Activity-driven sharpening of the retinotectal projection in goldfish: Development under stroboscopic illumination prevents sharpening

Blocking or synchronizing activity during regeneration of the retinotectal projection prevents both the sharpening of the retinotopic map recorded on tectum and the refinement of the structure of individual arbors within the plane of the map, and this refinement is triggered by N-methyl-d-aspartate (NMDA) receptors. We tested whether activity-driven refinement also occurs during development of the projection in larval and young adult goldfish. Shortly after hatching, larval goldfish were placed into tanks within light-tight chambers illuminated by a xenon strobe at 1 Hz for 14 h of each daily cycle. Fish were reared for 1.5–2 years, until large enough to record in our retinotectal mapping apparatus (6 cm length). Age- and size-matched controls had normal maps with multiunit receptive fields (MURFs) recorded at each tectal point of 10.8° (0.16 S.E.M., n = 5), whereas the strobe-reared fish had only roughly retino-topic maps with much enlarged MURFs averaging 26.7° (1.41 S.E.M., n = 5). This enlargement represents an abnormal convergence onto each tectal point, as the maps failed to sharpen during development. The arbors of individual retinal axons were stained with horseradish peroxidase (HRP) in larval fish and in adult strobereared and control fish. They were drawn with camera lucida from tectal whole mounts, and analyzed for spatial extent in the plane of the retinotopic map, order of branching, number of branch endings, depth of termination, and caliber of the parent axon. Arbors from larval fish (1–2 weeks) were small (approximately 50 × 40 μm) with less than 10 branches, occupied a single strata, and could not be separated into different classes by caliber of axon. The 87 arbors stained in control adult fish (6 cm long) were much like previously examined adult arbors, with those from fine, medium, and coarse axons averaging 115, 166, and 194 μm in extent, respectively, and having 17–24 branch endings. The 110 arbors from 12 strobe-reared fish were often abnormal. Although the fasciculation was normal, the extrafascicular routes were abnormal with reversing turns. The axons often had branches along their course, and these branches were scattered across a wider extent, rather than forming a distinct cluster. In contrast, neither the number of branches nor the depths of termination was significantly changed in any group. The coarse caliber arbors were most abnormal, being 64% longer and 30% wider than controls. The fine caliber arbors were also significantly larger by about 20%, but the medium caliber arbors were not enlarged. The enlarged arbors partially account for the unsharpened electrophysiological maps. Together the results show that during development, as well as during regeneration, the retinotectal map is subject to an activity-driven sharpening process. © 1993 John Wiley & Sons, Inc.     

Activity-driven sharpening of the regenerating retinotectal projection: Effects of blocking or synchronizing activity on the morphology of individual regenerating arbors

Both blocking activity with intraocular tetrodotoxin (TTX) and synchronizing activity with a xenon strobe light (1 Hz) prevent retinotopic sharpening of regenerating optic projection in goldfish (Meyer, 1983; Schmidt, 1985; Cook and Rankin, 1986). In this study, we tested, in both normal and regenerating projections, the effects of these two treatments on individual optic arbors. Arbors were stained via anterograde transport of HRP, drawn in camera lucida from tectal whole mounts, and analyzed for spatial extent in the plane of the retinotopic map, order of branching, number of branch endings, depth of termination, and the caliber of the parent axon. In normal tectum, fine, medium, and coarse caliber axons gave rise to small, medium, and large arbors, which averaged 127 μm, 211 μm and 275 μm in horizontal extent, and terminated at characteristic depths. All three classes averaged roughly 21 branch endings. Optic arbors that regenerated with normal patterns of activity returned to a roughly normal appearance by 6–11 weeks postcrush: the same three calibers of axons gave rise to the same three sizes of arbors at the same depths, but they were much less stratified and were on average about 16% larger in horizontal extent. At this time point, arbors regenerated under TTX or strobe were on the average 71 and 119% larger, respectively, than the control-regenerated arbors (larger in all classes), although they had approximately the same number of branch endings and were equally poorly stratified. Synapses formed under strobe were also normal in appearance. Thus the only significant effect of both strobe and TTX treatment was to enlarge the spatial extent of arbor branches. Arbors that were not regenerating were very slightly (but significantly) enlarged by TTX block of activity or strobe illumination. As previous staining showed that regenerating axons initially make widespread branches and later retract many of those branches (Schmidt, Turcotte, Buzzard, and Tieman, 1988; Stuermer, 1988), the present findings support the idea that blocking activity or synchronizing activity prevents retinotopic sharpening by interfering with the elimination of some of the errant branches.     

