Axon growth in embryonic chick and quail retinal whole mounts in vitro

Whole retinae of 4- to 10-day-old chick and quail embryos were spread on membrane filters and kept in culture for up to 4 days. Axon growth during culture was demonstrated by silver staining, anterograde labeling of fibers with RITC, time-lapse recording, and SEM. Fiber growth was observed in specimens from chick embryos up to 7 days old, with a growth maximum at E6 and from quail embryos up to E6 with the maximum at E5. Newly growing axons followed the optic fiber pattern already existing and, like axons in vivo, grew predominantly toward the optic fissure. Directional and orientational adaptation of newly growing axons to the preexisting fibers increased with the donor age. Retinae from donors up to E5 in chick and up to E4 in quail showed a high proportion of axons which crossed the optic fissure during the culture period and invaded the opposite retinal fiber layer. These fibers showed a correct radial orientation while growing in the opposite direction to normal. Likewise, in cultures from these young donors some fibers grew out initially in the diametrically opposite direction to normal toward the tissue periphery. Since all of the wrongly directed axons grew at the same rate as normal and adapted correctly to the already formed axon pattern, this suggests independent signals for the direction and orientation of growing fibers. Treatment of mounted retinae with collagenase or trypsin removed the vitreal retinal surface, leaving the existing axon pattern intact. Subsequently, new axons grew profusely in culture, but lost both their orientational and directional characteristics.     

A developmental and ultrastructural study of the optic chiasma in Xenopus

The structure of the optic chiasma in Xenopus tadpoles has been investigated by light and electron microscopy. Where the optic nerve approaches the chiasma, a tongue of cells protrudes from the periventricular cell mass into the dorsal part of the nerve. Glial processes from this tongue of cells ensheath fascicles of optic axons as they enter the brain. Coincident with this partitioning, the annular arrangement of axons in the optic nerve changes to the laminar organization of the optic tract. Beyond the site of this rearrangement, all newly growing axons accumulate in the ventral-most part of the nerve and pass into the region between the periventricular cells and pia which we have called the 'bridge'. This region is characterized by a loose meshwork of glial cell processes, intercellular spaces and the presence of both optic and nonoptic axons. In the bridge, putative growth cones of retinal ganglion cell axons are found in the intercellular spaces in contact with both the glia and with other axons. The newly growing axons from each eye cross in the bridge at the midline and pass into the superficial layers of the contralateral optic tracts. As the system continues to grow, previous generations of axon, which initially crossed in the existing bridge, are displaced dorsally and caudally, forming the deeper layers of the chiasma. At their point of crossing in the deeper layers, these fascicles of axons from each eye interweave in an intimate fashion. There is no glial segregation of the older axons as they interweave within the chiasma.     

Axonal pathfinding in organ-cultured embryonic avian retinae

Eye cups from stage 14-28 (E2 to E5) chick and quail embryos consisting of neural retina, lens, and vitreous body were cultured for 1 or 2 days. These eyes expanded by proliferation of the retinal cells and the surface areas of the retinae increased several-fold. The area covered by ganglion cells and axons also expanded in vitro. [3H]Thymidine labeling showed extensive proliferation of the neuroepithelial cells including the formation of new ganglion cells. Culturing eyes from embryos before stage 17 results, as in vivo, in the generation of the first ganglion cells of the retina, but unlike in the in vivo situation, the outgrowing axons always formed a random fiber net in the central portion of the retina. A defined axonal pattern identical to the in vivo developed only in specimens from embryos of stage 17 and older. Some aberrant axons, however, were also observed at the retinal periphery in specimens from embryos of more advanced stages (20-24), but only during the second day of culturing. Axons in retinae from embryos of stages 23 to 26 heading toward the optic fissure often crossed the fissure and, in contrast to the situation in vivo, invaded the opposite retinal side. These axons of wrong polarity followed the pathways of axons growing centripetally but in reverse direction. This suggests that the polarity of growing nerve fibers and their course are determined by different factors. Culturing the eyes of embryos from stages 20 to 25 in the presence of antibodies showed that the antibodies penetrated the entire retina with 6 hr. Neither anti-N-CAM nor the T-61 antibody--both recognizing membrane proteins of retinal cells--affected the growth of the eyes in vitro. The development of the axonal pattern in vitro was not affected by incubation with N-CAM-antibodies at concentrations up to 500 micron/ml, whereas the T-61 antibody which is known to block neurite extention in vitro (S. Henke-Fahle, W. Reckhaus, and R. Babiel (l984). "Developmental Neuroscience: Physiological, Pharmacological, and Clinical Aspects," pp. 393-398. Elsevier, Amsterdam/New York) showed inhibition of axonal growth in retina cultures at 50 micron/ml. These results indicate that the eye cultures can be used as a test system for antibodies against antigens which could be involved in axon extension and neurite pathfinding in situ.     

