Developmental changes in the fibre population of the optic nerve follow an avian/mammalian-like pattern in the turtle Mauremys leprosa

The changes in the axon and growth cone numbers in the optic nerve of the freshwater turtle Mauremys leprosa were studied by electron microscopy from the embryonic day 14 (E14) to E80, when the animals normally hatch, and from the first postnatal day (P0) to adulthood (5 years on). At E16, the first axons appeared in the optic nerve and were added slowly until E21. From E21, the fibre number increased rapidly, peaking at E34 (570,000 fibres). Thereafter, the axon number decreased sharply, and from E47 declined steadily until reaching the mature number (about 330,000). These observations indicated that during development of the retina there was an overproduction and later elimination of retinal ganglion cells. Growth cones were first observed in the optic nerve at as early as E16. Their number increased rapidly until E21 and continued to be high through E23 and E26. After E26, the number declined steeply and by E40 the optic nerve was devoid of growth cones. These results indicated that differentiation of the retinal ganglion cells occurred during the first half of the embryonic life. To examine the correlation between the loss of the fibres from the optic nerve and loss of the parent retinal ganglion cells, retinal sections were processed with the TUNEL technique. Apoptotic nuclei were detected in the ganglion cell layer throughout the period of loss of the optic fibres. Our results showed that the time course of the numbers of the fibres in the developing turtle optic nerve was similar to those found in birds and mammals.     

Observations on the early development of the optic nerve and tract of the mouse.

The early development of the retinofugal pathway of mice has been studied by light and electron microscopic methods in order to define the spatial distribution and the structure of the growth cones as they advance from the eye to the brain. We have studied the relationships of the growth cones to each other, to the glia and, in the older individuals, to the nerve fibers that are already terminating in the brain. We have looked at the rate of advance of the growth cones and have paid particular attention to the changing relationships of the growth cones as they approach the optic chiasm. We have also looked to see whether, at early stages, it is possible to recognise any characteristic features distinguishing the fibers destined to be the thickest in the adult, which come from ganglion cells that are generated among the earliest ganglion cells. In transverse sections through the optic stalk about 50-100 microns behind the eye, the first bundles of fibers are seen on embryonic day 12.5 (E12.5) as a mixture of thin (less than 0.5 micron) axons, thicker growth cones, and fine filopodial and foliopodial extensions. During the next two days, as these bundles in the intraorbital nerve increase in size and number, growth cones can be seen in all of the bundles and in all parts of the bundles. They show only a slight preference for one part of the nerve relative to another, and our material provides no evidence for the view that axons are particularly inclined to follow pre-existing bundles. The structure of the pathway changes significantly as it is traced towards the chiasm, and no section or small stretch of sections can be regarded as representative of the nerve as a whole. As the fibers approach the optic chiasm the growth cones come to lie predominantly close to the pial surface, with the deeper regions occupied almost entirely by fine axons. The change occurs in a region where the glial environment also changes, and where a characteristic neural tube-like organization first becomes recognizable. Here the glial cells lie in a periventricular position and send slender radial processes out towards the subpial surface. The newly invading axons in the early optic nerve taper from a broad growth cone back to an extremely slender axon, less than 0.5 micron in diameter. The tapered region is of the order of 100-300 microns in length and advances through the nerve at approximately 60 microns per hour.(ABSTRACT TRUNCATED AT 400 WORDS)     

Growth cone distribution patterns in the optic nerve of fetal monkeys: implications for mechanisms of axon guidance

