Enhanced labeling of a retinal protein during regeneration of optic nerve in goldfish.

Abstract

Goldfish retinas were examined for changes in the labeling pattern of protein during regeneration of retinal ganglion cell axons following unilateral optic nerve crush. At various times after optic nerve crush the normal retinas were incubated in vitro with [3H] Following intra-orbital crush of the optic nerve, in goldfish, the retinal ganglion cell must grow out a new axon several millimeters long. This process not only implies a substantial increase in the rate of protein synthesis but may also involve a selective increase in the synthesis of the proteins necessary for the renewal of axoplasm and replacement of structural elements of the axon. Formation of cell-specific markers involved in retinotectal recognition may be increased~during this period (10). There might, in addition, be an initial decrease in the synthesis of components necessary to maintain the functional integrity of the nerve ending until the growing nerve reaches the optic tectum (11).This report presents comparisons of the in vitro protein labeling pattern of normal goldfish retina with that of retinas removed at various times after optic nerve crush (postcrush retina). A double-labeling technique coupled with sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (12) revealed an increased amino-acid incorporation into a protein fraction with properties of the microtubule subunit, tubulin. MATERIALS AND METHODSGoldfish (Carassius auratus), 6-7 cm in body length, were anesthetized with tricaine methane sulfonate prior to an intraorbital crush of the right optic nerve. The left optic nerve remained intact so that the left retina served as a control. Following surgery, the goldfish were stored in groups of 50 to 80 in 30 gallon tanks at 20-22°, and were fed daily.Fish were dark-adapted for 30 min prior to removal of retinas in order to facilitate separation of the retina from the pigment epithelium. After hemisection of the eye, the retina was floated free from the pigment layer with 0.86% saline and detached by cutting at the optic disk. The retinas were then rinsed and stored in ice-cold saline or incubation medium for up to 1 hr prior to incubation.In were evaporated to dryness under nitrogen immediately before use. After evaporation, each was resuspended in a small volume of incubation medium. The purity of each isotopic preparation was checked by paper chromatography. The incubation medium was that of Dunlop et al. (13): N-2-bydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 25 mM; MgSO4, 1.3 mM; CaCl2, 2.6 mM; K2HPO4, 1.2 mM; KCI, 5.9 mM; NaCl, 106.5 mM; glucose, 12 mM; and NaOH to pH 7.4.Retinas (in groups of 5 to 20) were pre-incubated for 2 min at 25°in 1.9 ml of incubation medium. Labeled precursor (0.1 ml) was then added (1.0-4.0 ,uCi per retina) and incubation was continued in air in a Dubnoff metabolic shaker at 250. All incubations using double-labeled methionine were carried out for 1 hr. The reaction was stopped by dilution with 2 volumes of ice-cold incubation medium containing 2 mM L-methionine. After c...

Axotomy-induced changes in rabbit hindlimb nerves and the effects of chronic electrical stimulation

“…Axotomized nerve fiber caliber decreases (Gutmann and Sanders, 1943;Kreutzberg and Schubert, 197 1;Carlson et al, 1979;Risling et al, 1983) concurrent with a decline in conduction velocity (Cragg and Thomas, 196 1;Milner et al, 198 1;Gillespie and Stein, 1983). Molecular studies have more recently provided direct evidence for a switch in gene expression after axotomy: levels of messenger RNA and expression of neurotransmitters, rate limiting enzymes for transmitter synthesis and neurofilament (NF) protein decline following axotomy (Grafstein and McQuarrie, 1978;Gordon, 1983;Hoffman et al, 1984;Woolf et al, 1984;Tetzlaff et al, 1988a); other proteins associated with slow axonal transport, actin and tubulin, are upregulated (Hall et al, 1978;Hoffman and Lasek, 1980;Neumann et al, 1983;Sinicropi and McIlwain, 1983;Tetzlaff et al, 1988a), and the axotomized neurons express novel growth-associated proteins (Heacock and Agranoff, 1976;Skene and Willard, 198 1;Kalil and Skene, 1986;Tetzlaff et al, 1988aHoffman, 1989; for review, see Skene, 1989). Some of the biochemical changes have been directly correlated with physiological changes.…”

Phosphorylation of neurofilament proteins and chromatolysis following transection of rat sciatic nerve

“…Tubulin and actin are the major cytoskeletal components of growth cones and regenerating nerves (Peters and Vaughn, 1967;Fine and Bray, 197 1;Yamada et al, 1971;Marchisio et al, 1978) and undergo a marked enhancement in synthesis (Heacock and Agranoff, 1976;Burrell et al, 1979;Giulian et al, 1980;Sinicropi and McIlwain, 1983) and axonal transport (Lasek and Hoffman, 1976;Giulian et al, 1980;Hoffman and Lasek, 1980;Skene and Willard, 198 1) following nerve transection. By contrast, neurofilaments have a very limited presence, if any, in growth cones (Peters and Vaughn, 1967;Shaw et al, 198 1).…”

Homeobox genes are expressed in the retina and brain of adult goldfish.

“…These developmental and regenerative properties are not observed in the adult visual pathways of higher vertebrates. Thus, the goldfish visual pathway has emerged as an important model system to study molecular events associated with axonal growth and neurogenesis (9)(10)(11)(12).…”