GABAB receptor agonist baclofen promotes central nervous system remyelination

ORIGINAL RESEARCH ARTICLE GABA B receptor agonist baclofen promotes central nervous system remyelination Mari Paz Serrano-Regal 1,2 | Laura Bay on-Cordero 1,2,3 | Juan Carlos Chara Ventura 1,2,3 | Blanca I. Ochoa-Bueno 1,2 | Vanja Tepavcevic 1 | Carlos Matute 1,2,3 | María Victoria Sánchez-G omez 1,2,3 1 Laboratory of Neurobiology, Achucarro Basque Center for Neuroscience, Leioa, Spain 2 Department of Neurosciences, University of the Basque Country (UPV/EHU), Leioa, Spain 3 Centro de Investigaci on Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Leioa, Spain Correspondence María Victoria Sánchez-G omez and Carlos Matute, Department of Neurosciences, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940, Leioa, Spain. Email: [email protected]us and carlos. [email protected] Present address Mari Paz Serrano-Regal, Grupo de Neuroinmuno-Reparaci on, Hospital Nacional de Parapléjicos-SESCAM, Toledo, Spain. Funding information CIBERNED, Grant/Award Number: CB06/05/0076; Basque Government, Grant/Award Numbers: IT1203-19, IT702-13; Ministry of Economy and Competitiveness, Government of Spain, Grant/Award Numbers: SAF2015-74332-JIN, PID2019-109724RBI00, SAF2016-75292-R, SAF2013-45084-R Abstract Promoting remyelination is considered as a potential neurorepair strategy to prevent/ limit the development of permanent neurological disability in patients with multiple sclerosis (MS). To this end, a number of clinical trials are investigating the potential of existing drugs to enhance oligodendrocyte progenitor cell (OPC) differentiation, a process that fails in chronic MS lesions. We previously reported that oligodendroglia express GABA B receptors (GABA B Rs) both in vitro and in vivo,and that GABA B R-mediated signaling enhances OPC differentiation and myelin protein expression in vitro. Our goal here was to evaluate the pro-remyelinating potential of GABA B R agonist baclofen (Bac), a clinically approved drug to treat spasticity in patients with MS. We first demonstrated that Bac increases myelin protein production in lysolecithin (LPC)-treated cerebellar slices. Importantly, Bac administration to adult mice following induction of demyelination by LPC injection in the spinal cord resulted in enhanced OPC differentiation and remyelination. Thus, our results suggest that Bac repurposing should be considered as a potential therapeutic strategy to stimulate remyelination in patients with MS. KEYWORDS baclofen, GABA B receptor, multiple sclerosis, myelin, oligodendrocyte, remyelination 1|INTRODUCTION Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by disseminated demyelination (Dendrou et al., 2015). As a consequence of inflammatory demyelination, action potential conduction is disrupted and axons are deprived from metabolic and trophic support, which leads to axonal loss (Lee et al., 2012; Saab et al., 2016), the main correlate of permanent disability in patients with MS (Trapp et al., 1999). The majority of currently available treatments for MS target CNS inflammation and associated relapses, but do not prevent long-term disability (Kremer, Akkermann, et al., 2019). Thus, the development of therapies to prevent axonal and neuronal loss remains an unmet therapeutic need for patients with MS (Lubetzki et al., 2020). Remyelination is the spontaneous regeneration of myelin that prevents axonal degeneration both in animal models (Irvine & Blakemore, 2008; Mei et al., 2016) and Mari Paz Serrano-Regal and Laura Bay on-Cordero contributed equally to this work. Received: 27 February 2022 Revised: 25 July 2022 Accepted: 3 August 2022 DOI: 10.1002/glia.24262 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2022 The Authors. GLIA published by Wiley Periodicals LLC. Glia. 2022;1–15. wileyonlinelibrary.com/journal/glia 1 patients with MS (Kornek et al., 2000). However, in most patients, the efficiency of this process decreases