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Inward rectifying potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis in oligodendrocytes, the myelinating cells of the central nervous system (CNS). Prominent expression of Kir4.1 has been indicated in oligodendrocytes, but the extent of expression of other Kir subtypes is unclear. Here, we used qRT-PCR to determine expression of Kir channel transcripts in the mouse optic nerve, a white matter tract comprising myelinated axons and the glia that support them. A novel finding was the high relative expression of Kir7.1, comparable to that of Kir4.1, the main glial Kir channel. Significantly, Kir7.1 immunofluorescence labelling in optic nerve sections and in isolated cells was localised to oligodendrocyte somata. Kir7.1 are known as a K+transporting channels and, using patch clamp electrophysiology and the Kir7.1 blocker VU590, we demonstrated Kir7.1 channels carry a significant proportion of the whole cell potassium conductance in oligodendrocytes isolated from mouse optic nerves. Notably, oligodendrocytes are highly susceptible to ischemia/hypoxia and this is due at least in part to disruption of ion homeostasis. A key finding of this study is that blockade of Kir7.1 with VU590 compromised oligodendrocyte cell integrity and compounds oligodendroglial loss in ischemia/hypoxia in the oxygen–glucose deprivation (OGD) model in isolated intact optic nerves. These data reveal Kir7.1 channels are molecularly and functionally expressed in oligodendrocytes and play an important role in determining oligodendrocyte survival and myelin integrity.

Disruption or loss of oligodendrocytes (OLs) and myelin has devastating effects on CNS function and integrity, which occur in diverse neurological disorders, including Multiple Sclerosis (MS), Alzheimer’s disease and neuropsychiatric disorders. Hence, there is a need to develop new therapies that promote oligodendrocyte regeneration and myelin repair. A promising approach is drug repurposing, but most agents have potentially contrasting biological actions depending on the cellular context and their dose-dependent effects on intracellular pathways. Here, we have used a combined systems biology and neurobiological approach to identify compounds that exert positive and negative effects on oligodendroglia, depending on concentration. Notably, next generation pharmacogenomic analysis identified the PI3K/Akt modulator LY294002 as the most highly ranked small molecule with both pro- and anti-oligodendroglial concentration-dependent effects. We validated these in silico findings using multidisciplinary approaches to reveal a profoundly bipartite effect of LY294002 on the generation of OPCs and their differentiation into myelinating oligodendrocytes in both postnatal and adult contexts. Finally, we employed transcriptional profiling and signalling pathway activity assays to determine cell-specific mechanisms of action of LY294002 on oligodendrocytes and resolve optimal in vivo conditions required to promote myelin repair. These results demonstrate the power of multidisciplinary strategies in determining the therapeutic potential of small molecules in neurodegenerative disorders., Graphical Abstract ga1

Brain ageing is characterised by a decline in neuronal function and associated cognitive deficits. There is increasing evidence that myelin disruption is an important factor that contributes to the age‐related loss of brain plasticity and repair responses. In the brain, myelin is produced by oligodendrocytes, which are generated throughout life by oligodendrocyte progenitor cells (OPCs). Currently, a leading hypothesis points to ageing as a major reason for the ultimate breakdown of remyelination in Multiple Sclerosis (MS). However, an incomplete understanding of the cellular and molecular processes underlying brain ageing hinders the development of regenerative strategies. Here, our combined systems biology and neurobiological approach demonstrate that oligodendroglial and myelin genes are amongst the most altered in the ageing mouse cerebrum. This was underscored by the identification of causal links between signalling pathways and their downstream transcriptional networks that define oligodendroglial disruption in ageing. The results highlighted that the G‐protein coupled receptor Gpr17 is central to the disruption of OPCs in ageing and this was confirmed by genetic fate‐mapping and cellular analyses. Finally, we used systems biology strategies to identify therapeutic agents that rejuvenate OPCs and restore myelination in age‐related neuropathological contexts., Ageing reduces the number of OPCs and their ‘stemness’. Genomic analysis resolved key mechanisms underpinning the age related decline in OPCs, most notably reduced expression of Gpr17 in a subpopulation of differentiation committed OPCs. Pharmacogenomics identified small molecules that have the potential to rejuvenate OPCs and promote myelination and repair.

White matter (WM) is a highly prominent feature in the human cerebrum and is comprised of bundles of myelinated axons that form the connectome of the brain. Myelin is formed by oligodendrocytes and is essential for rapid neuronal electrical communication that underlies the massive computing power of the human brain. Oligodendrocytes are generated throughout life by oligodendrocyte precursor cells (OPCs), which are identified by expression of the chondroitin sulphate proteoglycan NG2 (Cspg4), and are often termed NG2-glia. Adult NG2+ OPCs are slowly proliferating cells that have the stem cell–like property of self-renewal and differentiation into a pool of ‘late OPCs’ or ‘differentiation committed’ OPCs(COPs) identified by specific expression of the G-protein-coupled receptor GPR17, which are capable of differentiation into myelinating oligodendrocytes. In the adult brain, these reservoirs of OPCs and COPs ensure rapid myelination of new neuronal connections formed in response to neuronal signalling, which underpins learning and cognitive function. However, there is an age-related decline in myelination that is associated with a loss of neuronal function and cognitive decline. The underlying causes of myelin loss in ageing are manifold, but a key factor is the decay in OPC ‘stemness’ and a decline in their replenishment of COPs, which results in the ultimate failure of myelin regeneration. These changes in ageing OPCs are underpinned by dysregulation of neuronal signalling and OPC metabolic function. Here, we highlight the role of purine signalling in regulating OPC self-renewal and the potential importance of GPR17 and the P2X7 receptor subtype in age-related changes in OPC metabolism. Moreover, age is the main factor in the failure of myelination in chronic multiple sclerosis and myelin loss in Alzheimer’s disease, hence understanding the importance of purine signalling in OPC regeneration and myelination is critical for developing new strategies for promoting repair in age-dependent neuropathology.

Oligodendrocytes are the myelin forming cells of the central nervous system (CNS) and are generated from oligodendrocyte progenitor cells (OPCs). Disruption or loss of oligodendrocytes and myelin has devastating effects on CNS function and integrity, which occurs in diverse neurological disorders, including Multiple Sclerosis (MS), Alzheimer’s disease (AD) and neuropsychiatric disorders. Hence, there is a need to develop new therapies that promote oligodendrocyte regeneration and myelin repair. A promising approach is drug repurposing, but most agents have potentially contrasting biological actions depending on the cellular context and their dose-dependent effects on intracellular regulatory pathways. Here, we have used a combined drug connectivity systems biology and neurobiological approach to identify compounds that exert positive and negative effects on oligodendroglia, depending on concentration. Notably, LY294002, a potent inhibitor of PI3K/Akt signalling, was the most highly ranked small molecule for both pro- and anti-oligodendroglial effects. We validated thesein silicofindings in multiplein vivoandex vivoneurobiological models and demonstrate that low and high doses of LY294002 have a profoundly bipartite effect on the generation of OPCs and their differentiation into myelinating oligodendrocytes. Finally, we employed transcriptional profiling and signalling pathway activity assays to determine cell-specific mechanisms of action of LY294002 on oligodendrocytes and resolve optimalin vivoconditions required to promote myelin repair. These results demonstrate the power of multifactorial neurobiological andin silicostrategies in determining the therapeutic potential of small molecules in neurodegenerative disorders.One-sentence summaryDrug discovery and CNS myelination

Inward rectifying potassium (Kir) channels comprise a large family with diverse biophysical properties. A predominant feature of central nervous system (CNS) glia is their expression of Kir4.1, which as homomers are weakly rectifying channels, but form strongly rectifying channels as heteromers with Kir2.1. However, the extent of Kir2.1 expression and their association with Kir4.1 in glia throughout the CNS is unclear. We have examined this in astrocytes and oligodendrocytes of the mouse optic nerve, a typical CNS white matter tract. Western blot and immunocytochemistry demonstrates that optic nerve astrocytes and oligodendrocytes express Kir2.1 and that it co-localises with Kir4.1. Co-immunoprecipitation analysis provided further evidence that Kir2.1 associate with Kir4.1 and, moreover, Kir2.1 expression was significantly reduced in optic nerves and brains from Kir4.1 knock-out mice. In addition, optic nerve glia express Kir5.1, which may associate with Kir2.1 to form silent channels. Immunocytochemical and co-immunoprecipitation analyses indicate that Kir2.1 associate with Kir5.1 in optic nerve glia, but not in the brain. The results provide evidence that astrocytes and oligodendrocytes may express heteromeric Kir2.1/Kir4.1 and Kir2.1/Kir5.1 channels, together with homomeric Kir2.1 and Kir4.1 channels. In astrocytes, expression of multiple Kir channels is the biophysical substrate for the uptake and redistribution of K+ released during neuronal electrical activity known as &lsquo, potassium spatial buffering&rsquo, Our findings suggest a similar potential role for the diverse Kir channels expressed by oligodendrocytes, which by way of their myelin sheaths are intimately associated with the sites of action potential propagation and axonal K+ release.

