摘要:

AbstractWe applied in situ hybridization to analyze the location and the developmental changes in the distribution of the transcripts for carbonic anhydrase II (CAII) and myelin basic protein (MBP) in mouse primary cultured glial cells. Both mRNAs were localized to the oligodendrocyte using double‐labeling in situ hybridization. No evidence for CAII transcripts in astrocytes was obtained, indicating that CAII is expressed only by oligodendrocytes in normal rodent glia. As early as 48 h after plating, CAII and MBP mRNAs are present in a few, small round cells. Message is present 2–4 days before levels of these proteins can be detected in similar primary glial cultures.The intensity of labeling for MBP and CAII mRNA positive cells increases significantly during the second week but then decreases after the end of the third week.Only the oligodendrocyte perikaryon and a few processes are positive during the first week. In contrast, at 14 days, a large number of cell processes in addition to the cell bodies are heavily stained for both mRNAs. Both mRNAs could be detected far away from the cell body, up to 250 μm in some cell processes. Some segments on a cell process accumulate higher levels of mRNA than other areas. These areas may correspond to the accumulation of free ribosomes and to starting points for the membrane sheets elaborated by cultured oligodendrocytes. The developmental profile for timing and distribution of these two messages mimics closely their in situ pat

ISSN:0894-1491    

DOI:10.1002/glia.440040102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractComponents of the extracellular matrix (ECM) have been implicated in the regulation of neuronal migration, axonal growth, and synaptogenesis. We have examined cultures of glial cells, Schwann cells, and schwannomas for the expression of two components of the ECM, laminin and s‐laminin, using immunohistochemical and Western blot techniques. Laminin is a potent promotor of neurite outgrowth in cultures of both central and peripheral neurons, and is present in all ECMs. In contrast, s‐laminin (for synaptic laminin), a recently described homolog of laminin, is highly localized at the neuromuscular synaptic cleft (Sanes and Chiu,Cold Spring Harbor Symp. Quant. Biol.1983;48:667–678; Chiu and Sanes,Dev. Biol.1984;103:456–467) and shows selective adhesivity for motor neurons (Hunter et al.Cell1989;59:905–913). While the distribution of these ECM components have been well documented in situ, the sources of these extracellular molecules are unclear. We report that astrocytes cultured in serum‐free medium maintain an organized ECM that only bears laminin immunoreactivity; s‐laminin appears to be sequestered intracellularly. However, both molecules are found in the astrocyte conditioned medium. Thus, under these growth conditions, astrocytes produce and release laminin and s‐laminin, but only incorporate the former into an ECM. In contrast, neither molecule is present in comparable cultures of oligodendrocytes. Although no established ECM is seen in cultures of Schwann cells or schwannomas, laminin and s‐laminin immunoreactivity are present within cells and in the conditioned media. These results indicate that certain populations of non‐neuronal support cells and cell lines can produce and release both synaptic and extrasynaptic components of the ECM. The assembly of these different molecules into an organized basal lamina may require the presence of additional factors or inter

ISSN:0894-1491    

DOI:10.1002/glia.440040103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractIn order to analyze intercellular communication between ependymal cells in mammalian brain, we have studied gap junctional communication of ependymal and glial cells in long term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus obtained in serum supplemented media with two complementary methods: (1) dye transfer of Lucifer Yellow CH after intracellular microinjection of the different cellular types, and (2) freeze‐fracture of the same cultured ependymal cells. In our culture conditions, we have shown that the GJIC capacity to transfer dye was very different according to cellular types microinjected with Lucifer Yellow CH in the following respects: (1) in ependymal cells, GJIC was always important: ciliated ependymal cells, which are numerous in hypothalamic ependymal cultures (10–120 coupled cells), choroidal ependymocytes in plexus cultures (15–250 coupled cells), and non‐choroidal ependymocytes in diencephalic roof cultures (10–30 coupled cells), and (2) in astroglial cells found in these primary cultures, no GJIC was observed in spite of the presence of well‐differentiated gap junctions revealed by freeze‐fracture replicas. All these results show a strong GJIC in ependymal cells and indicate the very good functional state of these c

