Electrophysiologically "complex" glial cells freshly isolated from the hippocampus are immunopositive for the chondroitin sulfate proteoglycan NG2

We have recently described a subgroup of isolated glial fibrillary acidic protein-positive (GFAP+) hippocampal astrocytes that predominantly express outwardly rectifying currents (which we term "ORAs" for outwardly rectifying astrocytes), which are similar to the currents already described for hippocampal GFAP- "complex glia." We now report that post-recording staining of cells that were first selected as "complex" by morphology and then confirmed by their electrophysiological characteristics were NG2+ approximately 90% of the time. Also, the morphology of freshly isolated NG2+ cells differs from that of isolated GFAP+ ORAs in having a smaller and round cell body with thinner processes, which usually are collapsed back onto the soma. Upon detailed examination, NG2+ cells were found to differ quantitatively in some electrophysiological characteristics from GFAP+ ORAs. The outward, transient K+ currents (IKa) in the NG2+ cells showed a slower decay than the IKa in ORAs, and their density decreased in NG2+ cells from older animals. The other two major cation currents, the voltage-activated Na+ and outwardly delayed rectifier K+ currents, were similar in NG2+ cells and ORAs. To further distinguish isolated complex cells from outwardly rectifying GFAP+ astrocytes, we performed immunocytochemistry for glial markers in fixed, freshly isolated rat hippocampal glia. NG2+ cells were negative for GFAP and also for the astrocytic glutamate transporters GLT-1 and GLAST. Thus the isolated hippocampal NG2+ glial cells, though having an electrophysiological phenotype similar to that of ORAs, are an immunologically and morphologically distinct glial cell population and most likely represent NG2+ cells in situ.     

Coexpression of lactosyl and gangliotetraosyl gangliosides in rat cerebellar radial glial cells in culture

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from rat embryonic cerebellar tissue was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. To distinguish the major neural cell types present in the cultures the expression of the following cell type-specific markers was investigated: neuron-specific enolase and microtubule-associated protein-2 (MAP-2) as probes for neuronal cells and the intermediate filament protein glial fibrillar acidic protein (GFAP) as a probe for astroglial cells. More than 80% of cells dissociated from cerebellar tissue of 15-day-old rat embryos (E15) are positive for the expression of GD3 and about 50% for the expression of GM1 and polysialosyl-GTC, but most are negative for the expression of neuron-specific enolase, MAP-2, and GFAP. After culturing for 4 days (E15 + 4) most cells that show characteristics of neuronal cells are positive for the expression of polysialosyl-GTC and "inactivate" the expression of GD3. Most cells with characteristics of radial and stellate glial cells are also positive for the expression of polysialosyl-GTC, but unlike neuron-like cells, they do not "inactivate" the expression of GD3.     

The interface between glial progenitors and gliomas

The mammalian brain and spinal cord contain heterogeneous populations of cycling, immature cells. These include cells with stem cell-like properties as well as progenitors in various stages of early glial differentiation. This latter population is distributed widely throughout gray and white matter and numerically represents an extremely large cell pool. In this review, we discuss the possibility that the glial progenitors that populate the adult CNS are one source of gliomas. Indeed, the marker phenotypes, morphologies, and migratory properties of cells in gliomas strongly resemble glial progenitors in many ways. We review briefly some salient features of normal glial development and then examine the similarities and differences between normal progenitors and cells in gliomas, focusing on the phenotypic plasticity of glial progenitors and the responses to growth factors in promoting proliferation and migration of normal and glioma cells, and discussing known mutational changes in gliomas in the context of how these might affect the proliferative and migratory behaviors of progenitors. Finally, we will discuss the “cancer stem cell” hypothesis in light of the possibility that glial progenitors can generate gliomas.     

