Goldfish Optic Nerve Research Articles

Neurolin expression in the optic nerve and immunoreactivity of Pax6-positive niches in the brain of rainbow trout (Oncorhynchus mykiss) after unilateral eye injury.

In contrast to astrocytes in mammals, fish astrocytes promote axon regeneration after brain injury and actively participate in the regeneration process. Neurolin, a regeneration-associated, Zn8-labeled protein, is involved in the repair of damaged optic nerve in goldfish. At 1 week after unilateral eye injury, the expression of neurolin in the optic nerve and chiasm, and the expression of Pax6 that influences nervous system development in various brain regions in the rainbow trout (Oncorhynchus mykiss) were detected. Immunohistochemical staining revealed that the number of Zn8+ cells in the optic nerve head and intraorbital segment was obviously increased, and the increase in Zn8+ cells was also observed in the proximal and distal parts of injured optic nerve. This suggests that Zn8+ astrocytes participate in optic nerve regeneration. ELISA results revealed that Pax6 protein increased obviously at 1 week post-injury. Immunohistochemical staining revealed the appearance of Pax6+ neurogenic niches and a larger number of neural precursor cells, which are mainly from Pax6+ radial glia cells, in the nuclei of the diencephalon and optic tectum of rainbow trout (Oncorhynchus mykiss). Taken together, unilateral eye injury can cause optic nerve reaction, and the formation of neurogenic niches is likely a compensation phenomenon during the repair process of optic nerve injury in rainbow trout (Oncorhynchus mykiss).

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TGR) mRNA increased in RGCs 1–6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1–40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3–10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.

Characterization of Pax2 Expression in the Goldfish Optic Nerve Head during Retina Regeneration

The Pax2 transcription factor plays a crucial role in axon-guidance and astrocyte differentiation in the optic nerve head (ONH) during vertebrate visual system development. However, little is known about its function during regeneration. The fish visual system is in continuous growth and can regenerate. Müller cells and astrocytes of the retina and ONH play an important role in these processes. We demonstrate that pax2a in goldfish is highly conserved and at least two pax2a transcripts are expressed in the optic nerve. Moreover, we show two different astrocyte populations in goldfish: Pax2+ astrocytes located in the ONH and S100+ astrocytes distributed throughout the retina and the ONH. After peripheral growth zone (PGZ) cryolesion, both Pax2+ and S100+ astrocytes have different responses. At 7 days after injury the number of Pax2+ cells is reduced and coincides with the absence of young axons. In contrast, there is an increase of S100+ astrocytes in the retina surrounding the ONH and S100+ processes in the ONH. At 15 days post injury, the PGZ starts to regenerate and the number of S100+ astrocytes increases in this region. Moreover, the regenerating axons reach the ONH and the pax2a gene expression levels and the number of Pax2+ cells increase. At the same time, S100+/GFAP+/GS+ astrocytes located in the posterior ONH react strongly. In the course of the regeneration, Müller cell vitreal processes surrounding the ONH are primarily disorganized and later increase in number. During the whole regenerative process we detect a source of Pax2+/PCNA+ astrocytes surrounding the posterior ONH. We demonstrate that pax2a expression and the Pax2+ astrocyte population in the ONH are modified during the PGZ regeneration, suggesting that they could play an important role in this process.

Sema-3A indirectly disrupts the regeneration process of goldfish optic nerve after controlled injury

Neurons of adult mammalian CNS are prevented from regenerating injured axons due to formation of a non-permissive environment. The retinal ganglion cells (RGC), which are part of the CNS, share this characteristic. In sharp contrast, the RGC of lower vertebrates, such as fish, are capable of re-growing injured optic nerve axons, and achieve, through a complex multi-factorial process, functional vision after injury. Semaphorin-3A (sema-3A), a member of the class 3 semaphorins known for its repellent and apoptotic activities, has previously been shown to play a key role in the formation of a non-permissive environment after CNS injury in mammalians. The expression of sema-3A and its effect on regenerative processes in injured gold fish retina and optic nerve were investigated in this study. Unilateral optic nerve axotomy or crush was induced in goldfish. 2 microl sema-3A was injected intraviterally 48 hours post injury. Neuronal viability was measured using the lipophilic neurotracer dye 4-Di-10-Asp. Axonal regeneration was initiated using the anterograde dye dextran. Retinas and optic nerves were collected at intervals of 2, 3, 7, 14 and 28 days after the procedure. Using Western blot and immunohistochemical analysis, the expression levels of semaphorin-3A, axonal regeneration, the removal of myelin debris and macrophage invasion were studied. We found a decrease in sema-3A levels in the retina at an early stage after optic nerve injury, but no change in sema-3A levels in the injured optic nerve. Intravitreal injection of sema-3A to goldfish eye, shortly after optic nerve injury, led to destructive effects on several pathways of the regenerative processes, including the survival of retinal ganglion cells, axonal growth, and clearance of myelin debris from the lesion site by macrophages. Exogenous administration of sema-3A in fish indirectly interferes with the regeneration process of the optic nerve. The findings corroborate our previous findings in mammals, and further validate sema-3A as a key factor in the generation of a non-permissive environment after transection of the optic nerve.