Inward Rectifying Potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis and neuronal excitability. The most recently described Kir subtype is Kir7.1, which is known as a K+ transporting subtype. Earlier studies localised Kir7.1 to subpopulations of neurones in the brain. However, the pattern of Kir7.1 expression across the brain has not previously been examined. Here, we have determined neuronal and glial expression of Kir7.1 in the adult mouse brain, using immunohistochemistry and transgenic mouse lines expressing reporters specific for astrocytes [glial fibrillary acidic protein‐enhanced green fluorescent protein (GFAP‐EGFP], myelinating oligodendrocytes (PLP‐DsRed), oligodendrocyte progenitor cells (OPC, Pdgfra‐creERT2/Rosa26‐YFP double‐transgenic mice) and all oligodendrocyte lineage cells (SOX10‐EGFP). The results demonstrate significant neuronal Kir7.1 immunostaining in the cortex, hippocampus, cerebellum and pons, as well as the striatum and hypothalamus. In addition, astrocytes are shown to be immunopositive for Kir7.1 throughout grey and white matter, with dense immunostaining on cell somata, primary processes and perivascular end‐feet. Immunostaining for Kir7.1 was observed in oligodendrocytes, myelin and OPCs throughout the brain, although immunostaining was heterogeneous. Neuronal and glial expression of Kir7.1 is confirmed using neurone‐glial cortical cultures and optic nerve glial cultures. Notably, Kir7.1 have been shown to regulate the excitability of thalamic neurones and our results indicate this may be a widespread function of Kir7.1 in neurones throughout the brain. Moreover, based on the function of Kir7.1 in multiple transporting epithelia, Kir7.1 are likely to play an equivalent role in the primary glial function of K+ homeostasis. Our results indicate Kir7.1 are far more pervasive in the brain than previously recognised and have potential importance in regulating neuronal and glial function.

Oligodendrocytes are the myelinating cells of the CNS, producing the insulating myelin sheath that facilitates rapid electrical conduction of axonal action potentials. Oligodendrocytes arise from oligodendrocyte progenitor cells (OPCs) under the control of multiple factors, including neurotransmitters and other neuron-derived factors. A significant population of OPCs persists in the adult CNS, where they are often referred to as NG2-glia, because they are identified by their expression of the NG2 chondroitin sulphate proteoglycan (CSPG4). In the adult brain, the primary function of NG2-glia is the life-long generation of oligodendrocytes to replace myelin lost through natural 'wear and tear' and pathology, as well as to provide new oligodendrocytes to myelinate new connections formed in response to new life experiences. NG2-glia contact synapses and respond to neurotransmitters and potassium released during neuronal transmission; to this end, NG2-glia (OPCs) express multiple neurotransmitter receptors and ion channels, with prominent roles being identified for glutamatergic signalling and potassium channels in oligodendrocyte differentiation. Myelinating oligodendrocytes also express a wide range of neurotransmitter receptors and ion channels, together with transporters and gap junctions; together, these have critical functions in cellular ion and water homeostasis, as well as metabolism, which is essential for maintaining myelin and axon integrity. An overriding theme is that oligodendrocyte function and myelination is not only essential for rapid axonal conduction, but is essential for learning and the long-term integrity of axons and neurones. Hence, myelination underpins cognitive function and the massive computing power of the human brain and myelin loss has devastating effects on CNS function. This chapter focuses on normal oligodendrocyte physiology.

Oligodendrocytes form the myelin that ensheaths CNS axons, which is essential for rapid neuronal signalling and underpins the massive computing power of the human brain. Oligodendrocytes and myelin also provide metabolic and trophic support for axons and their disruption results in axonal demise and neurodegeneration, which are key features of Alzheimer’s disease (AD). Notably, the brain has a remarkable capacity for regenerating oligodendrocytes, which is the function of adult oligodendrocyte progenitor cells (OPCs) or NG2-glia. White matter loss is often among the earliest brain changes in AD, preceding the tangles and plaques that characterize neuronal deficits. The underlying causes of myelin loss include oxidative stress, neuroinflammation and excitotoxicity, associated with accumulation of Aβ and tau hyperphosphorylation, pathological hallmarks of AD. Moreover, there is evidence that NG2-glia are disrupted in AD, which may be associated with disruption of synaptic signalling. This has led to the hypothesis that a vicious cycle of myelin loss and failure of regeneration from NG2-glia plays a key role in AD. Therapies that target NG2-glia are likely to have positive effects on myelination and neuroprotection in AD.

The name neuroglia is generally translated as nerve glue. In the recent past, this has been used to describe passive structural cells. Presently, this view has been challenged and the true dynamic and multifunctional nature of neuroglia is beginning to be appreciated. In the central nervous system, the main kinds of neuroglia are astrocytes (the primary homeostatic cells that ensure synaptic transmission), oligodendrocytes (which form the myelin that ensures rapid electrical transmission) and microglia (the main immune cells). In the peripheral nervous system, neuroglia comprise Schwann cells, satellite glia and enteric glia. These functionally diverse and specialised cells are fundamental to function at the molecular, cellular, tissue and system levels. Without nerve glue, the body cannot function and the future will begin to unlock their importance in higher cognitive functions that set humans apart from other animals and their true potential as therapeutic targets in neurodegenerative and other diseases.

Studies by Bruce Ransom and colleagues have made a major contribution to show that white matter is susceptible to ischemia/hypoxia. White matter contains axons and the glia that support them, notably myelinating oligodendrocytes, which are highly vulnerable to ischemic-hypoxic damage. Previous studies have shown that metabotropic GluRs (mGluRs) are cytoprotective for oligodendrocyte precursor cells and immature oligodendrocytes, but their potential role in adult white matter was unresolved. Here, we report that group 1 mGluR1/5 and group 2 mGluR3 subunits are expressed in optic nerves from mice aged postnatal day (P)8–12 and P30–35. We demonstrate that activation of group 1 mGluR protects oligodendrocytes against oxygen-glucose deprivation (OGD) in developing and young adult optic nerves. In contrast, group 2 mGluR are shown to be protective for oligodendrocytes against OGD in postnatal but not young adult optic nerves. The cytoprotective effect of group 1 mGluR requires activation of PKC, whilst group 2 mGluR are dependent on negatively regulating adenylyl cyclase and cAMP. Our results identify a role for mGluR in limiting injury of oligodendrocytes in developing and young adult white matter, which may be useful for protecting oligodendrocytes in neuropathologies involving excitoxicity and ischemia/hypoxia.

In the postnatal and adult central nervous system (CNS) the subventricular zone (SVZ) of the forebrain is the main source of neural stem cells (NSCs) that generate olfactory neurons and oligodendrocytes (OLs) the myelinating cells of the CNS. Here we provide evidence of a primary role for canonical Wnt/ß catenin signaling in regulating NSC fate along neuronal and oligodendroglial lineages in the postnatal SVZ. Our findings demonstrate that glutamatergic neuronal precursors (NPs) and oligodendrocyte precursors (OPs) are derived strictly from the dorsal SVZ (dSVZ) microdomain under the control of Wnt/ß catenin whereas GABAergic NPs are derived mainly from the lateral SVZ (lSVZ) microdomain independent of Wnt/ß catenin. Transcript analysis of microdissected SVZ microdomains revealed that canonical Wnt/ß catenin signaling was more pronounced in the dSVZ microdomain. This was confirmed using the ß catenin activated Wnt reporter mouse and by pharmacological stimulation of Wnt/ß catenin by infusion of the specific glycogen synthase kinase 3ß inhibitor AR A014418 which profoundly increased the generation of cycling cells. In vivo genetic/pharmacological stimulation or inhibition of Wnt/ß catenin respectively increased and decreased the differentiation of dSVZ NSCs into glutamatergic NPs and had a converse effect on GABAergic NPs. Activation of Wnt/ß catenin dramatically stimulated the generation of OPs but its inhibition had no effect indicating other factors act in concert with Wnt/ß catenin to fine tune oligodendrogliogenesis in the postnatal dSVZ. These results demonstrate a role for Wnt/ß catenin signaling within the dorsal microdomain of the postnatal SVZ in regulating the genesis of glutamatergic neurons and OLs. Stem Cells 2014;32:1301 1312.

Oligodendrocytes, the myelinating cells of the CNS, are derived postnatally from oligodendrocyte precursors (OPs) of the subventricular zone (SVZ). However, the mechanisms that regulate their generation from SVZ neural stem cells (NSC) are poorly understood. Here, we have examined the role of glycogen synthase kinase 3β (GSK3β), an effector of multiple converging signaling pathways in postnatal mice. The expression of GSK3β by rt-qPCR was most prominent in the SVZ and in the developing white matter, around the first 1–2 weeks of postnatal life, coinciding with the peak periods of OP differentiation. Intraventricular infusion of the GSK3β inhibitor ARA-014418 in mice aged postnatal day (P) 8–11 significantly increased generation of OPs in the dorsal microdomain of the SVZ, as shown by expression of cell specific markers using rt-qPCR and immunolabelling. Analysis of stage specific markers revealed that the augmentation of OPs occurred via increased specification from earlier SVZ cell types. These effects of GSK3β inhibition on the dorsal SVZ were largely attributable to stimulation of the canonical Wnt/β-catenin signaling pathway over other pathways. The results indicate GSK3β is a key endogenous factor for specifically regulating oligodendrogenesis from the dorsal SVZ microdomain under the control of Wnt-signaling.

