Optic Nerve Astrocytes Research Articles

Transient presence of GABA in astrocytes of the developing optic nerve.

Immunostaining and high-pressure liquid chromatography (HPLC) were used to study the developmental time course of astrocytic gamma-aminobutyric acid (GABA) expression in rat optic nerve. GABA immunostaining was carried out on cultured astrocytes, and on whole optic nerve. Confocal scanning laser microscopy was used to obtain optical sections in excised whole tissue in order to localize the cellular origins of GABA within the relatively intact optic nerve. GABA immunoreactivity was localized in astrocytes identified by GFAP staining; GABA staining was most intense in early neonatal optic nerve and attenuated over 3 weeks of postnatal development. The staining was pronounced in the astrocyte cell bodies and processes but not in the nucleus. There was a paucity of GABA immunoreactivity by postnatal day 20, both in culture and in whole optic nerve. A biochemical assay for optic nerve GABA using HPLC indicated a relatively high concentration of GABA in the neonate, which rapidly attenuated over the first 3 postnatal weeks. Immunoreactivity for the GABA synthesis enzyme glutamic acid decarboxylase (GAD) was pronounced in neonates but also attenuated with development. These results indicate that GABA and the GABA synthesis enzyme GAD are localized in astrocytes of optic nerve, and that their expression is transient during postnatal development.

Immunocytochemical localization of taurine and glial fibrillary acidic protein in human optic nerve.

Taurine immunoreactivity (IR) in 1-microns sections of glutaraldehyde-fixed human optic nerve was observed using light microscopy and an antibody raised in rabbit to taurine conjugated to bovine serum albumin. Throughout the nerve, taurine-IR was prominent in glial cells, in their perinuclear regions, and in their numerous branching processes, some of which extended to the pial septa. The peripheral glial mantle (glia limitans) was densely stained, whereas axons and the pial septa showed relatively little or no taurine-IR. Immunoreactivity for glial fibrillary acidic protein (GFAP), and astrocyte-specific marker, was evaluated on adjacent sections. The pattern of GFAP-IR was highly similar to that for taurine, suggesting that a subset of taurine-immunoreactive glial cells are optic nerve astrocytes. To our knowledge, this is the first localization of taurine and GFAP in human optic nerve.

Sodium channel expression in optic nerve astrocytes chronically deprived of axonal contact.

Immunocytochemical and electrophysiological methods were used to examine the effect of retinal ablation on the expression of sodium channels within optic nerve astrocytes in situ and in vitro. Enucleation was performed at postnatal day 3 (P3), and electron microscopy of the enucleated optic nerves at P28-P40 revealed complete degeneration of retinal ganglion axons, resulting in optic nerves composed predominantly of astrocytes. In contrast to control (non-enucleated) optic nerve astrocytes, which exhibited distinct sodium channel immunoreactivity following immunostaining with antibody 7493, the astrocytes in enucleated optic nerves did not display sodium channel immunoreactivity in situ. Cultures obtained from enucleated optic nerves consisted principally (greater than 90%) of glial fibrillary acidic protein (GFAP)+/A2B5- ("type-1") astrocytes, as determined by indirect immunofluorescence; GFAP+/A2B5+ ("type-2") astrocytes were not present, nor were GFAP-/A2B5+ (O-2A) progenitor cells. Sodium channel immunoreactivity was not present in GFAP+/A2B5- astrocytes obtained from enucleated optic nerves; in contrast, GFAP+/A2B5- astrocytes from control optic nerves exhibited 7493 immunostaining for the first 4-6 days in culture. Sodium current expression, studied using whole-cell patch-clamp recording, was attenuated in cultured astrocytes derived from enucleated optic nerves. Whereas 39 of 50 type-1 astrocytes cultured from intact optic nerves showed measurable sodium currents at 1-7 days in vitro, sodium currents were present in only 6 of 38 astrocytes cultured from enucleated optic nerves. Mean sodium current densities in astrocytes from the enucleated optic nerves (0.66 +/- 0.3 pA/pF) were significantly smaller than in astrocytes from control optic nerves (7.15 +/- 1.1 pA/pF). The h infinity-curves of sodium currents were similar in A2B5- astrocytes from enucleated and control rat optic nerves. These results suggest that there is neuronal modulation of sodium channel expression in type-1 optic nerve astrocytes, and that, following chronic loss of axonal association in vivo, sodium channel expression is down-regulated in this population of optic nerve astrocytes.