The distribution of growth cones was studied in the optic nerve of monkeys during the first half of prenatal development using quantitative electron microscopic methods. Our aim was to test the hypothesis that ganglion cell growth cones extend predominantly along the surfaces of the nerve, just beneath the pia mater. A complete census of growth cones in cross sections of the nerve during the early phase of axon ingrowth, from embryonic day 39 (E39) to E41, demonstrates that growth cones are scattered within the majority of fascicles, even those located far from the surface of the nerve. By E45, growth cones are concentrated around the nasal, dorsal, and ventral edge of the optic nerve. They are less concentrated in the core and around the temporal edge. However, even as late as E49, virtually all fascicles in the nerve, whether deep or superficial, contain growth cones. Growth cones are dispersed within single fascicles and are often located far from glia. Thus, the newest fibers penetrate deep parts of the pathway and push through centers of densely packed bundles of older axons. This finding is consistent with the vagrant paths of growing axons reported in previous work on embryonic monkey optic nerve (Williams and Rakic, 1985). Our data challenge the hypotheses that growth cones extend selectively along the basal lamina, the pia mater, or glial end feet. Gradients found at later stages of development in the nerve are not due to a particular affinity of growth cones for non-neuronal substrata. The pattern we observed is much more likely to result from central-to-peripheral gradients in ganglion cell generation and possible associations between growth cones originating from the same regions of the retina.     

The organization of the fibers in the optic nerve of normal and tectum-less Rana pipiens

We have examined the detailed order of retinal ganglion cell (RGC) axons in the optic nerve and tract of the frog, Ranapipiens. By using horseradish peroxidase (HRP) injections into small regions of theretina, the tectum, and at various points along the visual pathway, it hasbeen possible to follow labelled fibers throughout their course in the nerve and tract. Several surprising features in the order of fibers in the visual pathway were discovered in our investigation. The fascicular pattern of RGC axons in che retina is similar to that described in other vertebrates; however, immediately central to their entry into the optic nerve head, approximately half of the fibers from the nasal or temporal retina cross over to the opposite side of the nerve. Although the axons from the dorsal and ventral regions of the retina generally remain in the dorsal and ventral regions of the nerve, some fiber crossing occurs in those axons as well. The result of this seemingly complex rearrangement is that the optic nerve of Rana pipiens contains mirror symmetric representations of the retinal surface on either side of the dorsal ventral midline of the nerve. The fibers in each of these representation are arranged as semicircles representing the full circumference of the retina. This precise fiber order is preserved in the nerve until immediately periphearal to the optic chiasm, at which point age-related axon from both side of the nerve bundle together. Consequently, when a small pellet of HRP is placed in the chiasmic region of the nerve, an annualus of retinal ganglion cells and a corresponding annulus of RGC terminals in the tectum are la belled. As the age-related bundles of fibers emerge from the chiasm they split to form a medial bundle and a lateral bundle, which grow in the medial and lateral branches of the optic tract, respectively. Although the course followed by RGC axons in the visual pathv/ay is complex, we propose a model in which the organization of fibers in the nerve and tract can arise from a few rules of axon guidance. To determine whether the optic tecta, the primary retinal targets, play a role in the development and organization of the optic nerve and tract, we removed the tectal primordia in Rana embryos and examined the order in the nerve when the animals had reached larval stages. We found that the order in the nerve and tract was well preserved in tectumless frogs. Therefore, we propose that guidance factors independent of the target direct axon growth in the frog visual system.     

Distribution and morphology of retinal ganglion cells in the Japanese quail

A ganglion cell density map was produced from the Nissl-stained retinal whole mount of the Japanese quail. Ganglion cell density diminished nearly concentrically from the central area toward the retinal periphery. The mean soma area of ganglion cells in isodensity zones increased as the cell density decreased. The histograms of soma areas in each zone indicated that a population of small-sized ganglion cells persists into the peripheral retina. The total number of ganglion cells was estimated at about 2.0 million. Electron microscopic examination of the optic nerve revealed thin unmyelinated axons to comprise 69% of the total fiber count (about 2.0 million). Since there was no discrepancy between both the total numbers of neurons in the ganglion cell layer and optic nerve fibers, it is inferred that displaced amacrine cells are few, if any. The spectrum in optic nerve fiber diameter showed a unimodal skewed distribution quite similar to the histogram of soma areas of ganglion cells in the whole retina. This suggests a close correlation between soma areas and axon diameters. Retinal ganglion cells filled from the optic nerve with horseradish peroxidase were classified into 7 types according to such morphological characteristics as size, shape and location of the soma, as well as dendritic arborization pattern. Taking into account areal ranges of somata of each cell type, it can be assumed that most of the ganglion cells in the whole retinal ganglion cell layer are composed of type I, II and III cells, and that the population of uniformly small-sized ganglion cells corresponds to type I cells and is an origin of unmyelinated axons in the optic nerve.     

