Prenatal exposure to methadone or buprenorphine alters µ-opioid receptor binding and downstream signaling in the rat brain

(1)

Int J Dev Neurosci. 2020;80:443–453. wileyonlinelibrary.com/journal/jdn

|

⁴⁴³

R E S E A R C H A R T I C L E

Prenatal exposure to methadone or buprenorphine alters

µ-opioid receptor binding and downstream signaling in the rat brain

Mette Kongstorp

^1,2

| Inger Lise Bogen

^1,3

| Synne Steinsland

¹

| Elisabeth Nerem

¹

|

Triske Woshyar Salih

¹

| Tom Stiris

^2,4

| Jannike Mørch Andersen

^1,5

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2020 The Authors. International Journal of Developmental Neuroscience published by John Wiley & Sons Ltd on behalf of International Society for Developmental Neuroscience Abbreviations: CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; ERK, extracellular signal-regulated kinase; MOR, µ-opioid receptor; NMDAR, N-methyl-D-aspartate receptor; OMT, opioid maintenance treatment; PND, postnatal day.

1Department of Forensic Sciences, Oslo University Hospital, Oslo, Norway

2Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway

3Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway

4Department of Neonatal Intensive Care, Oslo University Hospital, Oslo, Norway

5Department of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway Correspondence

Mette Kongstorp, Department of Forensic Sciences, Oslo University Hospital, P.O.

Box 4950 Nydalen, N-0424 Oslo, Norway.

Email: [email protected] Funding information

This research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sectors.

Abstract

There is a growing concern related to the use of opioid maintenance treatment during pregnancy. Studies in both humans and animals have reported reduced cognitive functioning in offspring prenatally exposed to methadone or buprenorphine;

however, little is known about the neurobiological mechanisms underlying these impairments. To reveal possible neurobiological effects of such in utero exposure, we examined brain tissue from methadone- and buprenorphine-exposed rat offspring previously shown to display impaired learning and memory. We studied µ-opioid receptor (MOR) and N-methyl-D-aspartate receptor (NMDAR) binding in the rat offspring cerebrum during development and in the hippocampus at young adulthood.

Moreover, we examined activation of the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the extracellular signal-regulated kinase (ERK), which are central in the downstream signaling of these receptors. The methadone- and buprenorphine-exposed rat pups displayed reduced MOR binding up to two weeks after birth, whereas the NMDAR binding was unaffected. Prenatal exposure to methadone or buprenorphine also resulted in decreased activation of CaMKII and/or ERK during development, while young adult offspring displayed increased hippocampal ERK activation.

In conclusion, our findings suggest that prenatal exposure to exogenous opioids, such as methadone or buprenorphine, may disturb the endogenous opioid system during development, with long-term effects on proteins important for cognitive functioning.

K E Y W O R D S

buprenorphine, methadone, neurobiological development, opioid maintenance treatment, prenatal exposure, µ-opioid receptor

(2)

1 | INTRODUCTION

Opioid maintenance treatment (OMT) with the full µ-opioid receptor (MOR) agonist methadone or the partial MOR agonist buprenorphine is the recommended therapy for pregnant opioid-dependent women (WHO, 2014). Patients in OMT generally take better care of their health and are less likely to relapse to illicit opioid use compared with those not receiving such treatment (Fajemirokun-Odudeyi et al., 2006; Finnegan, Amass, Jones, & Kaltenbach, 2005; Minozzi, Amato, Bellisario, Ferri, & Davoli, 2013); however, OMT during pregnancy may not be without risk for the fetus, as both methadone and buprenorphine cross the placenta (Kongstorp, Bogen, Stiris, & Andersen, 2019; Nanovskaya, Deshmukh, Brooks, & Ahmed, 2002; Nanovskaya, Nekhayeva, Hankins,

& Ahmed, 2008).

Opioid receptors are highly expressed by developing neuronal cells (Hauser & Knapp, 2017), and endogenous opioids act as inhibitory growth factors regulating dendritic growth and spine formation (Hauser, McLaughlin, & Zagon, 1987;

Zagon & McLaughlin, 1987). As the endogenous opioid system plays a key role in brain development (Hauser &

Knapp, 2017; Sargeant, Miller, & Day, 2008), the fetus is probably particularly vulnerable to exogenous opioid exposure. Furthermore, methadone acts as a non-competitive antagonist at the N-methyl-D-aspartate receptor (NMDAR) (Ebert, Andersen, & Krogsgaard-Larsen, 1995) which also has been shown to be involved in brain development (Pearce, Cambray-Deakin, & Burgoyne, 1987).

A growing number of clinical studies indicate that children born to mothers in OMT have an increased risk of cognitive impairments and attentional and behavioral problems compared with non-exposed children (summarized in Andersen, Hoiseth, & Nygaard, 2020; Baldacchino, Arbuckle, Petrie, & McCowan, 2015; Lee, Bora, Austin, Westerman, & Henderson, 2020; Monnelly, Hamilton, Chappell, Mactier, & Boardman, 2019). In support of these clinical findings, our research group (Kongstorp, Bogen, Stiris, & Andersen, 2020) and others (Chen et al., 2015;

Jantzie et al., 2020; Peters, 1977; Van Wagoner, Risser, Moyer, & Lasky, 1980; Zagon, McLaughlin, &

Thompson, 1979) have reported impaired cognitive functioning in rat offspring prenatally exposed to methadone or buprenorphine. Furthermore, some studies describe that opioids affect various processes of brain development, such as neurogenesis, cell proliferation and myelination (Robinson, 2002b; Sanchez, Bigbee, Fobbs, Robinson, &

Sato-Bigbee, 2008; Vestal-Laborde, Eschenroeder, Bigbee, Robinson, & Sato-Bigbee, 2014; Wu et al., 2014); however, the knowledge concerning the neurobiological effects of prenatal methadone or buprenorphine exposure is limited.

The aim of the present study was therefore to examine neurobiological effects of prenatal exposure to methadone

or buprenorphine in brain tissue taken from young adult rats previously shown to display impaired cognitive performance (Kongstorp et al., 2020). We examined binding to the MOR and the NMDAR as well as activation of downstream signaling pathways during development and at young adulthood.