significantly with age and disease progression (Franklin and ffrench-Constant, 2017). Therefore, the development of novel treatments that enhance remyelination is a major goal of current MS research, and includes the repurposing of existing drugs (Kremer, Göttle, et al., 2019). A block in OPC differentiation (Kotter et al., 2006;Kuhlmann et al., 2008) and lack of myelin sheath formation by surviving mature oligodendrocytes (OLs) have been pointed out as important contributors to remyelination failure in MS (Duncan et al., 2018; Yeung et al., 2019;Heb et al., 2020;Franklinetal.,2021). Therefore, promoting OPC differentiation and improving OL myelination capacity are potential strategies for enhancing myelin repair and preventing neurodegenerationinthisdisease. Neurotransmitters are important mediators of OPC-neuron communication with a clear influence on OPC behavior (Domercq et al., 2010; Fannon et al., 2015; Hamilton et al., 2017; Li et al., 2013; Serrano-Regal, Luengas-Escuza, et al., 2020; Zonouzi et al., 2015). As OPCs receive both excitatory and inhibitory synaptic inputs, mediated by glutamate and GABA (Bergles et al., 2000; Kárad ottir et al., 2008; Kukley et al., 2008; Lin & Bergles, 2004), these molecules have been identified as key regulators of oligodendroglial maturation and myelination (Bai et al., 2021; Fannon et al., 2015; Gautier et al., 2015; Serrano-Regal, Bay on-Cordero, et al., 2020). Regarding myelin repair, GABAergic signaling through GABA A Rs has been associated with remyelination after focal demyelination in the rat corpus callosum (Kalakh & Mouihate, 2019), as well as in the caudal cerebellar peduncle (Cisneros-Mejorado et al., 2020). GABA B Rs have also been suggested as important modulators of myelination given that GABA B R antagonism increased OPC proliferation while decreasing their maturation and the production of myelin-related proteins in the developing rat cingulum (Pudasaini et al., 2022). However, the role of oligodendroglial GABA B Rs in myelin regeneration remains to be investigated. Baclofen (Bac), the best known GABA B R agonist, is currently used as a therapeutic agent for spasticity in MS, and can be administered either intrathecally or orally because it crosses the blood–brain barrier (Ertzgaard et al., 2017). We previously reported that GABA B R activation by Bac promotes differentiation and myelin protein expression in rat cortical OPC (Serrano-Regal, Luengas-Escuza, et al., 2020). Here, we investigated whether Bac modulates remyelination in lysolecithin (LPC)-demyelinated organotypic cerebellar slices as well as in LPC spinal cord lesions in adult mice. Our results demonstrate that Bac stimulates myelin protein production ex vivo and enhances remyelination in vivo, which suggests that this drug may also be a useful therapeutic agent to stimulate remyelination. 2|MATERIALS AND METHODS 2.1 |Animals All experiments were conducted with the approval of the ethical committee of the University of the Basque Country (UPV/EHU). Animals were handled in accordance with the European Directive 2010/63/ EU and were housed under standard conditions with a 12 h light–dark cycle and ad libitum access to food and water. All possible efforts were made to minimize animal suffering and the number of animals used. Sprague Dawley rats, C57BL/6 mice, and transgenic mice expressing fluorescence reporter DsRed under the control of the glialspecific proteolipid protein promoter (PLP-DsRed; Hirrlinger et al., 2005), generously provided by Prof. Dr. F. Kirchhoff (University of Saarland, Homburg, Germany), were used in this study. 