Ipsilaterally projecting retinal ganglion cells in Xenopus laevis: An HRP study

The routes of ipsilaterally projecting retinal ganglion cell axons in the visual pathway of young postmetamorphic Xenopus laevis were studied by anterograde and retrograde transport of horseradish peroxidase (HRP). In the retina, most cells heavily labeled from injections in ipsilateral thalamus are large multipolar ganglion cells. They are found exclusively in the posterior half of the retina, and their axons occupy a central position in the optic nerve head. Immediately behind the eye, axons of ipsilaterally projecting axons leave the core of the nerve and regroup around the circumference of the nerve. The nerve increases in diameter in the region where the fibers reorganize, and pigmented processes are seen in this region of the nerve. At the point where the optic nerve enters the brain case through the optic foramen, the fibers undergo a second reorganization which results in a laminar arrangement of ipsilaterally projecting axons at the ventral margin of the intracranial portion of the nerve. As soon as the nerve touches the brain, uncrossed axons begin to turn toward the ipsilateral side rather than proceeding further towards the midline of the chiasm. These uncrossed axons keep their internal topographical order at least at the beginning of the marginal tract. All ipsilaterally projecting axons run at the rostral edge of the marginal tract at the lateral-wall of the brain until they reach their terminal fields in the thalamic visual nuclei. © 1993 Wiley-Liss, Inc.     

Fibroblasts at the transection site of the injured goldfish optic nerve and their potential role during retinal axonal regeneration

The region at and around the site of optic nerve transection (ONS) in goldfish, topologically the equivalent of the glial scar in mammals, is reported to remain free of astrocytes over weeks, but its cellular constituents are unknown. To learn what type of cell occupies the site of injury and thus provides support for the rapidly regenerating retinal growth cones, immunostaining experiments at the light micro scopic level and electron microscopic examinations were undertaken. Between 2 and 30 days after ONS, an area up to 150 μm wide at the transection site exhibits intense anti-fibronectin immunoreactivity. This site contained cells and processes with ultrastructural characteristics of fibroblasts and abundant collagen fibrils. Moreover, on fibroblast cultures derived from regenerating optic nerves, retinal axons grew to considerable density in vitro. Since fibroblasts are constituents of the interfascicular spaces and outer nerve sheath of the normal goldfish optic nerve, the present data imply that fibroblasts of either source migrate into the lesion. Judging from fibronectin immunostaining they remain there during the passage of regenerating axons, and thus may provide physical and perhaps molecular support for axon growth. The fibroblasts are again restricted to interfascicular spaces after restoration of the astrocytic glia limitans around regenerated fascicles. © 1995 Wiley-Liss, Inc.     

Selectivity of antibody-mediated destruction of axons in the cat's optic nerve

The purpose of this study was to determine the selectivity with which polyspecific antibodies directed against large retinal ganglion cells destroy axons in the cat's optic nerve. Immune serum prepared against large ganglion cells isolated from ox retinas was injected into 1 eye of each of 2 cats. After more than 1 month, the cats were perfused with mixed aldehydes and the optic nerves were prepared for transmission electron microscopy. On the basis of a large sample of micrographs of transverse thin sections, we estimated that each of the nerves that issued from treated eyes contained approximately 62,500 necrotic fibers, amounting to 42–46% of the total fiber population in 1 case and 39–42% in the other. The diameters of 2900–5000 intact axons from each of 4 nerves (2 from immune-treated eyes and 2 from untreated eyes) were measured. Comparisons of histograms of axon diameter for nerves from treated and untreated eyes revealed that 90–100% of large axons with diameters above 3.5–4.0 μm were eliminated by the antibodies. Between 65 and 70% of medium-sized fibers were also eliminated. The number of small axons—those with diameters less than 1.2–1.6 μm — did not differ appreciably from normal. These results suggest that the immune serum destroyed virtually all α cell axons and a substantial fraction of β cell axons but did not reduce the number of small fibers that largely stem from the γ class of retinal ganglion cells.     

