Emerging concepts in oligodendrocyte and myelin formation, inputs from the zebrafish model.

Oligodendrocytes (OLs) are the myelinating cells of the central nervous system (CNS), which are derived from OL precursor cells. Myelin insulates axons allowing the saltatory conduction of action potentials and also provides trophic and metabolic supports to axons. Interestingly, oligodendroglial cells have the capacity to sense neuronal activity, which regulates myelin sheath formation via the vesicular release of neurotransmitters. Neuronal activity-dependent regulation of myelination is mediated by specialized interaction between axons and oligodendroglia, involving both synaptic and extra-synaptic modes of communications. The zebrafish has provided key advantages for the study of the myelination process in the CNS. External development and transparent larval stages of this vertebrate specie combined with the existence of several transgenic reporter lines provided key advances in oligodendroglial cell biology, axo-glial interactions and CNS myelination. In this publication, we reviewed and discussed the most recent knowledge on OL development and myelin formation, with a focus on mechanisms regulating these fundamental biological processes in the zebrafish. Especially, we highlighted the critical function of axons and oligodendroglia modes of communications and calcium signaling in myelin sheath formation and growth. Finally, we reviewed the relevance of these knowledge's in demyelinating diseases and drug discovery of pharmacological compounds favoring myelin regeneration.

An anti-inflammatory transcriptional cascade conserved from flies to humans.

Innate immunity is an ancestral process that can induce pro- and anti-inflammatory states. A major challenge is to characterize transcriptional cascades that modulate the response to inflammation. Since the Drosophila glial cells missing (Gcm) transcription factor has an anti-inflammatory role, we explored its regulation and evolutionary conservation. Here, we show that the murine Gcm2 (mGcm2) gene is expressed in a subpopulation of aged microglia (chronic inflammation) and upon lysophosphatidylcholine (LPC)-induced central nervous system (CNS) demyelination (acute inflammation). Moreover, mGcm2 conditional knockout mice show an increased inflammatory phenotype upon aging or LPC injection, and hGCM2 is expressed in active demyelinating lesions of patients with multiple sclerosis. Finally, Drosophila Gcm expression is induced upon aging and acute challenge, and its overexpression decreases the inflammatory phenotype. Altogether, these data indicate that the inducible Gcm cascade is conserved from flies to humans and represents a potential therapeutic target in the control of the inflammatory response.

p70S6 kinase regulates oligodendrocyte differentiation and is active in remyelinating lesions.

The p70 ribosomal S6 kinases (p70 ribosomal S6 kinase 1 and p70 ribosomal S6 kinase 2) are downstream targets of the mechanistic target of rapamycin signalling pathway. p70 ribosomal S6 kinase 1 specifically has demonstrated functions in regulating cell size in Drosophila and in insulin-sensitive cell populations in mammals. Prior studies demonstrated that the mechanistic target of the rapamycin pathway promotes oligodendrocyte differentiation and developmental myelination; however, how the immediate downstream targets of mechanistic target of rapamycin regulate these processes has not been elucidated. Here, we tested the hypothesis that p70 ribosomal S6 kinase 1 regulates oligodendrocyte differentiation during developmental myelination and remyelination processes in the CNS. We demonstrate that p70 ribosomal S6 kinase activity peaks in oligodendrocyte lineage cells at the time when they transition to myelinating oligodendrocytes during developmental myelination in the mouse spinal cord. We further show p70 ribosomal S6 kinase activity in differentiating oligodendrocytes in acute demyelinating lesions induced by lysophosphatidylcholine injection or by experimental autoimmune encephalomyelitis in mice. In demyelinated lesions, the expression of the p70 ribosomal S6 kinase target, phosphorylated S6 ribosomal protein, was transient and highest in maturing oligodendrocytes. Interestingly, we also identified p70 ribosomal S6 kinase activity in oligodendrocyte lineage cells in active multiple sclerosis lesions. Consistent with its predicted function in promoting oligodendrocyte differentiation, we demonstrate that specifically inhibiting p70 ribosomal S6 kinase 1 in cultured oligodendrocyte precursor cells significantly impairs cell lineage progression and expression of myelin basic protein. Finally, we used zebrafish to show in vivo that inhibiting p70 ribosomal S6 kinase 1 function in oligodendroglial cells reduces their differentiation and the number of myelin internodes produced. These data reveal an essential function of p70 ribosomal S6 kinase 1 in promoting oligodendrocyte differentiation during development and remyelination across multiple species.

Reduced Axon Calibre in the Associative Striatum of the Sapap3 Knockout Mouse.

Pathological repetitive behaviours are a common feature of various neuropsychiatric disorders, including compulsions in obsessive-compulsive disorder or tics in Gilles de la Tourette syndrome. Clinical research suggests that compulsive-like symptoms are related to associative cortico-striatal dysfunctions, and tic-like symptoms to sensorimotor cortico-striatal dysfunctions. The Sapap3 knockout mouse (Sapap3-KO), the current reference model to study such repetitive behaviours, presents both associative as well as sensorimotor cortico-striatal dysfunctions. Previous findings point to deficits in both macro-, as well as micro-circuitry, both of which can be affected by neuronal structural changes. However, to date, structural connectivity has not been analysed. Hence, in the present study, we conducted a comprehensive structural characterisation of both associative and sensorimotor striatum as well as major cortical areas connecting onto these regions. Besides a thorough immunofluorescence study on oligodendrocytes, we applied AxonDeepSeg, an open source software, to automatically segment and characterise myelin thickness and axon area. We found that axon calibre, the main contributor to changes in conduction speed, is specifically reduced in the associative striatum of the Sapap3-KO mouse; myelination per se seems unaffected in associative and sensorimotor cortico-striatal circuits.

Teriflunomide Promotes Oligodendroglial 8,9-Unsaturated Sterol Accumulation and CNS Remyelination.

BACKGROUND AND OBJECTIVES: To test whether low concentrations of teriflunomide (TF) could promote remyelination, we investigate the effect of TF on oligodendrocyte in culture and on remyelination in vivo in 2 demyelinating models. METHODS: The effect of TF on oligodendrocyte precursor cell (OPC) proliferation and differentiation was assessed in vitro in glial cultures derived from neonatal mice and confirmed on fluorescence-activated cell sorting-sorted adult OPCs. The levels of the 8,9-unsaturated sterols lanosterol and zymosterol were quantified in TF- and sham-treated cultures. In vivo, TF was administered orally, and remyelination was assessed both in myelin basic protein-GFP-nitroreductase (Mbp:GFP-NTR) transgenic Xenopus laevis demyelinated by metronidazole and in adult mice demyelinated by lysolecithin. RESULTS: In cultures, low concentrations of TF down to 10 nM decreased OPC proliferation and increased their differentiation, an effect that was also detected on adult OPCs. Oligodendrocyte differentiation induced by TF was abrogated by the oxidosqualene cyclase inhibitor Ro 48-8071 and was mediated by the accumulation of zymosterol. In the demyelinated tadpole, TF enhanced the regeneration of mature oligodendrocytes up to 2.5-fold. In the mouse demyelinated spinal cord, TF promoted the differentiation of newly generated oligodendrocytes by a factor of 1.7-fold and significantly increased remyelination. DISCUSSION: TF enhances zymosterol accumulation in oligodendrocytes and CNS myelin repair, a beneficial off-target effect that should be investigated in patients with multiple sclerosis.

Identification of First-in-Class Inhibitors of Kallikrein-Related Peptidase 6 That Promote Oligodendrocyte Differentiation.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) that causes severe motor, sensory, and cognitive impairments. Kallikrein-related peptidase (KLK)6 is the most abundant serine protease secreted in the CNS, mainly by oligodendrocytes, the myelin-producing cells of the CNS, and KLK6 is assumed to be a robust biomarker of MS, since it is highly increased in the cerebrospinal fluid (CSF) of MS patients. Here, we report the design and biological evaluation of KLK6's low-molecular-weight inhibitors, para-aminobenzyl derivatives. Interestingly, selected hit compounds were selective of the KLK6 proteolytic network encompassing KLK1 and plasmin that also participate in the development of MS physiopathology. Moreover, hits were found noncytotoxic on primary cultures of murine neurons and oligodendrocyte precursor cells (OPCs). Among them, two compounds (32 and 42) were shown to promote the differentiation of OPCs into mature oligodendrocytes in vitro constituting thus emerging leads for the development of regenerative therapies.

Oligodendrocyte Development and Regenerative Therapeutics in Multiple Sclerosis.

Myelination by oligodendrocytes (OLs) is an important biological process essential for central nervous system (CNS) development and functions. Oligodendroglial lineage cells undergo several morphological and molecular changes at different stages of their lineage progression into myelinating OLs. The transition steps of the oligodendrocyte progenitor cells (OPCs) to myelinating oligodendrocytes are defined by a specific pattern of regulated gene expression, which is under the control of coordinated signaling pathways. Any abnormal development, loss or failure of oligodendrocytes to myelinate axons can lead to several neurodegenerative diseases like multiple sclerosis (MS). MS is characterized by inflammation and demyelination, and current treatments target only the immune component of the disease, but have little impact on remyelination. Recently, several pharmacological compounds enhancing remyelination have been identified and some of them are in clinical trials. Here, we will review the current knowledge on oligodendrocyte differentiation, myelination and remyelination. We will focus on MS as a pathological condition, the most common chronic inflammatory demyelinating disease of the CNS in young adults.

Co-culture of exogenous oligodendrocytes with unmyelinated cerebella: Revisiting ex vivo models and new tools to study myelination.

Common in vitro models used to study the mechanisms regulating myelination rely on co-cultures of oligodendrocyte precursor cells (OPCs) and neurons. In such models, myelination occurs in an environment that does not fully reflect cell-cell interactions and environmental cues present in vivo. To avoid these limitations while specifically manipulating oligodendroglial cells, we developed a reliable ex vivo model of myelination by seeding OPCs on cerebellar slices, deprived of their endogenous oligodendrocytes. We showed that exogenous OPCs seeded on unmyelinated cerebella, efficiently differentiate and form compact myelin. Spectral confocal reflectance microscopy and electron microscopy analysis revealed that the density of compacted myelin sheaths highly increases all along the culture. Importantly, we defined the appropriate culture time frame to study OPC differentiation and myelination, using accurate quantification resources we generated. Thus, this model is a powerful tool to study the cellular and molecular mechanisms of OPC differentiation and myelination. Moreover, it is suitable for the development and validation of new therapies for myelin-related disorders such as multiple sclerosis and psychiatric diseases.

Key role for lipids in cognitive symptoms of schizophrenia.

Schizophrenia (SZ) is a psychiatric disorder with a convoluted etiology that includes cognitive symptoms, which arise from among others a dysfunctional dorsolateral prefrontal cortex (dlPFC). In our search for the molecular underpinnings of the cognitive deficits in SZ, we here performed RNA sequencing of gray matter from the dlPFC of SZ patients and controls. We found that the differentially expressed RNAs were enriched for mRNAs involved in the Liver X Receptor/Retinoid X Receptor (LXR/RXR) lipid metabolism pathway. Components of the LXR/RXR pathway were upregulated in gray matter but not in white matter of SZ dlPFC. Intriguingly, an analysis for shared genetic etiology, using two SZ genome-wide association studies (GWASs) and GWAS data for 514 metabolites, revealed genetic overlap between SZ and acylcarnitines, VLDL lipids, and fatty acid metabolites, which are all linked to the LXR/RXR signaling pathway. Furthermore, analysis of structural T1-weighted magnetic resonance imaging in combination with cognitive behavioral data showed that the lipid content of dlPFC gray matter is lower in SZ patients than in controls and correlates with a tendency towards reduced accuracy in the dlPFC-dependent task-switching test. We conclude that aberrations in LXR/RXR-regulated lipid metabolism lead to a decreased lipid content in SZ dlPFC that correlates with reduced cognitive performance.

Interneuron hypomyelination is associated with cognitive inflexibility in a rat model of schizophrenia.

Impaired cognitive functioning is a core feature of schizophrenia, and is hypothesized to be due to myelination as well as interneuron defects during adolescent prefrontal cortex (PFC) development. Here we report that in the apomorphine-susceptible (APO-SUS) rat model, which has schizophrenia-like features, a myelination defect occurred specifically in parvalbumin interneurons. The adult rats displayed medial PFC (mPFC)-dependent cognitive inflexibility, and a reduced number of mature oligodendrocytes and myelinated parvalbumin inhibitory axons in the mPFC. In the developing mPFC, we observed decreased myelin-related gene expression that persisted into adulthood. Environmental enrichment applied during adolescence restored parvalbumin interneuron hypomyelination as well as cognitive inflexibility. Collectively, these findings highlight that impairment of parvalbumin interneuron myelination is related to schizophrenia-relevant cognitive deficits.

Sox17 Regulates a Program of Oligodendrocyte Progenitor Cell Expansion and Differentiation during Development and Repair.

Sox17, a SoxF family member transiently upregulated during postnatal oligodendrocyte (OL) development, promotes OL cell differentiation, but its function in white matter development and pathology in vivo is unknown. Our analysis of oligodendroglial- and OL-progenitor-cell-targeted ablation in vivo using a floxed Sox17 mouse establishes a dependence of postnatal oligodendrogenesis on Sox17 and reveals Notch signaling as a mediator of Sox17 function. Following Sox17 ablation, reduced numbers of Olig2-expressing cells and mature OLs led to developmental hypomyelination and motor dysfunction. After demyelination, Sox17 deficiency inhibited OL regeneration. OL decline was unexpectedly preceded by transiently increased differentiation and a reduction of OL progenitor cells. Evidence of a dual role for Sox17 in progenitor cell expansion by Notch and differentiation involving TCF7L2 expression were found. A program of progenitor expansion and differentiation promoted by Sox17 through Notch thus contributes to OL production and determines the outcome of white matter repair.

Neuronal activity in vivo enhances functional myelin repair.

In demyelinating diseases such as Multiple Sclerosis (MS), demyelination of neuronal fibers impairs impulse conduction and causes axon degeneration. While neuronal activity stimulates oligodendrocyte production and myelination in normal conditions, it remains unclear whether the activity of demyelinated axons restores their loss-of-function in a harmful environment. To investigate this question, we established a model to induce a moderate optogenetic stimulation of demyelinated axons in the corpus callosum at the level of the motor cortex in which cortical circuit activation and locomotor effects were reduced in adult freely moving mice. We demonstrate that a moderate activation of demyelinated axons enhances the differentiation of oligodendrocyte precursor cells onto mature oligodendrocytes, but only under a repeated stimulation paradigm. This activity-dependent increase in the oligodendrocyte pool promotes an extensive remyelination and functional restoration of conduction, as revealed by ultrastructural analyses and compound action potential recordings. Our findings reveal the need of preserving an appropriate neuronal activity in the damaged tissue to promote oligodendrocyte differentiation and remyelination, likely by enhancing axon-oligodendroglia interactions. Our results provide new perspectives for translational research using neuromodulation in demyelinating diseases.

Ultrahigh field imaging of myelin disease models: Toward specific markers of myelin integrity?

Specific magnetic resonance imaging (MRI) markers of myelin are critical for the evaluation and development of regenerative therapies for demyelinating diseases. Several MRI methods have been developed for myelin imaging, based either on acquisition schemes or on mathematical modeling of the signal. They generally showed good sensitivity but validation for specificity toward myelin is still warranted to allow a reliable interpretation in an in vivo complex pathological environment. Experimental models of dys-/demyelination are characterized by various levels of myelin disorders, axonal damage, gliosis and inflammation, and offer the opportunity for powerful correlative studies between imaging metrics and histology. Here, we review how ultrahigh field MRI markers have been correlated with histology in these models and provide insights into the trends for future developments of MRI tools in human myelin diseases. To this end, we present the biophysical basis of the main MRI methods for myelin imaging based on T1 , T2 , water diffusion, and magnetization transfer signal, the characteristics of animal models used and the outcomes of histological validations. To date such studies are limited, and demonstrate partial correlations with immunohistochemical and electron microscopy measures of myelin. These MRI metrics also often correlate with axons, glial, or inflammatory cells in models where axonal degeneration or inflammation occur as potential confounding factors. Therefore, the MRI markers' specificity for myelin is still perfectible and future developments should improve mathematical modeling of the MR signal based on more complex systems or provide multimodal approaches to better disentangle the biological processes underlying the MRI metrics.

SOX17 transcription factor negatively regulates oligodendrocyte precursor cell differentiation.

Oligodendrocyte development is a critical process timely and spatially regulated to ensure proper myelination of the central nervous system. HMG-box transcription factors are key regulators of oligodendrocyte lineage progression. Among these factors, Sox17 was previously identified as a positive regulator of oligodendrocyte development. However, the role of Sox17 in oligodendroglial cell lineage progression and differentiation is still poorly understood. To define the functional role of Sox17, we generated new transgenic mouse models with inducible overexpression of Sox17, specifically in oligodendroglial cells. Here, we report that gain of Sox17 function has no effect on oligodendrocyte progenitor cells (OPCs) specification. During early postnatal development, Sox17 overexpression increases the pool of OPCs at the expense of differentiated oligodendrocytes. However, the oligodendroglial cell population, OPC proliferation and apoptosis remained unchanged in Sox17 transgenic mice. RNA sequencing, quantitative RT-PCR and immunohistochemical analysis showed that Sox17 represses the expression of the major myelin genes, resulting in a severe CNS hypomyelination. Overall, our data highlight an unexpected role for Sox17 as a negative regulator of OPC differentiation and myelination, suggesting stage specific functions for this factor during oligodendroglial cell lineage progression.

Dual Requirement of CHD8 for Chromatin Landscape Establishment and Histone Methyltransferase Recruitment to Promote CNS Myelination and Repair.

Disruptive mutations in chromatin remodeler CHD8 cause autism spectrum disorders, exhibiting widespread white matter abnormalities; however, the underlying mechanisms remain elusive. We show that cell-type specific Chd8 deletion in oligodendrocyte progenitors, but not in neurons, results in myelination defects, revealing a cell-intrinsic dependence on CHD8 for oligodendrocyte lineage development, myelination and post-injury remyelination. CHD8 activates expression of BRG1-associated SWI/SNF complexes that in turn activate CHD7, thus initiating a successive chromatin remodeling cascade that orchestrates oligodendrocyte lineage progression. Genomic occupancy analyses reveal that CHD8 establishes an accessible chromatin landscape, and recruits MLL/KMT2 histone methyltransferase complexes distinctively around proximal promoters to promote oligodendrocyte differentiation. Inhibition of histone demethylase activity partially rescues myelination defects of CHD8-deficient mutants. Our data indicate that CHD8 exhibits a dual function through inducing a cascade of chromatin reprogramming and recruiting H3K4 histone methyltransferases to establish oligodendrocyte identity, suggesting potential strategies of therapeutic intervention for CHD8-associated white matter defects.

Hypomyelination and Oligodendroglial Alterations in a Mouse Model of Autism Spectrum Disorder.

Autism spectrum disorders (ASDs) are neuropsychiatric diseases characterized by impaired social interaction, communication deficits, and repetitive and stereotyped behaviors. ASD etiology is unknown, and both genetic and environmental causes have been proposed. Different brain structures are believed to play a role in ASD-related behaviors, including medial prefrontal cortex (mPFC), hippocampus, piriform cortex (Pir), basolateral amygdala (BLA) and Cerebellum. Compelling evidence suggests a link between white matter modifications and ASD symptoms in patients. Besides, an hypomyelination of the mPFC has been associated in rodents to social behavior impairment, one of the main symptoms of ASD. However, a comparative analysis of myelination as well as oligodendroglial (OL)-lineage cells in brain regions associated to social behaviors in animal models of ASD has not been performed so far. Here, we investigated whether OL-lineage cells and myelination are altered in a murine model of ASD induced by the prenatal exposure to valproic acid (VPA). We showed an hypomyelination in the BLA and Pir of adult VPA-exposed mice. These results were accompanied by a decrease in the number of OL-lineage cells and of mature OLs in the Pir, in addition to the mPFC, where myelination presented no alterations. In these regions the number of oligodendrocyte progenitors (OPCs) remained unaltered. Likewise, activation of histone deacetylases (HDACs) on OL-lineage cells in adulthood showed no differences. Overall, our results reveal OL-lineage cell alterations and hypomyelination as neuropathological hallmarks of ASD that have been overlooked so far.

The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation.

Oligodendrocyte and myelin deficits have been reported in mental/psychiatric diseases. The p21-activated kinase 3 (PAK3), a serine/threonine kinase, whose activity is stimulated by the binding of active Rac and Cdc42 GTPases is affected in these pathologies. Indeed, many mutations of Pak3 gene have been described in non-syndromic intellectual disability diseases. Pak3 is expressed mainly in the brain where its role has been investigated in neurons but not in glial cells. Here, we showed that PAK3 is highly expressed in oligodendrocyte precursors (OPCs) and its expression decreases in mature oligodendrocytes. In the developing white matter of the Pak3 knockout mice, we found defects of oligodendrocyte differentiation in the corpus callosum and to a lesser extent in the anterior commissure, which were compensated at the adult stage. In vitro experiments in OPC cultures, derived from Pak3 knockout and wild type brains, support a developmental and cell-autonomous role for PAK3 in regulating OPC differentiation into mature oligodendrocytes. Moreover, we did not detect any obvious alterations of the proliferation or migration of Pak3 null OPCs compared to wild type. Overall, our data highlight PAK3 as a new regulator of OPC differentiation.