Homotopic and heterotopic transplantations of quail tectal primordia in chick embryos: Organization of the retinotectal projections in the chimeric embryos

To study the adaptative capabilities of the retinotectal system in birds, the primordium of one optic tectum from 12-somite embryos of Japanese quail was transplanted either homotopically, to replace the ablated same primordium, or heterotopically, to replace the ablated dorsal diencephalon in White Leghorn chick embryos of the same stage. The quail nucleolar marker was used to recognize the transplants. The cytoarchitecture of the tecta and the retinal projections from the eye contralateral to the graft were studied on the 17th or 18th day of incubation in the chimeric embryos by autoradiographic or horseradish peroxidase tracing methods. Morphometric analysis was applied to evaluate the percentage of the tectal surface receiving optic projections. It was observed that: (i) quail mesencephalic alar plate can develop a fully laminated optic tectum even when transplanted heterotopically; (ii) retinal ganglion cells from the chick not only recognize the tectal neurons of the quail as their specific targets in homotopic grafts, but the optic fibers deviate to innervate the heterotopically grafted tectum; (iii) in the presence of a graft, the chick retina is unable to innervate a tectal surface of similar or larger size than that of the control tectum; (iv) tectal regions devoid of optic projections, whether formed by donor or by host cells, always present an atrophic lamination; (v) the diencephalic supernumerary optic tectum competes with and prevails over the host tectum as a target for optic fiber terminals.     

Immunohistochemical localization of laminin,neural cell adhesion molecule,collagen type IV and T-61 antigen in the embryonic retina of the Japanese quail by in vivo injection of antibodies

Summary Antibodies against laminin (LN), fibronectin (FN), collagen type IV (Col IV), neural cell adhesion molecule (N-CAM), T-61 antigen, actin, tubulin and neurofilament protein were injected into the eyes of quail embryos (Coturnix coturnix japonica) of different ages. Twenty h after injection, the heads of the embryos were fixed and the antibodies visualized in sections with the use of fluorescein-isothiocyanate (FITC) or peroxidase-labeled second antibodies by light- and electron microscopy. Antibodies against cell surface molecules, such as N-CAM, LN, Col IV and T 61, labeled matrix and membrane components of the retinal cells in different antigen-specific patterns. Antibodies against intracellular antigens, such as actin, tubulin and neurofilament protein labeled nonspecifically the vitreous body and the inner basal lamina of the retina, but resulted in only a very weak and diffuse labeling of retinal cells. N-CAM was detected in high concentration in the optic fiber layer on the surface of axons and on the membranes of all retinal cells. Col IV, LN and T 61 antigen were found predominantly in the optic fiber layer. LN and Col IV were located on the surface of axons and the endfeet of ventricular (neuroepithelial) cells in a patchy distribution. The T-61 antigen was found in early stages in the cell-free space of the optic fiber layer, on the surface of ventricular cells and axons, and at later stages also in high-density patches between nerve fibers. The distribution of LN and T-61 antigen together with data from in vitro experiments suggests a crucial role of these proteins in axon extension in the avian retina during early development of the optic fiber layer.     

