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Thalamic astrocytes and oligodendrocytes are coupled via gap junctions and form panglial networks. Here, we show that these networks have a key role in energy supply of neurons. Filling an astrocyte or an oligodendrocyte in acute slices with glucose or lactate is sufficient to rescue the decline of stimulation-induced field post-synaptic potential (fPSP) amplitudes during extracellular glucose deprivation (EGD). In mice lacking oligodendroglial coupling, loading an astrocyte with glucose does not rescue the EGD-mediated loss of fPSPs. Monocarboxylate and glucose transporters are required for rescuing synaptic activity during EGD. In mice deficient in astrocyte coupling, filling of an oligodendrocyte with glucose does not rescue fPSPs during EGD. Our results demonstrate that, in the thalamus, astrocytes and oligodendrocytes are jointly engaged in delivering energy substrates for sustaining neuronal activity and suggest that oligodendrocytes exert their effect mainly by assisting astrocytes in metabolite transfer to the postsynapse., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the optic nerve, oligodendrocytes maintain axonal function by supplying lactate as an energy substrate. Here, we report that, in acute brain slices of the mouse corpus callosum, exogenous glucose deprivation (EGD) abolished compound action potentials (CAPs), which neither lactate nor pyruvate could prevent. Loading an oligodendrocyte with 20 mM glucose using a patch pipette prevented EGD-mediated CAP reduction in about 70% of experiments. Loading oligodendrocytes with lactate rescued CAPs less efficiently than glucose. In mice lacking connexin 47, oligodendrocyte filling with glucose did not prevent CAP loss, emphasizing the importance of glial networks for axonal energy supply. Compared with the optic nerve, the astrocyte network in the corpus callosum was less dense, and loading astrocytes with glucose did not prevent CAP loss during EGD. We suggest that callosal oligodendrocyte networks provide energy to sustain axonal function predominantly by glucose delivery, and mechanisms of metabolic support vary across different white matter regions., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The thalamus plays important roles as a relay station for sensory information in the central nervous system (CNS). Although thalamic glial cells participate in this activity, little is known about their properties. In this study, we characterized the formation of coupled networks between astrocytes and oligodendrocytes in the murine ventrobasal thalamus and compared these properties with those in the hippocampus and cortex. Biocytin filling of individual astrocytes or oligodendrocytes revealed large panglial networks in all 3 gray matter regions. Combined analyses of mice with cell type-specific deletion of connexins (Cxs), semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blotting showed that Cx30 is the dominant astrocytic Cx in the thalamus. Many thalamic astrocytes even lack expression of Cx43, while in the hippocampus astrocytic coupling is dominated by Cx43. Deletion of Cx30 and Cx47 led to complete loss of panglial coupling, which was restored when one allele of either Cxs was present. Immunohistochemistry revealed a unique antigen profile of thalamic glia and identified an intermediate cell type expressing both Olig2 and Cx43. Our findings further the emerging concept of glial heterogeneity across brain regions., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Poor neurological outcome in preterm infants is associated with periventricular white matter damage and hypomyelination, often caused by perinatal inflammation, hypoxia-ischemia, and hyperoxia. Minocycline has been demonstrated in animal models to protect the immature brain against inflammation and hypoxia-ischemia by microglial inhibition. Here we studied the effect of minocycline on white matter damage caused by hyperoxia. To mimic the 3- to 4-fold increase of oxygen tension caused by preterm birth, we have used the hyperoxia model in neonatal rats providing 24h exposure to 4-fold increased oxygen concentration (80% instead of 21% O2) from P6 to P7. We analyzed whether minocycline prevents activation of microglia and damage of oligodendroglial precursor cell development, and whether acute treatment of hyperoxia-exposed rats with minocycline improves long term white matter integrity. Minocycline administration during exposure to hyperoxia resulted in decreased apoptotic cell death and in improved proliferation and maturation of oligodendroglial precursor cells (OPC). Minocycline blocked changes in microglial morphology and IL-1β release induced by hyperoxia. In primary microglial cell cultures, minocycline inhibited cytokine release while in mono-cultures of OPCs, it improved survival and proliferation. Long term impairment of white matter diffusivity in MRI/DTI in P30 and P60 animals after neonatal hyperoxia was attenuated by minocycline. Minocycline protects white matter development against oxygen toxicity through direct protection of oligodendroglia and by microglial inhibition. This study moreover demonstrates long term benefits of minocycline on white matter integrity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this study, we have investigated the contribution of oligodendrocytic connexin47 (Cx47) and astrocytic Cx30 to panglial gap junctional networks as well as myelin maintenance and function by deletion of both connexin coding DNAs in mice. Biocytin injections revealed complete disruption of oligodendrocyte-to-astrocyte coupling in the white matter of 10- to 15-d-old Cx30/Cx47 double-deficient mice, while oligodendrocyte-to-oligodendrocyte coupling was maintained. There were no quantitative differences regarding cellular networks in acute brain slices obtained from Cx30/Cx47 double-null mice and control littermates, probably caused by the upregulation of oligodendrocytic Cx32 in Cx30/Cx47 double-deficient mice. We observed early onset myelin pathology, and ∼40% of Cx30/Cx47 double-deficient animals died within 42 to 90 d after birth, accompanied by severe motor impairments. Histological and ultrastructural analyses revealed severe vacuolization and myelination defects in all white matter tracts of the CNS. Furthermore, Cx30/Cx47 double-deficient mice exhibited a decreased number of oligodendrocytes, severe astrogliosis, and microglial activation in white matter tracts. Although less affected concerning motor impairment, surviving double-knock-out (KO) mice showed behavioral alterations in the open field and in the rotarod task. Vacuole formation and thinner myelin sheaths were evident also with adult surviving double-KO mice. Since interastrocytic coupling due to Cx43 expression and interoligodendrocytic coupling because of Cx32 expression are still maintained, Cx30/Cx47 double-deficient mice demonstrate the functional role of both connexins for interastrocytic, interoligodendrocytic, and panglial coupling, and show that both connexins are required for maintenance of myelin.

Gap junction channels are intercellular conduits that allow diffusional exchange of ions, second messengers, and metabolites. Human oligodendrocytes express the gap junction protein connexin47 (Cx47), which is encoded by the GJC2 gene. The autosomal recessive mutation hCx47M283T causes Pelizaeus-Merzbacher-like disease 1 (PMLD1), a progressive leukodystrophy characterized by hypomyelination, retarded motor development, nystagmus, and spasticity. We introduced the human missense mutation into the orthologous position of the mouse Gjc2 gene and inserted the mCx47M282T coding sequence into the mouse genome via homologous recombination in embryonic stem cells. Three-week-old homozygous Cx47M282T mice displayed impaired rotarod performance but unchanged open-field behavior. 10-15-day-old homozygous Cx47M282T and Cx47 null mice revealed a more than 80% reduction in the number of cells participating in glial networks after biocytin injections into oligodendrocytes in sections of corpus callosum. Homozygous expression of mCx47M282T resulted in reduced MBP expression and astrogliosis in the cerebellum of ten-day-old mice which could also be detected in Cx47 null mice of the same age. Three-month-old homozygous Cx47M282T mice exhibited neither altered open-field behavior nor impaired rotarod performance anymore. Adult mCx47M282T expressing mice did not show substantial myelin alterations, but homozygous Cx47M282T mice, additionally deprived of connexin32, which is also expressed in oligodendrocytes, died within six weeks after birth and displayed severe myelin defects accompanied by astrogliosis and activated microglia. These results strongly suggest that PMLD1 is caused by the loss of Cx47 channel function that results in impaired panglial coupling in white matter tissue., Competing Interests: The authors have declared that no competing interests exist.

