Morphine sulfate concomitantly decreases neuronal differentiation and opioid receptor expression in mouse embryonic stem cells

Accepted Manuscript Title: Morphine sulfate concomitantly decreases neuronal differentiation and opioid receptor expression in mouse embryonic stem cells Author: Sanjay L. Dholakiya Angela Aliberti Frank A. Barile PII: DOI: Reference:

S0378-4274(16)30010-8 http://dx.doi.org/doi:10.1016/j.toxlet.2016.01.010 TOXLET 9305

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Toxicology Letters

Received date: Revised date: Accepted date:

24-4-2015 21-12-2015 17-1-2016

Please cite this article as: Dholakiya, Sanjay L., Aliberti, Angela, Barile, Frank A., Morphine sulfate concomitantly decreases neuronal differentiation and opioid receptor expression in mouse embryonic stem cells.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2016.01.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Morphine sulfate concomitantly decreases neuronal differentiation and opioid receptor expression in mouse embryonic stem cells. Sanjay L. Dholakiya, Angela Aliberti, and Frank A. Barile

St. John’s University College of Pharmacy and Health Sciences Department of Pharmaceutical Sciences Queens, New York 11439

Text Pages:

39 (including abstract and references)

Figures:

8

Tables:

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Running Title: Morphine sulfate decreases neuronal differentiation and receptor expression. Address Correspondence to:

Frank A. Barile, Ph.D. Chairman and Professor of Department of Pharmaceutical Sciences St. John’s University College of Pharmacy and Health Sciences 8000 Utopia Parkway, Queens, NY 11439 Office: 1-718-990-2013 Fax: 1-718-990-1877 [email protected]

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Highlights: 

Morphine sulfate treatment decreases neuronal differentiation.

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Morphine has distinct effects on different stages of stem cell/neuronal differentiation (early stage vs. late stage of neuronal development).

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Morphine treatment interferes with neuronal differentiation via MOR activation.

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Abstract: Opioids have been shown to affect prenatal and postnatal neural development in mammals. The present study investigates the impact of morphine sulfate (MS) treatment on neuronal differentiation as well as -opioid receptor (MOR) expression in mouse embryonic stem (mES) cells. Stem cells were manipulated in culture to differentiate in 3 sequential stages: Stage 1, cell transformation to embryoid bodies (EB); Stage 2, EB cell differentiation to neural progenitor (NP) cells; and, Stage 3, NP cell differentiation to neurons/astrocytes co-cultured cells. Using RT-PCR and flow cytometry analyses, cell types were confirmed by monitoring expression of Oct4, nestin, microtubule-associated protein 2 (mtap-2), and glial fibrillary acidic protein (GFAP) as cell-specific markers for stem cells, NP cells, neurons, and astrocytes, respectively. Similarly, gene expression for MOR, κ-opioid receptor (KOR), and δ-opioid receptor (DOR) was confirmed in each cell type. In order to investigate the effects of MS on differentiation, cells were treated with MS (1, 10, 100 M) at either early (Stage 1) or late (Stage 3) stage of cellular differentiation. At Stage 1 exposure, MOR gene expression and neuroectoderm specific marker expression of nestin were down-regulated in both EB and NP cells. In addition, the opioid downregulated GFAP in differentiated neurons/astrocytes co-cultured cells. Late stage treatment with MS resulted in a down-regulation of mtap-2 and GFAP in differentiated neurons/astrocytes cocultured cells. Moreover, late stage treatment with MS and naltrexone inhibited the effect of MS on neuronal differentiation, suggesting that MS treatment interferes with differentiation via MOR activation. Together, the results show that MS exposure at early and late stage of cellular differentiation significantly decreases genotype and phenotype in differentiated neuronal cells. The results of this study have implications regarding the potential effect of opiates on fetal brain development. Abbreviations: bFGF, basic fibroblast growth factor; DMEM, Dulbecco’s Modified Eagle’s Medium; DMEM-ITS, Dulbecco’s Modified Eagle’s Medium supplemented with insulin, transferrin, and selenium; DOR, δ-opioid receptor; EB cells, embryoid body cells; EGF, epidermal growth factor; GFAP, glial fibirillary acidic protein; ITS, insulin-transferrinselenium; KOR, κ-opioid receptor; LIF, leukemia inhibitory factor; mES cells, mouse embryonic stem cells; MS, morphine sulfate; MOR, μ-opioid receptor; Mtap-2, microtubule associated protein 2; NP cells, neuronal progenitor cells; Oct4, octamerbinding transcription factor 4

Keywords: morphine, opioid receptors, mouse embryonic stem cells, neuronal differentiation

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1. Introduction Morphine is a potent analgesic drug that has been widely used in the management of clinical pain. However, its use for chronic pain conditions is limited by the development of tolerance, withdrawal, sensitization, and addiction (Hyman et al., 2001). The opioid exerts its pharmacological effect mainly through the activation of -opioid receptor (MOR), which is a member of the G protein-coupled receptor superfamily (Kieffer, 1995). After binding with the agonist, the receptor activates intracellular signaling through inhibitory G proteins, which affect several downstream signaling pathways, including inhibition of adenyl cyclase and voltage-gated calcium channels and activation of receptor-operated potassium channels (Law et al., 2000). The three major classes of opioid receptors, MOR, KOR (κ-opioid receptor), and DOR (δ-opioid receptor), have been characterized in mouse brain (Yoburn et al., 1993; Zhu et al., 1998). Additionally, expression of opioid receptors has been detected during early embryonic stages. MOR and KOR have been shown to be expressed in pluripotent stem cells and in differentiated neuronal progenitor cells (Kim et al., 2006). Although it is known that MOR is widely distributed throughout the nervous system during development, little is known about its involvement in neurogenesis. Embryonic stem cells differentiate into neuronal cells and thus represent an ideal model for studying the effect of exogenous stimuli during neuronal development. In vitro neuronal differentiation of embryonic stem cells has been accomplished using various protocols. One well-studied and powerful approach is the formation of EB cells in a suspension culture followed by culture in defined medium conditions (Doetschman et al., 1985; Okabe et al., 1996; Guan et al., 2001; Wobus et al., 2001). EB cells are three-dimensional structures similar to mouse embryo blastocysts and represent a valid in vitro model system to study early lineage specification and further organogenesis in mammals (Leahy et al., 1999). EB cells cultured in the presence of serum free conditions containing mitogens differentiate into nestin-positive neuronal progenitor (NP) cells. Following removal of growth factors from the medium, NP cells stop dividing and differentiate into mtap-2- and β-3 tubulin-positive neurons and GFAP-positive astrocytes (Okabe et al., 1996; Tropepe et al., 2001; Wiles and Johansson, 1999). Experimental evidence indicates that morphine negatively impacts prenatal as well as postnatal neural development in mammals. For example, morphine attenuates proliferation and neurogenesis in the adult rat hippocampus (Eisch et al., 2000). Maternal administration of 4

morphine during pregnancy causes neural tube defects (Nasiraei-Moghadama et al., 2005) and a significant reduction in neuronal packing density and cortical thickness in offspring (Sandraie et al., 2008; Seatriz and Hammer, 1993). In addition, recent studies have shown that prenatal morphine exposure impairs dendritic growth and synaptic plasticity in juvenile rats (Mei et al., 2009; Niu et al., 2009). Furthermore, chronic exposure to morphine impairs cognitive function (Yang et al., 2003; Yang et al., 2006; Gruber et al., 2007; Lu et al., 2010). Interestingly, opioidinduced alterations of hippocampal functions most likely results from neurogenesis inhibition (Eisch and Harburg, 2006). Human data reveal that infants exposed to morphine in-utero are underweight and display disturbances in the development of the central nervous system (Heshman and Stitzer, 1989; Hunt et al., 2008; Lester and Legasse, 2010). Although morphine exposure causes abnormal neurogenesis, little is known about the time course effect on neuronal inhibition as well as the molecular and cellular basis underlying its effect. In the present study, pluripotent mES cells were used as an in vitro model for neural development to determine the potential effect of early vs. late morphine sulfate treatment on neuronal differentiation as well as MOR expression.

2. Material and Methods: 2.1) Reagents Reagents were obtained as indicated: morphine sulfate (Spectrum Chemicals, Gardena, CA); naltrexone hydrochloride (Sigma-Aldrich, St. Louis, MO); primer to nestin and antibody to -3 tubulin from R&D Systems (Minneapolis, MN); primers to Oct4, GFAP, mtap-2, μ-receptor, κ-receptor, and δ-receptor from Qiagen (Valencia, CA); antibodies to nestin and GFAP from BD Bioscience (Sane Jose, CA); 1000 U/ml leukemia inhibitory factor (LIF) from Millipore (Temecula, CA); all cell culture reagents are from either Sigma-Aldrich (St. Lois, MO) or Invitrogen (Grand Island, NY).

2.2) mES cell culture and neuronal differentiation Mouse ES cells (ES-D3, ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 2 mM L-alanine-L-glutamine, 1000 U/ml leukemia 5

inhibitory factor (LIF), 3.5 g/L sodium bicarbonate, 4.5 g/L glucose, 0.1 mM 2-mercaptoethanol, 0.1% non-essential amino acids, and 15% fetal bovine serum (FBS) on collagen I-coated flasks as previously described (Barile, 2013; Calabro et al., 2008). Neuronal induction of mES cells was accomplished using the protocol established by Okabe et al. (1996) with some modification. The details of the protocol for differentiation of mES cells to neuronal cells, including time periods of each observation and the medium used are shown in Table 1. Briefly, EB cells were formed by suspension culture of undifferentiated mES cells using non-tissue culture-treated plates (2-2.5 x 104 cells per cm2) in DMEM containing 10% FBS without LIF for 4 days. On day 4, EB cells were seeded onto laminin-coated 6-well plates and cultured in DMEM containing 10% FBS without LIF overnight. On day 5, medium was replaced with serum free DMEM containing insulin-transferrin-selenium (ITS). On day 8, NP cells emanated from the periphery of EB cells, and were further proliferated by culturing in DMEM-ITS medium supplemented with 20 ng/ml of basic fibroblast growth factor (bFGF) and 20 ng/ml of epidermal growth factor (EGF) for 4 days. NP cells differentiate into neurons and astrocytes upon withdrawal of mitogens followed by additional growth in B-27 supplemented neurobasal medium for 7 days.

2.3) Morphine Sulfate exposures MS (1, 10, and 100 M) was included in the culture medium during either early stage (Stage 1, 4 days starting at day 0) or late stage (Stage 3, 7 days starting at day 12) of the neuronal differentiation protocol. Medium containing morphine was replenished every second day.

2.4) mRNA isolation and RT-PCR analysis Total RNA was isolated from cells at different stages of differentiation using RNeasy mini kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. Total RNA was treated with RNase-free DNase, quantified using Synergy H1 hybrid multi-mode microplate reader (Biotek, Winooski, VT), and subjected to RT-PCR using OneStep RT-PCR Kit (Qiagen, Valencia, CA). The PCR reaction was performed with a thermal cycler with the following 6

conditions: one cycle of reverse transcription at 50°C for 30 minutes; one cycle of DNA polymerase activation at 94°C for 15 minutes; 35 cycles of denaturing at 94°C for 45 seconds, annealing at 55°C for 45 seconds, and elongation at 72°C for 1 minute; and then one cycle of final extension at 72°C for 10 minutes. The amplified DNA fragments were quantified using Synergy H1 hybrid multi-mode microplate reader, and electrophoresed on 2% agarose gel containing 0.5 μg/ml ethidium bromide (50v X 120 minutes) (Dholakiya and Benzeroual, 2011). Bands were quantified by measuring integrated optical density using NIH ImageJ software. The corrected relative OD values for the bands of interest were calculated against the 18s rRNA, which was used as an internal standard control. 2.5) Characterization of cell specific markers using flow cytometry analysis Cells at various stages of differentiation were made into a single cell suspension by dissociation with 0.25% trypsin and aliquoted in samples of 1 x 106 cells. Cells were fixed with 4% paraformaldehyde for 15 minutes and then permeabilized by slowly adding ice-cold Perm Buffer III (BD Bioscience, San Jose, CA) followed by incubation on ice for 30 minutes. Cells were then incubated with fluorochrome-conjugated antibody (10 L of mouse nestin phycoerythrin monoclonal antibody, 10 L of -3 tubulin APC-conjugated antibody, or 5 L of alexa fluor 488 mouse anti-GFAP antibody) for 1 hour in the dark at room temperature. Cells were then centrifuged and washed twice with PBS. Negative or isotype controls were run simultaneously with each antibody. Acquisition of 30,000 events was performed using BD Accuri C6 flow cytometer and analyzed with CFlow software (BD Biosciences, San Jose, CA). 2.6) Statistical analysis Data are expressed as mean SEM. For comparison of mean of multiple groups, analysis of variance was used followed by Tukey’s post hoc test. All statistical analyses were performed using Sigmaplot Software (v.11). Statistical significance level was set as P<0.05. 3. Results 3.1) Characterization of mES cells, NP cells, and neurons/astrocytes co-cultured cells Phase contrast micrographs of EB cells, NP cells, and neurons/astrocytes co-cultured cells are shown in Figure 1. On day 8, NP cells emanate from the periphery of EB cells (Figure 7