2.2 |Cerebellar organotypic slice culture Slice cultures were made from cerebella of P5-P7 or P11-day-old Sprague Dawley rats and P11-day-old transgenic PLP-DsRed mice according to previously described procedures (Doussau et al., 2017; Dusart et al., 1997; Tan et al., 2018). Briefly, cerebella were cut with a tissue chopper (Mcllwain) into 350 μm parasagittal slices. Meninges were removed and slices were plated onto 0.4 μm pore size Millicell CM culture inserts (Millipore), containing 2–3 slices each. Rat slices were maintained in six-well plates for 13–15 days and mice cerebellar slices for 11 days in culture medium consisting of 50% basal medium with Earle's salt (BME), 25% Hank's Balanced Salt Solution (HBSS), 25% inactivated horse serum (all from ThermoFisher Scientific), 5 mg/ml glucose (Panreac), 0.0025 mM L -glutamine (Sigma-Aldrich) and antibiotic-antimycotic solution (100 U/ml of penicillin, 100 μg/ml of streptomycin and 0.25 μg/ml of amphotericin B; ThermoFisher Scientific) at 37C in a humidified atmosphere with 5% CO 2 . Culture medium was replaced every 2–3 days. Slices were treated with GABAergic drugs starting on the day 2 in vitro (Table 1). Lysolecithin (LPC)-induced demyelination experiments were carried out in cerebellar slices from P11 animals at day 7 in vitro by incubation for 16 h with 0.5 mg/ml LPC (Sigma-Aldrich) (Birgbauer et al., 2004). Treatments were performed at the same time as the LPC-stimulus. Slices were fixed in culture inserts with 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS; pH 7.4) for immunochemistry or processed for western blot analysis at 4 and 6 days after treatment. 2.3 |Optic nerve-derived organotypic slice culture Cultures were obtained from optic nerves of P11-day-old transgenic PLPDsRed mice. Optic nerves together with the retina were extracted in TABLE 1 GABAergic agonists and antagonists used in this study Product Reference Supplier Concentration (in vitro) GABA A2129 Sigma-Aldrich 100 μM Gabazine SR-95531 Sigma-Aldrich 50 μM Baclofen 0796 Tocris Bioscience 100 μM Muscimol 0289 Tocris Bioscience 100 μM 2SERRANO-REGAL ET AL. order to maintain tissue organization and cellular connections. Meninges and residual tissue were removed in supplemented (2 μl/ml gentamicin, 1 mg/ml bovine serum albumin, BSA and 2 mM L-glutamine) HBSS under the microscope, and the optic nerve-retina units were maintained in 0.4 μmporesizeMillicellCMcultureinserts (Millipore), containing one unit each. Explants were placed in six-well plates for 3 days in the culture medium as described above for cerebellar organotypic cultures and in the same conditions. To favor appropriate feeding of the optic nerve-retina unit, 50 μl of culture medium were added directly over the tissue (Azim & Butt, 2011). GABA B Rspecificagonist baclofen (100 μM) was added to the medium immediately after plating and maintained for 3 days with daily renewal. Optic nerves without retina were fixed with 4% PFA in PBS and whole-mounted on slides with Prolong™Gold antifade (Invitrogen). 2.4 |EdU labeling and detection 5-ethynyl-20-deoxyuridine (EdU; Invitrogen) (10 μM) was added to the organotypic medium at day 5 in vitro and left for 48 h, to label proliferating cells. EdU was revealed using Click-iT Alexa Fluor 647 Imaging Kit according to the manufacturer's instructions (Invitrogen). 2.5 |Demyelinating lesion induction Demyelinating lesions were induced in the spinal cord of 10-week-old female C57BL/6 mice by a stereotaxic injection of 0.5 μlof1%LPC (Sigma-Aldrich) in sterile 0.9% NaCl solution, as previously described (Tepavcevic et al., 2014). Mice were anesthetized by intraperitoneal injection (i.p.) of a solution of ketamine (90 mg/kg; Fatro)/xylazine (20 mg/kg; Calier). Buprenorphine (0.1 mg/kg; Dechra) was subcutaneously administered as postoperative analgesic treatment. Daily i.p. injections of vehicle (saline solution) or baclofen (8 mg/kg) were performed from 5 to 16 days post lesion (dpl). Mice were sacrificed at 12 or 16 dpl, and the tissue was processed for immunohistochemical (IHC) or transmission electron microscopy (TEM) analysis, respectively. 