Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: Glia. 2011 Oct 14;60(1):125–136. doi: 10.1002/glia.21253

Oligodendrocyte Responses to Buprenorphine Uncover Novel and Opposing Roles of μ-Opioid- and Nociceptin/Orphanin FQ Receptors in Cell Development: Implications for Drug Addiction Treatment During Pregnancy

Andrew C Eschenroeder 1,§, Allison A Vestal-Laborde 1,§, Emilse S Sanchez 1, Susan E Robinson 2, Carmen Sato-Bigbee 1,*
PMCID: PMC3217102  NIHMSID: NIHMS326977  PMID: 22002899

Abstract

While the classical function of myelin is the facilitation of saltatory conduction, this membrane and the oligodendrocytes, the cells that make myelin in the central nervous system (CNS), are now recognized as important regulators of plasticity and remodeling in the developing brain. As such, oligodendrocyte maturation and myelination are among the most vulnerable processes along CNS development. We have shown previously that rat brain myelination is significantly altered by buprenorphine, an opioid analogue currently used in clinical trials for managing pregnant opioid addicts. Perinatal exposure to low levels of this drug induced accelerated and increased expression of myelin basic proteins (MBPs), cellular and myelin components that are markers of mature oligodendrocytes. In contrast, supra-therapeutic drug doses delayed MBP brain expression and resulted in a decreased number of myelinated axons. We have now found that this biphasic-dose response to buprenorphine can be attributed to the participation of both the μ-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOP receptor) in the oligodendrocytes. This is particularly intriguing because the NOP receptor/nociceptin system has been primarily linked to behavior and pain regulation, but a role in CNS development or myelination has not been described before. Our findings suggest that balance between signaling mediated by (a) MOR activation and (b) a novel, yet unidentified pathway that includes the NOP receptor, plays a crucial role in the timing of oligodendrocyte maturation and myelin synthesis. Moreover, exposure to opioids could disrupt the normal interplay between these two systems altering the developmental pattern of brain myelination.

Keywords: myelination, myelin, oligodendrocytes, opioids and pregnancy, addiction treatment

INTRODUCTION

The use and abuse of opioids has significantly increased in the recent years (Compton and Volkow 2006; Hall et al. 2008), an alarming trend representing a problem of particular importance during pregnancy (Broussard et al. 2011). We showed previously that rat brain myelination is significantly altered by perinatal exposure to buprenorphine (Sanchez et al. 2008), a synthetic opioid that functions as MOR partial agonist and κ-opioid receptor (KOR) antagonist (Lewis 1985). The Food and Drug Administration approved the use of buprenorphine for opioid dependence treatment (Amass et al. 2004; Jaffe and O’Keeffe 2003) and furthermore, this drug is currently on clinical trials for the management of pregnant opioid addicts. Several clinical studies showed that buprenorphine effectively prevents the use of other opioids by pregnant addicts and reduces the incidence and severity of neonatal abstinence syndrome as compared to the approved treatment with methadone (Ebner and Wiedmann 2006; Jones et al. 2005; Jones et al. 2010a; Unger et al. 2010). However, most studies on these treatments are limited to early neurodevelopment (Jansson et al. 2011; Jones et al. 2010b), so there is a lack of detailed systematic studies on the long-term effects of either buprenorphine or methadone on human brain development. In addition, buprenorphine and methadone are not only used in replacement therapies but abuse of both is also on the rise (Maxwell and McCance-Katz 2010).

Potential effects on myelination and the myelin-forming oligodendrocytes are particularly important because the original perception of myelin as an inert structure, only responsible for axonal insulation and saltatory conduction, has radically changed to that of a complex membrane which is also implicated in the control of axonal extension and radial growth (Yin et al. 1998), axolemmal integrity (Bruce et al. 2010) and the establishment of nodal and paranodal structures and ion channel localization at the nodes of Ranvier (Rasband and Trimmer 2001). Furthermore, the oligodendrocytes appear to regulate axonal function by maintaining important bidirectional glial-neuronal interactions (Fields 2008). These highly specialized cells originate from simple bipolar proliferative progenitors that undergo several steps of differentiation and morphological changes prior to becoming post mitotic, complex multipolar cells capable of myelination. Each of these developmental steps is accompanied by the expression of stage-specific markers of differentiation (Butt and Ransom 1993; Campagnoni 1988; Pringle et al. 1992; Sommer and Schachner 1981), and this well-defined sequence of developmental changes implies the existence of highly controlled mechanisms that are still poorly understood. Interestingly, several lines of evidence support the idea that oligodendrocyte development may be modulated by the endogenous opioid system. Not only do these cells express opioid receptors (Knapp et al. 1998) but they are also capable of synthesizing endogenous opioids in a developmentally regulated manner (Knapp et al. 2001). While cultured bipolar progenitors have been shown to produce dynorphin, the expression of this opioid is lost when the cells differentiate into mature oligodendrocytes. On the other hand, proenkephalin-derived peptides were detected in both young and differentiated oligodendrocytes (Knapp et al. 2001). Activation of MOR in oligodendrocyte progenitor cultures was shown to result in elevated DNA synthesis while inhibition of KOR was accompanied by increased membrane extensions (Knapp et al. 1998). Although the in vivo role of opioids in oligodendrocytes is not clear, these observations raise the possibility that interference with the endogenous opioid system could affect oligodendrocyte maturation and the process of myelination in the developing CNS.

As indicated above, previously we have found that perinatal exposure to buprenorphine alters myelination in the developing rat brain (Sanchez et al. 2008). Analysis of pups from mothers treated with buprenorphine indicated that exposure to therapeutic doses resulted in accelerated and increased brain expression of myelin basic proteins (MBPs), cellular and myelin components that are markers of mature oligodendrocytes. In contrast, supra-therapeutic doses of buprenorphine delayed MBP expression and resulted in a decreased number of myelinated axons. Importantly, a recent imaging study pointed to the development of white matter alterations in children prenatally exposed to drugs of abuse, in particular opioids (Walhovd et al. 2010). The importance of understanding the effects of opioids and drug abuse treatments on myelination is further underscored by the fact that, in agreement with a large number of animal studies, there is evidence correlating increased myelin formation with experience and enhanced function of the infant brain (Als et al. 2004; Bengtsson et al. 2005; Pujol et al. 2006). It is also important to consider that opioid effects may not be limited to early child development since there is also active brain myelination during adolescence (Berns et al. 2009; Qiu et al. 2008), a highly vulnerable period for drug abuse.

Thus, there is a true compelling need for deciphering the roles of the endogenous opioid system in oligodendrocyte differentiation and myelin formation, and understanding the mechanisms by which opioid abuse and related treatments may interfere with these processes in the developing CNS. We have now found that buprenorphine indeed has direct actions on the oligodendrocytes, inducing dose-specific effects in their differentiation. Interestingly, these buprenorphine actions uncovered novel and opposing functions of MOR and the NOP receptor that underscore the complexity of mechanisms controlling oligodendrocyte development and myelination.

MATERIALS AND METHODS

Materials

Percoll, bovine pancreas DNAse, papain for cell isolation and cell culture medium components were from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F-12) (1:1) medium with high glucose and L-glutamine was obtained from Invitrogen (Grand Island, NY). Reduced-growth factor Matrigel™ was from Becton Dickinson (Franklin Lakes, NJ). Buprenorphine, methadone, and the MOR antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, [Disulfide Bridge: 2–7]) were purchased from Sigma-Aldrich (St. Louis, MO). The NOP receptor inhibitor J113397 (1-[(3R*,4R*)-1-(Cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) and nociceptin (Orphanin FQ) were from Tocris Bioscience (Ellisville, MO). All gel electrophoresis reagents and supplies were purchased from Bio-Rad Laboratories (Hercules, CA). Anti-β-actin and anti-MBP (82–87 region) antibodies were from Sigma-Aldrich and Millipore Corporation (Temecula, CA), respectively. The MOR and NOP receptor antibodies were from Neuromics (Edina, MN). The mouse O4 monoclonal antibody was kindly provided by Dr. Rashmi Bansal (University of Connecticut, Farmington, CT) and Dr. Babette Fuss (Virginia Commonwealth University, Richmond, VA). Super Signal West Dura chemiluminescence reagent was from Pierce (Rockford, IL). All appropriate secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Preparation of oligodendrocyte cultures

Sprague-Dawley rats were provided by Harlan Laboratories (Frederick, MD). Oligodendrocytes were directly isolated from rat brains as described previously (Colello and Sato-Bigbee 2001) with minor modifications. For this, the animals were sacrificed by decapitation and the brains rapidly dissected out, transferred to ice, and the meninges and main blood vessels removed by rolling the tissue on sterile filter paper. After mincing into 1–2 mm pieces, the tissue was incubated for 25 min. at 37°C in the presence of 1 unit/ml papain and 0.01 mg/ml DNAse. Following extensive washes and filtration through a 75μm pore size nylon mesh, the resulting cell suspension was centrifuged for 15 min at 30,000×g in an isotonic self-generated Percoll gradient. The band enriched in oligodendrocytes was then subjected to differential adhesion on tissue culture-treated Petri dishes to eliminate microglial cells and residual astrocytes. The floating oligodendrocytes were then plated in 48-well plates (Falcon) (for western blot analysis), or on 10mm cover slips in 24-well plates or 8-well slide Permanox chambers (for TUNEL assay and immunocytochemistry) all previously coated with 12.5 μL/well reduced growth factor-Matrigel™ extracellular matrix. Prior to use, the cells were maintained overnight in chemically defined medium (CDM) (DMEM/F-12 supplemented with 1 mg/mL fatty acid-free bovine serum albumin, 50 μg/mL transferrin, 5 μg/mL insulin, 30 nM sodium selenite, 0.11 mg/mL sodium pyruvate, 10 nM biotin, 20 nM progesterone, 100 μM putrescine, and 30 nM triiodothyronine). Astroglial contamination, as assessed by glial fibrillary acid protein staining, was less than 5%. When cells were exposed to buprenorphine, methadone or receptor inhibitors, the CDM was supplemented with the appropriate drug concentrations and the culture media changed every 48 hrs. Animal use and oligodendrocyte isolation were conducted in accordance with the guidelines from the National Institutes of Health and approved by the Virginia Commonwealth University Animal Care and Use Committee.

Western blot analysis

Oligodendrocyte cultures were lysed in 50–75 μL 60 mM Tris-HCl buffer (pH 6.8) containing 10% glycerol, 2% sodium dodecyl sulfate (SDS), and 5% 2-mercaptoethanol. Proteins in 10 μL samples were then separated by SDS-polyacrylamide gel electrophoresis in 15% acrylamide and electro-transferred to nitrocellulose. The membranes were then subjected to immunoblot analysis as previously reported (Saini et al. 2005), with minor modifications. Nonspecific antibody binding was blocked by incubation in 10 mM Na2HPO4, 2.7 mM KCl and 137 mM NaCl, pH 7.4, (PBS) containing 3% nonfat-dry milk and 0.05% Tween-20 (blocking solution), for 1 hr at room temperature. Blots were then incubated overnight with anti-MBP (dil. 1:100), anti-MOR (dil.1:500) or anti-NOP receptor (dil.1:500) antibodies. β-Actin levels detected with anti-β-actin antibody (dil. 1:2,000) were used as loading controls. After extensive rinsing with PBS, blots were incubated for 30 min in blocking solution, followed by incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 2 hrs. All antibodies were diluted in blocking buffer. After extensive washing the immunoreactive bands were detected by chemiluminescence 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 and divided by β-actin levels to correct for loading differences.

Detection of Apoptotic Cells

After fixation of the cells with 4% paraformaldehyde for 1 h at room temperature, the number of apoptotic oligodendrocytes was determined by detecting DNA fragmentation using a TUNEL assay (In Situ Cell Death Detection Kit; Roche Diagnostics, Indianapolis, IN), as reported previously (Saini et al. 2005). For each condition, at least 20 visual fields containing approximately 200 cells each were analyzed.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde in PBS and immunocytochemistry was carried out as previously reported (Sato-Bigbee et al. 1999). Non-specific antibody binding was blocked by incubation of the cultures for 1 hr in PBS containing 5% non-fat dry milk, 0.05% Tween-20, and 0.5% normal goat serum (blocking solution). The cells were then incubated overnight with anti-MBP (dil, 1:20) or a mixture of O4 (dil.1:3) and MBP (dil. 1:20) antibodies in blocking solution. After several washes in PBS, the cells were incubated for 30 min in blocking solution and for 2 hrs with Texas Red-conjugated anti-rat IgG (dil. 1:150) to visualize MBP labeling or Alexa 488-conjugated anti-mouse IgM (dil. 1:250) and Texas Red-conjugated anti-rat IgG (dil. 1:150) for O4-MBP double immunocytochemistry. For detection of MOR and NOP receptor, cells were incubated overnight in a mixture of O4 and anti-MOR (1:100) or anti-NOP receptor (1:100) antibodies in PBS containing 0.1% Triton X-100 and 1% normal goat serum. After extensive washing and a second 30 min blocking step, cells were incubated for 2 hrs with a mixture of Alexa 488-conjugated anti-mouse IgM (dil. 1:250) for O4 labeling and Texas Red-conjugated anti-rabbit IgG (dil. 1:150) for MOR and NOP receptor visualization. The cultures were then analyzed using a Nikon Eclipse 800M fluorescence microscope with ad hoc digital camera system.

Statistical Analysis

Statistical analysis was performed by one-way analysis of variance and Bonferroni post test, using the GraphPad Prism program. Differences were considered statistically significant when p values were <0.05.

RESULTS

Our previous studies indicated that perinatal exposure to buprenorphine causes dose-specific and developmentally dependent effects on brain myelination. While elevated MBP levels were observed at all studied ages in the brains of pups exposed to therapeutic drug doses, treatment with a supra-therapeutic buprenorphine dose was accompanied by delayed MBP expression and reduced number of myelinated axons (Sanchez et al. 2008). Because MBPs are considered to be markers of mature oligodendrocytes, these findings suggested that the differentiation of these cells is accelerated by therapeutic levels of buprenorphine but paradoxically, delayed by higher drug doses. If this is the case, altered oligodendrocyte maturation could be the result of direct actions of buprenorphine or, on the other hand, be mediated by drug effects on other cells, such as astrocytes or microglia, which could in turn influence the rate of oligodendrocyte development.

To begin to address these possibilities, we decided to study the potential direct effects of buprenorphine on the maturation of cultured oligodendrocytes. For this, we next investigated if treatment of the cells with buprenorphine could have any noticeable action on MBP expression. We began testing the response of cells directly isolated from 9-day-old rat brain. Cultures prepared from these cells are composed of postmitotic quiescent pre-oligodendrocytes, the majority of which already react with the O4 antibody (Sato-Bigbee et al. 1999). These cells represent a crucial and highly vulnerable developmental stage that immediately precedes the generation of mature oligodendrocytes capable of myelination.

As shown in Figure 1, similar to the results previously observed in the in vivo studies, direct exposure of the cells to buprenorphine also resulted in a biphasic dose-specific effect on MBP expression. Low buprenorphine doses elicited a significant increase in MBP levels, with maximal stimulation observed between 0.25 μM and 0.5 μM drug concentrations. However, this stimulatory action was not detected when the cells were exposed to 3 μM buprenorphine.

Figure 1. Direct treatment of immature oligodendrocytes with buprenorphine alters MBP expression in a dose-specific manner.

Figure 1

Open in a new tab

Cells isolated from 9-day-old rat brains were incubated for 4 days in CDM with or without 0.25, 0.5, 1.0, 3.0 μM buprenorphine. MBP levels were determined by western blotting using β-actin levels as loading controls. Figures correspond to representative experiments. Results in the bar graph are expressed as percentage of controls (0μM buprenorphine) +/− SEM from 5 experiments and correspond to the combined scanning of the four major MBP isoforms. **p<0.005, ***p<0.0001.

Since morphine exposure was shown to increase apoptosis of cultured oligodendrocytes (Hauser et al. 2009), we next carried out an analysis of cell death by TUNEL assay. However, we failed to detect any differences between treatment conditions, indicating that the differences in MBP expression could not be attributed to potential effects of buprenorphine on apoptosis (data not shown). On the contrary, the results presented so far indicated that buprenorphine does exert direct dose-dependent effects on oligodendrocyte differentiation. In support of this possibility, immunostaining of the cell cultures with anti-MBP antibody demonstrated that buprenorphine actions on MBP expression levels are also accompanied by dose-specific effects on the morphology of the cells (Figure 2). When treated with 0.5 μM buprenorphine, which as described above, induces maximal MBP stimulation, the cells showed significant increases in process outgrowth and membrane extension, a morphology that is indicative of highly mature oligodendrocytes. In contrast, in agreement with the lower MBP levels observed in the western blots, both control cultures and those exposed to 3 μM buprenorphine exhibited MBP positive cells with shorter processes and simpler morphology characteristic of less mature cells.

Figure 2. Exposure of pre-oligodendrocytes to buprenorphine alters cell process extension and membrane outgrowth in a dose-specific manner.

Figure 2

Open in a new tab

Developing oligodendrocytes isolated from 9-day-old rat brain were cultured for 4 days in CDM with or without buprenorphine (0.5μM, 3.0μM). Cells were then stained with anti-MBP antibody and visualized by fluorescent microscopy. The figure shows three representative panels corresponding to each of the treatment conditions. Note the extensive process and membrane extension in cells treated with 0.5μM buprenorphine. Scale bar: 10 μm.

Analysis of the cell cultures by double immunocytochemistry with O4 and anti-MBP antibodies (Figure 3) further supported the idea that the above observations reflect differences in the extent of cell differentiation. While both immature pre-oligodendrocytes and mature cells are known to react with the O4 monoclonal antibody, only differentiated oligodendrocytes are expected to be labeled by both the O4 and anti-MBP antibodies. In the presence of 0.5 μM buprenorphine the great majority of the cells are both highly O4 and MBP positive. On the other hand, controls grown in the absence of buprenorphine or cultures treated with the 3.0 μM dose concentration exhibited a significant number of cells that were only O4 positive but just few that were highly stained by both the O4 and MBP antibodies. Quantitation of these results (Figure 3, bar graph) indicated that in cultures exposed to 0.5 μM buprenorphine about 80% of the cells are labeled by both the O4 and MBP antibodies while this number is only about 35% in the case of controls and cells treated with 3.0 μM buprenorphine.

Figure 3. Treatment of O4 positive pre-oligodendrocytes with buprenorphine results in dose-specific differences in the capacity of the cells to transition into the mature MBP-expressing developmental stage.

Figure 3

Open in a new tab

Pre-oligodendrocytes isolated from 9-day-old rat brain were cultured for 4 days in CDM alone (control) or supplemented with 0.5μM buprenorphine (0.5 μM B) or 3.0μM buprenorphine (3.0 μM B). After 4 days of treatment, cells were analyzed by double immunocytochemistry with O4 (green) and anti-MBP (red) antibodies. Notice that cells which are both O4 and highly MBP positive are particularly increased in the cultures exposed to 0.5μM buprenorphine. Scale bar: 20 μm. The bar graph indicates the percentage of O4 positive cells that are also MBP positive under each experimental condition. The results represent the average +/− SEM from 5 different fields and at least 100 cells per condition. *p<0.001.

Taken together, the above observations are consistent with the original hypothesis that buprenorphine exerts direct effects on oligodendrocyte maturation. In addition, the bell shaped dose-dependent nature of the drug effects indicates the existence of a complex mechanism. Therefore, we next investigated the bases for these dose-specific effects.

Buprenorphine is a MOR partial agonist and KOR antagonist. However, as shown in Figure 4, we found that the positive effect of 0.5 μM buprenorphine on MBP expression could be mimicked by methadone, an opioid analogue that functions as a MOR specific agonist. Moreover, the stimulatory effects on MBP expression of methadone and 0.5 μM buprenorphine were both abolished by co-incubation with the highly specific MOR antagonist CTOP, supporting a novel role of this receptor as a positive regulator of oligodendrocyte maturation.

Figure 4. The stimulatory effect of buprenorphine is mimicked by methadone and both drug effects are abolished by the MOR antagonist CTOP.

Figure 4

Open in a new tab

Pre-oligodendrocytes from 9-day-old rat brain were cultured for 4 days in the presence or absence of 1μM methadone (1μM M) or 0.5μM buprenorphine (0.5μM B) with or without 1μM CTOP. MBP levels were determined by western blot analysis. (A) The picture shows a representative experiment. (B) The results in the bar graph correspond to the combined scanning of the four major MBP isoforms from at least 3 experiments and are expressed as percentage of controls (0μM buprenorphine) +/− SEM. *p<0.02, **p<0.0001.

While the results using methadone and CTOP pointed to 0.5 μM buprenorphine working through activation of MOR, these observations did not explain the differential effect of the higher 3 μM dose of buprenorphine. Similar to the dose-specific effects on MBP expression and oligodendrocyte development described above, others have shown that buprenorphine antinociceptive effects also exhibit a bell shaped dose-response. Low buprenorphine concentrations exert an analgesic effect but this antinociceptive action is significantly decreased by higher levels of the drug (Dum and Herz 1981; Lizasoain et al. 1991). Later investigations also found that antinociception induced by buprenorphine is mediated by MOR but higher doses counteract this effect by concomitant activation of the NOP receptor (Lutfy et al. 2003), a molecule that has about 75% homology with the three different opioid receptors (Mogil and Pasternak 2001).

The expression of the NOP receptor in oligodendrocytes has never been investigated before. However, western immunobloting and immunocytochemistry revealed that pre-oligodendrocytes isolated from 9-day-old rat brain indeed express both MOR and the NOP receptor (Figure 5). Moreover, the results depicted in Figure 6 suggest that an antagonistic functional relationship between these two receptors also exists in the oligodendrocytes and underlies the dose-specific effects of buprenorphine on these cells. Analysis of MBP expression showed that the biphasic dose-dependent effect of buprenorphine is abolished by inhibition of the NOP receptor. As shown in Figure 6A, cultures exposed to 3μM buprenorphine in the presence of J-113397, a highly specific NOP receptor inhibitor, exhibited significantly higher MBP levels than controls as well as cells exposed to 3μM buprenorphine alone. This is in contrast with the levels of MBP in cells treated with 3μM buprenorphine and CTOP, in which MBP levels are even lower than in controls (Figure 6B). Furthermore, treatment of the cells with the NOP receptor ligand nociceptin/orphanin FQ abrogates the stimulatory effect of 0.5 μM buprenorphine (Figure 6C) which as described above (Figure 4) implicates MOR activation.

Figure 5. Pre-oligodendrocytes express both MOR and the NOP receptor.

Figure 5

Open in a new tab

Cells isolated from 9-day-old rat brain were allowed to fully attach on the culture plates by overnight incubation and stained by double immunocytochemistry with O4 (green) together with anti-MOR or anti-NOP receptor antibodies (red). Scale bar: 20 μm. The Western blot shows MOR and NOP receptor expression in two different samples of developing oligodendrocytes directly isolated from 9-day-old rat brains.

Figure 6. The NOP receptor/nociceptin system downregulates MBP expression.

Figure 6

Open in a new tab

(A) Cells isolated from 9-day-old rat brain were cultured for 4 days in CDM in the presence or absence of 3μM buprenorphine (3μM B) and different concentrations of the NOP receptor inhibitor J-113397 (J). (B) Cells were cultured for 4 days in CDM in the presence or absence of 3μM buprenorphine with or without the MOR inhibitor CTOP. For both A and B, results in the bar graphs are expressed as percentage of controls (0μM buprenorphine) +/− SEM from at least 5 experiments and correspond to the combined scanning of the four major MBP isoforms. *p<0.02, **p<0.005. (C) Cells were cultured in the presence or absence of 0.5 μM buprenorphine (0.5 μM B) with or without 1 μM nociceptin. Results are expressed as percentage of controls (0μM buprenorphine) +/− SEM from 4 experiments and correspond to the combined scanning of the four major MBP isoforms. *p<0.05. MBP levels were determined by western blotting using β-actin levels as loading controls.

The affinity of buprenorphine for the NOP receptor is lower than its affinity for the MOR (Toll et al. 1998), and thus, our findings are consistent with a model in which buprenorphine exerts direct effects in the oligodendrocytes by binding to two different receptors with opposing roles on cell development (Figure 7). Because the affinity of buprenorphine for the NOP receptor is lower than its affinity for the MOR (Toll et al. 1998), it is logical to suppose that low drug levels activate the high affinity MOR while high buprenorphine concentrations are required to bind to both MOR and the low affinity NOP receptor. MOR activation results in stimulation of oligodendrocyte maturation while this effect is counteracted by an inhibitory effect induced by concomitant signaling through the NOP receptor. Therefore, it is tempting to hypothesize that balance between these two pathways may play a crucial role in “timing” oligodendrocyte differentiation and the beginning of myelin synthesis. Buprenorphine, methadone, and other opioid drugs could alter this interplay affecting myelination in the maturing brain.

Figure 7. Buprenorphine effects and proposed involvement of MOR and the NOP receptor in oligodendrocyte maturation.

Figure 7

Open in a new tab

Low buprenorphine doses activate the high affinity MOR while at high concentrations the drug is able to bind to both MOR and the low affinity NOP receptor. MOR activation results in stimulation of oligodendrocyte maturation while this effect is counteracted by an inhibitory effect induced by signaling through the NOP receptor. Balance between both receptor activities could modulate the timing of oligodendrocyte maturation.

DISCUSSION

Our previous studies showed that perinatal exposure to buprenorphine affects myelination in the developing rat brain and suggested that these actions may in part reflect alterations in the timing of oligodendrocyte development (Sanchez et al. 2008). We have now found that buprenorphine indeed exerts direct actions on the oligodendrocytes. These effects are highly dose-specific and furthermore, they reveal novel functions of the MOR and the NOP receptor as regulators of oligodendrocyte development. The present results demonstrate that exposure of the cells to low doses of buprenorphine not only results in elevation of MBP levels, but is also accompanied by increased morphological complexity and elevated proportion of O4-positive pre-oligodendrocytes that are able to progress into the mature MBP-expressing developmental stage. Thus, while potential effects of buprenorphine on the expression of other myelin components remain to be investigated, these observations suggest that the increased levels of MBP do not result from a specific up-regulation of this protein but are instead a reflection of low buprenorphine doses stimulating oligodendrocyte differentiation. Interestingly, exposure to high buprenorphine doses exerts an opposing effect on oligodendrocyte development.

Understanding of the molecular mechanisms underlying buprenorphine effects in the oligodendrocytes is complicated by the complex pharmacology of this drug. Buprenorphine is generally regarded as a partial MOR agonist (Yu et al. 1997) and KOR antagonist (Leander 1987) that could also exert both agonist and antagonist actions on the δ-opioid receptor (DOR) (Huang et al. 2001; Sadee et al. 1982). Initial studies have shown that the MOR is expressed very early in the oligodendroglial lineage, whereas the other two opioid receptors appear at later stages of development (Knapp et al. 1998; Tryoen-Toth et al. 2000). However, more recent analysis has indicated the presence of the three opioid receptors at all stages along the oligodendroglial lineage (Hauser et al. 2009). The present results support the idea that the observed stimulatory actions of low buprenorphine doses on the pre-oligodendrocytes are mediated by agonist effect on the MOR. Early studies implicated this receptor as a stimulator of proliferation for both neonatal oligodendrocyte progenitors (Knapp and Hauser 1996) and adult neuroprogenitors (Persson et al. 2003). The supposition of an in vivo role of the endogenous opioid system in controlling cell proliferation during CNS development is strengthened by the observation that synthesis of pro-opiomelanocortin and its processing into the MOR and DOR agonist β-endorphin in the rat brain are elevated at embryonic days and postnatal ages that coincide with periods of crucial proliferative activity of neuronal and glial progenitors (Angelogianni et al. 2000). Moreover, several lines of evidence support the idea that opioid signaling may not only be important during development but also play a role in maintaining adequate numbers of different cell populations in the adult brain. Treatment of cultured adult hippocampal progenitors with MOR and DOR antagonists decreases proliferation and neurogenesis (Persson et al. 2003). Additional work also has shown that incubation with β-endorphin preferentially stimulates oligodendrogenesis in a population of cultured rat adult hippocampal progenitors (Persson et al. 2006; Raynor et al. 1994). More recently, MOR and KOR were shown to modulate oligodendrogenesis from stem cell-derived neural progenitors via MAP kinases (Hahn et al. 2010). However, to our knowledge, the present study is the first to directly implicate the MOR in the last stages of oligodendrocyte development.

