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. Author manuscript; available in PMC: 2008 Nov 3.
Published in final edited form as: Glia. 2008 Jul;56(9):1017–1027. doi: 10.1002/glia.20675

Opioid Addiction and Pregnancy: Perinatal Exposure to Buprenorphine Affects Myelination in the Developing Brain

EMILSE S SANCHEZ 1, JOHN W BIGBEE 2, WAMBURA FOBBS 1, SUSAN E ROBINSON 3, CARMEN SATO-BIGBEE 1,*
PMCID: PMC2577583  NIHMSID: NIHMS74119  PMID: 18381654

Abstract

Buprenorphine is a μ-opioid receptor partial agonist and κ-opioid receptor antagonist currently on trials for the management of pregnant opioid-dependent addicts. However, little is known about the effects of buprenorphine on brain development. Oligodendrocytes express opioid receptors in a developmentally regulated manner and thus, it is logical to hypothesize that perinatal exposure to buprenorphine could affect myelination. To investigate this possibility, pregnant rats were implanted with minipumps to deliver buprenorphine at 0.3 or 1 mg/kg/day. Analysis of their pups at different postnatal ages indicated that exposure to 0.3 mg/kg/day buprenorphine caused an accelerated and significant increase in the brain expression of all myelin basic protein (MBP) splicing isoforms. In contrast, treatment with the higher dose caused a developmental delay in MBP expression. Examination of corpus callosum at 26-days of age indicated that both buprenorphine doses cause a significant increase in the caliber of the myelinated axons. Surprisingly, these axons have a disproportionately thinner myelin sheath, suggesting alterations at the level of axon-glial interactions. Analysis of myelin associated glycoprotein (MAG) expression and glycosylation indicated that this molecule may play a crucial role in mediating these effects. Co-immunoprecipitation studies also suggested a mechanism involving a MAG-dependent activation of the Src-family tyrosine kinase Fyn. These results support the idea that opioid signaling plays an important role in regulating myelination in vivo and stress the need for further studies investigating potential effects of perinatal buprenorphine exposure on brain development.

Keywords: myelin synthesis, oligodendrocytes, opioids, buprenorphine

INTRODUCTION

Epidemiological surveys indicate an increasing trend in the abuse of and the addiction to opioids (Compton and Volkow, 2006), a problem of critical importance during pregnancy. Early reports indicated that infants exposed in utero to opioids exhibit an increased risk of sudden infant death syndrome (Kandall et al., 1993) as well as a significant incidence of reduced head circumference, decreased attention, and altered fine motor coordination (Marcus et al., 1984; Rosen and Johnson, 1982). Longitudinal studies found that while children exposed to opioids prenatally do not appear to exhibit major cognitive deficits, they still show heightened activity, impulsivity, and reduced attention span (Hutchings, 1982), suggesting the existence of underlying neurological problems. However, the effects of prenatal exposure to opioids on nervous system development and function are difficult to assess as child outcomes are highly influenced by environmental and life style factors (Ornoy et al., 2001).

Methadone maintenance is the standard substitution therapy for opioid addiction during pregnancy, even though this μ-opioid agonist has been shown to cause upon discontinuation neonatal abstinence syndrome (NAS) in a significant percentage of newborns (Fischer et al., 2006; Lejeune et al., 2006). New options for pregnant opioid addicts include buprenorphine, a μ-opioid receptor partial agonist and κ-opioid receptor antagonist, that has been recently approved for the treatment of nonpregnant opioid-dependent-adults in the United States and is used experimentally in pregnant addicts in several countries. Different clinical trials indicated that buprenorphine can effectively prevent the use of “street opioids” by pregnant addicts and reduce the incidence and severity of NAS (Ebner and Wiedmann, 2006; Jones et al., 2005). However, there is a lack of information on the potential effects of this drug on child brain development. Importantly, perinatal exposure of rats to buprenorphine has been shown to delay the generation of cholinergic neurons (Robinson, 2002b) and to reduce the expression of nerve growth factor in the striatum (Wu et al., 2001), underscoring the importance of further research on the actions of this drug in nervous system formation.