2.6 |Perfusion and tissue processing Mice were euthanized with ketamine/xylazine and transcardially perfused with 2% PFA solution in PBS for IHC analysis or 4% glutaraldehyde in 0.1 M PB for TEM studies. For IHC analysis, spinal cords were post-fixed with the same PFA solution, cryoprotected in 15% sucrose solution (Panreac) and frozen in 7% gelatin (Sigma-Aldrich)/15% sucrose solution in PBS. Samples were cut using a cryostat CM3050 S (Leica) to obtain 12 μm-thick coronal sections. For TEM studies, spinal cords were postfixed overnight, washed in 0.1 M PB, and cut into 2 mm-thick blocks. The tissue was postfixed in 1% osmium solution in 0.1 M PB, dehydrated and embedded in epoxy resin (Sigma Aldrich). Semithin (1 μm-thick) and ultrathin (55 nm-thick) sections were cut with an ultramicrotome RMC Boeckeler. 2.7 |Immunochemistry Cerebellar slices were washed in PBS, permeabilized and blocked in 4% goat serum and 0.1% Triton X-100 in PBS (blocking buffer) for 1 h and incubated overnight at 4C with primary antibodies (Table 2). Slices were washed in PBS with 0.1% Triton X-100 and incubated with Alexa fluorophore-conjugated secondary antibodies (1:400; Invitrogen) in blocking buffer for 1 h at RT. Slides with cryostat spinal cord sections were air-dried for 1 h, rehydrated in Tris buffer saline (TBS; 20 mM Tris and 1.4 M NaCl in dH 2 O; pH 7.6) and pre-treated with absolute ethanol (Sharlab) for 15 min at 20C. For APC and Olig2 immunostaining, antigen retrieval was performed by heating the sections in low-pH retrieval buffer (Vector Laboratories) for 45 s using a microwave. After washing, samples were incubated in blocking buffer solution (1% BSA, 5% goat serum, and 0.1% Triton X-100) for 30 min at RT, and then with the primary antibodies diluted in blocking buffer overnight at 4C (Table 2). Sections were washed in TBS, and incubated with Alexa fluorophore-conjugated secondary antibodies (1:500; Invitrogen) in blocking solution for 1 h at RT. Cell nuclei were counterstained with DAPI (4 μg/ml, Sigma-Aldrich) and sections were mounted with Fluoromount-G (SouthernBiotech). TABLE 2 Antibodies used in this study for immunohistochemistry Antibody Host Dilution (rat tissue) Dilution (mouse tissue) Supplier Reference Anti-GABAR B1 Rabbit 1:200 1:200 Alomone labs AGB-001 Anti-GABAR B1 Mouse —1:200 Abcam #55051 Anti-GABAR B2 Rabbit 1:200 1:200 Alomone labs AGB-002 Anti-APC (clone CC1) Mouse 1:200 1:200 Calbiochem #OP80 Anti-Olig2 Mouse 1:200, 1:1000 1:500 Millipore #MABN50 Anti-MBP Chicken —1:200 Millipore #AB9348 Anti-PDGFRαRat —1:300 BD Biosciences #558774 Anti-Iba1 Guinea pig —1:200 Synaptic systems 234,004 Anti-Nkx2.2 Mouse —1:20 Developmental Studies Hybridoma Bank #Q4818001B Anti-GFAP Rabbit 1:100 Millipore #AB5804 SERRANO-REGAL ET AL.3 2.8 |Image acquisition and analysis Images from cerebellar organotypic slices and optic nerve explants were acquired using Zeiss LSM800 and/or Leica TCS SP8 laser scanning confocal microscopes. Cells in cerebellar slices were counted blindly along the z-stack using a 20objective in Leica TCS SP8 confocal microscope. At least 3 different fields from 2 slices per experiment were analyzed by using LAS AF Lite software (Leica). The fluorescence signal corresponding to the PLP-DsRed OLs was quantified by ImageJ software and data were expressed as arbitrary units of fluorescence for each experimental situation. Images from spinal cord sections were collected using Zeiss LSM800 and/or Leica TCS SP8 confocal microscope and imported to ImageJ software. Area lacking myelin basic protein (MBP) staining within the dorsal funiculus of the spinal cord (area of demyelination) was delimited as region of interest (ROI) and measured. Cells positive for the markers of interest were counted from at least 3 different slices per animal. Results are presented as percentage of positive cells per lesion area measured or percentage area of lesion occupied by the corresponding markers. Same settings were kept for all samples (control and treated) belonging to a specific experiment. All images are shown as projections from zstacks. For TEM studies, semi thin sections stained with Richardson's Blue were used to identify the lesion area. Ultrathin sections were cut and contrasted by incubation in 4% uranyl acetate and lead citrate solution for its visualization in Philips CM200 transmission electron microscope. Remyelinated axons were counted. Remyelination was determined as the percentage of OL and Schwann cell (SC)- remyelinated axons within the total numbers of axons initially demyelinated (those remyelinated +those demyelinated). 