Oligodendrocytes are the myelin-forming cells of the CNS and guarantee proper nerve conduction. Sphingosine, one major component of myelin, has recently been identified to activate TRPM3, a member of the melastatin-related subfamily of transient receptor potential (TRP) channels. TRPM3 has been demonstrated to be expressed in brain with unknown cellular distribution. Here, we show for the first time that TRPM3 is expressed in oligodendrocytes in vitro and in vivo. TRPM3 is present during oligodendrocyte differentiation. Immunohistochemistry of adult rat brain slices revealed staining of white matter areas, which co-localized with oligodendrocyte markers. Analysis of the developmental distribution revealed that, prior to myelination, TRPM3 channels are localized on neurons. On oligodendrocytes they are found after the onset of myelination. RT-PCR studies showed that the transcription of TRPM3 splice variants is also developmentally regulated in vitro. Ca(2+) imaging approaches revealed the presence of a sphingosine-induced Ca(2+) entry mechanism in oligodendrocytes - with a pharmacological profile similar to the profile published for heterologously expressed TRPM3. These findings indicate that TRPM3 participates as a Ca(2+)-permeable and sphingosine-activated channel in oligodendrocyte differentiation and CNS myelination.

According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole-cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction-permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin-Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GFAP. In the latter population, the majority expressed Olig2 and some NG2, markers for oligodendrocyte precursors. Oligodendrocytes are known to express Cx47, Cx32 and Cx29, astrocytes Cx43 and Cx30. In Cx47-deficient mice, the number of coupled cells was reduced by 80%. Deletion of Cx32 or Cx29 alone did not significantly reduce the number of coupled cells, but coupling was absent in Cx32/Cx47-double-deficient mice. Cx47-ablation completely abolished coupling of oligodendrocytes to astrocytes. In Cx43-deficient animals, oligodendrocyte-astrocyte coupling was still present, but coupling to oligodendrocyte precursors was not observed. In Cx43/Cx30-double deficient mice, oligodendrocyte-to-astrocyte coupling was almost absent. Uncoupled oligodendrocytes showed a higher input resistance. We conclude that oligodendrocytes in white matter form a functional syncytium predominantly among each other dependent on Cx47 and Cx32 expression, while astrocytic connexins expression can promote the size of this network., (Copyright 2010 Wiley-Liss, Inc.)

Background: Stem cells, having the property of self renewal, offer the promise of lifelong repair of damaged tissue. However, somatic tissue-committed primary stem cells are rare and difficult to expand in vitro. Genetically modified stem-like cells with the ability to expand conditionally provide a valuable tool with which to study stem cell biology, especially the cellular events of proliferation and differentiation. In addition, stem cells may be appropriate candidates for therapeutic applications., Methods: Double transgenic mice possesing SV40 T antigen (Tag) under the control of the reverse tetracycline-transactivator (rtTA) were used to establish cell lines. One brain cell line was partially characterized by DNA sequencing, morphology, antigen expression using flow cytometry, confocal microscopy, and electrophysiology using the patch clamp technique. Cell cycle analysis was performed using propidium iodide staining; cell viability and H3-thymidine incorporation assays. The ability of this cell line to differentiate was assessed by confocal microscopy following co-culture with stem cells secreting cytokines., Results: We report here the establishment and partial characterization of a cell line derived from the brain tissue of rtTA-SV40 Tag transgenic mice. Analysis of the morphology and antigen markers has shown that this cell line mimics some aspects of primary glial precursors. The results of electrophysiology are consistent with this and suggest that the cell line is derived from O2A glial precursor cells. Cell cycle progression of this cell line is doxycycline-dependent. In the absence of doxycycline, cells become apoptotic. Differentiation into mature type 2 astrocytes and (precursor) oligodendrocytes can be induced upon withdrawal of doxycycline and addition of epithelial stem cells secreting cytokine, such as hIL3 (human Interleukine 3) or hIL6 to the culture. In contrast, co-culturing with hCNTF (human Ciliary NeuroTrophic Factor)-secreting epithelial stem cells did not induce them to mature into progeny cell types., Conclusion: The differentiation of this O2A glial precursor line does not occur automatically in culture. Additional external help is required from the cell-based delivery of appropriate transgenic cytokines. Withdrawal of doxycycline from the culture medium removes the proliferation signals and induces a fatal outcome.

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis. It has been shown that unnatural sialylation within glial cell cultures can have distinct effects on their proliferation and antigenic profiles. These cultures metabolize N-propanoylmannosamine (N-propanoylneuraminic acid precursor=P-NAP), a synthetic non-physiological precursor of neuraminic acid, resulting in the expression of N-propanoylneuraminic acid in glycoconjugates of their cell membranes [Schmidt, C., Stehling, P., Schnitzer, J., Reutter, W. and Horstkorte, R. (1998) J. Biol. Chem. 273, 19146-19152]. To determine whether these biochemically engineered sialic acids influence calcium concentrations in cells of the oligodendrocyte lineage, mixed glial cultures of oligodendrocytes growing on top of an astrocyte monolayer were exposed to glutamate, histamine, adrenaline, gamma-aminobutyric acid (GABA), high potassium (high K(+)) and ATP. Calcium responses in P-NAP-treated oligodendrocytes were determined by confocal microscopy with the calcium indicator fluo-3 AM, and compared with control cultures. We showed that P-NAP differentially modulated the calcium responses of individual oligodendrocytes when GABA was applied. GABA induced calcium oscillations with up to four spikes per min in 60% of oligodendrocytes when treated with P-NAP.

Outwardly rectifying K(+) channels determine the membrane conductance and influence the proliferation rate of glial progenitor cells. To analyze the molecular identity and the functional role of K(+) channels in glial progenitors of mouse brain, expression of shaker-type Kv1 genes was studied at three levels: (1) presence of Kv1 mRNAs, (2) biosynthesis of channel proteins and (3) electrophysiological and pharmacological properties of K(+) currents. mRNA expression of Kv1.1 to Kv1.6 genes was studied by single-cell reverse transcription-mediated polymerase chain reaction (RT-PCR) using degenerate primers to amplify the six Kv1 transcripts. Most cells expressed several mRNA combinations simultaneously. In more than half of the cells, messages for Kv1.2, Kv1.5 and Kv1.6 were found, while Kv1.1, Kv1.3 and Kv1.4 were detected in only a minority of cells. In contrast, at the level of protein expression - employing immunocytochemistry with subtype-specific antibodies - Kv1. 2 and Kv1.3 were undetectable (<2%), while almost all cells expressed Kv1.4 (85%), Kv1.5 (99%) and Kv1.6 (99%). Kv1.1 was present in a minor cell population (10%). Functional contribution of Kv1 proteins to progenitor membrane conductance was determined by analyzing the voltage-dependence of K(+) current activation and inactivation as well as their current sensitivities to the subtype-preferring blockers and toxins tetraethylammonium (TEA), 4-aminopyridine (4-AP), charybdotoxin (CTX), alpha-dendrotoxin (DTX) and mast-cell degranulating peptide (MCDP). From these results, it is concluded: first, glial progenitor cells can express all transcripts of the six Kv1 genes, but do not express all proteins; second, Kv1.4, Kv1.5 and Kv1.6 proteins are most abundant and were found in the majority of cells; and third, K(+) currents flow predominantly either through heteromeric channel complexes or through homomeric Kv1.5 ion pores, but not through homomeric Kv1.4 or Kv1.6 channels.

