Differential taurine effect on outgrowth from goldfish retinal ganglion cells after optic crush or axotomy. Influence of the optic tectum

The interaction between innervated tissues, targets and nerves is crucial in the maintenance of physiological conditions, and the disturbance of this harmony causes the production of morphological and biochemical changes. After lesion of the optic nerve, several modifications take place in the retina, the optic tectum and the optic nerve. The influence of the tectum on the outgrowth from the goldfish retina and the possible role of taurine was studied. Ganglion retinal cells were identified by retrolabeling with Dil. Crushing the optic nerve 10 days prior to plating retinal cells, as compared with optic axotomy, did not affect the survival of cultured retinal cells, as well as the length of the neurites. However, the number of neurites per cell and the branching of the longest fiber were higher after axotomy than after crushing. The addition of taurine to the medium did not modify this response at 5 days in culture. At early periods in culture, the stimulatory effect on isolated ganglion cell outgrowth produced by taurine was enhanced after axotomy respecting crushing of the optic nerve, but was not affected in retinal explants. The addition of medium from cultured optic tectum several days post-crush of the optic nerve to retinal explants from intact retinas or coming from post-crush retina modified the outgrowth, being inhibitory or stimulatory in a time-dependent manner. The co-culture of optic tectum and retina also affected the outgrowth from the retina with a byphasic shape. The results support the differential response of the retina facing partial or complete interruption with the target and limit the effect of taurine to early periods in culture. In addition, the production of inhibitory factors from the tectum, plus the stimulatory ones, are strongly supported by this work.     

Medium requirements for neuritic outgrowth from goldfish retinal explants and the trophic effect of taurine

The use of culture media of known composition are necessary for studying the role of trophic molecules. Since most of the in vitro research on regeneration of the optic nerve has been performed in the presence of fetal calf serum, the aim of this study was to obtain a medium in which the neuritic outgrowth from post-crush goldfish retinal explants could take place without adding fetal calf serum. After the lesion of the optic nerve (10 days), the retina of goldfish was dissected and explants were cultured for 5 and 10 days in the absence or in the presence of fetal calf serum, at which time the neuritic outgrowth was determined. Various concentrations and combinations of glucose, albumin, calcium, HEPES and taurine were used. The highest neuritic outgrowth was observed in the presence of fetal calf serum, in which condition the amino acid taurine increased length and density of neurites. Media supplemented with albumin, calcium or HEPES did not modify the outgrowth of neurites from the explants. However, glucose favored the neuritic outgrowth in a bell-shaped manner, although fibers were thinner than those observed in the presence of fetal calf serum. Taurine did not stimulate outgrowth of neurites from explants growing in a medium with optimal concentrations of glucose, indicating that elements of the fetal calf serum are determinant for the trophic effect of taurine. The present results contribute to further studies, such as those related to the effect of taurine and of trophic factors derived from the optic tectum, which would be performed in the presence of a medium free of fetal calf serum.     

Taurine might be acting as a trophic factor in the retina by modulating phosphorylation of cellular proteins

Protein phosphorylation is involved in the regeneration of the nervous system. Taurine modulates the phosphorylation of specific proteins in the retina, and also increases outgrowth from ganglion cells. In order to test the possible role of protein phosphorylation on the outgrowth from the retina and on the trophic effect of taurine, in vitro studies were performed in the presence of phorbol and nonphorbol protein kinase C activators, the protein kinase C inhibitor tamoxifen, and phosphatase inhibitors. After crush of the optic nerve, explants of the goldfish retina were cultured and the outgrowth was evaluated by measuring the length and the density of neurites. The activation of protein kinase C decreased the outgrowth from the explants and impaired the stimulatory effect of taurine. Phosphatase inhibitors produced a similar effect on basal and taurine-modulated outgrowth. In certain concentrations, some of these drugs did not affect the emission of neurites in the absence of taurine, but decreased the effect of the amino acid. Tamoxifen also reduced the outgrowth, probably acting at other cellular levels or indicating that the regulation of outgrowth by phosphorylation is a complex and dual process. The response to the drugs, evaluated by length or density of fibers, was not the same, since rate of outgrowth was more affected than density, which suggests that both parameters are modulated at differential stages or sensitivities to the tested agents. J. Neurosci. Res. 53:377–384, 1998. © 1998 Wiley-Liss, Inc.     

