1521-0111/89/1/63–74$25.00 http://dx.doi.org/10.1124/mol.115.100594MOLECULAR PHARMACOLOGY Mol Pharmacol 89:63–74, January 2016Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Axon-to-Glia Interaction Regulates GABAA Receptor Expressionin Oligodendrocytes

Rogelio O. Arellano, María Victoria Sánchez-Gómez, Elena Alberdi, Manuel Canedo-Antelo,Juan Carlos Chara, Aitor Palomino, Alberto Pérez-Samartín, and Carlos MatuteAchucarro Basque Center for Neuroscience, Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas,and Departamento de Neurociencias, Universidad del País Vasco, Leioa, Spain (R.O.A., M.V.S.-G., E.A., M.C.-A., J.C.C.,A.P., A.P.-S., C.M.); and Instituto de Neurobiología, Laboratorio de Neurofisiología Celular, Universidad Nacional Autónomade México, Juriquilla, Querétaro, México (R.O.A.)

Received June 29, 2015; accepted November 3, 2015

ABSTRACTMyelination requires oligodendrocyte-neuron communication, andboth neurotransmitters and contact interactions are essential forthis process. Oligodendrocytes are endowedwith neurotransmitterreceptors whose expression levels and properties may changeduring myelination. However, only scant information is availableabout the extent and timing of these changes or how they areregulated by oligodendrocyte-neuron interactions. Here, we usedelectrophysiology to study the expression of ionotropic GABA,glutamate, andATP receptors in oligodendrocytes derived from theoptic nerve and forebrain cultured either alone or in the presence ofdorsal root ganglion neurons. We observed that oligodendrocytesfrom both regions responded to these transmitters at 1 day in

culture. After the first day in culture, however, GABA sensitivitydiminished drastically to less than 10%,while that of glutamate andATP remained constant. In contrast, the GABA response amplitudewas sustained and remained stable in oligodendrocytes coculturedwith dorsal root ganglion neurons. Immunochemistry and pharma-cological properties of the responses indicated that they weremediated by distinctive GABAA receptors and that in coculture withneurons, the oligodendrocytes bearing the receptors were those indirect contactwith axons. These results reveal thatGABAA receptorregulation in oligodendrocytes is driven by axonal cues and thatGABA signaling may play a role in myelination and/or during axon-glia recognition.

IntroductionMyelination of axons is critical for fast and efficient com-

munication among distant neurons. It begins early afterbirth and proceeds throughout life, not only to maintain thefunctions of established circuits but also to generate or re-inforce new connections formed during plastic phenomena,such as memory and learning (Zatorre et al., 2012). In ad-dition, remyelination after demyelination promotes tissuerepair and functional recovery in chronic and acute diseasesthat impair oligodendrocytes (OLGs) and myelin (Gallo andArmstrong, 2008; Franklin et al., 2012).The myelinating program involves a series of cues that are

transmitted between OLGs and neurons and that comprise

multiple steps initiated at the early stages of oligodendrocyteprecursor cell (OPC) differentiation. Among the signals thathave been proposed as cues for differentiation and myelinationare several neurotransmitters and growth factors. They prob-ably act as paracrine or autocrine signals in addition to theirrole in synaptic transmission, and they have diverse effectsin either stimulating or regulating specific membrane recep-tors expressed in the OPC and OLG membrane (Káradóttirand Attwell, 2007; Boulanger and Messier 2014). Thus, aden-osine, glutamate, GABA, and ATP can modulate the prolifer-ation, differentiation, and migration of OPC as well as OLGsurvival and myelination (e.g., Gallo et al., 1996; Gudz et al.,2006; Ishibashi et al., 2006; Domercq et al., 2010; Etxeberriaet al., 2010; Wake et al., 2011; Zonouzi et al., 2015). Inparticular, both OPCs and mature OLGs express the two mainsubtypes of GABA receptors: GABAA (Hoppe and Kettenmann1989; Von Blankenfeld et al., 1991; Berger et al., 1992; Cahoyet al., 2008) andGABAB (Luyt et al., 2007). Sensitivity toGABAin mature OLGs is greatly reduced (Berger et al., 1992), whichsuggests a specific role for GABA signaling in the developmentof the oligodendroglial lineage and during the initial stages ofmyelination and/or axon recognition (Vélez-Fort et al., 2012;Zonouzi et al., 2015). However, there is no detailed information

Some information reported herein was presented as follows: Arellano RO,Sánchez-Gómez MV, Alberdi E, Canedo M, Palomino A, Pérez-Samartín A, andMatute C (2015) Neuron and glia interaction regulates GABAA receptorexpression in the oligodendrocyte membrane. XII European Meeting on GlialCells in Health and Disease; on July 15 and 16, 2015; Bilbao, Spain. NetworkGlia, Berlin, Germany.

This study was funded by the Ministerio de Economía y Competitividad/Fondos Europeos de Desarrollo Regional [Grants SAF2010-21547 and SAF2013-45084-R] and the Centro de Investigación Biomédica en Red en EnfermedadesNeurodegenerativas [Grant PRY-15-404].

dx.doi.org/10.1124/mol.115.100594.

ABBREVIATIONS: b-CCB, butyl b-carboline-3-carboxylate; DIV, days in vitro; DMCM, 4-ethyl-6,7-dimethoxy-9H-pyrido(3,4-b)indole-3-carboxylicacid methyl ester; DRG, dorsal root ganglion; DZP, diazepam [7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2(1H)-one]; FZP, flunitrazepam[5-(2-fluorophenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one]; Glut; glutamine; HK1, high K1; loreclezole, (Z)-1-[2-chloro-2-(2,4-dichlorophenyl)ethenyl]-1H-1,2,4-triazole; MBP, myelin basic protein; OLG, oligodendrocyte; OPC, oligodendrocyte precursor cell; PBS,phosphate-buffered saline; PDGFRa, platelet-derived growth factor receptor-a; THIP, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol hydrochloride.

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about the status and fate of the different receptors and channelsin this specific timewindowwhenOLGs encounter axons. Here,we have studied the sensitivity to GABA (and other neuro-transmitters) of OLGs that were cocultured with dorsal rootganglion (DRG) neurons. We found that OLGs in contact withaxons robustly express functional GABAA receptors, which arelost in OLGs cultured alone. TheGABAA receptors expressed inOLGs showed unique characteristics that distinguished themfrom receptors present in neurons and astrocytes as well asOPCs in the adult brain.

Material and MethodsRat Optic Nerve OLG Cultures. Primary cultures of OLGs

derived from optic nerves of 12-day-old Sprague-Dawley rats wereobtained as described previously (Barres et al., 1992). Cells wereseeded on 24-well plates bearing 12-mm-diameter coverslips coatedwith poly-D-lysine (10 mg/ml) at a density of 104 cells per well. Cellswere maintained at 37°C and 5% CO2 in a chemically defined medium(Sato medium; Barres et al., 1992) consisting of Dulbecco’s modifiedEagle’s medium supplemented with 100 mg/ml transferrin, 60 ng/mlprogesterone, 40 ng/ml sodium selenite, 5 mg/ml insulin, 16 mg/mlputrescine, 100mg/ml bovine serumalbumin, triiodothyronine (30 ng/ml),and thyroxine (40 ng/ml). After 3–5 days in vitro (DIV), cultures werecomposed of at least 98% cells positive for O4 antigen andmyelin basicprotein (O41/MBP1). The majority of the remaining cells wereimmunostained by antibodies against glial fibrillary acidic protein.No A2B51 or microglial cells were detected in these cultures.