Oligodendrocytes are the myelinating cells of the CNS and, like neurons, are highly sensitive to ischemic damage. However, the mechanisms underlying cytotoxicity in oligodendrocytes during hypoxic/ischemic episodes are not fully understood. TASK-1 is a K+ leak channel that mediates hypoxic depolarisation in neurons. The expression and function of TASK-1 in oligodendrocytes had not previously been addressed. In this study, we investigate the expression of TASK-1 in oligodendrocytes and its role in white matter ischemic damage. Expression of TASK-1 in oligodendrocytes was investigated in the mouse brain using immunostaining. TASK-1 channel function was identified by established pharmacological and electrophysiological strategies, using the whole-cell patch clamp technique in cell cultures of oligodendrocytes from the optic nerve, a typical white matter tract. The role of TASK-1 in hypoxia was examined in isolated intact optic nerves subjected to oxygen glucose deprivation (OGD). Oligodendrocytes are strongly immunopositive for TASK-1 throughout the brain. Patch-clamp identified functional TASK-1-like leak currents in oligodendrocytes using two recognised means of inhibiting TASK-1, decreasing extracellular pH to 6.4 and exposure to the TASK-1 selective inhibitor anandamide. Incubation of optic nerves with methanandamide, a non-hydrolysable form of anandamide, significantly protected oligodendrocytes against hypoxic disruption and death in OGD. Our data demonstrate for the first time that oligodendrocytes express functional TASK-1 channels and provide compelling evidence they contribute to oligodendrocyte damage in hypoxia. Since oligodendrocyte damage is a key factor in ischemic episodes, TASK-1 may provide a potential therapeutic target in stroke and white matter disease., Highlights • TASK-1 subtype of K2P channel is involved in ischemic neuronal damage. • Oligodendrocytes, the myelinating cells of the CNS, are highly sensitive to ischemic damage. • We demonstrate functional expression of TASK-1 in oligodendrocytes. • Inhibition of TASK1 with anandamide significantly protected against oligodendrocyte damage in response to ischemia.

White matter (WM) tracts contain bundles of myelinated axons and the glia that support them, namely oligodendrocytes that form the myelin and astrocytes, which provide structural and homeostatic support, together with small populations of microglia and oligodendrocyte precursor cells. Neurotransmitter signaling is prominent in WM, and there is a significant role for adrenergic signaling, together with glutamatergic and purinergic mechanisms. The site of axoglial signaling in myelinated axons is the node of Ranvier, which is the site of action potential propagation and displays many properties of synapses. Neurotransmitters released during axonal electrical activity activate receptors on perinodal astrocytes, to stimulate their release of gliotransmitters that integrate function within axon–glial–vascular networks. One of the key functions of astroglial adrenergic signaling in WM is to maintain energy supply to axons and oligodendrocytes, ensuring efficient and rapid information transfer throughout the central nervous system and maintaining WM integrity in times of energy stress.

A key aim of therapy for multiple sclerosis (MS) is to promote the regeneration of oligodendrocytes and remyelination in the central nervous system (CNS). The present study provides evidence that the vitamin K-dependent protein growth arrest specific 6 (Gas6) promotes such repair in in vitro cultures of mouse optic nerve and cerebellum. We first determined expression of Gas6 and TAM (Tyro3, Axl, Mer) receptors in the mouse CNS, with all three TAM receptors increasing in expression through postnatal development, reaching maximal levels in the adult. Treatment of cultured mouse optic nerves with Gas6 resulted in significant increases in oligodendrocyte numbers as well as expression of myelin basic protein (MBP). Gas6 stimulation also resulted in activation of STAT3 in optic nerves as well as downregulation of multiple genes involved in MS development, including matrix metalloproteinase-9 ( MMP9), which may decrease the integrity of the blood–brain barrier and is found upregulated in MS lesions. The cytoprotective effects of Gas6 were examined in in vitro mouse cerebellar slice cultures, where lysolecithin was used to induce demyelination. Cotreatment of cerebellar slices with Gas6 significantly attenuated demyelination as determined by MBP immunostaining, and Gas6 activated Tyro3 receptor through its phosphorylation. In conclusion, these results demonstrate that Gas6/TAM signaling stimulates the generation of oligodendrocytes and increased myelin production via Tyro3 receptor in the adult CNS, including repair after demyelinating injury. Furthermore, the effects of Gas6 on STAT3 signaling and matrix MMP9 downregulation indicate potential glial cell repair and immunoregulatory roles for Gas6, indicating that Gas6-TAM signaling could be a potential therapeutic target in MS and other neuropathologies.

Oligodendrocytes are specialised glial cells that myelinate CNS axons. Myelinated axons are bundled together into white matter tracts that interconnect grey matter areas of the brain and are essential for rapid, integrated neuronal communication and cognitive function. Life-long generation of oligodendrocytes is required for myelination of new neuronal connections and repair of myelin lost through natural 'wear and tear'. This is the function of a substantial population of adult oligodendrocyte progenitors (OPs). Notably, there is white matter shrinkage and decreased myelination in the ageing brain, which is accelerated in dementia. The underlying causes of myelin loss in dementia are unresolved, but it implies a decline in the regenerative capacity of OPs. A feature of OPs is that they form neuron-glial synapses and respond to glutamate released by neurons via a range of glutamate receptors. Glutamate neurotransmission onto OPs is proposed to regulate their proliferation and differentiation into myelinating oligodendrocytes. Here, we discuss evidence that deregulation of glutamate neurotransmission in dementia and compromised generation of oligodendrocytes from OPs are key features of myelin loss and associated cognitive decline.

Glycogen synthase kinase 3β (GSK3β) is an essential integrating molecule for multiple proliferation and differentiation signals that regulate cell fate. Here, we have examined the effects of inhibiting GSK3β on the development of oligodendrocytes (OLs) from their oligodendrocyte precursors (OP) in vivo by injection into the lateral ventricle of postnatal mice and ex vivo in organotypic cultures of isolated intact rodent optic nerve. Our results show that a range of GSK3β inhibitors (ARA-014418, lithium, indirubin, and L803-mt) increase OPs and OLs and promote myelination. Inhibition of GSK3β stimulates OP proliferation and is prosurvival and antiapoptotic. The effects of GSK3β inhibition in OPs is via the canonical Wnt signaling pathway by stimulating nuclear translocation of β-catenin. However, direct comparison of the effects of Wnt3a and GSK3β inhibition in optic nerves shows that they have opposing actions on OLs, whereby GSK3β inhibition strikingly increases OL differentiation, whereas Wnt3a inhibits OL differentiation. Notably, GSK3β inhibition overrides the negative effects of Wnt3a on OLs, indicating novel GSK3β signaling mechanisms that negatively regulate OL differentiation. We identify that two mechanisms of GSK3β inhibition are to stimulate cAMP response element binding (CREB) and decrease Notch1 signaling, which positively and negatively regulate OL differentiation and myelination, respectively. A key finding is that GSK3β inhibition has equivalent effects in the adult and stimulates the regeneration of OLs and remyelination following chemically induced demyelination. This study identifies GSK3β as a profound negative regulator of OL differentiation that contributes to inefficient regeneration of OLs and myelin repair in demyelination. © 2011 Wiley-Liss, Inc.

NG2-glia are an abundant population of cells in the adult CNS that make up a novel glial cell type. Here, we have examined calcium signals in NG2-glia identified by expression of the fluorescent protein DsRed under the control of the NG2 promoter in the white matter of the mouse optic nerve. We focused on mice aged postnatal day (P)12-16, after the main period of oligodendrocyte generation. Using fluo-4 and fura-2 calcium imaging in isolated intact nerves, we show that glutamate and ATP evoke Ca(2+) signals in NG2-glia in situ, acting on AMPA-type glutamate receptors and P2Y(1) and P2X(7) purine receptors; NMDA evoked a weak Ca(2+) signal in a small proportion of NG2-glia. We show that axonal action potentials and mechanical stimulation of astrocytes effect the release of glutamate and ATP to act on NG2-glia; ATP alone evokes robust Ca(2+) signals, whereas glutamate did not unless AMPA receptor desensitization was blocked with cyclothiazide. We identify the precise contacts that NG2-glia form with axons at nodes of Ranvier, and the intricate bipartite sheaths formed between the processes of NG2-glia and astrocytes. In addition, we provide evidence that NG2-glia express synaptophysin, indicating they have mechanisms for transmitting as well as receiving signals. This study places NG2-glia within a neuron-glial network, and identifies roles for glutamate and ATP in communication with astrocytes as well as axons.