Chapter 8: The expression of sodium channels in astrocytes in situ and in vitro

Publisher Summary The chapter describes recent morphophysiological work, and these data provide evidence for the expression of sodium channels in astrocytes in vivo and demonstrate that different subpopulations of astrocytes in vitro display different sodium channel expression patterns and sodium current properties, which appears to be modulated differentially by interactions with neurons. Sodium channels are expressed in astrocytes within the optic nerve in situ , although at this time it has not been established whether all optic nerve astrocytes in situ express similar densities of sodium channels, or sodium channels with similar electrophysiological properties. Second, cultured astrocytes are a heterogeneous group of cells in terms of ion channel expression. A2B5 - and A2B5 + astrocytes derived from optic nerves exhibit different patterns of sodium channel expression, and different properties of sodium current. In turn, optic nerve astrocytes display different patterns of sodium channel expression from astrocytes derived from hippocampus. The expression of sodium channels in at least some astrocytes in vitro appears to be modulated by cell-cell interactions.

Reactive astrocytes in myelin-deficient rat optic nerve reveal an altered distribution of orthogonal arrays of particles (OAP).

Reactive astrocytes are a common feature of various pathological conditions within the CNS. Morphological changes of reactive astrocytes include an altered nucleus-cytoplasm relationship, nuclear indentations, an increased amount of intermediate filaments, and an immunologically immature phenotype. Additionally, the number of orthogonal arrays of particles (OAP) was found to be increased within parenchymal membranes of reactive astrocytes. This observation prompted us to investigate the distribution of astroglial OAP in the amyelinated CNS of the myelin-deficient (md) rat in which reactive astrocytes prevail. In the present freeze-fracture study, astroglial OAP were determined within endfoot and nonendfoot (parenchymal) membranes in the developing optic nerve of md rats and normal littermates at the age of 12, 23, 40, and 64 days postnatally (dpn). The endfoot OAP density in md astrocytes remained constant during the entire period of investigation. In myelinated littermates, OAP densities continuously increased up to adult values. In contrast, the parenchymal OAP density in md astrocytes increased during the entire period of investigation. In normal littermates, OAP densities remained constant during the first 40 dpn and thereafter increased rapidly. These observations suggest that the absence of myelinogenesis in the md mutant may be a stimulus for parenchymal membranes of reactive astrocytes to insert OAP or to assemble OAP subunits into complete arrays.

A CNS specific proteoglycan associated with astrocytes in rat optic nerve

A novel CNS specific keratan sulfate proteoglycan is identified by a monoclonal antibody (mAb) TED15. The antibody recognizes a carbohydrate epitope that is removed from the core protein following keratanase digestion. The relative molecular weight of the glycoprotein is not altered following digestion with heparinase, heparitinase or chondroitinase ABC. By immunoblot analysis, the TED15 antigen is differentially distributed in diencephalic derivatives, being present in the optic nerve but absent from the retina. Within the optic nerve, the TED15 antigen is associated with astrocytes, while in the retina no labeling of astrocytes or Müller cells is observed.

Two types of Na(+)-currents in cultured rat optic nerve astrocytes: changes with time in culture and with age of culture derivation.

Na+ channel expression was studied in cultures of rat optic nerve astrocytes using whole-cell voltage-clamp recordings. Astrocytes from postnatal day 7 rat optic nerve (RON) expressed two distinct types of Na+ currents, which had significantly different h infinity curves. Stellate, GFAP+/A2B5+ astrocytes showed currents with h infinity curve midpoints close to -65 mV, similar to Na+ currents in most neurons. In contrast, flat fibroblast-like GFAP+/A2B5- astrocytes showed Na+ currents with h infinity midpoints around -85 mV, almost 20 mV more hyperpolarized than in neurons or A2B5+ astrocytes. Interestingly, Na+ current expression was maintained in A2B5+ astrocytes but began to decrease in A2B5- astrocytes after 6 days in vitro (DIV) and fell to or below the level of detection (i.e., 1 pA/pF) at 12 DIV. Astrocytes cultured from neonatal rats (P0) are almost exclusively GFAP+/A2B5-. These cells did not display measurable Na+ currents when studied at 2 DIV; however, Na+ current was observed after 5 DIV in A2B5- astrocytes from these neonatal (P0) cultures. These findings were substantiated by immunocytochemical experiments using 7493, an antibody raised against purified rat brain Na+ channels; in P0-derived astrocyte cultures 7493 antibody staining was initially lacking (up to 3 DIV), but it was prominent in cultures after 5 DIV, suggesting that Na+ current expression in RON astrocytes occurs postnatally.

The design of the optic nerve in fish

Fish have large eyes, with short optic nerves that are continually flexed by compensatory eye movements during swimming. Here, I review the tissue construction of the fish optic nerve, to see how the glia and axons are adapted to withstand these mechanical stresses, which are not normally encountered by CNS tissue within the skull. As in other lower vertebrates, the optic nerve astrocytes are highly unusual: their intermediate filaments are composed of cytokeratins (Giordano et al., 1989), not GFAP. Their processes are linked together by desmosomes, forming thin transverse lace-like partitions, placed at quasi-regular intervals longitudinally (Maggs & Scholes, 1990). This accordion-like arrangement is interpreted as providing a flexible tissue-skeleton for the optic nerve. A new observation is that the optic axons run in coherent parallel waves. This pattern, which is complementary to that of the astroglia, reversibly accommodates limited axial stretches. The waves are equivalent to those underlying the optical banding of Fontana (1781) in peripheral nerves, but wavelength (30 microns) and amplitude (5 microns) are about an order of magnitude less, reflecting the much smaller average size of the optic axons. The pattern also occurs in mammals, and may be restricted to the visual pathway: if present elsewhere in the CNS, nerve-fiber waves are inconspicuous at best. In fish, the astroglial partitions occur in register with the waves, suggesting that steric interactions between developing axons and glia may help to establish, or stabilize, the regular longitudinal spacing. This may have functional as well as mechanical implications, since the astrocytes form perinodal associations and their pattern is one which strongly clusters the nodes of Ranvier.

Response of müller cells to growth factors alters with time in culture

We have developed an explant culture technique, using the retinae of newborn guinea pigs, that reliably yields cultures of Müller cells showing uniform morphology and phenotype. Since the guinea pig retina is avascular and lacks astrocytes, Müller cells are the only glial cell-type and the only vimentin-positive population present. Virtually all passaged cells (greater than 98%) contain vimentin-positive intermediate filaments and no glial fibrillary acidic protein (GFAP) has been detected using a range of GFAP antibodies known to label astrocytes in the guinea pig optic nerve. Most vimentin-positive cells were also labeled with an antibody to carbonic anhydrase II, an enzyme which in the retina is specific for Müller cells. Proliferating Müller cells were identified within the inner nuclear layer of retinal fragments as early as 2 days in culture using bromodeoxyuridine (BrdU) and vimentin double labeling. Cultured Müller cells change their growth characteristics with successive passaging. The length of the cell cycle increases from 25.4 h for cells at first passage, to 66.7 h for cells at fourth passage. Altered responses to mitogens were also observed with passaging. First-passage cultures responded to basic fibroblast growth factor (bFGF) but not to several other factors tested including interleukin-2 (IL-2). In contrast, older cultures were highly responsive to IL-2 but showed a minimal response to bFGF. The altered responsiveness to mitogens observed in vitro may be relevant to changes in growth control of Müller cells in the developing and mature retina. The guinea pig retina provides an ideal mammalian tissue for generating Müller cell cultures that are free of astrocytes, endothelial cells, and pericytes, the most frequent contaminants of retinal glial cultures. The monolayers obtained show a high degree of homogeneity and are well suited for studies of Müller cell function.

Glial fibrillary acidic protein in the fish optic nerve

The intermediate filament glial fibrillary acidic protein (GFAP) is the predominant cytoskeletal protein of mature glial cells in the mammalian nervous system. The nervous systems of lower vertebrates, such as fish, have been examined for the presence of GFAP and several investigators have shown that goldfish (Carassius auratus) brain contains GFAP-positive astrocytes. The same studies have demonstrated that, in contrast to the brain, the optic nerve of goldfish did not show any GFAP immunoreactivity, suggesting that this intermediate filament protein is not expressed in fish optic nerve astrocytes. The present study shows, however, that the monoclonal antibodies to porcine GFAP react with the optic nerve of carp (Cyprinus carpio), another member of the goldfish family. These antibodies to porcine GFAP cross react with rat brain and carp optic nerve, yielding a band of approximately 52 kDa in both species. Northern blot analysis using mouse GFAP DNA probe revealed that carp optic nerve RNA contains two transcripts of 2.3 and 2.1 kb, which hybridize with the mouse GFAP probe. Injury to the carp optic nerve was followed by a decrease of GFAP immunoreactivity from neural tissue and a strong expression around blood vessels and connective tissues. On the basis of these observations and within the limitation of the techniques it is reasonable to conclude that the carp optic nerve expresses GFAP immunoreactivity and that the pattern of expression of this intermediate filament protein is altered after injury. Such an alteration might be relevant to the process of regeneration.

A human glioma cell line retaining expression of GFAP and gangliosides, recognized by A2B5 and LB1 antibodies, after prolonged passage

A malignant astrocytoma was cultured from a tissue biopsy taken at surgical resection, and serially passaged 65 times over a period of 4 years. Early culture cells showed a variety of morphological types, but were all positively labelled with a polyclonal antibody directed against glial fibrillary acidic protein (GFAP). As the number of passages increased small, GFAP-positive cells became the predominant cell type. Flow cytometry demonstrated changes in GFAP expression between early and late passages; early passages showing greater fluorescence intensity than those at later passages. All cells, however, remained positive for GFAP. In addition, this line (IPSB-18) was both vimentin and glutamine synthetase positive and no change in the expression of these two proteins was detectable on continued sequential passaging. Fibronectin was not detectable at any stage. As the number of passages increased, the population doubling time was shortened from 72 h at passage 5, to 28 h at passage 30. This line is unusual in its retention of GFAP expression through 65 sequential passages; the majority of astrocytoma lines lose this capacity after a few passages in vitro. The monoclonal antibody, A2B5, which recognizes surface gangliosides, has been previously shown to identify sub-set type 2 astrocytes in normal neural tissue in vitro. The expression of A2B5 gangliosides by 5-20% of GFAP-positive IPSB-18 cells, and their co-expression with the ganglioside GD3 identified by the LB1 monoclonal antibody, shows that cells with a similar phenotype to the type 1 and type 2 astrocytes of optic nerve are present in cultured gliomas, even after long term sequential passaging.

Specificity of cell-cell coupling in rat optic nerve astrocytes in vitro.

Intercellular coupling was studied in cultured rat optic nerve astrocytes individually characterized by A2B5 antibody staining. The presence of cell coupling was assessed by injecting single cells with the low molecular weight fluorescent dye Lucifer yellow and noting dye passage into adjacent cells; cell coupling was also studied by analyzing the decay phase of current transients recorded in response to small voltage steps using whole-cell patch-clamp recording. Cell coupling was restricted to A2B5- astrocytes, the majority of which had a flat fibroblast-like appearance and was never observed in A2B5+ stellate-shaped astrocytes. Furthermore, A2B5- astrocytes showed coupling only to A2B5- and never to A2B5+ astrocytes. Analysis of current transients provided an additional indicator for cell coupling. Astrocytes that showed dye coupling to at least one neighboring cell required the sum of two exponential functions to fit current transients, whereas a single exponential function sufficed to fit transients in cells that were not dye coupled. The specificity of cell coupling in cultured rat optic nerve astrocytes suggests that predominantly A2B5- astrocytes comprise a coupled glial syncytium; this physiological feature of these cells may be a specialized adaptation for "spatial buffering," the transport of K+ away from areas of focal extracellular accumulation. On the other hand, A2B5+ astrocytes form an uncoupled subpopulation of rat optic nerve glial cells that may serve different functions.

Coexpression of vimentin and glial fibrillary acidic protein in astrocytes of the adult rat optic nerve

The localization of vimentin (VIM) and glial fibrillary acidic protein (GFAP) was studied immunohistochemically in adult rat optic nerve. Consecutive Epon semithin tissue sections were immunostained respectively for VIM or GFAP. With this method, both antigens were detected in consecutive sections of the same cell. According to our results, most astrocytes in the adult rat optic nerve showed coexpression of VIM and GFAP.

