal cortical 5-HTP content tended to be elevated in the rats exposed to methadone only pre- or postnatally, but not both (Tables 1 and 2). However, further analysis of the data failed to find that parietal cortical 5-HTP in the mm rats was significantly less than that in the mw group, F(1, 20) 5 4.56, p 5 0.046, or the wm group, F(1, 22) 5 4.77, p 5 0.040. Interactions of prenatal and postnatal treatment, F(1, 56) 5 14.52, p , 0.05, as well as of prenatal and postnatal treatments and sex, F(1, 56) 5 4.09, p , 0.05, were observed on 5-HIAA in the parietal cortex. Further analysis of the 5-HIAA data indicated that the interaction between pre- and postnatal treatment was restricted to the females, such that 5-HIAA content in the wm treatment group was significantly greater than that of the mm group (Table 1), F(1, 15) 5 6.21, p , 0.05, as well as greater than that of the ww control group (Table 1), F(1, 15) 5 6.59, p , 0.05. However, these treatments did not appear to affect the activity of the serotonergic neurons, as there were no main effects of treatment or interactions on the ratios of metabolite to neurotransmitter (5-HIAA/5-HT; Tables 1 and 2) or precursor to neurotransmitter (5-HTP/5-HT; Tables 1 and 2). There were few effects of perinatal methadone exposure on serotonergic neurons in the frontal cortex or striatum (Tables 1 and 2). No effects were observed on 5-HT or its precursor 5-HTP. A significant effect of postnatal treatment was observed on the 5-HT metabolite 5-HIAA in the frontal cortex, F(1, 61) 5 4.89, p , 0.05, such that rats exposed to methadone postnatally exhibited elevated 5-HIAA when the data are collapsed across sex (Fig. 4); however, statistically nonsignificant changes in 5-HT prevented significant changes in the ratio 5-HIAA/5-HT. The only significant treatment effect observed in the striatum was an effect of postnatal treatment on 5-HIAA, F(1, 59) 5 4.313, p , 0.05, with a slight, but statistically significant, increase in 5-HIAA content in the animals exposed to methadone postnatally when the data are collapsed across sex (Fig. 4, p , 0.05). However, the effect of postnatal treatment on the ratio 5-HIAA/5-HT failed to reach statistical significance, F(1, 58) 5 0.39, p . 0.05, due to nonsignificant changes in 5-HT content. DISCUSSION

Numerous behavioral abnormalities have been reported in subjects exposed prenatally to methadone or other opioids (5,14,16,31,44,48). Previous studies have found that perinatal exposure to opioids produces delays in brain development and changes in neurotransmitters, including cholinergic, dopaminergic, noradrenergic, and serotonergic systems (6,10, 13,27,31,37,43,45–47). However, several of those studies used dosing procedures producing significant maternal toxicity. In other studies it was unclear whether the observed effects resulted from opioid exposure or withdrawal from opioid exposure. In the present study, the four treatment groups (ww,

300

ROBINSON ET AL.

FIG. 4. Effect of postnatal methadone exposure on (A) striatal and (B) frontal cortical 5-HIAA in female and male 21-day-old rats collapsed across sex. Data are expressed as mean 6 SEM. *p , 0.05.