Models of central nervous system (CNS) function have historically been based on neurons and their synaptic contacts – the neuronal doctrine. This doctrine envisages glia as passive supportive cells. However, electrophysiological and imaging studies in brain slices show us that astrocytes, the most numerous cells in the brain, express a wide range of neurotransmitter receptors that are activated in response to synaptic activity. Furthermore, astrocytes communicate via calcium signals that are propagated over long distances by the release of ‘gliotransmitters’, the most abundant being adenosine triphosphate (ATP). This has led to the concept of the neuron–astroglial functional unit as the substrate of integration in the CNS. Recently, a novel glial cell type has been characterized by expression of the proteoglycan NG2. These NG2-glia receive presynaptic input from neurons and responds to neurotransmitters released at synapses. Now, studies on transgenic mice in which fluorescent proteins are specifically expressed by subclasses of glia are helping to address the question of where NG2-glia fit in the neuron–astroglial model of integrated brain function. NG2-glia, as well as astrocytes, have been shown to respond to neuronal and astroglial signals by raised intracellular calcium, which is a potential communications mechanism by which NG2-glia may be active partners in neuron–glial circuits. Moreover, a current concept of NG2-glia considers them to be ‘neural stem cells’ and an exciting prospect is that neuron–glial signalling may regulate the differentiation capacity of NG2-glia and their response to injury.

The purine ATP has a prominent regulatory role in CNS function and pathology due to its actions on glial cells - microglia, astrocytes and oligodendrocytes. ATP serves as an apparently ubiquitous ‘gliotransmitter’ that is released by astrocytes and other cells to activate purine receptors on neighbouring cells. In pathology, the release of ATP mediates both tissue damage and repair by its direct effects on glial cell integrity and survival. The actions of ATP on glia are mediated via a wide range of receptors, broadly divided into ionotropic P2X and metabotropic (G-protein coupled receptors (GPCR)) P2Y receptors, of which there are multiple subtypes (P2X 1 -P2X 7 and P2Y 1 -P2Y 14 ). ATP-mediated interactions between astrocytes and microglia are at the centre of immune responses in the CNS, with prominent roles for the P2X 4 , P2X 7 , P2Y 1 , P2Y 6 and P2Y 12 receptor subtypes. In oligodendrocytes, P2X 7 and P2Y 1 receptor subtypes have a bipartite function in respectively mediating oligodendrocyte destruction and protection. Purine receptors mediate glial pathology, with prominent roles in ischemia, neuroinflammation, Multiple Scelerosis, neuropathic pain and traumatic injury. Notably, glial ATP signalling may be altered with ageing and is implicated in impaired myelination and immunity in Alzheimer’s disease. Hence, glial purine receptors provide potential therapeutic targets in multiple neuropathologies, but the ‘Jeckyll and Hyde’ nature of purine signalling underscores the importance of further research and a comprehensive understanding of the roles of the different purine receptors in mediating tissue damage and repair.

Myelin formation by oligodendrocytes has been shown to be regulated by cyclic AMP (cAMP) signaling pathways and to depend on the resting membrane potential (RMP). We therefore examined whether cAMP regulates the RMP of myelin-forming oligodendrocytes in isolated intact optic nerves of rats. Oligodendrocytes exhibited a significant developmental shift in the RMP from -37 mV at postnatal day (P)6-8 to -67 mV at P21-30. The regulation of RMP was examined further in myelin-forming oligodendrocytes in nerves aged P15-20. Raising intracellular cAMP with dbcAMP or forskolin induced a significant hyperpolarization in myelin-forming oligodendrocytes by 10-15 mV. Inhibition of cAMP-dependent protein kinase (PKA) with KT5720 depolarized the oligodendroglial RMP -30 mV, which was only partly reversed by dbcAMP. In contrast, inhibition of cAMP specific phosphodiesterase with rolipram had no significant effect on the oligodendroglial RMP or the cAMP-mediated hyperpolarization. Blockade of Kir with 100 microM BaCl(2) depolarized the oligodendrocyte RMP to -25 mV and inhibited the hyperpolarizing action of dbcAMP. The RMP was unaffected by agents that modulated ATP-sensitive potassium channels. The results provide evidence of a predominant role for Kir in setting the oligodendroglial RMP and show that cAMP regulates the oligodendroglial RMP, at least partly by a PKA-mediated pathway, possibly by modulating the activity of Kir.

The function of oligodendrocytes is to myelinate CNS axons. Oligodendrocytes and the axons they myelinate are functional units, and neurotransmitters released by axons can influence all stages of oligodendrocyte development via calcium dependent mechanisms. Some of the clearest functional evidence is for adenosine, ATP, and glutamate, which are released by electrically active axons and regulate the migration and proliferation of oligodendrocyte progenitor cells and their differentiation into myelinating oligodendrocytes. Glutamate and ATP, released by both axons and astrocytes, continue to mediate Ca2+ signaling in mature oligodendrocytes, acting via AMPA and NMDA glutamate receptors, and heterogeneous P2X and P2Y purinoceptors. Physiological signalling between axons, astrocytes, and oligodendrocytes is likely to play an important role in myelin maintenance throughout life. Significantly, ATP- and glutamate-mediated Ca2+ signaling are also major components of oligodendrocyte and myelin damage in numerous pathologies, most notably ischemia, injury, periventricular leukomalacia, and multiple sclerosis. In addition, NG2-expressing glia (synantocytes) in the adult CNS are highly reactive cells that respond rapidly to any CNS insult by a characteristic gliosis, and are able to regenerate oligodendrocytes and possibly neurons. Glutamate and ATP released by neurons and astrocytes evoke Ca2+ signaling in NG2-glia (synantocytes), and it is proposed these regulate their differentiation capacity and response to injury. In summary, clear roles have been demonstrated for neurotransmitter-mediated Ca2+ signaling in oligodendrocyte development and pathology. A key issue for future studies is to determine the physiological roles of neurotransmitters in mature oligodendrocytes and NG2-glia (synantocytes). © 2006 Wiley-Liss, Inc.

Glia in the central nervous system (CNS) express diverse inward rectifying potassium channels (Kir). The major function of Kir is in establishing the high potassium (K+) selectivity of the glial cell membrane and strongly negative resting membrane potential (RMP), which are characteristic physiological properties of glia. The classical property of Kir is that K+ flows inwards when the RMP is negative to the equilibrium potential for K+ (Ek), but at more positive potentials outward currents are inhibited. This provides the driving force for glial uptake of K+ released during neuronal activity, by the processes of “K+ spatial buffering” and “K+ siphoning”, considered a key function of astrocytes, the main glial cell type in the CNS. Glia express multiple Kir channel subtypes, which are likely to have distinct functional roles related to their differences in conductance, and sensitivity to intracellular and extracellular factors, including pH, ATP, G-proteins, neurotransmitters and hormones. A feature of CNS glia is their specific expression of the Kir4.1 subtype, which is a major K+ conductance in glial cell membranes and has a key role in setting the glial RMP. It is proposed that Kir4.1 have a primary function in K+ regulation, both as homomeric channels and as heteromeric channels by co-assembley with Kir5.1 and probably Kir2.0 subtypes. Significantly, Kir4.1 are also expressed by oligodendrocytes, the myelin-forming cells of the CNS, and the genetic ablation of Kir4.1 are also expressed by Oligodendrocytes, the myelin-forming cells of the CNS, and the genetic ablation of Kir4.1 results in severe hypomyelination. Hence, Kir, and in particular Kir4.1, are key regulators of glial functions, which in turn determine neuronal excitability and axonal conduction.

Classic studies have recognized neurons and three glial elements in the central nervous system (CNS) – astrocytes, oligodendrocytes and microglia. The identification of novel glia that specifically express the NG2 chondroitin sulphate proteoglycan (CSPG) raises the possibility of a fifth element. Until recently, all NG2-expressing glia were considered to be oligodendrocyte precursor cells (OPCs) that persist in the adult CNS to generate oligodendrocytes throughout life. However, this narrow view of the function of ‘NG2-glia’ is being challenged. The majority of NG2-expressing glia in the adult CNS are a distinct class of cells that we have called ‘synantocytes’ (from the Greek synanto for contact). Synantocytes are stellate cells, with large process arborizations, and are exquisitely related to neurons. Individual cells traverse white and grey matter and form multiple contacts with neurons, astrocytes, oligodendrocytes and myelin. Synantocytes are an integral component of the ‘tetrapartite’ synapse, and provide a potential integrative neuron-glial communications pathway. Neuronal activity, glutamate and adenosine triphosphate (ATP) act on synantocyte receptors and evoke raised intracellular calcium. It remains to be seen whether this serves a physiological function, but synantocytes may be specialized to monitor signals from neurons and glia, and to respond to changes in the integrity of the CNS via their specific contacts and ion channel and receptor profiles. The general consequences of synantocyte activation are proliferation and phenotypic changes, resulting in glial scar formation, or regeneration of oligodendrocytes, and possibly neurons.

In the adult CNS, cells that express the NG2 chondroitin sulphate proteoglycan are considered oligodendrocyte progenitor cells (OPCs). However, adult NG2-glia are highly complex cells, suggesting they may be a mature glial cell type distinct from conventional OPCs. To test this possibility, we have determined the response of NG2-glia to axon loss in the neonatal rat optic nerve, which has been shown previously to result in an almost complete loss of OPCs. We show here that NG2-glia are not lost following enucleation of the neonatal or juvenile optic nerve and that at all ages NG2-glia become "reactive" in response to axon degeneration, in direct contrast to previous studies on OPCs. We provide evidence that NG2-glia with an adult morphological phenotype develop postnatally, at the same time as oligodendrocytes. In the neonatal nerve, we show that the loss of axons blocks the development of oligodendrocytes, although reactive NG2-glia are abundant and are able to generate oligodendrocytes when placed in culture. Conversely, axon transection did not result in the loss of mature oligodendrocytes, indicating that the dependence of oligodendrocytes on axon-derived survival factors decreases with differentiation. This study shows that NG2-glia are a highly reactive cell type and do not generate oligodendrocytes in vivo in the absence of appropriate axon-derived signals. This may underlie in part the failure of remyelination in chronic multiple sclerosis, despite the abundance of NG2+ expressing glial cells within demyelinated lesions. It is clear either that OPCs do not depend on axons for their survival, in total contradiction to previous studies, or that in vivo a population of NG2 expressing glia are not perinatal or adult OPCs in the accepted sense. We suggest that a population of NG2-glia diverge postnatally into a novel glial type that is functionally and behaviourally distinct from conventional OPCs.

Activation of purinoceptors by extracellular ATP is an important component of the glial response to injury in the central nervous system (CNS). ATP has been shown to evoke raised cytosolic [Ca(2+)] in astrocytes, oligodendrocytes, and microglia, the three major glial cell types in the CNS. Glial cells express a heterogenous collection of metabotropic P2Y and ionotropic P2X purinoceptors, which respectively mobilise Ca(2+) from intracellular stores and trigger Ca(2+) influx across the plasmalemma. It is likely that different receptors have distinct roles in glial cell physiology and pathology. Our studies on optic nerve glia in situ indicate that P2Y(1) and P2Y(2/4) receptors are activated at low ATP concentrations, suggesting they are the predominant purinoceptors mediating physiological Ca(2+) signalling. Glia also express P2X(1) and P2X(3) purinoceptors, which mediate fast, rapidly desensitising current and may also be important in signalling. At high concentrations, such as occur in CNS injury, ATP induces large and prolonged increases in glial [Ca(2+)](i) with a primary role for P2Y purinoceptors and inositol trisphosphate (IP(3))-dependent release of Ca(2+) from intracellular stores. In addition, we found that high concentrations of ATP activated a significant P2X component that did not desensitise or saturate and was dependent on extracellular Ca(2+). These are characteristic properties of the P2X(7) subtype, and we provide in situ evidence that application of the P2X(7) receptor agonist benzoyl-benzoyl ATP (BzATP) evokes raised [Ca(2+)](i) in optic nerve glia, and that the dye YO-PRO-1, which passes through pore-forming P2X(7) receptors, is taken up by astrocytes, oligodendrocytes and microglia. Glia also express P2X(2) and P2X(4) receptors that are also pore-forming in the presence of sustained high ATP concentrations and which may also be important in the glial injury response. There is evidence that activation of P2 purinoceptors is a key step in triggering reactive changes in glial cells, including expression of immediate early genes, induction of extracellular signal regulated kinase and cyclooxygenase-2, synthesis of phospholipase A(2), release of arachidonic acid, production of prostaglandins and release of interleukins. We show that the ATP-mediated increase in glial [Ca(2+)](i) is potentiated by arachidonic acid and reduced by the inhibition of phospholipase A(2) inhibition. Together, the results implicate ATP as a primary signalling molecule in glial cells and indicate specific roles for P2Y and P2X purinoceptors in glial cell pathology.

We present evidence that NG2+ glia are an integral part of an oligodendrocyte/synantocyte (OS) lineage stream the progenitors of which begin to produce both glial phenotypes at about birth. The NG2 CSPG is differentially distributed within the OS lineage, being expressed in progenitors and synantocytes but not in oligodendrocytes. All cells in the OS lineage, except the primordial stem cells, express O4. The oligodendrocyte line reacts with CD9, but synantocytes are CD9-. Nonetheless, synantocytes are morphologically complex and specialised glia which contact axolemma in myelinated fibres at nodes of Ranvier and synaptic terminals, and form >99% of all NG2+ glia in the adult CNS. Thus, the other NG2+ phenotype, the adult oligodendrocyte progenitor cell (AOPC), constitutes a small population of

The taiep rat is a myelin mutant in which initial hypomyelination is followed by progressive demyelination of the CNS. An in vitro study suggests that accumulation of microtubules within oligodendrocytes is the cause of the taiep myelin defects (Song et al., 1999). In this article, we analyze microtubule accumulation in relation to taiep myelin defects in vivo in the anterior medullary velum (AMV), a CNS tissue that enables entire oligodendrocyte units to be resolved. Immunohistochemical analysis demonstrated notably high levels of β-tubulin and the microtubule associated protein τ in the somata and processes of taiep oligodendrocytes. This was correlated with markedly reduced expression of the myelin proteins, proteolipid protein (PLP), myelin basic protein (MBP), 2′,3 -cyclic nucleotide 3′-phosphodiesterase, and both large (L) and small (S) isoforms of myelin-associated glycoprotein (MAG). Moreover, PLP and L-MAG, which are dependent on the microtubule system for intracellular transport, accumulated in the perinuclear cytoplasm of the taiep oligodendrocyte. The myelin deficit was most marked in the area of the AMV populated by the small somata oligodendrocytes that have fine long processes that support numerous myelin sheaths of small diameter axons. Type III/IV oligodendrocytes, which have large somata and short processes that support a small number of myelin sheaths of large diameter axons, were also affected to a certain degree in compact myelin sheath formation. These results support the hypothesis that myelin loss and oligodendrocyte disruption in the taiep mutant result from a defect in the microtubule system that transports myelin components from the somata to the myelin sheath. GLIA 33:1–11, 2001. © 2000 Wiley-Liss, Inc.

The rat anterior medullary velum (AMV) is representative of the brain and spinal cord, overall, and provides an almost two-dimensional preparation for investigating axon-glial interactions in vivo. Here, we review some of our findings on axon-oligodendrocyte unit relations in our adult, development, and injury paradigms: (1) adult oligodendrocytes are phenotypically heterogeneous, conforming to Del Rio Hortega's types I-IV, whereby differences in oligodendrocyte morphology, metabolism, myelin sheath radial and longitudinal dimensions, and biochemistry correlate with the diameters of axons in the unit; (2) oligodendrocytes derive from a common premyelinating oligodendrocyte phenotype, and divergence of types I-IV is related to the age they emerge and the presumptive diameter of axons in the unit; (3) during myelination, axon-oligodendrocyte units progress through a sequence of maturation phases, related to axon contact, ensheathment, establishment of internodal myelin sheaths, and finally the radial growth and compaction of the myelin sheath; (4) we provide direct in vivo evidence that platelet-derived growth factor-AA (PDGF-AA), fibroblast growth factor (FGF-2), and insulin-like growth factor-I (IGF-I) differentially regulate these events, by injecting the growth factors into the cerebrospinal fluid of neonatal rat pups; (5) in lesioned adult AMV, transected central nervous system (CNS) axons regenerate through the putatively inhibitory environment of the glial scar, but remyelination by oligodendrocytes is incomplete, indicating that axon-oligodendrocyte interactions are defective; and (6) in the adult AMV, cells expressing the NG2 chondroitin sulphate have a presumptive adult oligodendrocyte progenitor antigenic phenotype, but are highly complex cells and send processes to contact axolemma at nodes of Ranvier, suggesting they subserve a specific perinodal function. Thus, axons and oligodendrocyte lineage cells form interdependent functional units, but oligodendrocyte numbers, differentiation, phenotype divergence, and myelinogenesis are governed by axons in the units, mediated by growth factors and contact-dependent signals.

The in vivo effects of fibroblast growth factor-2 (FGF-2) and insulin-like growth factor-I (IGF-I) on oligodendrocytes and CNS myelination were determined in the postnatal rat anterior medullary velum (AMV) following injection of both cytokines into the cerebrospinal fluid. Either FGF-2, IGF-I, or saline were administered via the lateral ventricle, twice daily commencing at postnatal day (P) 6. At P9, AMV were immunohistochemically labeled with the Rip antibody, to enable analysis of the numbers of myelin sheaths and of promyelinating and myelinating oligodendrocytes; promyelinating oligodendrocytes are a recognisable immature phenotype which express myelin-related proteins prior to forming myelin sheaths. In parallel experiments, AMV were treated for Western blot analysis to determine relative changes in expression of the myelin proteins 2′, 3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and myelin oligodendrocyte glycoprotein (MOG), which, respectively, characterise early and late stages of myelin maturation. In FGF-2–treated AMV, the number of promyelinating oligodendrocytes increased by 87% compared to saline-injected controls. The numbers of myelinating oligodendrocytes and myelin sheaths were not decreased, but conspicuous unmyelinated gaps within fibre tracts were indications of retarded myelination following FGF-2 treatment. Western blot analysis demonstrated decreased expression of CNP and a near-total loss of MOG, confirming that FGF-2 decreased myelin maturation. In contrast, IGF-I had no effect on the number of promyelinating oligodendrocytes, but increased the numbers of myelinating oligodendrocytes and myelin sheaths by 100% and 93%, respectively. Western blot analysis showed that the amount of CNP was increased following IGF-I treatment, correlating with the greater number of oligodendrocytes, but that MOG expression was lower than in controls, suggesting that the increased number of myelin sheaths in IGF-I was not matched by increased myelin maturation. The results provide in vivo evidence that FGF-2 and IGF-I control the numbers of oligodendrocytes in the brain and, respectively, retard and promote myelination. J. Neurosci. Res. 57:74–85, 1999. © 1999 Wiley-Liss, Inc.