2 | MATERIALS AND METHODS 2.1 | Brain tissue harvesting and

preparation

The animal exposure and experiments were approved by the Norwegian Animal Research Authority (Norwegian Food Safety Authority, Oslo, Norway) and performed in accordance with the ARRIVE (Animals in Research: Reporting in Vivo Experiments) (Kilkenny, Browne, Cuthill, Emerson, &

Altman, 2010) and the laws and regulations controlling experiments on live animals in Norway. The brain tissue used in the present study was harvested from rat offspring (Sprague–

Dawley; n = 200) exposed through gestational treatment of the dams to racemic methadone–HCl (10 mg/kg/day; n = 10), buprenorphine–HCl (1 mg/kg/day; n = 10) or vehicle (sterile water; n = 10), as described in Kongstorp et al. (2019) and Kongstorp et al. (2020). The drugs were delivered by a 28-day osmotic minipump (2ML4; Alzet, Cupertino, CA) implanted subcutaneously in females 5 days prior to mating with drug- naïve males. We have previously shown that this exposure regimen provides blood concentrations of 0.25 ± 0.02 µM methadone and 5.65 ± 0.16 nM buprenorphine in the dams during gestation (Kongstorp et al., 2019), which are compa- rable to the concentrations reported in pregnant women in OMT (Bartu, Ilett, Hackett, Doherty, & Hamilton, 2012;

Concheiro, Jones, Johnson, Shakleya, & Huestis, 2010;

Gordon, Lopatko, Somogyi, Foster, & White, 2010). The neonatal outcomes, growth parameters (body weight and length), withdrawal symptoms (ultrasonic vocalizations and corticosterone levels) and long-term cognitive effects in the offspring included in this study and, their littermates, have been reported previously (Kongstorp et al., 2019, 2020).

The day of delivery was noted as postnatal day (PND) 0. On PND 1, 7, 14, and 21, the cerebrum from one or two randomly chosen offspring from each litter was isolated after decapitation. On PND 77, one or two offspring from each litter were decapitated and the hippocampus was dissected.

The brain tissue was weighed, snap-frozen in liquid nitro- gen, and stored at −80°C. For sample preparation, the tissue was thawed on ice, homogenized (600–900 rpm; glass-Tef- lon) in ice-cold 0.32 M sucrose (100 mg tissue/ml) containing 1× cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland) and batched. The tissue was stored at −80°C until further analysis.

(3)

2.2 | Receptor binding

The receptor binding studies were performed as previously described (Fjelldal et al., 2019; Kvello et al., 2019). In brief, cerebrum and hippocampus homogenates were diluted to 10 mg tissue/ml in ice-cold incubation buffer containing 50 mM Tris–HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, and 0.5 mM MgCl₂. The samples were centrifuged for 30 min (30,000 g, 4°C), and the pellets were resuspended in incubation buffer before incubation for 30 min at 37°C to remove endogenous opioids. The samples were then centrifuged for 30 min (30,000 g, 4°C) and the resulting pellets were resuspended in 0.5 ml of the incubation buffer. For MOR binding, the samples (duplicates, 100 µl) were preincubated at room temperature for 10 min followed by incubation with 2 nM [³H]-DAMGO ([D-Ala₂-N-Me-Phe₄-Gly-ol⁵]-Enkephalin;

PerkinElmer, Oslo, Norway) at room temperature for 60 min (final volume 200 µl). Non-specific binding was determined by adding 50 µM naloxone to parallel samples prior to incubation with [³H]-DAMGO (final volume 200 µl). For NMDAR binding, the samples (duplicates, 75 µl) were preincubated for 10 min at room temperature followed by incubation with 4 nM [³H]-ifenprodil (PerkinElmer) at room temperature for 60 min (final volume 200 µl). Non-specific binding was determined by adding 50 µM ifenprodil to parallel samples prior to incubation with [³H]-ifenprodil (final volume 200 µl).

The reactions were terminated by adding 4 ml of ice- cold 50 mM Tris–HCl (pH 7.4) to the samples, followed by rapid filtration through GF/B glass microfiber filters (1.0 µm; GE Healthcare Life Sciences, IL). The filters were washed 3 times with ice-cold Tris–HCl. UltimaGold (4 ml; PerkinElmer, MA) was added to each filter and the radioactivity was counted in a liquid scintillation analyzer (Tri-Carb 2810TR; PerkinElmer). Total tissue protein was measured as previously described (Lowry, Rosebrough, Farr,

& Randall, 1951). Specific receptor binding was calculated as total binding minus non-specific binding, and calculated as the amount of [³H]-DAMGO or [³H]-ifenprodil bound per mg tissue protein. The presence of methadone or buprenorphine in the prepared samples was measured by UHPLC-MS/

MS, as previously described (Kongstorp et al., 2019).

2.3 | Western blotting

Activation of the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the extracellular signal-regulated kinase (ERK) were studied by measuring phosphorylated and total CaMKII and ERK expression, respectively, by western blotting. The cerebrum and hippocampus homogenates were diluted to a final concentration of 14.3 mg tissue/ml in ice-cold 0.32 M sucrose containing 1× cOmplete protease

inhibitor cocktail (Roche). The samples were then added loading buffer to a final concentration of 0.06 M DTT and 0.96× Laemmli buffer (Bio-Rad laboratories, CA). After protein denaturation (100°C for 5 min), the samples were loaded into wells (10 µg protein/well for PND 1 and 7; 11 µg protein/well for PND 14; 15 µg protein/well for PND 21 and 77) on SDS-gels (12% Criterion TGX, 18 wells; Bio- Rad Laboratories) and separated by electrophoresis (200V;

PowerPac HC 300W, Bio-Rad Laboratories). Next, the samples were electrophoretically transferred to a nitrocellulose membrane, confirmed by Ponceau S staining. The mem- branes were incubated in blocking buffer (3% low-fat dry milk in 0.1 M Tris-buffered saline containing 0.05% Tween 20 [TBS-T]; pH 7.4) for 1 hr, and probed with primary anti- bodies against CaMKII (1:500, #3362, Cell Signaling, CA), pCaMKII (1:3000, #12716, Cell Signaling), ERK (1:750000, SC-153, Santa Cruz Biotechnology, TX), pERK (1:750, SC-7383, Santa Cruz Biotechnology), or β-actin (1:100000, A5316, Sigma Aldrich, MO) overnight at 4°C. The mem- branes were washed in TBS-T (4 × 5 min) and incubated with a HRP-conjugated secondary antibody for 2 hr at room temperature. Each blot was then washed in TBS-T (3 × 5 min) and TBS (5 min) before being exposed to a chemilumines- cent substrate (SuperSignal^TM, Thermo Scientific, MA) for 5 min. The blots were visualized using a ChemiDoc XRS im- ager (Bio-Rad Laboratories). The 50-kDa band was used to quantify CaMKII and pCaMKII, the 42-kDa band was used to quantify ERK and pERK, and the 43-kDa band was used to quantify β-actin (see Figures 3–5 for representative blots).

The blots were stripped with Reblot Plus Mild Antibody Stripping Solution (Millipore, MA) and proceeded with the next primary antibody after confirmation of complete stripping. Total tissue protein was measured as previously described (Lowry et al., 1951).