Nitric oxide‐cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish (Carassius auratus). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5-40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5-40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo. None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.

Pax2 in the optic nerve of the goldfish, a model of continuous growth

Pax2 is a well known transcription factor which participates in optic nerve development. It assures the correct arrival and package of the newly formed retinal axons and the adequate differentiation of the newly formed glial cells. Pax2 protein expression is continuous throughout adult life in the goldfish optic nerve. We have found two populations of astrocytes in the optic nerve: Pax2 + and Pax2 −. Moreover, we have observed that the Pax2 + astrocytes from the optic nerve head present differences in number and organization to those of the rest of the optic nerve. In the optic nerve head some Pax2 + astrocytes, principally localized in the glia limitans, have thin GFAP + processes and weak cytokeratin and ZO1 immunolabeling. Several Pax2 + astrocytes are in close association with the GFAP +/GS + Müller cell vitreal processes and with the growing Zn8 + retinal ganglion cell axons. However, in the intraorbital segment, Pax2 + astrocytes are more numerous and they have strongly cytokeratin +/ZO1 + processes and form part of the reticular astrocytes and the glia limitans. We also found Pax2 − astrocytes in both the optic nerve head and the intraorbital segment. In the intraorbital segment there are GS +/Pax2 − cells which are absent from the optic nerve head. We propose that the maintenance of Pax2 protein expression in adult goldfish optic nerve could be related to the continuous addition of new ganglion cell axons and new glial cells.

Involvement of retinoic acid signaling in goldfish optic nerve regeneration

Recently, we identified a retina-specific retinol-binding protein, purpurin, as a trigger molecule in the early stage of goldfish optic nerve regeneration. Purpurin protein was secreted by photoreceptors to injured ganglion cells, at 2–5 days after optic nerve injury. Purpurin bound to retinol induced neurite outgrowth in retinal explant cultures and retinoic acid (RA) had a comparable effect on neurite outgrowth. These results indicate that purpurin acts as a retinol transporter and facilitates conversion of retinol to RA. Intracellularly, RA is transported into the nucleus with cellular retinoic acid-binding protein IIb (CRABPIIb) and binds with retinoic acid receptor α (RARα) as a transcriptional regulator of target genes. Here, we investigated the RA signaling through RA synthesis to RARα in the goldfish retina during optic nerve regeneration by RT-PCR. Retinaldehyde dehydrogenase 2 (RALDH2; an RA synthetic enzyme) mRNA was increased by 2.7-fold in the retina at 7–10 days and then gradually decreased until 40 days after nerve injury. In contrast, cytochrome P450 26a1 (CYP26a1; an RA degradative enzyme) mRNA was decreased to less than half in the retina at 5–20 days and then gradually returned to the control level by 40 days after nerve injury. CRABPIIb mRNA was increased by 1.5-fold in the retina at 10 days after axotomy, RARαa mRNA was increased by 1.8-fold in the retina at 10 days after axotomy. The cellular changes in the RA signaling molecules after optic nerve injury were almost all located in the ganglion cells, as evaluated by in situ hybridization. The present data described for the first time that RA signaling through RALDH2 and CRABPIIb to RARα was serially upregulated in the ganglion cells at 7–10 days just after the purpurin induction. Therefore, we conclude that the triggering action of purpurin on optic nerve regeneration is mediated by RA signaling pathway.

