Connexin-Based Channels Contribute to Metabolic Pathways in the Oligodendroglial Lineage

RESEARCH ARTICLE Connexin-based channels contribute to metabolic pathways in the oligodendroglial lineage Jianqin Niu1,*, Tao Li1,*, Chenju Yi2, Nanxin Huang1, Annette Koulakoff2, Chuanhuang Weng3, Chengren Li1, Cong-Jian Zhao3, Christian Giaume2,‡ and Lan Xiao1,‡

ABSTRACT In the present study, we considered the first step of energy Oligodendrocyte precursor cells (OPCs) undergo a series of energy- consumption: energy substrate uptake through selective consuming developmental events; however, the uptake and transporters (Hirrlinger and Nave, 2014). It is well known that trafficking pathways for their energy metabolites remain unknown. astrocytes are the major energy source for neurons because they In the present study, we found that 2-NBDG, a fluorescent glucose take up glucose through Gluts and provide the metabolite ‘ analog, can be delivered between astrocytes and oligodendrocytes lactate for neurons through the MCT-mediated astrocyte neuron ’ through connexin-based gap junction channels but cannot be lactate shuttle (Pellerin and Magistretti, 1994, 2012). Similarly, transferred between astrocytes and OPCs. Instead, connexin oligodendrocytes can absorb extracellular glucose and/or lactate hemichannel-mediated glucose uptake supports OPC proliferation, through Glut1 (also known as SLC2A1) and MCT1, respectively and ethidium bromide uptake or increase of 2-NBDG uptake rate (Hirrlinger and Nave, 2014; Morrison et al., 2013; Rinholm et al., is correlated with intracellular Ca2+ elevation in OPCs, indicating a 2011; Saab et al., 2013). By contrast, OPCs do not express MCT1 Ca2+-dependent activation of connexin hemichannels. Interestingly, (Lee et al., 2012), and there is a lack of evidence showing the deletion of connexin 43 (Cx43, also known as GJA1) in astrocytes expression of other Gluts in OPCs. Therefore, our main question is inhibits OPC proliferation by decreasing matrix glucose levels without whether OPCs can obtain energy supply through other pathways impacting on OPC hemichannel properties, a process that also such as non-selective energy uptake channels. occurs in corpus callosum from acute brain slices. Thus, dual A typical feature of glial cells is their high expression of functions of connexin-based channels contribute to glucose supply in connexins, which can form gap junctions and/or hemichannels in oligodendroglial lineage, which might pave a new way for energy- different glial cell types. For instance, connexin 43 (Cx43, also metabolism-directed oligodendroglial-targeted therapies. known as GJA1) and connexin 30 (Cx30, also known as GJB6) are mainly expressed in astrocytes (Ransom and Giaume, 2013), KEY WORDS: Oligodendroglia, Connexin hemichannel, Glucose whereas Cx47, Cx32 and Cx29 (also known as GJC2, GJB1 and uptake, Intracellular Ca2+, Glial metabolism GJC3, respectively) are present in oligodendroglial cells (Parenti et al., 2010; Theis et al., 2005). Recently, pathological changes of INTRODUCTION oligodendroglia or demyelination found in transgenic mice with Oligodendrocyte precursor cells (OPCs) undergo proliferation, different subsets of connexins (i.e. a Cx43 and Cx30 double migration and dynamic interactions with axons before myelination knockout) suggest that glial connexins might participate in the (Kang et al., 2010; Richardson et al., 2011; Rivers et al., 2008). In regulation of the myelination or remyelination processes (Li et al., addition to myelination, oligodendrocytes also act as an energy 2014; Markoullis et al., 2012a,b, 2014). In astrocytes, Cx43 gap source for axons by actively participating in monocarboxylate junction channels and hemichannels are permeable to glucose, transporter 1 (MCT1, also known as SLC16A1)-mediated lactate lactate and other metabolic substrates (Giaume et al., 2013; delivery (Funfschilling et al., 2012; Lee et al., 2012). Recently, it Ransom and Giaume, 2013). At a more integrated level, the has been found that OPCs directly promote angiogenesis to support astroglial networking mediated by gap junctions sustains neuronal the highly energy-consuming myelination process (Yuen et al., activity through the intercellular trafficking of metabolites 2014), and oligodendroglial cells take up more energy substrates (Rouach et al., 2008). Based on these findings, it has been than neurons do at least in culture (Sanchez-Abarca et al., 2001). hypothesized that gap junction communication between astrocytes Thus, oligodendroglial cells require an extraordinary metabolic and oligodendrocytes might allow oligodendrocytes to obtain demand to support their development and function (Harris and energy supplies from astrocytes (Hirrlinger and Nave, 2014; Attwell, 2012; Nave, 2010). However, the detailed energy metabolism Morrison et al., 2013). However, it is not clear whether this gap- pathways of the oligodendroglial lineage are currently unclear. junction-mediated energy substrate pathway also exists between OPCs and astrocytes. Given that connexin-based channel functions are related to the development of astrocytes and 1Department of Histology and Embryology, Faculty of Basic Medicine, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Chongqing oligodendrocytes (Tress et al., 2012; Venance et al., 1995; Von 400038, China. 2Collegè de France, Center for Interdisciplinary Research in Blankenfeld et al., 1993), it is worthwhile exploring whether Biology (CIRB)/Institut National de la Santéet de la Recherche Médicale U1050, connexin-based channels also contribute to energy uptake in Paris 75231, Cedex 05, France. 3Southwest Eye Hospital, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. OPCs. *These authors contributed equally to this work To address these questions, we took advantage of the ability of

‡ the fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3- Authors for correspondence ([email protected]; [email protected]) diazol-4-yl)amino)-deoxyglucose (2-NBDG), which can permeate connexin-mediated gap junction channels and hemichannels

Received 17 August 2015; Accepted 14 March 2016 (Retamal et al., 2007b; Rouach et al., 2008), to help analyze the Journal of Cell Science

