Evidence for shifting connections during development of the chick retinotectal projection

The pattern in which optic axons invade the tectum and begin synaptogenesis was studied in the chick. The anterogradely transported marker, horseradish peroxidase, was injected into one eye of embryos between 5 and 16 days of development (E5 to E16). This labeled the optic axons in the brain. The first retinal axons arrived in the most superficial lamina of the tectum on E6. They entered the tectum at the rostroventral margin. During the next 6 days of development the axons grew over the tectal surface. First they filled the rostral tectum, the oldest portion of the tectum, and then they spread to the caudal pole. Shortly after the first axons entered the tectum on E6, labeled retinal axons were found penetrating from the surface into deeper tectal layers. In any given area of the tectum, optic axons were seen penetrating deeper layers shortly after arriving in that area. Electron microscopic examination showed that at least some of the labeled axons in rostral tectum formed synapses with tectal cells by E7. These results show two things which contrast with results from previous studies. First, there is no delay between the time the retinal axons enter the tectum and the time they penetrate into synaptic layers of the tectum. Second, the first retinotectal connections are formed in rostral tectum and not central tectum. Retrograde tracing showed the first optic axons that arrived in the tectum were from ganglion cells in central retina. Previous studies have shown that the ganglion cells of central retina project to the central tectum in the mature chick. This opens the possibility that the optic axons from central retina, which connect to rostral tectum in the young embryo, shift their connections to central tectum during subsequent development. As they enter the tectum the growth cones of retinal axons appear to be associated with the external limiting membrane. During the time that connections would begin to shift in the tectum a second population of axons appears at the bottom of stratum opticum, some with characteristics of growth cones. This late-appearing population may represent axons shifting their connections. These results have implications for theories on how the retinotopic pattern of retinotectal connections develops.     

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.     

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.     

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.     

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.     

Spatial arrangement of radial glia and ingrowing retinal axons in the chick optic tectum during development

Neuroanatomical tracing of retinal axons and axonal terminals with the fluorescent dye, DiI, was combined with immunohistochemical characterization of radial glial cells in the developing chick retinotectal system. Emphasis was placed on the mode of the tectal innervation by individual retinal axons and on the distribution and fate of the tectal radial glial cells and their spatial relation to retinal axons. It was obvious from fluorescent images obtained from anterogradely filled axons that these axons deserted the superficial stratum opticum (SO) to penetrate the stratum griseum et fibrosum superficiale (SGFS) by making right-angled turns within the SO. Frequently, axons which had invaded the SGFS were bifurcated and had a superficial branch which remained within the SO. Terminal axonal arborization occurred at various depths within the SGFS. Characterization of the tectal glial cells and their radial fibers by means of the anti-filament antibody, R5, and post-mortem staining with the fluorescent dye, DiI, revealed the following. (a) At least from day E8 to P1, tectal glial fibers traversed all tectal layers from the periventricular location of their somata to the superficial interface between SO and pia mater. In this interface they enlarged and formed characteristic endfeet. (b) Glial endfeet covered the whole tectal surface. They showed at early ages anterior-posterior differences having a higher density in the posterior tectum. These differences disappeared at embryonic day E13. (c) After innervation, glial endfeet of the anterior tectal third were arranged in rows parallel to the retinal fibers within the SO. This arrangement was not observed in eyeless embryos. (d) Radial glial fibers could be stained with R5 from day E8 to late embryonic stages throughout their entire length. (e) At the first posthatching days, only the segments of the radial glial fibers restricted to the thickness of the SO were R5-positive, although the fibers still traversed throughout the depth of the tectum. The results are discussed in context to the genesis of the retinotectal projection.     