The present study investigated the potential effect of buprenorphine on the formation of myelin. This multilamellar membrane insulates the axons and restricts the presence of Na+ channels to the nodes of Ranvier (Rasband and Trimmer, 2001), which provide for the rapid “saltatory” conduction of impulses that characterizes myelinated fiber tracks. The synthesis of myelin is known to be influenced by multiple hormonal and growth factor signals as well as complex cell–cell interactions (Simons and Trajkovic, 2006), making this a most vulnerable and critical process during nervous system development. Interestingly, oligodendrocytes, the cells that make the myelin membrane in the central nervous system (CNS), express opioid receptors in a developmentally regulated manner. μ-Opioid receptors are expressed by both immature and mature cells while κ-receptors are only expressed in differentiated oligodendrocytes (Knapp et al., 1998; Tryoen-Toth et al., 2000). Moreover, treatment with μ- and δ-opioid receptor antagonists has been shown to decrease the generation of oligodendrocytes from cultured adult rat hippocampal progenitors (Persson et al., 2003). Nevertheless, the roles of the opioid receptors in oligodendrocyte development and myelination in vivo remain unknown. However, the presence of these receptors in the oligodendrocytes raises the possibility that opioid abuse as well as opioid maintenance treatments during CNS development could affect myelin formation. Here we tested this hypothesis by using an animal model in which rat pups were pre- and postnatally exposed to different doses of buprenorphine. We found that buprenorphine affects the expression of myelin basic proteins (MBPs) in a developmental and dose-specific manner. Moreover, analysis of the corpus callosum at the end of the rapid period of myelination indicated significant alterations in the caliber of the myelinated axons and the thickness of the myelin membrane. Animals exposed to buprenorphine also showed changes in the expression and glycosylation levels of myelin-associated glycoprotein (MAG), a molecule thought to play a crucial role in axon-glial interactions, suggesting that buprenorphine alters critical signaling processes modulating oligodendrocyte development and myelination.

MATERIALS AND METHODS

Materials

Timed-pregnant Sprague Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN). Model 2ML4 Alzet osmotic minipumps were from Durect (Cupertino, CA). Anti-myelin basic protein (MBP) and anti-myelin associated glycoprotein (MAG) antibodies were from Chemicon (Temecula, CA). Antibodies against N-cadherin, Fyn, and actin were purchased from Zymed Laboratories (San Francisco, CA), Santa Cruz Biotechnology (Santa Cruz, CA), and Sigma-Aldrich (St. Louis, MO), respectively. Protease and phosphatase inhibitor cocktails were purchased from Sigma-Aldrich. Super Signal West Dura reagent was from Pierce (Rockford, IL). The Pro-Q Emerald 300 Glycoprotein Gel Stain kit was from Invitrogen (Carlsbad, CA). All electrophoresis reagents were from Bio-Rad Laboratories (Hercules, CA).

Buprenorphine Treatment

This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy Press, 1996) and under protocols approved by the Animal Care and Use Committee of Virginia Commonwealth University. Rats were exposed to buprenorphine pre- and postnatally. On day 7 of gestation, dams were implanted subcutaneously while under isoflurane anesthesia with 28-day osmotic minipumps to deliver sterile water (controls) or buprenorphine (0.3 or 1 mg/kg/day) in sterile water, as described earlier (Robinson and Wallace, 2001). In this way, drug exposure began immediately prior to development of the CNS. Drug exposure via maternal milk was continued until the time of sacrifice or until weaning at 21 days postnatal. Buprenorphine is known to cross into breast milk (Elkader and Sproule, 2005). In a similar exposure paradigm using the opioid methadone, methadone is present in pups suckling dams implanted with osmotic minipumps delivering the drug (Kunko et al., 1996). It is recognized that the rat is born at a stage of development equivalent to the end of the second trimester in the human and the early postnatal period in the rat is analogous to the third trimester in human pregnancy. The degree of drug exposure is comparable to that of humans during gestation in the case of the lower dose and an overexposure in the case of the higher one. Pups were sacrificed for analysis at postnatal days 12, 19, and 26, time points that correspond to the beginning, peak, and end of the rapid period of brain myelination in rodents.

Western Blot Analysis

For analysis of proteins, the brains were rapidly removed and homogenized in phosphate buffer saline (PBS, 9.1 mM Na2HPO4, 1.7 mM NaH2PO4, and 150 mM NaCl, pH 7.4) containing protease and phosphatase inhibitor cocktails. Total brain homogenates were subjected to western blot analysis as previously described (Afshari et al., 2002). Briefly, equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose. The membranes were then subjected to immunoblot analysis using the following primary antibodies: anti-MBP (1:100), anti-MAG (1:1,000), anti-N-cadherin (1:1,000), anti-Fyn (1:1,000), and antiactin (1:1,000). The immunoreactive bands were detected after incubation of the blots with the appropriate horse-radish peroxidase-conjugated secondary antibodies followed by chemiluminescent reaction with Super Signal West Dura reagent. The relative amount of immunoreactive protein in each band was determined by scanning densitometric analysis of the X-ray films using the NIH Image J program.