mw, mm, and wm) were included to attribute the cause of neurotransmitter changes to opioid exposure during different periods of development or to the occurrence of such phenomena as withdrawal or tolerance during the perinatal period. It becomes clear from the present data that there are no consistent effects of perinatal methadone exposure on the development of dopaminergic, noradrenergic, or serotonergic neurons throughout the brain; instead, the effects of methadone exposure on these neurons vary according to brain region and gender. Dopaminergic neurons in the frontal cortex are affected by perinatal methadone exposure in a largely gender-specific manner. In females exposed to methadone postnatally, there are increases in both DA and DOPAC content, such that no net change occurs in the ratio of metabolite to neurotransmitter. This suggests the possibility that postnatal methadone exposure increases the number or size or even the enzymatic capacity, but not the activity, of dopaminergic neurons projecting to the frontal cortex of females. On the other hand, the ratio DOPAC/DA is reduced in males exposed to methadone postnatally, which suggests that the activity of these neurons is reduced by this treatment. This may reflect a direct action of methadone on the dopamine neurons, as morphine has been reported to reduce DA turnover in the frontal cortex of adult male rats (7). There are few changes in the neurotransmitter DA in the striatum after perinatal methadone exposure. This is remarkable in light of the fact that this brain region is the one in which cholinergic neurons are the most profoundly affected after perinatal methadone (39). Reduced striatal DA metabolism, as would be reflected in a reduced DOPAC/DA ratio, has been observed in mw neonatal rats (38) as well as in adult rats in active withdrawal (1). The lack of an effect on striatal DA activity suggests that none of the weanling rats in methadone treatment groups are undergoing withdrawal. Clearly, perinatal opioid exposure does not affect overall dopaminergic function. The presence of opioid receptors may determine whether perinatal methadone influences certain dopaminergic neurons. Opioid receptors are located on or near the cell bodies of major dopaminergic projections to the forebrain, and opioid receptors are located in terminal regions of these projections, as well (26). m-Opioid receptors located in the ventral tegmental area are known to influence the

activity of the A10 dopaminergic neurons (11), which project to the frontal cortex, among other regions. On the other hand, the fact that striatal dopaminergic activity does not appear to be influenced by perinatal methadone exposure may reflect the fact that nigral m-opioid receptors are located on terminals of striatonigral neurons and not on the nigrostriatal dopaminergic neurons (24). Thus, the presence of m-opioid receptors in the vicinity of dopaminergic neurons does not guarantee an effect of perinatal methadone exposure, as it is possible that the m-opioid receptors are not located upon the dopaminergic neurons or upon other neurons synapsing upon those neurons. Noradrenergic function in the hippocampus is perturbed by perinatal methadone exposure, and part of this effect appears to be gender specific. This is of particular interest, because the effects of methadone on cholinergic activity in the hippocampus are also gender specific (39). The fact that the increase in the MOPEG/NE ratio was largely due to increased MOPEG content suggests that the neurons were capable of synthesizing enough NE to maintain levels of the neurotransmitter under conditions of increased release. One could speculate that the increased ratio MOPEG/NE, which suggests increased noradrenergic activity, in the hippocampus of male rats contributes to the increased acetylcholine turnover observed in males exposed to methadone perinatally, as noradrenergic neurons have been demonstrated to activate hippocampal cholinergic neurons (32,34,35). However, the pattern of activation of cholinergic and of noradrenergic neurons in the hippocampus of male rats is not identical. The ratio MOPEG/ NE is increased significantly in weanling males exposed to methadone prenatally (mw and mm), whereas acetylcholine turnover is increased in males exposed to methadone postnatally (mm and wm) (39). Although increases in cholinergic (39) and noradrenergic activity were the most profound in the male mm treatment group, hippocampal noradrenergic activity was not affected in the wm group, despite increased hippocampal cholinergic activity in that group. Therefore, it would appear that the cholinergic activation previously observed in the hippocampi of male rats exposed to methadone does not result from activation of noradrenergic neurons. However, changes in neurotransmitters in brain areas not studied in these animals may explain the apparent discrepancy between noradrenergic and cholinergic activities in male