Oligodendrocytes express two gap junction proteins, connexin32 (Cx32) and Cx45. To test for functional coupling between oligodendrocytes, cells were filled with the (Cx32-permeable) dyes Lucifer Yellow (LY) and Neurobiotin. Cells in slices from rat spinal cord were dialyzed via the patch pipette containing the dye while recording with the patch-clamp technique. The dye-labeled cells were identified as oligodendrocytes by their characteristic pattern of membrane currents and by morphology. In gray matter, 18% of the injected cells (N = 94) were coupled to more than three adjacent cells (slices from postnatal day 1 to 19). In contrast, in white matter, the dye was restricted to the injected cell (N = 63 for Lucifer Yellow injection only; N = 11 for LY and Neurobiotin) indicating a lack of functional coupling. Immunolabeling of Cx32 in mature oligodendrocytes of white matter revealed that the gap junction protein is localized on the cell bodies and abaxonal processes which occupy non-overlapping territories. In immature white matter and gray matter, Cx32 is mostly concentrated in the somatic region of the cells. In addition to Cx32, we have obtained immunocytochemical data that oligodendrocytes can express Cx45 with a labeling pattern different from the Cx32 expression. Two alternative interpretations of the coupling data are discussed: 1) that the presence of Cx32 in mature white matter oligodendrocytes does not serve for communication between cells, but rather for communication within oligodendrocytes in the sense of autocellular coupling, or 2) that the glial syncytium is furnished with a high degree of functional rectification at the oligodendrocytic side.

The major classes of glial cells, namely astrocytes, oligodendrocytes, and microglial cells were compared in parallel for their susceptibility to damage after combined hypoxia and hypoglycemia or hypoxia alone. The three glial cell types were isolated from neonatal rat brains, separated, and incubated in N2/CO2-gassed buffer-containing glucose or glucose substitutes, 2-deoxyglucose or mannitol (both nonmetabolizable sugars). The damage to the cells after 6 hours' exposure was determined at 0, 1, 3, 7 days based on release of lactate dehydrogenase and counting of ethidium bromide-stained dead cells, double-stained with cell-type specific markers. When 2-deoxyglucose replaced glucose during 6 hours of hypoxia, both oligodendrocytes and microglia rarely survived (18% and 12%, respectively). Astroglia initially increased the release of lactate dehydrogenase but maintained 98% to 99% viability. When mannitol, a radical scavenger and osmolarity stabilizer, replaced glucose during 6 hours of hypoxia, oligodendrocytes rarely survived (10%), astroglia survival remained at 99%, but microglia survival increased to 50%. After exposure to 6 and 42 hours, respectively, of hypoxic conditions alone, oligodendrocytes exhibited 10% survival whereas microglia and astroglia were only temporarily stressed and subsequently survived. In conclusion, oligodendrocytes, then microglia, are the most vulnerable glial cell types in response to hypoxia or hypoglycemia conditions, whereas astrocytes from the same preparations recover.

Oligodendrocyte precursor cells are purported to migrate over long distances into the various brain regions where they differentiate into oligodendrocytes and fulfill their appropriate tasks, i.e., myelination of axons. Here we characterize motile oligodendrocyte precursor cells in detail. Video-time lapse analysis was performed on isolated precursor cells in single cell cultures, in co-culture with cerebellar microexplants, and in living brain slices. Motility analysis of individual cells was combined with electrophysiological, immunological, and morphological characterizations. Translocation of the cell bodies was not continuous but occurred in waves. All motile cells exhibited a simple morphology and most, but not all, of them expressed the A2B5 epitope in vitro. Patch clamp analysis of the motile cells confirmed that they belong to the O-2A lineage. The percentage of motile cells, as well as their velocities, were enhanced on substrate-coated laminin in comparison to poly-L-lysine. Motility was not influenced by the presence of cerebellar microexplants. O-2A progenitor cells did not migrate strictly along neurite fascicles which were projected from the microexplants. Glial progenitor cells in situ also did not strictly migrate along the main direction of the axonal fibers of the corpus callosum but rather traversed the fibers with an overall direction toward the cortex. After Lucifer Yellow filling of the motile progenitor cells in situ, we could demonstrate that they were dye-coupled to yet unidentified cells of the corpus callosum.

Whole cell patch-clamp recordings of K+ currents from oligodendrocyte precursors in 10-day-old rats (P10) and, following myelination, in mature oligodendrocytes from 20-day-old rats (P20) were correlated with extracellular space (ECS) diffusion parameters measured by the local diffusion of iontophoretically injected tetramethylammonium ions (TMA+). The aim of this study was to find an explanation for the changes in glial currents that occur with myelination. Oligodendrocyte precursors (P10) in slices from corpus callosum were characterized by the presence of A-type K+ currents, delayed and inward rectifier currents, and lack of tail currents after the offset of a voltage jump. Mature oligodendrocytes in corpus callosum slices from P20 rats were characterized by passive, decaying currents and large tail currents after the offset of a voltage jump. Measurements of the reversal potential for the tail currents indicate that they result from increases in [K+]e by an average of 32 mM during a 20 msec 100 mV voltage step. Concomitant with the change in oligodendrocyte electrophysiological behavior after myelination there is a decrease in the ECS of the corpus callosum. ECS volume decreases from 36% (P9-10) to 25% (P20-21) of total tissue volume. ECS tortuosity lambda = (D/ADC)0.5, where D is the free diffusion coefficient and ADC is the apparent diffusion coefficient of TMA+ in the brain, increases as measured perpendicular to the axons from 1.53 +/- 0.02 (n = 6, mean +/- SEM) to 1.70 +/- 0.02 (n = 6). TMA+ non-specific uptake (k') was significantly larger at P20 (5.2 +/- 0.6 x 10(-3) s(-1), n = 6) than at P10 (3.5 +/- 0.4 x 10(-3) s(-1), n = 6). It can be concluded that membrane potential changes in mature oligodendrocytes are accompanied by rapid changes in the K+ gradient resulting from K+ fluxes across the glial membrane. As a result of the reduced extracellular volume and increased tortuosity, the membrane fluxes produce larger changes in [K+]e in the more mature myelinated corpus callosum than before myelination. These conclusions also account for differences between membrane currents in cells in slices compared to those in tissue culture where the ECS is essentially infinite. The size and geometry of the ECS influence the membrane current patterns of glial cells and may have consequences for the role of glial cells in spatial buffering.