Upregulation of retinal transglutaminase during the axonal elongation stage of goldfish optic nerve regeneration

Fish CNS neurons can repair their axons following nerve injury, whereas mammalian CNS neurons cannot regenerate, and become apoptotic within 1–2 weeks after the nerve lesion. One explanation for these differences is that one, or several molecules are upregulated in fish CNS neurons during nerve regeneration, and this same molecule is downregulated in mammalian CNS neurons before the development of apoptosis caused by nerve injury. A molecule satisfying these criteria might successfully rescue and repair the mammalian CNS neurons. In this study, we looked for such a candidate molecule from goldfish retinas. Transglutaminase derived from goldfish retina (TG R) was characterized as a regenerating molecule after optic nerve injury. A full-length cDNA for TG R was isolated from the goldfish retinal cDNA library prepared from axotomized retinas. Levels of TG R mRNA and protein increased only in the retinal ganglion cells (RGCs) between 10 and 40 days after optic nerve transection. Recombinant TG R protein enhanced neurite outgrowth from adult fish RGCs in culture. Specific interference RNA and antibodies for TG R inhibited neurite outgrowth both in vitro and in vivo. In contrast, the level of TG R protein decreased in rat RGCs within 1–3 days after nerve injury. Furthermore, the addition of recombinant TG R to retinal cultures induced striking neurite outgrowth from adult rat RGCs. These molecular and cellular data strongly suggest that TG R promotes axonal elongation at the surface of injured RGCs after optic nerve injury.

The 'lipid raft' microdomain proteins reggie-1 and reggie-2 (flotillins) are scaffolds for protein interaction and signalling.

Reggie-1 and reggie-2 are two evolutionarily highly conserved proteins which are up-regulated in retinal ganglion cells during regeneration of lesioned axons in the goldfish optic nerve. They are located at the cytoplasmic face of the plasma membrane and are considered to be 'lipid raft' constituents due to their insolubility in Triton X-100 and presence in the 'floating fractions'; hence they were independently named flotillins. According to our current view, the reggies subserve functions as protein scaffolds which form microdomains in neurons, lymphocytes and many other cell types across species as distant as flies and humans. These microdomains are of a surprisingly constant size of less than or equal to 0.1 mm in all cell types, whereas the distance between them is variable. The microdomains co-ordinate signal transduction of specific cell-surface proteins and especially of GPI (glycosylphosphatidylinositol)-anchored proteins into the cell, as is demonstrated for PrP(c) (cellular prion protein) in T-lymphocytes. These cells possess a pre-formed reggie cap scaffold consisting of densely packed reggie microdomains. PrP(c) is targeted to the lymphocyte reggie cap when activated by antibody cross-linking, and induces a distinct Ca(2+) signal. In developing zebrafish, reggies become concentrated in neurons and axon tracts, and their absence, after morpholino antisense RNA-knockdown, results in deformed embryos with reduced brains. Likewise, defects in Drosophila eye morphogenesis occur upon reggie overexpression in mutant flies. The defects observed in the organism, as well as in single cells in culture, indicate a morphogenetic function of the reggies, with emphasis on the nervous system. This complies with their role as scaffolds for the formation of multiprotein complexes involved in signalling across the plasma membrane.

Characterization of a chondroitin sultate proteoglycan associated with regeneration in goldfish optic tract.

Regeneration of goldfish optic nerve axons is accompanied by a major increase in axonally transported proteoglycans (21). To identify specific proteoglycans increased during regeneration, we have used proteoglycan preparations from regenerating goldfish optic tracts to produce monoclonal antibodies. Western blot analysis shows a 28-kD antigen reacting with our 1G4/G5 antibody is present in optic tract 21 days after nerve crush, but absent in nonregenerating tract. Treatment with chondroitinase AC removes IG4/G5 immunostaining of the 28-kD molecule. An anti-CS antibody further confirmed this molecule as a chondroitin sulfate proteoglycan. A slightly smaller core protein following chondroitinase AC treatment indicates a low level of glycosylation. The N-terminal amino acid sequence of its core protein is not similar to any known proteoglycans. The CSPG sediments through 1.4 M sucrose, indicative of an extracellular matrix localization. It is expressed during the outgrowth of regenerating axons. Cut nerve and retinal explant studies demonstrate that the 1G4/G5 CSPG is not axonal, suggesting a glial localization.