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.     

Growth cone morphology varies with position in the developing mouse visual pathway from retina to first targets

We have labeled the growth cones of retinal ganglion cell axons with HRP in intact mouse embryos. This has allowed us to visualize growth cone morphology during outgrowth along an entire CNS pathway from origin to target; to ask whether growth cone forms, and thus behaviors, differ at various points along the pathway; and to study the relationships of growth cones with the cellular environment. During the major period of axon outgrowth between embryonic day (E) 12 and 15, growth cones in the optic nerve are highly elongated (up to 40 microns) and have lamellopodial expansions, but the majority lack the microspikes or filopodia characteristic of many growth cones. Within the optic chiasm (E13-15), most growth cones shorten and spread, and project several short filopodia. In the optic tract, growth cones become more slender and again lack filopodia, resembling sleeker versions of optic nerve growth cones. Near the first target region (lateral geniculate nucleus), growth cones with filopodia arise from individual axon lengths and turn medially toward the target. Within target regions, the branches of immature axon arbors are tipped by minute swellings rather than by the enlarged growth cones prevalent during outgrowth toward targets. Electron-microscopic analysis of identified labeled growth cones in the optic nerve reveal intimate interactions between growth cones and glia or other growth cones in the form of invaginating contacts. In the optic nerve, growth cones contact immature glial (neuroepithelial) cells somewhere along their length, and also envelop bundles of neurites. In the chiasm, single growth cones simultaneously relate to many different profiles. These results demonstrate that in this single pathway from origin to targets, growth cone morphology varies systematically with position along the visual pathway. During outgrowth, simple growth cones are prominent when axons follow well-defined common pathways, and more elaborate filopodial forms appear when growth cones diverge, as they turn or come to decision regions. Together with observations in vitro and in nonmammalian nervous systems in situ, these data serve as reference points for testing to what extent growth cone form reflects intrinsic factors and interactions with the environment.     

Changing course of growing axons in the optic chiasm of the mouse

The distribution of retinofugal fibres has been studied by electron microscopy throughout the extent of the developing mouse optic nerve and chiasm at embryonic day (E) 16, in order to determine the course of fibre growth. Growth cones and mature axons, which are randomly distributed in bundles in the extracranial optic nerve, segregate in the juxtachiasmatic optic nerve. Here, growth cones accumulate in subpial regions amongst the endfeet of radial glia, whereas axons lie in the depths of the nerve. Surprisingly, however, growth cones move away from this region toward the ventricular zone in the lateral and midline parts of the chiasm, only to return to subpial regions once more before entering the optic tract, where fibres are again in an age-related order. Superficially, mature axons mingle with growth cones in the chiasm and near the beginning of the optic tract, suggesting that the age-related order begins to be reestablished before growth cones enter the tract. Deep and superficial regions of the pathway were examined in different planes of section. Specialised membrane relationships between retinofugal fibres and radial glial cells were also studied in deep and superficial regions of the lateral part of the chiasm. In addition, the distribution of retinofugal fibre bundles in the adult mouse was looked at by using light microscopy. The changing fibre positions noted in the embryo are maintained in the adult. J. Comp. Neurol. 379:495–514, 1997. © 1997 Wiley-Liss, Inc.     