The recognition molecule myelin-associated glycoprotein is expressed by oligodendrocytes, the myelinating cells of the central nervous system. The myelin-associated glycoprotein gene gives rise to two alternatively spliced transcript variants ("early" and "late" message) which are developmentally regulated. In this study, using mice, we investigated whether both transcripts can be expressed in an individual oligodendrocyte or whether different oligodendrocyte populations exist expressing either one or the other myelin-associated glycoprotein messenger RNA. For this purpose the cytoplasmic RNA content of single oligodendrocytes derived either from cultures of embryonic mouse brain or from the corpus callosum murine slice preparation was harvested during patch-clamping in the whole-cell recording mode by applying negative pressure to the patch pipette. After reverse transcription, cDNA fragments were amplified by the polymerase chain reaction and analysed by agarose gel electrophoresis and restriction enzyme maps. Expression of myelin-associated glycoprotein transcripts could first be detected in those oligodendrocytes which already had acquired a more mature developmental stage. This stage could electrophysiologically be characterized by the dominance of passive K+ currents. In addition to oligodendrocytes expressing only the late or the early transcript, many cells were found expressing simultaneously both transcripts with varying levels. The myelin-associated glycoprotein transcript expression is therefore found to be developmentally regulated at a stage when oligodendrocytes have already acquired the channel properties of the adult.

AMPA/kainate receptor activation in cultured oligodendrocyte precursor cells from embryonic mouse cortex leads to a blockade of delayed rectifying K+ currents. In the present study, we provide evidence using the patch-clamp technique in the whole-cell configuration that the mechanism linking kainate receptor activation and K+ conductance blockade is due to the receptor-mediated Na+ entry: 1) The blockade was not observed in Na(+)-free bathing solution nor when intracellular [Na+] was elevated by dialzying the cell with a pipette solution containing high [Na+]. 2) Elevation of intracellular [Na+] alone led to a blockade of outward currents in contrast to cells dialyzed by sucrose. High [Li+]i also reduced the outward currents, and in Li(+)-containing bathing solution the kainate-induced blockade of K+ channels was more pronounced. Probably, Li+ accumulates intracellularly after permeation through the receptor pore due to slower extrusion mechanisms. Experiments with GTP gamma S or GDP beta S and pertussis toxin indicated that GTP-binding protein-mediated mechanisms were not of importance for the kainate-induced K+ conductance blockade. Our data suggest that in glial precursor cells AMPA/kainate receptor activation leads to an intracellular [Na+] increase which blocks delayed rectifying K+ channels.

Replication-defective retroviruses expressing the t-neu oncogene, or a hybrid protein with the neu tyrosine kinase linked to the external region of the human epidermal growth factor receptor (egfr-neu), were used to establish lines of murine oligodendroglial precursor cells. Differentiation of the t-neu lines into myelin-associated glycoprotein (MAG)-positive oligodendrocytes was induced by dibutyryl cAMP, and the egfr-neu line showed limited differentiation in vitro upon withdrawal of epidermal growth factor. Cerebellar granule cell neurons expressed mitogens for the cell lines. Upon transplantation into demyelinated lesions, t-neu line cells engaged with the demyelinated axons whereas the egfr-neu line cells differentiated further and ensheathed the axons. These cell lines thus interact with neurons in vitro and in vivo and can be used as tools to define the molecules involved in different stages of neuron-glia interaction.

Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole-cell patch-clamp technique and the fura-2-based Ca++ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K+ currents, both delayed outward and inward rectifying. The inwardly rectifying K+ currents were often as large as the outward delayed rectifying K+ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4-aminopyridine, but not by 2 or 5 mM BaCl2. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A-type K+ currents. Both Ca++ and Na+ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABAA receptors and kainate receptors that are Ca(++)-permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes.

Active mitochondria were visualized with a laser scanning confocal microscope using the dye rhodamine 123, which is accumulated in mitochondria with a high respiratory activity. In glial precursor cells cultured from rabbit retina the fluorescence signal of active mitochondria was confined to the tips of processes, while in oligodendrocytes an even distribution was found. The developmental stage of cultured oligodendrocytes was determined by staining with O4 and O10 antibodies after the end of microfluorometric measurements. In glial precursor cells, the mitochondrial uncoupler CCCP generated an increase in the cytoplasmic calcium concentration ([Ca2+]i) in regions corresponding to those revealed by rhodamine 123 staining. In mature oligodendrocytes CCCP evoked [Ca2+]i elevation without significant spatial heterogeneity. However, electron microscopy did not reveal an uneven distribution of mitochondria in either immature or mature oligodendrocytes. We conclude that in the precursor cells, active mitochondria are confined to process tips and serve as a Ca2+ pool, while in oligodendrocytes such a spatial heterogeneity cannot be detected. This preferential localization of active mitochondria may play a role in the neurone-glia interaction during the formation of the contact between the myelinating glial process and the axonal membrane.

1. The subcellular characteristics of an ATP-induced elevation of the cytoplasmic free calcium concentration ([Ca2+]i) were studied in cultured cells of the oligodendrocyte lineage obtained from mouse cortex and rabbit retina, as well as in oligodendrocytes from mouse corpus callosum slices, using laser scanning confocal microfluorometry. 2. With the stage- and lineage-specific antibodies O4 and O10, three developmental stages within the oligodendrocyte lineage were distinguished prior to Ca2+ recording. 3. Bath application of 1-100 microM ATP induced a transient increase of [Ca2+]i in late precursors and oligodendrocytes but not in early glial precursor cells from retinal and cortical cultures and from corpus callosum slices. This effect of ATP was observed in Ca(2+)-free extracellular solution, suggesting that the ATP-mediated elevation of [Ca2+]i is due to a Ca2+ liberation from intracellular stores. 4. In both late precursors and oligodendrocytes from retina, the amplitude of ATP-induced [Ca2+]i transients was significantly higher in processes as compared with the soma; in cortical cultures such an uneven response was only observed in oligodendrocytes, while in immature cells responses in soma and processes were of similar amplitude. 5. The rank order of potency for the purine and pyrimidine nucleotides was UTP > or = ATP > ADP >> AMP = adenosine = Me-ATP for retinal oligodendrocytes, and ADP > or = ATP >> UTP = AMP = adenosine = Me-ATP for cortical oligodendrocytes. The response to ATP and related nucleotides was blocked by suramin, indicating the involvement of a P2-purinoreceptor in the ATP-mediated [Ca2+]i response. 6. ATP-induced elevation of the cytosolic Ca2+ concentration was inhibited by incubating cells with thapsigargin (10 microM) and by intracellular administration of heparin (1 microM). These findings indicate that ATP triggers a release of Ca2+ ions from InsP3-sensitive internal stores. 7. The ATP receptors may play a role in neuron-glial signal transfer; ATP is released as neurotransmitter, but also under pathological conditions from damaged cells.

We studied the distribution of voltage-gated Ca2+ channels in cells of the oligodendrocyte lineage from retinal and cortical cultures. Influx of Ca2+ via voltage-gated channels was activated by membrane depolarization with elevated extracellular K+ concentration ([K+]e) and local, subcellular increases in cytosolic free Ca2+ concentration ([Ca2+]in) could be monitored with a fluometric system connected to a laser scanning confocal microscope. In glial precursor cells from both retina and cortex, small depolarizations (with 10 or 20 mM K+) activated Ca2+ transients in processes indicating the presence of low-voltage-activated Ca2+ channels. Larger depolarizations (with 50 mM K+) additionally activated high-voltage-activated Ca2+ channels in the soma. An uneven distribution of Ca2+ channels was also observed in the mature oligodendrocytes; Ca2+ transients in processes were considerably larger. Recovery of Ca2+ levels after the voltage-induced influx was achieved by the activity of the plasmalemmal Ca2+ pump, while mitochondria played a minor role to restore Ca2+ levels after an influx through voltage-operated channels. During the development of white matter tracts, cells of the oligodendrocyte lineage contact axons to form myelin. Neuronal activity is accompanied by increases in [K+]e; this may lead to Ca2+ changes in the processes and the Ca2+ increases might be a signal for the glial precursor cell to start myelin formation.

Studies during the last few years have shown that glial cells can express a large repertoire of neurotransmitter receptors. In this study, we have characterized the properties of a glutamate receptor in oligodendrocytes and their precursor cells from cultures of mouse brain, using the patch-clamp technique to measure ligand-activated currents and a fura-2 imaging system to determine changes in free cytosolic Ca2+ concentration ([Ca2+]i). The precursor cells were identified by their characteristic morphology and their voltage-gated currents as described previously [Sontheimer H. et al. (1989) Neuron 2, 1135-1145]. The ligands kainate, domoate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), as well as L-glutamate but not trans-1-amino-1,3-cyclopentanedicarboxylate elicited inward currents at a holding potential of -70 mV and the antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the glutamate- and kainate-induced response reversibly, indicating the expression of an AMPA/kainate-type glutamate receptor. The response is due to the activation of a cationic conductance as revealed by analysing the reversal potential of the kainate-activated current. Receptor activation is accompanied by two additional responses: (i) an increase in [Ca2+]i mediated by depolarization and a subsequent activation of voltage-gated Ca2+ channels and (ii) a transient blockade of a delayed rectifying K+ current, but not of the A-type K+ current. The blockade of the K+ current was not due to the increase in [Ca2+]i since it was also observed in Ca(2+)-free bathing solution when no increase in [Ca2+]i was detectable after exposure to kainate. In contrast to precursor cells, oligodendrocytes responded weakly or not at all to glutamate or related ligands. We conclude that glutamate activates a complex pattern of physiological events in the glial precursor cells, which may play a role during the differentiation process of these cells.

The development of functional gap-junctional communication was studied in cells of the oligodendrocyte lineage. The presence of cell-cell coupling was determined by the passage of current between cells using a double whole-cell patch-clamp system or by injecting the low molecular weight dye Lucifer yellow into individual cells via a patch pepette and observing the diffusion of the dye into adjacent cells. The developmental stage of the cells under study was determined using antibodies to specific surface markers (04, 01, and 010) that characterize cells of sequential maturity along the oligodendrocyte lineage (Kuhlmann-Krieg et al., 1988; Sommer and Schachner, 1981; 1982). Both stages of precursor cells of this lineage, O4+ and O4-, almost never showed dye or electrical coupling, even though they were in close physical contact with other cells. The O1-positive oligodendrocytes with simple morphology were also noncoupled, with only few exceptions. In contrast, more than 40 percent of more mature, O10-positive oligodendrocytes showed cell-cell coupling detectable with both dye and current injection. Thus, the formation of gap junctions between cells of the oligodendrocyte lineage does not occur with, but some time after, the commitment of the cell to becoming an oligodendrocyte.

Whole-cell transmitter-activated currents were recorded with the patch-clamp technique from glial cells in thin frontal brain slices of the corpus callosum. In slices from 6- to 8-day-old mice, glioblasts were predominantly found, while oligodendrocytes were predominant in slices from 10- to 13-day-old mice. These developmental stages could be readily distinguished by their K+ channel pattern and their morphology and ultrastructural features. Both cell types expressed GABA and glutamate receptors in this in situ preparation. GABA responses showed similarities to those described for GABAA receptors, i.e., they were mimicked by muscimol, blocked by bicuculline, and enhanced by pentobarbital. Glutamate responses showed similarities to those of the kainate/quisqualate receptor subtype. The amplitude of GABA-activated currents recorded in oligodendrocytes was significantly smaller than that from glioblasts, while glutamate responses did not show marked differences in either cell type.

The electrical properties of oligodendrocytes during their development in cell culture were analyzed by combining two techniques: cell identification with cell-type and stage-specific antibodies and the patch-clamp technique. The transition from the bipotential precursor cell, which can still develop into astrocytes and oligodendrocytes, into an oligodendrocyte results in a marked change in the ion channel pattern. During this developmental transition, voltage-activated Na+ and several types of K+ currents disappear, whereas a comparatively passive, inwardly rectifying K+ current becomes dominant. GABAA receptor-mediated Cl- currents and a pH-activated Na+ current are down-regulated at this transition but are still present at all developmental stages. In contrast, electrical coupling develops only in oligodendrocytes. This change in the channel repertoire could reflect the transition of a cell in a mobile, mitotic, plastic state (the glial precursor) to a more differentiated specialized state (the oligodendrocyte).

Using whole cell patch-clamp recordings we have found that swelling cultured cerebrocortical astrocytes or mouse spinal cord oligodendrocytes by perfusing them with hypotonic medium induced inward currents at the normal resting potential of -60 mV. The currents in the oligodendrocytes were always less than for astrocytes. We examined the reversal potentials of these responses by rapidly jumping the holding potential to different values and measuring the currents. We found that the hypotonic medium-induced conductance increase was always preceded by a conductance decrease in the case of oligodendrocytes, but only sometimes preceded by a conductance decrease in cultured astrocytes. The reversal potential of the conductance increase for astrocytes was around -40 mV, while the conductance decrease had a more negative reversal potential of -60 mV or less. For oligodendrocytes the reversal potential for the conductance increase was around -50 mV while the conductance decrease had a reversal potential of -90 mV or less. This suggests that K+ conductance decreased in the initial phase, while the conductance increase was due to additional channel openings. Ion substitution experiments in the case of the astrocytes showed that the reversal potential was shifted to a more positive value when medium K+ was increased, but was unaffected when Na+ was substituted by N-methyl-D-glucamine or Cl- by D-glucuronate, when corrected for liquid junction potential changes. Thus, the channels opened in these cells are likely to include non-specific cation channels. It is of interest that the two cells show a difference in their responses, and in the case of astrocytes these are likely to be involved in the regulatory volume decrease processes documented in these cells.

We have studied the membrane potential responses of cultured oligodendrocytes and astrocytes during changes in the osmolarity of the bathing solution. Oligodendrocytes responded with a de- or hyperpolarization when changing to hypo- or hyperosmolar medium, consistent with dilution or concentration of intracellular [K+], respectively. Astrocytes responded with a similar hyperpolarization in hyperosmolar medium that could also be explained by an increase in intracellular [K+]. In hypoosmolar medium, however, a much larger depolarization of astrocytes was observed. These data imply that in astrocytes, in contrast to oligodendrocytes, swelling activates specific channels, which might be involved in volume control. Dual-impalement studies in single cells were used to measure effects of medium hypotonicity on cell input resistance, while dual-impalement studies on neighboring cells were used to measure changes in cell-cell coupling. In oligodendrocytes an increase in apparent cell input resistance and electrical coupling was found. In cultured astrocytes there was no change in apparent cell input resistance but an apparent decrease in cell-cell electrical coupling when cultures were exposed to the lowest medium osmolarity examined (-100 mM NaCl). However, because of the unavoidable contribution of cell-cell electrical coupling to the measurement of membrane impedance by current injection into a single cell a lack of change in apparent cell resistance could be due to offsetting changes in cell-cell coupling.