Demonstration of centrifugal fibers in the optic chiasma and optic nerves in the rat after lesions of the suprachiasmatic nucleus

After an unilateral destruction of the suprachiasmatic nucleus and with the use of several silver impregnation techniques, degenerating centrifugal fibers were found in both optic nerves. Centrifugal fibers to the retina originate from three different regions of the nucleus and their position in the chiasma are different. In large majority the degenerating fibers were located at the periphery of the optic nerve and were more frequent on the contralateral than on the ipsilateral side to the destroyed suprachiasmatic nucleus. The possibility that our experimental procedure demonstrates the existence of fibers originating not only in the suprachiasmatic nucleus but also in other structures whose efferent fibers pass at this level, is discussed.     

Fibre organization and reorganization in the retinotectal projection of Xenopus

This study concerns the retinotopic organization of the ganglion cell fibres in the visual system of the frog Xenopus laevis. HRP was used to trace the pathways taken by fibres from discrete retinal positions as they pass from the retina, along the optic nerve and into the chiasma. The ganglion cell fibres in the retina are arranged in fascicles which correspond with their circumferential positions of origin. Within the fascicles the fibres show little age-related layering and do not have a strict radial organization. As the fascicles of fibres pass into the optic nerve head there is some exchange of position resulting in some loss of the retinal circumferential organization. The poor radial organization of the fibres in the retinal fascicles persists as the fibres pass through the intraocular part of the nerve. At a position just behind the eye there is a major fibre reorganization in which fibres arising from cells of increasingly peripheral retinal locations are found to have passed into increasingly peripheral positions in the nerve. Thus, fibres from peripheral-most retina are located at the nerve perimeter, whilst fibres from central retina are located in the nerve core. It is at this point that the radial, chronotopic, ordering of the ganglion cell axons, found throughout the rest of the optic pathway, is established. This annular organization persists along the length of the nerve until a position just before the nerve enters the brain. Here, fibres from each annulus move to form layers as they pass into the optic chiasma. This change in the radial organization appears to be related to the pathway followed by all newly growing fibres, in the most superficial part of the optic tract, adjacent to the pia. Just behind the eye, where fibres become radially ordered, the circumferential organization of the projection is largely lost. Fibres from every circumferential retinal position, which are of similar radial position, are distributed within the same annulus of the nerve. At the nerve-chiasma junction where each annulus forms a single layer as it enters the optic tract, there is a further mixing of fibres from all circumferential positions. However, as the fibres pass through the chiasma some active pathway selection occurs, generating the circumferential organization of the fibres in the optic tract. Additional observations of the organization of fibres in the optic nerve of Rana pipiens confirm previous reports of a dual representation of fibres within the nerve. The difference in the organization of fibres in the optic nerve of Xenopus and Rana pipiens is discussed.     

Dolphin peripheral visual pathway in chronic unilateral ocular atrophy: Complete decussation apparent

Components of the peripheral visual pathway were examined in two bottlenose dolphins, Tursiops truncatus, each with unilateral ocular degeneration and scarring of 3 or more years' duration. In both animals, the optic nerve associated with the blind eye right eye in Tg419 and left eye in Tt038 had a translucent, gel-like appearance upon gross examination. This translucency was also evident in the optic tract contralateral to the affected eye. In Tg419, myelinated axons of varying diameters were apparent in the left optic nerve, whereas the right optic nerve, serving the blind eye, appeared to be devoid of axons. In Tt038, myelinated axons were associated with the right optic nerve (serving the functional eye) and left optic tract but were essentially absent in the left optic nerve and right optic tract. Examined by light microscopy in serial horizontal sections, the optic chiasm of Tt038 was arranged along its central plane in segregated, alternating pathways for the decussation of right and left optic nerve fibers. Ventral to this plane, the chiasm was comprised of fibers from the left optic nerve, whereas dorsal to the central plane, fibers derived from the right optic nerve. Because of this architectural arrangement, the right and left optic nerves grossly appeared to overlap as they crossed the optic chiasm with the right optic nerve coursing dorsally to the left optic nerve. At the light and electron microscopic levels, the optic nerves and tracts lacking axons were well vascularized and dominated by glial cell bodies and glial processes, an expression of the marked glial scarring associated with postinjury axonal degeneration. The apparent absence of axons in one of the optic tract pairs (right in Tt038 and left in Tg419) supports the concept of complete decussation of right and left optic nerve fibers at the optic chiasm in the bottlenose dolphin. © 1994 Wiley-Liss, Inc.     