Axon-oligodendrocyte relations of Rip-immunolabelled and dye-injected oligodendrocyte units are characterised in the adult rat anterior medullary velum (AMV). Each oligodendrocyte unit comprised the oligodendrocyte cell body, processes and the internodal myelin segments they support. Oligodendrocyte units corresponded to classically described type I/II or type III/IV unit phenotypes which respectively myelinated discrete populations of small and large diameter axons, delineated by a myelinated fire diameter of 2–4 μm (diameter of the axon plus its myelin sheath). Within units, mean fibre diameter was directly related to mean internodal length and inversely related to the number of myelin sheaths in the unit. The relationship between fibre diameter and internodal length was retained in units which myelinated axons of different diameters, indicating that axon diameter was an important determinant of the longitudinal dimensions of myelin sheaths. We also show that type III/IV units maintained a far greater volume of myelin than type I/II units. It was concluded that type I/II and III/IV oligodendrocytes represent two functionally and morphologically distinct phenotypes whose distribution densities were determined by the diameter and spatial dispersion of axons.

We have previously demonstrated differences in the expression of carbonic anhydrase II (CAII) in oligodendrocyte units myelinating small and large diameter fibres in the anterior medullary velum (AMV) of the adult rat (each unit comprises the cell body, processes and myelin sheaths). Others have indicated that myelin composition may also vary with respect to myelin basic protein (MBP) and proteolipid protein (PLP), and the small (S)- and large (L)-isoforms of myelin associated glycoprotein (MAG). In this study, we have determined the expression of myelin proteins in oligodendrocyte unit phenotypes I–IV, which myelinate fibres ranging in diameter from 0.3–12 μm diameter in the AMV, by using double immunolabelling for Rip, which labels entire units, and MBP, PLP, myelin oligodendrocyte glycoprotein (MOG), L-MAG and S-MAG. We show differences in the expression of L- and S-MAG in units which myelinate different diameter fibres: (1) type I/II units myelinating small diameter fibres had a L-MAG+/S-MAG−/CAII+ phenotype; (2) type II/III units myelinating different diameter fibres had a L-MAG+/S-MAG+/CAII+ phenotype; (3) type III/IV units myelinated large diameter fibres had a L-MAG+/S-MAG+/CAII− phenotype. All units, irrespective of fibre diameter, expressed Rip, MBP, PLP and MOG. The results indicate that type I–IV units may be variants of a single oligodendrocyte population and that phenotypic differences are determined by the diameter of fibres within the unit. The possible significance of metabolic and biochemical differences between oligodendrocytes myelinating small and large diameter axons are discussed with reference to the pathology of demyelination.

The AA dimeric form of platelet-derived growth factor (PDGF-AA) is implicated in the differentiation of cells of the oligodendrocyte lineage, which express PDGF receptors of the alpha subunit type (PDGF-alphaR). In the present study, we show that a single injection of PDGF-AA into the cerebrospinal fluid of neonatal rats delays oligodendrocyte differentiation and interrupts the progress of myelination in the anterior medullary velum (AMV), a white matter tract roofing the IVth ventricle of the brain. PDGF-AA or saline was injected intrathecally in postnatal day (P) 7 rats, and the AMV was subsequently removed and immunolabelled with the oligodendrocyte-specific antibody Rip, at P9, P12, and P21, corresponding to postinjection days (PID) 2, 5, and 14. At P9 (PID2), myelination was retarded in PDGF-AA-treated rats as opposed to saline-treated controls but progressed rapidly after P12 (PID5). Quantification supported the qualitative observations that PDGF-AA mediated an acute decrease in the number of Rip+ oligodendrocytes at P9-12, which largely recovered by P21, suggesting that PDGF-AA may have delayed recruitment of myelinating oligodendrocytes. However, the definitive number of Rip+ oligodendrocytes in the AMV was not increased, suggesting that its action as a promoter of early oligodendrocyte survival may not ultimately affect the definitive number of myelinating oliogdendrocytes in vivo. We discuss the possibilities that excess PDGF-AA may have acted on early oligodendrocytes (precursors or preoligodendrocytes) to either (1) delay their differentiation by maintaining them in the cell cycle or (2) accelerate their differentiation, which may result in premature cell death in the absence of synchronised survival signals. This study supports a role for PDGF-AA in the timing of oligodendrocyte differentiation in vivo, as has been shown in vitro.

Myelinogenesis was investigated in whole-mounted anterior medullary vela from rats aged postnatal day (P) 10–12, using double immunofluorescence labelling with Rip and anti-neurofilament 200 (NF200) antibodies, to identify oligodendrocytes and axons, respectively. A number of discrete phases of maturation of oligodendrocyte units were recognised. (1) Promyelinating oligodendrocytes co-expressed Rip and Myelin basic Protein and formed axonal associations, prior to ensheathment. (2) Transitional oligodendrocytes contained both ensheathing and non-ensheating processes. (3) Myelinating oligodendrocytes were established after a period of remodelling (in which non-ensheathing processes were lost), appearing as oligodendrocyte unit morphological phenotypes with a definitive number of incipient myelin sheaths. (4) Maturation of myelinating oligodendrocytes was defined as the establishment of internodal sheath lengths and the redistrubution of myelin basic protein from the cell somata and radial processes into the myelin sheaths only. Myelination was probably related to the maturational state of the axons, since it was initiated when the latter had attained a critical diameter of between ∼0.2 and 0.4 μm, coincident with the expression of NF200. Oligodendrocyte differentiation and myelination of the AMV were asynchronous and multifocal, and at P10: (1) axons which were destined to be of the largest calibre in the adult AMV were already myelinated by early developing oligodendrocytes, whilst those which were destined to be the smallest calibre in the adult were unmyelinated, but ultimately became ensheathed by late developing oligoendrocytes; (2) axons were sequentially ensheathed by early developing myelinating oligodendrocytes and late developing promyelinating oligodendrocytes; (3) all axons were small calibre; (4) oligodendrocyte units exhibited polymorphism. Thus, the development of oligodendrocyte morphological phenotypes was not related solely to either the physical dimension of axon calibre at the time of ensheathment, nor oligodendrocyte birth dates.

Platelet-derived growth factor (PDGF) is a growth-regulatory dimer with A and B subunits. PDGF-AA, acting via PDGF receptors of the alpha-unit subtype (PDGF-alphaR), is implicated in the differentiation of oligodendrocyte precursors and in the survival of newly formed oligodendrocytes, which gradually lose expression of PDGF-alphaR. However, it is unclear whether terminally differentiated oligodendrocytes express PDGF-alphaR in vivo. To address this question, and to help clarify the role of PDGF-AA in late oligodendrocyte differentiation, we have used double in situ hybridization with digoxigenin- and fluorescein-labeled riboprobes to relate PDGF-alphaR mRNA and myelin basic protein (MBP) mRNA expression in the isolated intact anterior medullary velum (AMV) of rats ages Postnatal Day (P) 10-12 and P30-32. In parallel experiments, AMV were immunolabeled with the oligodendrocyte-specific monoclonal antibody Rip to provide information on oligodendrocyte development and the extent of myelination. At P10, the AMV contained tracts in which axons ranged from unmyelinated to fully myelinated, whereas myelination was complete in P30-32 AMV. The first oligodendrocytes to express MBP mRNA or Rip were promyelinating oligodendrocytes, which had a "star-burst" morphology and had not yet begun to form myelin sheaths. As myelination proceeded, MBP mRNA became dispersed throughout oligodendrocyte units, comprising cell somata, processes, and internodal myelin sheaths. By P30-32, MBP mRNA had been redistributed to the myelin sheaths only, reflecting a change in the site of protein synthesis in mature myelinated axon tracts. At no stage of oligodendrocyte differentiation did we observe cellular coexpression of mRNA for PDGFalphaR and MBP. Our results indicated that oligodendrocytes lost the expression of PDGFalphaR prior to gaining that of myelin gene products, and preclude an action of PDGF-AA on Rip+/MBP+ star-burst promyelinating oligodendrocytes. The spatial and temporal expression of PDGF-alphaR mRNA in the AMV was inversely related to the pattern of maturation of both myelin and oligodendrocytes, and is consistent with PDGF-alphaR being expressed by pro-oligodendrocytes. A notable finding was the high level of expression of PDGF-alphaR mRNA in the AMV of juvenile rats, localized to cell bodies within the myelinated axon tracts, strongly suggesting that oligodendrocyte precursors persisted in the mature velum.