Na(+)-selective double-barrelled microelectrodes were used to measure the intracellular Na+ activity (aiNa) and membrane potential (Em) in oligodendrocytes from cultures of embryonic mouse spinal cord. In Na(+)-free solutions aiNa rapidly fell from its baseline of about 15 mM to values below 1 mM. Elevation of the K+ concentration in the bath ([K+]o) from 5.4 to 15 or 50 mM elicited an aiNa decrease of 4.7 or 9.0 mM, respectively. Ouabain blocked the aiNa decrease in response to 50 mM K+ by 37%. Bath application of 1 mM glutamate resulted in a membrane depolarization of 4.5 mV and a concomitant rise of aiNa by 8.6 mM. aiNa increased by approximately 11 mM after washout of a solution containing 20 mM NH4+. This aiNa increase was not blocked by amiloride, excluding a major contribution of a Na+/H+ antiporter. We conclude that, in cultured oligodendrocytes, transmembraneous Na+ movements are involved in pH regulation, glutamate causes an influx of Na+, and that the Na+/K+ pump and passive KCl uptake contribute to K+ accumulation.

Electrical coupling between cultured mouse oligodendrocytes was transiently blocked when pHi was decreased below about 6.5 using the NH4+ prepulse method. This uncoupling could, however, only be achieved if the dominant pHi regulating mechanism in these cells, the Na+/HCO3- cotransporter, was blocked by lowering bath [HCO3-]. Under this condition, an NH4+ prepulse caused pHi to decrease toward the passive distribution for H+ (i.e., about pH 6.2). In the presence of normal bath [HCO3-] an NH4+ prepulse did not decrease pHi below 6.5 even when the second pHi regulating mechanism, the Na+/H+ exchanger, was blocked by amiloride, and consequently oligodendrocytes could not be uncoupled. Increasing CO2, which uncouples glial cells in situ (Connors et al: J. Neurosci. 4:1324-1330, 1984), did not uncouple cultured oligodendrocytes in the presence of normal bath [HCO3-], but did cause uncoupling in low [HCO3-] solution. These results indicate that electrical coupling between cultured oligodendrocytes is sensitive to pHi; in normal bath [HCO3-], however, the pHi regulation of these cells is so effective that standard techniques for intracellular acidification are unable to lower pHi to levels which cause the closure of oligodendrocyte gap junctions.

Evidence of electrical and dye coupling between oligodendrocytes and astrocytes was sought in cultures of mouse spinal cord. Cell identity was verified using cell specific antigenic markers. In most experiments current was injected into oligodendrocytes while recording voltage in nearby astrocytes. Nine of 17 oligodendrocyte-astrocyte cell pairs showed weak electrical coupling; the average estimated coupling ratio was 0.03 +/- 0.06 (cf. 0.11 for oligodendrocyte-oligodendrocyte and 0.44 for astrocyte-astrocyte pairs; Kettenmann and Ransom: Glia, 1: 64-73, 1988). Application of 0.5 mM BaCl2 or 44.6 mM CsCl depolarized astrocytes and oligodendrocytes and was estimated to increase the coupling ratio between these cells 3-5-fold; these effects were rapid in onset and completely reversible. In 5 of 7 cases, oligodendrocyte-astrocyte pairs that appeared uncoupled in normal solution exhibited coupling during Ba++ or Cs+ exposure. The actions of these cations are believed to be mediated by blockade of glial K+ channels. Depolarization, per se, as induced by increasing [K+]o, did not increase coupling ratio. The fluorescent dye lucifer yellow (LY) was injected into 10 oligodendrocytes, 8 of which were electrically coupled to nearby astrocytes, and never passed into astrocytes in detectable quantities. Likewise, astrocytes injected with LY stained other astrocytes, but never oligodendrocytes. These findings document the presence of weak electrical coupling between astrocytes and oligodendrocytes, in the absence of dye coupling. Weak coupling of this sort could subserve metabolic interactions between these cells mediated by the passage of small but important molecules such as cyclic AMP, but would not allow strong electrical interactions. If such coupling among glial cells is widespread, it would constitute a "metabolic syncytium" that could serve to coordinate glial behavior.

gamma-Aminobutyric acid (GABA) depolarizes in a dose-dependent manner approximately one-third of all immunologically identified oligodendrocytes in cultures of mouse spinal cord. Measurements of [K+]o indicate that the response to GABA is not due to K+ released from active neurons. The depolarization is not accompanied by a change in cell input resistance. Replacement of sodium in the bathing solution abolishes the entire response, whereas ouabain only inhibits the repolarization phase. Current clamp experiments with two separate intracellular electrodes show that the depolarization increases at more positive potentials while the repolarization increases at more negative potentials. Bicuculline and picrotoxin but not nipecotic acid reduce the GABA effect. Pentobarbital and chlordiazepoxid also reduce the GABA-induced depolarization. Muscimol produces a depolarization similar to that of GABA. Heterogeneity in the oligodendrocyte population is indicated by the observation that some cells respond to both GABA and glutamate, while others respond only to one and some are not responsive to either.

Single channel currents were recorded from isolated membrane patches of cultured mouse oligodendrocytes using the giga-seal technique. The observed conductance of the channel was 71 +/- 34 pS. Isolated patches contained 1 to 4 channels with similar conductances and kinetics. Closed times of the channel varied from less than 1 msec to many minutes. The open state was always interrupted by flickering to the closed state. The kinetics of opening and closing appeared insensitive to voltage steps of up to +/- 75 mV from the resting level of the membrane potential, but could be affected by very large voltage steps. The observed changes in channel current in response to changes of potassium concentration on either side of the membrane indicate a high selectivity for potassium. The results show a membrane with constant macroscopic permeability that contains channels which open and close.

Eleven monoclonal antibodies (O1-O11) directed against the surface of oligodendrocytes were applied individually or in combination during measurement of membrane potential, input resistance and K+-pump activity in explant cultures of mouse spinal cord. Antibody binding to oligodendrocytes was verified by indirect immunofluorescence. None of the antibodies affected the electrical properties studied. On the basis of these observations, it is possible to identify oligodendrocytes immunocytologically prior to electrophysiological characterization.

Subpopulations of cultured oligodendrocytes from mouse spinal cord respond with depolarization to glutamate and GABA. Heterogeneity in the oligodendrocyte population was indicated by the observation that some cells respond to both GABA and glutamate, while others respond to only one and some are not responsive to either. Depolarizations are not mediated by an increase of [K+]o released from neurons. The response to GABA was blocked in Na+-free solution and is not accompanied by a change in input resistance. Several other neurotransmitters did not induce changes in membrane potential.