2.9 |Western blot After treatments, cerebellar slices were directly resuspended in sodium dodecyl sulfate sample buffer on ice to enhance the lysis process and avoid protein degradation. Samples were boiled at 99C for 8 min, size-separated by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) in 4%–20% Criterion TGX Precast gels and transferred to Trans-Blot Turbo Midi PVDF Transfer Packs (Bio-Rad, Hercules). Membranes were blocked in 5% BSA (Sigma-Aldrich) in Tris-buffered saline/ 0.05% Tween-20 (TBS-T) and proteins were detected with specific primary antibodies (Table 3). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000; Sigma-Aldrich) and were developed by using an enhanced chemiluminescence detection kit according to the manufacturer's instructions (Supersignal West Dura or Femto; ThermoFisher Scientific). Protein bands were detected with a ChemiDocXRSImagingSystem(Bio-Rad) and quantified by volume using ImageLab software (version 3.0; Bio-Rad). 2.10 |Statistical analysis All data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism statistical software (version 8.0; GraphPad software). Comparisons between multiple experimental groups were made using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. For comparisons between two groups, we used the two-tailed Student's t-test assuming equal variance. In all instances, statistical differences were considered significant where p< .05. All the images shown represent the data obtained from at least three independent experiments. 3|RESULTS 3.1 |Baclofen treatment increases myelin protein levels in organotypic slice cultures We first validated the role of the GABAergic signaling in regulating oligodendroglial differentiation and myelination in organotypic cultures obtained from P5-P7 rats. We investigated GABA B1 and GABA B2 receptor-subunit expression during myelination ex vivo, and found that oligodendroglial cells–labeled using anti-Olig2 antibody–, and more specifically mature OLs–labeled using anti APC antibody–, express the two GABA B R subunits (Figure 1a,b), as we previously observed in OLs in vitro and in vivo (Serrano-Regal, Luengas-Escuza, et al., 2020). Then, we were interested in evaluating whether GABA agonists could also modulate myelin-protein expression levels in cerebellar organotypic cultures. Exposure to 100 μM GABA or 100 μM muscimol (Mus; GABA A R specific agonist) did not change the levels of expression of myelin-associated glycoprotein (MAG), 20,30-cyclic nucleotide30phosphodiesterase (CNPase) and MBP, compared with control slices (Supplementary Figures S2 and S3). However, treatment with 100 μM Bac (Figure 1c) induced a significant increase in the expression of MAG and MBP myelin proteins (2.0 ± 0.34 Bac vs. 1.0 ± 0.19 control for MAG, Figure 1d; and 1.29 ± 0.14 Bac vs. 1.0 ± 0.12 control for MBP, Figure 1f), together with a non-significant increase in the expression of CNPase (1.23 ± 0.11 Bac vs. 1.0 ± 0.14 control for CNPase, Figure 1e). This increase in MBP and MAG is similar to the effectofBacinculturedOPCs,andbecomesabrogatedinthepresenceof GABA B R specific antagonist CGP55845 (Supplementary Figure S1). To investigate whether this effect of Bac was associated with changes in oligodendroglial proliferation, cerebellar organotypic cultures were exposed to EdU for 48 h, in the absence or presence of GABA or Bac (100 μM). Neither GABA nor Bac modified the percentageofmatureOLs(APC + Olig2 + ) TABLE 3 Antibodies used