2.4 | Data and statistical analysis

All data are presented as mean ± SEM. A maximum of 2 offspring (males and females) from each litter were included in the study, and each animal was counted as n = 1. The receptor binding results from the methadone- and buprenorphine-exposed animals were normalized to the average of the vehicle-exposed animals on the respective PND. For the western blot studies, each gel was run with samples from 5 to 6 offspring from the different treatment groups. The expression of each individual methadone- or buprenorphine sample was normalized to the average of the vehicle-treated samples on the respective blot (% of vehicle). For each sample, 3–6 technical replicates were performed and the average value (in

% of vehicle) was reported as n = 1. The results were ana- lyzed by a one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. Statistical analyses were performed

(4)

using SPSS version 25 (SPSS, Chicago, IL). A value of p < .05 was considered statistically significant.

3 | ^RESULTS

The brain tissue used in the present study was taken from offspring that have been characterized in two previous studies focusing on pharmacokinetic parameters and birth outcomes (Kongstorp et al., 2019) and behavioral effects (Kongstorp et al., 2020). In Kongstorp et al. (2019), we reported that prenatal exposure to methadone or buprenorphine did not affect the litter size, the number of stillborn offspring, or the sex ratio. Prenatal exposure to methadone resulted in a small re- duction in the birth weight (Kongstorp et al., 2019), whereas no differences were revealed in growth parameters from PND 3 to 21 (Kongstorp et al., 2020). The methadone- and buprenorphine-exposed offspring showed impaired cognitive functioning at young adulthood (Kongstorp et al., 2020).

3.1 | Offspring brain weight

A significant effect of the treatment was found on the brain weight in 14-day-old pups [F_(2,37) = 4.98, p = .013]. Prenatal exposure to methadone reduced the brain weight by 9 ± 3 and 13 ± 3% compared with the vehicle- and buprenorphine- exposed offspring, respectively (p < .05; Figure 1). No significant differences were observed in offspring brain weight between the treatment groups on PND 1, 7, or 21.

3.2 | Receptor binding 3.2.1 | ^MOR

The binding of DAMGO to MORs in cerebrum homogenate (fmol/mg protein) from vehicle-exposed pups decreased during the three first weeks after birth [F_(3,38) = 5.33, p = .004, Figure 2a]. Prenatal exposure to methadone or buprenorphine significantly reduced the cerebral MOR binding in rat pups on PND 1 [F_(2,25) = 5.78, p = .009], PND 7 [F_(2,32) = 6.16, p = .006], and PND 14 [F_(2,33) = 3.86, p = .032]. On PND 1, the MOR bindings in the methadone- and buprenorphine- exposed offspring were 59.3 ± 3.5 and 61.9 ± 8.3%, respectively, of those of the vehicle-exposed animals (p < .05;

Figure 2b). On PND 7, the corresponding numbers were 81.3 ± 3.4 and 82.7 ± 3.5% for the methadone- and buprenorphine-exposed animals, respectively (p < .05). The buprenorphine-exposed offspring also displayed a reduced MOR binding on PND 14 which accounted for 84.8 ± 3.1%

of the vehicle group (p < .05). No differences in MOR binding were observed between the treatment groups on PND 21.

Prenatal exposure to methadone or buprenorphine did not affect MOR binding in the hippocampus from young adult rat offspring (PND 77; Figure 2b).

3.2.2 | ^NMDAR

The binding of ifenprodil to NMDARs in cerebrum homogenate (fmol/mg protein) from vehicle-exposed pups decreased during the three first weeks after birth [F_(3,28) = 7.30, p = .001, Figure 2c]. Prenatal exposure to methadone or buprenorphine did not affect NMDAR binding when measured on PND 1, 7, 14, and 21 (Figure 2d). Furthermore, the NMDAR binding in the hippocampus from young adult offspring did not differ between the three treatment groups (PND 77; Figure 2d).

Chemical analyses confirmed that no methadone or buprenorphine were present in the brain samples prepared for receptor binding studies (data not shown).

3.3 | Expression of CaMKII and ERK

Prenatal exposure to methadone or buprenorphine significantly reduced the expression of pCaMKII in 7-day-old rat pups [F_(2,25) = 5.08, p = .015]. The pCaMKII expression in the buprenorphine-exposed pups was 53.4 ± 10.8% of the control group (p < .05; Figure 3a). The methadone-exposed animals also showed a tendency to reduced pCaMKII expression on PND 7, displaying 70.2 ± 10.1% of the level in the vehicle- exposed group (p = .06; Figure 3a). There was no significant effect of the treatment on the expression of pCaMKII on PND

FIGURE 1 Offspring brain weight (g) following prenatal exposure to methadone or buprenorphine. The whole brain weight from rat pups was measured on postnatal days 1, 7, 14, and 21.

Values are shown as mean ± SEM. n = 11–16 offspring from 7 to 9 L. *p < .05 compared with vehicle, ^†p < .005 compared with buprenorphine, one-way ANOVA followed by Tukey‘s post hoc test

(5)

1, 14, and 21. The total CaMKII expression did not differ between the three treatment groups at any time point; however, there was a tendency of increased expression in the methadone- exposed offspring on PND 21 (p =.06; Figure 3b). The expression of pCaMKII and total CaMKII in the hippocampus from young adult offspring were not affected by prenatal exposure to methadone or buprenorphine (PND 77; Figure 3a,b).

Prenatal exposure to methadone or buprenorphine significantly reduced the expression of pERK in 14-day-old rat pups [F_(2,16) = 5.36, p = .019]. The pERK expression in the methadone-exposed pups was 75.9 ± 5.1% of the control group, which was significantly lower than the expression in both the vehicle- and the buprenorphine-exposed animals (p < .01

and p < .05; Figure 4a). No significant effects on the pERK expression were observed in the opioid-exposed animals on PND 1, 7, and 21. The total ERK expression in the cerebrum did not differ between the treatment groups during development (Figure 4b).

Young adult rats prenatally exposed to methadone or buprenorphine exhibited increased ERK phosphorylation in the hippocampus [F_(2,15) = 14.9, p < .001]. The expression of pERK in the methadone- and buprenorphine-exposed offspring were 123 ± 4 and 140 ± 6% of the control group, respectively (p < .05 and p < .001; Figure 4a), whereas no differences were observed in the expression of total ERK (Figure 4b).

FIGURE 2 Receptor binding in rat offspring prenatally exposed to methadone or buprenorphine. (a) Timeline of µ-opioid receptor (MOR) binding by [³H]-DAMGO (fmol/mg tissue protein) in cerebrum from vehicle-exposed rat offspring on postnatal day (PND) 1, 7, 14, and 21. (b) MOR binding by [³H]-DAMGO in the cerebrum during development (PND 1–21) and in the hippocampus from young adult offspring (PND 77) prenatally exposed to vehicle, methadone, or buprenorphine. The values were normalized to the average of the vehicle-exposed offspring (% of vehicle) and are shown as mean ± SEM. (c) Timeline of N-methyl-D-aspartate receptor (NMDAR) binding by [³H]-ifenprodil (fmol/mg tissue protein) in cerebrum from vehicle-exposed rat offspring on PND 1, 7, 14, and 21. (d) NMDAR binding by [³H]-ifenprodil in the cerebrum during development (PND 1–21) and in the hippocampus from young adult offspring (PND 77) prenatally exposed to vehicle, methadone, or buprenorphine. The values were normalized to the average of the vehicle-exposed offspring (% of vehicle) and are shown as mean ± SEM. PND 1, n = 7–10 offspring from 7 to 9 litters; PND 7, n = 7–15 offspring from 6 to 8 litters; PND 14, n = 7–12 offspring from 7 to 9 litters; PND 21, n = 7–8 offspring from 6 to 8 litters; PND 77; n = 4–5 offspring from 4 to 5 litters. *p < .05, **p < .01 compared with vehicle, one-way ANOVA followed by Tukey‘s post hoc test

(6)

Prenatal exposure to methadone or buprenorphine did not affect the expression of β-actin neither in the cerebrum during development (PND 1–21) nor in the hippocampus at young adulthood (PND 77; Figure 5). We did not observe any differences in total tissue protein between the methadone-, buprenorphine- and vehicle-exposed offspring (data not shown).

4 | DISCUSSION

In the present study, we examined possible neurobiological changes induced by prenatal exposure to methadone or buprenorphine in a rat model. We have previously reported that this exposure regimen used leads to long-term impairments in cognitive functioning in young adult offspring (Kongstorp et al., 2020). Here, we show that the opioid-exposed pups displayed reduced cerebral MOR binding and decreased activation of CaMKII and ERK the first two weeks after birth.

Moreover, we found an increased activation of ERK in the hippocampus of young adult animals.

The MOR binding was significantly reduced up to one and two weeks after birth in the methadone- and

buprenorphine-exposed offspring, respectively, with more prominent reductions in the newborns compared with the 7- and 14-day-old offspring. This shows that the MOR binding gradually normalized after the exposure ceased.

In the buprenorphine-exposed offspring, this normaliza- tion was delayed which could possibly be explained by the somewhat slower elimination of buprenorphine from the neonatal brain as compared with methadone (Kongstorp et al., 2019). There are also other studies showing decreased MOR binding in fetuses and rat pups after prenatal exposure to methadone, buprenorphine, or morphine (Belcheva et al., 1994, 1998; Darmani, Schnoll, Pandey,

& Martin, 1992; Hammer, 1991; Hou et al., 2004; Tempel, Habas, Paredes, & Barr, 1988; Tsang & Ng, 1980). In some of these studies, the binding was found to be normalized already one week after birth (Belcheva et al., 1994;

Darmani et al., 1992; Hou et al., 2004). The deviating findings may be due to differences in the opioid doses and exposure regimen used; however, as pharmacokinetic data are missing it is difficult to draw conclusions. While binding affinities were not examined in the present work, previous studies have reported normal binding affinity

FIGURE 3 Expression of (a) phosphorylated Ca²⁺/calmodulin-dependent protein kinase II (pCaMKII) and (b) total CaMKII in the cerebrum during development (PND 1–21) and in the hippocampus from young adult offspring (PND 77) prenatally exposed to vehicle (Veh), methadone (Met), or buprenorphine (Bup). The value of each of the methadone and buprenorphine sample was normalized to the average of the vehicle-exposed samples on the blot (% of vehicle) and are shown as mean ± SEM. n = 5–9 offspring from 4 to 8 litters. The right panel shows representative examples of western blots with the protein bands of 60 (upper) and 50 kDa (lower). The 50-kDa band was used for quantification.

*p < .05 compared with vehicle, ^#p = .06 compared with vehicle, one-way ANOVA followed by Tukey‘s post hoc test

(7)

to the MOR in 1- and 7-day-old rat pups prenatally exposed to buprenorphine (Belcheva et al., 1994, 1998).

This suggests that the reduced MOR binding observed in

the present study is a result of fewer MOR binding sites.

As no methadone or buprenorphine was detected in the prepared homogenates, it is unlikely that the reduced

FIGURE 4 Expression of (a) phosphorylated extracellular signal-regulated kinase (pERK) and (b) total ERK in the cerebrum during development (PND 1–21) and in the hippocampus from young adult offspring (PND 77) prenatally exposed to vehicle (Veh), methadone (Met), or buprenorphine (Bup). The value of each methadone and buprenorphine sample was normalized to the average of the vehicle-exposed samples on the same blot (% of vehicle) and are shown as mean ± SEM. n = 5–9 offspring from 4 to 8 litters. The right panel shows representative examples of western blots with the protein bands of 44 (upper) and 42 kDa (lower). The 42-kDa band was used for quantification. *p < .05, **p < .01,

***p < .001 compared with vehicle, ^†p < .05 compared with methadone, one-way ANOVA followed by Tukey‘s post hoc test

FIGURE 5 Expression of β-actin in the cerebrum during development (PND 1–21) and in the hippocampus from young adult offspring (PND 77) prenatally exposed to vehicle (Veh), methadone (Met), or buprenorphine (Bup). The value of each methadone and buprenorphine sample was normalized to the average of the vehicle-exposed samples on the same blot (% of vehicle) and are shown as mean ± SEM. n = 5–9 offspring from 4 to 8 litters. The right panel shows representative examples of western blots with the protein band of 43 kDa that was used for quantification

(8)

DAMGO binding was caused by remaining opioids in the samples.

Neither methadone nor buprenorphine affected the cerebral NMDAR binding in the offspring. Recently, we reported similar findings in the cerebellum from chicken embryos exposed to methadone on embryonic days 13 and 14 (Fjelldal et al., 2019). However, we also found that continuous gestational exposure to buprenorphine, but not methadone, reduced the cerebellar expression of the NMDAR subunit GluN2B in 14-day-old rat offspring (Fjelldal et al., 2019). This was surprising as buprenorphine does not bind to the NMDAR, while methadone acts as an NMDAR antagonist (Garrido &

Troconiz, 1999; Robinson, 2002a). It is well known that MOR and NMDAR co-localize on single neurons in many regions of the CNS (Rodriguez-Munoz, Sanchez-Blazquez, Vicente- Sanchez, Berrocoso, & Garzon, 2012; Trujillo, 2002), and previous studies indicate that some of the adverse effects seen in offspring prenatally exposed to methadone or morphine can be prevented by co-administration of the NMDAR antagonist dextromethorphan (Chiang et al., 2015; Tao, Yeh, Su, &

Wu, 2001). Although we did not observe any changes in the NMDAR binding, these previous findings indicate that the NMDAR might be involved in some of the effects of prenatal exposure to methadone or buprenorphine, probably through cross-talk with MORs.