1902 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731 glucose trafficking in oligodendroglial lineage cells. Here, we transferred between oligodendrocytes and astrocytes but not demonstrate that 2-NBDG can be taken up by OPCs through between OPCs and astrocytes. This result raised the question of connexin hemichannels in a Ca2+-dependent manner, whereas it is how glucose enters OPCs. transferred between astrocytes and oligodendrocytes through gap junction channels. We also show that connexin-hemichannel-mediated Functional hemichannels contribute to glucose analog glucose uptake supports OPC proliferation. Taken together, our uptake in oligodendroglial cells findings indicate that connexin-based channels contribute to energy As functional gap junctions between astrocytes and OPCs were not uptake pathway in oligodendroglial lineage cells. detected in the early developmental stages of oligodendroglial cells (Fig. 2) whereas Cx29 and Cx47 are already expressed, we RESULTS examined their hemichannel function by performing an ethidium Oligodendroglial cells express connexins during bromide (EtBr) uptake assay in cultured OPCs (Giaume et al., development 2012). As illustrated in Fig. 3, OPCs (PDGFRa+) exhibited Oligodendroglial cells pass through distinguished development hemichannel activity in normal culture conditions (solution with stages with specific biomarkers expression. Briefly, platelet-derived 1mMCa2+), which could be blocked by CBX and La3+ but not by growth factor a (PDGFRa) is used as an early developmental marker the Glut1 inhibitor STF31. This hemichannel activity was increased to identify OPCs. Immature oligodendrocytes are identified in Ca2+-free condition known to trigger hemichannel opening by O4 (marker specific for the oligodendroglial lineage; Sommer (Fig. 3A). The uptake assay was also performed on mature and Schachner, 1981; Bansal et al., 1989) or CNPase (also known as oligodendrocytes (CC1+) under the same condition, which CNP, 2′,3′-cyclic nucleotide 3′-phosphodiesterase), and MBP and showed that EtBr uptake activity in oligodendrocytes was less CC1 (also known as APC, adenomatous polyposis coli) are both than that in OPCs (Fig. 3A,a1,B,b1). To test whether hemichannels mature oligondendrocyte markers (Emery, 2010). In our study, most in OPCs and oligodendrocytes were permeable to glucose, we OPCs (PDGFRa positive) differentiate into immature performed a dye uptake assay with 2-NBDG (Retamal et al., 2007a). oligodendrocytes (O4 positive) on the third day, and into mature The uptake ratio for this fluorescent glucose analog was similar to oligodendrocytes (MBP positive) on the sixth day after being induced that monitored by the uptake of EtBr, indicating that both OPCs and to differentiate in vitro (Fig. 1). Double immunostaining results oligodendrocytes uptake glucose from the extracellular medium showed that only Cx29 and Cx47, but not Glut1, were detected in through connexin hemichannels; however, hemichannel-dependent OPCs and immature oligodendrocytes (Fig. 1A,B). At more advanced glucose analog uptake was more pronounced in OPCs than that in stages of differentiation, namely after 6 days differentiation in culture, oligodendrocytes (Fig. 3A,a2,B,b2). In addition, 2-NBDG uptake in all three connexins (i.e. Cx29, Cx32 and Cx47) and Glut1 expression OPCs could be blocked by hemichannel inhibitors but not by the were observed in mature oligodendrocytes (Fig. 1C). The percentages Glut1 inhibitor STF31 or Cytochalasin B, which has also been of oligodendroglial cells positive for connexins at different time in shown to inhibit Gluts (Griffin et al., 1982) (Fig. 3A,B; Fig. S2). culture showed that PDGFRa+ OPCs dominantly express Cx47 and Thus, hemichannels might be the main contributor for glucose Cx29 (Fig. 1D), and the same expression pattern was also observed in uptake in oligodendroglial cells, specifically at the OPC stage. the corpus callosum of postnatal developing mice (Fig. S1). The expression levels of connexins and Glut1 were further determined by Intracellular Ca2+ signaling actives hemichannels in OPCs 2+ 2+ western blot analysis as well as quantitative PCR (qPCR) upon Because it has been reported that intracellular Ca ([Ca ]i) differentiation of OPCs in cultures (Fig. 1E,F). Moreover, Glut2 and elevation triggers the opening of Cx43 and Cx32 hemichannels in Glut3 (neuronal specific) were not found in OPCs (Fig. S1D). Based other cell types (De Vuyst et al., 2006; Wang et al., 2013), we on the different expression patterns of connexins and Gluts between wondered whether hemichannel activity in the oligodendroglial OPCs and oligodendrocytes, we hypothesized that oligodendroglial lineage was also dependent on Ca2+. Thus, we monitored 2+ connexins might differently contribute to metabolic pathways (i.e. cytoplasmic [Ca ]i in OPCs and oligodendrocytes in normal glucose uptake) in oligodendroglial lineage cells. culture conditions following Rhod-2 loading. Real-time recordings showed that most OPCs exhibited spontaneous ‘oscillatory’-like Glucose analog can be exchanged between Ca2+ signaling with peak and plateau transients, whereas oligodendrocytes and astrocytes, but not between OPCs and oligodendrocytes showed ‘flat’ Ca2+ signaling (Fig. 4A,B). As 2+ astrocytes OPCs were characterized by higher [Ca ]i signal and hemichannel To examine the possibility of metabolic coupling between activity compared to oligodendrocytes, we focused our 2+ astrocytes and oligodendroglial lineage cells, we studied glucose investigation on OPCs. The cytoplasmic [Ca ]i and the trafficking in an astrocyte-oligodendroglia co-culture system by dye hemichannel activity were monitored under different conditions, coupling (Giaume et al., 2012). Briefly, the cells were patched including chelating cytoplasmic Ca2+ with BAPTA-AM or 2+ with a whole-cell recording pattern, and the intercellular diffusion increasing [Ca ]i with ionomycin. We found that treatment of of the dye was monitored after 20 min of recording. At the BAPTA-AM inhibited the spontaneous oscillatory-like Ca2+ beginning of the whole-cell configuration (1 min), 2-NBDG or signaling and reduced the 2-NBDG or EtBr uptake in OPCs sulforhodamine B (SRB) filled up CC1+ mature oligodendrocytes. (Fig. 4C–E). However, the relative 2-NBDG uptake rate was After 20 min, these probes diffused from oligodendrocytes to the significantly increased in the ionomycin-treated group, and this astrocyte layer underneath (Fig. 2A). Specifically, 2-NBDG was effect was inhibited by CBX treatment (Fig. 4D). Taken together, time dependently transferred to astrocytes (Fig. 2B), and this these results indicate that hemichannel activity depends on intercellular diffusion was blocked by carbenoxolone (CBX) intracellular Ca2+ elevation in OPCs. (Fig. 2A). However, when the same experiment was performed on OPCs (PDGFRa+), 2-NBDG and SRB only filled up the recorded Inhibition of hemichannel activity impacts OPC proliferation OPCs but were not detected in the underlying astrocytes after Glucose is considered as the most important energy source in the

