Further studies on the development of the isthmo-optic nucleus with special reference to the occurrence and fate of ectopic and ipsilaterally projecting neurons.

Using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) and the fluorescent dyes true blue and nuclear yellow, we have reex-amined the time of arrival at the retina of the centrifugal fibers from the contralateral isthmo-optic nucleus (ION), and have followed, quantitatively, the appearance and fate of other neurons that can be retrogradely labeled from the ipsilateral and contralateral eyes. The earliest age at which ION neurons can be retrogradely labeled is on the ninth day of incubation; the cells labeled at this stage are located in the ventrolateral part of the nucleus on the contralateral side. Over the course of the next 48 hours of development more and more cells can be labeled in a distinct ventrolateral to dorsomedial progression within the nucleus. Since this labeling sequence parallels the time course of generation of the ION neurons it is suggested that the axons of the first ION cells to be generated are the first to reach the contralateral retina, and that in this system there is a close relationship between the time that neurons withdraw from the cell cycle and the time that their axons reach their target area. In addition to the neurons in the ION, about 1,500 cells outside of the nucleus can be retrogradely labeled by WGA-HRP injected into the contra-lateral eye in posthatched chicks. This is slightly less than half the number of “ectopic isthmo-optic neurons” that can be similarly labeled on the 13th day of incubation (when the ION is numerically complete). The reduction in the number of ectopic ION neurons occurs over the same period as the phase of naturally occurring cell death in the ION itself–between the 13th and 17th days of incubation. Long-term labeling experiments with true blue indicate that the disappearance of about 53% of the ectopic ION cells is due to their death rather than the elimination of axon collaterals. In their morphology the ectopic neurons resemble the cells in the ION at early stages in their development, and there is indirect evidence that the ectopic ION neurons which survive also receive an input from the optic tectum through the-tecto-isthmal tract. On these and other grounds it is suggested that the ectopic neurons are indeed “misplaced” ION cells. Between the tenth and 13th days of incubation a small and rather variable number of neurons (58–178) was retrogradely labeled from the ipsilateral eye. Of these neurons with aberrant, ipsilaterally projecting axons, an average of just under 30 lay within the interior of the ION, about 33 were along its borders, and 38 were observed scattered among the ectopic ION cells. Double labeling experiments indicate that early in their development some of these neurons have axon collaterals which project to the contralateral eye. In a large number of animals injected with WGA-HRP after the 17th day of incubation, only a single neuron was seen in the interior of the ipsilateral ION, but, on average, about nine labeled neurons were found along its borders and about 20 were ectopically placed. The numbers of ectopic ION neurons and ION cells with ipsilaterally projecting axons that we have observed are substantially higher than those reported in a previous study (Clarke and Cowan, '76) in which it was also suggested the both classes of cells were effectively eliminated between the 13th and 17th days of incubation. The observed differences are attributable to the greater sensitivity of the WGA-HRP method when used with the chromogen tetramethyl benzidine. It is now clear that although cells in the ION with aberrantly projecting axons (or axon collaterals) may be selected against during the later stages of development, the fate of the ectopic ION neurons is not significantly different from that of the cells in the ION itself, but since they lie outside the nucleus their dendritic processes are not subject to the same morphogenetic influences.