Investigations on the development and topographic order of retinotectal axons: anterograde and retrograde staining of axons and perikarya with rhodamine in vivo

Rhodamine-B-isothiocyanate (RITC) is shown to be a convenient and advantageous fluorescence tracer both for anterograde staining of retinal ganglion cell axons on the tectum and for retrograde staining of ganglion cell bodies in the retina of chick embryos. After intravitreal injection the dye is taken up by ganglion cells of the retina from the extracellular space and is transported anterogradely at about 10 mm/day up to the axonal growth cones on the tectum. RITC can be taken up by growing axons on the tectum and it is transported retrogradely at about 5 mm/day to the cell bodies in the retina. Local staining can be achieved if RITC is applied in its crystalline form. RITC is nontoxic for the cells and their axons, is resistant to histological fixation procedures, and allows quick observation in vivo and on dissection stained tissue. Local application of RITC to distinct retinal areas allows examination of the position of the corresponding stained fibers along the retinotectal pathway. Fibers which arise from the central temporal retina occupy deeper layers, whereas fibers from the peripheral temporal retina occupy more superficial layers in the optic tract and in the stratum opticum on the anterior tectum. The growth cones of early retinal fibers growing directly on the tectal surface show a different morphology to later growth cones growing on top of the stratum opticum on the tectum.     

Development of the transient ipsilateral retinotectal projection in the chick embryo: A numerical flourescence-microscopic analysis

The ipsilateral retinotectal projection in the developing chick was examined by using rhodamine-B-isothiocyanate (RITC)as an anterograde and retrograde vital marker for the retinal ganglion cells and their axons. Staining of the entire retina following intravitreal RITC injection between incubation days 3 and 16 revealed a small number of anterogradely labeled fibers in the optic tract and the anterior half of the optic tectum ipsilateral to the injection site. The total number of ipsilaterally projecting fibers was estimated to be about 2,000 on developmental day 9. The ipsilateral projection totally disappeared after day 15. The arrangement of fibers within the ipsilateral projection was examined by local anterograde RITC staining of localized retinal regions between days 9 and 10. The projection was retinotopically organized along the dorsoventral axis such that fibers of dorsal retinal origin projected on the ventral tectal half, whereas fibers of ventral retinal orgin projected on the dorsal tectal half. The localization of ipsilaterally projecting ganglion cell bodies was examined by retrograde RITC staining during days 9 and 15. Ganglion cells of all four quadrants of the central retina contributed to the production of the ipsilateral projection. The ipsilaterally growing retinotectal fibers did not represent collaterals of contralaterally projecting retinotectal axons. We assume that the tendency of early growing retinotectal axons to grow straight, as well as the ability of axonal growth cones to “sample” the environment, lead to a crossing of axons to the contralateral side. Ipsilateral projections would therefore represent “pathfinding errors.” Explanations for the elimination of the ipsilateral retinotectal projection are discussed.     

Development of the retinotectal system in normal quail embryos: cytoarchitectonic development and optic fiber innervation

The development of the optic tectum and the establishment of retinotectal projections were investigated in the quail embryo from day E2 to hatching day (E16) with Cresyl violet-thionine, silver staining and anterograde axonal tracing methods. Both tectal cytodifferentiation and retinotectal innervation occur according to a rostroventral-caudodorsal gradient. Radial migration of postmitotic neurons starts on day E4. At E14, the tectum is fully laminated. Optic fibers reach the tectum on day E5 and cover its surface on day E10. 'Golgi-like' staining of optic fibers with HRP injected in vitro on the surface of the tectum reveals that: growing fronts are formed exclusively by axons extending over the tectal surface; fibers penetrating the outer tectal layers are always observed behind the growing fronts; the penetrating fibers are either the tip of the optic axons or collateral branches; as they penetrate the tectum, optic fibers give off branches which may extend for long distances within their terminal domains; the optic fiber terminal arbors acquire their mature morphology by day E14. The temporal sequence of retinotectal development in the quail was compared to that already established for the chick, thus providing a basis for further investigation of the development of the retinotectal system in chimeric avian embryos obtained after xenoplastic transplantation of quail tectal primordia into the chick neural tube.     