ISSN:0894-1491    

DOI:10.1002/glia.440040104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractGlial cells in the rat optic nerve were visualized by intracellular injections of horseradish peroxidase (HRP). A novel class of cell was encountered that was presumed to be an oligodendrocyte on the basis of arguments related to its light microscopic appearance after intracellular staining (Butt and Ransom,Glia1989; 2:470–475). These cells had 10–20 parallel processes 200–300 μm long that were oriented exclusively along the long axis of the optic nerve; the parallel processes were connected to the cell body by thin branches 15–30 μm long. To determine if these HRP‐filled cells were oligodendrocytes, they were examined ultrastructurally; all cells examined in this way were unequivocally found to be myelin‐producing oligodendrocytes. The oligodendrocytes contained intracellular organelles that were characteristic of this cell type, including abundant Golgi profiles and microtubules. In addition, HRP was found to fill the inner and outer tongue processes of myelin sheaths and the paranodal loops at nodes of Ranvier, proving that the entire cytoplasmic border surrounding the myelin sheath rapidly communicates by intracellular diffusion with the cell body. This electron microscopic study demonstrates that oligodendrocytes in the rat optic nerve can be positively identified by their distinctive light microscopic appearance after intracellular dye injection, and provides light microscopic criteria for establishing the number, distribution, and dimensions of the myelin segments provided by individual oli

ISSN:0894-1491    

DOI:10.1002/glia.440040105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe ability of sciatic nerve grafts to support in vivo regeneration of retinal ganglion cell axons in the adult rat raises the question of which peripheral nerve constituents may be required to promote this unexpected central regenerative response. Prime candidates for this role include the surface of the Schwann cell and components of extracellular matrix present in peripheral nerve trunks. To determine the relative importance of Schwann cells and their basal lamina in promoting retinal ganglion cell axon regeneration in the mammalian visual system, we have used an in vitro model. This approach allowed analysis of the abilities of defined peripheral nerve constituents to promote in vitro outgrowth of neurites from explants of adult rat retina harvested 7 to 10 days after in vivo optic nerve crush. Neurite outgrowth was assessed by neurofilament immunofluorescence after 3 to 20 days in vitro. Culture substrata, consisting of isolated Schwann cells (SC), Schwann cells with their assembled extracellular matrix (SC + ECM), or isolated extracellular matrix from which the Schwann cells had been removed (ECM), were prepared by first co‐culturing rat Schwann cells with embryonic dorsal root ganglion neurites on a layer of type I collagen, and then manipulating the cultures to produce the desired substrata. Type I collagen alone did not support neurite growth from adult rat retina. SC and SC + ECM supported regeneration of axons from retinal explants at average growth rates of 18 and 30 μm/h, respectively. Isolated ECM was a poor substrate for retinal neurite growth; the few neurites that gained access to this material grew at rates averaging less than 3 μm/h. These observations suggest that regeneration of adult mammalian retinal ganglion cell axons through peripheral nerve grafts (in vivo) is primarily dependent on neurite‐promoting factors present on the surface of Schwann cells and does not require organized extracellular m