In this paper we make the surprising observation that intracellular injection of horseradish peroxidase (HRP) into a single myelinating oligodendrocyte also resulted in localised HRP labelling at the nodes of Ranvier of some axons of the unit. It appeared that HRP had been transferred to the nodal axoplasm from the paranodal loops of the HRP-filled oligodendrocyte. Three HRP-filled oligodendrocytes from isolated optic nerves of 14-day-old rats were analysed by serial section electron microscopy, and HRP was observed in the axonal cytoplasm at three of the nodes of Ranvier delineated by one of the cells. At labelled nodes, HRP was of a uniform intensity throughout the nodal axoplasm. Axonal labelling gradually diminished along the paranodal regions and was not evident in the contiguous internodal axoplasm beyond 20 microns from the node. The myelin sheaths, paranodal loops, and axons appeared normal at labelled nodes, and the paranodal loops and astrocyte perinodal processes adjacent to those of the HRP-filled oligodendrocyte unit did not contain HRP. There was no evidence of extracellular HRP or tissue damage in the surrounding neuropil, and axons neighbouring those enwrapped by the HRP-filled oligodendrocyte did not contain HRP. The possibility that axonal labelling was an artefact of either iontophoretic injection or tissue preparation is discussed. This provocative finding is not definite proof of exchange, but the balance of evidence supports the possibility that there was transcellular exchange of HRP at paranodes between the labelled oligodendrocyte and some of the axons in the unit. The rarity of HRP transfer to axons suggests that it may be a transient or labile event. It is not clear whether oligodendrocyte to axon macromolecular exchange has real physiological and/or pathological significance.

FGF2 is considered a key factor in the generation of oligodendrocytes (OLs) derived from neural stem cells (NSCs) located within the subventricular zone (SVZ). Here, we have examined FGF2 signaling in the forebrain of postnatal and adult mice. Using qPCR of microdissected microdomains of the dorsal SVZ (dSVZ) and lateral SVZ (lSVZ), and prominin1-sorted NSCs purified from these microdomains, we show that transcripts for FGF receptor 1 (FGFR1) and FGFR2 are enriched in the dSVZ, from which OLs are largely derived, whereas FGFR3 are significantly enriched within prominen1-sorted NSC of the lSVZ, which mainly generate olfactory interneurons. We show that direct administration of FGF2 into the lateral ventricle increased the generation of oligodendrocyte progenitors (OPCs) throughout the SVZ, both within the dSVZ and ectopically in the lSVZ and ependymal wall of the SVZ. Furthermore, FGF2 stimulated proliferation of neural progenitors (NPs) and their differentiation into OPCs. The results indicate that FGF2 increased specification of OPCs, inducing NPs to follow an oligodendrocyte developmental pathway. Notably, FGF2 did not block OPC differentiation and increased the number of oligodendrocytes in the periventricular white matter (PVWM) and cortex. However, FGF2 markedly disrupted myelination in the PVWM. A key finding was that FGF2 had equivalent actions on the generation of OPCs and myelin disruption in postnatal and adult mice. This study demonstrates a central role for FGF2 in promoting oligodendrocyte generation in the developing and adult brain.

The three-dimensional morphology of astrocytes and oligodendrocytes was analysed in the isolated intact mature mouse optic nerve, by correlating laser scanning confocal microscopy and camera lucida drawings of single cells, dye-filled with lysinated rhodamine dextran or horseradish peroxidase, respectively. These techniques enabled the entire process field of single dye-filled cells to be visualized in all planes and resolved the fine details of glial morphology. Morphometric analysis showed that the processes of all astrocytes had branches ending at the pial surface, on blood vessels, and freely in the nerve; branches ending in the nerve were described to end at nodes of Ranvier in the accompanying paper. Astrocytes were classified into a single morphological population in which each cell subserved multiple functions. The results of this study do not support the contention that astrocytes can be subdivided into two morphological and functional subtypes, namely type-1 and type-2, which have process ending either at the glia limitans or at nodes, respectively. Three-dimensional analysis of oligodendrocyte units, defined as the oligodendrocyte, its processes and the axons it ensheaths, showed the provision of single myelin segments for an average of 19 nearby axons (range 12-35) with a mean internodal length of 138 microns (range 50-350 microns). Mouse optic nerve oligodendrocytes were a homogeneous population and were markedly similar to those in the rat optic nerve. The results of our analysis of oligodendrocyte morphology are consistent with the view that the number and internodal length of myelin sheaths supported by a single oligodendrocyte are related to the diameter of the ensheathed axons.

NG2 cells, oligodendrocyte precursors, play a critical role in myelination during postnatal brain maturation, but a pool of these precursors is maintained in the adult and recruited to lesions in demyelinating diseases. NG2 cells in immature animals have recently been shown to receive synaptic inputs from neurons, and these have been assumed to persist in the adult. Here, we investigated the GABAergic synaptic activity of NG2 cells in acute slices of the barrel cortex of NG2-DsRed transgenic mice during the first postnatal month, which corresponds to the period of active myelination in the neocortex. Our data demonstrated that the frequency of spontaneous and miniature GABAergic synaptic activity of cortical NG2 cells dramatically decreases after the second postnatal week, indicating a decrease in the number of synaptic inputs onto NG2 cells during development. However, NG2 cells still receive GABAergic inputs from interneurons in the adult cortex. These inputs do not rely on the presence of functional synapses but involve a form of GABA spillover. This GABA volume transmission allows interneurons to induce phasic responses in target NG2 cells through the activation of extrasynaptic GABAAreceptors. Hence, after development is complete, volume transmission allows NG2 cells to integrate neuronal activity patterns at frequencies occurring duringin vivosensory stimulation.

Glial cells in the rat optic nerve were visualized by intracellular injections of horseradish peroxidase (HRP). A novel class of cell was encountered that was presumed to be an oligodendrocyte on the basis of arguments related to its light microscopic appearance after intracellular staining (Butt and Ransom, Glia 1989;2:470-475). These cells had 10-20 parallel processes 200-300 microns long that were oriented exclusively along the long axis of the optic nerve; the parallel processes were connected to the cell body by thin branches 15-30 microns long. To determine if these HRP-filled cells were oligodendrocytes, they were examined ultrastructurally; all cells examined in this way were unequivocally found to be myelin-producing oligodendrocytes. The oligodendrocytes contained intracellular organelles that were characteristic of this cell type, including abundant Golgi profiles and microtubules. In addition, HRP was found to fill the inner and outer tongue processes of myelin sheaths and the paranodal loops at nodes of Ranvier, proving that the entire cytoplasmic border surrounding the myelin sheath rapidly communicates by intracellular diffusion with the cell body. This electron microscopic study demonstrates that oligodendrocytes in the rat optic nerve can be positively identified by their distinctive light microscopic appearance after intracellular dye injection, and provides light microscopic criteria for establishing the number, distribution, and dimensions of the myelin segments provided by individual oligodendrocytes.

In vitro studies indicate that fibroblast growth factor 2 (FGF2) has diverse effects on cells of the early and late oligodendrocyte lineage. Here, we have examined this in vivo by comparing the actions of FGF2 on the developing and developed anterior medullary velum (AMV) of postnatal rats. FGF2, or saline vehicle in controls, was administered into the cerebrospinal fluid of anaesthetised rats between postnatal day (P)6 and P9 either for 1 day (1d), 2d, or 3d, and AMV were analysed at P8 or P9. Immunolabelling for NG2 was used to identify oligodendrocyte progenitor cells (OPCs) and Rip for premyelinating and myelin-forming oligodendrocytes. At P6-9, the AMV was clearly demarcated into myelinated caudal and premyelinated rostral areas. The caudal AMV was populated by differentiated myelin-forming oligodendrocytes and ‘adult’ OPCs, whilst the rostral AMV contained mixed populations of ‘perinatal’ OPCs, and both premyelinating and myelin-forming oligodendrocytes. Administration of FGF2 resulted in the accumulation of OPCs in both the developing and developed AMV. Notably, FGF2 had a bipartite action on premyelinating oligodendrocytes, at first dramatically expanding their population throughout the premyelinated and myelinated AMV, but subsequently causing the loss of these previously generated cells. In addition, FGF2 induced the loss of existing myelin-forming oligodendrocytes in the developed AMV, and arrested the generation of new myelin-forming cells in the developing AMV. This study provides evidence that FGF2 has opposing positive and negative actions on early and late oligodendrocyte lineage cells in vivo.

We have previously shown that fibroblast growth factor 2 (FGF2) disrupts myelin formation by oligodendrocytes in vivo. Here, we have investigated the possibility that this is associated with changes in the expression of tau, a major microtubule-associated protein (MAP) involved in the production of myelin membranes by oligodendrocytes. FGF2, or saline vehicle in controls, was delivered into the brain ventricles of deeply anaesthetised young rats, and their actions were examined on the anterior medullary velum (AMV), a thin sheet of tissue that roofs part of the ventricular system underlying the cerebellum. The results show that the FGF2-induced loss of myelin is associated with increased immunostaining for tau within oligodendrocyte somata. Immunohistochemical and Western blot analyses demonstrate a 50% decrease in myelin-forming oligodendrocytes, axonal myelin sheaths, and levels of myelin-related proteins, with a correlative 100% increase in the level of tau. The results identify a potential mechanism by which FGF2-mediated accumulation of tau disrupts the transport of myelin-related gene products, resulting in disruption and eventual loss of oligodendrocytes and myelin, which are features of ischemia and a variety of demyelinating and neurodegenerative diseases.

Oligodendrocytes are the myelin-forming cells of the CNS and are lost in demyelinating diseases such as multiple sclerosis (MS). A role for fibroblast growth factor 2 (FGF2) has been proposed in the pathogenesis of demyelination and the failure of remyelination in experimental models of MS. However, the in vivo effects of FGF2 on oligodendrocytes and oligodendrocyte progenitors (OPCs) in the adult CNS had not previously been determined. To address this, FGF2 was delivered into the cerebrospinal fluid (CSF) of the IVth ventricle and its actions were examined on the anterior medullary velum (AMV), a thin tissue that partly roofs the IVth ventricle and is bathed by CSF. FGF2 was administered twice daily for 3 days and AMV were analysed using immunohistochemical labelling; saline was administered in controls. The results show that raised FGF2 induces severe disruption of mature oligodendrocytes and a marked loss of myelin. At the same time, FGF2 treatment resulted in the aberrant accumulation of immature oligodendrocytes with a premyelinating phenotype, together with NG2-expressing OPCs. Axons are patent within demyelinated lesions, and they are contacted but not ensheathed by surviving oligodendrocytes, newly formed premyelinating oligodendrocytes and OPCs. These results demonstrate that raised FGF2 induces demyelination in the adult CNS, and support a role for FGF2 in the pathogenesis of demyelination and regulation of remyelination in MS.

Deletion studies in transgenic mice indicate that the potassium inward rectifying channel Kir4.1 is crucial for oligodendrocyte differentiation and has a special role in regulation of extracellular potassium (K(+)), a major function of astrocytes. However, there are conflicting reports on whether Kir4.1 is expressed by white matter astrocytes and oligodendrocytes, raising doubts over its functions. Here, we have examined Kir4.1 expression in astrocytes and oligodendrocytes of the rat optic nerve, a typical central nervous system white matter tract. Single and double immunofluorescence labelling was performed on frozen sections from optic nerves aged postnatal day (P)5, 10, 15, 20 and adult, using anti-Kir4.1 antibodies and the glia-specific antibodies glial fibrillary acidic protein (GFAP, astrocytes), carbonic anhydrase II (CAII, oligodendrocyte somata and processes) and myelin basic protein (MBP, oligodendrocyte myelin sheaths). The results demonstrate Kir4.1 expression in rows of glial cells as early as P5, and this pattern persisted throughout development and into adulthood, consistent with early expression of Kir4.1 on developing oligodendrocytes. Clear co-expression of Kir4.1 and GFAP is first evident at P10 and increases to adult levels by P15 and P20, which correlates with the development of K(+) regulation between P15 and P20. Astrocyte expression of Kir4.1 is localized to perivascular end-feet and fine processes within the fascicles of myelinated axons, consistent with a role in K(+) spatial buffering between nodes of Ranvier and blood vessels. By contrast, Kir4.1 is concentrated in the cell bodies of oligodendrocytes, and there is no apparent co-expression with MBP(+) myelin sheaths, suggesting oligodendroglial Kir4.1 channels are not involved in K(+) regulation. The results support roles for Kir4.1 in both oligodendrocyte differentiation and K(+) regulation by astrocytes.

Axon regeneration is arrested in the injured central nervous system (CNS) by axon growth-inhibitory ligands expressed in oligodendrocytes/myelin, NG2-glia, and reactive astrocytes in the lesion and degenerating tracts, and by fibroblasts in scar tissue. Growth cone receptors (Rc) bind inhibitory ligands, activating a Rho-family GTPase intracellular signaling pathway that disrupts the actin cytoskeleton inducing growth cone collapse/repulsion. The known inhibitory ligands include the chondroitin sulfate proteoglycans (CSPG) Neurocan, Brevican, Phosphacan, Tenascin, and NG2, as either membrane-bound or secreted molecules; Ephrins expressed on astrocyte/fibroblast membranes; the myelin/oligodendrocyte-derived growth inhibitors Nogo, MAG, and OMgp; and membrane-bound semaphorins (Sema) produced by meningeal fibroblasts invading the scar. No definitive CSPG Rc have been identified, although intracellular signaling through the Rho family of G-proteins is probably common to all the inhibitory ligands. Ephrins bind to signalling Ephs. The ligand-binding Rc for all the myelin inhibitors is NgR and requires p75(NTR) for transmembrane signaling. The neuropilin (NP)/plexin (Plex) Rc complex binds Sema. Strategies for promoting axon growth after CNS injury are thwarted by the plethora of inhibitory ligands and the ligand promiscuity of some of their Rc. There is also paradoxical reciprocal expression of many of the inhibitory ligands/Rc in normal and damaged neurons, and NgR expression is restricted to a limited number of neuronal populations. All these factors, together with an incomplete understanding of the normal functions of many of these molecules in the intact CNS, presently confound interpretive acumen in regenerative studies.

Glial cells are the most abundant cells in the nervous system and have the overall function of ensuring the integrity of neurons and neuronal signaling. There are many different types of glial cells and they are involved in all pathologies of the nervous system. As such, glia are important targets in novel therapeutic strategies.The 10th European Meeting on Glial Cells in Health and Disease is the largest conference of its kind in the world, specialising in the most recent advances in glial cell biology. This meeting brought together the leading international scientists studying the physiology and pathology of glial cells in a 5-day conference, with eight plenary lectures, 32 symposia with 128 lectures from world-renowned experts, poster sessions with over 480 posters summarizing new experimental data, plus workshops and special lectures on state-of-the-art techniques.

Fibroblast growth factor-2 (FGF-2) controls in part the timely differentiation of oligodendrocytes into the myelin-producing cells of the CNS. However, although differentiated oligodendrocytes express FGF receptors (R), the effect of FGF-2 on myelin-producing oligodendrocytes in vivo was unknown. In the present study, we show that delivery of FGF-2 into the cerebrospinal fluid of anaesthetized rat pups, aged postnatal day (P) 6 to 9, induced a severe loss of myelin in the caudal anterior medullary velum (AMV). Furthermore, we show that the caudal AMV was myelinated at the time of treatment, so the effects of FGF-2 represent a loss of myelin and not delayed differentiation. This was confirmed by injection of platelet-derived growth factor-AA (PDGF-AA), a factor known to affect the differentiation of PDGF-αR expressing oligodendrocyte progenitors, but which did not induce myelin loss in the caudal AMV and did not affect differentiated oligodendrocytes, which do not express PDGF-αR. Compared to controls treated with saline or PDGF-AA, FGF-2 induced an accumulation of PLP protein and MBP mRNA within the somata of myelin-producing oligodendrocytes. The results indicate that FGF receptor signalling disrupts myelin production in differentiated oligodendrocytes in vivo and interrupted the transport of myelin-related gene products from the oligodendrocyte cell body to their myelin sheaths.

A role for fibroblast growth factor-2 (FGF-2) has been proposed in mediating the glial response to injury in the central nervous system (CNS). We have tested this possibility in vivo, by injecting FGF-2 into the cerebrospinal fluid (CSF) of the brain ventricles of young rats and analysing glial cells in the anterior medullary velum (AMV), which partly roofs the IVth ventricle. FGF-2 was administered at two different doses, low FGF-2 (500 ng mL(-1) CSF) and high FGF-2 (10 microg mL(-1) CSF), and saline vehicle was injected in controls. Injections were performed twice daily for three days, commencing at postnatal day (P) 6, and AMV were analysed at P9, using immunohistochemistry and Western blotting. Glial cells were unaffected by treatment with saline or low FGF-2, whereas high FGF-2 induced reactive changes in glial cell types: (1) there was increased GFAP expression in astrocytes, demonstrated by Western blot and immunohistochemistry, and astrocytes appeared hypertrophic, with increased process thickness and number; (2) the number of ED1 labelled microglia/macrophages was doubled, from 47 +/- 6 to 114 +/- 17 cells per field (0.75 mm2; values are mean +/- SEM), and microglia appeared activated, with a multipolar and granular appearance; (3) NG2 positive glial cells appeared more fibrous and there was increased density of processes, although there was no significant increase in their number; (4) oligodendrocyte somata were enlarged and there was a loss of myelin sheaths. The results show that at high CSF titres of FGF-2 induce glial reactivity in vivo and support a role for FGF-2 in the pathology of CNS injury and EAE.