Rules of order in the retinotectal fascicles of goldfish

Individual fascicles of retinal axons were labeled in the goldfish tectum with horseradish peroxidase (HRP). The contralateral retina was later processed for HRP histochemistry to mark the cells that had axons in the fascicles. Labeled cells were found in a partial half anulus in ventral hemiretina, centered on the optic disk. The distance of the partial anulus from the disk depended on which tectal fascicle had been labeled; the more rostrocentral the fascicle, the smaller was the annular radius. The angular subtense of the partial anulus with respect to the disk depended on where (along its tectal course) the fascicle had been labeled; the more rostral the label site, the longer was the angular subtense. These results were interpreted in the context of retinotectal growth, and it was inferred that the axons followed two rules: (1) grow in along the edge of the tectum and (2) exit and terminate in order, axons from temporal retina first, nasal retina last. These rules would produce a retinotopic projection in peripheral tectum, but they require that some of the terminals already in place must shift as the tectum grows.     

Thy-1 expression in the retinotectal system of the chick

Previous investigations into the occurrence of Thy-1 in the chick retina have not clearly defined when the antigen first appears and have not adequately described its expression during the relatively early phases of retinal ontogeny. We have investigated these issues, using improved immunohistochemical procedures and show that Thy-1 is associated with the retinal ganglion cells from the time they begin to differentiate by extending their axonal projections. In addition, we have found that its expression reflects the growth of the optic fibre layer and the elaboration of the ganglion cell dendritic processes into the inner plexiform layer. For the first time we describe the appearance and the developmental expression of Thy-1 in the chick tectum. We have found that Thy-1 is associated with retinal axons from the time of their arrival at the tectum and that its expression reflects the elaboration of the stratum opticum. Within the tectum proper Thy-1 appears first in 3 distinct layers all of which are plexiform in nature. By the time that tectal histogenesis is essentially complete the antigen is expressed by all the layers of the tectum. The implications of these findings are discussed in terms of the development of the individual tissues and with respect to the elaboration of the retinotectal pathway.     

Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order

The retinotectal projection is organized in a precise retinotopic manner. We find, though, that during development the growth and arborization of temporal retinal axons within the optic tectum of chick embryos is initially imprecise. Axonal targeting errors occur along the rostral-caudal and medial-lateral tectal axes, and arbors are formed at topographically inappropriate positions. Subsequent course corrections along both tectal axes and large-scale axonal remodeling lead to the retinotopic ordering of terminal arborizations characteristic of the mature projection. The trajectories and branching patterns of temporal retinal axons labeled with Dil or DiO were determined in whole mounts of retina and tectum from chicks ranging in age from embryonic day 9 to posthatching. Within the retina, labeled retinofugal axons travel in a compact bundle but do not maintain strict neighbor relations, as they course to the optic fissure. The axons enter the contralateral tectum at its rostral edge and grow caudally. Many extend well past their appropriate terminal zone within rostral tectum; a proportion of these later reverse their direction of growth. Many axons grow onto the tectum at incorrect positions along the medial-lateral tectal axis. Some correct this error in a directed manner by altering their trajectory or extending collateral branches at right angles. About 80% of the positional changes of this type are made in the direction appropriate to correct axon position, and thus are likely a response to tectal positional cues. After maturation of retinotopic order, about half of the axons that project to a mature terminal zone have made abrupt course corrections along one or both tectal axes, indicating that initially mistargeted axons can establish appropriately positioned arbors and survive. The development of temporal axons within the tectum is characterized by 3 phases: elongation, branch and arbor formation, and remodeling. After considerable rostrocaudal elongation, an axon typically develops numerous side branches and arbors, many at inappropriate locations. Most arbors are formed by side branches that develop as interstitial collaterals; few axons grow directly to their appropriate terminal zone and arborize. Aberrant arbors, and axons and axon segments that fail to form arbors in the appropriate terminal zone, are rapidly eliminated over about a 2 d period. Axon degeneration appears to play a role in this remodeling process.     

Axons added to the regenerated visual pathway of goldfish establish a normal fiber topography along the age-axis

Throughout a goldfish's life, new generations of ganglion cells are added on the retinal margin and their axons extend centrally to occupy predictable positions in the retinotectal pathway, adjacent to their predecessors and subjacent to the pia. The stacking of successive generations of axons defines the age-axis of the pathway. This study examined whether an ordered array of predecessor axons is a prerequisite for the patterned growth of new axons. One optic nerve was crushed intraorbitally and the fish was injected with 3H-thymidine to label the proliferating cells on the retinal margin. The ring of 3H-thymidine-labeled cells separated retina that was present at the time of nerve crush (inside the ring) from new retina added afterward (outside). After a period of 14-16 months postcrush, both tectal lobes received two punctate applications of horseradish peroxidase (HRP), one in the central and the other in peripheral tectum, to retrogradely label contralateral retinal ganglion cell bodies and their axons. The pattern of HRP labeling from the control tectum confirmed earlier work: axons on the central tectum had somata in the central retina, and axons on the peripheral tectum had somata in the peripheral retina. The labeled cells and axons were both in predictable patterns. The somata that were backfilled from applications to the center of the experimental tectum lay inside the radioactive ring and had therefore regenerated their axons. The patterns of their labeled axons in the optic pathway and of their somata in the retina were typical of the regenerated condition as described in earlier studies. The somata backfilled from the periphery of the experimental tectum were outside the radioactive ring and had been added after the optic nerve crush. The patterns of their labeled axons and somata were comparable to the normal pattern. These observations indicate that new axons do not depend on an ordered array of predecessors to reestablish normal order along the age-axis of the pathway.     

Growth cone-target interactions in the frog retinotectal pathway

The growth cones of retinal ganglion cell axons were studied in the optic tract and tectum with horseradish peroxidase (HRP) histochemistry and electron microscopy. The ganglion cell growth cones has many morphological features similar to those described in vitro and in other in vivo systems. However, we found that some processes formed highly differentiated terminal arborizations, while retaining growth cones on many of their branches. In addition, ultrastructural examination of the tectal neuropil revealed that many ganglion cell axonal processes had characteristics of both growth cones and presynaptic endings. These findings are discussed in the context of the hypothesis of shifting connections and the evidence that retinotectal map formation involves several mechanisms, including a process that depends on the action potential activity in the optic fibers.     

Evidence for the stability of positional markers in the goldfish tectum

Positional markers in the tectum, which are thought to guide growing axons to their target sites, have been proposed to be induced by axons, to be only transiently associated with the tectal cells, and then lost after long-term denervation periods (Schmidt: J. Comp. Neurol. 177:279-300, '78). To further investigate this concept, retinal axons were induced to regenerate into ipsilateral tecta which had been deprived of their retinal afferents for shorter (0-4 months) and longer periods (4-8 months). The paths of HRP-labeled regenerating axons of known retinal origin were traced and used as an operational test to decide whether the axons might navigate under the influence of positional markers. Two different kinds of experiments were performed: 1. The axons from a subpopulation of all ganglion cells in the retina were labeled by applying a small crystal of HRP at defined retinal regions. Independent of the denervation period of the tectum, the labeled regenerating axons traveled in abnormal but nonrandom routes. In early regeneration stages, axons exhibited signs of exploratory growth. They extended branches equipped with growth cones and filopodia into various regions of the tectum. In late regeneration stages, the axons lost these branches, exhibited U-turns and bends, and ended in terminal arbors in the retinotopic target region. These findings suggest that the axons travel under the influence of tectal positional markers and that these markers are not transient. 2. Axons from a surgically created temporal hemiretina were labeled by application of HRP to the optic nerve to test whether the temporal axons might expand into the caudal tectum in long-term-denervated tecta. The HRP-labeled axons coursed over rostral and midtectal regions. Instead of invading the caudal tectum they bent and terminated in the rostral tectal half. These results add further support for the conclusion that the path of regenerating retinal axons is governed by long-lasting positional markers.     

Cross-species collapse activity of polarized radial glia on retinal ganglion cell axons

Inhibition of incorrect axonal outgrowth has been shown to be a crucial guidance mechanism during the development of the nervous system. Within the visual system of chick and rat, extension of retinal ganglion cell axons is essentially restricted to distinct layers of the retina and distinct brain regions such as the tectum opticum. In addition, populations of ganglion cells from defined retina locations project topographically to defined tectal areas, their growth possibly being inhibited by radial glia in incorrect tectal regions. In the current study, we aimed to analyse potential inhibitory activity of retinal glia during outgrowth of ganglion cell axons of embryonic chick and rat. The response of ganglion cell axons originating from different retina locations when exposed to purified retinal radial glia cell membranes were monitored in collapse assays by time lapse video recording. The interaction of axons growing on purified glial somata or glial endfeet was analysed in outgrowth assays. Our results indicate that (1) nasal and temporal chick growth cones are equally induced to collapse by cell membranes from retinal radial glia: 75% nasal and 72% temporal. (2) The collapse inducing component of radial glia can be inactivated by defined heat treatment, reducing collapsing activity to 6% nasal and 5% temporal. (3) Rat growth cones respond in a similar way to chick radial glia. (4) Rat axons grow perfectly on endfeet but not on somata of radial glia of the chick. In summary, the data suggest that radial glia are functionally polarized with permissive endfeet and inhibitory somata based on heat-labile proteins. Glia polarization is likely to inhibit aberrant growth of ganglion cell axons into outer retina layers. However, retinal radial glia are unlikely to participate in preordering axons within the retina and therefore do not affect the topographic projection. Finally, the inhibitory function of radial glia is conserved between birds and mammals and represents possibly a fundamental mechanism for structuring the central nervous system. GLIA 25:143–153, 1999. © 1999 Wiley-Liss, Inc.     

Topography of the retinal projection to the superficial pretectal parvicellular nucleus of goldfish: a cobaltous-lysine study

The retinal projection to the superficial pretectal parvicellular nucleus (SPp) of goldfish was examined by filling select groups of optic axons with cobaltous-lysine. The tracer was applied intraocularly to peripheral retinal slits in some fish. In other fish, it was applied to optic axons from an intact hemiretina after one-half of the retina was ablated and the corresponding optic axons had degenerated. The results indicated that SPp is a folded structure, having a dorsal surface innervated by axons from temporal retinal ganglion cells and a ventral surface innervated by axons from nasal retinal ganglion cells. Peripheral retina innervates the anterodorsal and anteroventral edges of SPp, while central retina innervates the posterior genu. Dorsal retina innervates lateral SPp and ventral retina innervates medial SPp. Thus, although SPp is a folded nucleus, the topography of the retino-SPp projection is similar to the topography of the retinotectal projection. That is, the relative position of optic axons within SPp mirrors the retinal location of the ganglion cells that project to SPp. Retino-SPp axons occupy the center of the main optic tract before it divides into the two optic brachia. These axons are topographically arranged, with temporal retino-SPp axons being flanked on both sides by nasal retino-SPp axons. Retino-SPp axons arborize within SPp and then continue to enter the superficial tectal retino-recipient lamina. Thus, these axons innervate both SPp and the optic tectum. These findings are discussed with respect to chemospecific and morphogenetic views of visual system topography.     

Trajectories of regenerating retinal axons in the goldfish tectum: I. A comparison of normal and regenerated axons at late regeneration stages

To visualize and compare the intratectal path of normal and regenerated retinal axons, HRP was applied to localized sites in the dorsotemporal and dorsonasal retina in normal goldfish and in goldfish at 3-12 months after optic nerve section. The anterogradely labeled axons were traced in tectal whole mounts. In normal animals the axons were confined to the appropriate ventral hemitectum. Therein they ran in very orderly routes (Stuermer and Easter: J. Neurosci. 4:1045-1051, '84) and terminated in regions retinotopic to the labeled ganglion cells in the retina. The terminal arbors of dorsotemporal axons resided in the ventrorostral tectum and those of dorsonasal axons in the ventrocaudal tectum. In regenerating animals the terminal arbors also resided at retinotopic regions, where they sometimes formed two separate clusters. In contrast to normal axons, the regenerating ones traveled in abnormal routes through the appropriate and inappropriate hemitectum. From various ectopic positions, they underwent course corrections to redirect their routes toward the retinotopic target region. In their approach toward their target sites, dorsotemporal and dorsonasal axons behaved differently in that the vast majority of dorsotemporal axons coursed over the more rostral tectum whereas dorsonasal axons progressed into the caudal tectal half. This differential behavior of regenerating dorsonasal and dorsotemporal axons was substantiated by a quantitative evaluation of axon numbers and orientations.     