Vitamin D receptor-retinoid X receptor heterodimer signaling regulates oligodendrocyte progenitor cell differentiation.

The mechanisms regulating differentiation of oligodendrocyte (OLG) progenitor cells (OPCs) into mature OLGs are key to understanding myelination and remyelination. Signaling via the retinoid X receptor γ (RXR-γ) has been shown to be a positive regulator of OPC differentiation. However, the nuclear receptor (NR) binding partner of RXR-γ has not been established. In this study we show that RXR-γ binds to several NRs in OPCs and OLGs, one of which is vitamin D receptor (VDR). Using pharmacological and knockdown approaches we show that RXR-VDR signaling induces OPC differentiation and that VDR agonist vitamin D enhances OPC differentiation. We also show expression of VDR in OLG lineage cells in multiple sclerosis. Our data reveal a role for vitamin D in the regenerative component of demyelinating disease and identify a new target for remyelination medicines.

Liver X receptors alpha and beta promote myelination and remyelination in the cerebellum.

The identification of new pathways governing myelination provides innovative avenues for remyelination. Liver X receptors (LXRs) α and ß are nuclear receptors activated by oxysterols that originated from the oxidation of cholesterol. They are crucial for cholesterol homeostasis, a major lipid constituent of myelin sheaths that are formed by oligodendrocytes. However, the role of LXRs in myelin generation and maintenance is poorly understood. Here, we show that LXRs are involved in myelination and remyelination processes. LXRs and their ligands are present in oligodendrocytes. We found that mice invalidated for LXRs exhibit altered motor coordination and spatial learning, thinner myelin sheaths, and reduced myelin gene expression. Conversely, activation of LXRs by either 25-hydroxycholesterol or synthetic TO901317 stimulates myelin gene expression at the promoter, mRNA, and protein levels, directly implicating LXRα/ß in the transcriptional control of myelin gene expression. Interestingly, activation of LXRs also promotes oligodendroglial cell maturation and remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Together, our findings represent a conceptual advance in the transcriptional control of myelin gene expression and strongly support a new role of LXRs as positive modulators in central (re)myelination processes.

Alteration of synaptic connectivity of oligodendrocyte precursor cells following demyelination.

Oligodendrocyte precursor cells (OPCs) are a major source of remyelinating oligodendrocytes in demyelinating diseases such as Multiple Sclerosis (MS). While OPCs are innervated by unmyelinated axons in the normal brain, the fate of such synaptic contacts after demyelination is still unclear. By combining electrophysiology and immunostainings in different transgenic mice expressing fluorescent reporters, we studied the synaptic innervation of OPCs in the model of lysolecithin (LPC)-induced demyelination of corpus callosum. Synaptic innervation of reactivated OPCs in the lesion was revealed by the presence of AMPA receptor-mediated synaptic currents, VGluT1+ axon-OPC contacts in 3D confocal reconstructions and synaptic junctions observed by electron microscopy. Moreover, 3D confocal reconstructions of VGluT1 and NG2 immunolabeling showed the existence of glutamatergic axon-OPC contacts in post-mortem MS lesions. Interestingly, patch-clamp recordings in LPC-induced lesions demonstrated a drastic decrease in spontaneous synaptic activity of OPCs early after demyelination that was not caused by an impaired conduction of compound action potentials. A reduction in synaptic connectivity was confirmed by the lack of VGluT1+ axon-OPC contacts in virtually all rapidly proliferating OPCs stained with EdU (50-ethynyl-20-deoxyuridine). At the end of the massive proliferation phase in lesions, the proportion of innervated OPCs rapidly recovers, although the frequency of spontaneous synaptic currents did not reach control levels. In conclusion, our results demonstrate that newly-generated OPCs do not receive synaptic inputs during their active proliferation after demyelination, but gain synapses during the remyelination process. Hence, glutamatergic synaptic inputs may contribute to inhibit OPC proliferation and might have a physiopathological relevance in demyelinating disorders.