PERINATAL METHADONE AND NEUROTRANSMITTERS rats exposed to methadone perinatally. For example, ventral tegmental dopaminergic neurons terminating in the septum are known to inhibit cholinergic activity in the hippocampus, and removal of septal DA innervation increases hippocampal acetylcholine turnover (33). If the activity of these dopaminergic neurons were reduced in the wm group, one would expect increased hippocampal acetylcholine turnover even in the absence of increased noradrenergic activity. Although dopaminergic activity was not assessed in the septum, it was studied in the frontal cortex, which, like the septum, receives dopaminergic input from the ventral tegmental area. The fact that the ratio DOPAC/DA is reduced in the frontal cortex of males exposed to methadone postnatally suggests that the activity of dopaminergic projections to the septum may be reduced in the wm group. Of course, appropriate studies must be performed to support this speculation. m-Opioid receptors are located: 1) within the locus coeruleus (26), the source of noradrenergic projections to the hippocampus, septal and basal forebrain regions, striatum, and cortex (9,23); 2) in the vicinity of many of the lateral tegmental noradrenergic nuclei, which project to basal forebrain regions (26,29); and 3) in terminal regions, as well (26). These are all possible sites through which methadone can affect noradrenergic function. It remains unclear why some neurons are affected by perinatal opioid exposure and others are not. m-Opioid agonists have been demonstrated to inhibit NE release from cortical neurons in vitro (12), yet no significant effect of postnatal methadone has been observed on noradrenergic neurons in the cortical regions sampled in the present experiment. Perhaps additional neuronal circuits are at work to oppose changes in noradrenergic activity in the intact animal. On the other hand, MOPEG levels in the hippocampus, but not the cortex, have been shown to increase following acute morphine administration to young adult rats (42). The pattern of changes of NE metabolism in this latter study is similar to the pattern of changes observed in prenatally exposed males, but unfortunately this does not provide a mechanism of action, as no methadone should remain in the brains of the rats exposed to methadone only prenatally, and MOPEG levels in the rats exposed to methadone only postnatally did not differ from controls. Although there were effects of perinatal methadone exposure on levels of the neurotransmitter 5-HT and the 5-HT metabolite 5-HIAA in the parietal cortex, no significant effects of perinatal methadone exposure were observed on the ratios of the neurotransmitter to its metabolite. Therefore, it is unlikely that the activity of serotonergic neurons was significantly affected by methadone exposure. Because changes in 5-HT, 5-HIAA, and 5-HTP content occurred roughly in parallel, one may speculate that perinatal methadone exposure influences the number or size, but not the firing rate, of serotonergic neurons in the parietal cortex. It is interesting that the serotonergic neurons of animals exposed to methadone both pre- and postnatally are affected less than those animals exposed only pre- or postnatally. This difference may indicate that animals exposed both pre- and postnatally develop tolerance to this effect on serotonergic neurons. The increased 5-HIAA content in the striatum and frontal cortex of postnatally exposed rats could reflect a reduction in removal of 5-HIAA from the brain; however, if this were the case, 5-HIAA levels should be increased across all brain areas. However, a morphine-induced increase in striatal 5-HIAA has been previously reported in adult rats (18), consistent with the findings in the postnatal methadone exposure group, suggesting a possible direct action of the drug.

301

m-Opioid receptors are located within the dorsal and median raphe nuclei, the main sources of serotonergic innervation of the forebrain, as well as in the forebrain terminal regions (26). Dorsal raphe serotonergic dopaminergic neurons project to the striatum, cortex, and, to a lesser extent, the hippocampus. On the other hand, serotonergic neurons from the median raphe project to the hippocampus, as well as to the cortex (2). The fact that postnatal methadone increases 5-HIAA content in the frontal cortex and striatum, but not in the hippocampus, suggests an involvement of the dorsal raphe nucleus, but not the median raphe nucleus. The fact that the effects of methadone on neurotransmitters vary across brain regions suggests that perinatal methadone does not exert a generalized action on dopaminergic, noradrenergic, or serotonergic neurons. The response pattern may reflect the distribution of opioid receptors upon which methadone may act, and through which it influences neuronal development. Thus, the consequences of opioid exposure could reflect either changes in development of the neurons upon which opioid receptors are located or changes in the expression or coupling of those receptors. However, as is obvious from the lack of effects on striatal DA and NE and hippocampal 5-HT, the presence of m-opioid receptors in the vicinity of dopaminergic, noradrenergic, or serotonergic neurons does not guarantee an effect of perinatal methadone exposure. Several gender-specific effects of perinatal methadone exposure were observed. Gender-related differences in responses to opioids are well known (3). The existence of sex steroid binding sites in many of these brain areas may contribute to these differences. For example, there are sexual dimorphisms in the effect of perinatal methadone on noradrenergic activity in the hippocampus, and both the hippocampus and septum contain sex steroid binding sites (30) that may influence the actions of methadone. Several other researchers have investigated the actions of perinatal opioid exposure on the development of neurotransmitters. Only Rech et al. (31) studied brain regional biogenic amines in 21-day-old animals. Using a different exposure paradigm that pooled together rats receiving the same prenatal exposure, but different postnatal exposures, they found slight, but statistically significant, reductions in striatal DA, DOPAC, and 5-HIAA content in weanling rats. This contrasts with the present study in which no effect was observed on striatal DA and DOPAC content and an opposite effect was observed on striatal 5-HIAA content. However, there are several differences between the two dosing regimens, which may explain the discrepancies between the two studies. For example, the dosing paradigm of Rech et al. (31) may have not have been sufficient to produce physical dependence, yet may have intermittently exposed the fetuses to toxic levels of methadone. On the other hand, Vathy et al. (45) studied the effects of prenatal morphine exposure on catecholamine turnover in 70–80day-old male and (ovariectomized) female rats. Similar to the present results with methadone, no effect of prenatal morphine exposure was observed on DA or NE content or turnover in the striatum or frontal cortex of either male or female rats in that study. From the above studies, it is clear that perinatal methadone exposure influences the development or activity of dopaminergic, noradrenergic, and serotonergic neurons in a brain region-specific manner. Some of the effects in the postnatal exposure groups may be attributed to direct actions of the drug. Changes in neuronal activity cannot be strictly correlated with neonatal withdrawal; in fact, continued exposure to the drug throughout the perinatal period in some instances