It has been shown that prenatal exposure to methadone or buprenorphine decreases the level of brain-derived neu- rotrophic factor and affects neurotransmitter biosynthesis, neurogenesis, cell proliferation, and myelination in the rat pup brain the first two weeks after birth (Robinson, 2002b;

Sanchez et al., 2008; Vestal-Laborde et al., 2014; Wu et al., 2014). In the present study, we examined the effect of prenatal opioid exposure on the activation of CaMKII and ERK, two proteins that are central in the downstream signaling pathways of the MOR (Al-Hasani & Bruchas, 2011;

Williams, Christie, & Manzoni, 2001) and critically involved in neuronal and glial maturation (Kennedy, 2000; Rongo &

Kaplan, 1999; Samuels, Saitta, & Landreth, 2009), as well as in the processes of learning and memory (Frankland, O'Brien, Ohno, Kirkwood, & Silva, 2001; Peng, Zhang, Zhang, Wang, & Ren, 2010). The buprenorphine-exposed pups displayed reduced activation of CaMKII one week after birth. Similar findings have been reported in the hippocampus from 4-week-old rats prenatally exposed to morphine (Nasiraei-Moghadam et al., 2013).

The methadone-exposed offspring displayed reduced phosphorylation of ERK in the cerebrum at two weeks of age. No such effect was observed in the buprenorphine-exposed offspring, which is different from the findings of Hung et al. (2013) who reported reduced pERK in the cortex of 3-week-old rat pups prenatally exposed to daily injections of buprenorphine. Wu and colleagues have reported reduced phosphorylation of ERK in prefrontal cortex in 7-week-old

rat offspring after maternal exposure to daily injections of buprenorphine during gestation (Wu et al., 2017). Our studies in the hippocampus of young adult rat offspring previously shown to display impaired cognitive functioning (Kongstorp et al., 2020) revealed increased activation of ERK. To date, a possible relationship between increased hippocampal ERK phosphorylation and cognitive impairments remains unex- plored. However, excessive ERK activation has been associated with increased GABA release and reduced long-term potentiation in mice (Costa et al., 2002; Cui et al., 2008), which could be a possible mechanism involved in the observed learning and memory deficits in the methadone- and buprenorphine-exposed rats.

In accordance with previous studies (Field, McNelly, &

Sadava, 1977; Zagon & McLaughlin, 1977a, 1977b, 1978), we found that prenatal exposure to methadone reduced brain weight in two-week-old rat pups. While we did not observe any effect of buprenorphine, other studies have reported reduced offspring brain weight also following maternal exposure to daily injections of buprenorphine (Hung et al., 2013;

Wu et al., 2017). In humans, neuroimaging studies of children born to mothers using opioids during pregnancy have indicated reduced brain volume compared with non-exposed children (Walhovd et al., 2007; Yuan et al., 2014), which has been associated with poorer cognitive performance later in life (Peterson et al., 2000).

One limitation of the present study is the use of the cerebrum to study receptor binding and protein expression during development, as this does not allow for detection of regional specific alterations, as previously reported for MOR binding (Vathy, Slamberova, Rimanoczy, Riley, &

Bar, 2003), and expression of myelin proteins in rats prenatally exposed to opioids (Oberoi et al., 2019). Another limitation is that the number of animals did not allow us to examine sex differences, which have been reported by others after in utero exposure to opioids, both in respect to neurobiological mechanisms and behavioral effects (Daly, Hughes,

& Woodward, 2012; Gholami et al., 2020; Rimanoczy &

Vathy, 1995; Vathy et al., 2003).

In conclusions, the present study shows that prenatal exposure to methadone or buprenorphine affects opioid receptors and downstream signaling in the rat offspring brain. We report decreased MOR binding and reduced activation of CaMKII and ERK the first two weeks after birth. Moreover, the ERK activation was increased in the hippocampus at young adult age. Our findings suggest that prenatal exposure to exogenous opioids such as methadone or buprenorphine may disturb the endogenous opioid system during development, with long-term effects on proteins important for cognitive functioning.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

(9)

AUTHOR CONTRIBUTIONS

Participated in research design: MK, ILB, SS, EN, TS, and JMA. Conducted the experiments: MK, SS, EN, TWS, and JMA. Performed data analysis: MK, SS, and EN. Wrote or contributed to the writing of the manuscript: MK, ILB, SS, EN, TWS, TS, and JMA. All Authors have contributed to and approved the final manuscript.

DATA AVAILABILITY STATEMENT Research data are not shared.

ORCID

Mette Kongstorp https://orcid.

org/0000-0002-2115-053X

Inger Lise Bogen https://orcid.org/0000-0003-2877-0624 Triske Woshyar Salih https://orcid.

org/0000-0003-2819-2043

Jannike Mørch Andersen https://orcid.

org/0000-0002-8425-9743 REFERENCES

Al-Hasani, R., & Bruchas, M. R. (2011). Molecular mechanisms of opioid receptor-dependent signaling and behavior. Anesthesiology, 115, 1363–1381. https://doi.org/10.1097/ALN.0b013 e3182 38bba6

Andersen, J. M., Hoiseth, G., & Nygaard, E. (2020). Prenatal exposure to methadone or buprenorphine and long-term outcomes: A meta-analysis. Early Human Development, 143, 104997. https://doi.

org/10.1016/j.earlh umdev.2020.104997

Baldacchino, A., Arbuckle, K., Petrie, D. J., & McCowan, C. (2015).

Erratum: Neurobehavioral consequences of chronic intrauterine opioid exposure in infants and preschool children: A systematic review and meta-analysis. BMC Psychiatry, 15, 134. https://doi.

org/10.1186/s1288 8-015-0438-5

Bartu, A. E., Ilett, K. F., Hackett, L. P., Doherty, D. A., & Hamilton, D. (2012). Buprenorphine exposure in infants of opioid-dependent mothers at birth. Australian and New Zealand Journal of Obstetrics and Gynaecology, 52, 342–347. https://doi.

org/10.1111/j.1479-828X.2012.01424.x

Belcheva, M. M., Bohn, L. M., Ho, M. T., Johnson, F. E., Yanai, J., Barron, S., & Coscia, C. J. (1998). Brain opioid receptor adapta- tion and expression after prenatal exposure to buprenorphine.