Adult human spinal cord harbors neural precursor cells that generate neurons and glial cells in vitro

Adult human and rodent brains contain neural stem and progenitor cells, and the presence of neural stem cells in the adult rodent spinal cord has also been described. Here, using electron microscopy, expression of neural precursor cell markers, and cell culture, we investigated whether neural precursor cells are also present in adult human spinal cord. In well-preserved nonpathological post-mortem human adult spinal cord, nestin, Sox2, GFAP, CD15, Nkx6.1, and PSA-NCAM were found to be expressed heterogeneously by cells located around the central canal. Ultrastructural analysis revealed the existence of immature cells close to the ependymal cells, which display characteristics of type B and C cells found in the adult rodent brain subventricular region, which are considered to be stem and progenitor cells, respectively. Completely dissociated spinal cord cells reproducibly formed Sox2(+) nestin(+) neurospheres containing proliferative precursor cells. On differentiation, these generate glial cells and gamma-aminobutyric acid (GABA)-ergic neurons. These results provide the first evidence for the existence in the adult human spinal cord of neural precursors with the potential to differentiate into neurons and glia. They represent a major interest for endogenous regeneration of spinal cord after trauma and in degenerative diseases.     

Unique expression and localization of aquaporin-4 and aquaporin-9 in murine and human neural stem cells and in their glial progeny

Aquaporins (AQP) are water channel proteins that play important roles in the regulation of water homeostasis in physiological and pathological conditions. AQP4 and AQP9, the main aquaporin subtypes in the brain, are expressed in the adult forebrain subventricular zone (SVZ), where neural stem cells (NSCs) reside, but little is known about their expression and role in the NSC population, either in vivo or in vitro. Also, no reports are available on the presence of these proteins in human NSCs. We performed a detailed molecular and phenotypical characterization of different AQPs, and particularly AQP4 and AQP9, in murine and human NSC cultures at predetermined stages of differentiation. We demonstrated that AQP4 and AQP9 are expressed in adult murine SVZ-derived NSCs (ANSCs) and that their levels of expression and cellular localization are differentially regulated upon ANSC differentiation into neurons and glia. AQP4 (but not AQP9) is expressed in human NSCs and their progeny. The presence of AQP4 and AQP9 in different subsets of ANSC-derived glial cells and in different cellular compartments suggests different roles of the two proteins in these cells, indicating that ANSC-derived astrocytes might maintain in vitro the heterogeneity that characterize the astrocyte-like cell populations in the SVZ in vivo. The development of therapeutic strategies based on modulation of AQP function relies on a better knowledge of the functional role of these channels in brain cells. We provide a reliable and standardized in vitro experimental model to perform functional studies as well as toxicological and pharmacological screenings.     

Molecular and electrophysiological evidence for the expression of BK channels in oligodendroglial precursor cells

Changes in intracellular Ca(2+) play a key role in regulating gene expression and developmental changes in oligodendroglial precursor cells (OPCs). However, the mechanisms by which Ca(2+) influx in OPCs is controlled remains incompletely understood. Although there are several mechanisms that modulate Ca(2+) influx, in many systems the large-conductance, voltage- and Ca(2+) -activated K(+) channel (BK channel) plays an important role in regulating both membrane excitability and intracellular Ca(2+) levels. To date, the role of the BK channel in the regulation of intracellular Ca(2+) in oligodendroglial lineage cells is unknown. Here we investigated whether cells of the oligodendroglial lineage express BK channels and what potential role they play in regulation of Ca(2+) influx in these cells. In oligodendrocytes derived from differentiated adult neural precursor cells (NPCs, obtained from C57bl6 mice) we observed outward currents that were sensitive to the BK channel blocker iberiotoxin (IbTx). Further confirmation of the expression of the BK channel was obtained utilizing other blockers of the BK channel and by confocal immunofluoresence labelling of the BK channel on oligodendroglia. Using Fura-2AM to monitor intracellular Ca(2+) , it was observed that inhibition of the BK channel during glutamate-induced depolarization led to an additive increase in intracellular Ca(2+) levels. Electrophysiological difference currents demonstrated that the expression levels of the BK channel decrease with developmental age. This latter finding was further corroborated via RT-PCR and Western blot analysis. We conclude that the BK channel is involved in regulating Ca(2+) influx in OPCs, and may potentially play a role during differentiation of oligodendroglial lineage cells.     