Neurites outgrowth and amino acids levels in goldfish retina under hypo-osmotic or hyper-osmotic conditions

Amino acids are known to play relevant roles as osmolytes in various tissues, including the retina. Taurine is one of these active molecules. In addition, taurine stimulates outgrowth from the goldfish retina by mechanisms that include extracellular matrix, calcium fluxes and protein phosphorylation. The present report aims to explore the effect of medium osmolarity on goldfish retinal outgrowth and the possible modifications produced by changing eye osmolarity on amino acid levels in the retina. Goldfish retinal explants were obtained 10 days after crush of the optic nerve and cultured under iso-, hypo- or hyper-osmotic conditions. Hypo-osmotic medium was prepared by diluting the solutions 10% twice, preserving fetal calf serum concentration. Hyper-osmotic medium was done by adding 50 or 100 mM urea or mannitol. Evaluation of length and density of neurites was performed 5 days after plating. Outgrowth was reduced in hypo- and in hyper-osmotic conditions. Taurine, 4 mM, increased length and density of neurites in iso-osmotic, and produced stimulatory effects under both hyper-osmotic conditions. The in vivo modification of osmolarity by intraocular injection of water or 100 mM urea modified levels of free amino acids in the retina. Taurine and aspartate retinal levels increased in a time-dependent manner after hypo- and hyper-osmotic solution injections. Serine, threonine, arginine, γ-aminobutyric acid, alanine and tyrosine were elevated in hyper-osmotic conditions. Outgrowth in vitro, after in vivo osmolarity changes, was higher in the absence of taurine, but did not increase in the presence of the amino acid. The fact that certain outgrowth took place in these conditions support that the impairment was not due to tissue damage. Rather, the effects might be related to the cascade of kinase events described during osmolarity variations. The time course under these conditions produced adjustments in ganglion cells probably related to taurine transporter, and phosphorylation of specific proteins.     

Outgrowth and maintenance of neurites from cultured goldfish retinal ganglion cells

Single retinal ganglion cells obtained from goldfish retina are shown to support outgrowth of neurites. Processes up to 1 mm in lenght grow out over a two week period from cells identified as retinal ganglion cells on the basis of morphological criteria, in the absence of supporting elements. Prior crush of the optic nerve significantly enhances neuritic outgrowth. Fibers emerging from single cells often associate to form a single process. A tendency for characteristic clockwise patterning of neurite outgrowth from single cells is seen, similar to that obtained from retinal explants.     

Serotonin inhibits outgrowth of goldfish retina and impairs the trophic effect of taurine

The regeneration of explants prepared from goldfish retinas with a prior crush of the optic nerve is stimulated by the sulphur amino acid, taurine. Serotonin has been reported to modify survival, proliferation, and outgrowth of nervous tissue. In the present work we evaluated the effect of serotonin and some serotonergic agonists on the neuritic outgrowth from goldfish retinal explants. Serotonin, its precursor, 5-hydroxytryptophan, and the 5HT_1A receptor agonists, 8-hydroxy-2-(di-n-propylamino)tetralin and busprione, inhibited the outgrowth. The blockers of serotonin uptake, imipramine and citalopram, were also inhibitors of neurite sprouting. Imipramine favoured the inhibitory effect of serotonin at 10 days in culture. The concentration of serotonin and its metabolite, 5-hydroxyindoleacetic acid, decreased in the retina at 3 and 5 days after the crush of the optic nerve. Serotonin levels started to recover after 5 days post-lesion, and the metabolite also increased. This indicates that the lesion increases the turnover rate of serotonin and this may be related to its role in regeneration. Serotonin concentration was elevated by the intraocular administration of its precursor, 5-hydroxytryptophan, indicating that the capacity for synthesis was preserved after the crush, but that it was smaller in the post-lesioned retinas. The trophic effect of taurine was impaired by a low concentration of serotonin, probably by opposing the final effect on growth via different targets. These results support a role of serotonin in the regeneration of goldfish retina probably through 5HT_1A receptors. © 1994 Wiley-Liss, Inc.     