Detection of mRNAGFAP by post-embedding EM in situ hybridization

We have developed a technique that allows mRNA molecules to be localized within cells at extremely high (nanometer) resolution. Using a cDNA for glial fibrillary acidic protein (GFAP forms lOnm diameter intermediate filaments), we have localized the mRNAGFAP in three different cell types (optic nerve astrocytes, retinal astrocytes and retinal Miiller cells). One retina was experimentally detached to stimulate GFAP expression in retinal Miiller cells. Cells containing GFAP were identified by immunoelectron microscopy. Cat retinas and optic nerves (n=7) were fixed with 2% or 4% paraformaldehyde for 1.0 hr, dehydrated with dimethylformamide, and embedded in Lowicryl K4M resin. Thin sections, placed on nickel grids, were probed with biotinylated cDNAGFAP (overnight at 37°C) followed by streptavidin-gold conjugate (1.0 hr, 23°C).In each of the three cell types that contain GFAP, the localization of the mRNAGFAP was the same. In the nuclei gold spheres were localized over amorphous, electron-dense regions within the euchromatin (presumably precursor mRNA).

Changes in astroglial scar formation in rat optic nerve as a function of development

Although astroglial scar formation is a common response to almost any type of injury to the adult central nervous system, lesions in fetal and neonatal rats have been reported to induce little or no scar formation. To examine this developmental difference further, rats ranging in age from 1 to 65 days postnatal were unilaterally enucleated, a surgical procedure that causes the axons in the optic nerve to degenerate. The optic nerves were processed for light and electron microscopy at times ranging from 7 to 365 days postenucleation. Pronounced and permanent glial scars were formed in every age group examined, including the neonates. However, the time course for removal of the degenerating axonal debris and formation of a compact, debris-free glial scar varied as a function of developmental age. In neonatal rats, a compact glial scar formed in 1-2 weeks whereas 3-5 months were required for compact glial scar formation in juveniles and adults. Changes in cross-sectional area were also associated with optic nerve degeneration and glial scar formation. Whereas lesioned neonatal optic nerves underwent little change in area, there was a substantial decrease in area in the juvenile and adult. Morphometric analysis showed that irrespective of the age of the animal at the time of enucleation, the final area of the compact glial scar was 10-20% of the unlesioned adult control. These results suggest that conflict in the literature over the ability of neonatal astrocytes to form a glial scar may be due to the nature of the lesion or the method of detection since astrocytes in the neonatal rat optic nerve clearly have the capacity to become reactive and form a glial scar.

Gliogenesis in rat optic nerve: Astrocytes are generated in a single wave before oligodendrocytes

The time of origin for astrocytes in the rat optic nerve was investigated to determine whether this cell type is generated in two waves, a first wave which occurs before the formation of oligodendrocytes and a second wave which occurs after the peak period of oligodendrocyte formation. To answer this question, multiple injections of radioactive thymidine were administered to rats after the peak period of oligodendrocyte production in the optic nerve and the animals were sacrificed several weeks after the first injection. Thymidine-labeled cells in the optic nerve were identified with the electron microscope. Of the labeled cells, >80% are oligodendrocytes, 4% are microglia, 2% are astrocytes, and the remainder are unclassifiable. The thymidine-labeled cells in the nerve were not immunostained for glial fibrillary acidic protein (GFAP), a marker characteristic of astrocytes. The number of thymidine-labeled glia generated after the second postnatal week is a small fraction of the total number of glia generated neonatally. No evidence exists for a second wave of astrocyte formation in the rat optic nerve as has been suggested in a study by Miller et al. (1985, Dev. Biol. 111, 35–41) ; rather, the vast majority of astrocytes are generated during the first 2 postnatal weeks and these data are in keeping with classical studies of gliogenesis. The question of whether astrocytes in the rat optic nerve arise directly from division of an undifferentiated, common progenitor cell or from a cell committed to the astrocyte lineage was addressed by combining thymidine autoradiography with GFAP immunocytochemistry. Rats were sacrificed 1 hr after an injection of thymidine and their nerves were processed for GFAP immunocytochemistry and autoradiography. During the first postnatal week, many thymidine-labeled cells are immunostained for GFAP. These observations demonstrate that cells committed to the astrocyte lineage divide neonatally and give rise to additional astrocytes.