The morphological development of neurons derived from EGF- and FGF-2-driven human CNS precursors depends on their site of integration in the neonatal rat brain

Neural precursor cells derived from the developing human brain were expanded in vitro under the influence of fibroblast growth factor-2 (FGF-2) and epidermal growth hormone (EGF), and were then transplanted into different regions of the neonatal rat brain. Four weeks later neurons were seen to have developed from human embryonic precursors, using a human-specific antibody to tau (htau). There were morphological differences between implanted neurons developing in the hippocampus, striatum and neocortex, which were confirmed by cell volume measurements, although no specific neurochemical phenotypes were identified. Htau-positive fibres were seen to project extensively along fibre pathways appropriate for the site of neuronal integration. This study demonstrates that, following cell division in vitro, neurons differentiating from human precursor cell populations retain the ability to respond appropriately to regional determinants present in the neonatal rat brain. This is important for the application of such cells in CNS repair strategies, in particular neural transplantation.     

Modelling large areas of demyelination in the rat reveals the potential and possible limitations of transplanted glial cells for remyelination in the CNS

Transplantation of myelin-forming glial cells may provide a means of achieving remyelination in situations in which endogenous remyelination fails. For this type of cell therapy to be successful, cells will have to migrate long distances in normal tissue and within areas of demyelination. In this study, 40 Gy of X-irradiation was used to deplete tissue of endogenous oligodendrocyte progenitors (OPCs). By transplanting neonatal OPCs into OPC-depleted tissue, we were able to examine the speed with which neonatal OPCs repopulate OPC-depleted tissue. Using antibodies to NG-2 proteoglycan and in situ hybridisation to detect platelet-derived growth factor alpha-receptor Ralpha (PDGFRalpha) mRNA to visualise OPCs, we were able to show that neonatal OPCs repopulate OPC-depleted normal tissue 3-5 times more rapidly than endogenous OPCs. Transplanted neonatal OPCs restore OPC densities to near-normal values and when demyelinating lesions were made in tissue into which transplanted OPCs had been incorporated 1 month previously, we were able to show that the transplanted cells retain a robust ability to remyelinate axons after their integration into host tissue. In order to model the situation that would exist in a large OPC-depleted area of demyelination such as may occur in humans; we depleted tissue of its endogenous OPC population and placed focal demyelinating lesions at a distance (< or =1 cm) from a source of neonatal OPCs. In this situation, cells would have to repopulate depleted tissue in order to reach the area of demyelination. As the repopulation process would take time, this model allowed us to examine the consequences of delaying the interaction between OPCs and demyelinated axons on remyelination. Using this approach, we have obtained data that suggest that delaying the time of the interaction between OPCs and demyelinated axons restricts the expression of the remyelinating potential of transplanted OPCs.     

Insulin-like growth factor actions during development of neural stem cells and progenitors in the central nervous system

Insulin-like growth factor-I (IGF-I) plays a key role in normal development. Recent studies show that IGF-I exerts a wide variety actions in the central nervous system during development as well as in adulthood. This report reviews recent developments on IGF-I actions and its mechanisms in the central nervous system, with a focus on its actions during the development of neural stem cells and progenitors. Available data strongly indicate that IGF-I shortens the length of the cell cycle in neuron progenitors during embryonic life and has an influence on the growth of all neural cell types. The phosphatidylinositol-3 kinase/Akt and mitogen-activated protein kinase pathways seem to be the predominant mediators of IGF-I-stimulated neural cell proliferation and survival. IGF-I actions, however, likely depend on cell type, developmental stage, and microenvironmental milieu.     