Developmental Brain Research, 111, 35–42. https://doi.org/10.1016/

s0165 -3806(98)00117 -5

Belcheva, M. M., Dawn, S., Barg, J., McHale, R. J., Ho, M. T., Ignatova, E., & Coscia, C. J. (1994). Transient down-regulation of neonatal rat brain mu-opioid receptors upon in utero exposure to buprenorphine. Developmental Brain Research, 80, 158–162. https://doi.

org/10.1016/0165-3806(94)90100 -7

Chen, H. H., Chiang, Y. C., Yuan, Z. F., Kuo, C. C., Lai, M. D., Hung, T.

W., … Chen, S. T. (2015). Buprenorphine, methadone, and morphine treatment during pregnancy: Behavioral effects on the offspring in rats. Neuropsychiatric Disease and Treatment, 11, 609–618. https://

doi.org/10.2147/NDT.S70585

Chiang, Y. C., Ye, L. C., Hsu, K. Y., Liao, C. W., Hung, T. W., Lo, W. J., … Tao, P. L. (2015). Beneficial effects of co-treatment with dextromethorphan on prenatally methadone-exposed offspring.

Journal of Biomedical Science, 22, 19. https://doi.org/10.1186/

s1292 9-015-0126-2

Concheiro, M., Jones, H., Johnson, R. E., Shakleya, D. M., & Huestis, M. A. (2010). Confirmatory analysis of buprenorphine, norbu- prenorphine, and glucuronide metabolites in plasma by LCMSMS.

Application to umbilical cord plasma from buprenorphine-main- tained pregnant women. Journal of Chromatography B, 878, 13–20.

https://doi.org/10.1016/j.jchro mb.2009.11.005

Costa, R. M., Federov, N. B., Kogan, J. H., Murphy, G. G., Stern, J., Ohno, M., … Silva, A. J. (2002). Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1. Nature, 415, 526–530. https://doi.org/10.1038/natur e711

Cui, Y., Costa, R. M., Murphy, G. G., Elgersma, Y., Zhu, Y., Gutmann, D. H., … Silva, A. J. (2008). Neurofibromin regulation of ERK signaling modulates GABA release and learning. Cell, 135, 549–560.

https://doi.org/10.1016/j.cell.2008.09.060

Daly, F. M., Hughes, R. N., & Woodward, L. J. (2012). Subsequent anxiety-related behavior in rats exposed to low-dose methadone during gestation, lactation or both periods consecutively. Pharmacology, Biochemistry and Behavior, 102, 381–389. https://doi.org/10.1016/j.

pbb.2012.05.010

Darmani, N. A., Schnoll, S. H., Pandey, U., & Martin, B. R. (1992).

Chronic prenatal methadone exposure alters central opioid mu-receptor affinity in both fetal and maternal brain. Neurotoxicology and Teratology, 14, 265–271. https://doi.org/10.1016/0892- 0362(92)90006 -v

Ebert, B., Andersen, S., & Krogsgaard-Larsen, P. (1995). Ketobemidone, methadone and pethidine are non-competitive N-methyl-D-aspartate (NMDA) antagonists in the rat cortex and spinal cord. Neuroscience Letters, 187, 165–168. https://doi.org/10.1016/0304-3940(95) 11364 -3

Fajemirokun-Odudeyi, O., Sinha, C., Tutty, S., Pairaudeau, P., Armstrong, D., Phillips, T., & Lindow, S. W. (2006). Pregnancy outcome in women who use opiates. European Journal of Obstetrics, Gynecology, and Reproductive Biology, 126, 170–175. https://doi.

org/10.1016/j.ejogrb.2005.08.010

Field, T., McNelly, A., & Sadava, D. (1977). Effect of maternal methadone addiction on offspring in rats. Archives Internationales de Pharmocodynamie et de Thérapie, 228, 300–303.

Finnegan, L. P., Amass, L., Jones, H., & Kaltenbach, K. (2005).

Addiction and pregnancy. Heroin Addiction and Related Clinical Problems, 7, 5–21.

Fjelldal, M. F., Hadera, M. G., Kongstorp, M., Austdal, L. P. E., Sulovic, A., Andersen, J. M., & Paulsen, R. E. (2019). Opioid receptor-medi- ated changes in the NMDA receptor in developing rat and chicken.

International Journal of Developmental Neuroscience, 78, 19–27.

https://doi.org/10.1016/j.ijdev neu.2019.07.009

Frankland, P. W., O'Brien, C., Ohno, M., Kirkwood, A., & Silva, A.

J. (2001). Alpha-CaMKII-dependent plasticity in the cortex is re- quired for permanent memory. Nature, 411, 309–313. https://doi.

org/10.1038/35077089

Garrido, M. J., & Troconiz, I. F. (1999). Methadone: A review of its pharmacokinetic/pharmacodynamic properties. Journal of Pharmacological and Toxicological Methods, 42, 61–66. https://doi.

org/10.1016/s1056 -8719(00)00043 -5

Gholami, M., Saboory, E., Ahmadi, A. A., Asouri, M., Nasirikenari, M., & Rostamnezhad, M. (2020). Long-time effects of prenatal morphine, tramadol, methadone, and buprenorphine exposure on seizure and anxiety in immature rats. International Journal of Neuroscience, 1–8. https://doi.org/10.1080/00207 454.2019.1709841

(10)

Gordon, A. L., Lopatko, O. V., Somogyi, A. A., Foster, D. J., &

White, J. M. (2010). (R)- and (S)-methadone and buprenorphine concentration ratios in maternal and umbilical cord plasma following chronic maintenance dosing in pregnancy. British Journal of Clinical Pharmacology, 70, 895–902. https://doi.

org/10.1111/j.1365-2125.2010.03759.x

Hammer, R. P., Jr., Seatriz, J. V., & Ricalde, A. R. (1991). Regional dependence of morphine-induced mu-opiate receptor down-regulation in perinatal rat brain. European Journal of Pharmacology, 209, 253–256. https://doi.org/10.1016/0014-2999(91)90178 -s

Hauser, K. F., & Knapp, P. E. (2017). Opiate drugs with abuse liability hijack the endogenous opioid system to disrupt neuronal and glial maturation in the central nervous system. Frontiers in Pediatrics, 5, 294. https://doi.org/10.3389/fped.2017.00294

Hauser, K. F., McLaughlin, P. J., & Zagon, I. S. (1987). Endogenous opioids regulate dendritic growth and spine formation in developing rat brain. Brain Research, 416, 157–161. https://doi.org/10.1016/0006- 8993(87)91509 -5