Characterisation of tectal ephrin-A2 expression during optic nerve regeneration in goldfish: implications for restoration of topography

EphA receptors and their ligands the ephrin-As, expressed as retinal and tectal gradients, are required for the development of retino-tectal topography [Neuron 25 (2000) 563] and its restoration during goldfish optic nerve regeneration [Mol. Cell. Neurosci. 25 (2004) 56]. We have reported previously that, during regeneration, a transient EphA3/A5 gradient is formed by differential expression across the entire retinal ganglion cell (RGC) population [Neurosci. Abs. 33 (2003) 358.2; Exp. Neurol. 183 (2003) 593]. In retino-recipient tectal layers, ephrin-A2 is normally expressed by only a sub-population of cells, but during regeneration, there is a graded increase with more expressing cells caudally than rostrally [Exp. Neurol. 166 (2000) 196]. Here, we examine the characteristics of tectal ephrin-A2 expression during regeneration. We report that the level of ephrin-A2 expression is comparable for all ephrin-A2-positive cells in normal animals and during regeneration. Using double-labelling immunohistochemistry for ephrin-A2 and specific cell markers (NeuN for neurons, GA5 for astrocytes, NN-1 for microglia/endothelial cells and 6D2 for oligodendrocytes), we demonstrate that ephrin-A2-expressing cells, as in normal animals, are exclusively neuronal. Moreover, double labelling with BrdU showed that ephrin-A2 is expressed in resident cells and not those generated during optic nerve regeneration [Brain Res. 854 (2000) 178, 153 (1978) 345].

Axonal regeneration of fish optic nerve after injury.

Since Sperry's work in the 1950s, it has been known that the central nervous system (CNS) neurons of lower vertebrates such as fish and amphibians can regenerate after axotomy, whereas the CNS neurons of mammals become apoptotic after axotomy. The goldfish optic nerve (ON) is one of the most studied animal models of CNS regeneration. Morphological changes in the goldfish retina and tectum after ON transection were first researched in the 1970s-1980s. Many biochemical studies of neurite outgrowth-promoting substances were then carried out in the 1980s-1990s. Many factors have been reported to be active substances that show increased levels during fish ON regeneration, as shown by using various protein chemistry techniques. However, there are very few molecular cloning techniques for studying ON regeneration after injury. In this review article, we summarize the neurite outgrowth-promoting factors reported by other researchers and describe our strategies for searching for ON regenerating molecules using a differential hybridization technique in the goldfish visual system. The process of goldfish ON regeneration after injury is very long. It takes about half a year from the start of axonal regrowth to complete restoration of vision. The process has been classified into three stages: early, middle and late. We screened for genes with increased expression during regeneration using axotomized goldfish retinal and tectal cDNA libraries and obtained stage-specific cDNA clones that were upregulated in the retina and tectum. We further discuss functional roles of these molecules in the regeneration processes of goldfish ON.

Na,K-ATPase alpha3 subunit in the goldfish retina during optic nerve regeneration.

The goldfish optic nerve can regenerate after injury. To understand the molecular mechanism of optic nerve regrowth, we identified genes whose expression is specifically up-regulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened by differential hybridization with cDNA probes derived from axotomized or normal retina. Of six cDNA clones isolated, one clone was identified as the Na,K-ATPase catalytic subunit alpha3 isoform by high- sequence homology. In northern hybridization, the expression level of the mRNA was significantly increased at 2 days and peaked at 5-10 days, and then gradually decreased and returned to control level by 45 days after optic nerve transection. Both in situ hybridization and immunohistochemical staining have revealed the location of this transient retinal change after optic nerve transection. The increased expression was observed only in the ganglion cell layer and optic nerve fiber layer at 5-20 days after optic nerve transection. In an explant culture system, neurite outgrowth from the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (50-100 nm ) completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that up-regulation of the Na,K-ATPase alpha3 subunit is involved in the regrowth of ganglion cell axons after axotomy.

Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting, April 29???May 4, 2001

Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting, April 29???May 4, 2001

Slow recovery of goldfish retinal ganglion cells’ soma size during regeneration

The goldfish optic nerve regenerates after sectioning. Recently both short-term (30 days) and long-term (4 months) recovery of various goldfish behaviors were observed after optic nerve section. Using intracellular injection of Lucifer Yellow (LY) the morphology of regenerating ganglion cells in goldfish retina after optic nerve section over a 4 month period have been investigated. In normal retinas, most cells (96-98%) were 7-10 microm in soma diameter which increased with increasing distance from the optic disc. Only two or three short, thin processes could be traced with LY. The remaining cells (2-4%) were 13-16 microm in soma diameter and all of the long dendritic trees could be traced with LY. The most conspicuous morphological change observed was cellular hypertrophy, which occurred for 20-90 days after axotomy. Neuronal processes were also hypertrophic in this period. The percentage increase in hypertrophy of the central ganglion cells tended to be slightly higher compared to cells from other regions. These morphological changes peaked at 60 days after axotomy and fully disappeared by 120 days after axotomy. The slow recovery of ganglion cells' soma size may reflect the slow return to the normal number of optic axon terminals in the tectum during regeneration.