Dynamic aspects of retinotectal map formation revealed by a vital-dye fiber-tracing technique

In the visual system of Xenopus laevis, the axons from the retinal ganglion cells of the eye form a topographic projection onto the optic tectum. Many studies have focused on revealing the mechanisms responsible for this precise and regular projection pattern. In contrast to the static view of the system that one might expect from examining the regularity of the projection, recent work on its regeneration and its changes during larval development indicate that part of the patterning process involves the dynamic behavior of optic fibers. Typically, anatomical and electrophysiological techniques have been used to obtain static views of the developing retinotectal projection which then must be complied to provide a glimpse of any dynamic behavior. Here we report on experiments using a newly developed fiber tracing technique to directly follow the emergence of topography in the developing retinotectal projection. Defined halves of the developing eyebud were labeled with a vital fluorescent dye which fills the growing axons, and the projection of the labeled cells was followed for up to 2 weeks in individual animals. The experiments confirm that dorsal and ventral optic nerve fibers sort out into an ordered projection early in development. In contrast, nasal and temporal fibers initially overlap, and the same sets of prelabeled fibers then sort out into the adult topography over a period of days.     

Retinohypothalamic projection in the mouse: Electron microscopic and iontophoretic investigations of hypothalamic and optic centers

The problem of the direct retinohypothalamic projection in mammals (Moore, 1973) was reinvestigated in the laboratory mouse by electron microscopy and cobalt chloride-iontophoresis. The time-course of the axonal degeneration in the suprachiasmatic nucleus was studied 3, 6 and 12 h, 1, 2, 4, 6, 9 and 12 days after unilateral retinectomy. Specificity of the degenerative changes was controlled by investigation of the superficial layers of the superior colliculus. The ratio of crossed to uncrossed optic fibers could could be determined by counting degenerating structures (axons and terminals) in the optic chiasma and the ipsilateral and contralateral areas of the optic tract, the suprachiasmatic nucleus, and the superior colliculus. The number of degenerating axons in the suprachiasmatic nucleus showed a maximum one day after unilateral retinectomy and was, at all stages studied, two to three times higher in the contralateral than in the ipsilateral nuclear area. In the optic tract and in the superior colliculus the number of degenerating profiles was three times higher in the contralateral than in the ipsilateral area. Retinohypothalamic connections and crossing pattern of retinal fibers were studied light microscopically using impregnation with cobalt sulfide in whole mounts of brains. Most of the optic fibers in the laboratory mouse are crossed crossed (70-80%). A bundle of predominantly crossed optic fibers runs to the suprachiasmatic nucleus.     

The polysialic acid moiety of the neural cell adhesion molecule is involved in intraretinal guidance of retinal ganglion cell axons

We have characterized the antigen recognized by mab10, a monoclonal antibody that has been shown to modify outgrowth of thalamic and cortical axons in vitro, and investigated the influence of this antibody on axonal growth in the chicken retina in vivo. Immunopurification, peptide sequencing, and biochemical characterization proved the epitope recognized by mab10 to be polysialic acid (PSA), associated with the neural cell adhesion molecule (NCAM). Intravitreal injections of antibody-secreting hybridoma cells were combined with whole-mount studies using the fluorescent tracer 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI). Pathfinding at the optic fissure was affected, resulting in a failure of axons to exit into the nerve. Misprojections also occurred in more peripheral areas of the retina; however, axons eventually oriented toward the center. Similar projection errors were observed after enzymatic removal of PSA by injecting endoneuraminidase N (endo N). Quantitative measurements of the optic nerve diameter as well as the width of the optic fiber layer confirmed that many axons failed to leave the retina and grew back in the optic fiber layer of the retina. Our findings suggest that NCAM-linked PSA is involved in guiding ganglion cell axons in the retina and at the optic fissure.     

The relationship of acetylcholinesterase activity to optic fiber projections in the tiger salamander Ambystoma tigrinum

In the tiger salamnder the distribution of optic fibers, as revealed by the Fink-Heimer method, is compared with the localization of acetylcholinesterase, as revealed by histochemical methods. AChE activity coincides with optic nerve axons in the optic fiber layer of the retina, in the optic nerve, in the optic tracts and in every optic projection. Reginons of optic fiber terminals show heavier activity than optic fibers of passage. Comparison with other vertebrates is also made.     

Studies on induction of polydactyly in rats with cytosine arabinoside.

The development of the retino-tectal projection in Rana pipiens has been studied by the intraocular injection of small amounts of [³H]proline at late embryonic and at several larval stages. After survival periods varying from 1–24 hr the distribution of the radioactively labeled proteins in the axons of the retinal ganglion cells was studied autoradiographically. It is evident from the appearance of labeled proteins in the optic nerve and chiasm at late embryonic and early larval stages that there is a rapid phase of axonal transport at these stages and that some fraction of the materials transported in this phase are distributed to the tips of the growing axons.The first retinal fibers reach the contralateral optic tectum at embryonic Stage 22; at this stage they are confined to the rostrolateral portion of the tectum where the first tectal neurons are generated. At successively later stages the fibers appear to grow across the surface of the tectum in a general rostrolateral to caudomedial direction, reaching the dorsal part of the mid-tectum at larval Stage II and the lateral part of its caudal third by Stage V. However, it is not until relatively late larval stages (XVIII) that the fibers reach the caudomedial region of the tectum, and it is only at the time of metamorphosis (Stage XXV) that the retinal projection appears to cover the entire tectum.     

Retina mediators in fresh-water mollusc Lymnaeae stagnalis

Retrograde staining of the Lymnaeae stagnalis retina with neurobiotin has shown that most photoreceptor cells send axons to optic nerve without intermediate contacts. A part of these photoreceptors have immunireactivity to glutamate that possibly provides synaptic transmission of visual signal to central neurons. Other photoreceptors stained through optic nerve seem to have different transmitter systems. In some retina cell, but not in optic nerve fibers, immunoreactivity to pigment-dispersing hormone has been revealed. In tissues surrounding the eye cup numerous serotonin-containing fibers are present, a part of them penetrating the retina basal layer. Some of them belong to central neurons responsible for efferent innervation of the pond snail eye. It is suggested that the serotoninergic innervation as well as the cell containing the pigment-dispersing hormone are included in the mechanism of regulation of light sensitivity of the mollusc eye.     