Hou, Y., Tan, Y., Belcheva, M. M., Clark, A. L., Zahm, D. S., & Coscia, C. J. (2004). Differential effects of gestational buprenorphine, naloxone, and methadone on mesolimbic mu opioid and ORL1 receptor G protein coupling. Developmental Brain Research, 151, 149–157.

https://doi.org/10.1016/j.devbr ainres.2004.05.002

Hung, C. J., Wu, C. C., Chen, W. Y., Chang, C. Y., Kuan, Y. H., Pan, H. C., … Chen, C. J. (2013). Depression-like effect of prenatal buprenorphine exposure in rats. PLoS One, 8, e82262. https://doi.

org/10.1371/journ al.pone.0082262

Jantzie, L. L., Maxwell, J. R., Newville, J. C., Yellowhair, T. R., Kitase, Y., Madurai, N., … Allan, A. (2020). Prenatal opioid exposure: The next neonatal neuroinflammatory disease. Brain, Behavior, and Immunity, 84, 45–58. https://doi.org/10.1016/j.

bbi.2019.11.007

Kennedy, M. B. (2000). Signal-processing machines at the postsynap- tic density. Science, 290, 750–754. https://doi.org/10.1126/scien ce.290.5492.750

Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., & Altman, D.

G. (2010). Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. PLoS Biology, 8, e1000412.

https://doi.org/10.1371/journ al.pbio.1000412

Kongstorp, M., Bogen, I. L., Stiris, T., & Andersen, J. M. (2019). High accumulation of methadone compared with buprenorphine in fetal rat brain after maternal exposure. Journal of Pharmacology and Experimental Therapeutics, 371, 130–137. https://doi.org/10.1124/

jpet.119.259531

Kongstorp, M., Bogen, I. L., Stiris, T., & Andersen, J. M. (2020).

Prenatal exposure to methadone or buprenorphine impairs cognitive performance in young adult rats. Drug and Alcohol Dependence, 212, 108008. https://doi.org/10.1016/j.druga lcdep.2020.108008 Kvello, A. M. S., Andersen, J. M., Oiestad, E. L., Steinsland, S., Aase,

A., Morland, J., & Bogen, I. L. (2019). A monoclonal antibody against 6-acetylmorphine protects female mice offspring from adverse behavioral effects induced by prenatal heroin exposure.

Journal of Pharmacology and Experimental Therapeutics, 368, 106–115. https://doi.org/10.1124/jpet.118.251504

Lee, S. J., Bora, S., Austin, N. C., Westerman, A., & Henderson, J.

M. T. (2020). Neurodevelopmental outcomes of children born to opioid-dependent mothers: A systematic review and meta-analysis. Academic Pediatrics, 20, 308–318. https://doi.org/10.1016/j.

acap.2019.11.005

Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951).

Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.

Minozzi, S., Amato, L., Bellisario, C., Ferri, M., & Davoli, M. (2013).

Maintenance agonist treatments for opiate-dependent pregnant women. Cochrane Database of Systematic Reviews, CD006318.

https://doi.org/10.1002/14651 858.CD006 318.pub3

Monnelly, V. J., Hamilton, R., Chappell, F. M., Mactier, H., & Boardman, J. P. (2019). Childhood neurodevelopment after prescription of maintenance methadone for opioid dependency in pregnancy: A systematic review and meta-analysis. Developmental Medicine and Child Neurology, 61, 750–760. https://doi.org/10.1111/dmcn.14117 Nanovskaya, T., Deshmukh, S., Brooks, M., & Ahmed, M. S. (2002).

Transplacental transfer and metabolism of buprenorphine. Journal of Pharmacology and Experimental Therapeutics, 300, 26–33.

https://doi.org/10.1124/jpet.300.1.26

Nanovskaya, T. N., Nekhayeva, I. A., Hankins, G. D., & Ahmed, M. S. (2008). Transfer of methadone across the dually perfused preterm human placental lobule. American Journal of Obstetrics and Gynecology, 198(1), 126.e1–126.e4. https://doi.org/10.1016/j.

ajog.2007.06.073

Nasiraei-Moghadam, S., Sherafat, M. A., Safari, M. S., Moradi, F., Ahmadiani, A., & Dargahi, L. (2013). Reversal of prenatal morphine exposure-induced memory deficit in male but not female rats. Journal of Molecular Neuroscience, 50, 58–69. https://doi.

org/10.1007/s1203 1-012-9860-z

Oberoi, R., Chu, T., Mellen, N., Jagadapillai, R., Ouyang, H., Devlin, L.

A., & Cai, J. (2019). Diverse changes in myelin protein expression in rat brain after perinatal methadone exposure. Acta Neurobiologiae Experimentalis, 79, 367–373. https://doi.org/10.21307 /ane- 2019-034

Pearce, I. A., Cambray-Deakin, M. A., & Burgoyne, R. D. (1987).

Glutamate acting on NMDA receptors stimulates neurite outgrowth from cerebellar granule cells. FEBS Letters, 223, 143–147. https://

doi.org/10.1016/0014-5793(87)80525 -2

Peng, S., Zhang, Y., Zhang, J., Wang, H., & Ren, B. (2010). ERK in learning and memory: A review of recent research. International Journal of Molecular Sciences, 11, 222–232. https://doi.org/10.3390/ijms1 1010222

Peters, M. A. (1977). The effect of maternally administered methadone on brain development in the offspring. Journal of Pharmacology and Experimental Therapeutics, 203, 340–346.

Peterson, B. S., Vohr, B., Staib, L. H., Cannistraci, C. J., Dolberg, A., Schneider, K. C., … Ment, L. R. (2000). Regional brain volume abnormalities and long-term cognitive outcome in preterm infants.

JAMA, 284, 1939–1947. https://doi.org/10.1001/jama.284.15.1939 Rimanoczy, A., & Vathy, I. (1995). Prenatal exposure to morphine alters

brain mu opioid receptor characteristics in rats. Brain Research, 690, 245–248. https://doi.org/10.1016/0006-8993(95)00638 -7

Robinson, S. E. (2002a). Buprenorphine: An analgesic with an expand- ing role in the treatment of opioid addiction. CNS Drug Reviews, 8, 377–390. https://doi.org/10.1111/j.1527-3458.2002.tb002 35.x Robinson, S. E. (2002b). Effects of perinatal buprenorphine and meth-

adone exposures on striatal cholinergic ontogeny. Neurotoxicology and Teratology, 24, 137–142. https://doi.org/10.1016/s0892 -0362(01)00185 -4

Rodriguez-Munoz, M., Sanchez-Blazquez, P., Vicente-Sanchez, A., Berrocoso, E., & Garzon, J. (2012). The mu-opioid receptor and the NMDA receptor associate in PAG neurons: Implications in

(11)

pain control. Neuropsychopharmacology, 37, 338–349. https://doi.

org/10.1038/npp.2011.155

Rongo, C., & Kaplan, J. M. (1999). CaMKII regulates the density of central glutamatergic synapses in vivo. Nature, 402, 195–199.

https://doi.org/10.1038/46065

Samuels, I. S., Saitta, S. C., & Landreth, G. E. (2009). MAP'ing CNS development and cognition: An ERKsome process. Neuron, 61, 160–167. https://doi.org/10.1016/j.neuron.2009.01.001

Sanchez, E. S., Bigbee, J. W., Fobbs, W., Robinson, S. E., & Sato- Bigbee, C. (2008). Opioid addiction and pregnancy: Perinatal exposure to buprenorphine affects myelination in the developing brain.