Precocious invasion of the optic stalk by transient retinopetal axons

This study demonstrates that the fetal optic nerve contains a conspicuous population of transient retinopetal axons. Implants of the carbocyanine dye, DiI, were made into the retina or diencephalon of fetal ferrets to label the retinopetal axons retrogradely or anterogradely, respectively, and sections were immunostained for β-tubulin to label the early differentiating axons in the optic nerve. Dye implants into the optic nerve head, but not the retinal periphery, retrogradely labeled somata in the ventrolateral diencephalon, provided the implants were made before embryonic day (E) 30. When dye implants were made into the ventrolateral diencephalon, these same retinopetal axons were anterogradely labeled, coursing through the optic nerve but never invading the retina. The axons course as 2–5 fascicles from their cells of origin and turn laterally to enter the optic nerve where it joins the future hypothalamus. The retinopetal cells can be retrogradely labeled as early as E20, before optic axons have left the retina. The optic nerve and fiber layer are immunoreactive for β-tubulin on E24 and thereafter, whereas on E20 and E22, they are immunonegative. Yet at these early embryonic ages, immunopositive fascicles of axons course from the diencephalon into the optic stalk, confirming the precocious nature of the retinopetal projection. Implants of dye made into the future optic nerve head at these very early stages also retrogradely label retinopetal cells in the future chiasmatic region. These cells are distributed primarily on the side ipsilateral to the midline, but a few can be found contralateral to it. Both these, as well as the retinopetal axons arising from the ventrolateral diencephalon, may serve a transient guidance function for later developing optic axons. © 1995 Wiley-Liss, Inc.     

The ability of axons to regenerate their growth cones depends on axonal type and age, and is regulated by calcium, cAMP and ERK

The processes activated at the time of axotomy and leading to the formation of a new growth cone are the first step in regeneration, but are still poorly characterized. We investigated this event in an in vitro model of axotomy performed on dorsal root ganglia and retinal explants. We observed that the dorsal root ganglion axons and retinal ganglion cell axons, which had grown out on a poly d-lysine/laminin substrate at the time of culture preparation greatly differed in their regenerative response after a subsequent in vitro lesion made far from the cell body. The majority of axons of adult dorsal root ganglia but only a small percentage of axons of adult retinal ganglion cells regenerated new growth cones within four hours after in vitro axotomy, though both kinds of axons were growing before the lesion. The depletion of extracellular calcium and the inhibition of extracellular-signal regulated kinase 1,2 (ERK) and protein kinase A (PKA) at the time of injury significantly impaired the capacity of dorsal root ganglia axons to re-initiate growth cones without affecting growth cone motility. Pharmacological treatments directed at increasing the level of cAMP promoted growth cone regeneration in adult retinal ganglion cell axons in spite of the low regenerative potential exhibited in normal conditions. Understanding the cellular mechanisms activated at the time of lesion and leading to the formation of a new growth cone is necessary for devising treatments aimed at enhancing the regenerative response of injured axons.     

European Neuroscience Association The changing pattern of fibre bundles that pass through the optic chiasm of mice

The organization of retinofugal fibres in the developing and adult mouse has been studied with transmission electron microscopy, autoradiography and the Bodian silver method. It has previously been shown that all retinal ganglion cell axons are in glial-wrapped bundles in the developing and adult optic nerve, but are not in similar bundles close to the chiasm. In the embryonic mouse this region shows a transition in glial morphology from an interfascicular to a radial type and here retinofugal fibres begin to form a new order related to their age. Growth cones become concentrated at the pial surface of the juxtachiasmatic nerve and older fibres are restricted to deeper regions. This same age-related order is also evident in the optic tract. However, the age-related order is lost within the chiasm, where growth cones, young and old fibres are again mingled in distinct bundles as they cross the mid-line. This study is particularly concerned with the structure of the mid-line bundles. These fibre bundles cross each other at right angles, and are recognizable in fetal and adult mice. In the adult, monocular injections of H³ proline followed by autoradiographic study show that the individual mid-line bundles are monocular and that they fuse again, losing the fascicular structure as they leave the chiasm and enter the tract. In the fetus and in the adult, the bundles generally lack a complete glial wrapping so that growth cones can lie in intimate contact with two crossing bundles, one coming from the left eye, the other from the right. The interesting question about the mechanisms that keep growth cones from entering the wrong bundles when they are in this position remains to be addressed.     

Time of ganglion cell genesis in relation to the chiasmatic pathway choice of retinofugal axons.