Ethanol exposure during embryogenesis decreases the radial glial progenitorpool and affects the generation of neurons and astrocytes

Prenatal ethanol exposure induces functional abnormalities during brain development affecting neurogenesis and gliogenesis. We have previously reported that alcohol exposure during embryogenesis disrupts radial glia (RG) and gliogenesis. Taking into account the new role of RG as neural progenitors, we have investigated whether ethanol affects RG as a neural stem cell. We found that in utero ethanol exposure impairs cell proliferation and decreases neurons and astrocytes generated in cultured RG and in embryonic cerebral cortex. Telencephalic cultures obtained at E12 from ethanol-treated rats displayed a reduction in the proportion of actively dividing RG progenitors, as demonstrated by 5-bromo-2'-deoxyuridine incorporation, and in the percentage of brain lipid binding protein-positive RG. Consistently, neurosphere formation assay from E12 telencephalon showed a reduced number of multipotent progenitor cells in cultures isolated from ethanol-treated rats in comparison with pair-fed control group. Moreover, levels of activated Notch1 and fibroblast growth factor receptor 2, which regulate the maintenance of the progenitor state of RG, are decreased by prenatal ethanol exposure. These findings demonstrate that ethanol reduces the telencephalic RG progenitor pool and its transformation into neurons and astrocytes, which may contribute to an explanation of the defects in brain function often observed in fetal alcohol syndrome.     

Dynamic expression of de novo DNA methyltransferases Dnmt3a and Dnmt3b in the central nervous system

To explore the role of DNA methylation in the brain, we examined the expression pattern of de novo DNA methyltransferases Dnmt3a and Dnmt3b in the mouse central nervous system (CNS). By comparing the levels of Dnmt3a and Dnmt3b mRNAs and proteins in the CNS, we showed that Dnmt3b is detected within a narrow window during early neurogenesis, whereas Dnmt3a is present in both embryonic and postnatal CNS tissues. To determine the precise pattern of Dnmt3a and Dnmt3b gene expression, we carried out X-gal histochemistry in transgenic mice in which the lacZ marker gene is knocked into the endogenous Dnmt3a or Dnmt3b gene locus (Okano et al. [1999] Cell 99:247-257). In Dnmt3b-lacZ transgenic mice, X-gal-positive cells are dispersed across the ventricular zone of the CNS between embryonic days (E) 10.5 and 13.5 but become virtually undetectable in the CNS after E15.5. In Dnmt3a-lacZ mice, X-gal signal is initially observed primarily in neural precursor cells within the ventricular and subventricular zones between E10.5 and E17.5. However, from the newborn stage to adulthood, Dnmt3a X-gal signal was detected predominantly in postmitotic CNS neurons across all the regions examined, including olfactory bulb, cortex, hippocampus, striatum, and cerebellum. Furthermore, Dnmt3a signals in CNS neurons increase during the first 3 weeks of postnatal development and then decline to a relatively low level in adulthood, suggesting that Dnmt3a may be of critical importance for CNS maturation. Immunocytochemistry experiments confirmed that Dnmt3a protein is strongly expressed in neural precursor cells, postmitotic CNS neurons, and oligodendrocytes. In contrast, glial fibrillary acidic protein-positive astrocytes exhibit relatively weak or no Dnmt3a immunoreactivity in vitro and in vivo. Our data suggest that whereas Dnmt3b may be important for the early phase of neurogenesis, Dnmt3a likely plays a dual role in regulating neurogenesis prenatally and CNS maturation and function postnatally.     

Synaptic transmission in the hippocampus: Critical role for glial cells

The importance of glial cells in controlling the neuronal microenvironment has been increasingly recognized. We now demonstrate that glial cells play an integral role in hippocampal synaptic transmission by using the glial-specific metabolic blocker fluoroacetate (FAC) to selectively inhibit glial cell function. FAC inhibits evoked intracellular postsynaptic potentials (PSPs; IC₅₀ = 39 μM) as well as population PSPs (IC₅₀ = 65 μM) in field CA1 of the guinea pig hippocampal slice. Spontaneous synaptic transmission is concurrently decreased. These effects are time and dose dependent. ATP concentrations in glial but not neuronal elements are also significantly reduced with FAC treatment. Simultaneous application of the metabolic substrate isocitrate with FAC prevents both the reduction in glial ATP concentrations and the decrease in evoked PSPs. Given that isocitrate is selectively taken up by glia, these data further support a glial specific metabolic action of FAC. Additionally, FAC has no postsynaptic effects as peak responses to iontophoretically applied glutamate are unchanged. However, the decay of both iontophoretic and evoked PSPs are prolonged following FAC treatment suggesting inhibition of glutamate uptake may contribute to the FAC-induced depression of synaptic potentials. These results show, for the first time, that glial cells are critical for maintenance of synaptic transmission and suggest a role for glial cells in the modulation of synaptic efficacy. © 1994 Wiley-Liss, Inc.     