ISSN:0894-1491    

DOI:10.1002/glia.440040106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractStimulation‐evoked transient changes in extracellular potassium ([K+]e) and pH (pHe) were studied in the neonatal rat spinal cords isolated from 3–13‐day‐old pups. In unstimulated pups the [K+]ebaseline was elevated and pHewas more acid than that in Ringer's solution (3.5 mM K+, pH 7.3–7.35). The [K+]eand pHein 3–6‐day‐old pups was 3.91 ± 0.12 mM and pHe7.19 ± 0.01, respectively, while in 10–13‐day‐old pups it was 4.35 ± 0.15 mM and 7.11 ± 0.01, respectively. The [K+]echanges evoked in the dorsal horn by a single electrical stimulus were as large as 1.5–2.5 mM. Such changes in [K+]eare evoked in the adult rat spinal cord with stimulation at a frequency of 10–30 Hz. The maximal changes of 2.1–6.5 mM were found at a stimulation frequency of 10 Hz in 3–6‐day‐old animals. In older animals the [K+]echanges progressively decreased. The poststimulation K+‐undershoot was found after a single stimulus as well as after repetitive stimulation.In 3–8‐day‐old pups, the stimulation evoked an alkaline shift, which was followed by a smaller poststimulation acid shift when the stimulation was discontinued. In pups 3–4‐days‐old the stimulation evoked the greatest alkaline shifts, i.e., by as much as 0.05 pH units after a single pulse and by about 0.1 pH units during stimulation at a frequency of 10 Hz. In 5–8‐day‐old pups, the alkaline shift became smaller and the poststimulation acid shift increased. Stimulation in 10–13‐day‐old pups produced an acid shift of 0.03–0.07 pH units, which was preceded by a scarcely discernible alkaline shift. MgCl2(20 mM) reversibly reduced the alkaline but not the acid shifts by 50–60%. Bath application of the carbonic anhydrase inhibitor acetazolamide had no effect on the alkaline shift, while the acid shift decreased by 70–80%. The superfusion of the cord with 10 mM KCl resulted in acid shifts of 0.10–0.14 pH units.We conclude that the [K+]eceiling level and the character of pHetransients in the spinal cord are closely related to gliogenesis. Our results suggest that glial cells buffer the activity

ISSN:0894-1491    

DOI:10.1002/glia.440040107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe convulsant methionine sulfoximine is a potent glycogenic agent in the central nervous system of rodents in vivo. This investigation was undertaken to look for the basic mechanism underlying this property. Astrocytes were cultivated from newborn rat neopallium and glycogen was studied by both biochemical and ultrastructural methods. When the astrocytes were incubated in a medium containing 5.55 mM glucose, methionine sulfoximine (0.55 mM) induced a significant increase in their glycogen content. Glucose content did not change in astrocytes, but it diminished in the medium in all cases. When the decrease in glucose level in the medium was limited, the same glycogenic effects of methionine sulfoximine were observed, but the glycogen contents were higher. The augmentation of the concentration of the convulsant enhanced its glycogenic effect, but this was not directly dose dependent. When the flat and polygonal astrocytes were transformed into process‐bearing astrocytes by dibutyryl cyclic AMP methionine sulfoximine always induced an increase in glycogen content. In this case, the values of glycogen contents were lower. In electron microscopy, no glycogen particles were present in the astrocytes even after methionine sulfoximine treatment, contrary to the case in vivo. These results show that the convulsant does not need the presence of neuronal cells to induce glycogen accumulation and that astrocytes may be the direct cell targets. The apparent discrepancy between the biochemical and ultrastructural data is probably due to the relatively low concentration of glycogen in cultured astrocyte