Particularly interesting is the observation that pre-oligodendrocytes exhibit increased differentiation when exposed to low concentrations of buprenorphine but not in response to elevated doses of the drug. A potential explanation to the observed bell-shaped dose-dependent effect of buprenorphine on oligodendrocyte maturation and MBP expression could be receptor desensitization by high drug concentrations. However, our present observations indicated that buprenorphine dose-specific effects in the oligodendrocytes implicate a delicate balancing act between MOR and the NOP receptor. This finding is particularly intriguing because the NOP/nociceptin system has been primarily linked to behavior and pain regulation but a role in CNS development or myelination has not been described before.

Similar to the biphasic bell-shaped curve observed in our present studies, different lines of evidence have demonstrated that while low doses of buprenorphine exert an analgesic effect, this antinociceptive action is decreased by higher drug concentrations (Dum and Herz 1981; Lizasoain et al. 1991). Later studies found that that while buprenorphine antinociceptive effects are mediated by the MOR, this action is counteracted by the concomitant activation of NOP receptors (Lutfy et al. 2003). In those studies, the co-administration of J-113397, the specific NOP receptor inhibitor used in our present experiments, not only enhanced the antinociceptive effect of buprenorphine but also eliminated the characteristic bell-shaped response. Moreover, the biphasic response was not observed in NOP receptor knockout mice. The results of the present study suggest for the first time that an interaction between the MOR and NOP receptor-mediated signaling plays a crucial role timing oligodendrocyte maturation.

It remains to be determined the extent to which this type of dose-specific receptor activation is responsible for the biphasic effects on myelination that we previously observed in the brains of rats perinatally exposed to buprenorphine (Sanchez et al. 2008). It is important to consider that the in vivo effects may not be entirely explained by the direct effects of buprenorphine on the oligodendrocytes. The ubiquity of opioid receptors in the CNS and the importance of neuron-glial interactions in the developmental coordination of myelination raise the possibility of combined drug effects in different cell populations. In this regard, previous observations found that perinatal exposure of rodents to buprenorphine delays the generation of cholinergic neurons and reduces the expression of nerve growth factor in the striatum (Robinson 2002; Wu et al. 2001). In support of additional effects on neuron-glia interactions, we also found before that regardless of the dose, pups perinatally exposed to buprenorphine exhibited a significant increase in the caliber of the myelinated axons (Sanchez et al. 2008). Surprisingly, these axons were characterized by having a disproportionately thinner myelin sheath. These changes were also accompanied by increased levels of the myelin associated glycoprotein (MAG), a molecule that has been implicated in glial-axonal communication (Yin et al. 1998). Exposure to therapeutic low buprenorphine doses also exhibited increased MAG glycosylation and interaction with the Src-family tyrosine kinase Fyn. Interestingly, others have implicated both MAG and Fyn as key molecules potentially mediating bidirectional signals between neurons and oligodendrocytes (Biffiger et al. 2000). Based on these observations we hypothesized that opioid signaling may be part of the molecular mechanisms that coordinate axonal radial growth with myelination (Sanchez et al. 2008). Altogether, our previous and present findings suggest that interference with the endogenous opioid system during development may have significant consequences on oligodendrocyte maturation and myelination.

Although perinatal opioid exposure is the most immediate clinical correlate to this study, drug effects on oligodendrocytes and myelin formation may not be limited to early brain maturation. It is now known that human cognitive development and memory, as well as learning experience beyond early infancy, are positively correlated with late white matter deposition in functionally related areas of the brain (Bengtsson et al. 2005; Deutsch et al. 2005; Niogi and McCandliss 2006; Rimrodt et al. 2010; Takeuchi et al. 2010; Uhlhaas et al. 2010; Whitaker et al. 2008). A major concern in this regard is the existence of active myelination during adolescence (Pfefferbaum et al. 1994; Qiu et al. 2008; Whitaker et al. 2008), a highly vulnerable period for drug abuse. This is also a critical developmental window for circuit refining and changes in the brain reward system and the magnitude of these structural and functional changes is evident both physically and behaviorally (Paus et al. 2008). The importance of myelin in the later phases of brain maturation is further supported by a growing number of neurological diseases and psychotic disorders being associated to white matter pathology during adolescence, including, among others, schizophrenia (Chambers and Perrone-Bizzozero 2004; Kerns et al. 2010; Kubicki et al. 2005; Walterfang et al. 2005; Whitford et al. 2010), bipolar disorder (Brambilla et al. 2009; Chambers and Perrone-Bizzozero 2004; Mahon et al. 2010), and autism (O’Hearn et al. 2008). It would therefore be important to consider that opioid addiction at adolescence may critically affect myelination resulting in long-lasting behavioral and cognitive problems.

In conclusion, the present results indicate that both buprenorphine and methadone exert direct specific effects on the oligodendrocytes. Together with the previous observations, the present findings further support an important role for opioid signaling in regulating brain maturation and, in particular, oligodendrocyte development and myelination. The novel finding of opposing actions of the MOR and NOP receptor further emphasize the intricate mechanisms underlying opioid function in glial biology. Future studies investigating the molecular mechanisms by which buprenorphine and methadone affect myelination and neuro-glial interactions should provide deeper understanding into these developmental processes and new and better strategies for the managing of both pregnant addicts and drug addiction in adolescence.

Acknowledgments

Supported by NIH grant R21DA027099-01 and NMSS grant RG 3432A2.