Reactive astrogliosis induces astrocytic differentiation of adult neural stem/progenitor cells in vitro

Neural stem cells reside in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells (NSPCs) isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Recent studies have shown that endogenous or grafted NSPCs are activated after an injury and migrate toward lesioned areas. In these areas, reactive astrocytes are present and secrete numerous molecules and growth factors; however, it is not currently known whether reactive astrocytes can influence the lineage selection of NSPCs. We investigated whether reactive astrocytes could affect the differentiation, proliferation, and survival of adult NSPCs by modelling astrogliosis in vitro, using mechanical lesion of primary astrocytes. Initially, it was found that conditioned medium from lesioned astrocytes induced astrocytic differentiation of NSPCs without affecting neuronal or oligodendrocytic differentiation. In addition, NSPCs in coculture with lesioned astrocytes also displayed increased astrocytic differentiation and some of these NSPC-derived astrocytes participated in glial scar formation in vitro. When proliferation and survival of NSPCs were analyzed, no differential effects were observed between lesioned and nonlesioned astrocytes. To investigate the molecular mechanisms of the astrocyte-inducing activity, the expression of two potent inducers of astroglial differentiation, ciliary neurotrophic factor and leukemia inhibitory factor, was analyzed by Western blot and shown to be up-regulated in conditioned medium from lesioned astrocytes. These results demonstrate that lesioned astrocytes can induce astroglial differentiation of NSPCs and provide a mechanism for astroglial differentiation of these cells following brain injury.     

HERG- and IRK-like Inward Rectifier Currents are Sequentially Expressed During Neuronal Development of Neural Crest Cells and their Derivatives

Quail neural crest cells were cultured in a differentiative medium to study the inward K⁺ channel profile in neuronal precursors at various stages of maturation. Between 12 and 24 h of culture, neural crest-derived neurons displayed, in addition to the previously described outward depolarization-activated K⁺ currents, an inward current enhanced in high K⁺ medium. A biophysical and pharmacological analysis led us to conclude that this inward K⁺ current is identical to that previously demonstrated in mouse and human neuroblastoma cell lines (I_IR). This current (quail I_IR or ql_lR), which is active at membrane potentials positive to -35 mV, was blocked by Cs⁺ and by class Ill antiarrhythmic drugs, thus resembling the K⁺ current encoded by the human ether-a-go-go-related gene (HERG). At later stages of incubation (>48 h), neural crest-derived neurons underwent morphological and biochemical differentiation and expressed fast Na⁺ currents. At this stage the cells lost qll_R, displaying instead a classical inward rectifier K⁺ (IRK) current (quail I_IRK= qI_IRK). This substitution was reflected in the resting potential (V_REST), which became hyperpolarized by >20 mV compared with the 24 h cells. Neurons were also harvested from peripheral ganglia and other derivatives originating from the migration of neural crest cells, viz. ciliary ganglia, dorsal root ganglia, adrenal medulla and sympathetic chain ganglia. After brief culture following harvesting from young embryos, ganglionic neurons always expressed qi_lR. On the other hand, when ganglia were explanted from older embryos (7–12 days), briefly cultured neurons displayed the IRK-like current. Again, in all the above derivatives the ql_lR substitution by ql_lRK was accompanied by a 20 mV hyperpolarization of V_REST. Together, these data indicate that the V_REST of normal neuronal precursors is sequentially regulated by HERG- and IRK-like currents, suggesting that HERG-like channels mark an immature and transient stage of neuronal differentiation, probably the same stage frozen in neuroblastomas by neoplastic transformation.