Activity-driven sharpening of the regenerating retinotectal projection: effects of blocking or synchronizing activity on the morphology of individual regenerating arbors.

Both blocking activity with intraocular tetrodotoxin (TTX) and synchronizing activity with a xenon strobe light (1 Hz) prevent retinotopic sharpening of regenerating optic projection in goldfish (Meyer, 1983; Schmidt, 1985; Cook and Rankin, 1986). In this study, we tested, in both normal and regenerating projections, the effects of these two treatments on individual optic arbors. Arbors were stained via anterograde transport of HRP, drawn in camera lucida from tectal whole mounts, and analyzed for spatial extent in the plane of the retinotopic map, order of branching, number of branch endings, depth of termination, and the caliber of the parent axon. In normal tectum, fine, medium, and coarse caliber axons gave rise to small, medium, and large arbors, which averaged 127 microns, 211 microns and 275 microns in horizontal extent, and terminated at characteristic depths. All three classes averaged roughly 21 branch endings. Optic arbors that regenerated with normal patterns of activity returned to a roughly normal appearance by 6-11 weeks postcrush: the same three calibers of axons gave rise to the same three sizes of arbors at the same depths, but they were much less stratified and well on average about 16% larger in horizontal extent. At this time point, arbors regenerated under TTX or strobe were on the average 71 and 119% larger, respectively, than the control-regenerated arbors (larger in all classes), although they had approximately the same number of branch endings and were equally poorly stratified. Synapses formed under strobe were also normal in appearance. Thus the only significant effect of both strobe and TTX treatment was to enlarge the spatial extent of arbor branches. Arbors that were not regenerating were very slightly (but significantly) enlarged by TTX block of activity or strobe illumination. As previous staining showed that regenerating axons initially make widespread branches and later retract many of those branches (Schmidt, Turcotte, Buzzard, and Tieman, 1988; Stuermer, 1988), the present findings support the idea that blocking activity or synchronizing activity prevents retinotopic sharpening by interfering with the elimination of some of the errant branches.     

Rotation of the tectal primordium reveals plasticity of target recognition in retinotectal projection

Retinotectal projection is precisely organized in a retinotopic manner. In normal projection, temporal retinal axons project to the rostral part of the tectum, and nasal axons to the caudal part of the tectum. The two-dimensional relationship between the retina and the tectum offers a useful experimental system for analysis of neuronal target recognition. We carried out rotation of the tectal primordium in birds at an early stage of development, around the 10-somite stage, to achieve a better understanding of the characteristics of target recognition, especially the rostrocaudal specificity of the tectum. Our results showed that temporal retinal axons projected to the rostral part of the rotated tectum, which was originally caudal, and that nasal axons projected to the caudal part of the rotated tectum, which was originally rostral. Therefore, the tectum that had been rotated at the 10-somite stage received normal topographic projection from the retinal ganglion cells. Rostrocaudal specificity of the tectum for target recognition is not determined by the 10-somite stage and is acquired through interactions between the tectal primordium and its surrounding structures.     

金鱼再生的视神经在顶盖投射精确化的DiI顺行标记研究

本文用微量显微注射法,在金鱼视网膜的背侧用亲脂类荧光染料DiI标记少量神经节细胞,通过顺行标记研究了视神经再生过程中视网膜顶盖投射的精确化过程。在损伤视神经后的不同时期观察了再生视神经纤维在顶盖整装片上的分布。在再生早期它们以超出正常的途径由背腹两侧进入顶盖,广泛分布。但其中大部分仍分布于顶盖腹侧的靶区。在再生晚期通过精确化,重建如正常鱼一样精确的视网膜顶盖投射。这个精确化过程表现在以下三方面:(1)再生于顶盖错误区域的再生视神经纤维的消失;(2)再生早期视神经纤维主干上生长的侧部分支的消失;(3)到达靶区的再生视神经纤维形成重迭的终末分支。由以上结果推测,顶盖中可能存在两类不同的因子:一类是普通诱向因子,存在于整个顶盖中,它在再生早期引导再生的视神经纤维长入顶盖。另一类是神经营养因子,它具区域特异性,在再生晚期引导视神经纤维到达顶盖靶区,形成精确的视网膜顶盖投射。     