Flotillin 2 is distinct from epidermal surface antigen (ESA) and is associated with filopodia formation

ECS-1, a monoclonal antibody (MoAb) raised to cultured human keratinocytes, stains the intercellular glycocalyx with a pemphigus-like pattern and recognizes a 35-kDa epidermal surface antigen (ESA) on Western blotting of keratinocyte extracts. When ECS-1 MoAb was used to screen a keratinocyte expression library, a unique cDNA was identified that predicted a 42-kDa globular protein of unknown function. This putative ESA was conserved between mice and humans and was encoded by a gene on chromosome 17q11–12 in linkage with neurofibromin. Homology between the cDNA sequence has been reported with flotillin 1, a caveolae associated protein, as well as Reggie 1 and 2, neuronal proteins expressed during axonal regeneration present in activated GPI-anchored cell adhesion molecules in non-caveolar-associated micropatches. In order to determine whether the cDNA predicted protein and ECS-1 antigen were identical, we compared ECS-1 with the immunoreactivity of a new antibody raised to the cDNA fusion protein in epidermis and cultured cells. The cDNA fusion protein was expressed in bacteria and in cos cells with his, FLAG, and EGFP reporter tags and by stable transfection as an EGFP fusion protein. The fusion protein and native protein of 42 kDa were detected by the new antibody, but not by the original ECS-1. Thus, the ECS-1 antigen, ESA (35 kDa), is clearly distinct from the protein predicted by the cDNA (renamed flotillin 2). Stable transfection of ESA/flotillin 2 fusion protein in cos cells induced filopodia formation and changed epithelial cells to a neuronal appearance. Thus, the function of flotillin 2 may resemble that of the goldfish optic nerve neuronal regeneration proteins, Reggie 1 and 2. J. Cell. Biochem. 75:147–159, 1999. © 1999 Wiley-Liss, Inc.

A vulnerable period of colchicine toxicity during goldfish optic nerve regeneration

The effects of intraocular (i.o.) administration of the alkaloid colchicine on visual recovery following axotomy of the goldfish optic nerve were investigated. Under the experimental conditions used, control goldfish recovered vision, measured behaviorally, within 5-7 weeks of retro-orbital optic nerve crush. Fish injected i. o. with 0.1 microg of colchicine within 3 days of optic nerve crush (post-crush; PC) recovered vision after some delay relative to control fish, while injection with colchicine between 7 and 14 days PC produced a much more profound inhibition of recovery of vision, in most cases a complete block for the duration of the study (98 days). Further evidence for a delayed susceptibility of the regenerating optic nerve to colchicine following crush was reflected in a suppression of neurite outgrowth normally seen in explanted retinal tissue taken from PC goldfish. In addition, retrograde transport of the fluorescent dye 4-(4-didecylaminostyryl)-N-methylpyridinium iodide from the optic tectum to the retina as a measure of axonal continuity revealed substantially less labeling following i.o. administration of colchicine 1 week PC when compared to retinas from fish receiving colchicine at the time of optic nerve crush. Histological sections of the retina showed no evidence of residual retinal damage resulting from the colchicine injections or from interactions of axotomy and the drug administration. These results indicate a period of increased vulnerability of the regenerating visual system to the toxic effects of i.o. administered colchicine, beginning 3-5 days PC, and remaining until regenerating optic nerve fibers have begun to reach the tectum. While colchicine has many known effects on nerve function, it is proposed that the delayed susceptibility to disruption of regeneration observed in these experiments is largely, if not entirely, attributable to a colchicine-induced accumulation of tubulin heterodimers, which are known to block microtubule assembly and to participate in a feedback inhibition of tubulin synthesis. Thus, it is during the maximal induction of tubulin synthesis and of microtubule formation which normally occurs several days following axotomy that colchicine has its greatest effect. The results suggest that colchicine may be especially neurotoxic during neural development and regeneration.