The interaction of substrate and taurine modulates the outgrowth from regenerating goldfish retinal explants

Retinal growth from explants attached to different substrates was evaluated in the absence and the presence of various concentrations of taurine. Fiber thickness was higher in poly-L-lysine, collagen and laminin than in the other substrates tested. Explants on wheatgerm, soybean, and red kidney bean lectins did not show emission of neurites at 5 days in culture; growth on laminin was very poor at this period of time. By day 10 nerve growth index was possible to evaluate on soybean and laminin. Outgrowth from retinal explants on wheatgerm lectin was only observed in the presence of taurine. The percentage of growing explants on laminin was increased by the amino acid. Emission of neurites from explants on concanavalin A and lentil lectin was constant from 5 to 10 days in culture and was not accelerated or increased by taurine. Growth on poly-L-lysine and collagen was stimulated by the addition of taurine in a concentration-dependent manner. The amino acid stimulates outgrowth from explants attached to peanut lectin in a bell-shaped concentration dependency similar to that observed on poly-L-lysine. The effect of taurine on retinal explants is substrate-dependent, as an indication of the interaction of soluble molecular components plus charge and structure of the extracellular matrix in order to modulate the outgrowth from regenerating tissue.     

Adult olfactory ensheathing glia promote the long-distance growth of adult retinal ganglion cell neurites in vitro

In vivo, transplanted adult olfactory ensheathing glia (OEG) and adult Schwann cells (SC) can support the regrowth of at least some transected axons within adult CNS neuropil. In the present study, we developed an in vitro adult rat retinal explant model to explore the influence of primary adult SC and OEG on retinal ganglion cell (RGC) neurite regrowth in the presence of glial cells endogenous to the retina. Retinal quadrants were plated RGC-side down onto aclar hats coated with either pure collagen (type 1), collagen with OEG, collagen with SCs, or collagen coated with both OEG and SCs. Regrowing retinal neurites extended onto the pure collagen substrate, largely in association with astrocytes that migrated out from the explants (mean number of neurites: 144+/-65 SEM). The additional presence of OEG (669+/-122), but not SCs (97+/-41), supported the regrowth of significantly greater numbers of RGC neurites. Furthermore, this OEG-stimulated regeneration was over significantly greater distances; >68% of neurites extended >500 microm from the explant, compared with explants plated onto SCs or collagen alone (15% and 29%, respectively). When OEG and SCs were co-cultured the number of regenerating neurites was reduced (397+/-81) compared with the pure OEG treatment. Analysis of explants on pure collagen substrates fed with media conditioned by purified OEG or SC showed no increase in neurite outgrowth compared with control treatments, suggesting that the enhanced growth in the presence of OEG is a contact-mediated effect. The observed differences between the abilities of OEG and SC to support the growth of CNS-derived fibers in the presence of astrocytes support the suggestion that OEG may be better suited for direct transplantation into CNS neuropil following injury.     

Different cell surface areas of polarized radial glia having opposite effects on axonal outgrowth

During neuronal development neurites are likely to be specifically guided to their targets. Within the chicken retina, ganglion cell axons are extended exclusively into the optic fibre layer, but not into the outer retina. We investigated, whether radial glial cells having endfeet at the optic fibre layer and somata in the outer retina, might be involved in neurite guidance. In order to analyse distinct cell surface areas, endfeet and somata of these glial cells were purified. Glial endfeet were isolated from flat mounted retina by a specific detachment procedure. Glial somata were purified by negative selection using a monoclonal antibody/complement mediated cytolysis of all non-glial cells. Retinal tissue strips were explanted either onto pure glial endfeet or onto glial somata. As revealed by scanning and fluorescence microscopy, essentially no ganglion cell axons were evident on glial somata, whereas axonal outgrowth was abundant on glial endfeet. However, when glial somata were heat treated and employed thereafter as the substratum, axon extension was significantly increased. Time-lapse video recording studies indicated that purified cell membranes of glial somata but not of endfeet induced collapse of growth cones. Collapsing activity was destroyed by heat treatment of glial membranes. The collapsing activity of retinal glia was found to be specific for retinal ganglion cell neurites, because growth cones from dorsal root ganglia remained unaffected. Employing four different kinase inhibitors revealed that the investigated protein kinase types were unlikely to be involved in the collapse reaction. The data show for the first time that radial glial cells are functionally polarized having permissive endfeet and inhibitory somata with regard to outgrowing axons. This finding underscores the pivotal role of radial glia in structuring developing nervous systems.     

Regeneration of axons from adult rat retinal ganglion cells on cultured Schwann cells is not dependent on basal lamina.

The 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 microns/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 microns/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 matrix.     

Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina

Despite evidence that glial cell surfaces and components of the extracellular matrix (ECM) support neurite outgrowth in many culture systems, the relative contributions of these factors have rarely been compared directly. Specifically, it remains to be determined which components of peripheral nerve support growth of central nerve fibers. We have directly compared neurite outgrowth from embryonic day 15 rat retinal explants placed onto beds of (1) Schwann cells without ECM, (2) Schwann cells expressing ECM (including a basal lamina), (3) cell-free ECM prepared from neuron-Schwann cell cultures, (4) nonglial cells (fibroblasts), and (5) 2 isolated ECM components, laminin and type I collagen. From the first day in culture, retinal explants extended neurites when placed on Schwann cells without ECM. Outgrowth on Schwann cells expressing ECM was also extensive, but not obviously different form that on Schwann cells alone. Ultrastructural study revealed that 95% of retinal neurites in ECM-containing cultures contacted other neurites and Schwann cell surfaces exclusively. On cell-free ECM prepared from neuron-Schwann cell cultures, neurite extension was poor to nonexistent. No neurite outgrowth occurred on fibroblasts. Retinal explants also failed to extend neurites onto purified laminin and ammoniated type I collagen substrata; however, growth was rapid and extensive on air-dried type I collagen. In cultures containing islands of air-dried type I collagen on a laminin-coated coverslip, retinal explants attached and extended neurites on collagen, but these neurites did not extend off the island onto the laminin substratum. We conclude from these experiments that neurite extension from embryonic rat retina is supported by a factor found on the surface of Schwann cells and that neither organized nor isolated ECM components provide this neurite promotion. These findings are discussed in relation to possible species differences in growth requirements for retinal ganglion cell neurites and to the specificity of response of different CNS neurites to ECM substrata.     

Effects of retinal pigment epithelial cell-secreted factors on neonatal rat retinal explant progenitor cells

This study demonstrates the effects of conditioned media from transformed neonatal rat retinal pigment epithelial cells (tnrRPE-CM) in a culture system consisting of neonatal rat retinal explants. For this study, retinal explants from postnatal day 2 (PN2) normal rats were cultured for over 3 weeks on a poly-D -L -ornithine-coated surface in RPE-CM only, 10% serum, or a serum-free defined media, and then examined by phase-contrast and scanning electron microscopy and immunocytochemistry. After 2 days in vitro, long ganglion cell-like neurites projected from retinal explants grown in tnrRPE-CM. These neurites increased in number and length with prolonged time in culture. In addition, by 5 days, round cells were observed adjacent to neonatal explants grown in tnrRPE-CM. By day 10, these round cells had increased in number and were seen along the neurites, in massive clusters immediately adjacent to these explants and dispersed throughout the culture-plate surface. Media conditioned by primary cultures of normal neonatal rat RPE cells caused a similar, but less robust, cellular response in retinal explants when compared to tnrRPE-CM. At 10 days, retinal explants grown in 10% serum showed only a few short processes, but no round cells, while those explants grown in defined media appeared to be degenerating. The round migrating cells are classified as retinal progenitor cells since they immunostained for opsin and interphotoreceptor retinoid-binding protein (IRBP), two photoreceptor cell markers, and a few for cellular retinaldehyde binding protein (CRALBP), a Muller cell marker. Neurite outgrowth and retinal progenitor cell production from explants were eliminated when the tnrRPE-CM was subjected to trypsin or heat treatment, indicating that the factor(s) responsible for promoting these cellular events was most likely proteinaceous. Growth factors, including basic fibroblast growth factor, were unable to generate long neurite outgrowth or progenitor cell production as observed in RPE-CM-supplemented explant cultures. We report that CM from cultures of primary and transformed neonatal rat RPE cells promoted ganglion cell-like neurites and the production of migrating retinal progenitor cells that primarily expressed photoreceptor-specific markers, from neonatal rat retinal explants. This evidence further confirms the important role of RPE in retinal development. The production of large numbers of progenitor cells by an RPE-secreted factor(s) may have important implications for possible therapeutic approaches to help correct retinal disease states by replacing lost cells through transplantation technology. © 1996 Wiley-Liss, Inc.     