The time of generation of retinal ganglion cells in fetal cats has been related to the course taken later by their axons in the optic chiasm. The ganglion cells were labelled with tritiated thymidine either on embryonic day (E) 26 or on E-30. When the cats were mature, ganglion cells were retrogradely labelled with horseradish peroxidase injected into one optic tract. The distribution of double-labelled cells showed that cells in the temporal retina generated on E-26 all have axons that take an uncrossed course in the chiasm, whereas, of the cells generated on E-30 in the temporal retina, some take a crossed course and others take an uncrossed course. The uncrossed axons of the E-26 cohort come from cells having a central distribution on the retina. For the E-30 cohort, the uncrossed axons come from cells having a relatively peripheral distribution, whereas the crossed axons come from more central cells. The present results suggest that the mechanism which serves to direct temporal retinal axons into the ipsilateral optic tract weakens as development proceeds. In principle, the change may occur in either a chiasmatic signal, read by temporal but not nasal optic axons, or in a retinal label, carried by temporal but not nasal cells and their processes. Since temporal retinal cells born concurrently at different places can project to opposite optic tracts, a retinal signal that deteriorates with time in a centroperipheral fashion is favored by the present results.     

Studies of the early stages of optic axon regeneration in the goldfish

We have studied the early stages (4-14 days) of axonal regeneration following intraorbital optic nerve crush in the goldfish. We used 3H-proline autoradiography to anterogradely label and visualize the growing axons and wheat germ agglutinin-conjugated horseradish peroxidase (WGA:HRP) for retrograde labeling to determine the cells of origin of the earliest projections. The first retinal ganglion cells (RGCs) that could be retrogradely filled from the optic tract, following optic nerve crush, were in the central retina and were seen at 8 days postoperative. More peripheral cells were only labeled with longer postcrush survival periods. Thus, the first axons to regenerate past the lesion were from central RGCs. The axons of these cells extended into the cranial nerve stump between 4 and 5 days postcrush and entered the nerve as a fascicle, which travelled just beneath its surface. Studies of nerve cross sections from animals at 5-8 days postoperative demonstrated that initial outgrowth was not confined to any particular locale within the nerve although the early fibers appeared to avoid its temporal aspect. When the regenerating axons reached the optic tract they remained in fascicles but left the surface to run along the medial, deep portion of the tract, immediately adjacent to the diencephalon and pretectum. The positions occupied by the earliest-regenerating axons in the optic nerve were variable and not always appropriate for their central retinal origin. However, the abrupt change in growth trajectory as the fibers entered the optic tract brought them into the areas of the visual paths that are occupied by central axons in intact animals. We suggest that this change in position is related to both changes in the structural organization of the intracranial visual paths and to possible axon guidance signals in the region of the nerve-tract juncture.     

Development of the crossed retinocollicular projection in the mouse

Changes in the distribution of axons of the crossed retinal projection within the superior colliculus of the developing mouse were studied by means of normal fiber and Golgi impregnations and by anterograde horseradish peroxidase labelling. Retinal axons advance along the optic tract from gestational days E12 to E14 and first invade the superior colliculus on E15. Over the subsequent days until birth (E19), the retinal axons extend within rostrocaudally oriented fascicles that distribute through the full thickness of the uppermost collicular layer, the stratum superficiale (SS). A dramatic transformation of this fiber stratification pattern into the mature pattern occurs over the first postnatal week. The fiber bundles are progressively cleared from the upper half of SS, identified as the future stratum griseum superficiale (SGS). Concurrently, the fiber bundles in the deep SS, identified as the stratum opticum (SO), give rise to individual, nonfasciculated fibers, which arborize within SGS. The contralateral retinal origin of the transient population of axons in SGS as well as the majority of axons that persist in SO is evident from the observation that they degenerate following neonatal enucleation. The number of fiber bundles lost is estimated to be 40-50% of the total population present in the superficial layers at birth. The combined set of observations indicates that axon elimination plays a major role in shaping the laminar pattern of retinal innervation of the colliculus. Retinal ganglion cell death, and not axon pruning, is proposed as the most probable mechanism by which axon fascicles are eliminated from SGS.     

Increased NCAM-180 immunoreactivity and maintenance of L1 immunoreactivity in injured optic fibers of adult mice

The injury related expression of two axon-growth promoting cell adhesion molecules (CAMs), NCAM-180 which is developmentally downregulated and L1 which is regionally restricted, were compared in optic fibers in the adult mouse. The neuron-specific isoform of NCAM (NCAM-180) is present at very low levels in unlesioned adult optic axons. At 7 days after nerve crush, immunoreactivity was strongly and uniformly increased in optic axons within the nerve and throughout retina. Reactivity in surviving axons had returned to control levels at 4 weeks. To induce regrowth of adult retinal ganglion cell axons retinal explants were placed in culture. Strong NCAM-180 staining was observed on these regenerating optic axons. The neuronal cell adhesion molecule L1 is restricted to retina and to the unmyelinated segment of the optic nerve near the optic nerve head in unlesioned adult animals. Following nerve crush, L1 immunoreactivity was retained within retina and proximal nerve and novel staining was detected in the more distal segment of the optic nerve up to the lesion site where it persisted for at least eight months. The capacity of optic fibers to show increased NCAM-180 immunoreactivity and maintain L1 expression after a lesion may explain why these fibers exhibit relatively good potential for regeneration.     

Postnatal innervation of the rat superior colliculus by axons of late-born retinal ganglion cells

Rat retinal ganglion cells (RGCs) are generated between embryonic day (E) 13 and E19. Retinal axons first reach the superior colliculus at E16/16.5 but the time of arrival of axons from late-born RGCs is unknown. This study examined (i) whether there is a correlation between RGC genesis and the timing of retinotectal innervation and (ii) when axons of late-born RGCs reach the superior colliculus. Pregnant Wistar rats were injected intraperitoneally with bromodeoxyuridine (BrdU) on E16, E18 or E19. Pups from these litters received unilateral superior colliculus injections of fluorogold (FG) at ages between postnatal (P) day P0 and P6, and were perfused 1-2 days later. RGCs in 3 rats from each BrdU litter were labelled in adulthood by placing FG onto transected optic nerve. Retinas were cryosectioned and the number of FG, BrdU and double-labelled (FG+/BrdU+) RGCs quantified. In the E16 group, the proportion of FG-labelled RGCs that were BrdU+ did not vary with age, indicating that axons from these cells had reached the superior colliculus by P0/P1. In contrast, for the smaller cohorts of RGCs born on E18 or E19, the proportion of BrdU+ cells that were FG+ increased significantly after birth; axons from most RGCs born on E19 were not retrogradely FG-labelled until P4/P5. Thus there is a correlation between birthdate and innervation in rat retinotectal pathways. Furthermore, compared to the earliest born RGCs, axons from late-born RGCs take about three times longer to reach the superior colliculus. Later-arriving axons presumably encounter comparatively different growth terrains en route and eventually innervate more differentiated target structures.     

Changes in morphology and behaviour of retinal growth cones before and after crossing the midline of the mouse chiasm – a confocal microscopy study

The growth of retinal axons was investigated in different regions of the optic chiasm in C57 pigmented mouse embryos aged embryonic day 13 (E13) to E15. Individual retinal axons and their growth cones were labelled anterogradely by DiI and imaged using a confocal imaging system. In aldehyde-fixed embryos, retinal growth cones display a simple form in the optic nerve and become more complex in morphology in the chiasm. The complex form is particularly prominent in those axons that turn to the ipsilateral tract in the premidline region of chiasm. Moreover, complex growth cones are also commonly found in axons in the postmidline chiasm, which are markedly different in morphology from those axons in the premidline region, suggesting that the postmidline chiasm contains a novel environment for the pathfinding of retinal axons. In another experiment, the dynamic growth of retinal axons is studied in a brain slice preparation of the living retinofugal pathway. Retinal axons show an intermittent growth across the premidline and postmidline chiasm. Extensive remodelling of growth cone form followed by a shift in growth direction is commonly seen during the pause periods, indicating that signals that guide axon growth across the chiasm are not restricted to the midline, but are laid down throughout the chiasm. Moreover, dramatic changes in axon trajectory are noted first at the premidline chiasm where the uncrossed axons segregate from the crossed axons, and second at the postmidline chiasm where specific sorting of retinal axons according to their position in the dorsal ventral retinal axis and their ages are known to take place. These results show that there are two distinct environments, separated by the midline in the chiasm, where axons show different responses to local guidance cues and develop the distinct fibre orders.