Dynamic changes in optic fiber terminal arbors lead to retinotopic map formation: an in vivo confocal microscopic study

Dynamic remodeling of retinal ganglion cell terminal arbors has been proposed to contribute to formation of the topographically ordered retinotectal projection. To test this directly, the growth of individual terminal arbors was observed in live X. laevis tadpoles using a confocal microscope to visualize their complex three-dimensional structure. During initial development, nasal and temporal retinal arbors covered overlapping tectal areas. Despite subsequent remodeling, the dimensions and positions of the temporal arbors remained relatively stable. In contrast, the nasal arbors grew caudally, as they extended caudal branches and retracted rostral branches. These results suggest that differences in the remodeling of the nasal and temporal arbors lead to the emergence of retino-topography along the rostrocaudal axis of the tectum. All the terminal arbors were dynamic, including those with stable dimensions, suggesting that continual remodeling of arbors may be a universal feature of neuronal projections.     

Goldfish retinal axons respond to position-specific properties of tectal cell membranes in vitro

Using a special in vitro assay, we tested whether retinal ganglion cell axons in an adult vertebrate, the goldfish (which can regenerate a retinotopic projection after optic nerve section), recognize position-specific differences in cell surface membranes of their target, the tectum opticum. On a surface consisting of alternating stripes of membranes from rostral and caudal tectum, temporal axons accumulate on membranes derived from their retinotopically related rostral tectal half. Nasal axons grow randomly over both types of membranes. Nasal and temporal axons can elongate on both rostral and caudal membranes. A quantitative growth test, however, revealed that caudal membranes are less permissive substrates for the outgrowth of temporal axons than rostral membranes, and than rostral or caudal membranes for nasal axons.     

The development of ipsilateral retinal projections into the tectum in the cichlid fish Haplochromis burtoni: a Dil study in fixed tissue.

The normal development of the retinal projection was studied in a bony fish with Dil. Between 5.5 and 10 days postfertilization the contralateral retinal projection grows from the rostral pole of the tectum across its center. A maximum of 15 retinal fibers reaches the ipsilateral tectum. In 33-day-old juvenile animals, less than 15 ipsilateral fibers terminate in the entire tectum. Ipsilaterally projecting ganglion cells (maximal number = 20 cells) are scattered throughout the entire retina, and the location of ganglion cells in the retina and axonal terminations in the tectum display a large interindividual variability. This suggests that the small adult contingent of ipsilateral fibers in this bony fish develops without an initial exuberant ipsilateral retinal projection that is later pruned back.     

Alterations in the Xenopus retinotectal projection by antibodies to Xenopus N-CAM

The patterned neural projection from the eye to the optic tectum of lower vertebrates (the retinotectal projection) has been proposed to be ordered by interactions between the optic nerve fibers and their surrounding tissues. To investigate the role of one such defined cell interaction, agarose implants containing antibodies to the neural cell adhesion molecule, N-CAM, were inserted into the tectum of the African clawed frog, Xenopus laevis. Both monoclonal and polyclonal antibodies against N-CAM reversibly and specifically distorted the pattern of the retinotectal projection, decreasing the precision of the projection as determined by electrophysiological techniques as well as decreasing the density of retinal innervation of the tectum and the branching of single axons as determined by horseradish peroxidase tracing. The anatomical effects became maximal at 4 to 6 days after implantation and returned to undetectable levels by 2 weeks, whereas the physiological effects became maximal by 8 to 10 days and a normal physiological map was reestablished within 4 weeks. The results are consistent with the hypothesis that anti-N-CAM antibodies perturb the ongoing growth and retraction of the terminal arbors of the optic nerve fibers, such that a region of the tectum becomes largely denuded of fibers. The physiological defects may then be a consequence both of the initial retraction of optic nerve terminals and of the rapid ingrowth of the perturbed and neighboring optic nerve fibers into the denuded region after the antibodies were cleared from the tectum. These results support the concept of a major role for N-CAM-mediated adhesion during map regeneration and maintenance.     