Reticular astrocytes in the fish optic nerve: macroglia with epithelial characteristics form an axially repeated lacework pattern, to which nodes of Ranvier are apposed

Astroglia in lower vertebrate optic nerves are unusual: as shown recently (Giordano et al., 1989; Rungger-Brändle et al., 1989), they express abundant Type II cytokeratin, not glial fibrillary acidic protein (GFAP), the cytoskeletal marker for astrocytes elsewhere. To determine the implications for the glial cells of these epithelial-type cytoskeletons, which are linked up by desmosomal junctions, we analyzed the tissue patterning of fish optic nerve astroglia, which we term reticular astrocytes on account of their uniquely specialized arrangement. The processes of the reticular astrocytes fasciculate extensively with one another in a pattern stabilized by the desmosomes, forming a network laid out in thin planar sheets, or partitions. These are arranged transversely, that is at right angles to the optic fibers and are repeated at regular intervals of about 15 microns longitudinally throughout the optic nerve. They merge periodically forming a 3-dimensional framework whose pattern we speculate provides a flexible tissue skeleton for the optic nerve, capable of accommodating eye movements. Virtually all fibers in mature regions of the optic nerve are myelinated, and we show that nodes of Ranvier mostly occur in register with the partitions, displaying perinodal astrocytic associations resembling those found at CNS nodes in mammals. This clustering may account for the unexpectedly high observed incidence of neighbor pairs of nodes. Among other peculiarities associated with the reticular astrocytic network, one is that up to 20% of all cells comprise foamy macrophages not found elsewhere in the CNS.

Rat astrocytes express interferon-γ immunoreactivity in normal optic nerve and after nerve transection

Astrocytes are pivotal components of immune reactions in the CNS. We further support this notion by the localization of the lymphokine interferon-γ (IFN-γ), which plays an important role during immune responses, to astrocytes in rat optic nerve (ON). Astrocytes identified by glial fibrillary acidic protein immunoreactivity were IFN-γ positive in normal and transected ON while oligodendrocytes did not express IFN-γ immunoreactivity. These findings indicate that astrocytes can generate important signals which orchestrate immunoinflammatory responses in the brain.

Immunocytochemical Localization of Taurine Within Glial Cells in the Optic Nerve of Adult Albino Rats

Taurine immunoreactivity in one micron sections of the adult rat optic nerve was observed using light microscopy. Prominent staining was seen in glial cell somata in the perinuclear region and in their processes throughout the nerve, particularly in endfeet on capillaries and in the peripheral basal lamina (glia limitans). Axons and blood vessels showed relatively little or no staining in the mature nerve. The pattern of glial fibrillary acidic protein immunoreactivity on subsequent sections was similar in many respects to that of taurine, suggesting that a sub-population of taurine immunoreactive glial cells are optic nerve astrocytes.

Immuno-ultrastructural localization of sodium channels at nodes of Ranvier and perinodal astrocytes in rat optic nerve

Immuno-electron microscopic localization of sodium channels at nodes of Ranvier within adult optic nerve was demonstrated with polyclonal antibody 7493. The 7493 antisera, which is directed against purified sodium channels from rat brain, recognizes a 260 kDa protein in immunoblots of the crude glycoprotein fraction from adult rat optic nerve. Intense immunoreactivity with 7493 antisera was observed at nodes of Ranvier. Axon membrane at the node was densely stained, whereas paranodal and internodal axon membrane did not exhibit immunoreactivity. The axoplasm beneath the nodal membrane displayed variable immunostaining. Neither terminal paranodal oligodendroglial loops nor oligodendrocyte plasmalemma were immunoreactive with 7493 antisera. However, perinodal astrocyte processes exhibited intense immunoreactivity with the anti-sodium channel antisera. Optic nerves incubated with pre-immune sera, or with 7493 antisera that had been pre-adsorbed with purified sodium channel protein, displayed no immunoreactivity. These results demonstrate localization of sodium channels at high density at mammalian nodes of Ranvier and in some perinodal astrocyte processes. The latter observation offers support for an active role for perinodal astrocyte processes in the aggregation of sodium channels within the axon membrane at the node of Ranvier.