1b, day 8). Further proliferation of NP cells in the presence of EGF and bFGF shows rosette structural organization (Figure 1c, day 12), which is similar to neural tube formation in vivo (Tomooka et al., 1993). Upon withdrawal of bFGF and EGF and subsequent addition of B-27 supplemented neurobasal medium, NP cells differentiate into neuronal and astroglial cells (Figure 1d, day 20). Cells at each stage of differentiation were further characterized by semi-quantitative RTPCR and flow cytometry analyses. Cells were subjected to RT-PCR for expression of mES cell-, NP cell-, and neuron-, and astrocyte-specific gene markers on days 1, 8, and 20. As shown in Figure 2a, mES cell pluripotency marker Oct4 was expressed in mES cells, but not in either NP cells or differentiated neurons/astrocytes co-cultured cells. Similarly, nestin was expressed in NP cells, only; while, mtap-2 and GFAP were expressed in neurons/astrocytes co-cultured cells, only. The expression of cell specific protein at each stage of neuronal differentiation was detected by flow cytometry analysis. As shown in Figure 2b, histograms show the protein expression of nestin in NP cells and -3 tubulin and GFAP in neurons/astrocytes co-cultured cells. Each histogram also included the appropriate negative control or isotype control for each cell specific marker to account for background or non-specific fluorescence. MOR expression in mES cells was also determined using immunocytochemistry (Supplementary Figure 1). Furthermore, in order to investigate what types of neuronal cell types are present in the differentiated cultures, mRNA expression of neuronal lineage specific markers, such as choline acetyl transferase (Chat), glutamic acid decarboxylase 1 (Gad1), and tyrosine hydroxylase (Th), was assessed in neurons/astrocytes co-cultured cells (Figure 2c). The results show the presence of all three neuronal lineage specific markers, suggesting that differentiated cells are a mixture of midbrain and forebrain neuronal cells. 3.2) Opioid receptor gene expression in mES cells, NP cells, and in neurons/astrocytes cocultured cells The three major classes of opioid receptors, MOR, KOR, and DOR, have been characterized in the mouse brain (Zhu et al., 1998). Expression of all three opioid receptors in p19 mouse embryonal carcinoma cells has also been reported (Chen et al., 1999), while the expression of MOR and KOR has been demonstrated in mES cells and in mES derived NP cells 8

(Kim et al., 2006). In addition, both DOR and MOR subtypes are expressed strongly in the subventricular zones during gestational weeks 11 to 16 followed by decreased expression by week 20 (Tripathi et al., 2008). This current study shows that all three genes (MOR, KOR, and DOR) are expressed in mES cells, NP cells, and neurons/astrocytes co-cultured cells (Figure 3a). The specificity of MOR primer was confirmed by detecting the MOR mRNA expression in rat heart and rat spleen tissue (Figure 3b). Moreover, morphine sulfate has also been shown to inhibit forskolin-stimulated cAMP production in mES cells (Supplementary Figure 2). In addition, these results suggest that MOR, KOR, and DOR genes are constitutively expressed in mES cells and then decrease in expression during the early phase of neuronal differentiation followed by their subsequent increase in terminally differentiated neuronal cells. The presence of opioid receptors at each stage of differentiation further confirms that mES cells are a valid in vitro model system to study the effect of exogenous stimuli on the regulation of opioid receptors at the cellular and molecular levels during neuronal differentiation. 3.3) Effect of early phase morphine treatment on nestin and MOR gene expression in EB cells Nestin, a type VI intermediate filament protein, is a marker for neuroectoderm and is dramatically down-regulated when neural progenitor cells differentiate and become postmitotic (Wiese et al., 2004). Nestin mRNA expression has been reported during the first four days of mouse EB cell formation by using a suspension culture method (Mogi et al., 2009). In this study, EB cells were formed in the presence of morphine sulfate and RT-PCR analysis was performed to assess the effect of morphine on nestin and MOR gene expression. RT-PCR results show that morphine sulfate causes significant down-regulation of both nestin and MOR gene expression after treatment with morphine sulfate during EB cells formation compared to control cells (Figure 4a and 4b, respectively). DNA band intensity is presented as percentage of the relative ratio of nestin (Figure 4c) and MOR (Figure 4d) mRNA to 18s rRNA. Relative to the percentage of control MOR mRNA level, the MOR mRNA levels of 1 μM, 10 μM, and 100 μM morphine sulfate treatment were down-regulated to 51.3 ± 10.1%, 33.9 ± 7.6%, and 19.7 ± 5.4%, respectively (P<0.05). This suggests a concentration dependent decreased in MOR mRNA expression. Similarly, the nestin mRNA levels of 1 μM, 10 μM, and 9