Neurotrophic stimulation of fetal rat retinal explant neurite outgrowth and cell survival: age-dependent relationships

Serum-free tissue culture conditions have been defined where stimulation of neurite outgrowth from fetal rat retinal explants occurred only in the presence of an active fraction (BE) prepared from a pig brain extract purification procedure. Under these conditions, 18-20-day fetal retinal explants survived and continued to extend long radial neurites for at least 3 weeks in the presence of BE. However, if fibronectin was not equilibrated onto the basic collagen/poly-L-lysine substrate the neurite outgrowth was restricted to a short halo about the circumference of the explant. In addition, a dose-response relationship was demonstrated in the presence of increasing concentrations of BE with respect to the neurite growth index. The half-maximal response for BE was estimated to be between 5 and 10 micrograms/ml. In addition a number of important age-dependent relationships were observed with respect to BE stimulation of retinal neurite outgrowth and cell survival. An inverse relationship was demonstrated between increased developmental age and responsiveness to BE. After 1 week in culture, there was a 3-fold reduction in retinal neurite length measured from the 2-day neonatal explant when compared to that of the 18-day fetus. There was also a significant inverse relationship demonstrated between the length of time before BE was added to the culture medium and the ability of 20-day fetal explants to extend neurites onto the culture substrate. If BE was added as late as 2 weeks after initial explant culture, the various neurite outgrowth indices were significantly lower than in those situations where BE was added at the time of culture or 1 week later. These results imply that BE not only is required for stimulating neurite outgrowth from fetal rat retinal explants, but may be important in survival and maturation of developing retinal neurons. This hypothesis was confirmed when morphometric analysis was performed on 16- and 20-day explants cultured for a week in the presence or absence of BE. The number of necrotic cells in the developing retinal ganglion plexiform-cell layer of 20-day fetal explants was significantly lower when treated with BE. Conversely, the density of identifiable differentiating retinal ganglion-like cells was significantly greater in response to BE treatment in both 16- and 20-day retinal explants.     

Explant culture of adult goldfish retina: A model for the study of CNS regeneration

Conditions are described for culture of retinal explants of adult goldfish which favour outgrowth of neuritic processes onto a substratum. A growth index to quantitate the outgrowth was developed. If the optic nerve is crushed several days prior to explanation, a marked enhancement of neuritic outgrowth is seen relative to control retinas. Histological examination of the explants revealed that retinal ganglion cells in explants from unoperated eyes became hypertrophied in vitro with a time course similar to that observed in vivo following optic nerve crush. Experiments with hemiaxotomized retinas indicate that the perikaryal regenerative response is mediated intracellularly.     

Is the Capacity for Optic Nerve Regeneration Related to Continued Retinal Ganglion Cell Production in the Frog?

In the central nervous system of fish and frogs, some, but not all, axons can regenerate. Retinal ganglion cells are among those that can. The retinae of fish and frogs produce new retinal neurons, including ganglion cells, for months or years after hatching. We have evaluated the hypothesis that retinal axonal regeneration is obligatorily linked to continued production of new ganglion cells. We used bromodeoxyuridine immunocytochemistry to assess retinal neurogenesis in juvenile, yearling, and 10 year old Xenopus laevis. Retinal ganglion cell genesis was vigorous in the marginal retina of the juveniles, but in the yearlings and the 10 year olds, no new ganglion cells were produced there. Cellular proliferation in the central retina was evident at all three ages, but none of the cells produced centrally were in the ganglion cell layer. Regeneration was examined in vivo by cutting one optic nerve and then, weeks later, injecting the eye with tritiated proline. Autoradiographs of brain sections showed that the optic nerves of all three ages regenerated. Regeneration in vitro was assessed using retinal explants from frogs of all three ages. In all cases, the cultures produced neurites, with some age-specific differences in the patterns of outgrowth. We conclude that retinal axonal regeneration is not linked obligatorily to maintained neurogenesis.     

Citicoline has a protective effect on damaged retinal ganglion cells in mouse culture retina

Some clinical reports indicate that exogenous CDP-choline (citicoline) may have a therapeutic effect in patients with glaucoma. However, the precise effect of citicoline on damaged retinal ganglion cells (RGCs) remains to be explained. We performed tissue culture of mouse retinal explants and investigated the effect of citicoline on damaged RGCs by the quantitative analysis of TdT-dUTP terminal nick-end labeling (TUNEL) staining and the assessment of the number of regenerating neurites. The TUNEL-positive ratio in 0.1-10 micromol/l citicoline-treated retina was very low, and the number of regenerating neurites increased more than in control retina. Our findings suggest that citicoline has a protective effect on damaged RGCs in tissue culture of retina.     