20 min recording (Fig. 2C), indicating that glucose can be brain and OPC development might rely on it. To determine the role Journal of Cell Science

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Fig. 1. Oligodendroglial cells express connexins during development in vitro. (A) Double immunostaining of Cx29, Cx47, Cx32 and Glut1 (red) with the OPC-specific bio-marker PDGFRa (PDGRaR, green). (B) Double immunostaining of connexins and Glut1 (red) with the immature oligodendrocyte-specific bio- marker O4 (green). (C) Double immunostaining of connexins and Glut1 (red) with the oligodendrocyte-specific bio-marker MBP (green). Note: only Cx29 and Cx47 can be detected in OPCs (PDGFRa+) and immature oligodendrocytes (O4+), and all three connexins and Glut1 can be observed in mature oligodendrocytes (MBP+). Arrows highlight representative positive cells. (D) Quantification of connexin- or Glut1-positive oligodendroglial cells at different time in culture. Development of oligodendroglia is identified by PDGFRa, O4 and MBP, respectively at the indicated time points. (E) Western blot showing the expression patterns of connexins and Glut1 during oligodendroglial differentiation in vitro. (F) qPCR showing the connexins and Glut1 expression levels at indicated time points in culture. Values are mean±s.e.m., three independent experiments were performed in triplicate. **P<0.01 compared to day 0 (unpaired t-test). of connexin-hemichannel-mediated glucose uptake in supporting glucose (0, 0.75 and 1.5 mg/ml). Usually 1.5 mg/ml is considered OPC development, we firstly tested the effect of glucose on OPC as the normal extracellular glucose concentration. Olig2 is an proliferation. Purely cultured OPCs were fed with OPC- oligodendroglial lineage marker which is expressed through the proliferation media containing three different concentrations of whole development process, and we use Ki67 and Olig2 double- Journal of Cell Science

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proliferating OPCs were significantly decreased in a concentration-dependent manner (Fig. 5A,B). Moreover, in the presence of a normal glucose concentration, the blockade of connexin hemichannels in OPCs with CBX or La3+ resulted in a significant decrease of OPC proliferation, indicated by decreased viable cell numbers or Ki67 and Olig2 double-positive OPC numbers (Fig. 5C–E). These results indicate that glucose uptake through hemichannels contributes to OPC proliferation.

Astrocytic Cx43 depletion decreases glucose concentration in the extracellular medium and inhibits OPC proliferation, which can be compensated by glucose supply Cx43 is highly expressed by astrocytes, and works as hemichannels and/or homomeric gap junction channels (Cx43–Cx43) between astrocytes (Evans et al., 2013; Mitterauer, 2015; Ransom and Giaume, 2013; Ye et al., 2009), and also forms heteromeric gap junctions (Cx43–Cx47) between astrocytes and oligodendrocytes (Orthmann-Murphy et al., 2007; Theis et al., 2005). It has been reported that in astrocytes, Cx43 deletion increases glucose uptake by a compensatory upregulation of Glut transporters and/or glucose metabolism enzymes (Gangoso et al., 2012). To determine the influence of astroglial glucose over-consumption on the oligodendroglial lineage, we used the astrocyte Cx43 conditional knockout mouse (hGFAPCre/+:Cx43fl/fl, Cx43-KO). Interestingly, it was found that the glucose concentration in the medium collected from Cx43-KO astrocytes was significantly lower than that from the wild-type astrocytes (Fig. 6A). Similar results were found in wild- type astrocytes treated with the connexin-based channel blocker CBX (Fig. 6B). In addition, we performed an EtBr uptake assay in corpus callosum of acute brain slices from postnatal day (P)14 mice or astrocyte–OPC co-cultures to detect hemichannel function in OPCs under different conditions. We found that the hemichannel function in OPCs was blocked by CBX and increased in Ca2+-free solution, but was not affected by Cx43 deletion in astrocytes (Fig. 6D,E). However, decreased OPC proliferation was found in the corpus callosum of postnatal Cx43-KO mice as well as OPCs co-cultured with astrocytes from Cx43-KO mice (Fig. 6F,G; Fig. S3), whereas Fig. 2. Glucose analog is transferred between astrocytes and no significant difference in PDGF, bFGF, CNTF and lactate levels oligodendrocytes, but not between astrocytes and OPCs. In a co-culture were found after Cx43 deletion in astrocytes (Fig. S4). Moreover, system, astrocytes show flat cell bodies, thick processes, and form a confluent the expression of oligodendroglial connexins was not significantly monolayer; and OPCs show typical bipolar processes, round cell bodies, while altered by Cx43 knockout in astrocytes (Fig. 6C). oligodendrocytes (OL) show multipolar processes above the astrocytes Finally, we performed rescue experiments and found that the monolayer. (A) Intercellular diffusion test in oligodendrocyte and astrocyte decrease in OPC proliferation observed after Cx43 deletion in co-cultures. At the beginning (1 min) of the whole-cell patch, 2-NBDG astrocytes could be compensated by an external glucose supply in (342.3 g/mol) and sulforhodamine B (SRB, 558.7 g/mol,) fill up the multi-polar – processes of oligodendrocytes and diffuse from oligodendrocytes to astrocyte OPC co-cultures (Fig. 7A). To further confirm that neighboring astrocytes after 20 min. 2-NBDG and SRB diffusion can be astrocytes affect OPC proliferation in a non-cell-autonomous blocked by CBX (50 µM). Oligodendrocytes are positive for CC1 (red, white manner, we designed sandwich co-cultures in which OPCs were arrowheads) immunostaining, and the underlying astrocytes are shown by seeded on coverslips in contact with astrocyte medium but not phase contrast imaging (black arrows). (B) Timecourse of 2-NBDG diffusion directly on astrocyte monolayers, as shown in the diagram (Fig. 7B). from one oligodendrocyte to neighboring oligodendrocytes and underlying In these sandwich co-cultures, similar results were obtained as that astrocytes (green arrows show the cells containing newly diffused 2-NBDG). – (C) Intercellular diffusion test in OPCs co-cultured with astrocytes. 2-NBDG in the astrocyte OPC co-cultures (Fig. 7C,D). Taken together, these and SRB only fill up the typical bipolar processes of OPCs but cannot be results indicate that Cx43 deletion in astrocytes affects glucose level detected in neighboring astrocytes after 20 min. OPCs are immunostained by in the extracellular medium but does not impact on the connexin PDGFRa (PDGRaR, red, white arrowheads), and the underlying astrocytes hemichannel function of OPCs. are shown by phase contrast imaging (black arrows). More than six injections were performed for each group. DISCUSSION OPCs and oligodendrocytes need to go through a series of energy- positive cells to mark the proliferating OPCs as described consuming developmental events, including proliferating to enrich previously (Niu et al., 2012b). When exposed to media with the population in central nervous system (CNS) tissues, migrating reduced concentrations of glucose (0 mg/ml and 0.75 mg/ml), the and/or distributing into their destinations and wrapping axons to number of viable cells and Ki67 and Olig2 double-positive form myelin (Kang et al., 2010; Richardson et al., 2011; Rivers Journal of Cell Science