References

  1. Als H, Duffy FH, McAnulty GB, Rivkin MJ, Vajapeyam S, Mulkern RV, Warfield SK, Huppi PS, Butler SC, Conneman N, et al. Early experience alters brain function and structure. Pediatrics. 2004;113(4):846–857. doi: 10.1542/peds.113.4.846. [DOI] [PubMed] [Google Scholar]
  2. Amass L, Ling W, Freese TE, Reiber C, Annon JJ, Cohen AJ, McCarty D, Reid MS, Brown LS, Clark C, et al. Bringing buprenorphine-naloxone detoxification to community treatment providers: the NIDA Clinical Trials Network field experience. Am J Addict. 2004;13(Suppl 1):S42–66. doi: 10.1080/10550490490440807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Angelogianni P, Li HL, Gianoulakis C. Ontogenesis of proopiomelanocortin and its processing to beta-endorphin by the fetal and neonatal rat brain. Neuroendocrinology. 2000;72(4):231–241. doi: 10.1159/000054592. [DOI] [PubMed] [Google Scholar]
  4. Bengtsson SL, Nagy Z, Skare S, Forsman L, Forssberg H, Ullen F. Extensive piano practicing has regionally specific effects on white matter development. Nat Neurosci. 2005;8(9):1148–1150. doi: 10.1038/nn1516. [DOI] [PubMed] [Google Scholar]
  5. Berns GS, Moore S, Capra CM. Adolescent engagement in dangerous behaviors is associated with increased white matter maturity of frontal cortex. PLoS One. 2009;4(8):e6773. doi: 10.1371/journal.pone.0006773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Biffiger K, Bartsch S, Montag D, Aguzzi A, Schachner M, Bartsch U. Severe hypomyelination of the murine CNS in the absence of myelin-associated glycoprotein and fyn tyrosine kinase. J Neurosci. 2000;20(19):7430–7437. doi: 10.1523/JNEUROSCI.20-19-07430.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brambilla P, Bellani M, Yeh PH, Soares JC, Tansella M. White matter connectivity in bipolar disorder. Int Rev Psychiatry. 2009;21(4):380–386. doi: 10.1080/09540260902962172. [DOI] [PubMed] [Google Scholar]
  8. Broussard CS, Rasmussen SA, Reefhuis J, Friedman JM, Jann MW, Riehle-Colarusso T, Honein MA. Maternal treatment with opioid analgesics and risk for birth defects. Am J Obstet Gynecol. 2011;204(4):314, e311–314, e311. doi: 10.1016/j.ajog.2010.12.039. [DOI] [PubMed] [Google Scholar]
  9. Bruce CC, Zhao C, Franklin RJ. Remyelination - An effective means of neuroprotection. Horm Behav. 2010;57(1):56–62. doi: 10.1016/j.yhbeh.2009.06.004. [DOI] [PubMed] [Google Scholar]
  10. Butt AM, Ransom BR. Morphology of astrocytes and oligodendrocytes during development in the intact rat optic nerve. J Comp Neurol. 1993;338(1):141–158. doi: 10.1002/cne.903380110. [DOI] [PubMed] [Google Scholar]
  11. Campagnoni AT. Molecular biology of myelin proteins from the central nervous system. J Neurochem. 1988;51(1):1–14. doi: 10.1111/j.1471-4159.1988.tb04827.x. [DOI] [PubMed] [Google Scholar]
  12. Chambers JS, Perrone-Bizzozero NI. Altered myelination of the hippocampal formation in subjects with schizophrenia and bipolar disorder. Neurochem Res. 2004;29(12):2293–2302. doi: 10.1007/s11064-004-7039-x. [DOI] [PubMed] [Google Scholar]
  13. Colello RJ, Sato-Bigbee C. Purification of oligodendrocytes and their progenitors using immunomagnetic separation and Percoll gradient centrifugation. Curr Protoc Neurosci. 2001;Chapter 3(Unit 3):12. doi: 10.1002/0471142301.ns0312s03. [DOI] [PubMed] [Google Scholar]
  14. Compton WM, Volkow ND. Major increases in opioid analgesic abuse in the United States: concerns and strategies. Drug Alcohol Depend. 2006;81(2):103–107. doi: 10.1016/j.drugalcdep.2005.05.009. [DOI] [PubMed] [Google Scholar]
  15. Deutsch GK, Dougherty RF, Bammer R, Siok WT, Gabrieli JD, Wandell B. Children’s reading performance is correlated with white matter structure measured by diffusion tensor imaging. Cortex. 2005;41(3):354–363. doi: 10.1016/s0010-9452(08)70272-7. [DOI] [PubMed] [Google Scholar]
  16. Dum JE, Herz A. In vivo receptor binding of the opiate partial agonist, buprenorphine, correlated with its agonistic and antagonistic actions. Br J Pharmacol. 1981;74(3):627–633. doi: 10.1111/j.1476-5381.1981.tb10473.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ebner E, Wiedmann M. Transdermal buprenorphine during pregnancy. Schmerz. 2006;20(4):334–337. doi: 10.1007/s00482-005-0445-4. [DOI] [PubMed] [Google Scholar]
  18. Fields RD. Oligodendrocytes changing the rules: action potentials in glia and oligodendrocytes controlling action potentials. Neuroscientist. 2008;14(6):540–543. doi: 10.1177/1073858408320294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hahn JW, Jagwani S, Kim E, Rendell VR, He J, Ezerskiy LA, Wesselschmidt R, Coscia CJ, Belcheva MM. Mu and kappa opioids modulate mouse embryonic stem cell-derived neural progenitor differentiation via MAP kinases. J Neurochem. 2010;112(6):1431–1441. doi: 10.1111/j.1471-4159.2009.06479.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hall AJ, Logan JE, Toblin RL, Kaplan JA, Kraner JC, Bixler D, Crosby AE, Paulozzi LJ. Patterns of abuse among unintentional pharmaceutical overdose fatalities. JAMA. 2008;300(22):2613–2620. doi: 10.1001/jama.2008.802. [DOI] [PubMed] [Google Scholar]
  21. Hauser KF, Hahn YK, Adjan VV, Zou S, Buch SK, Nath A, Bruce-Keller AJ, Knapp PE. HIV-1 Tat and morphine have interactive effects on oligodendrocyte survival and morphology. Glia. 2009;57(2):194–206. doi: 10.1002/glia.20746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huang P, Kehner GB, Cowan A, Liu-Chen LY. Comparison of pharmacological activities of buprenorphine and norbuprenorphine: norbuprenorphine is a potent opioid agonist. J Pharmacol Exp Ther. 2001;297(2):688–695. [PubMed] [Google Scholar]
  23. Jaffe JH, O’Keeffe C. From morphine clinics to buprenorphine: regulating opioid agonist treatment of addiction in the United States. Drug Alcohol Depend. 2003;70(2 Suppl):S3–S11. doi: 10.1016/s0376-8716(03)00055-3. [DOI] [PubMed] [Google Scholar]