302

ROBINSON ET AL.

produces further disruption of neuronal function. However, changes in activity of these three neurotransmitters may contribute to effects of perinatal methadone exposure on the activity of other neurons, such as cholinergic neurons, as well as to some of the behavioral effects of perinatal opioid exposure.

ACKNOWLEDGEMENTS

This research was funded by NIDA grants DA05274 and DA07027. The authors acknowledge the technical assistance of Mr. Morayo Omojokun and Ms. Dionne Otey.

REFERENCES 1. Attila, L. M. J.; Ahtee, L.: Retardation of cerebral dopamine turnover after morphine withdrawal and its enhanced acceleration by acute morphine administration in rats. Naunyn Schmiedebergs Arch. Pharmacol. 327:201–207; 1984. 2. Azmitia, E. C.: The serotonin-producing neurons of the midbrain median and dorsal raphe nuclei. In: Iversen, L. L.; Iversen, S. D.; Snyder, S. H., eds. Handbook of psychopharmacology, vol. 9. Chemical pathways in the brain. New York: Plenum Press; 1978: 233–314. 3. Bodnar, R. J.; Romero, M.-T.; Kramer, E.: Organismic variables and pain inhibition: Roles of gender and aging. Brain Res. Bull. 21:947–953; 1988. 4. Darmani, N. A.; Saady, J. J.; Schnoll, S. H.; Martin, B. R.: Demonstration of physical dependence following chronic continuous methadone delivery via osmotic minipumps in pregnant rats. Neurotoxicol. Teratol. 13:627–630; 1991. 5. Davis, D. D.; Templer, D. I.: Neurobehavioral functioning in children exposed to narcotics in utero. Addict. Behav. 13:275–283; 1988. 6. De Montis, G. M.; Devoto, P.; Angioni, R. M.; Currelli, V.; Tagliomonte, A.: In utero exposure to methadone produces a stable decrease of the cortex 5-HT transport system in rats. Eur. J. Pharmacol. 90:57–63; 1983. 7. Deyo, S. N.; Swift, R. M.; Miller, R. J.: Morphine and endorphins modulate dopamine turnover in rat median eminence. Proc. Natl. Acad. Sci. USA 76:3006–3009; 1979. 8. Enters, E. K.; Guo, H.; Pandey, U.; Ko, D.; Robinson, S. E.: The effect of prenatal methadone exposure on development and nociception during the early postnatal period of the rat. Neurotoxicol. Teratol. 13:161–166; 1991. 9. Graybiel, A. M.; Ragsdale, C. W.: Biochemical anatomy of the striatum. In: Emson, P. C., ed. Chemical neuroanatomy. New York: Raven Press; 1983:427–504. 10. Guo, H.; Enters, E. K.; McDowell, K. P.; Robinson, S. E.: The effect of prenatal exposure to methadone on neurotransmitters in neonatal rats. Dev. Brain Res. 57:296–298; 1990. 11. Gysling, K.; Wang, R. Y.: Morphine-induced activation of A10 dopamine neurons in the rat. Brain Res. 277:119–127; 1983. 12. Hagan, R. M.; Hughes, I. E.: Opioid receptor subtypes involved in the control of tranbsmitter release in the brain of the rat. Neuropharmacology 23:491–495, 1984. 13. Hammer, R. P.; Ricalde, A. A.; Seatriz, J. V.: Effects of opiates on brain development. Neurotoxicology 10:475–484; 1989. 14. Hutchings, D. E.: Behavioral teratology: A new frontier in neurobehavioral research. In: Johnson, E. M.; Kochhar, D. M., eds. Handbook of experimental pharmacology, vol. 65. Teratogenesis and reproductive pharmacology. Berlin: Springer-Verlag; 1983: 207–235. 15. Hutchings, D. E.: Issues of methodology and interpretation in clinical and animal behavioral teratology studies. Neurobehav. Toxicol. Teratol. 7:639–642; 1985. 16. Hutchings, D. E.; Zmitrovich, A. C.; Brake, S. C.; Malowany, D.; Church, S. H.; Nero, T. J.: Prenatal administration of methadone using the osmotic minipump: Effects on maternal and offspring toxicity, growth, and behavior in the rat. Neurotoxicol. Teratol. 14: 65–71; 1992. 17. Jarvis, M. A. E.; Schnoll, S. H.: Methadone treatment during pregnancy. J. Psychoactive Drugs 26:155–161; 1994. 18. Johnston, C. A.; Moore, K. E.: The effect of morphine on 5-hydroxytryptamine synthesis and metabolism in the striatum, and several discrete hypothalamic regions of the rat brain.. J. Neural Transm. 57:65–73; 1983. 19. Kirk, R. E.: Experimental design. Monterey, CA: Brooks/Cole Publishing Co.; 1982.

