Astrocytes In Rat Optic Nerve Research Articles

HCO3−-independent pH Regulation in Astrocytes in Situ Is Dominated by V-ATPase

The mechanisms of HCO3(-)-independent intracellular pH (pHi) regulation were examined in fibrous astrocytes within isolated neonatal rat optic nerve (RON) and in cultured cortical astrocytes. In agreement with previous studies, resting pHi in cultured astrocytes was 6.82 ± 0.06 and inhibition of the V-ATPase H(+) pump by Cl(-) removal or via the selective inhibitor bafilomycin had only a small effect upon resting pHi and recovery following an acid load. In contrast, resting pHi in RON astrocytes was 7.10 ± 0.04, significantly less acidic than that in cultured cells (p < 0.001), and responded to inhibition of V-ATPase with profound acidification to the 6.3-6.5 range. Fluorescent immuno-staining and immuno-gold labeling confirmed the presence V-ATPase in the cell membrane of RON astrocyte processes and somata. Using ammonia pulse recovery, pHi recovery in RON astrocyte was achieved largely via V-ATPase with sodium-proton exchange (NHE) playing a minor role. The findings indicate that astrocytes in a whole-mount preparation such as the optic nerve rely to a greater degree upon V-ATPase for HCO3(-)-independent pHi regulation than do cultured astrocytes, with important functional consequences for the regulation of pH in the CNS.

Nitric Oxide Increases the Expression of Aquaporin-4 Protein in Rat Optic Nerve Astrocytes through the Cyclic Guanosine Monophosphate/Protein Kinase G Pathway

Aims: Nitric oxide (NO) is associated with neuroinflammation in the central nervous system. We determined whether NO increases the expression of aquaporin-4 (AQP4) in optic nerve astrocytes of rats. Methods: Isolated astrocytes were incubated under normoxic or hypoxic conditions with or without glucose (5.5 m<smlcap>M</smlcap>). The astrocytes were also exposed to different concentrations of S-nitroso-N-acetyl-DL-penicillamine (SNAP, 1.0-100 μ<smlcap>M</smlcap>), an NO donor. The expression of AQP4 was determined by Western blot analyses, and NO formation was measured by the Griess reaction. The changes in astrocytic cellular volumes were determined by flow cytometry. Results: Hypoxia and glucose deprivation increased AQP4 expression and NO formation. Inhibition of NO synthetase (NOS) significantly suppressed these changes. SNAP caused a significant increase in AQP4 expression, and the increase was significantly suppressed by carboxy-PTIO, a scavenger of NO. Incubation with 8-Br-cyclic guanosine monophosphate (cGMP) mimicked the effects of SNAP, while the addition of either 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; inhibitor of soluble guanylate cyclase) or KT5823 (protein kinase G inhibitor) suppressed the SNAP-induced increase in AQP4 significantly. SNAP also caused a significant increase in astrocytic cellular volume through the AQP4 channels. Conclusions: NO increased the AQP4 expression of optic nerve astrocytes through the cGMP/protein kinase G pathway and enlarged their volume.

Hydrostatic Pressure–Induced Release of Stored Calcium in Cultured Rat Optic Nerve Head Astrocytes

Elevated intraocular pressure is associated with glaucomatous optic nerve damage. Other investigators have shown functional changes in optic nerve head astrocytes subjected to elevated hydrostatic pressure (HP) for 1 to 5 days. Recently, the authors reported ERK1/2, p90(RSK) and NHE1 phosphorylation after 2 hours. Here they examine calcium responses at the onset of HP to determine what precedes ERK1/2 phosphorylation. Cytoplasmic calcium concentration ([Ca(2+)](i)) was measured in cultured rat optic nerve astrocytes loaded with fura-2. The cells were placed in a closed imaging chamber and subjected to an HP increase of 15 mm Hg. Protein phosphorylation was detected by Western blot analysis. The increase of HP caused an immediate slow increase in [Ca(2+)](i). The response persisted in calcium-free solution and when nickel chloride (4 mM) was added to suppress channel-mediated calcium entry. Previous depletion of the ER calcium stores by cyclopiazonic acid abolished the HP-induced calcium level increase. The HP-induced increase persisted in cells exposed to xestospongin C, an inhibitor of IP3R-mediated calcium release. In contrast, ryanodine receptor (RyR) antagonist ruthenium red (10 microM) or dantrolene (25 microM) inhibited the HP-induced calcium increase. The HP-induced calcium increase was abolished when ryanodine-sensitive calcium stores were pre-depleted with caffeine (3 mM). HP caused ERK1/2 phosphorylation. The magnitude of the ERK1/2 phosphorylation response was reduced by ruthenium red and dantrolene. Increasing HP causes calcium release from a ryanodine-sensitive cytoplasmic store and subsequent ERK1/2 activation. Calcium store release appears to be a required early step in the initial astrocyte response to an HP increase.

Ouabain-induced stimulation of sodium-hydrogen exchange in rat optic nerve astrocytes

Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cytoplasmic pH fell abruptly, then gradually recovered toward baseline. Ouabain (1 microM) did not change cell sodium content, but the rate of pH recovery increased by 68%. Ouabain speeded pH recovery both in the presence and absence of bicarbonate. In bicarbonate-free medium, dimethylamiloride, an NHE inhibitor, eliminated the effect of 1 microM ouabain on pH recovery. Western blot analysis showed an NHE1 immunoreactive band but not NHE2, NHE3, or NHE4. Immunoprecipitation studies showed phosphorylation of NHE1 in cells treated with 1 microM ouabain. Ouabain evoked an increase of cAMP, and the effect of 1 microM ouabain on pH recovery was abolished by H-89, a protein kinase A inhibitor. 8-Bromoadenosine-cAMP increased the pH recovery rate, and this recovery was not further increased by ouabain. Although 1 microM ouabain did not alter cytoplasmic calcium concentration, it stimulated calcium entry after store depletion, a response abolished by 2-APB. Ouabain-induced stimulation of pH recovery was suppressed by inhibitors of capacitative calcium entry, SKF-96365, and 2-APB, as well as the cytoplasmic calcium chelator BAPTA. The cAMP increase in ouabain-treated cells was abolished by BAPTA and 2-APB. Taken together, the results are consistent with increased capacitative calcium entry and subsequent cAMP-PKA-dependent stimulation of NHE1 in ouabain-treated cells.

Inhibition of capacitative calcium entry suppresses ouabain‐induced stimulation of sodium‐hydrogen exchange in rat optic nerve astrocytes

Objective: Earlier we showed that 1 μM ouabain increased capacitative calcium entry (CCE) but did not increase cell sodium1. Here we studied cytoplasmic pH recovery after an acid load in ouabain‐treated cells and tested whether pH response were linked to altered calcium entry.Methods: Ammonium chloride (20mM) was used to acid load the cells. BCECF and FURA‐2 were used to measure intracellular pH and calcium.Results: Ammonium chloride removal causes the pHi to fall abruptly, followed by gradual recovery. lμM ouabain increased the rate of pHi recovery both in the presence and in the absence of bicarbonate in the bathing medium. NHE inhibitor DMA reduced the rate of pHi recovery. Ouabain did not detectably alter cytoplasmic Ca2+ (Ca2+i) but CCE inhibitors SKF96365 and 2APB reduced both the basal and ouabain‐stimulated rates of pHi recovery. Calcium chelator BAPTA‐AM also inhibited basal and ouabain‐stimulated pHi recovery. Western blot showed presence of NHE1 but not NHE2, NHE3 or NHE4.Conclusion: Ouabain‐induced stimulation of pHi recovery was due, at least in part, to stimulation of NHE1. Although lμM ouabain failed to alter Ca2+i, the findings suggest the NHE response may be dependent on CCE.Funding: NIH Grant EY014069

Activation of the Epidermal Growth Factor Receptor in Optic Nerve Astrocytes Leads to Early and Transient Induction of Cyclooxygenase-2

The epidermal growth factor receptor (EGFR) appears in astrocytes after neural injury. The authors' laboratory has reported the presence of EGFR in glaucomatous optic nerves. The activation of EGFR is often associated with induction of cyclooxygenase (COX)-2. In this study, the induction of COX-2 pathway in rat optic nerve astrocytes was investigated. Induction of COX-2 was determined by immunoblot and immunocytochemistry in optic nerve astrocytes stimulated with EGF. EGF-induced prostaglandin (PG)E(2) release into the culture medium was assayed by ELISA. The effects of the EGFR tyrosine kinase inhibitor, AG1478, were studied on COX-2 expression and PGE(2) synthesis. In rat optic nerve transection and a rat optic nerve explant culture model, the relationship between the expression of COX-2 and activation of EGFR was examined. Activation of EGFR caused the rapid and transient induction of COX-2 in optic nerve astrocytes. The level of COX-2 was rapidly upregulated in optic nerves after axotomy and in an optic nerve explant culture model. When induced, COX-2 localized to the nuclear membrane of the astrocytes. When COX-2 was induced in response to activation of EGFR, the activated astrocytes produced and released the proinflammatory mediator, PGE(2), in a time-dependent manner. EGF-stimulated induction of COX-2 protein and synthesis of PGE(2) were abolished by the EGFR tyrosine kinase inhibitor AG1478. The stimulatory action of EGF on release of PGE(2) was inhibited by the COX-2-selective inhibitor NS398. The data demonstrate that the activation of EGFR in optic nerve astrocytes leads to the induction of the immediate early gene COX-2 and subsequent signaling through the synthesis of PGE(2). This early signal of neural tissue damage may be important in setting up secondary events in the damaged tissue.

Guidance of oligodendrocytes and their progenitors by substratum topography

Oligodendrocyte progenitors arise in subventricular zones and migrate extensively during development before differentiating into mature oligodendrocytes, which myelinate nerve tracts in the central nervous system. We have used microfabricated substrata, containing periodic patterns of contours similar to those of central nervous system axons to assess the influence in vitro of substratum topography on oligodendrocytes isolated from 7 day rat optic nerve. Antiganglioside antibody A2B5 positive oligodendrocyte-type 2 astrocyte progenitors, and galactocerebroside positive and myelin basic protein positive oligodendrocytes, were highly aligned by surface contours as small as 100 nm depth and 260 nm repeat spacing. Rat optic nerve astrocytes also aligned on surface contours, but rat hippocampal and cerebellar neurons were unresponsive. Oligodendrocytes demonstrated enhanced parallel extension of their processes on narrow repeating topography in an arrangement similar to that found in the intact optic nerve. This is in marked contrast to the phenotype displayed by this cell type on planar substrata. Neither oligodendrocytes nor oligodendrocyte-type 2 astrocyte progenitors showed high-order F-actin cytoskeletal networks; thus their alignment on gratings is unlikely to result from deformation of actin cables and focal contacts. In contrast, aligned astrocytes showed striking arrangements of actin stress fibres. These results establish glial cells as potentially the most topographically sensitive cell types within the central nervous system. Furthermore, the topographical pattern inducing maximal alignment of oligodendrocyte lineage cells corresponds to the diameters of single axons within the 7 day optic nerve. Thus the migration of oligodendrocyte-type 2 astrocyte progenitors and axonal ensheathment by oligodendrocytes may be guided by axonal topography within the developing nerve.

Astrocytes from adult rat optic nerves are nonpermissive for regenerating retinal ganglion cell axons

We have directly compared the abilities of astrocytes from newborn and adult rats to support or inhibit the growth of regenerating axons in vitro. Astrocytes prepared from newborn rats were able to promote retinal ganglion cell (RGC) axon growth from embryonic and adult rat and from adult fish retinal explants. Retinal axons from E16 rat retinae grew significantly faster on astrocytes from neonatal rats than those from E18 or adult rat retinae with growth rates comparable to RGC axons from adult fish retinae. RGC regeneration from adult rat retinae was almost completely inhibited on adult rat optic nerve astrocytes. Only axons from adult fish retinae were able to extend onto monolayers from these reactive astrocytes, although their growth rates were significantly reduced. We conclude that the failure of mammalian RGC axons to regrow within the lesioned optic nerve environment is, at least in part, due to nonpermissive aspects of adult “reactive” optic nerve astrocytes. However, the cell intrinsic growth potential of RGCs also appears to influence their ability to extend axons on cellular substrates.