  24. Jansson LM, Dipietro JA, Velez M, Elko A, Williams E, Milio L, O’Grady K, Jones HE. Fetal neurobehavioral effects of exposure to methadone or buprenorphine. Neurotoxicology and teratology. 2011;33(2):240–243. doi: 10.1016/j.ntt.2010.09.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jones HE, Johnson RE, Jasinski DR, O’Grady KE, Chisholm CA, Choo RE, Crocetti M, Dudas R, Harrow C, Huestis MA, et al. Buprenorphine versus methadone in the treatment of pregnant opioid-dependent patients: effects on the neonatal abstinence syndrome. Drug Alcohol Depend. 2005;79(1):1–10. doi: 10.1016/j.drugalcdep.2004.11.013. [DOI] [PubMed] [Google Scholar]
  26. Jones HE, Kaltenbach K, Heil SH, Stine SM, Coyle MG, Arria AM, O’Grady KE, Selby P, Martin PR, Fischer G. Neonatal abstinence syndrome after methadone or buprenorphine exposure. N Engl J Med. 2010a;363(24):2320–2331. doi: 10.1056/NEJMoa1005359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jones HE, O’Grady KE, Johnson RE, Velez M, Jansson LM. Infant neurobehavior following prenatal exposure to methadone or buprenorphine: results from the neonatal intensive care unit network neurobehavioral scale. Subst Use Misuse. 2010b;45(13):2244–2257. doi: 10.3109/10826084.2010.484474. [DOI] [PubMed] [Google Scholar]
  28. Kerns D, Vong GS, Barley K, Dracheva S, Katsel P, Casaccia P, Haroutunian V, Byne W. Gene expression abnormalities and oligodendrocyte deficits in the internal capsule in schizophrenia. Schizophr Res. 2010;120(1–3):150–158. doi: 10.1016/j.schres.2010.04.012. [DOI] [PubMed] [Google Scholar]
  29. Knapp PE, Hauser KF. mu-Opioid receptor activation enhances DNA synthesis in immature oligodendrocytes. Brain Res. 1996;743(1–2):341–345. doi: 10.1016/s0006-8993(96)01097-9. [DOI] [PubMed] [Google Scholar]
  30. Knapp PE, Itkis OS, Zhang L, Spruce BA, Bakalkin G, Hauser KF. Endogenous opioids and oligodendroglial function: possible autocrine/paracrine effects on cell survival and development. Glia. 2001;35(2):156–165. doi: 10.1002/glia.1080. [DOI] [PubMed] [Google Scholar]
  31. Knapp PE, Maderspach K, Hauser KF. Endogenous opioid system in developing normal and jimpy oligodendrocytes: mu and kappa opioid receptors mediate differential mitogenic and growth responses. Glia. 1998;22(2):189–201. doi: 10.1002/(sici)1098-1136(199802)22:2<189::aid-glia10>3.0.co;2-u. [DOI] [PubMed] [Google Scholar]
  32. Kubicki M, Park H, Westin CF, Nestor PG, Mulkern RV, Maier SE, Niznikiewicz M, Connor EE, Levitt JJ, Frumin M, et al. DTI and MTR abnormalities in schizophrenia: analysis of white matter integrity. Neuroimage. 2005;26(4):1109–1118. doi: 10.1016/j.neuroimage.2005.03.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Leander JD. Buprenorphine has potent kappa opioid receptor antagonist activity. Neuropharmacology. 1987;26(9):1445–1447. doi: 10.1016/0028-3908(87)90112-2. [DOI] [PubMed] [Google Scholar]
  34. Lewis JW. Buprenorphine. Drug Alcohol Depend. 1985;14(3–4):363–372. doi: 10.1016/0376-8716(85)90067-5. [DOI] [PubMed] [Google Scholar]
  35. Lizasoain I, Leza JC, Lorenzo P. Buprenorphine: bell-shaped dose-response curve for its antagonist effects. Gen Pharmacol. 1991;22(2):297–300. doi: 10.1016/0306-3623(91)90452-c. [DOI] [PubMed] [Google Scholar]
  36. Lutfy K, Eitan S, Bryant CD, Yang YC, Saliminejad N, Walwyn W, Kieffer BL, Takeshima H, Carroll FI, Maidment NT, et al. Buprenorphine-induced antinociception is mediated by mu-opioid receptors and compromised by concomitant activation of opioid receptor-like receptors. J Neurosci. 2003;23(32):10331–10337. doi: 10.1523/JNEUROSCI.23-32-10331.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mahon K, Burdick KE, Szeszko PR. A role for white matter abnormalities in the pathophysiology of bipolar disorder. Neurosci Biobehav Rev. 2010;34(4):533–554. doi: 10.1016/j.neubiorev.2009.10.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Maxwell JC, McCance-Katz EF. Indicators of buprenorphine and methadone use and abuse: what do we know? Am J Addict. 2010;19(1):73–88. doi: 10.1111/j.1521-0391.2009.00008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mogil JS, Pasternak GW. The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family. Pharmacol Rev. 2001;53(3):381–415. [PubMed] [Google Scholar]
  40. Niogi SN, McCandliss BD. Left lateralized white matter microstructure accounts for individual differences in reading ability and disability. Neuropsychologia. 2006;44(11):2178–2188. doi: 10.1016/j.neuropsychologia.2006.01.011. [DOI] [PubMed] [Google Scholar]
  41. O’Hearn K, Asato M, Ordaz S, Luna B. Neurodevelopment and executive function in autism. Dev Psychopathol. 2008;20(4):1103–1132. doi: 10.1017/S0954579408000527. [DOI] [PubMed] [Google Scholar]
  42. Ornoy A, Segal J, Bar-Hamburger R, Greenbaum C. Developmental outcome of school-age children born to mothers with heroin dependency: importance of environmental factors. Dev Med Child Neurol. 2001;43(10):668–675. doi: 10.1017/s0012162201001219. [DOI] [PubMed] [Google Scholar]
  43. Paus T, Keshavan M, Giedd JN. Why do many psychiatric disorders emerge during adolescence? Nat Rev Neurosci. 2008;9(12):947–957. doi: 10.1038/nrn2513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Persson AI, Bull C, Eriksson PS. Requirement for Id1 in opioid-induced oligodendrogenesis in cultured adult rat hippocampal progenitors. Eur J Neurosci. 2006;23(9):2277–2288. doi: 10.1111/j.1460-9568.2006.04764.x. [DOI] [PubMed] [Google Scholar]
  45. Persson AI, Thorlin T, Bull C, Zarnegar P, Ekman R, Terenius L, Eriksson PS. Mu- and delta-opioid receptor antagonists decrease proliferation and increase neurogenesis in cultures of rat adult hippocampal progenitors. Eur J Neurosci. 2003;17(6):1159–1172. doi: 10.1046/j.1460-9568.2003.02538.x. [DOI] [PubMed] [Google Scholar]
  46. Pfefferbaum A, Mathalon DH, Sullivan EV, Rawles JM, Zipursky RB, Lim KO. A quantitative magnetic resonance imaging study of changes in brain morphology from infancy to late adulthood. Arch Neurol. 1994;51(9):874–887. doi: 10.1001/archneur.1994.00540210046012. [DOI] [PubMed] [Google Scholar]
  47. Pringle NP, Mudhar HS, Collarini EJ, Richardson WD. PDGF receptors in the rat CNS: during late neurogenesis, PDGF alpha-receptor expression appears to be restricted to glial cells of the oligodendrocyte lineage. Development. 1992;115(2):535–551. doi: 10.1242/dev.115.2.535. [DOI] [PubMed] [Google Scholar]