Rat Forebrain OLG Cultures. Primary mixed glial cultureswere prepared from newborn (P0–P2) Sprague-Dawley rats accord-ing to the modified technique of McCarthy and de Vellis (1980).Briefly, forebrains were removed from the skulls, and the corticeswere isolated and digested by incubation (15 minutes, 37°C) inHank’s balanced salt solution containing 0.25% trypsin and 0.4%DNAse. The cells were dissociated by passage through needles(21 G and 23 G), centrifuged, and resuspended in Iscove’s modi-fied Dulbecco’s medium supplemented with 10% fetal bovine serum(Hyclone; Thermo Scientific, Waltham, MA) and antibiotic/antimycoticsolution (Sigma-Aldrich, St. Louis, MO). Cells were seeded into poly-D-lysine–coated 75 cm2 flasks and maintained in culture at 37°C and 5%CO2. After 10–15 days in culture, the flasks were shaken (400 rpm,2hours, 37°C) to remove loosely adherentmicroglia. The remainingOPCspresent on top of the confluent monolayer of astrocytes were dislodged byshaking overnight at 400 rpm. The cell suspension was then filteredthrough a 10-mm nylon mesh and preplated on bacterial grade Petridishes for 2 hours. The nonadherent OPCs that remained in suspensionwere recoveredandplatedagainonbacterial gradePetri dishes for 1hour.The resulting enriched forebrain OPC cell suspension was centrifugedand resuspended in the Sato medium described above for OLG cultures.Cells were plated onto poly-D-lysine–coated 12-mm-diameter coverslipsin 24-well culture dishes at a density of 104 cells/well, and the purity ofoligodendroglial cultures was assessed by examining the characteristiccell morphologies under phase-contrast microscopy and confirmed byimmunostaining with cell type–specific antibodies. After 1 day in culture,platelet-derived growth factor receptora1 (PDGFRa1) OPCs represented976 5% of the total cells, and after 3 days in Sato medium, at least 98%were MBP1 cells.

Coculture of DRG Neurons with OLG. DRGs were dissectedfrom E15 rat embryos, dissociated with 0.25% trypsin and 0.4%DNAse at 37°C for 45 minutes, and mechanically triturated until asingle-cell suspension was obtained. The dissociated cells werepelleted and resuspended in DRG medium composed of Neurobasalmedium (Gibco; Thermo Scientific, Waltham,MA) supplemented with10% fetal bovine serum (Gibco), 50 ng/ml nerve growth factor (Invitrogen,Carlsbad, CA), and 2% B27 supplement (Invitrogen). The cells wereplated onto poly-D-lysine–coated Petri dishes for 30 minutes to remove

most of the fibroblasts and Schwann cells, which attach to the surface ofthe dish. The supernatant with neurons was removed and centrifuged,and the cells were plated onto poly-D-lysine–laminin coated coverslips ata density of 5� 104 cells per coverslip (50-ml drops). The cells were left inthe incubator overnight, after which they were covered with 450 ml ofDRG medium. To remove contaminating fibroblasts and glial cells, thecultures were pulsed twice with 10 mM cytosine arabinoside for 2 dayseach time. Following the last round of purification, the DRG neuronswere cultured for at least 2 weeks in DRG medium alone, with freshmedium added every 3 days.

For DRG cocultures, isolated OLGs (either from the optic nerve orforebrain) were plated on a 2- to 3-week-old DRG neuron culture at adensity of 2 � 104 cells/coverslip. The medium was changed to a 50:50mixture of Sato and DRG medium, without nerve growth factor, andthe cultures were left for different times in this medium to allow theoligodendroglial cells to associate with the DRG axons before theexperiments were done.

Immunocytochemistry. To characterize isolated OLGs or OLGscocultured with DRG neurons, the cells were immunostained withantibodies against oligodendroglial or neuronal cell-specific markers:mouse anti-O4 (10 mg/ml, Ref. MAB345; Chemicon International,Temecula, CA); rabbit anti-PDGFRa (1:200, Ref. 338; Santa CruzBiotechnology, Santa Cruz, CA); mouse anti-MBP to visualize myelinformation (1:100, Ref. SMI-99; Covance, Princeton, NJ); and rabbitantineurofilament to visualize neurites (1:200, Ref. 2837; Cell SignalingTechnology, Danvers, MA). In all cases except for the O4 antigen, thecells were fixed in 4% paraformaldehyde in phosphate-buffered saline(PBS) for 20 minutes at room temperature. The fixed cultures werepermeabilized with 0.1% Tween 20, blocked with 5% goat serum in PBSfor 30 minutes, and incubated overnight at 4°C, with the antibodiesdiluted in PBS containing 5% goat serumand 0.1%Tween 20. Then, thecells were rinsed and incubated for 2 hours at room temperature with1:200anti-mouse or anti-rabbit IgGs conjugatedwithAlexa 488 orAlexa594 (Molecular Probes, Eugene, OR). Coverslips were mounted on glassslides with fluorescent mounting medium (Glycergel; Dako, Glostrup,Denmark), and the preparationswere visualized under a laser scanningconfocalmicroscope (TCSSP8X; LeicaMicrosystems Inc., BuffaloGrove,IL) with a 40� objective.

For immunostaining of the O4 antigen, live cells were incubated for30minutes at 37°Cwithmouse anti-O4monoclonal antibodies dilutedin Satomedium. After being rinsed with PBS, the cells were fixed with4% paraformaldehyde before incubating for 2 hours with secondaryAlexa-594 anti-mouse IgM (1:200; Molecular Probes) in PBS contain-ing 5% goat serum. Then, the coverslips were washed, mounted onslides, and evaluated under a confocal microscope.

For immunostaining of surface GABAA receptor subunits, livecells were incubated with anti-GABAA antibodies (1:100; Alomone,Jerusalem, Israel) in culture medium at 37°C for 5 minutes. Doublelabeling with antibodies to specific subunits and oligodendrocytemarkers was done as described above for single staining by addingboth primary antibodies together. The absence of nonspecific interac-tions of secondary antibodies was verified by omitting the primaryantibodies.

Electron Microscopy. Ultrastructural localization of GABAA

receptors was carried out using pre-embedding immunohistochemis-try. Adult (10 weeks) Sprague-Dawley rats were deeply anesthetizedand transcardially perfused with 4% paraformaldehyde and 0.1%glutaraldehyde in 0.1 M PBS (pH 7.4) and postfixed in the samesolution for 2 hours. Vibratome sections (50 mm) of the rat brain werepreincubated in 10% goat serum in PBS (blocking solution) for 1 hour.Sections were then incubated with rabbit anti-GABAA antibodies(1:250; Alomone) in blocking solution for 2 days at 4°Cwith continuousshaking. After several washes in PBS, tissue sections were in-cubated with biotinylated horse anti-rabbit (1:200) and processed forimmunoperoxidase staining as indicated by the supplier (Vector;Palex Biomedical, Barcelona, Spain). Subsequently, sections wereincubated in 1% osmium tetroxide for 30 minutes, dehydrated, andembedded in Epon Resin 812 (Electron Microscopy Sciences, Hatfield,

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PA). Ultrathin sections were counterstained with lead citrate andexamined using a Philips EM208S (Philips Innovation Services,Eindhoven, The Netherlands) electron microscope at the AnalyticMicroscopy Service of the University of the Basque Country. Photo-graphs were taken using a digital camera coupled to the electronmicroscope, and minor adjustments in contrast and brightness of theimages weremade using Adobe Photoshop (Adobe Systems,MountainView, CA).