Analysis of In Vivo Myelination

To determine the extent of myelination in vivo, 26-day-old pups were anesthetized by intraperitoneal injection with 2.5% Avertin and then perfused transcardially with PBS containing 4% paraformaldehyde as previously described (Dupree et al., 2005). The brains were removed and fixed overnight in 4% paraformaldehyde and 1% glutaraldehyde. Regions containing the anterior portion of the corpus callosum were dissected, postfixed with 1% osmium tetroxide in 100 mM sodium cacodylate buffer, pH 7.3, and plastic embedded. One micron cross-sections were stained with toluidine blue and six representative fields from each section were photographed at 1,000× magnification. Myelinated axons in each field were counted and totaled for each treatment group. For electron microscopic analysis, thin sections stained with uranyl acetate and lead citrate were examined with a Jeol JEM-1230 transmission electron microscope. Corresponding fields from each treatment group were photographed at 1,200× magnification and analyzed for the number of unmyelinated and myelinated axons, axon diameter, and myelin thickness. A minimum of 750 axons were analyzed for each treatment group. For each axon, the diameter was calculated as the average of the maximal and minimal diameters. G-ratios to assess myelin thickness were calculated from the corresponding myelinated axons and fiber diameters (diameter of axon/diameter of axon + myelin).

Analysis of Protein Glycosylation

To analyze MAG or N-cadherin glycosylation, brains were homogenized in RIPA buffer (PBS containing 1% NP-40, 0.5% deoxycholate, and 0.1% SDS), supplemented with a protease and phosphatase inhibitor cocktail. After 30 min on ice, the lysates were centrifuged at 10,000×g for 15 min and the supernatants used in immunoprecipitation assays as previously reported, with minor modifications (Sato-Bigbee et al., 1994). Depending on the age of the pups and the protein target, supernatants equivalent up to 5 mg of protein were subjected to immunoprecipitation by 3 h incubation with anti-MAG (1:60) or anti-N-cadherin (1:60) antibodies, followed by 3 h incubation with protein A/G PLUS-agarose (Santa Cruz Biotechnology). The immunocomplexes were collected by centrifugation, extensively washed with 20 mM Tris-HCl, 1% Triton X-100, pH 7.4; resuspended in 60 mM Tris-HCl, 10% glycerol, 2% SDS, 5% of β-mercaptoethanol, pH 6.8, and subjected to SDS-PAGE. Glycoproteins were detected using the Pro-Q Emerald 300 Glycoprotein Gel Stain kit, following the manufacture’s recommendations. Briefly, gels were fixed in 50% methanol for 30 min and washed twice with 3% acetic acid for 10–15 min each. The glycoproteins were then oxidized by incubation in 1% periodic acid for 30 min and stained with Pro-Q Emerald 300 dye solution for 2 h. Afterwards the gels were washed twice with 3% acetic acid for 15 min each. The reactive bands were detected by ultra violet illumination at 302 nm using a Fluor-Chem Image Scanner (Alpha Innotech) and quantified using the NIH Image J program.

Analysis of MAG-Fyn Interaction

The interaction of MAG with Fyn was analyzed by co-immunoprecipitation followed by western blot analysis. For this, total brain homogenates were first subjected to immunoprecipitation with anti-MAG antibody. The immunocomplexes were then analyzed by western blotting with anti-MAG or anti-Fyn antibodies as described earlier.

Statistical Analysis

Statistical analysis was performed by one-way analysis of variance. Differences were considered statistically significant when P-values were <0.05.

RESULTS

Perinatal Exposure to Buprenorphine Alters the Levels and Developmental Expression of all MBP Isoforms

As indicated earlier, the presence of opioid receptors in oligodendrocytes raises the question of whether perinatal exposure to buprenorphine could alter the differentiation of these cells and the process of myelination. To address this possibility, we first investigated the developmental expression of MBPs, proteins which are essential for myelin formation (Readhead et al., 1987) and comprise about 30% of all myelin protein in the CNS (Boggs, 2006). The different MBP isoforms are generated by alternative splicing of a single gene (de Ferra et al., 1985) and both MBP gene activity and splicing are developmentally regulated (Campagnoni, 1988). Their differential localization in oligodendrocytes and myelin suggests that some MBP isoforms may play a role in cell differentiation while others are mainly structural components of the myelin membrane (Pedraza et al., 1997).