The development of ipsilateral retinal projections into the tectum in the cichlid fish Haplochromis burtoni: A dil study in fixed tissue

The normal development of the retinal projection was studied in a bony fish with Dil. Between 5.5 and 10 days postfertilization the contralateral retinal projection grows from the rostral pole of the tectum across its center. A maximum of 15 retinal fibers reaches the ipsilateral tectum. In 33-day-old juvenile animals, less than 15 ipsilateral fibers terminate in the entire tectum. Ipsilaterally projecting ganglion cells (maximal number = 20 cells) are scattered throughout the entire retina, and the location of ganglion cells in the retina and axonal terminations in the tectum display a large interindividual variability. This suggests that the small adult contingent of ipsilateral fibers in this bony fish develops without an initial exuberant ipsilateral retinal projection that is later pruned back. © 1992 John Wiley & Sons, Inc.     

Computational modeling of retinotopic map development to define contributions of EphA-ephrinA gradients, axon-axon interactions, and patterned activity

The topographic projection of retinal ganglion cell (RGC) axons to mouse superior colliculus (SC) or chick optic tectum (OT) is formed in three phases: RGC axons overshoot their termination zone (TZ); they exhibit interstitial branching along the axon that is topographically biased for the correct location of their future TZ; and branches arborize preferentially at the TZ and the initial exuberant projection refines through axon and branch elimination to generate a precise retinotopic map. We present a computational model of map development that demonstrates that the countergradients of EphAs and ephrinAs in retina and the OT/SC and bidirectional repellent signaling between RGC axons and OT/SC cells are sufficient to direct an initial topographic bias in RGC axon branching. Our model also suggests that a proposed repellent action of EphAs/ephrinAs present on RGC branches and arbors added to that of EphAs/ephrinAs expressed by OT/SC cells is required to progressively restrict branching and arborization to topographically correct locations and eliminate axon overshoot. Simulations show that this molecular framework alone can develop considerable topographic order and refinement, including axon elimination, a feature not programmed into the model. Generating a refined map with a condensed TZ as in vivo requires an additional parameter that enhances branch formation along an RGC axon near sites that it has a higher branch density, and resembles an assumed role for patterned neural activity. The same computational model generates the phenotypes reported in ephrinA deficient mice and Isl2-EphA3 knockin mice. This modeling suggests that gradients of counter-repellents can establish a substantial degree of topographic order in the OT/SC, and that repellents present on RGC axon branches and arbors make a substantial contribution to map refinement. However, competitive interactions between RGC axons that enhance the probability of continued local branching are required to generate precise retinotopy.     

Retinal projections in the chain pickerel (Esox niger Lesueur)

Summary The retinal projections inEsox niger, as determined with the aid of a modified cobalt-lysine method, are considerably more extensive in the diencephalon and pretectum than in other teleost fishes so far examined. Although most retinal axons terminate contralaterally, rare fibers can be traced to the same aggregates ipsilaterally. The retinohypothalamic projection appears larger than hitherto reported in teleosts, and the dorsomedial optic tract issues fibers to a series of cell clusters extending from the rostral thalamus to mid-torus levels. A retinal projection to a presumed ventrolateral optic nucleus (VLO) is described for the first time in a teleost. Other targets of retinal fibers include the nucleus geniculatus lateralis ipse of Meader (GLI), the pretectal nucleus (P), the cortical nucleus and a well-developed ventromedial optic nucleus (VMO). The projection to the optic tectum is principally to the stratum fibrosum et griseum superficiale (SFGS) and stratum marginale (SM), but a considerable number of axons also course through the stratum album centrale (SAC) before terminating there or piercing the stratum griseum centrale (SGC) and terminating in SFGS. Rare terminal arborizations of retinal fibers were also observed in stratum griseum centrale (SGS) and in the stratum griseum periventriculare (SGC) in restricted portions of the tectum. Because of the relatively large size of the visual structures inE. niger it is a potentially useful model for future experimental studies on the visual system.     

