Abstract. Axons derived from young ganglion cells in the periphery of the retinae of larval and adult goldfish are known to fasciculate with one another and their immediate forerunners, creating the typical age-related order in the retinotectal pathway. Young axons express the E587 antigen, a member of the L1 family of cell adhesion molecules. Repeated injections of Fab fragments from a polyclonal E587 antiserum (E587 Fabs) into the eye of 3.4 cm goldfish disrupted the orderly fascicle pattern of RGC axons in the retina which was preserved in controls. Instead of bundling tightly, RGC axons crossed one another, grew between fascicles and arrived at the optic disk in a broadened front. When added to RGC axons growing in vitro, E587 Fabs neutralized the preference of growth cones to elongate on lanes of E587 protein, caused defasciculation of axons which normally prefer to grow along each other when explanted on polylysine, and prevented clustering of E587 antigen at axon-axon contact sites. Monoclonal E587 antibody disturbed axonal fasciculation moderately but led to a 30% reduction in growth velocities when axons tracked other axons.Therefore we conclude that E587 antigen mediates axonal recognition, selective fasciculation and the creation of the age-related order in the fish retina.

To determine whether netrin receptors (DCC, UNC5H1, UNC5H2) and netrin-1 are present in the adult rat retina and may affect regeneration of retinal ganglion cell (RGC) axons into peripheral nerve (PN) grafts, in situ hybridization (ISH) and immunostaining experiments were performed in normal and operated rats. Netrin-1 expression was not found in the optic nerve head of adult rats, normal and postlesion, but appeared transiently at 7 and 14 days after PN grafting. ISH signals of netrin receptors, however, disappeared from RGCs within 2 days after lesion and remained absent after PN grafting except for UNC5H2, which transiently occurred in a few RGCs. Netrin-1 expression was observed in the optic nerve head of adult fish, normal and postlesion, and the netrin-1 Fc fusion protein bound to young growing and all regenerating axons. Thus, the netrin-1-dependent guidance system continues to function in fish but apparently no longer operates in adult rats.

The optic disk–directed growth of retinal ganglion cell axons is markedly disturbed in the presence of polyclonal antineurolin antibodies, which mildly affect fasciculation (Ott, H., M. Bastmeyer, and C.A.O. Stuermer, 1998. J. Neurosci. 18:3363–3372).New monoclonal antibodies (mAbs) against goldfish neurolin, an immunoglobulin (Ig) superfamily cell adhesion/recognition molecule with five Ig domains, were generated to assign function (guidance versus fasciculation) to specific Ig domains. By their ability or failure to recognize Chinese hamster ovary cells expressing recombinant neurolin with deletions of defined Ig domains, mAbs were identified as being directed against Ig domains 1, 2, or 3, respectively. Repeated intraocular injections of a mAb against Ig domain 2 disturb the disk-directed growth: axons grow in aberrant routes and fail to reach the optic disk, but remain fasciculated. mAbs against Ig domains 1 and 3 disturb the formation of tight fascicles.mAb against Ig domain 2 significantly increases the incidence of growth cone departure from the disk-oriented fascicle track, while mAbs against Ig domains 1 and 3 do not. This was demonstrated by time-lapse videorecording of labeled growth cones.Thus, Ig domain 2 of neurolin is apparently essential for growth cone guidance towards the disk, presumably by being part of a receptor (or complex) for an axon guidance component.

The properties of glial cells in lesioned nerves contribute quite substantially to success or failure of axon regeneration in the CNS. Goldfish retinal axons regenerate after optic nerve lesion (ONS) and express the L1-like cell adhesion protein E587 antigen on their surfaces. Goldfish oligodendrocytes in vitro also produce E587 antigen and promote growth of both fish and rat retinal axons. To determine whether glial cells in vivo synthesize E587 antigen, in situ hybridizations with E587 antisense cRNA probes and light-and electron microscopic E587 immunostainings were carried out. After lesion, the goldfish optic nerve/tract contained glial cells expressing E587 mRNA, which were few in number at 6 days after ONS, increased over the following week and declined in number thereafter. Also, E587-immunopositive elongated cells with ultrastructural characteristics of oligodendrocytes were found. Thus, glial cells synthesize E587 antigen in spatiotemporal correlation with retinal axon regeneration. To determine the functional contribution of E587 antigen, axon-oligodendrocyte interactions were monitored in co-culture assays in the presence of Fab fragments of a polyclonal E587 antiserum. E587 Fabs in axon-glia co-cultures prevented the normal tight adhesion of goldfish retinal growth cones to oligodendrocytes and blocked the preferential growth of fish and rat retinal axons on the oligodendrocyte surfaces. The ability of glia in the goldfish visual pathway to upregulate the expression of E587 antigen and the growth supportive effect of oligodendrocyte-associated E587 antigen in vitro suggests that this L1-like adhesion protein promotes retinal axon regeneration in the goldfish CNS.

The properties of glial cells in lesioned nerves contribute quite substantially to success or failure of axon regeneration in the CNS. Goldfish retinal axons regenerate after optic nerve lesion (ONS) and express the L1-like cell adhesion protein E587 antigen on their surfaces. Goldfish oligodendrocytes in vitro also produce E587 antigen and promote growth of both fish and rat retinal axons. To determine whether glial cells in vivo synthesize E587 antigen, in situ hybridizations with E587 antisense cRNA probes and light-and electron microscopic E587 immunostainings were carried out. After lesion, the goldfish optic nerve/tract contained glial cells expressing E587 mRNA, which were few in number at 6 days after ONS, increased over the following week and declined in number thereafter. Also, E587-immunopositive elongated cells with ultrastructural characteristics of oligodendrocytes were found. Thus, glial cells synthesize E587 antigen in spatiotemporal correlation with retinal axon regeneration. To determine the functional contribution of E587 antigen, axon-oligodendrocyte interactions were monitored in co-culture assays in the presence of Fab fragments of a polyclonal E587 antiserum. E587 Fabs in axon-glia co-cultures prevented the normal tight adhesion of goldfish retinal growth cones to oligodendrocytes and blocked the preferential growth of fish and rat retinal axons on the oligodendrocyte surfaces. The ability of glia in the goldfish visual pathway to upregulate the expression of E587 antigen and the growth supportive effect of oligodendrocyte-associated E587 antigen in vitro suggests that this L1-like adhesion protein promotes retinal axon regeneration in the goldfish CNS.