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Fig. 3. Functional hemichannels contribute to glucose analog uptake in oligodendroglial cells. (A) EtBr (394.3 g/ mol, red) uptake (a1) or 2-NBDG (green) uptake (a2) in PDGFRa+ OPCs (PDGRaR, green) or oligodendrocytes (CC1+, green) under different conditions, including normal culture medium and culture medium pre-incubated with CBX, La3+ ions, Glut1 inhibitor (STF31) or Ca2+-free solution. OL, oligodendrocyte. (B) Quantification of the EtBr uptake ratio (b1) or 2-NBDG uptake ratio (b2). Both EtBr and 2-NBDG uptake can be blocked by CBX (50 µM) and La3+ (200 µM) but not by STF31 (5 µM), whereas the uptake ratio is significantly increased in Ca2+-free solution. Note: both OPCs and oligodendrocytes take up EtBr or 2-NBDG from the extracellular medium through connexin hemichannels in normal conditions, and OPCs can take up more EtBr or 2-NBDG than oligodendrocytes do. Values are mean±s.e.m., three independent experiments were performed in triplicate. *P<0.05, **P<0.01 compared to normal conditions (unpaired t-test).

et al., 2008). However, the mechanisms underlying their energy astroglial connexin channels (gap junction channels and metabolism, in particular the pathways involved in energy hemichannels) are permeable to glucose derivatives (2-NBDG) in metabolite uptake and trafficking remain unidentified. Functional both in vitro and ex vivo models (Blomstrand and Giaume, 2006; tests have shown that astrocytes and oligodendrocytes are connected Retamal et al., 2007a), thus they provide the basis to form metabolic by gap junctions to form panglial networks (Griemsmann et al., intercellular networks (Rouach et al., 2008). Interestingly, gap

2014; Ransom and Giaume, 2013; Tress et al., 2012). Moreover, junction channels are ∼65% less permeable to the phosphorylated Journal of Cell Science

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Fig. 4. Intracellular Ca2+ signaling affects hemichannel activity in OPCs. 2+ 2+ (A) Intracellular Ca signaling ([Ca ]i)in oligodendroglial [OPC and oligodendrocyte (OL)] cells is monitored by Rhod-2 loading. Spontaneous intracellular Ca2+ signals (ΔF/F) in OPCs and oligodendrocytes are shown in a2, respectively, and their morphologies are shown in a1. (B) Percentages of cells with ‘ ’ 2+ oscillatory -like [Ca ]i in OPCs and oligodendrocytes. **P<0.01 between two groups (unpaired t-test). (C) Real-time 2+ [Ca ]i in OPCs with different conditions. Arrowhead indicates the starting point of the treatment. (D) Relative 2-NBDG uptake per second. Note that the 2-NBDG uptake rate is repressed by BAPTA-AM (25 µM) but increased by ionomycin (1 µM) and that this effect can be inhibited by CBX (50 µM). (E) EtBr (red) uptake in OPCs with or without BAPTA-AM (25 µM) treatment. Values are the means±s.e.m., more than 10 cells were tested independently in each group. *P<0.05, **P<0.01 compared to vehicle; ##P<0.01 between two groups (unpaired t-test).

2-NBDG-6P than to 2-NBDG (Rouach et al., 2008), indicating that to adulthood (Parenti et al., 2010). Here, we used OPC cultures to the phosphorylated form of the fluorescent glucose analog is further demonstrate the dynamic expression pattern of connexins restricted in the glial cells. As glial hemichannels exhibit similar during oligodendroglial development in vitro, suggesting a role for permeability properties with gap junction channels (Giaume et al., connexin-based channels in this process (Li et al., 2014). Although 2013), it is expected that once absorbed in OPCs by hemichannels, the combination of different connexins between oligodendrocytes 2-NBDG is phosphorylated and then stays trapped within the cells and astrocytes (Cx47–Cx43, Cx47–Cx30, Cx32–Cx30 and Cx32– for further metabolic requirement, such as glycolysis. Based on Cx26) has been identified, and a heterotypic gap junction activity these findings, and to further understand the metabolic role of glial has been confirmed by dye coupling and/or electrical measurements connexins in metabolic supply, we investigated whether connexin- (Magnotti et al., 2011), the types of metabolic substrates that are based channels contribute to glucose trafficking pathways in exchanged between these two glial cell types have not been oligodendroglial cells. Here, we provide evidence demonstrating investigated yet. Current data have shown that this ‘panglial’ that a glucose analog cannot exchange between OPCs and networking of oligodendrocytes and astrocytes serves to spatially astrocytes due to the absence of gap junction, whereas buffer K+, allow water transport and serve as bi-directional channels hemichannel activity supports glucose uptake in OPCs, which is for the spreading of Ca2+ waves (Kamasawa et al., 2005; Menichella essential for their proliferation. In addition, for the first time, we et al., 2006; Parys et al., 2010; Wallraff et al., 2006). Here, we show that a glucose analog can be transferred through provide direct evidence that oligodendrocyte–astrocyte gap junctions oligodendrocyte–astrocyte gap junctions, which provides the basis are also permeable to the fluorescent glucose analog 2-NBDG. for a panglial metabolic route that previously had only been Considering the capacity of astrocytes to uptake, store and supply suggested but not demonstrated (Morrison et al., 2013). energy substrates (Rouach et al., 2008), it is likely that glucose During brain development, oligodendroglial-specific Cx47 and and/or its metabolites might be transferred from astrocytes to Cx32 are detected at embryonic stages, whereas Cx29 appears at P0. oligodendrocytes through oligodendrocyte–astrocyte gap junctions Cx47 expression increases successively in regions populated with as recently hypothesized (Morrison et al., 2013). In this regard, our developing oligodendroglia and declines from early postnatal stage findings might provide a new understanding of a connexin-channel- Journal of Cell Science