The electrical properties of immunocytologically identified oligondendrocytes from embryonic mouse spinal cord maintained in culture for 3 to 6 weeks studied by passing current and recording potential changes with two separate intracellular electrodes. The average input resistance was 3.3 M omega and ranged from 0.7 to 16 M omega (n = 35). The input resistance increased by 19% with depolarization and decreased by 9% with hyperpolarization of 25 mV. The membrane time constant determined from the slope of the late exponential tail was 3.45 +/- 2.5 ms SD (n = 15). The specific membrane resistance of three cells was determined by a simplified square pulse analysis combined with measurement of membrane area. Membrane area was estimated from photomicrographs of cells injected with Lucifer Yellow CH and stained with the cell surface-reactive antibody 04 and from electron micrographs. An average specific membrane resistance of 1.3 X 10(3) omega cm2 and specific capacitance of 1.7 mu F/cm2 were calculated. Increasing [K+]o depolarized the cells and decreased the input resistance and the time constant.

The K+ uptake processes of immunologically identified oligodendrocytes from embryonic mouse spinal cord were studied in primary culture by injecting ions and recording membrane potential changes and, in some experiments, K+ ion activity with intracellular electrodes. When Na+ was injected [K+]i decreased. Immediately before and after current injection the membrane potential was close to the K+ equilibrium potential (EK) and this finding was used to study K+ uptake following its depletion by intracellular ionophoresis. The uptake of K+ following Na+ injection was blocked by ouabain and unaffected by removal of extracellular Cl- or Cl- transport blockers. This suggests that recovery comes about mostly through the activity of the Na+/K+ -ATPase stimulated by either the increase in [Na+]i or the decrease in [K+]i. Pump current could be determined by clamping at different membrane potentials and was found to increase in proportion to the depolarization of the cell resulting from [K+]i depletion. The time course of recovery of membrane potential following either Li+ or tetramethylammonium (TMA+) injection was similar to that after Na+ injection, indicating that injection of these ions to produce a comparable decrease in [K+]i leads to a similar stimulation of the Na+/K+ -ATPase. In addition, the recovery of membrane potential following injection of TMA+, but not of Na+ or Li+, was blocked when the external Na+ was removed. Internal Na+ or Li+ appears necessary for Na+/K+ -ATPase-activity, but under conditions of normal or low [Na+]i the rate of Na+/K+ -ATPase activity seems to be sensitive to [K+]i and/or membrane potential.

1. Intracellular pH (pHi) and the mechanism of pHi regulation have been investigated in cultured oligodendrocytes from mouse spinal cord using double-barrelled neutral-carrier H+-selective microelectrodes. The distribution of H+ was not in electrochemical equilibrium. The pHi was more alkaline than the pH of the bathing medium (pHo), namely 7.5 at pHo 7.2 at 7.6 at pHo 7.4. 2. Removal of HCO3- from the bathing medium reduced the steady-state pHi by 0.4 units. An increase in extracellular K+ caused, with a delay, an increase in pHi. A decrease in pHo to 6.2 caused an acidification of pHi by 0.5 units. 3. The pHi regulation was studied by applying and subsequently removing NH4+ which resulted in an acidification of the cell. The subsequent recovery of pHi could then be analysed. The recovery from an acidification by 1 pH unit lasted 3-10 min. In HCO3- -free solution pHi recovery was slowed. 4. In HCO3- -free solution pHi recovery was completely blocked when either Na+ was removed or when amiloride was applied indicating an exclusive activation of the Na+-H+ exchanger. 5. In the presence of HCO3-, removal of Na+ also completely blocked pHi recovery. When Na+ was readded, pHi recovered. In HCO3- -containing solution amiloride slightly slowed, but did not block pHi recovery. 6. Removal of Cl- or application of SITS, DIDS or furosemide, blockers of Cl- -coupled transport mechanisms, did not affect the pHi recovery in the presence of HCO3-. 7. In conclusion, oligodendrocytes possess two mechanisms regulating pHi, a Na+-H+ exchanger and a Na+-HCO3- co-transporter while the latter is clearly more potent. It follows that pHi regulation of oligodendrocytes is dependent on the transmembrane Na+ gradient and is strictly separated from regulation of internal Cl-.

When the blue fluorescing dye SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid disodium salt) is injected into one of a pair of electrically and dye-coupled oligodendrocytes it does not cross the intercellular junctions but remains in the injected cell. Moreover, the fluorescent dye Lucifer Yellow CH (LY), which normally crosses these intercellular junctions after injection, does not diffuse into a SITS-injected cell. Thus, intracellular SITS injection leads to dye-uncoupling. SITS injection does not eliminate electrical-coupling.

Membrane currents in cultured murine oligodendrocytes and their precursors were characterized using the patch-clamp technique. Prior to recording, cells were identified by immunofluorescence using monoclonal antibodies characteristic of two types of precursor cells and two differentiation stages of oligodendrocytes. The most immature, A2B5 antigen-positive glial precursors, expressed four types of voltage-activated K+ currents and tetrodotoxin-sensitive Na+ currents. The more differentiated cells, O4 antigen-positive glial precursors, expressed similar K+ currents, but Na+ currents were recorded in only a minority of cells. In differentiated O1 and O10 antigen-positive oligodendrocytes the channels characteristic of precursor cells were no longer observed, but an inwardly rectifying K+ current was apparent. Thus, channel expression by cells of the oligodendrocyte lineage correlates with differentiation stage and is more complex in precursor cells than in oligodendrocytes.

The characteristics of electrical coupling between astrocytes and between oligodendrocytes were analyzed in cell cultures derived from rodent central nervous system. Experiments were carried out by impaling one member of a glial pair with separate voltage recording and current passing electrodes (cell 1) and the other cell, a measured distance from the first, with a voltage-recording electrode (cell 2). Astrocyte pairs within 300 microns of one another were always coupled. The coupling ratio was determined for 23 astrocytic pairs various distances apart, and decreased with distance in a roughly exponential manner. The average coupling ratio of astrocytes within 100 microns of each other was 0.44 +/- 0.32. Oligodendrocytes were less strongly coupled to each other than astrocytes. Even cells immediately adjacent to one another were often uncoupled. Among coupled oligodendrocytes within 100 microns of each other, the average coupling ratio was 0.11 +/- 0.1. Current passage between pairs of astrocytes and pairs of oligodendrocytes was nonrectifying. Application of 0.5 mM BaCl2 or 44.6 mM CsCl (substituted for NaCl) depolarized and increased the input resistance of astrocytes and oligodendrocytes. These ions also increased the coupling ratio in astrocyte pairs and oligodendrocyte pairs; this effect was rapid in onset and completely reversible. Ba++ and Cs+ appear to block resting K+ conductance in glia and probably increase the coupling ratio by increasing the effective length constant of the glial membrane without any direct effect on junctional resistance. In three cases, oligodendrocyte pairs that appear uncoupled in normal solution exhibited coupling in the presence of BaCl2 or CsCl. This suggests that oligodendrocytes may be widely coupled by junctions that provide only weak electrical interaction; such junctions might be important for the exchange of small metabolically active molecules. The strong electrical coupling among astrocytes, in concert with their K+-selective membrane conductance, would provide for an electrical syncytium well designed to transport K+ away from areas of focal extracellular accumulation (i.e., the spatial buffer mechanism), and these cells, more than oligodendrocytes, may provide this function.