Topographic targeting and pathfinding errors of retinal axons following overexpression of ephrinA ligands on retinal ganglion cell axons

In the retinotectal projection, the Eph receptor tyrosine kinase ligands ephrinA2 and ephrinA5 are differentially expressed not only in the tectum, but also in a high-nasal-to-low-temporal pattern in the retina. Recently, we have shown that retrovirally driven overexpression of ephrinA2 on retinal axons leads to topographic targeting errors of temporal axons in that they overshoot their normal termination zones in the rostral tectum and project onto the mid- and caudal tectum. The behavior of nasal axons, however, was only marginally affected. Here, we show that overexpression of ephrinA5 affects the topographic targeting behavior of both temporal and nasal axons. These data reinforce the idea that differential ligand expression on retinal axons contributes to topographic targeting in the retinotectal projection. Additionally, we found that ectopic expression of ephrinA2 and ephrinA5 frequently leads to pathfinding errors at the chiasm, resulting in an increased stable ipsilateral projection.     

Regenerated retinal ganglion cell axons form normal numbers of boutons but fail to expand their arbors in the superior colliculus

Regenerated retinal ganglion cell (RGC) axons can re-form functional synapses with target neurons in the superior colliculus (SC). Because preterminal axon branching determines the size, shape and density of innervation fields, we investigated the branching patterns and bouton formation of individual RGC axons that had regrown along peripheral nerve (PN) grafts to the SC. Within the superficial layers of the SC, the regenerated axons formed terminal arbors with average numbers of terminal boutons that were similar to the controls. However, axonal branches were shorter than normal so that the mean area of the regenerated arbors was nearly one-tenth that of control arbors and the resulting fields of innervation contained greater than normal numbers of synapses concentrated in small areas of the target. Our results have delineated a critical defect in the reconstitution of retino-collicular circuitry in adult mammals: the failure of terminal RGC branches to expand appropriately. Because recent studies have documented that brain-derived neurotrophic factor (BDNF) can specifically lengthen RGC axonal branches not only during development in the SC but also within the adult retina after axotomy, the present quantitative studies should facilitate experimental attempts to correct this deficit of the regenerative response. © 1998 Chapman and Hall     

Evidence for a driving role of ingrowing axons for the shifting of older retinal terminals in the tectum of fish.

In amphibians and teleosts, retina and tectum grow incongruently. In order to maintain the retinotopy of the retinotectal projection, Gaze, Keating, and Chung (1974) postulated a shifting of terminals throughout growth. In order to test the possibility that ingrowing retinal fibers are the driving force for this shifting, we induced a permanent retinal projection into the ipsilateral tectum in juveniles of the cichlid fish Haplochromis burtoni. The surface of the tectum had increased (11-18 months later) 2.5-5.8 times, and the surface of the retina 8.6-14 times. Filling of ganglion cells with horseradish peroxidase (HRP) retrogradely from the tectum showed ipsilaterally regenerating ganglion cells only in the center of the retina. The position of ganglion cells indicated that the ipsilateral projection derived only from axotomized and regenerating retinal ganglion cells but not from those newly born. Ipsilaterally projecting retinal fibers showed terminals only in the rostral half of the tectum. Comparison of area of terminations of ipsilaterally projecting ganglion cells at various times after the crush provided no evidence for expansion or a shift into caudal tectal areas throughout the period of growth. These findings are compatible with the idea that newly ingrowing fibers induce older terminals to move caudally.     

Evidence for a driving role of ingrowing axons for the shifting of older retinal terminals in the tectum of fish

In amphibians and teleosts, retina and tectum grow incongruently. In order to maintain the retinotopy of the retinotectal projection, Gaze, Keating, and Chung (1974) postulated a shifting of terminals throughout growth. In order to test the possibility that ingrowing retinal fibers are the driving force for this shifting, we induced a permanent retinal projection into the ipsilateral tectum in juveniles of the cichlid fish Haplochromis burtoni. The surface of the tectum had increased (11–18 months later) 2.5–5.8 times, and the surface of the retina 8.6–14 times. Filling of ganglion cells with horseradish peroxidase (HRP) retrogradely from the tectum showed ipsilaterally regenerating ganglion cells only in the center of the retina. The position of ganglion cells indicated that the ipsilateral projection derived only from axotomized and regenerating retinal ganglion cells but not from those newly born. Ipsilaterally projecting retinal fibers showed terminals only in the rostral half of the tectum. Comparison of area of terminations of ipsilaterally projecting ganglion cells at various times after the crush provided no evidence for expansion or a shift into caudal tectal areas throughout the period of growth. These findings are compatible with the idea that newly ingrowing fibers induce older terminals to move caudally.     