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Fig. 5. Inhibition of the hemichannel activities impacts on OPC proliferation. (A) CCK-8 assay shows the viable cell numbers of OPCs in proliferation medium with different concentrations of glucose at the indicated time points. The lower glucose concentrations (0 and 0.75 mg/ml) significantly decreased the viable cell numbers in comparison with the normal medium containing 1.5 mg/ml glucose. (B) The cell count of proliferating OPCs (Ki67+ and Olig2+) shows that reduced glucose concentrations decrease OPC proliferation at 24 h. Values are the means±s.e.m., three independent experiments were performed in triplicate, *P<0.05, **P<0.01 compared to normal conditions (unpaired t-test). (C) Pure cultured OPCs were treated with CBX (20 µM) or La3+ (200 µM) for 24 h; proliferating OPCs are labeled with Ki67 (red) and Olig2 (green). Arrows show the double-positive cells. (D) Quantification of Ki67 and Olig2 double- positive cells shows that the percentage of proliferating OPCs is significantly decreased in the CBX or La3+ treatment groups. (E) The CCK-8 assay shows that a 12-h treatment with CBX or La3+ significantly decreased the viable cell numbers in comparison with vehicle group. Values are the means±s.e.m., three independent experiments were performed in triplicate. *P<0.05, **P<0.01 compared to vehicle (unpaired t-test).

mediated metabolic supply alternative pathway besides Glut- or brain areas such as the hippocampus (Chever et al., 2014) and the MCT-mediated pathways in the oligodendroglial lineage. olfactory bulb (Roux et al., 2015). Moreover, they are triggered to However, using 2-NBDG, we could not observe such gap- open by some stimulation, such as inflammation, mechanical stress junction-mediated glucose transport between OPCs and adjacent or ischemia (Batra et al., 2012; Johansen et al., 2011; Karpuk et al., astrocytes. Instead, we showed for the first time that connexin-based 2011). In our study, however, we demonstrated for the first time hemichannels in OPCs are responsible for glucose analog uptake that connexin-mediated hemichannels are responsible for glucose from the extracellular environment, though there is still a lack of uptake in OPCs, implying a role for oligodendroglial connexin evidence to show which connexin protein plays the dominant role in hemichannels under physiological conditions. We further revealed this process. These in vitro results imply that a functional gap that the activation of hemichannels in the oligodendroglial lineage junction channel might not exist at early OPC developmental stages depends on intracellular Ca2+ signaling, which can trigger connexin until they differentiate into GalC-positive oligodendrocytes hemichannel opening in other cell types (De Vuyst et al., 2006). (Venance et al., 1995). Consistently, it has been demonstrated that Interestingly, we observed that OPCs exhibit stronger spontaneous cells positive for the cell surface ganglioside A2B5, and NG2 glial Ca2+ oscillationsthan oligodendrocytes do (Fig. 4) as recently reported cells do not electrically or dye couple with astrocytes whereas (Cheli et al., 2015), which might explain why more glucose uptake mature oligodendrocytes do (Bergles et al., 2000; Lin and Bergles, through connexin hemichannels occurred in OPCs compared to 2004; Xu et al., 2014). Importantly, given that neither Gluts (i.e. oligodendrocytes (Fig. 3). In this regard, our data shed light on Glut1, Glut2 and Glut3) nor MCT1 was detectable in OPCs in our different energy substrate uptake mechanisms and pathways between studies and in studies by other (Lee et al., 2012), whereas 2-NBDG OPCs and oligodendrocytes. In OPCs, connexin hemichannels uptake in OPCs can be blocked by CBX but not by Glut inhibitors provide a main route for glucose entry, whereas in oligodendrocytes, such as STF31 and Cytochalasin B, it is likely that connexin- glucose and lactate transporters, supplemented by the gap junctions mediated hemichannels serve as a major metabolic supply pathway between astrocytes and oligodendrocytes, as well as their in OPCs. Thus, the crucial developmental step of myelination might hemichannels contribute to the energy substrate uptake and traffic. depend on a connexin-channel-mediated glucose supply (Rinholm As elegantly stated by A. Harris (2007), “Although connexin- et al., 2011; Yan and Rivkees, 2006). based channels are considered as poorly selective channels leading Normally it isthought that glial hemichannels are either kept closed, to an idea that they are ‘non-specific large conductance channels’, to maintain cellular integrity (Spray et al., 2006), oractive, as in certain numbers of studies demonstrate that there are dramatic, Journal of Cell Science

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Fig. 6. Cx43 deletion in astrocytes decreases glucose concentration in the extracellular medium and inhibits OPC proliferation. (A) Glucose assay of the astrocytic culture medium. The concentration of glucose in the Cx43-KO group is lower than that in the wild-type group. (B) In pure cultured astrocytes, CBX (50 µM) treatment reduces the glucose concentration in the culture medium. (C) qPCR shows the connexin mRNA levels in P7 mice brain, there is no significant difference between wild-type and Cx43-KO mice. (D) EtBr (red) uptake assay and quantification in corpus callosum from acute brain slices of P14 wild-type or Cx43-KO mice. Arrows highlight OPCs in corpus callosum. (E) EtBr (red) uptake assay and quantification in astrocyte (AST) and OPC co- cultures. OPCs are labeled by anti-PDGFRa (PDGRaR, green). Note that hemichannel function in OPCs is blocked by CBX and increased with Ca2+-free solution but is not impacted by Cx43 deletion in astrocytes. (F) Quantification of PDGFRa+ (OPCs) or Ki67 and Olig2 double-positive cells. Cx43 deletion in astrocytes significantly decrease the number of OPCs and Ki67 and Olig2 double-positive cells during the development of brain white matter. (G) In astrocyte–OPC co-cultures, the lack of Cx43 in astrocytes reduces the number of OPCs, and the number of Ki67 and Olig2 double- positive proliferating OPCs. Values are the means±s.e.m., more than 4 mice (P14) were tested in each group; for co-cultures three independent experiments were performed in triplicate. *P<0.05, **P<0.01 (unpaired t-test).