20. Kunko, P. M.; Smith, J. A.; Wallace, M. J.; Maher, J. R.; Saady, J. J.; Robinson, S. E.: Perinatal methadone exposure produces physical dependence and altered behavioral development in the rat. J. Pharmacol. Exp. Ther. 277:1344–1351; 1996. 21. Lauder, J. A.; Bloom, F. E.: Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. J. Comp. Neurol. 155:469–481; 1974. 22. Lichtblau, L.; Sparber, S. B.: Opioids and development: A perspective on experimental models and methods. Neurobehav. Toxicol. Teratol. 6:3–8; 1984. 23. Lindvall, O.; Björklund, A.: Organization of catecholamine neurons in the rat central nervous system. In: Iversen, L. L.; Iversen, S. D.; Snyder, S. H., eds. Handbook of psychopharmacology, vol. 9. Chemical pathways in the brain. New York: Plenum Press; 1978:139–231. 24. Llorens-Cortes, C.; Pollard, H.; Schwartz, J. C.: Localization of opiate receptors in substantia nigra evidenced by lesion studies. Neurosci. Lett. 12:321–326; 1979. 25. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. 26. Mansour, A.; Fox, C. A.; Akil, H.; Watson, S. J.: Opioid-receptor mRNA expression in the rat CNS: Anatomical anbd functional implications. Trends Neurosci. 18:22–29; 1995. 27. McGinty, J. F.; Ford, D. H.: Effects of prenatal methadone on rat brain catecholamines. Dev. Neurosci. 3:224–234; 1980. 28. Misra, A. L.; Mulé, S, J.; Bloch, R.; Vadlamani, N. L.: Physiological disposition and metabolism of levo-methadone-1-3H in nontolerant and tolerant rats. J. Pharmacol. Exp. Ther. 185:287–299; 1973. 29. Moore, R. Y.; Bloom, F. E.: Central catecholamine neuron systems: Anatomy and physiology of the norepinephrine and epinephrine systems. Annu. Rev. Neurosci. 2:113–168; 1979. 30. Pfaff, D.; Keiner, M.: Atlas of estradiol-concentrating cells in the central nervous system of the female rat. J. Comp. Neurol. 151: 121–158; 1973. 31. Rech, R. H.; Lomuscio, G.; Algeri, S.: Methadone exposure in utero: Effects on brain biogenic amines and behavior. Neurobehav. Toxicol. 2:75–78; 1980. 32. Robinson, S. E.; Cheney, D. L.; Costa, E.: Effect of nomifensine and other antidepressant drugs on acetylcholine turnover in various regions of rat brain. Naunyn Schmiedebergs Arch. Pharmacol. 304:263–269; 1978. 33. Robinson, S. E.; Malthe-Sorenssen, D.; Wood, P. L.; Commissiong, J.: Dopaminergic regulation of the cholinergic septal hippocampal pathway. J. Pharmacol. Exp. Ther. 208:476–479; 1979. 34. Robinson, S. E.: Contribution of the dorsal noradrenergic bundle to the effect of amphetamine on acetylcholine turnover. In: Hanin, I., ed. Dynamics of cholinergic function. New York: Plenum Press; 1986:991–998. 35. Robinson, S. E.: 6-Hydroxydopamine lesion of the ventral noradrenergic bundle blocks the effect of amphetamine on hippocampal acetylcholine. Brain Res. 397:181–184; 1986. 36. Robinson, S. E.; Rice, M. A.; Hambrecht, K. L.: Effect of intrastriatal injection of DFP on acetylcholine, dopamine, and serotonin metabolism. J. Neurochem. 46:1632–1638; 1986. 37. Robinson, S. E.; Guo, H.; Enters, E. K.; McDowell, K. P.; Pascua, J. R.: Prenatal exposure to methadone affects central cholinergic neuronal activity in the weanling rat. Dev. Brain Res. 64:183–188; 1991. 38. Robinson, S. E.; Guo, H.; Maher, J. R.; McDowell, K. P. Kunko, P. M.: Postnatal methadone exposure does not prevent prenatal methadone-induced changes in striatal cholinergic neurons. Dev. Brain Res. 95:118–121; 1996. 39. Robinson, S. E.; Mo, Q.; Maher, J. R., Wallace, M. J.; Kunko, P. M.:

PERINATAL METHADONE AND NEUROTRANSMITTERS

40.

41. 42.

43.

Perinatal exposure to methadone affects central cholinergic activity in the weanling rat. Drug Alcohol Depend. 41:116–126; 1996. Shannon, N. J.; Gunnet, J. W.; Moore, K. E.: A comparison of biochemical indices of 5-hydroxytryptaminergic neuronal activity following electrical stimulation of the dorsal raphe nucleus. J. Neurochem. 47:958–965; 1986. Slotkin, T. A.; Whitmore, W. L.; Salvaggio, M.; Seidler, F. J.: Perinatal methadone addiction affects brain synaptic development of biogenic amine systems in the rat. Life Sci. 24:1223–1230; 1979. Tanaka, M.; Kohno, Y.; Tsuda, A; Nakagawa, R.; Ida, Y.; Iimori, K.; Hoaki, Y.; Nagasaki, N.: Differential effects of morphine on noradrenaline release in brain regions of stressed and nonstressed rats. Brain Res. 275:105–115; 1983. Tsang, D.; Ng, S. C.: Effect of antenatal exposure to opiates on the development of opiate receptors in rat brain. Brain Res. 188:199–206; 1980.

303

44. Van Baar, A. L.; Fleury, P.; Soepatmi, S.; Ultee, C. A.; Wesselman, P. J. M.: Neonatal behavior after drug dependent pregnancy. Arch. Dis. Child. 64:235–240; 1989. 45. Vathy, I.; Rimanoczy, A.; Eaton, R. C.; Katay, L.: Modulation of catecholamine turnover rate in brain regions of rats exposed prenatally to morphine. Brain Res. 662:209–215; 1994. 46. Wang, C.; Pasulka, P.; Perry, B.; Pizzi, W. J.; Schnoll, S. H.: Effect of perinatal exposure to methadone on brain opioid and a2-adrenergic receptors. Neurobehav Toxicol. Teratol. 8:399–402; 1986. 47. Zagon, I. S.; McLaughlin, P. J.: Perinatal methadone exposure and brain development: A biochemical study. J. Neurochem. 31:49–54; 1978. 48. Zmitrovich, A. C.; Brake, S. C.; Liu, P. R.; Hamowy, A. S.; Hutchings, D. E.: Prenatal administration of methadone in the rat: Acoustic startle amplitude and the rest–activity cycle at 30 days of age. Neurotoxicol. Teratol. 16:251–255; 1994.