ISSN:0894-1491    

DOI:10.1002/glia.440040108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractTwo factors that may influence the course of axonal regeneration in the central nervous system (CNS) are extracellular matrix (ECM) and cell surface molecules that may enhance or inhibit neurite outgrowth. Whereas cultured astrocytes have been reported to be a good substratum for neurite outgrowth, there is recent evidence that cultured oligodendrocytes are inhibitory. To test the influences of (1) ECM components, (2) the L1 adhesion molecule, and (3) the inhibitory potential of mature oligodendrocytes in the astrocytic environment, we have utilized a culture system in which neurites from embryonic rat retina grow vigorously on astrocyte monolayers. The major ECM components were assembled in neonatal rat cortical astrocyte‐retina co‐cultures only when the medium contained serum. In electron microscopic studies of serum containing cultures, retinal neurites were seen to be related to astrocyte surfaces but rarely were found in contact with ECM; in serum‐free medium the association between neurites and astrocytes was similar. In addition, the growth of neurites was vigorous whether ECM was present or absent. Presence of antibodies against the cell surface adhesion molecule L1 did not inhibit retinal neurite elongation on glial fibrillary acidic protein‐positive astrocytes, When oligodendrocytes from adult rat spinal cord were combined with the astrocytes, retinal neurites grew as well on the mixed glial population as on astrocytes alone. Immunostaining for galactocerebroside showed many oligodendrocyte processes to be aligned in the direction of neurite growth, suggesting association between the two cell types. This association was verified by electron microscopy. Furthermore, retinal explants extended neurites among myelin basic protein‐positive oligodenrocytes cultured without astrocytes.Thus, the astrocyte surface is a strong promoter of neurite growth from embryonic rat retina. This growth did not depend upon either ECM or the L1 adhesion molecule. Because neurites grew on astrocytes in the presence of mature oligodendrocytes or among oligodendrocytes alone, we conclude that oligodendrocytes do not inhibit neurite growth under certain c

ISSN:0894-1491    

DOI:10.1002/glia.440040109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractTaurine is stored in and released from astroglial cells. We have investigated whether taurine is stored in multiple subcellular compartments and whether taurine is released from a distinct pool. Taurine compartmentation was examined by determining the subcellular distribution of labelled taurine and by comparing the specific radioactivities of taurine in cells with that of the taurine released into the superfusion medium during receptor stimulation. Three observations indicate that taurine is found in a single subcellular pool. First, labelled taurine was localized exclusively within a freely exchangeable pool and not sequestered in membrane‐bound compartments. Second, endogenous and newly acquired [14C]taurine was released identically by the β‐adrenergic agonist isoproterenol (IPR). Third, measurements of the specific radioactivity of taurine indicate that accumulated [14C]taurine mixes homogeneously with endogenous taurine and both are simultaneously released from the same pool. Chemical analysis revealed net uptake of taurine from the medium during loading and a net loss during IPR‐stimulated release. Taurine was selectively released by IPR; five other amino acids found in the superfusion medium were unaffected. One possible mechanism capable of explaining these results is that receptor‐mediated taurine release from astroglia occurs by a selective transporter moving taurine from a single cytoplasmic pool to the extracellul

ISSN:0894-1491    

DOI:10.1002/glia.440040110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe function of astrocytes in cerebral cortex may be studied by comparing the properties of conventional, astrocyte‐rich cultures with astrocyte‐poor cultures in which astrocyte proliferation has been stringently suppressed. Exposure of astrocytepoor, but not astrocyte‐rich, cultures to fresh medium containing 2 mM glutamine resulted in the death of most neurons within 24 h. This study was undertaken to understand the basis for the apparent toxicity of glutamine in astrocyte‐poor cultures. The toxicity of glutamine was found to be mediated by glutamate, which demonstrated an LD50 as a neurotoxin in astrocyte‐poor cultures of 2 μM. Exposure of astrocyte‐poor (but not astrocyte‐rich) cultures to fresh medium containing glutamine for 17.5–24 h resulted in the accumulation of substantial quantities of glutamate (255 ± 158 μM; mean ± standard deviation) coincident with the death of neurons in the cultures. Exposure of astrocyte‐poor cultures to glutamate in the absence of glutamine did not result in the accumulation of extracellular glutamate. Both the neuronal death and the extracellular glutamate accumulation in astrocyte‐poor cultures exposed to glutamine could be blocked by N‐methyl‐D‐aspartate (NMDA) antagonists. These observations suggest that astrocytes as well as glutamine may play an important role in the pathogenesis of glutamate neurotoxicity

ISSN:0894-1491    

DOI:10.1002/glia.440040111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY