The role of nitric oxide in development of topographic precision in the retinotectal projection of chick

Abstract

The axonal projection from the retina to the tectum exhibits a precise topographic order in the mature chick such that neighboring ganglion cells send axons to neighboring termination zones in the contralateral tectum. The initial pattern formed during development is much less organized and is refined to the adult pattern during a discrete period of development. Refinement includes elimination of radically aberrant projections, such as those from the temporal side of the retina to posterior regions of the tectum, as well as a more subtle improvement in the topographic precision of the projection. The enzyme that synthesizes nitric oxide is expressed at high levels in the tectum during the developmental period in which the topography improves. Pharmacological blockade of nitric oxide synthesis during this period prevented elimination of topographically inappropriate retinotectal projections in a dose-dependent manner. This effect could not be duplicated by treatment of embryos with a vasoconstrictor, indicating that vascular changes were not a factor. These results show that nitric oxide is involved in refinement of the topography of the retinotectal projection as well as in other aspects of refinement of this projection in developing chick.

Fig. 1.

Improvement in the topographic precision of the retinotectal projection during normal development. The position of retrogradely labeled ganglion cells is indicated by dotson these enlarged tracings of retinal whole mounts from untreated embryos of different ages. The embryonic age at which each retina was fixed is indicated. Twenty-four hours before preparation of these whole mounts, fluorescent latex microspheres were microinjected into a small spot in the posterior region of the tecta contralateral to these retinas. The precision of the topography progressively improves through a process of refinement from E12 to E17. The nasal side of each retina is on theleft, and the temporal side is on theright. The ★ indicates the position of the optic fissure. Scale bar, 2 mm.

Fig. 2.

Dose-dependent reduction in NO synthase activity in tectum after treatment with l-NoArg. NO synthase activity was assayed in homogenates of tectum by measuring the conversion of [³H]-arginine to [³H]-citrulline. Twelve hours before harvesting the tecta for analysis, embryos were treated systemically with saline or l-NoArg at the doses indicated. Error bars indicate SE;n = 6 for each data point.

Fig. 3.

Incomplete refinement of retinotectal topography with inhibition of NO synthesis. The position of retrogradely labeled ganglion cells is indicated by dots on these enlarged tracings of retinal whole mounts. Embryos were treated daily from E9 through E16 with 10 μmol of d-NAME (A, control), 2 μmol of l-NoArg (B), or 10 μmol of l-NAME (C). On E16, 24 hr before preparation of these whole mounts, fluorescent latex microspheres were microinjected into a small spot in the posterior region of the tecta contralateral to these retinas. The small insets to the right of each retina are traces of whole mounts of the tecta with the injection sites indicated. Unlike controls, retrogradely labeled ganglion cells were distributed across much of the retina in drug-treated embryos. This indicates that the precision of the topography of the retinotectal projection did not undergo refinement in the absence of NO synthesis. The nasal side of each retina is on the left, and the temporal side is on the right. The ★ indicates the position of the optic fissure. Scale bars, 2 mm.

Fig. 4.

Scatter analysis of retrogradely labeled cells. The graph shows the number of retrogradely labeled ganglion cells with increasing distance from the area of the retina with the highest concentration of labeled cells. Labeled cells in retinas from embryos treated with (●) 2 μmol of l-NoArg during the period of refinement were significantly more scattered than in retinas from (▪) embryos treated with saline during the same period (p = 0.0001), indicating that inhibition of NO synthesis prevents complete refinement of topography. Unit of distance is a radial increment of 1.3 mm of retina.

Fig. 5.

Dose-dependent change in retinotectal topography with inhibition of NO synthesis. The graph shows the number of retrogradely labeled ganglion cells on the temporal side of E17 retinas after microspheres were microinjected into a small spot in the posterior region of the tecta contralateral to the retinas analyzed. Experimental embryos were treated daily from E9 to E16 with doses ofl-NAME as shown. Control embryos were treated withd-NAME, saline or were untreated. Another group of embryos was treated with 10 nmol of phenylephrine HCl to test the effect of vasoconstriction on refinement. The division between nasal and temporal retina was deemed to be the optic fissure and a line that extended from the optic fissure into the dorsal retina. This cut to divide the two sides of the retina was made before the retinas were removed from the sclera. The ★ indicates results significantly different from each of the controls (p < 0.001). Error bars indicate SE; n = 5 for each treatment group.

Fig. 6.

Reduction in the percentage of cells with topographically correct projections with inhibition of NO synthesis. Fluorescent micrographs show all cells in the ganglion cell layer stained with DAPI (blue) and cells retrogradely labeled (red) from an injection of microspheres in the contralateral tectum. The micrographs show the area of the retinas with the highest density of retrogradely labeled cells. Retinas were from embryos treated with 2 μmol of l-NoArg (A) or saline (B) during the period of refinement. Scale bar, 25 μm. C, A lower percentage of cells were retrogradely labeled after inhibition of NO synthesis (p = 0.002). Error bars indicate SE.

Fig. 7.

NO synthase activity in the retina and tectum during development. NO synthase activity was assayed in homogenates of retina and tectum from embryos ranging in age from E6 to P0 by measuring the conversion of [³H]-arginine to [³H]-citrulline. Aliquots of the cytosolic fraction of tissue samples containing 250 μg of total protein were assayed. NO synthase levels are at background levels in the retina through much of the refinement period, whereas levels in the tectum peak during this period. Results are presented as the mean liquid scintillation spectroscopy reading in disintegrations per min (DPM) from three samples. Five retinas were pooled for each retinal sample. One tectum was used for each tectal sample. Results for tectum at each age were statistically different from one another (p < 0.001). Error bars indicate SE. There was no significant difference in the results for E6, E9, and E10 retina (p > 0.05); results for E17 and P0 retina were significantly different from each of the younger ages (p < 0.01 and p < 0.001, respectively). There was no significant difference in the result for E6 retina and E6 tectum (p > 0.05); for all other ages, results for retina and tectum were significantly different (p < 0.0001).