The astrocyte response to gamma-aminobutyric acid attenuates with age in the rat optic nerve.

There is increasing evidence that glial cells respond to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), and astrocytes have been shown to possess GABAA receptors both in vivo and in vitro. A recent study by Sakatani et al. (Proc. R. Soc. Lond. B247, 155 (1992)) demonstrated the transient expression of functional GABAA receptors in the developing rat optic nerve, but axonal and glial components of the response were not distinguished. To help address this problem, we have determined the electrophysiological response to GABA in astrocytes of the isolated intact optic nerves from neonatal rats, identified morphologically following intracellular injection of horseradish peroxidase. Astrocytes responded to GABA by a GABAA receptor-mediated depolarization which attenuated gradually during post-natal development; astrocytes in 21-day-old nerves were not observed to respond to GABA. The results indicate the transient presence of functional GABAA receptors in developing rat optic nerve astrocytes in situ, and we speculate upon a role for GABA in glial signalling and the organization of axonglial interrelations during development.

The beta 1 subunit mRNA of the rat brain Na+ channel is expressed in glial cells.

Although the molecular characteristics of glial Na+ channels are not well understood, recent studies have shown the presence of mRNA for rat brain Na+ channel alpha subunits in astrocytes and Schwann cells. In this study, we asked whether the mRNA for the rat brain Na+ channel beta 1 subunit is expressed in glial cells. We performed in situ hybridization using a complementary RNA probe for the coding regions of the rat brain Na+ channel beta 1 subunit mRNA and detected beta 1 subunit mRNA in cultured rat optic nerve astrocytes and sciatic nerve Schwann cells. The beta 1 subunit was amplified by reverse transcription-polymerase chain reaction in rat optic and sciatic nerves, which lack neuronal somata but contain astrocytes and Schwann cells, respectively. Doublet bands of the beta 1 subunit mRNA were amplified from both optic and sciatic nerves. Through the cloning and sequencing of these bands, we confirmed the amplification of a mRNA highly homologous to the previously cloned rat brain Na+ channel beta 1 subunit (beta 1.1) and a novel form of the beta 1 subunit mRNA (beta 1.2), which is closely homologous to beta 1.1 but contains an additional 86-nucleotide insert in 3' noncoding regions. Two beta 1 subunit mRNAs were also amplified from rat brain and skeletal muscle, but not from rat liver or kidney. These results indicate that rat brain Na+ channel beta 1 subunit mRNAs are expressed in glial cells as well as in neurons.

Response of astrocytes to γ-aminobutyric acid in the neonatal rat optic nerve

The electrophysiological response to γ-aminobutyric acid (GABA) was determined in astrocytes of the isolated intact optic nerves of rats aged 8 to 12 days old, identified morphologically following intracellular injection of horseradish peroxidase. At this age, astrocytes had a mean (± S.E.M.) resting membrane potential of −662.25 ± 1.9 mV ( n = 32), and responded to GABA by a depolarization characterized by an initial peak of mean 9.1 ± 0.6 mV, which was not sustained and fell to a plateau level. The effect of GABA was mimicked by the GABA A-receptor agonist muscimol, but not by the GABA B-receptor agonist baclofen, and was reduced in the presence of the GABA A-receptor antagonist bicuculline. Astrocytes responded to 10 mM [K +] o by a single phase depolarization of 16 ± 2 mV ( n = 5). It is concluded that GABA acts directly on astrocytes and its effect is not mediated by K + released by axons. This study indicates the presence of functional GABA A receptors in neonatal rat optic nerve astrocytes in situ. The results suggest immature astrocytes may be the source of the GABA response described in two recent studies on the rat whole optic nerve preparation. The astrocyte response to GABA may be important in axon-glial signalling during development.

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.

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.

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.

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.