in this study for western blot analysis Antibody Host Dilution Supplier Reference Anti-MAG Mouse 1:500 Santa Cruz SC-376145 Anti-CNPase Mouse 1:1000 Sigma-Aldrich #C5922 Anti-MBP Mouse 1:1000 Biolegend #SMI 99 Anti-GAPDH Mouse 1:1000 Millipore #MAB374 Anti-β-tubulin Mouse 1:5000 abcam AB7291 4SERRANO-REGAL ET AL. among total oligodendroglial cells, nor Olig2 + cells that underwent proliferation in this time period (Olig2 + Edu + )(Figure1g–i), suggesting that GABA B R activation promotes myelin generation by mature OLs without affecting OPC proliferative capacity. Additionally, we examined the effect of Bac in optic nerve explants of transgenic PLP-DsRed mice (Figure 1j). Quantification of PLP-DsRed-fluorescent signal (Figure 1k) revealed a significant increase in those optic nerves treated with Bac compared to controls (27.82 ± 2.27 for Bac vs. 19.62 ± 2.59 for control; Figure 1l). Together, these results show that Bac enhances myelin protein production in murine organotypic cultures and in optic nerve explants, confirming our previous observations in isolated OLs (Serrano-Regal, Luengas-Escuza, et al., 2020)in a complex environment more similar to physiological conditions. FIGURE 1 Legend on next page. SERRANO-REGAL ET AL.5 3.2 |GABA B R activation elevates the levels of major myelin proteins during remyelination ex vivo Since Bac promoted myelin protein synthesis in organotypic slices, we next studied the impact of GABA B R activation under experimental conditions mimicking damage to myelin. P11 rat-derived cerebellar slices were maintained for 7 days to allow myelination ex vivo and then exposed to LPC for 16 h. In the first set of experiments, slices were daily treated with GABA (100 μM) or Bac (100 μM) for 6 days after LPC exposure (Supplementary Figure S4A, B) and MAG and MBP proteins were analyzed by western blot. We found that LPC induced a significant decrease in both proteins (0.99 ± 0.11 LPC vs. 1.44 ± 0.11 fold control for MAG, and 1.00 ± 0.08 LPC vs. 1.50 ± 0.09 fold control for MBP). Bac treatment post-LPC significantly increased MAG levels (1.44 ± 0.11 Bac vs. 0.99 ± 0.11 LPC; Supplementary Figure S4C) while MBP levels were not affected (Supplementary Figure S4D). We then investigated whether previous application of these agonists during demyelinating phase may be more effective in recovering the levels of myelin protein expression after exposure to LPC. We exposed cerebellar slices to LPC concomitantly to GABA or Bac (100 μM) application. After LPC removal, GABA or Bac was maintained in the medium for 6 more days (Figure 2a). Under this experimental paradigm, we observed a significant increase in the expression levels of both MAG and MBP (2.75 ± 0.35 GABA and 2.81 ± 0.26 Bac vs. 1.0 ± 0.26 LPC for MAG, and 3.39 ± 0.52 GABA and 2.82 ± 0.29 Bac vs. 1.0 ± 0.37 LPC for MBP; Figure 2b–d). We also used immunofluorescence to investigate the effect of GABA B R-mediated signaling on OL differentiation and myelin protein production ex vivo by taking advantage of PLP-DsRed reporter mice, in which changes in PLP-associated endogenous fluorescence can be exploited to track changes in PLP-expression. We prepared organotypic cerebellar slices and maintained these in culture for 7 days, after which we exposed the slices to LPC in combination with drug treatments for 4days. We applied GABA (100μM), Bac (100 μM), and GABA plus gabazine (50 μM)–aGABA A R antagonist–, in order to study the effect of GABA directly over GABA B Rs, or gabazine alone (50 μM) (Figure 2e, f). As shown in Figure 2g, the PLP-DsRed fluorescent signal increased significantly in Bacand GABA plus gabazine-treated slices compared to those exposed to LPC without treatment (10.42 ± 0.65 Bac and 20.74 ± 5.32 GABA plus gabazine vs. 4.5 ± 0.38 LPC; Figure 2g), indicating that GABA B receptor stimulation in these slices increases PLP production. Overall, these results indicate that GABA B Ractivation with Bac favors remyelination ex vivo in cerebellar organotypic slices. 3.3 |Baclofen administration promotes OPC differentiation in adult mouse CNS We then aimed to assess the effect of Bac administration on CNS remyelination in vivo. We first validated the expression of GABA B R subunits in OPCs in normal spinal cord tissue of adult mice (Figure 3a), and observed expression of both B1 and B2. We then confirmed that this expression was maintained after induction of demyelination by LPC injection in the dorsal funiculus, both on reactive OPCs (Figure 3b) and by newly generated OLs (Figure 3c), which suggested these cells could be targeted by Bac. To investigate the effect of Bac on remyelination, at 5 days post lesion (dpl), daily i.p. injections of vehicle or Bac (8 mg/kg/day) were initiated and administered during 7 days. Bac administration was initiated at 5 dpl to ensure that the treatment would not affect the extent of demyelination, as demyelination in LPC model is complited by 2 dpl. OPC differentiation was investigated at 12 dpl, given that the peak of OPC differentiation occurs during the second week post demyelination (Figure 4a). Then, we explored the changes induced by Bac administration in OPC numbers and microglia/macrophage response at 12 dpl using anti-PDGFRαantibody as OPC marker and anti-Iba1 antibody as microglia/macrophage marker (Figure 4b). Quantification of PDGFRα + cells per mm 2 (578.2 ± 117.3 Bac vs. 461.3 ± 49.93 vehicle; Figure 4c) or the percentage of lesion area occupied by Iba-1 (60.10 ± 5.52% Bac vs. 49.87 ± 11.84% vehicle; Figure 4d) revealed no variations between control vs Bac-treated mice. In addition, we used antiNkx2.2 antibody to label reactive OPCs and anti-GFAP antibody as FIGURE 1 Baclofen increases myelin-related protein synthesis in organotypic cultures without altering the proliferation ratio of oligodendroglial lineage. (a) Oligodendroglial cells, distinguished as Olig2 + cells (red), are positive for GABA B1 and GABA B2 subunits (green) of GABA B Rs in cerebellar slices of P5-P7 rats. (b) Mature oligodendrocytes (OLs), identified as APC + cells (red), express GABA B1 , and GABA B2 subunits (green) of GABA B Rs in the same preparations. Arrows indicate double-stained cells and arrowheads show the cell magnified in the corresponding inset. Scale bars =20 μm. (c) Representative western blot image-showing expression of myelin-associated glycoprotein (MAG), CNPase, and MBP proteins in control and baclofen-treated cerebellar slices. Quantification of MAG (d), CNPase (e), and myelin basic protein (MBP) (f) expression normalized to GAPDH values. *p< .05 and ** p< .01 versus control; paired Student's t-test. (g) Representative images showing immunofluorescence of mature OLs (APC + , red) and total oligodendroglial cells (Olig2 + , green) co-labeled with EdU (cyan) to identify mature OLs (APC + ) and Olig2 + cells in cerebellar slices in the indicated condition. Arrows indicate mature OLs (APC + Olig2 + ) or newly generated oligodendroglial cells (Olig2 + EdU + ). Scale bar =50 μm. Quantification of (h) percentage of mature OL from total oligodendroglial cell pool (APC + Olig2 + /Olig2 + ) and (i) density of newly formed oligodendroglial cells (Olig2 + EdU + ), in the indicated conditions. One-way ANOVA followed by Tukey's post-test. (j) Optic nerve-retina unit from P11 PLP-DsRed transgenic mice. Scale bar =500 μm. (k) Optic nerves explants cultured in control conditions (left) or in presence of baclofen (right) showing DsRed fluorescent signal. Scale bars =100 μm; higher magnification scale bar =315 μm. (l) Quantification of DsRed fluorescent signal in control and treated optic nerve explants. *p< .05 versus control; unpaired Student's t-test. (a–f): Control and bac 6 slices from different animals. (g–i): Control 15, GABA 18, and bac 18 images from cerebellar slices. (j–l): Control 27, bac 24 images from optic nerve explants 6SERRANO-REGAL ET AL. FIGURE 2 GABA B Rs modulate