Distribution of [ 3H]RNA in goldfish optic tectum following intraocular or intracranial injection of [ 3H]uridine. Evidence of axonal migration of RNA in regenerating optic fibers

Abstract

The distribution of [ 3H]RNA in the goldfish optic tectum following either intra-ocular or intracranial injection of [ 3H]uridine during optic fiber regeneration has been studied by light (LMA) and electron (EMA) microscopic autoradiography. In one group of 4 fish both optic nerves were crushed, and 18 days later [ 3H]uridine was injected into the right eye. A second group of 5 fish, in which only one optic nerve had been crushed, received intracranial injections of [ 3H]uridine 18 or 22 days after the crush. All fish were sacrificed 24 days after crushing the optic nerves, a time when regenerating optic fibers have entered the tectum and are establishing functional reconnections. Tecta were fixed in situ with glutaraldehyde, dissected out, and samples were processed for LMA and EMA. Controls were carried out to ensure that [ 3H]RNA was the only radioactive component present in the tissue after fixation. The distribution of silver grains related to [ 3H]RNA in intraocularly injected goldfish was different from that following intracranial injection. Following intraocular injection virtually all the [ 3H]RNA was located in the layers of the left optic tectum (contralateral to the side of intraocular injection) where the regenerating optic fibers course and terminate, whereas virtually no radioactivity was present in the right optic tectum. EMA quantitative analysis of the labeled layers of the left optic tectum revealed that perikarya of cells, most of which are glial cells, had a density of grains related to [ 3H]RNA of 20–28 g/100 sq.μm; axonal growth cones had a density of 14–24 g/100 sq.μm. Grain densities over non-axonal cell structures were markedly lower, ranging between 3 and 6 g/sq.μm. Grains located over axons and growth cones accounted for 50–60% of all counted grains. In intracranially injected goldfish, either 2 or 6 days after injection, silver grains were clustered over leptomeninges as well as vessels and parenchymal cells of the tectal strata containing the regenerating optic fibers. In the stratum opticum a high grain density was seen over glial cells, whereas virtually no grains were present over the fascicles of regenerating axons. EMA quantitative analysis revealed a grain density over glial and other parenchymal cells of the stratum opticum of 67 g/100 sq.μm, whereas densities over growth cones and regenerating axons were 1.3 g/100 sq.μm and 1.8 g/100 sq.μm respectively. Grains located over axons and growth cones accounted for 3.3% of all counted grains. On the basis of the present and previous findings it is suggested that following intraocular injection of [ 3H]uridine the [ 3H]RNA present inside regenerating optic axons is transported from the ganglion cells of the retina; on the other hand, the [ 3H]RNA present in surrounding glial cells is the result of local utilization of [ 3H]RNA precursors which also migrate from the retina along with the [ 3H]RNA. It is also concluded that 2 and 6 days following intracranial injection of [ 3H]uridine no substantial tranfer of [ 3H]RNA from glial cells to regenerating optic fibers occurs in the goldfish optic tectum.