Double labeling methods were used to identify changes in the complement of proteins synthesized in the retinal ganglion cells and transported down the optic nerve during the process of axonal regeneration. Eight to 62 days after goldfish underwent a unilateral optic nerve crush, one eye was labeled with [3H]-, the other with [14C]proline. Control and regenerating optic nerves were dissected out and homogenized together after 5 hr, a time which allowed us to examine selectively membrane-bound components which migrate in the rapid phase of axoplasmic transport. Proteins from the two sides were so-purified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the 3H and 14C incorporation patterns along the gels revealed a radical shift away from the normal labeling spectrum during regeneration, with selective changes in labeling at particular molecular weights varying over a 3-fold range. Eight days after crushing the optic nerve, the greatest increases in labeling were seen for material with apparent molecular weights of 24,000 to 27,000, 44,000, and 210,000 daltons. These peaks declined thereafter, and on days 29 to 39, the most prominent increases were at 110,000 to 140,000 daltons. These studies indicate a continuously changing pattern in the synthesis and/or degradation of proteins that are rapidly transported down the optic nerve during regeneration and point to molecular species potential significance in the establishment of the visual map upon the brain.
A class of tectal cells whose mitotic activity is enhanced by optic nerve regeneration in adult goldfish has been identified as radial neuroglia. These mitotic glial cells are morphologically distinct cells with prominent radial processes which extend through the entire depth of the tectal layers. The radial glia incorporate exogenous [3H]thymidine ([3H]TdR) into DNA as early as 2 hr after systemic injection. The plane of cell division for the mitotic radial glia is always aligned to the equator of the cell body, perpendicular to the direction of the radial process. In a few cases, radially adjacent pairs of labeled daughter radial glial cells are observed as early as 12 hr and also as late as 51 days after [3H]TdR injection. In other cases, however, only one labeled daughter radial glial cell is identified, and the other daughter cell cannot be traced. These observations suggest that radial glial cells of the adult goldfish tectum can be induced (presumably by mitogenic effects of regenerating optic nerve fibers) to undergo mitosis.
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