remyelination in lysolecithin (LPC)-treated cerebellar organotypic slices. (a) Time course showing the experimental design in LPC-induced demyelination in cerebellar organotypic cultures obtained from P11 rats. (b) Representative western blot image showing in duplicates the effect of GABA and baclofen in modulating myelin-related protein restoration in LPC-treated organotypic cultures following the paradigm shown in a. (c, d) Quantification of myelin-associated glycoprotein (MAG) (c) and myelin basic protein (MBP) (d) levels in indicated conditions. **p< .01 and ****p< .0001 versus control, ## p< .01 and ### p< .001 versus LPC; one-way ANOVA followed by Tukey's post-test. (e) Representative images of cerebellar slices from P11 PLP-DsRed transgenic mice showing DsRed fluorescence in indicated conditions. Scale bar =100 μm. (f) Treatments were added to the slices in conjunction with LPC for 16 h and maintained thereafter for 4 days after. GABA B Rs were selectively activated with baclofen or with GABA plus the GABA A R antagonist gabazine. (g) Quantification of DsRed fluorescent signal in indicated conditions. ****p< .0001 versus control, # p< .05 and #### p< .001 versus LPC; one-way ANOVA followed by Tukey's post-test. (b–d): Control 9, LPC 8, GABA 9, bac 8 slices from different animals. (e–g): Control 27, LPC 25, GABA 8, bac 19, Gbz 10, GABA +Gbz 10 images from optic nerve explants SERRANO-REGAL ET AL.7 astrocyte marker (Figure 4e). We observed no significant differences in Nkx2.2 + cells per mm 2 (437.4 ± 46.8 Bac vs. 496.5 ± 65.5 control; Figure 4f) nor in the percentage of lesion area occupied by GFAP (21.2 ± 4.0 Bac vs. 26.9 ± 2.0 control; Figure 4g) between vehicle versus Bac-treated animals. Finally, we investigated whether Bac administration accelerates OPC differentiation in the lesions by analyzing the numbers of mature OLs (APC + Olig2 + ) relative to the total number of oligodendroglial cells (Olig2 + ) (Figure 4h). The percentage of APC + among total Olig2 + cells was significantly increased in Bac-treated LPC-injected mice (41.91 ± 2.29% Bac vs. 27.95 ± 1.46% vehicle; Figure 4i), without altering the total numbers of Olig2 + cells (928.0 ± 100.7 Bac vs. 814.8 ± 50.13 vehicle; Figure 4j). These results demonstrate that Bac treatment promotes differentiation of OPCs in LPCinduced demyelinating lesions. 3.4 |Baclofen administration accelerates remyelination We next analyzed whether Bac administration accelerates remyelination. Vehicle and Bac injections were initiated at 5 dpl, and the proportion of remyelinated axons was analyzed at 16 dpl (Figure 5a), given that the onset of remyelination in the LPC model takes place at 14 dpl and is near completion at 21 dpl. At this time point, OL remyelination–identified as thin myelin sheaths–was found predominantly around lesion borders, while SC remyelination, a wellrecognized feature of the LPC lesion in the spinal cord (Jeffery & Blakemore, 1995), was observed in the lesion center. TEM analysis revealed that the percentage of remyelinated axons (Figure 5b, c) was higher following Bac treatment (36.95 ± 2.18% Bac vs. 22.29 ± 1.85% FIGURE 3 GABA B receptors are expressed by oligodendrocyte progenitor cell (OPCs) and mature oligodendrocytes from the mouse spinal cord. (a) Confocal images showing OPCs (Nkx2.2 + , gray) expressing GABA B1 (red) and GABA B2 (green) subunits of GABA B Rs in the dorsal funiculus of the spinal cord of unlesioned mice. (B, C) Confocal images showing OPCs (Nkx2.2 + , red) (b) and mature OLs (APC + , red) (c) expressing GABA B1 (green, top) and GABA B2 (green, bottom) subunits of GABA B Rs in the dorsal funiculus of the spinal cord of control lysolecithin (LPC)- injected mice. White dash line indicates lesion border. Arrowheads point at cells shown at higher magnification in each photograph. Scale bars =50 μm. Higher magnification =10 μm 8SERRANO-REGAL ET AL. FIGURE 4 Legend on next page. SERRANO-REGAL ET AL.9