Developing retinotectal projection in larval goldfish

The retinotectal projection in larval goldfish was studied with the aid of anterograde filling of optic fibers with HRP applied to the retina. The results show that optic fibers have already reached the tectum and begun to form terminal arbors in newly hatched fish. The projection is topographic in that fibers from local regions of the retina project to discrete patches of tectum, with the smallest patch covering 3.5% of the total surface area of tectal neuropil. Many fibers in young larvae have numerous short side branches along their length and only some of them show evidence of terminal sprouting. The arbors are approximately elliptical in shape and average about 1,500 microns 2. Growth cones are seen frequently. In older larvae, terminal arbors are larger and more highly branched, and they have begun to resemble those in adult fish. Fibers terminate in two strata; those in the upper layer are smaller (1,800 microns 2 on average) than those in the deeper stratum (4,000 microns 2 on average). The fraction of tectal surface area covered by individual arbors (the "tectal coverage") ranges from 1.5% to 3% of the total surface area of the tectal neuropil. In contrast, the tectal coverage of individual arbors in young adult goldfish is much smaller, ranging from 0.02% to 0.42% of tectal surface area (Stuermer, '84, and unpublished). This apparent increase in precision of the map in older animals is not due to retraction of arbors, which are slightly larger in adults, but is accounted for by overall tectal growth: the tectal neuropil in goldfish increases in area by about 250-fold during this period (Raymond, '86).     

Regulation of retinal ganglion cell axon arbor size by target availability: Mechanisms of compression and expansion of the retinotectal projection

The ability of pre- and postsynaptic populations to achieve the proper convergence ratios during development is especially critical in topographically mapped systems such as the retinotectal system. The ratio of retinal ganglion cells to their target cells in the optic tectum can be altered experimentally either by early partial tectal ablation, which results in an orderly compression of near-normal numbers of retinal projections into a smaller tectal area, or by early monocular enucleation, which results in the expansion of a reduced number of axons in a near-normal tectal volume. Our previous studies showed that changes in cell death and synaptic density consequent to these manipulations can account for only a minor component of this compensation for the population mismatch. In this study, we examine other mechanisms of population matching in the hamster retinotectal system. We used an in vitro horseradish peroxidase labeling method to trace individual retinal ganglion cell axons in superior colliculi partially ablated on the day of birth, as well as in colliculi contralateral to a monocular enucleation. We found that individual axon arbors within the partially lesioned tectum occupy a smaller area, with fewer branches and fewer terminal boutons, but preserve a normal bouton denstiy. In contrst, ipsilaterally projecting axon arbors in monoculary enucleated animals occupy a greater area than in the normal condition, with a much larger arbor length and greater number of boutons and branches compared with normal ipsilaterally projecting cells. Alteration of axonal arborization of retinalganglion cells is the main factor responsible for matching the retinal and tectal cell populations within the tectum. This process conserves normal electrophysiological function over a wide range of convergence ratios and may occur through strict selectivity of tectal cells for their normal number of inputs. © 1994 Wiley-Liss, Inc.     

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.     

Mode of growth of retinal axons within the tectum of Xenopus tadpoles, and implications in the ordered neuronal connection between the retina and the tectum

Retinal axons of Xenopus tadpoles at various stages of larval development were filled with horseradish peroxidase (HRP), and their trajectories and the patterns of branching within the tectum were analyzed in wholemount preparations. To clarify temporal and spatial modes of growth of retinal axons during larval development, special attention was directed to labeling a restricted regional population of retinal axons with HRP, following reported procedures (H. Fujisawa, K. Watanabe, N. Tani, and Y. Ibata, Brain Res. 206:9-20, 1981; 206:21-26, 1981; H. Fujisawa, Dev. Growth Differ 26:545-553, 1984). In developing tadpoles, individual retinal axons arrived at the tectum, without clear sprouting. Axonal sprouting first began when growing tips of each retinal axon had arrived at the vicinity of its site of normal innervation within the tectum. Thus, the terminals of the newly added retinal axons were retinotopically aligned within the tectum. The retinotopic alignment of the terminals may be due to an active choice of topographically appropriate tectal regions by growth cones of individual retinal axons. The stereotyped alignment of the newly added retinal axons was followed by widespread axonal branching and preferential selection of those branches. Each retinal axon was sequentially bifurcated within the tectum, and old branches that had inevitably been left at ectopic parts of the tectum (owing to tectal growth) were retracted or degenerated in the following larval development. The above mode of axonal growth provides an adequate explanation of cellular mechanisms of terminal shifting of retinal axons within the tectum during development of retinotectal projection. Selection of appropriate branches may also lead to a reduction in the size of terminal arborization of retinal axons, resulting in a refinement in targeting.     

Axonal arborization in the developing chick retinotectal system

The growth and arborization of chicken retinal ganglion cell axons have been investigated by means of an intraaxonally transported fluorescent marker in the developing retinotectal system. The fluorescent dye D282 or diI from the carbocyanine group of dyes is taken up by ganglion cells and labels the axon as well as the axonal growth cones and the terminal arborizations on the tectum. Branching and arborization start in the chick retinotectal system on embryonic day 9 (E9). At this stage retinal axons leave the stratum opticum (SO) and invade the stratum griseum et fibrosum superficiale (SGFS), where arborization takes place. On day E12 several axons were found to arborize in the SGFS. At this stage arbors appear to have small branches with less than 4 branching points. The extension of terminal arbors in the anterior/posterior (A/P) and in the dorsal/ventral (D/V) direction was determined for 50 axonal trees at days E13-14 and for 24 arbors at days E15-16. Few axonal terminals were investigated at day E18. The mean A/P extent of axonal terminal trees increases from 0.23 +/- 0.12 to 0.36 +/- 0.22 mm from E13-14 to E15-16 and seems to stay at this order of magnitude on E18. The mean D/V extent increases from 0.23 +/- 0.17 to 0.30 +/- 0.18 mm in the same embryonic period of development. The number of branching points calculated from the same number of axonal trees increases from 7.50 +/- 2.98 at E13-14 to 11.70 +/- 4.10 at E15-16. This number seems to increase further after day E16 achieving values of about 20 to 25 at E18. This was, however, not quantifiable by the technique used here and represents an approximate value estimated from 6 completely labeled terminal fields at E18. The data presented here suggest that the modeling of the final branching pattern in the chick retinotectal system takes place within a relatively short period of embryonic development. Prior to the beginning of terminal arborization two important events contribute to the formation of a retinotopic projection. One event is the change of the D/V position by a minority of axons lying ectopic in terms of retinotopy. Some axons turn at right angles and change their D/V position. The other event is the appearance of side branches along the A/P axis.     

Glial environment in the developing superior colliculus of hamsters in relation to the timing of retinal axon ingrowth

We have examined the developmental changes of glial cell organization in the superior colliculus of embryonic and neonatal hamsters in reference to the known sequence of retinal axon ingrowth and arborization in the midbrain. Immunolocalization of vimentin, a marker for neuronal and glial cell precursors, reveals a uniform distribution of radially oriented cells, with perikarya located at the ventricular surface and thin, elongated processes fanning out toward the pia. These vimentin-positive cells, referred to as the lateral radial cells, are present in the tectum from embryonic day (E) 10 (earliest day examined) until approximately postnatal day (P) 5. Vimentin expression in the lateral radial cells decreases markedly during the second week of postnatal life: application of DiI to the ventricular surface reveals that the pial attachment of the lateral radial cells is withdrawn and that the radial processes are gradually pulled back toward the ventricular zone. By P14, virtually no vimentin-positive radial cells are detectable in the superior colliculus. At no time during development are the lateral radial cells immunopositive for the glial fibrillary acidic protein (GFAP); however, shorter, vimentin-positive astrocytic profiles can be seen in the tectum, around the time the radial fibers have been withdrawn, suggesting that at least some radial cells are transformed into astrocytes that will colonize the mature colliculus. At approximately E12, a second group of cells, referred to as the midline radial glia, is detected at the tectal midline. These cells are tightly bundled, forming a raphe in the tectum. They are intensely vimentin positive from E13 until at least P14. From the time of birth, the midline radial cells also exhibit intense immunoreactivity for GFAP. The lateral radial cells are present in the superior colliculus prior to and during the period of neurogenesis but remain well past the time when collicular neuronal migration is completed. Pial processes of the lateral radial cells are present within the superficial tectal layers during the time retinal axons are entering this target; they may be involved in directing the growth and initial collateralization of retinotectal axons. Their withdrawal from retinorecipient collicular zones begins at about the time arbors are being elaborated on retinal axons. In constrast, the midline glia become distinct just prior to the time retinal axons enter the superior colliculus and persist during the time retinotectal projections are being fully established. These raphe glia may be involved in maintaining the laterality of the retinotectal projection. © 1995 Wiley-Liss, Inc.     

Outgrowth and directional specificity of fibers from embryonic retinal transplants in the chick optic tectum

Retinal pieces taken from known positions of 6-day chick embryos were vitally labeled with the fluorescent dye Rhodamine-B-isothiocyanate (RITC). They were then transferred onto the surfaces of optic tecta following early bilateral removal of the embryo's optic vesicles. One to 5 days after transplantation the tecta were fixed and transplants that issued fibers were examined on tectal whole-mounts or were sectioned and viewed with a fluorescence microscope. Retinal fibers growing out from transplants on day E6 tecta showed a capacity for changing their initial outgrowth directions and for reorienting themselves towards their specific retinotopic projection area. Frequently, changes in growth direction appeared in a right-angled pattern. The capacity for turning was strongest for fibers of nasal retinal origin, less strong for fibers of temporal origin, and occurred rarely but unquantifiably in the case of fibers of ventral retinal origin. Fibers of all investigated retinal quadrants were found to reach their corresponding projection areas and to arborize there, that is, fibers of nasal retinal transplants in the posterior tectum, of temporal transplants in the anterior tectum, and of ventral transplants in the dorsal tectum. Furthermore, once in their target region, the fibers left the outer layer of the tectum and turned, again in right angles, to invade deeper layers. Capacity of fibers to turn towards their projection area was not observed for fibers issued from transplants placed on the tectum later than day E8. We suggest that there is a specific guidance of retinal axons on the tectum.     

Retinotopic organization of the developing retinotectal projection in the zebrafish embryo

Developing retinal axons in the zebrafish embryo were stained with HRP or with the fluorescent dyes dil and diO to study the formation of the retinotectal projection. Retinal axons leave the eye at 34-36 hr postfertilization (PF), invade the tectum at 46-48 hr PF, and innervate the tectal neuropil at 70-72 hr PF. Dorsal and ventral axons occupy separate aspects of the optic nerve and tract and pass into their retinotopically appropriate ventral and dorsal hemitectum, respectively. Nasal and temporal axons are segregated in the nerve, mixed in the tract, and are coextensive over the rostral half of tectum until 56 hr PF. They then segregate again, due to the progression of nasal axons into the open caudal tectum. Thus, at 70-72 hr PF, dorsal and ventral as well as temporal and nasal axons occupy their retinotopically appropriate tectal quadrants. After ablation of the temporal retina prior to the time of axonal outgrowth, the nasal axons bypass the vacant rostral tectum to terminate in the caudal tectal half. Temporal axons in the absence of nasal axons remain restricted to their appropriate rostral tectal half, suggesting that nasal and temporal axons possess a preference for their retinotopically appropriate tectal domains. Measurements of individual terminal arbors and the tectal areas in embryos and in adult zebrafish showed that individual arbors are large with respect to the embryonic tectum but are about 14-15 times smaller than in the adult. However, the proportion of tectum covered by embryonic arbors is about 7 times larger than in the adult, suggesting that a higher precision of the adult projection is achieved as a result of a greater enlargement of the tectum than of the arbors.     

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.     

Peripheral nerve grafts to the frog optic tectum: a morphological study of foreign axon regeneration in the central nervous system

The proximal stump of a transected mandibular nerve was grafted onto the rostrodorsal surface of the optic tectum in adult Rana pipiens to investigate the morphologic characteristics of nonspecific axonal regeneration in a highly organized region of central nervous system (CNS). Within the first 3 weeks postgraft surgery (WPS), the nerve-tectum interface became firmly established. Concomitant with this was an invasion of the host tectum by a small number of fine "pioneerlike" axons from the nerve. By 6 WPS there developed a concerted instreaming of a large number of peripheral fibers. Once within the CNS, the foreign axons distributed themselves throughout the rostrocaudal extent of the tectum, but primarily its dorsal aspect within superficial layers 8 and 9. Presence of intact optic fibers at the time of mandibular fiber invasion served somewhat to restrict the regenerating aberrant axons in their course through layer 9. This restriction could be avoided by removal of the optic input either before or during peripheral ingrowth. However, once peripheral fibers had entered and established themselves in the host environment, no subsequent manipulation of the retinotectal projection had any effect. The aberrant growth pattern, which appeared remarkably stable after 6 WPS, consisted of a plexus of medium- and fine-caliber peripheral axons. Many of these fibers had numerous branches and "en passant" varicosities, the latter encompassing a variety of shapes and sizes. Terminal swellings and arborizations were also found. When comparing the regeneration of optic and mandibular nerve fibers in the tectum, two distinctions were made. Whereas optic axons revealed a fascicular and layered organization, mandibular axons showed a highly segregated and disordered growth pattern. These characteristic differences were maintained even when the two fiber systems were allowed to coregenerate into the same target tectum. Thus, each of the two groups of axons interacts with the tectal substrate in a distinct manner, apparently independent of the other.     