unanticipated and connexin-specific differences in the channel have highly specific interactions within connexin pores that enable permeability to cytoplasmic molecules, particularly among those surprising degrees of selective permeability that cannot be predicted thought to directly mediate intercellular signaling (i.e. cAMP, from simple considerations of pore width or charge selectivity”

IP3,…). The data strongly indicate that certain biological molecules (Harris, 2007). Based on these statements, it seems reasonable to Journal of Cell Science

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OPC hemichannel properties. Moreover, this effect can be rescued by a compensatory extracellular glucose supply, indicating the importance of astrocytes in maintaining the glucose levels of the CNS extracellular matrix. Another point that needs caution is that lactate is also an important metabolic substrate for the brain during the early postnatal period (Barros, 2013; Rinholm et al., 2011). Given the extensive permeability of hemichannels for various metabolites (Giaume et al., 2013), there is no evidence to exclude the uptake of lactate or other energy substrates through hemichannels in OPCs. In fact, it has already been reported that gap junction channels in astrocytes are permeable to lactate in vitro and ex vivo (Tabernero et al., 1996; Rouach et al., 2008). Given that various connexin channels exhibit different functional properties, including size selectivity, charge selectivity and voltage or chemical gating, we still have no evidence to show that connexin channels in OPCs are permeable or impermeable to other substrates, such as Ca2+,K+ and metabolites. Because OPCs need to go through a series of highly energy- consuming developmental events (Barateiro and Fernandes, 2014) and likely cannot get enough energy support from Glut1, MCT1 or gap junctions as oligodendrocytes do, they might rely more on the hemichannel-mediated energy supply pathway (Fig. 8). This dependence might partly explain why OPCs are more vulnerable to oxygen-glucose deprivation than oligodendrocytes (Fern and Moller, 2000; Ziabreva et al., 2010). Moreover, likely due to deficient Cx47- based hemichannel properties, Cx47-null mice exhibit decreased oligodendroglial lineage cell numbers at P14, but gene knockdown does not influence myelination in adult mice (Tress et al., 2012). Finally, abnormal connexin expression is involved in limiting OPC recruitment and consequent remyelination failure in multiple sclerosis patients and in autoimmune encephalomyelitis mice (a model of multiple sclerosis) (Markoullis et al., 2012a,b, 2014).

Concluding remarks In this study, we focused on connexin-mediated energy supply Fig. 7. The reduced OPC proliferation caused by the lack of Cx43 in astrocytes is compensated by increased glucose supply. (A) Quantification pathways in oligodendroglial lineage cells, providing direct of PDGFRa+ (PDGRaR, green) OPCs in different conditions. OPC proliferation evidence that glucose can be delivered between astrocytes and −/− is reduced when grown with CX43-KO astrocytes (ASTCx43 ), and oligodendrocytes through gap junction channels, whereas supplementary glucose significantly reversed this proliferation deficit. (B) The diagram shows the sandwich co-culture system. OPCs are seeded on cover slips placed on top of an astrocyte monolayer. (C) Proliferating OPCs in sandwich co-cultures are double-labeled with anti-Ki67 (red) and anti-PDGFRa (green) under different conditions. Arrows show the double-positive cells. (D) Cell counting of Ki67 and PDGFRa double-positive OPCs illustrates that the −/−– decrease of OPC proliferation in the ASTCx43 OPC group can be reversed by adding glucose (3 mg/ml) to the culture medium. Values are the means± s.e.m., three independent experiments were performed in triplicate.*P<0.05, **P<0.01 between the indicated groups (unpaired t-test). consider that although we show here that astrocyte–oligodendrocyte gap junction channels and OPCs hemichannels are permeable to 2-NBDG, it does not automatically imply that all cytoplasmic or extracellular signaling molecules with small molecular mass can permeate through these channels. Finally, we used a conditional astrocytic Cx43 knockout (Cx43- KO) mouse as a glucose-deprivation model to confirm the Fig. 8. Schematic of the hemichannel and gap junction contribute to importance of connexin-channel-based energy support on the glucose supply in oligodendroglial lineage cells. Glucose can be OPC proliferation in both in vivo and co-culture systems. In these transferred between astrocytes and mature oligodendrocytes (OL) through gap mice, glucose uptake in Cx43 lacking astrocytes is increased by junction channels when functional gap junction channels are not formed at the compensatory upregulation of Glut1, Glut3 and type I/II early stage of oligodendroglial development. However, a connexin- hemichannel-based glucose supply from the extracellular microenvironment hexokinase expression (Gangoso et al., 2012; Tabernero et al., maintains OPC proliferation. Deletion of Cx43 in astrocytes (Cx43-KO) leads to 2006). As predicted, we observed that OPC proliferation was glucose over consumption by compensatory upregulation of Glut1 and Glut3, inhibited by Cx43 deletion in astrocytes without impacting on and results in the suppression of OPC proliferation. Journal of Cell Science

