Imaging of regenerating optic fibers in living adult goldfish was used to visualize arbor restructuring during activity‐dependent refinement. A small number of neighboring retinal ganglion cells were labeled with DiI and observed in the tectum of the living animal for 5–7 hours during the period of activity‐dependent refinement. In contrast to earlier stages of regeneration, many optic arbors were surprisingly stable, showing little or no change. The observed changes were mainly retractions, and these were affected by retinotopic position and activity. Axon branches in retinotopic positions changed by much smaller amounts than ectopic axons, but in fish with retinal tetrodotoxin impulse blockade, no systematic difference was observed as a function of tectal position. Otherwise, impulse blockade had no notable effects. J. Comp. Neurol. 406:548–562, 1999. © 1999 Wiley‐Liss, Inc.

Regenerating optic fibers in goldfish make large-scale errors when they invade tectum and subsequently correct these to generate a projection with moderate retinotopic order by 1 month. The behavior of fibers underlying these extensive rearrangements is not well understood. To clarify this, we have imaged optic fibers in living adult goldfish at 2-4 weeks of regeneration. A small number of neighboring retinal ganglion cells were labeled with microinjections of DiI and imaged in the dorsal tectum with a cooled CCD camera on a fluorescence microscope for 5 to 8 hours. Nearly all fibers were simple unbranched processes and had endings that were highly dynamic showing both growth and retraction. Fibers from dorsal retina that normally innervate ventral tectum were frequently observed in dorsal tectum. These ectopic fibers oscillated more frequently between growth and retraction and retracted more often than ventral optic fibers. Like retinotopic fibers, ectopic fibers exhibited net growth but they showed no apparent directional preference toward their retinotopic position. In contrast, large errors along the anterior-posterior axis corresponding to nasal-temporal retina were rare and there was no differential behavior that distinguished these fibers.

The dynamic behavior of axons in systems that normally regenerate may provide clues for promoting regeneration in humans. When the optic nerve is severed in adult goldfish, all axons regenerate back to the tectum to reestablish accurate connections. In adult mammals, regeneration can be induced in optic and other axons but typically few fibers regrow and only for short distances. These conditions were mimicked in the adult goldfish by surgically deflecting 10-20% of optic fibers from one tectum into the opposite tectum which was denervated of all other optic fibers by removing its corresponding eye. At 21-63 days, DiI was microinjected into retina to label a few fibers and the fibers were visualized in the living fish for up to 5-7 hours. The dynamic behavior and morphology of these regenerating deflected fibers were analyzed and compared to those regenerating following optic nerve crush. At 3-4 weeks, deflected fibers were found to form more branches and to maintain many more branches than crushed fibers. Although both deflected and crushed fibers exhibited stochastic growth and retraction, deflected fibers spent more time growing but grew for less distance. At 2 months, both deflected and crushed fibers became much more stable. These results show the morphology and behavior of fibers regenerating into the same target tissue can be substantially altered by the injury conditions, that is, they show state dependent plasticity. The morphology and behavior of the deflected fibers suggests they were impaired in their capacity to grow to their correct targets.

A great deal of effort has been invested in using trophic factors and other bioactive molecules to promote cell survival and axonal regeneration in the adult central nervous system. Far less attention has been paid to investigating potential effects that trophic factors may have that might interfere with recovery. In the visual system, BDNF has been previously reported to prevent regeneration. To test if BDNF is inherently incompatible with regeneration, BDNF was given intraocularly during optic nerve regeneration in the adult goldfish. In vivo imaging and anatomical analysis of selectively labeled axons were used as a sensitive assay for effects on regeneration within the tectum. BDNF had no detectable inhibitory effect on the ability of axons to regenerate. Normal numbers of axons regenerated into the tectum, exhibited dynamic growth and retractions similar to controls, and were able to navigate to their correct target zone in the tectum. However, BDNF was found to have additional effects that adversely affected the quality of regeneration. It promoted premature branching at ectopic locations, diminished the growth rate of axons through the tectum, and resulted in the formation of ectopic collaterals. Thus, although BDNF has robust effects on axonal behavior, it is, nevertheless, compatible with axonal regeneration, axon navigation and the formation of terminal arbors.