A comparison of the normal and regenerated retinotectal pathways of goldfish

This is a light and electron microscopic study of the retinotectal pathway: intact and after regeneration of the optic nerve. The spatiotemporal pattern of axonal outgrowth and termination was studied with the methods of proline autoradiography, horseradish peroxidase (HRP) labeling, and fiber degeneration. The spatial order of optic fibers in the normal and regenerated pathways was assessed by labeling small groups intraretinally with HRP and then tracing them to the tectum. The labeled fibers occupied a greater fraction of the cross section of the regenerated than the normal optic tract. At the brachial bifurcation, roughly 20% of the regenerated fibers chose the incorrect brachium vs. less than 1% of the normals. In tectum, the regenerated optic fibers reestablished fascicles in stratum opticum, but they were less orderly than in the normals. The retinal origins of the fibers in the fascicles were established by labeling individual fascicles with HRP and then, following retrograde transport, finding labeled ganglion cells in whole-mounted retinas. Labeled cells were more widely scattered over the previously axotomized retinas than over the normal ones. A similar result was obtained when HRP was applied in the tectal synaptic layer. All of these results indicate that the pathway of the regenerated optic fibers is less well ordered than the intact pathway. Both autoradiography and HRP showed that the regenerating optic fibers invaded the tectum from the rostral end, and advanced from rostral to caudal and from peripheral to central tectum, along a front roughly perpendicular to the tectal fascicles. Synapses of retinal origin were noted electron microscopically in the tectum at the same sites where autoradiography indicated that the fibers had arrived. No retinal terminals were seen where grain densities were at background levels. Fiber ingrowth and synaptogenesis apparently occurred simultaneously. The synapses were initially smaller and sparser than in normals, but were in the normal tectal strata and contacted the same classes of post synaptic elements as in normals.     

Developmental expression of cannabinoid receptors in the chick retinotectal system

The cannabinoid system has been suggested to participate in processes such as antinociception, cognition, motor control, and, more recently, development of the nervous system. This study describes the expression of the CB1 cannabinoid receptor in the developing chick retina and optic tectum by means of conventional immunoperoxidase protocols. CB1 immunoreactivity was initially detected around the embryonic day 4 (E4) in both the retina and tectum. In the retina, CB1 immunoreactivity was first observed in presumptive ganglion cells and, subsequently, in the inner plexiform layer and two populations of neurons of the inner nuclear layer. The post-hatched chick exhibited a pattern of staining that included four sublayers of the inner plexiform layer, a few stained cells in the ganglion cell layer, and labeled neurons both in the inner and central parts of the inner nuclear layer. The latter two types of neurons appear to be amacrine and bipolar cells, respectively. In the tectum, CB1 first appeared in its most superficial zone and later in several tectal laminae, including a white matter layer (stratum album centrale; Cajal's layer 14). There was a remarkable and transient increase of labeling at E10, followed by a continuous reduction of staining until E18. In the post-hatched chick, tectal staining was mostly confined to layers 2-3 and 5-6. Stained perikarya were seldom observed in the tectum at any stage. These data are in agreement with a possible developmental function of CB1, as it is expressed several days before synaptogenesis ensues and exhibits transient expression in the optic tectum.     

Evidence for centripetally shifting terminals on the tectum of postmetamorphic Rana pipiens

In larval frogs the retina and tectum grow in topologically dissimilar patterns: new cells are added as peripheral annuli in the retina and as caudal crescents in the tectum. Retinotopy is maintained by the continual caudalward shifting of the terminals of the optic axons. After metamorphosis the pattern of growth changes. The retina continues to add new ganglion cells peripherally, but there is no neurogenesis in the tectum. To maintain retinotopy in postmetamorphic frogs, the terminals of the optic axons must continually shift toward the central tectum. We tested the proposal of centripetally shifting axons by making punctate injections of horseradish peroxidase (HRP) in the tectum of adult Rana pipiens and observing the patterns of filled cells in the contralateral retina, as was done in the goldfish (Easter and Stuermer, '84). Punctate applications of HRP in the tectum should be taken up: 1) by fascicles, and label a partial anulus of cells, 2) by terminals, and label a cluster of cells in the corresponding retinotopic site, and 3) by the extrafascicular axonal segments, and label a band of cells connecting the partial annulus to the cluster. If the terminals have shifted centripetally, the band of cells labeled through their extrafascicular segments should have a spoke-like orientation, with the center of the retina as the hub. As the tectal site moves from rostral to caudal, this band of cells should move, pendulum-like, from temporal to nasal retina. In general, the patterns of HRP-filled retinal cells we observed were consistent with our predictions. In addition, HRP taken up by the oldest (rostral) tectal axons produced more complex patterns of filled cells that indicated that these axons had shifted both caudally before metamorphosis and centripetally after.     