  48. Pujol J, Soriano-Mas C, Ortiz H, Sebastian-Galles N, Losilla JM, Deus J. Myelination of language-related areas in the developing brain. Neurology. 2006;66(3):339–343. doi: 10.1212/01.wnl.0000201049.66073.8d. [DOI] [PubMed] [Google Scholar]
  49. Qiu D, Tan LH, Zhou K, Khong PL. Diffusion tensor imaging of normal white matter maturation from late childhood to young adulthood: voxel-wise evaluation of mean diffusivity, fractional anisotropy, radial and axial diffusivities, and correlation with reading development. Neuroimage. 2008;41(2):223–232. doi: 10.1016/j.neuroimage.2008.02.023. [DOI] [PubMed] [Google Scholar]
  50. Rasband MN, Trimmer JS. Developmental clustering of ion channels at and near the node of Ranvier. Dev Biol. 2001;236(1):5–16. doi: 10.1006/dbio.2001.0326. [DOI] [PubMed] [Google Scholar]
  51. Raynor K, Kong H, Hines J, Kong G, Benovic J, Yasuda K, Bell GI, Reisine T. Molecular mechanisms of agonist-induced desensitization of the cloned mouse kappa opioid receptor. J Pharmacol Exp Ther. 1994;270(3):1381–1386. [PubMed] [Google Scholar]
  52. Rimrodt SL, Peterson DJ, Denckla MB, Kaufmann WE, Cutting LE. White matter microstructural differences linked to left perisylvian language network in children with dyslexia. Cortex. 2010;46(6):739–749. doi: 10.1016/j.cortex.2009.07.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Robinson SE. Effects of perinatal buprenorphine and methadone exposures on striatal cholinergic ontogeny. Neurotoxicol Teratol. 2002;24(2):137–142. doi: 10.1016/s0892-0362(01)00185-4. [DOI] [PubMed] [Google Scholar]
  54. Sadee W, Rosenbaum JS, Herz A. Buprenorphine: differential interaction with opiate receptor subtypes in vivo. J Pharmacol Exp Ther. 1982;223(1):157–162. [PubMed] [Google Scholar]
  55. Saini HS, Coelho RP, Goparaju SK, Jolly PS, Maceyka M, Spiegel S, Sato-Bigbee C. Novel role of sphingosine kinase 1 as a mediator of neurotrophin-3 action in oligodendrocyte progenitors. J Neurochem. 2005;95(5):1298–1310. doi: 10.1111/j.1471-4159.2005.03451.x. [DOI] [PubMed] [Google Scholar]
  56. Sanchez ES, Bigbee JW, Fobbs W, Robinson SE, Sato-Bigbee C. Opioid addiction and pregnancy: perinatal exposure to buprenorphine affects myelination in the developing brain. Glia. 2008;56(9):1017–1027. doi: 10.1002/glia.20675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sato-Bigbee C, Pal S, Chu AK. Different neuroligands and signal transduction pathways stimulate CREB phosphorylation at specific developmental stages along oligodendrocyte differentiation. J Neurochem. 1999;72(1):139–147. doi: 10.1046/j.1471-4159.1999.0720139.x. [DOI] [PubMed] [Google Scholar]
  58. Sommer I, Schachner M. Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system. Dev Biol. 1981;83(2):311–327. doi: 10.1016/0012-1606(81)90477-2. [DOI] [PubMed] [Google Scholar]
  59. Takeuchi H, Sekiguchi A, Taki Y, Yokoyama S, Yomogida Y, Komuro N, Yamanouchi T, Suzuki S, Kawashima R. Training of working memory impacts structural connectivity. J Neurosci. 2010;30(9):3297–3303. doi: 10.1523/JNEUROSCI.4611-09.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Toll L, Berzetei-Gurske IP, Polgar WE, Brandt SR, Adapa ID, Rodriguez L, Schwartz RW, Haggart D, O’Brien A, White A, et al. Standard binding and functional assays related to medications development division testing for potential cocaine and opiate narcotic treatment medications. NIDA Res Monogr. 1998;178:440–466. [PubMed] [Google Scholar]
  61. Tryoen-Toth P, Gaveriaux-Ruff C, Labourdette G. Down-regulation of mu-opioid receptor expression in rat oligodendrocytes during their development in vitro. J Neurosci Res. 2000;60(1):10–20. doi: 10.1002/(SICI)1097-4547(20000401)60:1<10::AID-JNR2>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  62. Uhlhaas PJ, Roux F, Rodriguez E, Rotarska-Jagiela A, Singer W. Neural synchrony and the development of cortical networks. Trends Cogn Sci. 2010;14(2):72–80. doi: 10.1016/j.tics.2009.12.002. [DOI] [PubMed] [Google Scholar]
  63. Unger A, Jung E, Winklbaur B, Fischer G. Gender issues in the pharmacotherapy of opioid-addicted women: buprenorphine. J Addict Dis. 2010;29(2):217–230. doi: 10.1080/10550881003684814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Walhovd KB, Westlye LT, Moe V, Slinning K, Due-Tonnessen P, Bjornerud A, van der Kouwe A, Dale AM, Fjell AM. White matter characteristics and cognition in prenatally opiate- and polysubstance-exposed children: a diffusion tensor imaging study. AJNR Am J Neuroradiol. 2010;31(5):894–900. doi: 10.3174/ajnr.A1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Walterfang M, Wood SJ, Velakoulis D, Copolov D, Pantelis C. Diseases of white matter and schizophrenia-like psychosis. Aust N Z J Psychiatry. 2005;39(9):746–756. doi: 10.1080/j.1440-1614.2005.01678.x. [DOI] [PubMed] [Google Scholar]
  66. Whitaker KJ, Kolind SH, MacKay AL, Clark CM. Quantifying development: Investigating highly myelinated voxels in preadolescent corpus callosum. Neuroimage. 2008;43(4):731–735. doi: 10.1016/j.neuroimage.2008.07.038. [DOI] [PubMed] [Google Scholar]
  67. Whitford TJ, Kubicki M, Schneiderman JS, O’Donnell LJ, King R, Alvarado JL, Khan U, Markant D, Nestor PG, Niznikiewicz M, et al. Corpus callosum abnormalities and their association with psychotic symptoms in patients with schizophrenia. Biol Psychiatry. 2010;68(1):70–77. doi: 10.1016/j.biopsych.2010.03.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wu VW, Mo Q, Yabe T, Schwartz JP, Robinson SE. Perinatal opioids reduce striatal nerve growth factor content in rat striatum. Eur J Pharmacol. 2001;414(2–3):211–214. doi: 10.1016/s0014-2999(01)00807-x. [DOI] [PubMed] [Google Scholar]
  69. Yin X, Crawford TO, Griffin JW, Tu P, Lee VM, Li C, Roder J, Trapp BD. Myelin-associated glycoprotein is a myelin signal that modulates the caliber of myelinated axons. J Neurosci. 1998;18(6):1953–1962. doi: 10.1523/JNEUROSCI.18-06-01953.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Yu Y, Zhang L, Yin X, Sun H, Uhl GR, Wang JB. Mu opioid receptor phosphorylation, desensitization, and ligand efficacy. J Biol Chem. 1997;272(46):28869–28874. doi: 10.1074/jbc.272.46.28869. [DOI] [PubMed] [Google Scholar]

RESOURCES