A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue

Carbocyanine dyes, fluorescent lipophilic substances used for optical recordings of membrane voltage and for studies of membrane fluidity, have recently been shown to provide intense and long-lasting staining of neurones in vivo and in vitro (Schwartz & Agranoff, 1981; Honig & Hume, 1985, 1986; Catsicas, Thanos & Clarke, 1986; Landmesser & Honig, 1986; Thanos & Bonhoeffer, 1987). We report here that two of these dyes, diI (1,1',dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate) and diO (3,3'-dioctadecyloxacarbocyanine perchlorate), can also label neurones in embryonic mouse and chicken brain tissue that has been previously fixed in aldehyde fixatives. Neuronal processes and perikarya can be labelled along considerable distances in both anterograde and retrograde directions. The staining of processes and cells, including their finest extensions is smooth and clear, rivalling intracellular injections of HRP or Lucifer Yellow. The appearance and time course of progression of the staining along axons suggest that the staining in fixed tissue occurs due to a process of diffusion of dyes along the plasma membranes of cells. This technique has allowed us to study the first stages in the development of optic fibres in mouse embryos, especially at the optic chiasm. The early retinal projection (E13-E13 1/2) is mainly crossed, but some optic fibres grow to the ipsilateral side of the brain at the outset. Retrogradely labelled ganglion cells from the dorsocentral area of the retina participate in the formation of both the ipsilateral and the contralateral projection. Thus, at early stages, crossed and uncrossed projections arise from identical subregions of the retina and the partition of the retina with respect to the laterality of its projection arises later.     

Up-regulation of protein kinase C in regenerating optic nerve fibers of goldfish: Immunohistochemistry and kinase activity assay

Protein kinase C (PKC) activation has been associated with synaptic plasticity in many projections, and manipulating PKC in the retinotectal projection strongly affects the activity-driven sharpening of the retinotopic map. This study examined levels of PKC in the regenerating retinotectal projection via immunostaining and assay of activity. A polyclonal antibody to the conserved C2 (Ca²⁺ binding) domain of classical PKC isozymes (anti-panPKC) recognized a single band at 79–80 kD on Western blots of goldfish brain. It stained one class of retinal bipolar cells and the ganglion cells in normal retina, as shown previously. Strong staining was not present in the optic fiber layer of retina or in optic nerve, optic tract, or terminal zone in tectum, with the exception of a single fascicle of optic nerve fibers that by their location and by L1 (E587) staining were identified as those arising from newly added ganglion cells at the retinal margin. Normal tectal sections showed dark staining of a subclass of type XIV neuron with somas at the top of the periventricular layer and an apical dendrite ascending to stratum opticum. In regenerating retina, swollen ganglion cells stained darkly and stained axons were seen in the optic fiber layer. In regenerating optic nerve (2–11 weeks postcrush), all fascicles of optic fibers stained darkly for both PKC and L1(E587). At 5 weeks postcrush, PKC staining could also be seen in the medial and lateral optic tracts and stratum opticum at the front half of the tectum and very lightly over the terminal zones. PKC activity was measured in homogenized tissues dissected from a series of fish with unilateral nerve crush from 1 to 5 weeks previously. Activity levels stimulated by phorbols and Ca²⁺ were measured by phosphorylation of a specific peptide and referred to levels measured in the opposite control side. Regeneration did not increase overall PKC activity in retina or tectum, but in optic nerve there was an 80% rise after the first week. The increased activity verifies that the increased staining in nerve represented an up-regulation of functional PKC during nerve regeneration. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 315–324, 1998     

Mutations affecting retinotectal axonal pathfinding in Medaka, Oryzias latipes

We screened for mutations affecting retinotectal axonal projection in Medaka, Oryzias latipes. In wild-type Medaka embryos, all the axons of retinal ganglion cells (RGCs) project to the contralateral tectum, such that the topological relationship of the retinal field is maintained. We labeled RGC axons using DiI/DiO at the nasodorsal and temporoventral positions of the retina, and screened for mutations affecting the pattern of stereotypic projections to the tectum. By screening 184 mutagenized haploid genomes, seven mutations in five genes causing defects in axonal pathfinding were identified, whereas mutations affecting the topographic projection of RGC axons were not found. The mutants were grouped into two classes according to their phenotypes. In mutants of Class I, a subpopulation of the RGC axons branched out either immediately after leaving the eye or after reaching the midline, and this axonal subpopulation projected to the ipsilateral tectum. In mutants of Class II, subpopulations of RGC axons branched out after crossing the midline and projected aberrantly. These mutants will provide clues to understanding the functions of genes essential for axonal pathfinding, which may be conserved or partly divergent among vertebrates.