1910 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1902-1914 doi:10.1242/jcs.178731 spontaneous intracellular Ca2+ signaling triggers connexin St Louis, MO, USA), La3+ ions (lanthanum, Sigma), Glut1 inhibitor hemichannel activity that enables glucose uptake in OPCs that (STF31, Tocris Bioscience, Bristol, UK), Cytochalasin B (Sigma), 2+ supports their proliferation. Identification of the dual functions of Cytochalasin D (Enzo Life Sciences, East Farmingdale, NY) or Ca - connexin-based channels in the oligodendroglial lineage, along free solution. Then, the cells were fixed with 4% PFA for 15 min at room with their metabolic roles, might provide valuable insights into the temperature. Immunofluorescence pictures were captured using a confocal laser-scanning microscope (Olympus, IV1000) at an energy supply pathways of oligodendroglial cells and might result in appropriate wavelength (EtBr, 561 nm; 2-NBDG, 488 nm). The new therapeutic strategies for energy-related diseases. EtBr fluorescent signal in the nuclei and the 2-NBDG fluorescent signal in cell bodies were analyzed using Mac Imaris 7.4.0 software. The 3 MATERIALS AND METHODS mean fluorescence intensity per µm was quantified in arbitrary units Animals (AUs) (Giaume et al., 2012). Cx43flox/flox and hGFAPCre/+ mice were purchased from the Jackson Laboratory. These two mouse strains were paired to produce offspring, Immunofluorescence staining and quantification either hGFAPCre/+:Cx43fl/fl (Cx43-KO) or hGFAP+/+:Cx43fl/fl (wild type). Mouse pups of different ages (P7, P14, P21) were anesthetized with 1% The offspring were genotyped by PCR for Cre and floxed Cx43 alleles. pentobarbital and transcardially perfused with 4% PFA. Brains were More than four animals were tested in each group. All animal studies were dissected and cryoprotected in 30% sucrose at 4°C. Serial coronal conducted with the approval of the Laboratory Animal Welfare and Ethics sections (20 μm) were obtained using a cryostat microtome (MS 1900, Committee of the Third Military Medical University (TMMU) Leica, Wetzlar, Germany). OPCs on the cover slips were fixed as Administrative Panel on Laboratory Animal Care. previously described (Niu et al., 2012a). Brain sections or cell cultures were blocked with 0.5% bovine serum albumin (BSA) and 0.2% Triton X-100 for 1 h and then incubated with primary antibodies overnight at 4° Acute brain slices, EtBr uptake and immunofluorescence C followed by the fluorescence-conjugated secondary antibodies at room staining temperature for 1 h. Cell nuclei were stained with 4′,6-diamidino-2- Acute brain slices were prepared from brains of P14 Cx43-KO and phenylindole (DAPI, 0.1 μg/ml, ThermoFisher). Rabbit anti-PDGFRa wild-type littermate mice using a vibroslicer (Thermo Scientific, (1:200, cat. no. sc-338; Santa Cruz, Dallas, TX, USA) or rat anti- Microm HM650V) as previously described (Geiger et al., 2002). PDGFRa (1:100, cat. no. ab61219; Abcam, Cambridge, UK) antibodies Slices were incubated with the hemichannel-permeable fluorescent were used to label OPCs. Mouse anti-O4 (1:100, cat. no. O7139; Sigma) tracer ethidium bromide (EtBr) (394.3 g/mol, 4 µM final concentration, antibody was used to label immature oligodendroglial cells, and goat ThermoFisher, Grand Island, NY, USA) in oxygenated artificial anti-MBP (1:300, cat. no. sc-13914; Santa Cruz Biotechnology) or cerebro-spinal fluid (ACSF) for 10 min at room temperature. After mouse anti-CC1 (1:1000, cat. no. OP80; Millipore, Billerica, MA) fixation with 4% paraformaldehyde (PFA), the slices were incubated antibodies were used to label mature oligodendrocytes. Mouse anti-Olig2 with anti-platelet-derived growth factor a receptor (PDGFRa) primary (1:200, cat. no. MABN50; Millipore) antibody was used to label all antibody (1:200, cat. no. sc-338; Santa Cruz Biotechnology, Dallas, oligodendroglial lineage cells. Rabbit anti-Ki67 (1:1000, cat. no. RM- TX, USA) overnight at 4°C after pre-incubation in the blocking buffer 9106; Thermo Scientific) antibody was used to detect proliferative cells. for 1 h (containing 0.2% gelatin and 1% Triton X-100), followed by Rabbit anti-Cx29 (1:100, cat. no. sc-68377; Santa Cruz Biotechnology), incubation in fluorescent-conjugated secondary antibody at room rabbit anti-Cx32 (1:100, cat. no. C3595; Sigma), rabbit anti-Cx47 (1:200, temperature for 1 h. Images were taken using a confocal laser- cat. no. 364700; Life Technologies; or 1:100, sc-30335-R; Santa Cruz scanning microscope (Olympus, IV1000) at the selected specific Biotechnology), mouse anti-Glut1 (1:500, cat. no. ab40084; Abcam), wavelength (561 nm). More than eight images were captured in each goat anti-Glut2 (1:100, cat. no. sc-7580; Santa Cruz Biotechnology) and group for statistical analysis. mouse anti-Glut3 (1:100, cat. no. ab41525; Abcam) antibodies were used to detect connexin proteins and Glut in oligodendroglial cells. Mouse Astrocyte and oligodendroglial cell cultures anti-Cx43 (1:100, cat. no. 610062; BD, Erembodegem, Belgium) Primary astrocyte cultures were prepared from brain hemispheres of antibody was used to label Cx43 in astrocytes. Appropriate Alexa- Cx43-KO and wild-type littermate mice pups (P1–P3) as previously Fluor-conjugated secondary antibodies included donkey anti-mouse, described (Giaume et al., 2012). The purified astrocytes were plated into rabbit and goat IgG (1:1000, Life Technologies). The immunofluorescent six-well plates (105 cells per well). The OPC cultures were prepared as signal was determined using a fluorescence microscope (Olympus BX- previously described (Niu et al., 2012b). Briefly, the mixed glial cells 60) or a confocal laser-scanning microscope (Olympus, IV 1000) with were isolated from cortex of P1–P3 animals and enriched in OPC growth excitation wavelengths appropriate for Alexa Fluor 488 (488 nm, Life medium followed by a two-passage purifying processes. The purified Technologies), Alexa Fluor 568 (568 nm) or Alexa Fluor 647 (647 nm). OPCs were induced to differentiate by using OPC differentiation medium Cell counting and fluorescence intensity analyses were conducted on nine [DMEM/F12+1%N2supplement+5g/mlNAC+1%FBS;Dulbecco’s randomly chosen fields under a 20× objective lens for each sample using modified Eagle’s media/F12 (DMEM/F12, cat. no. SH30023, Hyclone, an Image Pro Plus image analysis system. Logan, Utah, USA), N2 supplement (cat. no. 17502048, Life Technologies), fetal bovine serum (FBS, cat. no. SV30087, Hyclone), Western blotting N-acetyl-l-cysteine (NAC, cat. no. 0LA0011, AMERSCO, Solon, OH, SDS-PAGE western blotting was performed as previously described (Niu USA)]. For co-cultures, OPCs were seeded either on an astrocyte et al., 2010). Proteins were transferred to polyvinylidenedifluoride monolayer to set up astrocyte–OPC co-cultures or on poly-D-lysine- membranes and visualized by chemiluminescence (ECL Plus, GE coated glass coverslips placed above the astrocytes to set up a sandwich Healthcare, Marlborough, MA, USA) after incubation with antibodies. co-culture system. We used 4.5×104 cells per well in 24-well plates and β-actin (1:1000, cat. no. sc- 47778; Santa Cruz Biotechnology) was used as 106 cells per well in 60-mm dishes. the loading control. Quantification of band intensity was analyzed using the Image Pro Plus software. The primary antibodies included: rabbit anti- Dye uptake and quantification PDGFRa (1:500, cat. no. sc-338; Santa Cruz Biotechnology), goat OPCs or oligodendrocytes were incubated with 4 µM EtBr or 500 µM anti-MBP (1:1000, cat. no. sc-13914; Santa Cruz Biotechnology), mouse 2-NBDG (342.3 g/mol, neutral, Life Technologies, Carlsbad, CA) for anti-Olig2 (1:1000, cat. no. MANB50; Millipore), rabbit anti-Cx29 (1:500, 30 min at room temperture in different conditions (Giaume et al., 2012), cat. no. sc-68377; Santa Cruz Biotechnology), rabbit anti-Cx32 (1:500, including normal culture medium (OPCs are in OPC proliferation cat. no. C3595; Sigma), rabbit anti-Cx47 (1:1000, cat. no. 364700; Life medium and oligodendrocytes are in OPC differentiation medium) or Technologies; 1:500, cat. no. sc-30335-R; Santa Cruz Biotechnology) and culture medium pre-incubated with carbenoxolone (CBX, Sigma, mouse anti-Glut1 (1:2000, cat. no. ab40084; Abcam). Journal of Cell Science