Plasticity in the chick ventral lateral geniculate nucleus

The chick ventral lateral geniculate nucleus (GLv) receives topographically corresponding projections from the retina and optic tectum. Tectal lesions produced on the day of hatching removed the tectogeniculate input to the GLv region corresponding to the tectal lesion and also severed some retinotectal axons. Following a survival period of 3 to 10 weeks, a patch of augmented retinogeniculate projection was noted in the GLv segment that corresponds topographically to the damaged area of the tectum. Changing the site of the tectal lesion led to changes in the locus of heavy retinal projection to the GLv predictable from topographic maps. Nuclei which received retinal but not tectal projections did not appear to have regions of augmented retinal termination nor did nuclei which received tectal but not retinal innervation. It is unlikely that the increased retinogeniculate termination is due to rerouting of growing retinotectal axons since the chick retinofugal pathway is well established by the time of hatching. Furthermore, there was no evidence of a projection from the ipsilateral eye to the affected GLv. On the basis of these light microscopic studies, it would appear that retinogeniculate terminals have sprouted in the GLv and that competition for terminal space, conservation of terminal space, proximity, and perhaps other factors are necessary for the augmented projection to occur.     

Recovery of regenerating goldfish retinal ganglion cells is slowed in the absence of the topographically correct synaptic target

When the axons of goldfish retinal ganglion cells are severed the cell bodies undergo a series of changes as the axons regenerate. These changes begin to reverse when the axons start to innervate the tectum and by 3 months after the lesion the cell bodies have nearly returned to normal. When the axons projecting to the caudal tectum were severed by a mediolateral transection of the tectum, only retinal ganglion cells in the nasal portion of the contralateral retina underwent the changes normally associated with regeneration, followed by a speedy return to normal. Because the injured fibers probably did not fully retract from the tectum, these results indicated that: (1) the complete removal of the axons from the tectal milieu was not essential for initiating the cell body changes, and (2) close proximity to the target sites would speed the recovery of the cells. When the caudal portion of the tectum was ablated the retinal ganglion cells of the nasal retina remained enlarged significantly longer than after tectal transection. During the time the cells remained enlarged the electrophysiological projection onto the remaining rostral part of the tectum revealed no significant 'compression' of the visual field. Compression of the visual field onto the rostral portion of the tectum can be accelerated if the caudal tectal ablation is accompanied by an optic nerve crush. However, under this condition the recovery of ganglion cells in the nasal retina was significantly slower than the recovery of cells in the temporal retina. This may reflect an element of topographical specificity in the regulation of the recovery of the cell body from axonal injury.     

Target regulation of synaptic number in the expanded retinotectal projection of goldfish: the half-retinal preparation

The nature of the expansion of the visual field projection was studied in goldfish in which size disparities were created between the retina and the tectum. After removal of one-half of the retina, the remaining retinal ganglion cells expand their projections so that the entire contralateral optic tectum is encompassed (Schmidt et al.1978). We wished to determine whether this expansion is accompanied by increased arborization including proliferation of synaptic terminals by the spared retinal ganglion axons or whether field expansion is accomplished by increased arborization without changes in synaptic number. Portions of the retina were ablated and the animals were allowed to survive for at least 5 months, the time at which expansion can be demonstrated, before sacrifice. We mapped retinotectal projections to determine the extent of the expanded visual fields and used stereological and morphometric analyses of synaptic contacts in the retinal target lamina, the stratum fibrosum et griseum superficialis (SFGS), in the optic tectum to estimate synaptic number. Numbers of synaptic terminals in the SFGS contralateral to the lesioned retina were not different from numbers in the comparable portion of control tecta. These observations indicate that the surviving retinal axons increased the number of synaptic contacts on tectal target cells in response to removal of other retinal ganglion cells.