100 μM MS treatment were decreased to 61.6 ± 3.8%, 63.4 ± 7.9%, and 43.2 ± 7.6%, respectively (P<0.05), relative to nestin mRNA level of control. Using flow cytometry analysis, we further confirmed that nestin protein expression was also significantly decreased in morphine-treated EB cells compared with control EB cells (Figure 4e). Mean fluorescence intensity (MFI) of control cells was set at 100% and the fraction of signal remaining after 1 μM, 10 μM, and 100 μM morphine treatment was calculated. The results show 60.9 ± 7.2%, 52.5 ± 7.1%, and 63.2 ± 8.6% of nestin protein expression in 1 μM, 10 μM, and 100 μM morphine sulfate-treated EB cells, respectively, compared to control EB cells (P<0.05). 3.4) Effect of early phase morphine treatment on nestin and MOR gene expression in NP cells We next examined the impact of morphine treatment on NP cell differentiation (Stage 2) by treating EB cells. The expression level of nestin was significantly decreased in a concentration dependent manner in NP cells as compared to control (Figure 5a). In concordance with the decrease in nestin mRNA expression, NP cells also had significantly decreased MOR mRNA expression in a concentration dependent manner (Figure 5b). DNA band intensity is presented as percentage of the relative ratio of nestin (Figure 5c) and MOR (Figure 5d) mRNA to 18s rRNA. There was a significant decrease in nestin protein expression observed using flow cytometry analysis (Figure 5e). Relative to the percentage of control nestin mRNA level, the nestin mRNA levels of 1 μM, 10 μM, and 100 μM morphine sulfate treatment were down-regulated to 68.1 ± 8.4%, 55.9 ± 12.7%, and 61.2 ± 5.8%, respectively (P<0.05). Similarly, relative to the percentage of control MOR mRNA level, the MOR mRNA levels of 1 μM, 10 μM, and 100 μM morphine sulfate treatment were decreased to 53 ± 4.4%, 65.8 ± 3.3%, and 73.9 ± 6.8%, respectively (P<0.05).

3.5) Effect of early phase morphine treatment on GFAP expression in differentiated neurons/astrocytes co-cultured cells A decrease in nestin, a neuroectodemal intermediate filament protein, is observed in both EB and NP cells after morphine treatment during EB cell formation; therefore, we investigated 10

whether this early stage treatment was also involved in the inhibition of NP cell differentiation into neurons/astrocytes co-cultured cells. Our RT-PCR results show that there was no significant difference in mtap-2 expression between the control and morphine treated cells (data not shown). However, the expression level of GFAP is significantly decreased in morphine-treated neurons/astrocyte co-cultured cells in comparison to that of control neurons/astrocytes cocultured cells (Figure 6b). We found a 69.4 ± 3.9%, 45.6 ± 2.2% and 37.7 ± 3.9% of GFAP mRNA expression in 1 μM, 10 μM and 100 μM morphine sulfate-treated cells, respectively, compared to control neurons/astrocytes co-cultured cells (P<0.05). In addition, despite the downregulation of MOR gene expression in EB and NP cells after morphine treatment during EB cell formation, our results show no significant change in MOR gene expression in neurons/astrocytes co-cultured cells (Supplementary Figure 3).

3.6) Effect of late phase morphine treatment on GFAP, mtap-2, and β-3 tubulin expression in differentiated neurons/astrocytes co-cultured cells In order to characterize the effect of morphine on neuronal differentiation of NP cells, we performed RT-PCR analysis for neuronal marker mtap-2 and astrocyte marker GFAP. Under neuronal differentiating conditions, we evaluated the effect of NP cell treatment with morphine (1, 10, and 100 μM). As shown in Figure 7a and 7b, morphine significantly decreased the percentage of both GFAP- and mtap-2-positive astrocytes and neurons in a concentrationdependent manner. Results showed 86 ± 3.7%, 78 ± 3.3% and 64.3 ± 2.7% of mtap-2 mRNA expression in 1 μM, 10 μM and 100 μM morphine sulfate-treated cells, respectively, compared to control neurons/astrocytes co-cultured cells (P<0.05). Moreover, the mRNA level of GFAP was decrease to 68.8 ± 4.6% at 100 μM (P<0.05). No significant difference was observed at 1 μM and 10 μM in GFAP mRNA levels. To further investigate the negative effect of morphine on neuronal differentiation of NP cells, cells were differentiated in vitro in the presence of vehicle, 100 μM morphine, 100 μM naltrexone, or both morphine and naltrexone. Thereafter, flow cytometry analysis was performed using dual staining with antibodies for glial cell-specific protein GFAP and neuron-specific protein β-3 tubulin. 11

As shown in Figure 8b, we found 76.6% of both β-3 tubulin- and GFAP-positive neurons/astrocytes co-cultured cells in control vehicle treatment. Morphine negatively regulated neuronal differentiation since it significantly decreased both GFAP- and β-3 tubulin-positive cells to 51.2% compared with vehicle treatment (Figure 8c). Importantly, naltrexone alone had no effect on neuronal differentiation (Figure 8d); however, it prevented the adverse effect of morphine on neuronal differentiation (Figure 8e). Overall, these data suggest that morphine inhibits neuronal differentiation via MOR activation. 4. Discussion In the past few decades, the prevalence of opioid use among women of reproductive age is associated with an increased risk of adverse outcomes (Ailes et al., 2015). The occurrence of anomalies of the central nervous system often results from exposure of opioids during the first trimester of pregnancy, which is a critical period for organogenesis and the formation of the neural tube in the early embryo. Yazdy et al. (2013) has reported that opioid intoxication in the periconceptional period is associated with increased risk of neural tube defects. Neonatal abstinence syndrome and psychomotor and cognitive impairment has also been reported in infants exposed to opioids in utero (Hunt et al., 2008; Patrick et al., 2012). Neurogenesis occurs throughout life in rodents, primates, and humans and it is crucial in the regulation of early development and in the late maintenance and homeostasis of the brain (Altman and Das, 1965; Kaplan and Hinds, 1977; Gage, 2000). Early phase of neurogenesis includes neural stem cell differentiation and proliferation, as well as neuronal vs. glial fate commitment and migration; whereas, late phase of neurogenesis involves morphological and synaptic development and survival of newly formed neurons (Pathania et al., 2010). However, limited focus has been placed on understanding whether early exposure to opioids has bearing on neuronal cell development. Understanding the molecular and cellular basis of opioids on neuronal development is crucial in pregnant and pediatric populations where opioids are regularly prescribed for analgesia. The present study investigates the impact of early vs. late morphine sulfate treatment on neuronal differentiation as well as MOR expression in mES cells. Early stage treatment with morphine impairs differentiation of mES cells to neural progenitor cells, which is shown by down-regulation of nestin expression in both EB and NP 12