In these experiments, MBP brain expression was investigated by western blot analysis with an antibody that recognizes the four major MBPs, which consist of 14.0, 17.0, 18.5, and 21.5 kDa isoforms. Analysis of these proteins was carried out at 12, 19, and 26 days postnatal, ages that correspond to the beginning, peak and end of the rapid period of brain myelination, respectively. Interestingly, all MBP isoforms were significantly increased, at all studied ages, in the brain of pups from dams treated with 0.3 mg/kg/day buprenorphine (see Fig. 1). In contrast, Figure 1A shows that at 12 days of age, all MBPs were dramatically decreased in the pups exposed to 1 mg/kg/day buprenorphine. However, at 19 and 26 days postnatal, this treatment group reached MBP values comparable to those in the control pups (Fig. 1, panels B and C). Thus, these results indicate that while a dose of 0.3 mg/kg/day buprenorphine increases MBP levels throughout the entire period of rapid rat brain myelination, the higher dose of the drug causes a delay in MBP expression.

Fig. 1.

Fig. 1

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Perinatal exposure to buprenorphine alters the levels and developmental expression of all MBP isoforms. Perinatal exposure to buprenorphine (0.3 or 1 mg/kg/day) was carried out as indicated under “Methods.” Controls were treated with water. Western blot analysis was used to investigate the expression of the 14, 17, 18.5, and 21.5 kDa myelin basic protein (MBP) isoforms in total brain homogenates at (A) postnatal day 12, (B) postnatal day 19, and (C) postnatal day 26. Western blots on the left correspond to a film exposure time adequate to show all MBP isoforms. For correct quantification of individual MBP isoforms, film exposure times were adjusted to maintain linear detection of the bands. β-Actin levels were used as loading controls. The figures show representative western blots. Results are expressed as % of control values and are the mean ± SEM from at least 12 brains from three different litters/group. *P < 0.05, **P < 0.005, ***P < 0.0005, as compared with controls.

High Doses of Buprenorphine Reduce the Number of Myelinated Axons in the Corpus Callosum

The results presented so far suggested that perinatal exposure to buprenorphine affects brain myelination. Thus, we next investigated the state of myelin formation at the end of the myelination period. As shown in Figure 2, analysis of the corpus callosum of 26-day-old animals indicated that while pups exposed to 0.3 mg/kg/day buprenorphine exhibited normal numbers of myelinated axons, the numbers were decreased by 25% in the animals exposed to the higher dose of the drug.

Fig. 2.

Fig. 2

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Buprenorphine alters the number of myelinated axons in a dose dependent manner. The number of myelinated axons in the corpus callosum was determined in samples from 26-day-old rats after staining of the brain sections with toluidine blue. The micrographs show representative fields from (A) controls, (B) animals exposed to 0.3 mg/kg/day buprenorphine, or (C) animals exposed to 1 mg/kg/day buprenorphine. (D) Number of myelinated axons. Results represent the mean ± SEM from three different animals from three different litters per group. *P < 0.005, as compared with controls.

Buprenorphine Exposure Increases the Caliber of Myelinated Axons and Results in a Disproportionately Thinner Myelin Sheath

Examination of the corpus callosum by electron microscopy (see Fig. 3) showed that, regardless of the number of myelinated axons in each treatment group, exposure to either dose of buprenorphine altered the normal size distribution of the myelinated axons, resulting in a significant increase in the proportion of those with larger diameters (Fig. 3A). In contrast, neither dose of buprenorphine induced any significant changes in the size distribution of the nonmyelinated axons (Fig. 3B). In control animals, 60% of the myelinated axons had diameters that were either smaller or equal to 0.8 μm while 40% had diameters larger than 0.8 μm (Fig. 3C). However, this proportion was reversed in both buprenorphine groups in which only about 40–45% of the myelinated axons were smaller or equal to 0.8 μm (Fig. 3C) while 60% had diameters larger than 0.8 μm (Fig. 3D).

Fig. 3.

Fig. 3

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Buprenorphine treatment increases the caliber of myelinated axons. The diameter of myelinated and nonmyelinated axons in the corpus callosum was determined by electron microscopic examination of samples from of 26-day-old controls and buprenorphine-exposed animals. The micrographs show representative fields. (A) Size distribution of myelinated axons. (B) Size distribution of nonmyelinated axons. (C) Percent of myelinated axons with diameters equal or less than 0.8 μm. (D) Percent of myelinated axons with diameters larger than 0.8 μm. The results represent the mean ± SEM from three different animals from three different litters/group. Scale bar, 1 μm. *P < 0.0005, as compared with controls.

Interestingly, analysis of G ratios indicated that in both buprenorphine groups, the increased diameter of the myelinated axons was accompanied by a disproportionately thinner myelin sheath (see Fig. 4). This inverse of the normal condition suggests that regardless of the dose, buprenorphine exposure appears to alter the normal balance between axonal growth and myelin thickness.

Fig. 4.

Fig. 4

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Buprenorphine exposure results in reduced relative thickness of the myelin sheath. The myelin thickness of axons in the corpus callosum was determined by electron microscopic examination of samples from of 26-day-old controls and buprenorphine-exposed animals (See Fig. 3). G-ratios were calculated from the corresponding myelinated axons and fiber diameters (g ratio: diameter of axon/diameter of axon +myelin). The results represent the mean ± SEM from three different animals from three different litters/group. *P < 0.0005, as compared with controls.

Buprenorphine Affects the Expression of MAG in a Developmental and Dose-Specific Manner

The results presented so far led us to investigate the possible effect of buprenorphine exposure on the expression of MAG, a glycoprotein particularly enriched in the periaxonal layers of the myelin sheath. Several lines of evidence indicate that this protein mediates axon-glial interactions and plays a role in controlling both the initiation of myelination and the growth of the myelinated axons (Quarles, 2007).

As shown in Figure 5, we found that brains from 12-day-old pups exposed to 1 mg/kg/day buprenorphine exhibited a lower content of MAG compared with control and 0.3 mg/kg/day buprenorphine-treatment groups. However, at 26 days of age, the brains of animals exposed to either dose of buprenorphine reached levels of MAG that significantly exceeded those in the controls, with the highest levels of MAG being observed in the pups treated with a dose of 0.3 mg/kg/day. A developmental comparison of MAG levels between the different experimental groups is shown in Figure 5B.

Fig. 5.

Fig. 5

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Buprenorphine affects the expression of MAG in a developmental and dose-specific manner. MAG expression was investigated in total brain homogenates from controls and buprenorphine-exposed pups at postnatal days 12, 19, and 26. (A) Representative western blots and relative MAG levels for each age group. (B) Developmental pattern of MAG expression in controls and treated animals (results are expressed as percent of 12-day-old controls). β-Actin levels were used as loading controls. Results represent the mean ± SEM of at least 12 brains per group. Control vs. treated, **P < 0.005; 0.3 vs. 1 mg/kg/day buprenorphine, *P < 0.05.

Effect of Buprenorphine on MAG Glycosylation

We next investigated whether exposure to buprenorphine could affect MAG glycosylation. This protein has a high carbohydrate content, and it has been suggested that changes in MAG glycosylation at the beginning of the myelination process may be important for its function (Quarles, 1976).

As shown in Figure 6, the brains of 12-day-old animals exposed to 0.3 mg/kg/day buprenorphine had a significant increase in MAG glycosylation. On the contrary, the animals exposed to 1 mg/kg/day buprenorphine exhibited a reduced content of glycosylated MAG. This decrease, however, appears to reflect the lower content of MAG, which as indicated earlier, was observed at this age and at this high dose of drug treatment. In spite of these differences in the youngest animals, neither dose of buprenorphine caused significant changes in the levels of MAG glycosylation at later stages of development. A developmental comparison of glycosylated MAG levels between the different experimental groups is shown in Figure 6B.

Fig. 6.

Fig. 6

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Effect of buprenorphine on MAG glycosylation. Levels of glycosylated MAG were determined after immunoprecipitation with anti-MAG antibody, separation by SDS-PAGE and “in gel” detection of carbohydrates as indicated under “Methods.” (A) The figures show representative glycoprotein-stained gels for each age group, the bars are the mean ± SEM from at least 12 brains/group. (B) Developmental pattern of glycosylated MAG expression in controls and treated animals (results are expressed as percent of 12-day-old controls). *P < 0.0005, as compared with controls.

The observed changes in the levels of glycosylated MAG at the beginning of myelination could either reflect the actions of buprenorphine on myelin synthesis or alternatively, result from a general effect of this drug on protein glycosylation. To discern between these possibilities, we investigated the potential effect of buprenorphine on the glycosylation of N-cadherin, another heavily glycosylated adhesion molecule. Unlike MAG, N-cadherin is not specific to myelin and oligodendrocytes, but is widely distributed in the CNS. In support of a specific effect of buprenorphine on MAG glycosylation, analysis of 12-day-old rat brains indicated that neither dose of buprenorphine induced any significant changes in either N-cadherin protein levels or glycosylation (data not shown).

Buprenorphine Affects the Interaction of MAG with the Src-Family Tyrosine Kinase Fyn

The results described earlier led us to hypothesize that MAG could play a significant role in the mechanisms by which buprenorphine affects myelination. This possibility is based on several lines of evidence supporting the idea that binding of this glycoprotein to extracellular ligands triggers MAG interaction with the Src-family tyrosine kinase Fyn, resulting in the activation of downstream signaling pathways in the oligodendrocytes (Fujita et al., 1998; Jaramillo et al., 1994; Umemori et al., 1994). Furthermore, co-localization and immunoprecipitation studies showed that MAG interaction with Fyn is temporally restricted to the beginning of myelination (Umemori et al., 1994).

Thus, we next investigated whether buprenorphine effects on MAG expression and glycosylation could also be accompanied by changes at the level of MAG-Fyn interaction. For this, total brain homogenates from 12-day-old pups were subjected to immunoprecipitation with anti-MAG antibody and the resulting pellets analyzed by western blotting to determine Fyn levels associated to MAG. As shown in Figure 7A, neither treatment with buprenorphine altered the expression levels of Fyn in total brain homogenates. However, analysis of samples from animals exposed to 0.3 mg/kg/day buprenorphine indicated increased levels of Fyn co-immunoprecipitated with MAG (Fig. 7B). Moreover, calculation of relative Fyn/MAG ratios (Fig. 7C) indicated a significantly augmented interaction of Fyn with MAG. In contrast with these results, the interaction between both proteins was not affected in the pups exposed to a 1 mg/kg/day dose of buprenorphine. However, since MAG levels in this treatment group are reduced, it can be assumed that these animals have a decreased number of Fyn/MAG complexes.

Fig. 7.

Fig. 7

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Buprenorphine affects the interaction of MAG with the Src-family tyrosine kinase Fyn. Total brain homogenates were prepared from 12-day-old controls and pups exposed to buprenorphine. (A) Western blot analysis of Fyn levels in total brain homogenates. (B) Fyn interaction with MAG was determined in pellets obtained by immunoprecipitation of total homogenates with anti-MAG antibody, as indicated under “Methods.” Fyn and MAG levels in the immunoprecipitates were determined by western blot analysis with their respective antibodies. The bar graph indicates levels of Fyn (C) Relative Fyn/MAG ratio in the immunoprecipitates. Results are expressed as percent of the controls and represent the mean ± SEM of from at least 12 brains/group. *P < 0.05.

In summary, these results suggest that exposure to a dose of 0.3 mg/kg/day buprenorphine results in a significant increase in the interaction of Fyn with MAG. In contrast, while a dose of 1 mg/kg/day buprenorphine does not affect Fyn/MAG interaction it causes a significant reduction in the total number of Fyn/MAG complexes.

DISCUSSION

The present study shows that perinatal exposure to buprenorphine could have profound effects on the myelination of the developing brain (a summary of findings is shown in Table 1). Elevated levels of all MBP isoforms were observed at all studied ages in the brains of animals exposed to 0.3 mg/kg/day buprenorphine, a level of drug comparable to that used for the management of pregnant opioid addicts. In contrast, 1 mg/kg/day buprenorphine, a dose equivalent to overexposure levels, delayed the expression of all MBPs at the beginning of the active period of myelination. These age- and dose-specific effects on MBP expression are also accompanied by alterations in the number and caliber of myelinated axons in the maturing brain and appear to result in an abnormal balance between myelin thickness and axonal growth.

TABLE 1.

Summary of Effects Induced by Perinatal Exposure to Buprenorphine

Buprenorphine 0.3 mg/kg/day
Buprenorphine 1 mg/kg/day
12 days 19 days 26 days 12 days 19 days 26 days
MBPs ↑ ↑ ↑ ↓ NS NS
MAG NS NS ↑ ↓ NS ↑
Glycosylated MAG ↑ NS NS ↓ NS NS
Fyn/MAG ratio ↑ NS
Theoretical MAG/Fyn complexes ↑ ↓
Myelinated axons NS ↓
Axons <0.8 μm ↓ ↓
Axons >0.8 μm ↑ ↑
Myelin thickness ↓ ↓
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The table summarizes the effects detected at different postnatal ages following perinatal exposure to different doses of buprenorphine. Results are compared with respect to controls. ↑, denotes increased values ↓, denotes decreased values; NS, no significant differences.

It is generally accepted that buprenorphine is a μ-opioid receptor partial agonist and a κ-opioid receptor antagonist. Studies on cultured oligodendrocytes indicated that these cells express opioid receptors in a developmentally regulated manner (Knapp et al., 1998). While μ-opioid receptors are expressed by both immature and mature cells, κ-receptors are only expressed in differentiated oligodendrocytes. Activation of μ-opioid receptors was shown to result in elevated DNA synthesis while inhibition of κ-receptors was accompanied by increased membrane extensions (Knapp et al., 1998). Based on these findings in cultured cells, the increased levels of MBP expression in animals exposed to 0.3 mg/kg/day buprenorphine could result from elevated number of oligodendrocytes due to the agonist effect of this drug on μ-opioid receptors. As the cells mature, this would be followed by increased myelin synthesis due to an antagonistic effect of buprenorphine on κ-receptors. However, this extrapolation of in vitro results to an in vivo system does not explain the results obtained in the animals exposed to 1 mg/kg/day buprenorphine.

The results in the higher dose buprenorphine treatment group may in fact reflect the complex pharmacological profile of buprenorphine. The exhibition of biphasic dose-response curves is a hallmark of the pharmacology of buprenorphine, especially with effects mediated by μ-opioid receptors (Robinson, 2002a). In addition to its actions on μ and κ receptors, buprenorphine effects may be modulated by concomitant activation of opioid receptor-like-1 receptors (Lutfy et al., 2003) as well as antagonism of δ-opioid receptors (Lee et al., 1999). Moreover, understanding of mechanisms underlying the actions of buprenorphine is further complicated by the potential effects of its active metabolite, norbuprenorphine (Robinson, 2002a). The effects of buprenorphine on oligodendrocyte differentiation and myelination could also be mediated by other cells in the CNS, in particular neurons since perinatal exposure to buprenorphine was also shown to delay the generation of cholinergic neurons (Robinson, 2002a) and caused reduced expression of nerve growth factor in the striatum (Wu et al., 2001).

Regardless of these possibilities, the finding that buprenorphine alters the expression and glycosylation levels of MAG suggests that this protein is a key player in the mechanisms by which this drug affects both the expression of the MBPs and the caliber of the myelinated axons. Although MAG is a minor myelin component, several studies indicate that this protein plays important roles during CNS development. The early developmental expression of MAG and its specific localization in the periaxonal membranes of actively myelinating oligodendrocytes suggest a potential role in axon-glial interactions at the initial stages of myelination (Bartsch et al., 1989; Sternberger et al., 1979; Trapp et al., 1989) In agreement with this possibility, optic nerves in developing MAG mutant mice exhibit a significant delay in myelin formation (Montag et al., 1994). Later on, MAG appears to play a crucial role in the maintenance of myelin and myelinated axons as the protein is still expressed at relatively high levels in wild-type adult nervous system and its absence in MAG knockout mice correlates with alterations in axonal integrity (Fruttiger et al., 1995).