Optic nerve transection decreases 2-[I]iodomelatonin binding in the chick optic tectum

The distribution of specific 2-[¹²⁵I]iodomelatonin binding sites in the various layers of the chick optic tectum was analyzed using quantitative receptor autoradiography. Following unilateral optic nerve transection, binding in the optic fiber layer and superficial retinorecipient layers of the contralateral tectum was significantly decreased at 7 and 14 days, but not at 1 day, following transection. The results are consistent with the presence of presynaptic melatonin receptors on axon terminals of retinotectal fibers.     

Persistence of graded EphA/Ephrin-A expression in the adult frog visual system

Many studies have demonstrated the involvement of the EphA family of receptor tyrosine kinases and their ligands, ephrin-A2 and -A5, in the development of the temporonasal axis of the retinotectal/collicular map, but the role of these molecules in optic nerve regeneration has not been well studied. Noting that the characteristic gradients of the EphA/ephrin-A family that are expressed topographically in the retina and tectum of embryonic chicks and mice tend to disappear after birth, we took as our starting point an analysis of EphA and ephrin-A expression in leopard frogs (Rana pipiens and utricularia), species capable of regenerating the retinotectal map as adults. For the EphA family to be involved in the regeneration, one would expect these topographic gradients to persist in the adult or, if downregulated after metamorphosis, to be reexpressed after optic nerve injury. Using EphA3 receptor and ephrin-A5 ligand alkaline phosphatase in situ affinity probes (RAP and LAP, respectively) in whole-mount applications, we report that reciprocally complementary gradients of RAP and LAP binding persist in the optic tract and optic tectum of postmetamorphic frogs, including mature adults. EphA expression in temporal retinal axons in the optic tract was significantly reduced after nerve section but returned during regeneration. However, ephrin-A expression in the tectal parenchyma was not significantly elevated by either eye removal, with degeneration of optic axons, or during regeneration of the retinotectal projection. Thus, the present study has demonstrated a persisting expression of EphA/ephrin-A family members in the retinal axons and tectal parenchyma that may help guide regenerating fibers, but we can offer no evidence for an upregulation of ephrin-A expression in conjunction with optic nerve injury.     

The monoclonal antibody E587 recognizes growing (new and regenerating) retinal axons in the goldfish retinotectal pathway

E587 is a new monoclonal antibody against a 200 kDa cell-surface glycoprotein in the fish retinotectal pathway. The E587 antigen probably belongs to the class of cell adhesion molecules, and more specifically, to the family of L1-like molecules. The immunopurified protein is recognized by the antibody against the HNK1/L2 sugar epitope (associated with most cell adhesion molecules) and by a polyclonal antiserum against chick G4, which is related to the cell adhesion molecule L1 in mouse. Moreover the NH2-terminal sequence of E587 shows similarity with L1 and Ng-CAM. The E587 immunostaining pattern in the fish retinotectal pathway suggests that the E587 antigen is a growth-associated molecule on fish retinal axons. In fish embryos, all retinal axons are labeled. In adult fish, however, only the young axons from newly added ganglion cells carry E587 staining. After optic nerve transection (ONS) and retinal axonal regeneration, all axons reexpress the E587 antigen into their terminal processes in the tectal retinorecipient layers. The reexpression of the E587 antigen is temporally regulated, and E587 immunoreactivity declines by 7 months and disappears at 12 months after ONS. We hypothesize that the E587 antigen may mediate axon-axon associations. In its restricted appearance on young axons in normal adult fish, it may contribute to the selective fasciculation of the newest axons with young axons and thus participate in the creation of the age-related fiber organization in the fish optic nerve.