Electrophysiology. Whole-cell recordings were performed atroom temperature using the Axon 700B amplifier (Axon Instruments,Molecular Devices, Sunnyvale, CA). Currents were recorded at a holdingmembrane potential of 280 mV (unless otherwise stated), digitized,and stored for analysis using the A/D Converter Digidata1400 (AxonInstruments) and pClamp10 software (Axon Instruments). In mostcases, peak currents generated at 280 mV by drug superfusion wereused for the analysis. Current-voltage (I/V) relationships were built bychanging the membrane potential from2140 to140mV in 20-mV steps(250 milliseconds), the peak membrane current values at the begin-ning for each step were plotted. In some instances, cells were held at adesiredmembrane potential while GABAwas superfused, and the peakcurrents generated were I/V plotted. The extracellular bath solutionwith a pH of 7.3 contained the following (in mM): 140 NaCl, 5.4 KCl,2 CaCl2, 1 MgCl2, and 10 HEPES (Attali et al., 1997). Patch-clamppipettes (3–5 MV) were filled with an internal solution at pH 7.3containing the following (inmM): 140KCl, 2CaCl2, 2MgCl2, 10HEPES,11EGTA, 2Na-ATP, and 0.2GTP. The highK1 (HK1) external solutionwas prepared by replacing 20 mM NaCl with KCl in the externalsolution. Perforated whole-cell recording was performed using 5 mg/mlgramicidin added to the internal solution from a fresh stock solutionof gramicidin prepared in dimethylsulfoxide. Agonists and otherdrugs were added to the external solution from stock solutions.When necessary [e.g., diazepam (DZP; [7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2(1H)-one]) and loreclezole ((Z)-1-[2-chloro-2-(2,4-dichlorophenyl)ethenyl]-1H-1,2,4-triazole)], dimethylsulfoxidewas no more than 0.1% in the final solution. In some experiments, cellsunder patch-clamp recording were loaded with Lucifer yellow (2 mM)added to the internal solution for subsequentvisualization using confocalmicroscopy. OLGs in coculture were chosen for an electrical recordingbased on their close apposition to axons in areas thatwere otherwise freeof other cells. The OLG electrophysiological profile was confirmedsubsequently.

Fluorometry. Intracellular Ca21 concentration ([Ca21]i) changewas determined according to the method described previously(Grynkiewikcz et al., 1985). OLGs were loaded with Fluo4-AM(5 mM; Invitrogen) in culture medium for 30 minutes at 37°C. Cellswere washed in Hank’s balanced salt solution containing 20 mMHEPES, pH 7.4, 10 mM glucose, and 2 mM CaCl2 (incubation buffer)for 5 minutes at room temperature. Experiments were performed ina coverslip chamber continuously perfused with incubation buffer at1 ml/min. The perfusion chamber was mounted on the stage of a Zeiss(Oberkochen, Germany) inverted epifluorescence microscope(Axiovert 35) equipped with a 150 W xenon lamp Polychrome IV(T.I.L.L. Photonics, Martinsried, Germany) and a Plan Neofluar 40�oil immersion objective (Zeiss). Cells were visualized with a high-resolution digital black/white charge-coupled device camera (ORCA;Hamamatsu Photonics Iberica, Barcelona, Spain), and image acquisi-tion and data analysis were performed using the AquaCosmos softwareprogram (Hamamatsu Photonics Iberica). Data were analyzed withExcel (Microsoft, Seattle, WA) and Prism (GraphPad Software, SanDiego, CA) software.

Substances. Diazepam, 5-(2-fluorophenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one; 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol hydrochloride (THIP), loreclezole,4-ethyl-6,7-dimethoxy-9H-pyrido(3,4-b)indole-3-carboxylic acid methyl ester (DMCM),butyl b-carboline-3-carboxylate (b-CCB), and N-methyl-N-(3-[3-(2-thienylcarbonyl)pyrazolo(1,5-a)pyrimidin-7-yl]phenyl)acetamide(indiplon), were all from Tocris Bioscience (Bristol, UK). All saltsas well as cytosine arabinoside, GABA, glutamate, ATP, adenosine,

Fig. 1. Inward current responses elicited by neurotransmitters and HK+

solution of isolatedOLGs in culture. (A) Current responses elicited byGABA,Glut,ATP,and adenosine (ADE) (all at 1mM) aswell asHK+ solution in opticnerve OLGs maintained alone for 1 DIV (top row). In this and subsequentrecords, drugs orHK+were applied during the times indicated by bars at thetop, and cells were held at280mV. A similar protocol of response activationmonitored in OLGsmaintained for 3 DIV is illustrated in the bottom row. (B)Average (mean 6 S.E.M.) amplitude current response elicited by agonists(1 mM) and HK+ solution superfusion of OLGs (23–39 OLGs for each case)maintained alone for 1–3 DIV. (C) Inward current responses elicited inforebrainOLGs cultured alone in differentiationmediummonitored at 1DIV(top row) or 3 DIV (bottom row). Agonist concentrations were as in (A).

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tetrodotoxin, verapamil, bicuculline, gramicidin, and picrotoxin werefrom Sigma-Aldrich.

Statistical Analysis. Data are presented as the mean 6 S.E.M.The significance of differences between two datasets was tested withthe unpaired Student’s t test. For multiple comparisons, we usedanalysis of variance (ANOVA) (Tukey’s post hoc). In all instances,values with P , 0.05 were considered significant.

ResultsResponses to GABA Decrease Dramatically with

Time in Isolated OLGs In Vitro. Both optic nerve andforebrain OLGs were monitored electrophysiologically usingthe patch-clamp technique in cultured cells. Responses elicitedby the application of the neurotransmitters glutamate (Glut),ATP, adenosine, and GABA (all at 1 mM) were studied from 1to 3 DIV in cells held at280 mV (Fig. 1, A and B). In addition,the responses toHK1 solutionwere alsomonitored as an indexfor potassium current generation through inwardly rectify-ing K1 channels (Fig. 1) since they were fully blocked byextracellular 100 mM Ba21 (data not shown). As reportedearlier, at 1 DIV, OLGs responded to GABA-, Glut-, or ATP-generating inward currents (Berger et al., 1992; Matute et al.,2007). Applying adenosine instead did not elicit any electricalresponse (16 and 13 OLGs from the optic nerve and forebrain,respectively). The HK1 solution elicited stronger inwardcurrents at 1 DIV in optic nerve OLGs (476 6 123 pA; n 5 23)than in OLGs derived from the forebrain (38 6 25 pA; n 5 17)(Fig. 1C), a result that is consistent with previous studies(Sontheimer et al., 1989; Attali et al., 1997). Responsesgenerated by GABA, Glut, or ATP were associated with anincrease in membrane conductance, and their kinetic charac-teristics suggested the activation of their respective receptorchannels. With time in culture, OLGs from the optic nerveshowed a gradual and strong decrease in their sensitivity toGABA, from 868.8 6 76 pA at 1 DIV to 26.7 6 11 pA at 3 DIV(n 5 22 each). However, the responses to Glut and ATProughly maintained both their amplitude and general char-acteristics (40 6 12 pA and 15 6 3 pA, respectively) whileremaining electrically unresponsive to adenosine (Fig. 1, Aand B). Similarly, forebrain OLG responses to GABA de-creased (from 4386 87 to 166 4 pA; n5 16 in each case) after2 to 3 DIV, whereas Glut responses were unchanged, andcurrents generated by HK1 increased gradually with time(Fig. 1C). Thus, OLGs from the optic nerve and forebraincultured alone lost their sensitivity to GABA between 2 and 3DIV and did not recover it during the time monitored (up to 5DIV).GABA Responses Are Maintained in OLGs Cocultured

with DRG Neurons. OLGs grown in isolation may lackaxonal signals needed to sustain GABA signaling. To testthis possibility, we cocultured OLGs together with DRGneurons. Under these conditions, both cell types showedsimilar Glut and ATP responses that were stable with timein culture (data not shown) and had an amplitude and kineticssimilar to those observed in freshly isolated OLGs, regard-less of their origin. OLGs retained GABA sensitivity in thepresence of neurons (Fig. 2A). Indeed, the amplitude of theresponses to GABA tended to increase with time in culture.Thus, in four different cell preparations of optic nerve OLG-neuron cocultures, at 1 DIV, GABA responses had an averageamplitude of 11356 156 nA (n5 25), whereas at 3 DIV, it was

28246 303 nA (n5 41). Similarly, forebrain OLGs coculturedwithDRGneurons remained sensitive toGABA over time, andresponses at 1 and 3 DIV had an average amplitude of 583 695 pA (n 5 14) and 1998 6 227 pA (n 5 53), respectively (Fig.2A). This effect was maintained when current responses werenormalized for cell capacitance (Fig. 2B). Notably, coculturedoptic nerve or forebrain OLGs continued to have robust GABAresponses at 16 DIV (23406 560 pA; n5 8) or 11 DIV (17366349 pA; n 5 13), respectively.To explore the possibility that soluble factors released by

neurons sustained the expression and function of GABA recep-tors inOLG-neuron cocultures, we used amedium that had beenconditioned by DRG neurons for 7 days to culture OLGs andfound their GABA responses were similarly downregulated(Fig. 2B). Moreover, OLGs (n 5 11) cocultured at 3 DIV withneurons in the presence of 1 mM tetrodotoxin generatedresponses to 1mMGABA as robust as those in control cultures(1856 6 347 versus 2049 6 287 pA, corresponding to 72 6 13versus 78 6 11 pA/pF, respectively), which suggests that theexpression of receptors in OLGs was independent of basalaxonal electrical activity.Myelin Protein Expression and I/V Relationship in

OLGs Cocultured with DRG Neurons. Previous studiesshowed that OLGs engage in the myelination of axons whencocultured with DRG neurons (e.g., Chan et al., 2004; Wakeet al., 2011). To assess this feature in the cocultures used, we

Fig. 2. Inward current responses in OLGs cocultured with DRG neurons.(A) Membrane current responses elicited by 1 mM GABA or HK+ solutionin optic nerve and forebrain OLGs that were cocultured together with DRGneurons. Recordings were made on the third day in vitro. (B) Columnscorrespond to the average current in response toGABA (1mM) for cells 1–3DIV, normalized against the membrane capacitance in each condition:OLG cultured alone (2 DRG neurons), cocultured with DRG neurons(+ DRG neurons), OLGs cultured 3 DIV in control nonconditionedmedium (CtM), or OLGs cultured for 3 days in medium conditioned byDRG neurons for 7 days (CoM).

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analyzed the expression ofMBP as an index of maturation andevaluated the impact of myelination on the I/V relationship inOLGs in the critical period for GABA receptor expression (1and 3 DIV).At 1 DIV the vast majority of cells (.95%) in the cul-

tures derived from the optic nerve were O41, a marker forpremyelinatingOLGs, whereas the antibody against PDGFRalabeled .95% of cells in OLG cultures derived from theforebrain (Fig. 3A), indicating that at this stage (1 DIV), these

cultures contained mostly OPCs. Both OLG cultures pre-sented a robust expression of MBP at 3 DIV and thereafter,either when cultured alone or together with neurons (Fig. 3A).In the latter cultures, MBP1 processes were frequently seen incontact with axons, as visualized by neurofilament labeling.It is known that K1 currents in OLGs change during

maturation. Thus, OPCs present a nonlinear I/V relationshipdue to the expression of voltage-dependent K1 channels andfew or no inwardly rectifying K1 channels (Sontheimer et al.,

Fig. 3. Expression ofmarkers and I/V relation profileof OLGs cocultured with DRG neurons. (A) Imagesillustrate the expression of markers in optic nerveand forebrain OLGs cultured alone or cocultured withDRG neurons. The vast majority of isolated cells wereO4+ (optic nerve OLGs) or PDGFRa+ (forebrainOLGs) after 1 DIV (in red, first column). In both celltypes cultured alone, MBP (in red) was stronglyexpressed starting at 2 to 3 DIV (second column). Incocultures with DRG neurons (third and fourthcolumns), expression of MBP (in green) in OLGswas also observed at 2 to 3 DIV. Colabeling of axons[neurofilaments (NF-L) in red] and MBP (green)suggested a close interaction based on the extensiveoverlay of the myelinating protein signal with NF-L+

axons. Bar = 30 mm. (B) Graphs illustrate the I/Vrelationships of optic nerve OLGs cocultured withDRG neurons recorded during the first (d) and third(s) DIV. Traces are examples for each case of thevoltage-step protocol applied from2140 to +40 mV incells held at 280 mV. Data points are the averagepeak current (6 S.E.M.) recorded in 9–12 cells in eachcondition. (C) I/V relationships obtained in the sameconditions as in (B) for forebrain OLGs coculturedwith DRG neurons.

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1989; Berger et al., 1991; Attali et al., 1997). To assess thisproperty, OLGs were cocultured with DRG neurons andmonitored electrically by applying voltage steps from 2140to 140 mV. The resulting I/V relationships were built asillustrated in Fig. 3. Most optic nerve OLGs at 1 DIV showed anear-linear I/V relationship (Fig. 3B), whereas forebrainOLGs (Fig. 3C) displayed a strong rectification with low con-ductance for inward currents. Then, gradually, with longertimes in coculture (at 3 DIV) with neurons, the inwardcurrents increased in both OLG preparations and their I/Vrelationships became more linear. This change correlateddirectly with the current increase elicited by HK1 superfusionbetween 1 and 3DIV (1176 65%and 2986 81% for optic nerveand forebrain OLGs, respectively; n 5 31 in each case). Inaddition, forebrain OLG cultures, which at 1 DIV typicallypresented voltage-dependent outward currents with inacti-vating kinetics characteristic of voltage-dependent K1 chan-nels (Attali et al., 1997), progressively lost the inactivatingphase at 3 DIV (Fig. 3C). Together, these results indicate thatas OLGs mature, their myelinating and electrical propertiesbecome similar regardless of their regional origin.GABA Responses Elicited in OLGs in Close Contact

with Axons Versus OLGs without Contact. In cocultureswith neurons, many OLGs establish close contacts with axons,whereas others do not (Fig. 3). We wondered whether thesetwo populations of cells were similarly sensitive to GABA. Tothat end, we recorded the maximal response to GABA (1 mM)in cells contacting axons or not during the interval of 3–11DIV. Interestingly, cells in contact with axons had a strongresponse to GABA (n 5 54 and n 5 42 for optic nerve andforebrain OLGs, respectively), which was absent in cellsgrowing without direct apposition to the axons (n 5 23 andn 5 18 for optic nerve and forebrain OLGs, respectively; Fig.4A). These results clearly indicate that a direct neuron-gliainteraction is required to maintain the sensitivity to GABA.To characterize the morphology of GABA responding and

nonresponding cells, a group of OLGs was loaded with Luciferyellow during recording (Fig. 4B). We observed that OLGs incontact with axonal bundles typically developed parallelprocesses in contact with several individual axons, whereasOLGs without axon contacts had a generally more radialappearance. However, both subpopulations of cells showedrobust responses to HK1 solution and similar linear I/Vrelationships (Fig. 4C).Properties of GABA Receptors in OLGs Cocultured

with Neurons. Responses elicited by GABA in OLGs cocul-turedwith neuronswere studied inmore detail to characterizeand compare them with those reported previously in variousdifferent experimental conditions. GABA responses in OLGs(3–5DIV) were dose dependent and had anEC50 of 796 12mM(Fig. 5A). The current response inverted at136 2mV (n5 13)using the standard whole-cell patch-clamp configuration (Fig.5B). However, using the gramicidin-perforated whole-cellconfiguration, the inversion potential was at 224 6 3.5 mV(n 5 8) (Fig. 5B). This suggests that GABA generated celldepolarization, which was confirmed by monitoring the mem-brane potential in gramicidin-perforated cells. These cellsshowed an averagemembrane potential of2746 8mV, whichdepolarized to2326 6 mV (n5 13) in the presence of 100 mMGABA (Fig. 5C).GABA (100 mM) responses were mimicked by muscimol

but with greater potency. They were fully antagonized by

bicuculline, with an IC50 close to 1 mM (n5 9 and n5 6 for theoptic nerve and forebrain OLG, respectively, for both drugs)and blocked by 100 mM picrotoxin (n 5 6 OLGs) (Fig. 5, D–F).We also tested the effect of THIP (100–300 mM), a GABAanalog and potent agonist of GABAA receptors containingthe d subunit (Meera et al., 2011), and observed that 300 mMTHIP elicited weak responses of 48 6 17 pA (n 5 23), which

Fig. 4. GABA responses in OLGs with or without contact with axons. (A)Current response to 1 mMGABA was monitored in OLGs either in contact(+axon) or not (2axons) with axons. Conditions correspond to cells grownon the same coverslips. Each data point represents the response in a cellrecorded between 3 and 11 DIV. The average current response and S.E.M.are also shown for each group. Amplitude differences in the –axon groupwere significantly different from those in the +axon group (P , 0.05). (B)Images illustrate OLGs in contact with axons or without axonal contact.The OLGs were loaded with Lucifer yellow during a whole-cell electricalrecording. Bar = 20 mm. (C) Current responses displayed by cells shown in(B). The I/V relationship corresponds to voltage steps from2200 to +20mVin cells held at 280 mV.

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were 2.46 0.36% of those elicited byGABA (300mM) (Fig. 5G).Thus, GABA receptors in OLGs cocultured with neurons corre-sponded to the GABAA subtype, which was similar to thosedescribed in freshly isolated OLGs (Von Blankenfeld et al.,1991; see also, Bronstein et al., 1998), OPCs (Williamson et al.,1998), and OLGs from both optic nerve and corpus callosumslices (Berger et al., 1992) as well as in those expressed inNG21 cells from an adult brain (Lin and Bergles 2004).Next, we carried out an in-depth characterization of the

pharmacology of the GABAA receptors expressed in OLGscocultured with neurons (Fig. 6). GABAA currents (30 mM

GABA) were not sensitive to 10 mM indiplon (n 5 25), apyrazolopyrimidine and a positive allosteric modulator thatacts at the benzodiazepine-binding site (Petroski et al., 2006),suggesting that the GABAA receptor in OLGs did not containthe g2 subunit. Control experiments performed with DRGneurons showed that 10 mM indiplon potentiated their GABA(10 mM) response by 187.8 6 19% (n 5 7).Since sensitivity to indiplon is also highly determined by

the a subunit involved, we tested two classic benzodiaze-pines, DZP and flunitrazepam (FZP [5-(2-fluorophenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one]), and

Fig. 5. Dose-response (D-R) curve in response toGABAand I/V relationship in culturedOLGs. (A)The GABA response was elicited by applying thedistinct concentrations of the agonist as illus-trated, and the current recorded was normalizedagainst the current obtained at 1 mM for eachcell. The data points represent the averagenormalized current response. Recordings weredone in optic nerve OLGs cultured alone duringthe first day in vitro as well as in OLGscocultured with DRG neurons in the intervalfrom 1 to 8 DIV (n = 8). No difference in thepotency of GABA was observed either betweenthe later OLG groups or when the D-R curveswere built in similar experiments recordingforebrain OLGs. D-R curve adjustment parame-ters were EC50 = 796 12 mM andH = 1.1. (B) I/Vcurves were built for the GABA responses hold-ing the OLG membrane at distinct potentials asillustrated in each trace. Currents were moni-tored using the standard whole-cell recording(n = 13) or by “perforated” voltage-clamp usinggramicidin (n = 8), and the average peak currentwas plotted for each potential. Note that inperforated patch-clamp conditions, the GABAresponse inverted around 224 mV. (C) In perfo-rated patch-clamp recording, GABA elicited de-polarization of the resting membrane potential(black trace) in OLGs. The current response inthe same cell is also shown (gray trace) and wasdrawn inverted to compare the time courses. (D)The GABA response in OLGs was mimicked bymuscimol (gray traces), which was more potentthan GABA (black trace). (E) Bicuculline stronglyantagonized generation of the GABA response.Two different concentrations of the antagonistare illustrated (gray traces; GABA control re-sponse in black). (F) Picrotoxin (gray trace)blocked the generation of GABA responses (inblack). (G) The GABAA receptor agonist THIP(300 mM, gray trace) did not mimic the GABA(300 mM) current response.

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found that DZP (10 mM) produced a potentiation of 159.5 65% (n 5 27) when coapplied with 30 mM GABA. Likewise,FZP (10 mM) potentiated the response to 30 mM GABA by196.2 6 13.8% (n 5 10). However, potentiation elicited by 1–10 mM DZP was reduced or absent when it was tested at 50–100 mM GABA, a concentration around the EC50, in whichcase responses were 98 6 3% (n 5 25). Also, drugs derivedfrom b-carboline, which exert their effects through binding tothe benzodiazepine site affected the GABA response inOLGs; thus, DMCM and b-CCB (both at 10 mM), two drugsthat frequently act as inverse agonists, produced responseinhibition (67.7 6 6%, n 5 17) and potentiation (237 6 21%,n5 13), respectively. These results indicated that in OLGs, theGABAA receptor contains a binding site for benzodiazepines.On the other hand, loreclezole (10mM) increased the current

to 30 mM GABA, producing a modest but consistent currentpotentiation to 135 6 8% (n 5 15), suggesting that b2/b3subunits contribute to native GABAA receptors in OLGs.Finally, we found that Zn21 strongly inhibited the GABA(100 mM) response, with an IC50 of 8.35 6 1.9 mM (n 5 11),whereas coapplication of 100mMLa31 had no significant effect(97.5 6 2% compared with the control response; n 5 6). Thepharmacological characteristics of OLGs in coculture withneurons at 3–15 DIV were similar to those displayed by OLGscultured alone and monitored during the first day in vitro.Increase of Ca21 Elicited by GABAA Receptor in

Myelinating OLGs. GABAstimulationofOPCselicitsa [Ca21]iincrease when cultured alone (Kirchhoff and Kettenmann1992). Accordingly, we observed that GABA (0.1–1 mM) in-duced a rise of basal Ca21 levels in OLGs from the optic nerveand the forebrain at 1 DIV, a response that was absent at laterstages in culture (2–5 DIV). Responses at 1 DIV were observedin 34 6 10% of the cells and were inhibited when GABA wascoapplied with bicuculline (Fig. 7, A and B) or picrotoxin. Incontrast, OLGs cocultured with DRG neurons had a robust[Ca21]i increase (80 6 3% of the cells were responsive) inresponse to GABA (100 mM), which was not lost with timein culture (1–6 DIV). Thus, these data indicated that OLGs

coculturedwithneurons preserve themachinery that generatesthe Ca21 response elicited by GABA. Moreover, the [Ca21]iincreasewas absent inmedium devoid of Ca21 (n5 15; Fig. 7, Cand D). Verapamil (100 mM; n5 15) strongly reduced the Ca21

increase (Fig. 7E), confirming amajor role of voltage-dependentCa21 channels in the response, and the increase was abolishedby picrotoxin and bicuculline (Fig. 7F).Expression of GABAA Subunits in OLGs In Vitro and

In Situ. Freshly isolated OPCs as well as myelinating andnonmyelinating OLGs express high levels of GABAA receptora1 and a3 subunit transcripts and intermediate to low levelsof b and g subunits (Cahoy et al., 2008). Accordingly, we foundthat OLGs cultured alone expressed a1 and a3 subunits at 1DIV by immunofluorescence; however, the presence of thesesubunits decreased below detection levels at later stages inculture (3–5 DIV; Fig. 8A). In contrast, in neuron-OLGcocultures (5 DIV or more), levels of a1 and a3 subunitsremained stable in OLGs that were in close apposition toaxons but not in those lacking such an interaction (Fig. 8B).These immunocytochemical data are consistent with thefunctional studies described above using electrophysiologyandCa21 imaging as well as with electronmicroscopy showingintense immunoperoxidase staining of a1 and a3 subunitsin interfascicular OLGs in situ in subcortical white matter(Fig. 9).

DiscussionIn this study, we provide evidence that OLGs are endowed

with functional GABAA receptors, which are readily lost whenOLGs are cultured in isolation, but can be maintained bydirect axon-to-OLG interaction in cocultures with neurons.The GABA responses observed in our study are similar tothose described in isolated OLGs (Von Blankenfeld et al.,1991) and their precursors (Williamson et al., 1998) as well asin OLGs in optic nerve and corpus callosum slices (Bergeret al., 1992). In addition, theGABAA receptor-mediated currents

Fig. 6. Pharmacology of GABAA receptors expressed in OLGs in coculture with DRG neurons. (A) Traces illustrate the effect provoked by distinct drugscoapplied with 30mMGABA (around the EC30 of the response). Each set of traces included the control response elicited by GABA alone or in coapplicationwith a drug (10 mM) and the recovery of the GABA response after washing. The interval between applications was 80–100 seconds of continuous externalsolution superfusion. (B) The graph shows the effect of the drugs illustrated in (A) on the GABA response, and the effects elicited by FZP and THIP (as inFig. 5G). All the effects were normalized against the corresponding response to 30 mM GABA alone (13–19 OLGs for each drug), with the exception of300 mMTHIP (n = 6), which was normalized against the current obtained by 300 mMGABA in the same cells. All groups, with the exception of indiplon,were significantly different from the GABA control response (P , 0.05).

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described here resemble those reported in NG21 cells from theadult brain (Lin and Bergles 2004).GABAA receptors in OLGs undergo a strong downregulation

as the cells mature (e.g., Berger et al., 1992), a feature that isconfirmed by our data in isolated OLGs. However, in apparentcontradiction with that data, we found that GABAA receptorexpression and function remain stable over time in OLGs thatare in contact with axons, and that a subunits contributing tonative GABAA receptors are present in interfascicular OLGsof the adult brain.Neuron-OLG cocultures have been extensively used to

study the mechanisms leading to myelination of the axons(e.g., Chan et al., 2004; Laursen et al., 2009;Wake et al., 2011).Nevertheless, the properties of the neurotransmitter recep-tors in OLGs have not been thoroughly studied. In the DRGneuron–OLG in vitro preparation used in our study, weobserved that GABAA receptor expression required a closeinteraction between OLGs and axons since OLGs withoutaxonal contacts in the same cultures lost their sensitivity toGABA, and the addition of neuronal conditioned medium toisolated OLGs did not result in the recovery of the GABAresponses. The electrophysiological and pharmacologicalproperties of GABAA receptors in OLGs cocultured for severaldays were similar to those recorded in isolated cells at 1 DIV,suggesting that the subunit composition remains similar or

identical during the period maintained in culture. In addition,the increase in [Ca21]i generated by GABA stimulation wasalso well maintained in OLGs in coculture. The pharmacologyof the Ca21 response strongly suggested that it was due to theactivation of voltage-dependent Ca21 channels triggered byGABA-mediated depolarization, similar to that shown pre-viously in isolated OLGs (Kirchhoff and Kettenmann, 1992).This indicates that both a depolarizing Cl2 gradient andexpression of voltage-dependent Ca21 channels are present inmyelinating OLGs.GABAA receptors are heteromeric in nature and formed

by five subunits encoded by 19 known genes that are ro-bustly expressed in the central nervous system. There are nopharmacological tools that conclusively distinguish amongdistinct possible combinations and therefore we used a batteryof ligands to make a preliminary characterization of theGABAA receptors expressed in OLGs in isolation (1 DIV) orin contact with axons (2–7 DIV). We found that the pharma-cology of the GABA responses was similar in all the culturesassayed, which suggested that OLGs may all have a specificsubtype of GABAA receptors, regardless of their origin (fore-brain or optic nerve) or culture conditions (alone or togetherwith DRG neurons). We also confirmed that the GABAA

receptors in OLGs may have a unique molecular composition(see, e.g., Velez-Fort et al., 2012). First, we observed a very

Fig. 7. GABA-evoked [Ca2+]i increase in OLGscocultured with DRG neurons. Changes in thefluorescence ratio, as an indication of [Ca2+]i, wererecorded during the application of different ago-nists and drugs to isolated OLGs or in coculturewith DRG neurons. In (A), isolated cells (1 DIV)were exposed to GABA 100 mM and Ca2+ levelsweremeasured in control conditions and (B) in thepresence of bicuculline 100 mM. GABA induced arapid increase in [Ca2+]i, an effect that wasblocked by the inhibitor. Data are presented asthe mean 6 S.E.M. of 10 cells on the samecoverslip. (C) Similar recordings were made inOLGs in coculture with DRG neurons (3–6 DIV).GABA (100 mM) recordings were obtained with anexternal solution containing 2 mM Ca2+. Pairedresponses to GABA were obtained from the samecells and were separated by 60-second intervals.(D) Application of GABA (100 mM) in Ca2+-freesolution eliminated the response. (E) Coapplica-tion of verapamil and GABA (100 mM) alsoinhibited the Ca2+ response. (F) The [Ca2+]i in-crease during the second application of GABA indifferent cells was normalized against the peakresponse. The average amplitude of this responsewas also monitored in the presence of the GABAAchannel blocker picrotoxin (100 mM) or the specificantagonist bicuculline (100 mM) (*P, 0.05; ***P,0.01).

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weak response to THIP, indicating absence of the d subunit(Meera et al., 2011). This is consistent with the fact that the dsubunit confers sensitivity to inhibition by lanthanum (Saxenaet al., 1997), and this polycation had no effect on the OLGGABAA receptor. In sharp contrast to the lack of effect byTHIP in OLGs, it has been shown that OPCs in the adulthippocampus do respond to this analog (Lin and Bergles,2004). Second, potentiation by loreclezole strongly suggests aninvolvement of the b2/b3 subunits since receptors containingthe b1 subunit are insensitive to loreclezole (Wafford et al.,1994). Third, the sensitivity to benzodiazepines (DZP andFZP) favors the idea of g subunit contribution (Olsen andSieghart, 2008); however, the response to GABA is insensitiveto indiplon and it is inhibited by Zn21 (Hosie et al., 2003),both of which suggest that the benzodiazepine-binding siteexpressed in OLGs may not involve the g2 subunit. We alsoobserved that DZP potentiation only occurs at low GABA(#EC30) concentrations. This effectmight explain the apparentdiscrepancy observed in previous studies with respect to thebenzodiazepine sensitivity of cells from the oligodendrogliallineage since different GABA concentrations were applied.In one case using # EC30 (Von Blankenfeld et al., 1991),potentiationwas reported for FZP, and in other cases applying

GABA $ EC50, no effect was observed for FZP (Williamsonet al., 1998) or DZP (Bronstein et al., 1998) (see also Vélez-Fortet al., 2012). Nonetheless, the presence of the benzodiazepinesite in GABAA receptors in OLGs is further supported by theeffects of b-carbolines, which are postulated to be endogenousbenzodiazepine site modulators (Peña et al., 1986). Thus,DMCM reduced the GABA response as a typical inverseagonist, in agreement with its known properties in otherbenzodiazepine sites, whereas b-CCB acted to increase theOLG GABA response, an effect that deserves further study(Peña et al., 1986; Rigo et al., 1994).It is noteworthy that either g1 or g3 subunits confer

benzodiazepine sensitivity to receptors in heterologous ex-pression studies (Knoflach et al., 1991; Puia et al., 1991), andthat receptors with these subunits have also been shown to beinsensitive to other pyrazolopyrimidine molecules, such aszolpidem (e.g., Puia et al., 1991), in a way similar to thatshown here for indiplon. Thus, potentiation by benzodiaze-pines and insensitivity to indiplon are consistent with theinvolvement of g1 or g3 subunits in the GABAA receptorexpressed in OLGs. An inconsistency seems to emerge withthe inhibitory effect of Zn21 since there is no evidenceindicating that receptors with g1 or g3 subunits might be

Fig. 8. Immunocytochemistry of GABAA-a subunits inOLGs maintained in vitro. (A) OLGs maintained for 1 DIVshowed reactivity to antibodies against the a1 or a3 subunits(in green) of GABAA receptors. O4 antibodywas used to label(in red) premyelinating OLGs at 1 DIV, whereas MBP (inred) expression was observed from 3 to 5 DIV. Immunor-ecognition of a1 and a3 subunits (in green) was lost in OLGscultured alone for 3–5 DIV. Bar = 20 mm. (B) Labeling ofsubunits a1 and a3 (in green) was maintained in OLGscoculturedwithDRGneurons in cell cultures kept over 5DIVin contact with (+) axons. Most of these cells also showedstrong MBP immunoreactivity (in red). OLGs in the samecoculture without axonal contact lost the label for a subunitsbut not that for MBP. Bar = 20 mm.

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sensitive to Zn21, mainly because the residues involved inZn21 binding site disruption are conserved in the three gsubunits of GABAA receptors (Hosie et al., 2003; Trudell et al.,2008); clearly, further analysis is required to solve thisimportant issue.It is also important to note that these OLG receptors

presented a rather high EC50 for GABA (close to 80 mM)compared withmany other receptors expressed in the nervoussystem. This characteristic suggests an involvement of the a3subunit (Williamson et al., 1998) since in heterologous ex-pression studies, this subunit confers a low sensitivity toGABA receptors when coexpressed with either b2 (Sigel et al.,1990) or b3 subunits (Böhme et al., 2004).On the other hand, the pharmacological profile of the

GABAA receptors observed in our study corresponds wellwith the functional genomics analysis carried out in OLGs,which shows a high expression of a1 and a3, along withmoderate levels of b2 or b3 subunits and low levels of g1 andg2 subunits (Cahoy et al., 2008). Accordingly, immunofluo-rescence studies in vitro demonstrated the presence of a1 anda3 subunits in OLGs cocultured with neurons, and immuno-peroxidase staining of adult brain sections showed intenselabeling of these subunits in interfascicular OLGs. Togetherwith functional studies, our results suggest a contribution ofa3, b2/b3, and g1/g3 subunits to the GABAA receptors inOLGs. However, this does not exclude the involvement ofother subunits since the final pharmacological propertiesresult from a complex interplay conferred by multisubunitparticipation in binding sites formation for either GABA,benzodiazepine, or Zn21 (Olsen and Sieghart, 2008; Sieghart,2015). In addition, during the period of postnatal develop-ment, extensive changes in the expression for different

GABAA subunits in the nervous system occur (Laurie et al.,1992). Thus, the characteristics for GABAA receptors expressedby OLGs in culture conditions at this age must be confirmed inmore integral preparations.The axon-to-OLG signals regulating the stabilization

of OLG GABAA receptors remain unknown. Blockade ofneuronal activity with tetrodotoxin did not change OLGsensitivity to GABA, which suggests that basal neuronalactivity was not necessary to drive GABAA receptor expres-sion in OLGs. Conversely, chronic activation of GABAA

receptors with GABA or their blockade by either Zn21 orbicuculline in cerebellar organotypic cultures did not alterMBP expression levels (data not shown). Nevertheless, GABAA

receptors in OLGs and their precursors may be crucial forproper myelination and remyelination after injury since dis-ruption of GABAergic communication in NG2-expressing pro-genitor cells during cerebellar development results in a delay ofOLG maturation and dysmyelination (Zonouzi et al., 2015).In summary, we have shown here that premyelinating and

myelinating OLGs from the optic nerve and forebrain incontact with axons have a robust sensitivity to GABA, whichacts on a GABAA receptor subtype, with distinctive functionaland pharmacological characteristics. Expression of GABAA

receptors in OLGs was controlled by a mechanism thatdepended on their contactwith axons during the premyelinatingstage. GABAA receptor activation causes depolarization ofOLGs, and an increase in [Ca21]i that may be relevant formyelination and remyelination after injury.

Acknowledgments

The authors thank Saioa Marcos and Dr. Edith Garay for technicalassistance, and Dr. Dorothy D. Pless for editing the manuscript. R.O.A.was supported by Ikerbasque, Bizkaia Talent, and the Programa deApoyos para la Superación del Personal Académico de la UniversidadNacional Autónoma de México.

Authorship contributions

Participated in research design: Arellano, Matute, Sánchez-Gómez.Conducted experiments: Arellano, Matute, Sánchez-Gómez,

Alberdi, Canedo-Antelo, Pérez-Samartín, Chara, Palomino.Performed data analysis: Arellano, Matute, Sánchez-Gómez,

Alberdi, Canedo-Antelo, Chara, Palomino.Wrote or contributed to the writing of the manuscript: Arellano,

Matute, Sánchez-Gómez, Pérez-Samartín.

References

Attali B, Wang N, Kolot A, Sobko A, Cherepanov V, and Soliven B (1997) Charac-terization of delayed rectifier Kv channels in oligodendrocytes and progenitor cells.J Neurosci 17:8234–8245.

Barres BA, Hart IK, Coles HS, Burne JF, Voyvodic JT, Richardson WD, and Raff MC(1992) Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70:31–46.

Berger T, Schnitzer J, and Kettenmann H (1991) Developmental changes in themembrane current pattern, K1 buffer capacity, and morphology of glial cells in thecorpus callosum slice. J Neurosci 11:3008–3024.

Berger T, Walz W, Schnitzer J, and Kettenmann H (1992) GABA- and glutamate-activated currents in glial cells of the mouse corpus callosum slice. J Neurosci Res31:21–27.

Böhme I, Rabe H, and Lüddens H (2004) Four amino acids in the a subunits de-termine the g-aminobutyric acid sensitivities of GABAA receptor subtypes. J BiolChem 279:35193–35200.

Boulanger JJ and Messier C (2014) From precursors to myelinating oligodendrocytes:contribution of intrinsic and extrinsic factors to white matter plasticity in the adultbrain. Neuroscience 269:343–366.

Bronstein JM, Hales TG, Tyndale RF, and Charles AC (1998) A conditionally im-mortalized glial cell line that expresses mature myelin proteins and functionalGABA(A) receptors. J Neurochem 70:483–491.

Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y,Lubischer JL, Krieg PA, and Krupenko SA et al. (2008) A transcriptome databasefor astrocytes, neurons, and oligodendrocytes: a new resource for understandingbrain development and function. J Neurosci 28:264–278.

Fig. 9. Electronmicroscopy immunolabeling of GABAA-a1 andGABAA-a3subunits in mature OLGs of adult rats. (A and B) Subcortical white matterOLGs stained with immunoperoxidase against the GABAA a1 or a3subunit, respectively. Note the abundant electron-dense precipitate(insets) in the perykarion and membrane and the numerous myelinatedaxons surrounding the OLG cell bodies.

GABAA Receptor Expression in Myelinating Oligodendrocytes 73

at ASPE

T Journals on D

ecember 23, 2020

molpharm

.aspetjournals.orgD

ownloaded from

Chan JR, Watkins TA, Cosgaya JM, Zhang C, Chen L, Reichardt LF, Shooter EM,and Barres BA (2004) NGF controls axonal receptivity to myelination by Schwanncells or oligodendrocytes. Neuron 43:183–191.

Domercq M, Perez-Samartin A, Aparicio D, Alberdi E, Pampliega O, and Matute C(2010) P2X7 receptors mediate ischemic damage to oligodendrocytes. Glia 58:730–740.

Etxeberria A, Mangin JM, Aguirre A, and Gallo V (2010) Adult-born SVZ progenitorsreceive transient synapses during remyelination in corpus callosum. Nat Neurosci13:287–289.

Franklin RJ, ffrench-Constant C, Edgar JM, and Smith KJ (2012) Neuroprotectionand repair in multiple sclerosis. Nat Rev Neurol 8:624–634.

Gallo V and Armstrong RC (2008) Myelin repair strategies: a cellular view. CurrOpin Neurol 21:278–283.