Flotillin 2 is distinct from epidermal surface antigen (ESA) and is associated with filopodia formation

ECS-1, a monoclonal antibody (MoAb) raised to cultured human keratinocytes, stains the intercellular glycocalyx with a pemphigus-like pattern and recognizes a 35-kDa epidermal surface antigen (ESA) on Western blotting of keratinocyte extracts. When ECS-1 MoAb was used to screen a keratinocyte expression library, a unique cDNA was identified that predicted a 42-kDa globular protein of unknown function. This putative ESA was conserved between mice and humans and was encoded by a gene on chromosome 17q11-12 in linkage with neurofibromin. Homology between the cDNA sequence has been reported with flotillin 1, a caveolae associated protein, as well as Reggie 1 and 2, neuronal proteins expressed during axonal regeneration present in activated GPI-anchored cell adhesion molecules in non-caveolar-associated micropatches. In order to determine whether the cDNA predicted protein and ECS-1 antigen were identical, we compared ECS-1 with the immunoreactivity of a new antibody raised to the cDNA fusion protein in epidermis and cultured cells. The cDNA fusion protein was expressed in bacteria and in cos cells with his, FLAG, and EGFP reporter tags and by stable transfection as an EGFP fusion protein. The fusion protein and native protein of 42 kDa were detected by the new antibody, but not by the original ECS-1. Thus, the ECS-1 antigen, ESA (35 kDa), is clearly distinct from the protein predicted by the cDNA (renamed flotillin 2). Stable transfection of ESA/flotillin 2 fusion protein in cos cells induced filopodia formation and changed epithelial cells to a neuronal appearance. Thus, the function of flotillin 2 may resemble that of the goldfish optic nerve neuronal regeneration proteins, Reggie 1 and 2.

Fast and slow recovery phases of goldfish behavior after transection of the optic nerve revealed by a computer image processing system

As the goldfish is a common experimental animal for vision research, including psychophysical behavior, it is very important to quantitatively score fish behavior. We have previously developed a computer image processing system which can acquire the positional coordinates of goldfish moving freely in an aquarium and determine turning directions (go straight, right or left turn). In the present study, an algorithm to determine tilting angles of moving goldfish was constructed. We also made histograms for quantifying the interaction between pairs of goldfish (two-point distance). By using these histograms, we estimated the time-course of behavioral regeneration after optic nerve transection in goldfish. Control goldfish showed an equal percentage of right or left turns and maintained an upright position in a dorsoventral axis. When the optic nerve of a goldfish was unilaterally sectioned, the goldfish showed predominant turning and slight tilting toward the intact eye. The abnormal turning and tilting behaviors lasted for 10–14 days and then gradually decreased, returning to control behaviors by one month after the unilateral transection. When the optic nerve of a single goldfish was bilaterally sectioned, it did not show any preferential turning and tilting behavior, which is similar to what was observed in control goldfish. However, the trace maps showed that, after bilateral sectioning, fish preferred to cross the center of the tank, which was unlike control fish. In control pairs, one goldfish chased the other with a fixed small range of two-point distances. However, in pairs of goldfish with bilateral transection of the optic nerve, the blind goldfish behaved independently of each other, with a long two-point distance. The long two-point distance of the blind goldfish lasted for at least two months and then slowly returned to control two-point distance by four months after bilateral transection. Such fast and slow recovery in goldfish behaviors evoked after unilateral and bilateral transection of the optic nerve is discussed with respect to reconnection of regenerating optic nerves in the fish central nervous system. This computer image processing system is a useful tool with which we can quickly and easily quantify fish behavior.

Large-scale synaptic errors during map formation by regenerating optic axons in the goldfish

During the formation of visual maps, growing axons initially form a map by using topographically distributed cues that direct their growth and branching to the appropriate target region. This initial map is typically roughly retinotopic and is subsequently refined through activity-dependent rearrangement or cell death. Although synaptic connections are thought to be rearranged during the later refinement phase, there is no clear evidence that synapses are being formed during the initial targeting phase of development. Also, because optic fiber growth can be accurately directed during normal development, it is unclear whether regenerative fibers that have more pathway disorder would behave similarly. This issue was addressed by using optic fibers of goldfish that have the capacity to regenerate a retinotopic projection and can reestablish a rough retinotopic order without impulse activity. The optic nerve of goldfish was crushed, and at various times later, a small number of optic fibers in ventronasal retina was labeled with wheatgerm agglutinin-horseradish peroxidase. The tectum was then processed for electron microscopy to look at the distribution of labeled synapses during regeneration. At 3 weeks, synapses were observed at the far anterior end of the tectum and none were yet seen at the correct posterior retinotopic position. At 4-5 weeks, synapses were seen in nearly equal numbers at the incorrect anterior end and at both correct (retinotopic) and incorrect posterior positions. At late stages of regeneration, synapses were restricted to their correct posterior retinotopic position in the tectum, as they were in normal fish. These findings show that the formation of global retinotopic order entails the formation and subsequent elimination of a large number of highly ectopic synapses. Synaptic rearrangement is a major feature of targeting in this system and may be required for the regeneration of a retinotopic projection.