Different findings support the idea that MAG interacts with a variety of molecules present in neurons and oligodendrocytes, thus working as a communicator between both cell types. MAG contains five extracellular immunoglobulin (Ig)-like domains (Lai et al., 1987; Salzer et al., 1987) and is a member of the Siglec family of sialic acid-binding proteins (Crocker, 2002). It has been suggested that MAG could mediate axonal stability by binding of the extracellular domains to the gangliosides GD1a and GT1b in the axonal membrane (Pan et al., 2005). Binding of MAG to extracellular ligands could also translate into signaling to the oligodendrocytes by interaction of its cytoplasmic domain with different molecules, including the Src-family tyrosine kinase Fyn. Co-immunoprecipitation studies using brain lysates from mice of different postnatal ages revealed that the interaction of MAG with Fyn is restricted to the early stages of myelination, an observation further supported by immunocytochemical studies indicating that prior to myelination, MAG and Fyn are highly co-localized in nerve fibers and oligodendrocytes (Umemori et al., 1994). Thus, the present observation that brain samples from 12-day-old animals exposed to a therapeutic dose of buprenorphine exhibit increased MAG interaction with Fyn is particularly interesting. Equally intriguing is the finding that at this same age, animals treated with an overexposure dose of buprenorphine have decreased levels of MAG that could result in reduced number of MAG/Fyn complexes. In support of a role of Fyn in myelination, analysis of Fyn-deficient mice indicated that while these animals are able to make myelin, their brains exhibit a 40–50% reduction in the content of myelin when compared with age-matched wild-type mice (Umemori et al., 1994). Furthermore, the absence of both MAG and Fyn results in an even more severe hypomyelination (Biffiger et al., 2000). Importantly, fewer oligodendrocytes develop from glial cells isolated from newborn Fyn−/− mice, suggesting that the decreased myelination may reflect a decreased oligodendrocyte maturation (Sperber et al., 2001). In support of this hypothesis, morphological differentiation of oligodendrocytes appears to require the activation of Fyn (Osterhout et al., 1999) and promoter studies suggested that transactivation of the MBP gene by Fyn may be important for myelination (Umemori et al., 1999). Thus, it is possible to hypothesize that the elevated levels of MBP expression in the animals exposed to 0.3 mg/kg/day buprenorphine may in part be due to the increased MAG/Fyn interaction that was observed in these animals at the beginning of the myelination process. In contrast, the apparent delay in MBP expression observed in 12-day-old rats exposed to 1 mg/kg/day buprenorphine may be attributed to a decreased level of MAG/Fyn complexes.

It is tempting to suggest that the increased interaction of MAG with Fyn detected in samples from animals exposed to the lower dose of buprenorphine may be the result of the observed increase in MAG glycosylation. MAG is heavily glycosylated and early experiments indicated that in rapidly myelinating rat brain, MAG contains more sialic acid-rich oligosaccharide units than in adult brain, suggesting that changes in glycosylation may play a role in regulating MAG function along development (Quarles, 1976).

An intriguing finding of these studies is the fact that 26-day-old rats that were exposed to either therapeutic or overexposure doses of buprenorphine exhibit myelinated axons with larger caliber than controls. In contrast, no significant changes in axonal caliber were detected at the level of the nonmyelinated axons. Interestingly, at this age, both buprenorphine treatment groups also achieve higher levels of MAG than control rats. As indicated earlier, several lines of evidence support the idea that MAG may not only play a role in the early stages of myelination but may also have a crucial function in modulating axonal cytoskeleton and integrity. Myelinated axons in MAG-null mice have diminished neurofilament spacing and phosphorylation as well as reduced caliber (Yin et al., 1998). Therefore, it is possible that elevated levels of MAG in the 26-day-old animals exposed to either dose of buprenorphine may be responsible for the increased caliber of the myelinated axons. Surprisingly, the analysis of G ratios indicated that the increased diameter of the myelinated axons in both groups of animals is accompanied by a disproportionately thinner myelin sheath. Thus, it appears that buprenorphine, regardless of the dose, causes an abnormal balance between myelin thickness and axonal growth. In the peripheral nervous system, the amount of myelin formed by Schwann cells may be regulated by the levels of axonal neuregulin-1 type III (Michailov et al., 2004). However, the mechanisms involved in coordinating axonal growth with myelin formation in the CNS remain unknown. Our findings that this interaction is altered by exposure to buprenorphine may provide important clues to understanding these processes. It remains to be investigated if the abnormalities detected in the corpus callosum extend to other areas of the CNS and whether similar alterations occur in the peripheral nervous system.

In conclusion, the present results further support a role for opioid signaling in regulating brain maturation and, in particular, oligodendrocyte development and myelination. The observation that buprenorphine alters myelination and axonal growth in the developing rat brain stresses the need for further studies and the strict control for the use of this drug in the treatment of pregnant opioid addicts. Future studies investigating how buprenorphine modifies the expression of myelin proteins by the oligodendrocytes and the mechanisms by which perinatal exposure to this drug alters axon-glial interactions should provide deeper understanding into these developmental processes and new strategies for the managing of pregnant addicts.

Acknowledgments

The authors would like to thank Drs. Kurt F. Hauser, Pamela E. Knapp, and Rochelle P. Coelho for their helpful suggestions and discussions.

Grant sponsors: A.D. Williams Foundation, DA005274.

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