Glia, 56, 1017–1027. https://doi.org/10.1002/glia.20675

Sargeant, T. J., Miller, J. H., & Day, D. J. (2008). Opioidergic regulation of astroglial/neuronal proliferation: Where are we now? Journal of Neurochemistry, 107, 883–897. https://doi.

org/10.1111/j.1471-4159.2008.05671.x

Tao, P. L., Yeh, G. C., Su, C. H., & Wu, Y. H. (2001). Co-administration of dextromethorphan during pregnancy and throughout lactation significantly decreases the adverse effects associated with chronic morphine administration in rat offspring. Life Sciences, 69, 2439–

2450. https://doi.org/10.1016/s0024 -3205(01)01316 -9

Tempel, A., Habas, J., Paredes, W., & Barr, G. A. (1988). Morphine- induced downregulation of mu-opioid receptors in neonatal rat brain. Brain Research, 469, 129–133. https://doi.org/10.1016/0165- 3806(88)90176 -9

Trujillo, K. A. (2002). The neurobiology of opiate tolerance, dependence and sensitization: Mechanisms of NMDA receptor-dependent synaptic plasticity. Neurotoxicity Research, 4, 373–391. https://doi.

org/10.1080/10298 42029 0023954

Tsang, D., & Ng, S. C. (1980). Effect of antenatal exposure to opiates on the development of opiate receptors in rat brain. Brain Research, 188, 199–206. https://doi.org/10.1016/0006-8993(80)90568 -5 van Wagoner, S., Risser, J., Moyer, M., & Lasky, D. (1980). Effect of

maternally administered methadone on discrimination learning of rat offspring. Perceptual and Motor Skills, 50, 1119–1124. https://

doi.org/10.2466/pms.1980.50.3c.1119

Vathy, I., Slamberova, R., Rimanoczy, A., Riley, M. A., & Bar, N. (2003).

Autoradiographic evidence that prenatal morphine exposure sex-de- pendently alters mu-opioid receptor densities in brain regions that are involved in the control of drug abuse and other motivated behaviors.

Progress in Neuro-Psychopharmacology and Biological Psychiatry, 27, 381–393. https://doi.org/10.1016/s0278 -5846(02)00355 -x Vestal-Laborde, A. A., Eschenroeder, A. C., Bigbee, J. W., Robinson, S.

E., & Sato-Bigbee, C. (2014). The opioid system and brain development: Effects of methadone on the oligodendrocyte lineage and the early stages of myelination. Developmental Neuroscience, 36, 409–421. https://doi.org/10.1159/00036 5074

Walhovd, K. B., Moe, V., Slinning, K., Due-Tonnessen, P., Bjornerud, A., Dale, A. M., … Fischl, B. (2007). Volumetric cerebral

characteristics of children exposed to opiates and other substances in utero. NeuroImage, 36, 1331–1344. https://doi.org/10.1016/j.neuro image.2007.03.070

Williams, J. T., Christie, M. J., & Manzoni, O. (2001). Cellular and synaptic adaptations mediating opioid dependence. Physiological Reviews, 81, 299–343. https://doi.org/10.1152/physr ev.2001.81.1.299 World Health Organization. (2014). Guidelines for the identification

and management of substance use and substance use disorders in pregnancy (pp. 1–224). Geneva, Switzerland: World Health Organization.

Wu, C. C., Hung, C. J., Lin, S. Y., Wang, Y. Y., Chang, C. Y., Chen, W. Y., … Chen, C. J. (2017). Treadmill exercise alleviated prenatal buprenorphine exposure-induced depression in rats. Neurochemistry International, 110, 91–100. https://doi.org/10.1016/j.

neuint.2017.09.012

Wu, C. C., Hung, C. J., Shen, C. H., Chen, W. Y., Chang, C. Y., Pan, H. C., … Chen, C. J. (2014). Prenatal buprenorphine exposure decreases neurogenesis in rats. Toxicology Letters, 225, 92–101.

https://doi.org/10.1016/j.toxlet.2013.12.001

Yuan, Q., Rubic, M., Seah, J., Rae, C., Wright, I. M. R., Kaltenbach, K.,

… Oei, J. L. (2014). Do maternal opioids reduce neonatal regional brain volumes? A pilot study. Journal of Perinatology, 34, 909–913.

https://doi.org/10.1038/jp.2014.111

Zagon, I. S., & McLaughlin, P. J. (1977a). Effect of chronic maternal methadone exposure on perinatal development. Biology of the Neonate, 31, 271–282. https://doi.org/10.1159/00024 0975

Zagon, I. S., & McLaughlin, P. J. (1977b). Methadone and brain development. Experientia, 33, 1486–1487. https://doi.org/10.1007/

BF019 18824

Zagon, I. S., & McLaughlin, P. J. (1978). Perinatal methadone exposure and brain development: A biochemical study. Journal of Neurochemistry, 31, 49–54. https://doi.org/10.1111/j.1471-4159.1978.tb124 31.x Zagon, I. S., & McLaughlin, P. J. (1987). Endogenous opioid systems

regulate cell proliferation in the developing rat brain. Brain Research, 412, 68–72. https://doi.org/10.1016/0006-8993(87)91440 -5

Zagon, I. S., McLaughlin, P. J., & Thompson, C. I. (1979). Learning ability in adult female rats perinatally exposed to methadone.

Pharmacology, Biochemistry and Behavior, 10, 889–894. https://

doi.org/10.1016/0091-3057(79)90063 -7

How to cite this article: Kongstorp M, Bogen IL, Steinsland S, et al. Prenatal exposure to methadone or buprenorphine alters µ-opioid receptor binding and downstream signaling in the rat brain. Int J Dev Neurosci. 2020;80:443–453. https://doi.org/10.1002/

jdn.10043