Growth stimulation and chemotropic attraction of rat retinal ganglion cell axons in vitro by co-cultured optic nerves, astrocytes and astrocyte conditioned medium

The effects of explants of optic nerves of different ontogenetic ages (P0-P14, adult), and of cultured astrocytes of various ages on the neurite regeneration of rat retinal ganglion cells (RGC) were assessed in vitro, using a three-dimensional culture system which allows the co-cultivation of various explants. Both co-cultured P0-P12 optic nerves and astrocyte cultures from P2 cerebral cortex stimulated the regeneration of neurites from the retinal explants after 3 days in culture. By contrast, P14 and older explants of the optic nerve, astrocytes from P17 optic nerve and astrocytes that had previously been grown in culture for more than 6 weeks had no effect on RGC neurite outgrowth. Moreover, both the P0–P12 optic nerve explants and the astrocytes from P2 cerebral cortex also seemed to have a chemotropic effect on the regenerating neurites, because the latter were longer on the side facing the co-explantat. The absence of a cellular bridge between retinal and optic nerve explants suggests that the effects are mediated by astroglia-derived diffusible neurite growth promoting factors. Accordingly, astrocyte-conditioned medium from P2 astrocytes also stimulated the outgrowth of neurites from the retinal explants. These findings show that immature astrocytes of a limited ontogenetic period release as yet unknown diffusible neurite growth-promoting factors which stimulate the regeneration of neurites from retinal explants.     

Spatial distribution of glutamate, taurine and GABA in teleosts and mammals retina: in vivo and in vitro study

Teleost and amphibian retina grows throughout life at the circumferential margin distal to the center. Retinas of two teleosts, goldfish and Eugerres plumieri; and two mammals, rat and rabbit were dissected into concentric regions; center and periphery; or center, intermediate and periphery. The concentration of glutamic acid, taurine, and gamma-aminobutyric acid was determined in these areas. A non-significant difference was found between the center and periphery of retinas dissected into two regions. By dissecting the teleost retina in three concentric regions a progressive increase of gamma-aminobutyric acid and taurine for center to periphery was observed, which was statistically significant for taurine. Rat and rabbit retina did not present this difference. The concentration of these amino acids was also determined in two concentric regions of goldfish retina 24 hr after crush of the optic nerve. Taurine content significantly increased in the center of the regenerating retina, but not in the periphery. Outgrowth was measured in explants prepared from central and peripheral retina in the presence of exogenous taurine. The amino acid produced an increase of the nerve growth index of central explants, but inhibited the growth from peripheral explants. This report presents a correlation between taurine concentration in a region of the retina that could be considered as embryonic tissue, suggesting that this amino acid plays a role in the formation of new cells. Moreover, the results from post-crush and cultured retina, with respect to taurine concentration or regenerating effect, suggest a saturation of the taurine-enriched zone.     

Rapid initiation of neurite outgrowth onto laminin from explants of adult mouse retina induced by optic nerve crush

One optic nerve of an adult mouse was crushed in the orbit. After 8 days, both retinas were explanted onto laminin-coated coverslips. Within 24 h, neurites grew out onto this substrate from explants with prior crush and by 48 h two-thirds of explants had neurites. On polylysine or plain glass, outgrowth was generally not seen until 48 h and this was significantly reduced compared to laminin. Explants without optic nerve crush did not extend neurites until about 5 days later. We suggest that axotomy induces a time-dependent regenerative response in adult mammalian retinal ganglion cells which includes the expression of laminin responsiveness.     

Intraretinal pathfinding of ganglion cell axons is perturbed by a monoclonal antibody specific for a G4/Ng-CAM-like cell adhesion molecule.

To identify molecular components involved in directed axonal outgrowth and in neural pattern formation, hybridoma technology was employed using the visual system of the chicken as a model system. Using cell surface protein fractions as immunogens, we obtained the monoclonal antibody mAb C4, which binds to a 135 kDa cell surface glycoprotein of the high-mannose or complex type. Within the retina, the C4 antigen is found exclusively in the optic fiber layer. Immuno-double labeling of retinal whole mounts with a glial marker and mAb C4 suggests that the C4 antigen is restricted to ganglion cell axons but not found on Müller glial endfeet. Biochemical and histological data reveal similarities between the C4-antigen and G4/NgCAM. Addition of mAb C4 to retina explants cultured on a striped carpet of tectal cell membranes leads to defasciculation of outgrowing axons, suggesting that the C4 antigen serves as an axon cell adhesion molecule (Ax-CAM). Axon elongation on neighboring axons can be also inhibited by the application of mAb C4 to embryonic retina whole mounts in vitro. The aberrant axon growth into incorrect retina layers observed under these conditions suggests that the C4 antigen functions as a guiding cue for the generation of the retinal optic fiber layer.