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CCK-8 assay baseline 2-NBDG fluorescence intensity; S, time (seconds) from start point The number of viable cells was estimated using the CCK-8 assay, which of treatment to the end time point of experiment]. provides an effective and reproducible measurement of OPC proliferation 4 (Xiao et al., 2008). Briefly, OPCs were cultured at a density of 1.5×10 Quantification real-time polymerase chain reaction cells per well in poly-D-lysine-coated 96-well plates containing 100 µl Total ribonucleic acid (RNA) was isolated from brains or cultured astrocytes OPC proliferation medium [DMEM/F12 + 1% N2 supplement + 10 ng/ml of wild-type and Cx43-KO mice using TRIzol (Life Technologies) and the bFGF + 10 ng/ml PDGF-AA; PDGF-AA (cat. no. 100-13A, Peprotech, RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Real-time quantitative Rocky Hill, NJ, USA), bFGF (cat. no. 100-18B, Peprotech)]. After 12 h, PCR (qPCR) was performed with the C1000 Touch™ Real-time PCR the cells were subjected to different conditions, and at the indicated time Detection System (Bio-Rad) and GoTaq® qPCR Master Mix (Promega, points, 10 µl of CCK-8 solution reagent (Dojindo, Cell Counting Kit-8) Sunnyvale, CA, USA). The oligonucleotide primers, amplification was added to each well according to the manufacturer’s instructions, procedure, and melt curve analysis were performed. For each sample, three followed by a 4 h incubation. The relative viable cell numbers were independent repeats were performed. Each sample was tested in triplicate. determined by measuring the absorbance at 450 nm using a microplate reader (Bio-Rad, Model 680). Three independent experiments were Statistical analysis performed in triplicate. Statistical significance between groups was determined with GraphPad Prism 5 software. Statistical analyses were performed by one-way analysis Glucose assay of variance (ANOVA) followed by Tukey’s post-hoc test. Comparisons Glucose concentrations were determined using a glucose assay kit (Abcam) between two experimental groups were made using an unpaired t-test. A according to the manufacturer’s instructions. Supernatants from 48 h probability of P<0.05 was considered statistically significant. cultured astrocytes in different conditions were collected for the glucose assay. Treatment with CBX (50 µM, 48 h) was used to mimic Cx43 deletion Acknowledgements in astrocytes. The optical density (OD) value was measured according to the The authors wish to thank Jia Lou for her assistance in preparing the figures. absorbance at 570 nm using a microplate reader. Competing interests The authors declare no competing or financial interests. Intercellular dye trafficking Astrocyte–OPC co-cultures were used for dye-trafficking experiments. Author contributions Recorded cells were patched in current-clamp mode using an Axon L.X., C.G., J.N. and T.L. designed experiments and wrote the manuscript; J.N., T.L., Instruments system (Axopatch 200B). The oligodendroglial cells were C.Y., N.H. and C.W. conducted the experiments; C.L. and C.-J.Z. collected and patched with a whole-cell recording technique, using 5–10 MΩ glass analyzed the data; A.K. reviewed and edited the manuscript. electrodes filled with an internal solution as previously described (Giaume et al., 2012; Rouach et al., 2008). To monitor intercellular diffusion, 2- Funding NBDG (2 mg/ml) and sulforhodamine B (SRB) (558.7 g/mol, 2 negative This work is in part supported by the National Natural Science Foundation of China charges, 1 mg/ml, ThermoFisher) were injected into either oligodendrocytes (NSCF) [grant numbers 31171046, 31300906]; Chongqing Scientific and Technical Innovation Foundation of China [grant number CSTCKJCXLJRC07]; and the or OPCs during whole-cell recordings and images were captured after 1 and China-France Joint Program YUANPEI 2013 PROJECT [grant numbers 26038XE]. 20 min to confirm the diffusion of dye among cells. The connexin channel blocker CBX was applied in astrocyte-oligodendrocyte co-cultures to verify Supplementary information connexin-channel-dependent glucose transport. Images of intercellular Supplementary information available online at diffusion of these fluorescent dyes were captured after 1 or 20 min, and the http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.178731/-/DC1 coupling of these cells was analyzed with an Olympus CCD and Clampex 10.0 software. Dye-trafficking experiments were carried out for more than References six cells in each group. Bansal, R., Warrington, A.E., Gard, A.L., Ranscht, B. and Pfeiffer, S.E. (1989). Multiple and novel specificities of monoclonal antibodies O1, O4, and R-mAb used in the analysis of oligodendrocyte development. J. Neurosci. Res. 24, 2+ Intracellular Ca imaging and glucose analog uptake imaging 548-557. OPCs were cultured on glass-bottomed dishes in OPC proliferation medium Barateiro, A. and Fernandes, A. (2014). Temporal oligodendrocyte lineage and then loaded with the fluorescent Ca2+-sensitive dye Rhod-2 (5 μM, Life progression: in vitro models of proliferation, differentiation and myelination. 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