Gallo V, Zhou JM, McBain CJ, Wright P, Knutson PL, and Armstrong RC (1996)Oligodendrocyte progenitor cell proliferation and lineage progression are regulatedby glutamate receptor-mediated K1 channel block. J Neurosci 16:2659–2670.

Grynkiewicz G, Poenie M, and Tsien RY (1985) A new generation of Ca21 indicatorswith greatly improved fluorescence properties. J Biol Chem 260:3440–3450.

Gudz TI, Komuro H, and Macklin WB (2006) Glutamate stimulates oligodendrocyteprogenitor migration mediated via an alphav integrin/myelin proteolipid proteincomplex. J Neurosci 26:2458–2466.

Hosie AM, Dunne EL, Harvey RJ, and Smart TG (2003) Zinc-mediated inhibition ofGABA(A) receptors: discrete binding sites underlie subtype specificity. Nat Neu-rosci 6:362–369.

Hoppe D and Kettenmann H (1989) GABA triggers a Cl- efflux from cultured mouseoligodendrocytes. Neurosci Lett 97:334–339.

Ishibashi T, Dakin KA, Stevens B, Lee PR, Kozlov SV, Stewart CL, and Fields RD (2006)Astrocytes promote myelination in response to electrical impulses. Neuron 49:823–832.

Káradóttir R and Attwell D (2007) Neurotransmitter receptors in the life and deathof oligodendrocytes. Neuroscience 145:1426–1438.

Kirchhoff F and Kettenmann H (1992) GABA triggers a [Ca21]i increase in murineprecursor cells of the oligodendrocyte lineage. Eur J Neurosci 4:1049–1058.

Knoflach F, Rhyner T, Villa M, Kellenberger S, Drescher U, Malherbe P, Sigel E,and Möhler H (1991) The g 3-subunit of the GABAA-receptor confers sensitivity tobenzodiazepine receptor ligands. FEBS Lett 293:191–194.

Laurie DJ, Wisden W, and Seeburg PH (1992) The distribution of thirteen GABAAreceptor subunit mRNAs in the rat brain. III. Embryonic and postnatal develop-ment. J Neurosci 12:4151–4172.

Laursen LS, Chan CW, and ffrench-Constant C (2009) An integrin-contactin complexregulates CNS myelination by differential Fyn phosphorylation. J Neurosci 29:9174–9185.

Lin SC and Bergles DE (2004) Synaptic signaling between GABAergic interneuronsand oligodendrocyte precursor cells in the hippocampus. Nat Neurosci 7:24–32.

Luyt K, Slade TP, Dorward JJ, Durant CF, Wu Y, Shigemoto R, Mundell SJ, VáradiA, and Molnár E (2007) Developing oligodendrocytes express functional GABA(B)receptors that stimulate cell proliferation and migration. J Neurochem 100:822–840.

McCarthy KD and de Vellis J (1980) Preparation of separate astroglial and oligo-dendroglial cell cultures from rat cerebral tissue. J Cell Biol 85:890–902.

Matute C, Torre I, Pérez-Cerdá F, Pérez-Samartín A, Alberdi E, Etxebarria E, ArranzAM, Ravid R, Rodríguez-Antigüedad A, and Sánchez-Gómez M et al. (2007) P2X(7)receptor blockade prevents ATP excitotoxicity in oligodendrocytes and amelioratesexperimental autoimmune encephalomyelitis. J Neurosci 27:9525–9533.

Meera P, Wallner M, and Otis TS (2011) Molecular basis for the high THIP/gaboxadolsensitivity of extrasynaptic GABA(A) receptors. J Neurophysiol 106:2057–2064.

Olsen RW and Sieghart W (2008) International Union of Pharmacology. LXX. Sub-types of gamma-aminobutyric acid(A) receptors: classification on the basis of

subunit composition, pharmacology, and function. Update. Pharmacol Rev 60:243–260.

Peña C, Medina JH, Novas ML, Paladini AC, and De Robertis E (1986) Isolationand identification in bovine cerebral cortex of n-butyl b-carboline-3-carboxylate,a potent benzodiazepine binding inhibitor. Proc Natl Acad Sci USA 83:4952–4956.

Petroski RE, Pomeroy JE, Das R, Bowman H, Yang W, Chen AP, and Foster AC(2006) Indiplon is a high-affinity positive allosteric modulator with selectivity fora1 subunit-containing GABAA receptors. J Pharmacol Exp Ther 317:369–377.

Puia G, Vicini S, Seeburg PH, and Costa E (1991) Influence of recombinantg-aminobutyric acid-A receptor subunit composition on the action of allostericmodulators of g-aminobutyric acid-gated Cl- currents. Mol Pharmacol 39:691–696.

Rigo JM, Belachew S, Lefebvre PP, Leprince P, Malgrange B, Rogister B,Kettenmann H, and Moonen G (1994) Astroglia-released factor shows similareffects as benzodiazepine inverse agonists. J Neurosci Res 39:364–376.

Saxena NC, Neelands TR, and MacDonald RL (1997) Contrasting actions of lan-thanum on different recombinant g-aminobutyric acid receptor isoforms expressedin L929 fibroblasts. Mol Pharmacol 51:328–335.

Sieghart W (2015) Allosteric modulation of GABAA receptors via multiple drug-binding sites. Adv Pharmacol 72:53–96.

Sigel E, Baur R, Trube G, Möhler H, and Malherbe P (1990) The effect of subunitcomposition of rat brain GABAA receptors on channel function. Neuron 5:703–711.

Sontheimer H, Trotter J, Schachner M, and Kettenmann H (1989) Channel expres-sion correlates with differentiation stage during the development of oligodendro-cytes from their precursor cells in culture. Neuron 2:1135–1145.

Trudell JR, Yue ME, Bertaccini EJ, Jenkins A, and Harrison NL (2008) Molecularmodeling and mutagenesis reveals a tetradentate binding site for Zn21 in GABA(A) alphabeta receptors and provides a structural basis for the modulating effect ofthe g subunit. J Chem Inf Model 48:344–349.

Vélez-Fort M, Audinat E, and Angulo MC (2012) Central role of GABA in neuron-gliainteractions. Neuroscientist 18:237–250.

Von Blankenfeld G, Trotter J, and Kettenmann H (1991) Expression and de-velopmental regulation of a GABAA receptor in cultured murine cells of the oli-godendrocyte lineage. Eur J Neurosci 3:310–316.

Wafford KA, Bain CJ, Quirk K, McKernan RM, Wingrove PB, Whiting PJ, and KempJA (1994) A novel allosteric modulatory site on the GABAA receptor b subunit.Neuron 12:775–782.

Wake H, Lee PR, and Fields RD (2011) Control of local protein synthesis and initialevents in myelination by action potentials. Science 333:1647–1651.

Williamson AV, Mellor JR, Grant AL, and Randall AD (1998) Properties of GABA(A)receptors in cultured rat oligodendrocyte progenitor cells. Neuropharmacology 37:859–873.

Zatorre RJ, Fields RD, and Johansen-Berg H (2012) Plasticity in gray and white:neuroimaging changes in brain structure during learning. Nat Neurosci 15:528–536.

Zonouzi M, Scafidi J, Li P, McEllin B, Edwards J, Dupree JL, Harvey L, Sun D,Hübner CA, and Cull-Candy SG et al. (2015) GABAergic regulation of cerebellarNG2 cell development is altered in perinatal white matter injury. Nat Neurosci 18:674–682.

Address correspondence to: Dr. Rogelio O. Arellano, Instituto de Neuro-biología, Universidad Nacional Autónoma de México, Boulevard Juriquilla 3001,Juriquilla Querétaro, C.P. 76230, México. E-mail: arellano.ostoa@comunidad.unam.mx or Dr. Carlos Matute, Departamento Neurociencias, Universidad delPaís Vasco, E-48940 Leioa, Spain. E-mail: carlos.matute@ehu.es

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