cells. Furthermore, early stage treatment also impairs differentiation of NP cells into astrocytes, which is shown by a decrease in expression of GFAP in neurons/astrocytes co-cultured cells. Hahn et al. (2010) have shown that MOR and KOR agonists inhibit differentiation of neural progenitor cells to glial cells via extracellular-signal regulated kinase (ERK) pathway. In addition, studies in mature astrocytes suggest that endogenous opioids (Stiene-Martin and Hauser, 1990), as well as morphine (Hauser and Stiene Martin, 1991; Stiene Martin et al., 1991), alter astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Hauser et al. (1996) demonstrate that morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca2+-dependent mechanism in mature astrocytes (Hauser et al., 1996). MOR manages a key role in opioid mediated motivational and rewarding behavior in various brain regions. MOR knockout mice models have shown the absence of pharmacological effects of morphine, suggesting that the activities of morphine depend on MOR (Loh et al., 1998). Our data suggest that opioid receptors are present in the three cell types, including stem, NP, and neuronal cells. In addition, the results demonstrate down-regulation of MOR in both EB and NP cells following treatment with morphine at early stage of differentiation. MOR activity is regulated at different levels, including transcriptional (Wei and Loh, 2011), post-transcriptional (Kim et al., 2008), translational (Song et al., 2009), and epigenetic (Hwang et al., 2009). In order to investigate the down-regulation of MOR at transcriptional levels after morphine treatment, we measured MOR mRNA expression in both EB and NP cells. MOR mRNA levels are significantly decreased in both cell types, indicating that receptor regulation at the transcriptional level is involved in the mechanisms for down-regulation of MOR following morphine treatment. These results are consistent with those reported by Yu et al. (2003), suggesting that MOR downregulation results from either increased receptor degradation or decreased receptor synthesis. Morphine treatment during late neuronal development inhibits the differentiation of NP cells to both neurons and astrocytes, as indicated by the down-regulation of both mtap-2 and GFAP mRNA expression. We further confirmed the down-regulation of β-3 tubulin and GFAP protein expression in differentiated neurons/astrocytes co-cultured cells after treatment of NP cells. In addition, co-treatment with naltrxone, an opioid receptor antagonist, in the presence of morphine completely blocks the down-regulation of β-3 tubulin and GFAP protein, indicating 13

that morphine’s effect on neuronal inhibition is receptor mediated. However, RT-PCR results do not show change in MOR mRNA expression in neurons/astrocytes co-cultured cells during late stage of neuronal differentiation. This indicates that the opioid mediates distinct molecular events that are involved in the regulation of MOR at early stage and late stage of neuronal differentiation. Eisch et al. (2000), Kahn et al. (2005), and Arguello et al. (2008 and 2009) have shown that administration of opioids inhibits proliferation of neural progenitor cell populations in the subgranular zone, as well as alters the progenitor cell cycle. Also, Hahn et al. (2010) have shown that activation of opioid receptors blocks the differentiation of NP cells to both astrocytes and neurons via ERK signaling and p38 signaling pathways, respectively. A deficit in circulation of stem/progenitor cells and neural stem/progenitor cells was reported in opioid-dependent patients and chronic opioid treated animals (Arguello et al., 2009; Eisch et al., 2000; Reece and Davidson, 2007; Seidler et al., 1982). Taken together, our findings emphasize that morphine has distinct effects on different stages of stem cell/neuronal differentiation. Persistence of neurogenesis occurs throughout adult mammalian life, and it is crucial for neural tube formation during the early embryonic period and for learning and memory formation in the adult brain. In addition, alteration in neurogenesis is associated with neurobehavioral effects and has relevance for the development of neurological and psychological impairments observed in infants born to mothers addicted to opioids. However, the effects of opioids on brain development are complex and also influenced by duration, dose, route, and timing of opioid exposure (early stage vs. late stage of neuronal development). Future work could address this hypothesis by evaluating potential long-term effects of neurobehavioral alterations in the infants after prenatal morphine exposure.

5. References: Ailes, E.C., Dawson, A.L., Lind, J.N., Gilboa, S.M., Frey, M.T., Broussard, C.S., and Honein, M.A. (2015). Opioid prescription claims among women of reproductive age -- United States, 2008-2012. Morbidity and Mortality Weekly Report 64: 37-41. 14

Arguello, A.A., Fischer, S.J., Schonborn, J.R., Markus, R.W., Brekken, R.A., and Eisch, A.J. (2009). Effect of chronic morphine on the dentate gyrus neurogenic microenvironment. Neuroscience 159: 1003-1010. Altman, J., and Das, G. D. (1965). Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. Journal of Comparative Neurology 124: 319-335. Arguello, A.A., Harburg, G.C., Schonborn, J.R., Mandyam, C.D., Yamaguchi, M., and Eisch, A.J. (2008). Time course of morphine’s effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons. Neuroscience 157: 70-79. Barile, F.A. (2013). Principles of Toxicology testing, 2nd edition. CRC press: Boca Raton, FL.ISBN 9781842145289. Calabro, A.R., Konsoula, R., and Barile, F.A. (2008). Evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse embryonic stem cells. Toxicology in Vitro 22: 1273-1284. Chen, H.C., Wei, L.N., and Loh, H.H. (1999). Expression of μ-, κ- and δ-opioid receptors in P19 mouse embryonal carcinoma cells. Neuroscience 92: 1143-1155. Dholakiya, S.L., and Benzeroual, K.E. (2011). Protective effect of diosmin on LPS-induced apoptosis in PC12 cells and inhibition of TNF-alpha expression. Toxicology in Vitro 25: 10391044. Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. (1985). The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. Journal of Embryology and Experimental Morphology 87: 2745. Eisch A.J., Barrot, M., Schad, C.A., Self, D.W., and Nestler, E.J. (2000). Opiates inhibit neurogenesis in the adult rat hippocampus. Proc Natl Acad Sci USA 97: 7579-7584. Eisch, A.J., and Harburg G.C. (2006). Opiates, psychostimulants, and adult hippocampal neurogenesis: Insights for addiction and stem cell biology. Hippocampus 16: 271-286. 15

Gage, F.H. (2000). Mammalian neural stem cells. Science 287: 1433-1438. Gruber, S.A., Silveri, M.M., and Yurgelun-Todd, D.A. (2007). Neuropsychological consequences of opiate use. Neuropsychology Review 17: 299-315. Guan, K., Chang, H., Rolletschek, A., and Wobus, A.M. (2001). Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells. Cell and Tissue Research 305: 171-176. Hahn, J.W., Jagwani, S., Kim, E., Rendell, V.R., He, J., Ezerskiy, L.A., Wesselschmidt, R., Coscia, C.J., and Belcheva, M.M. (2010). Mu and kappa opioids modulate mouse embryonic stem cell-derived neural progenitor differentiation via MAP kinases. Journal of Neurochemistry 112: 1431-1441. Hauser, K.F., and Stiene-Martin, A. (1991). Characterization of opioid-dependent glial development in dissociated and organotypic cultures of mouse central nervous system: Critical periods and target specificity. Developmental Brain Research 62: 245-255. Hauser, K.F., Stiene-Martin, A., Mattson, M.P., Elde, R.P., Ryan, S.E., and Godleske, C.C. (1996). mu-Opioid receptor-induced Ca2 mobilization and astro- glial development: Morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca2+-dependent mechanism. Brain Research 720: 191-203. Heshman, S.J., and Stitzer, M.L. (1989). Acute opioid physical dependence in humans: Effect of varying the morphine naloxone interval. Journal of Pharmacology and Experimental Therapeutics 250: 485-491. Hwang, C.K., Song, K.Y., Kim, C.S., Choi, H.S., Guo, X.H., Law, P.Y., Wei, L.N., and Loh, H.H. (2009). Epigenetic programming of mu opioid receptor gene in mouse brain is regulated by MeCP2 and Brg1 chromatin remodeling factor. Journal of Cellular and Molecular Medicine 13: 3591-3615 Hunt, R.W., Tzioumi, D., Collins, E., and Jeffery, H.E. (2008). Adverse neurodevelopmental outcome of infants exposed to opiate in-utero. Early Human Development 84: 29-35.

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Hyman, S.E. and Malenka, R.C. (2001). Addiction and the brain: the neurobiology of compulsion and its persistence. Nature Reviews Neuroscience. 2: 695-703. Kahn, L., Alonso, G., Normand, E., and Manzoni, O.J. (2005). Repeated morphine treatment alters polysialylated neural cell adhesion mole- cule, glutamate decarboxylase-67 expression and cell proliferation in the adult rat hippocampus. European Journal of Neuroscience 21: 493-500. Kaplan, M.S., and Hinds, J.W. (1977). Neurogenesis in the adult rat: Electron microscopic analysis of light radioautographs. Science 197: 1092-1094. Kieffer, BL. (1995). Recent advances in molecular recognition and signal transduction of active peptides: receptors for opioid peptides. Cellular and Molecular Neurobiology 15: 615-635. Kim, C.S., Hwang, C.K., Song, K.Y., Choi, H.S., Kim do, K., Law, P.Y., Wei, L.N., and Loh, H.H. (2008). Novel function of neuron-restrictive silencer factor (NRSF) for posttranscriptional regulation. Biochimica et Biophysica Acta 1783: 1835-1846 Kim, E., Clark, A.L., Kiss, A., Hahn, J.W., Wesselschmidt, R., Coscia, C.J., and Belcheva, M.M. (2006). Mu- and kappa-opioids induce the differentiation of embryonic stem cells to neural progenitors. The Journal of Biological Chemistry 281: 33749-33760. Law, P.Y., Wong, Y.H., and Loh, H.H. (2000). Molecular mechanisms and regulation of opioid receptor signaling. Annual Review of Pharmacology and Toxicology 40: 389-430. Leahy, A., Xiong, J.W., Kuhnert, F., and Stuhlmann, H. (1999). Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. Journal of Experimental Zoology 284: 67-81. Lester, B.M., and Lagasse L.L. (2010). Children of addicted women. Journal of Addictive Disease 29: 259-276. Loh, H.H., Liu, H.C., Cavalli, A., Yang, W., Chen, Y.F., and Wei, L.N. (1998). mu Opioid receptor knockout in mice: effects on ligand-induced analgesia and morphine lethality. Molecular Brain Research 54: 321-326.

17

Loh, H.H. (2008). Novel function of neuron-restrictive silencer factor (NRSF) for posttranscriptional regulation. Biochimica et Biophysica Acta 1783: 1835-1846 Lu, G., Zhou, Q.X., Kang, S., Li, Q.L., Zhao, L.C., Chen, J.D., Sun, J.F., Cao, J., Wang, Y.J., Chen, J., Chen, X.Y., Zhong, D.F., Chi, Z.Q., Xu, L., and Liu, J.G. (2010). Chronic morphine treatment impaired hippocampal long-term potentiation and spatial memory via accumulation of extracellular adenosine acting on adenosine A1 receptors. The Journal of Neuroscience 30: 5058-5070. Mandyam, C.D., Norris, R.D., and Eisch, A.J. (2004). Chronic morphine induces premature mitosis of proliferating cells in the adult mouse subgranular zone. Journal of Neuroscience Research 76: 783-794. Mandyam, C.D., Harburg, G.C., and Eisch, A.J. (2007). Determination of key aspects of precursor cell proliferation, cell cycle length and kinetics in the adult mouse subgranular zone. Neuroscience 146:108-122. Mei, B., Niu, L., Cao, B., Huang, D., and Zhou, Y. (2009). Prenatal morphine exposure alters the layer II/III pyramidal neurons morphology in lateral secondary visual cortex of juvenile rats. Synapse 63: 1154-1161. Mogi, A., Ichikawa, H., Matsumoto, C., Hieda, T., Tomotsune, D., Sakaki, S., Yamada, S., and Sasaki, K. (2009.) The method of mouse embryoid body establishment affects structure and developmental gene expression. Tissue and Cell 41: 79-84. Nasiraei-Moghadam, S., Hedayat, S., Bahadoran, H., Sadooghi, M., Salimi, S.H., Kaka, G.R., Imani, H., Mahdavi-Nasab, H., and Dashtnavard, H. (2005). Effects of maternal oral morphine consumption on neural tube development in Wistar rats. Developmental Brain Research 159: 12-17. Niu, L., Cao, B., Zhu, H., Mei, B., Wang, M., Yang, Y., and Zhou, Y. (2009). Impaired in vivo synaptic plasticity in dentate gyrus and spatial memory in juvenile rats induced by prenatal morphine exposure. Hippocampus 19: 649-657.

18

Okabe, S., Forsberg-Nilsson, K., Spiro, A., Segal, M., and McKay, R. (1996). Development of neuronal precursor cells and functional post- mitotic neurons form embryonic stem cells in vitro. Mechanisms of Development 59: 89-102. Pathania, M., Yan, L.D., and Bordey, A. (2010). A symphony of signals conducts early and late stages of adult neurogenesis. Neuropharmacology 58: 865-876. Patrick, S.W., Schumacher, R.E., Benneyworth, B.D., Krans, E.E., McAllister, J.M., and Davis, M.M. (2009). Neonatal abstinence syndrome and associated health care expenditures: United States, 2000-2009. Journal of the American Medical Association 307: 1934-40 Reece, A.S., and Davidson, P. (2007). Deficit of circulating stem-progenitor cells in opiate addiction: a pilot study. Substance Abuse Treatment, Prevention, and Policy 2: 19. Sadraie, S.H., Kaka, G.R., Sahraei, H., Dashtnavard, H., Bahadoran, H., Mofid, M., Nasab, H.M., and Jafari, F. (2008). Effects of maternal oral administration of morphine sulfate on developing rat fetal cerebrum: A morphometrical evaluation. Brain Research 1245: 36-40 Seatriz J.V., and Hammer R.P. (1993). Effects of opiates on neuronal development in the rat cerebral cortex. Brain Research Bulletin 30: 523-527. Seidler, F.J., Whitmore, W.L., and Slotkin, T.A. (1982). Delays in growth and biochemical development of rat brain caused by maternal methadone administration: are there alterations in synaptogenesis and cellular maturation independent of reduced maternal food intake? Developmental Neuroscience 5: 13-18. Song, K.Y., Choi, H.S., Hwang, C.K., Kim, C.S., Law, P.Y., Wei, L.N., and Loh, H.H. (2009). Differential use of an in-frame translation initiation codon regulates human mu-opioid receptor (OPRM1). Cellular and Molecular Life Sciences 66: 2933-2942. Stiene-Martin, A. and Hauser, K.F. (1990). Opioid-dependent growth of glial cultures: Suppression of astrocyte DNA synthesis by met-enkephalin. Life Sciences 46: 91-98. Stiene-Martin, A., Gurwell, J.A., and Hauser, K.F. (1991). Morphine alters astrocyte growth in primary cultures of mouse glial cells: evidence for a direct effect of opiates on neural maturation. Developmental Brain Research 60: 1-7. 19

Tomooka, Y., Kitani, H., Jing, N., Matsubara, M., and Sakakura, T. (1993). Reconstruction of neural tube-like structures in vitro from primary neural precursor cells. Proc Natl Acad Sci USA 90: 9683-9687. Tripathi, A., Khurshid, N., Kumar, P., and Iyengar, S. (2008). Expression of delta- and muopioid receptors in the ventricular and subventricular zones of the developing human neocortex. Neuroscience Research 61: 257-270. Tropepe, V., Hitoshi, S., Sirard, C., Mak, T.W., Rossant, J., van der Kooy, D. (2001). Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 30: 65-78. Wei, L.N., and Loh, H.H. (2011). Transcriptional and epigenetic regulation of opioid receptor genes: present and future. Annual Review of Pharmacology and Toxicology 51: 75-97. Wiese, C., Rolletschek, A., and Kania, G., Blyszczuk, P., Tarasov, K.V., Tarasova, Y., Wersto, R.P., Boheler, K.R., and Wobus, A.M. (2004). Nestin expression--a property of multi-lineage progenitor cells? Cellular and Molecular Life Sciences 61: 2510-2522. Wiles, M.V., and Johansson, B.M. (1999). Embryonic stem cell development in a chemically defined medium. Experimental Cell Research 247: 241-248. Wobus, A.M., Guan, K., and Pich, U. (2001). In vitro differentiation of embryonic stem cells and analysis of cellular phenotypes. Methods in Molecular Biology 158: 263-286. Yang, S.N., Huang, L., Wang, C., Chen, W., Yang, C., Lin, S., Lai, M., Chen, S., and Tao, P. (2003). Prenatal administration of morphine decreases CREBSerine-133 phosphorylation and synaptic plasticity range mediated by glutamatergic transmission in the hippocampal CA1 area of cognitive-deficient rat offspring. Hippocampus 13: 915-921. Yang, S.N., Liu, C.A., Chung, M.Y., Huang, H.C., Yeh, G.C., Wong, C.S., Lin, W.W., Yang, C.H., and Tao, P.L. (2006). Alterations of postsynaptic density proteins in the hippocampus of rat offspring from the morphine-addicted mother: Beneficial effect of dextromethorphan. Hippocampus 16: 521-530.

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Yazdy, M.M., Mitchell, A.A., Tinker, S.C., Parker, S.E., and Werler, M.M. (2013). Periconceptional use of opioids and the risk of neural tube defects. Obstetrics and Gynecology 122: 838-44. Yoburn, B.C., Billings, B., and Duttaroy, A. (1993). Opioid receptor regulation in mice. Journal of Pharmacology and Experimental Therapeutics 265: 314-320. Yu, X., Mao, X., Blake, A.D., Li, W.X., and Chang, S.L. (2003). Morphine and endomorphins differentially regulate μ-opioid receptor mRNA in SHSY-5Y human neuroblastoma cells. Journal of Pharmacology and Experimental Therapeutics 306: 447-454. Zhu, Y., Hsu, M.S., and Pintar, J.E. (1998). Developmental expression of the mu, kappa, and delta opioid receptor mRNAs in mouse. Journal of Neuroscience 18: 2538-2549.

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Figure 1. Differentiation of mES cells into neuronal cells. (a) mES cells form EB cells in a suspension culture. (b) EB cells cultured in serum free medium for 4 days show NP cell differentiation at periphery. (c) By 12 days, NP cells proliferating in the presence of bFGF and EGF show radial rosette-like structure. (d) Further culture of NP cells in the presence of neurobasal medium supplemented with B-27 for 1 week results in differentiation into neurons/astrocytes co-cultured cells. Magnification: 40X (a) and 100X (b, c, and d). Data are representative of at least three independent experiments.

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Figure 2. Expression of cell specific markers at different stages of neuronal differentiation. (a) At day 1, 8, and 20, mRNA was analyzed by RT-PCR for expression of mES cell-, NP cell-, neuron-, and glial cell-specific gene markers in mES, NP, and neurons/astrocytes co-cultured cells: mES cell pluripotency marker Oct4 (lane 1), NP cell specific marker nestin (lane 2), astrocyte specific marker GFAP (lane 3), and neuron specific marker mtap-2 (lane 4). (b) Flow cytometry analysis of nestin protein expression in NP cells, -3 tubulin protein expression in neurons, and GFAP protein expression in astrocytes. (c) mRNA expression of neuronal lineage specific markers (Chat, Gad1, Th) in neurons/astrocytes co-cultured cells. Data are representative of at least three independent experiments. Abbreviation: Chat, choline acetyl transferase; Gad1, glutamic acid decarboxylase 1; GFAP, glialfabirillary acidic protein; mtap-2, microtubule associated protein 2; Oct4, octamer-binding transcription factor 4; Th, tyrosine hydroxylase.

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Figure 3. RT-PCR analysis of μ-, κ-, and δ-opioid receptor mRNA expression. (a) μ(lane 1), κ- (lane 2), and δ- (lane 3) opioid receptor mRNA expression in mES cells, NP cells, and neurons/astrocytes co-cultured cells. (b) μ-opioid receptor mRNA expression in rat heart and spleen tissue.

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Figure 4. Down-regulation of nestin and MOR mRNA expression in EB cells by morphine sulfate. mES cells were was treated for 4 days with morphine sulfate (0, 1, 10, and 100 μM) (Stage 1). EB cells were collected at day 4 of the differentiated protocol and total RNA was isolated and subjected to RT-PCR for the measurement of nestin, MOR, and 18s rRNA mRNA expression. Representative gels showing nestin expression (a) and MOR expression (b). Analyses of band intensity on gels are presented as percentage of the relative ratio of nestin (c) and MOR (d) mRNA to 18s rRNA. (e) Flow cytometry expression of nestin protein expression in control- and morphine sulfate-treated EB cells. Data presented are mean  S.E.M of three independent experiments. *P<0.05 compared with control (one-way ANOVA and Tukey’s post hoc test).

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Figure 5. Down-regulation of nestin and MOR mRNA expression in NP cells by morphine sulfate. mES cells were treated for 4 days with morphine sulfate (0, 1, 10, and 100 μM) (Stage 1). NP cells were collected at day 12 of the differentiation protocol and total RNA was isolated and subjected to RT-PCR for the measurement of nestin, MOR, and 18s rRNA mRNA expression. Representative gels showing nestin expression (a) and MOR expression (b). Analyses of band intensity on gels are presented as percentage of the relative ratio of nestin (c) and MOR (d) mRNA to 18s rRNA. (e) Flow cytometry expression of nestin in control and NP cells differentiated from morphine sulfate-treated EB cells. Data presented are mean ± S.E.M of three independent experiments. *P<0.05 compared with control (one-way ANOVA and Tukey’s post hoc test).

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Figure 6. Early stage effect of morphine sulfate on differentiation of NP cells into neurons and astrocytes. mES cells were was treated for 4 days with morphine sulfate (0, 1, 10, and 100 μM) (Stage 1). Neurons/astrocytes co-cultured cells were collected at day 20 of the differentiation protocol and total RNA was isolated and subjected to RT-PCR for the measurement of GFAP and 18s rRNA mRNA expression. (a) Representative gel showing GFAP expression in neurons/astrocytes co-cultured cells. (b) Relative intensity of individual bands from gel was quantified using ImageJ software. Levels of GFAP mRNA expression were normalized to 18s rRNA and expressed as percentage relative to control. Data presented are mean  S.E.M of three independent experiments. *P<0.05 compared with control (one-way ANOVA and Tukey’s post hoc test).

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Figure 7. Late stage effect of morphine sulfate on differentiation of NP cells into neurons and astrocytes. NP cells were differentiated into neurons/astrocytes co-cultured cells in the presence of morphine sulfate (0, 1, 10, and 100 M). Neurons/astrocytes co-cultured cells were collected at day 20 of the differentiation protocol and total RNA was isolated and subjected to RT-PCR for the measurement of mtap-2, GFAP, and 18s rRNA mRNA expression. Representative gels showing mtap-2 (a) and GFAP (b) expression in neurons and astrocytes. Relative intensity of individual bands was quantified using ImageJ software (c and d, respectively). Levels of mtap-2 and GFAP mRNA expression were normalized to 18s rRNA and expressed as percentage relative to control. Data presented are mean  S.E.M of three independent experiments. *P<0.05 compared with control (one-way ANOVA and Tukey’s post hoc test).

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Figure 8. Flow cytometry analysis of effect of morphine on differentiation of NP cells into neurons and astrocytes. Neurons/astrocytes co-cultured cells were double stained for β-3 tubulin and GFAP (b). Note the decrease of both β-3 tubulin- and GFAP-positive cells after 100 μM morphine treatment at Stage 3 of the neuronal differentiation protocol (c). Naltrexone alone had no effect on neuronal differentiation (d). Co-treatment of naltrexone plus morphine abolished the deleterious effect of morphine on neuronal differentiation (e). Percentage of expression using flow cytometry of both β-3 tubulin and GFAP in control- and morphine-treated neurons/astrocytes co-cultured cells (f). Data presented are mean  S.E.M of three independent experiments. *P<0.05 compared with control (one-way ANOVA and Tukey’s post hoc test).

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Table 1. Schematic of protocol for differentiation of mES cells into neurons/astrocyte cocultured cells. Approximate time periods of each observation and the medium used are indicated. Differentiation was induced by platting EB cells onto 6-well plates in DMEM-ITS medium containing mitogens. Medium was changed every other day. Differentiation of NP cells into neurons and astrocytes was induced by withdrawal of mitogens and in the presence of defined culture conditions. RT-PCR and flow cytometry analysis were performed at days 1, 8, and 20. Abbreviation: DMEM-ITS, Dulbecco’s Modified Eagle’s Medium supplemented with insulin, transferrin, and selenium; EB cells, embryoid body cells; mES cells, mouse embryonic stem cells; NP cells, neuronal progenitor cells.

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