Membrane potential, conductance, and intracellular potassium concentration were measured in oligodendrocytes in 3- to 10-week-old cultures of embryonic mouse spinal cord. After intracellular recording the cells were first injected with Lucifer Yellow and then stained by immunofluorescence using rhodamine-labeled monoclonal antibody 01 specific for oligodendrocyte cell surfaces. The membrane potential of these identified oligodendrocytes was in mV -66 +/- 4.3 SD; it could be reversibly reduced almost to zero by the addition of ouabain. Changes in external K+ but not Na+, Ca++, or Cl- changed the membrane potential. A 10-fold increase in extracellular potassium concentration ([K+]0) depolarized the cell by about 52 mV. This is less than the 61 mV predicted by the Nernst equation for a K+ electrode assuming a constant intracellular potassium concentration ([K+]i). However, when [K+]i was measured with an ion-selective electrode during the increase in [K+]0 it was found to rise. The Nernst equation for K+ accurately predicts the oligodendrocyte membrane potential when the increase in [K+]i is taken into account. Oligodendrocytes may be described as accurate K+ electrodes with a variable reference solution.

In the present study we have analyzed the membrane currents of mature oligodendrocytes in cultures from dissociated fetal mouse cerebral hemispheres and explant cultures from fetal mouse spinal cord. Both types of oligodendrocytes showed large voltage-dependent, but time-independent inward and outward currents that were partially blocked by Ba2+. In addition, time- and voltage-dependent inward and outward currents were observed in a minority of oligodendrocytes from spinal cord. All voltage-dependent currents were completely blocked by Ba2+, and inward currents were completely blocked by Cs+, suggesting that they are mediated by K+ channels. Current-voltage curves of mouse spinal cord oligodendrocytes varied from being linear to outwardly or inwardly rectifying. In contrast, oligodendrocytes cultured from mouse brain always showed an inward rectification of the current voltage relation and a lack of time-dependent currents. It thus appears that mature oligodendrocytes in explant cultures of mouse spinal cord, in contrast to oligodendrocytes from dissociated brain, consist of different cell populations that are distinguished by their expression or active state of K+ channels.

Cultured oligodendrocytes take up K+ triggered by an increase in [K+]o. Simultaneously [Cl-]i increases in the majority of the oligodendrocytes. This KCl uptake, which is not furosemide sensitive, can be explained by the following model. The first event is the entry of Cl- into the cell driven by the discrepancy between the membrane and Cl- equilibrium potential. As a consequence of the movement of negative charge across the membrane, K+ is driven into the cell. The prerequisites of this model, a passive Cl- distribution at resting membrane potential and a Cl- conductance of the membrane were found to exist in most cultured oligodendrocytes. The chloride equilibrium potential (-61 mV, SD +/- 10 mV) was slightly more positive than the membrane potential (-64 +/- 8 mV). Since cell input resistance determined with two independent electrodes increased by 11% (SD +/- 0.07) when [Cl-]o was reduced to 10 mM, part of the membrane conductance appears to be mediated by Cl-. Differences between membrane potential and Cl- equilibrium potential therefore will lead to Cl- fluxes across the membrane. In contrast with oligodendrocytes, [Cl-]i in astrocytes is significantly increased (from 20 to 40 mM) above the equilibrium distribution owing to the activity of an inward directed Cl- pump; this suggests a different mechanism of K+ uptake in these cells.

gamma-Aminobutyric acid (GABA) has been shown to depolarize the membranes of astrocytes and oligodendrocytes taken from different tissues and species. The mechanism mediating this depolarization was identified, in cultured rat brain astrocytes, as an activation of GABA receptor-linked Cl- channels. A subpopulation of cultured oligodendrocytes from mouse spinal cord also responded to GABA with a membrane depolarization. In the present study we demonstrate that, in oligodendrocytes, the depolarization was accompanied by a decrease in intracellular Cl- activity [( Cl-]i) as measured with ion-selective microelectrodes. At rest, [Cl-]i was elevated above the passive distribution, and upon application of GABA, [Cl-]i decreased towards the level of passive distribution. Furosemide blocked the Cl- inward carrier which led to a passive Cl- distribution; in the presence of furosemide, GABA no longer elicited a membrane depolarization or a change in [Cl-]i. We conclude that oligodendrocytes, which were depolarized by GABA, expressed GABA-activated Cl- channels. Oligodendrocytes which were unresponsive to GABA demonstrated a non-passive Cl- distribution indicating that they did express inward-directed Cl- carriers, but no GABA-activated Cl- channels.

Neurons and oligodendrocytes, but not type I astrocytes and Schwann cells, generate large Na+ currents in response to a step increase of [H+]. Proton-activated Na+ channels are the first cationic channels expressed in neuronal precursor cells from the mammalian brain. Glial precursor cells cultured from mouse brain are also capable of generating Na+ currents in response to step acidification (INa(H]. With further development along the oligodendrocyte lineage, this property is retained, whereas voltage-activated Na+ and K+ currents disappear. Comparing INa(H) of oligodendrocytes with INa(H) of their precursor cells did not reveal a difference in current amplitude, suggesting a higher density of INa(H) channels on the (smaller) precursor cells. The properties of INa(H) in glial precursor cells and oligodendrocytes are similar to those of neurons, with respect to activation conditions, time course, and the effect of extracellular Ca2+ concentrations. The results are consistent with previous observations which showed that oligodendrocytes partially preserve their chemically activated, but completely lose their voltage-activated, ion channels.

We studied the steady state and the regulation of intracellular Cl- activity (aCl-i) and the mechanisms of KCl uptake in cultured oligodendrocytes from mouse spinal cord using Cl(-)-selective microelectrodes. The majority of oligodendrocytes actively accumulated Cl- above passive distribution (2-3 mM), few cells showed a passive Cl- distribution. To identify the carriers mediating Cl- uptake, oligodendrocytes were maintained in a solution with low extracellular Cl- concentration ([Cl-]o) which resulted in a rapid decrease in aCl-i. The recovery of aCl-i above its passive distribution in normal [Cl-]o was blocked in the absence of Na+ or in the presence of furosemide and of bumetanide, which has been reported to inhibit Na+/K+/Cl- cotransport. We therefore conclude that Cl- uptake is primarily due to the activity of a Na+K+/Cl- transport system. Cl- uptake above passive distribution was not affected in HCO3(-)-free solution or in the presence of SITS and DIDS, indicating that Cl-/HCO3- exchange is not involved in Cl- uptake by oligodendrocytes. Elevation of [K+]o induced an increase in aCl-i and, as shown earlier, intracellular K+ activity. This K+-induced Cl- uptake was not blocked by bumetanide, furosemide, SITS, or DIDS, suggesting that under conditions of raised [K+]o the combined uptake of K+ and Cl- is not mediated by a carrier, but can be explained by the entry through channels driven by Donnan forces.

Cultured mouse oligodendrocytes were superfused by pressure application to the rear-end of a 3-20 microns micropipette filled with normal bathing fluid. Input resistance was determined during superfusion with two separate electrodes. The input resistance increased reversibly by 90% in 26 cells tested and was unaffected in 39 cells. When pairs of oligodendrocytes were electrically coupled, coupling decreased in a reversible manner during superfusion. Therefore, the flow from a micropipette can uncouple cells, and studies involving application of substances by pressure ejection from microelectrodes must be evaluated with care.