Presynaptic protein kinase C controls maturation and branch dynamics of developing retinotectal arbors: Possible role in activity‐driven sharpening

Visual activity refines developing retinotectal maps and shapes individual retinal arbors via an NMDA receptor‐dependent mechanism. As retinal axons grow into tectum, they slow markedly and emit many transient side branches behind the tip, assuming a “bottlebrush” morphology. Some branches are stabilized and branch further, giving rise to a compact arbor. The dynamic rate of branch addition and deletion is increased twofold when MK801 is used to block NMDA receptors, as if this prevents release of a stabilizing signal such as arachidonic acid (AA) from the postsynaptic neuron. In optic tract, AA mediates NCAM and L1 stimulation of axon growth by activating presynaptic protein kinase C (PKC) to phosphorylate GAP‐43 and stabilize F‐actin, and, if present in tectum, this growth control pathway could be modulated by postsynaptic activation. To test for the effects on arbor morphology of blocking PKC or AA release, we examined DiO‐labeled retinal axons of larval zebrafish with time‐lapse videomicroscopy. Bath application of the selective PKC inhibitor bisindolylmaleimide from 2 or 3 days onward doubled the rate at which side branches were added and deleted, as seen with MK801, and also prevented maturation of the arbor so that it retained a “bottlebrush” morphology. In order to selectively block the PKC being transported to retinal terminals, we injected the irreversible inhibitor calphostin C into the eye from which the ganglion cells were labeled, and this produced both effects seen with bath application. In contrast, there were no effects of control injections, which included Ringers into the same eye and the same dose into the opposite eye (actually much closer to the tectum of interest), to rule out the possibility that the inhibitor leaked from the eye to act on tectal cells. For comparison, we examined arbors treated with the NMDA blocker MK801 at half‐hour time‐lapse intervals, and detected the twofold rise in rates of branch addition and deletion previously reported in Xenopus larvae, but not the structural effect seen with the PKC inhibitors. In addition, we could produce both effects seen with PKC inhibitors by using RHC80267 to block AA release from DAG lipase, indicating that AA is the main drive for PKC activation. Thus, the results show a distinct role of AA and presynaptic PKC in both maturation of arbor structure and in the dynamic control of branching. The effects on branch dynamics were present regardless of the level of maturity of arbor structure. The fact that they mimicked those of MK801 suggests that presynaptic PKC may be involved in the NMDA receptor‐driven stabilization of developing retinal arbors. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 328–340, 2004     

Restoration of retino-tectal connections in frogs during regeneration of the optic nerve

At various times after unilateral division of the optic nerve in the frogRana temporaria L. evoked potentials in response to electrical stimulation of the optic nerve were investigated in a segment distal to the site of operation, spike activity was recorded from endings of regenerating and intertectal axons when stimuli of different shapes were placed in the field of vision, and the distribution of axonal bulbs of growth by depth in the tectum mesencephal was studied electron-microscopically. During regeneration of the axons the responses of the retinal ganglionic cells to visual stimuli retained most of their individual features. Myelinated axons of the retinal ganglionic cells regenerate first (starting on the 21st day after operation). Myelination of these fibers lags significantly behind their growth and is complete more than 100 days after the operation. Unmyelinated axons of the retinal ganglionic cells grow up toward the tectum mesencephali after myelinated axons (80 or more days after the operation). Axonal bulbs of growth in the initial periods after the operation are located close to the pial surface and the level of spread of the myelinated axons of the retinal ganglionic cells differs significantly from their normal level of localization. Intertectal connections persist after division of the nerve and are activated by visual stimuli during regeneration of the axons of the retinal ganglionic cells. Connections were found mainly between intertectal fibers terminating superficially and retinal ganglionic cells belonging to class 1 and 2 detectors. Axons of the retinal ganglionic cells grow up toward the caudal region of the tectum mesencephali later than toward the rostral region.A. N. Severtsov Institute of Evolutionary Morphology and Ecology of Animals, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 5, No. 6, pp. 611–620, November–December, 1973.     

Dynamic responses of Xenopus retinal ganglion cell axon growth cones to netrin-1 as they innervate their in vivo target

Netrin-1 influences retinal ganglion cell (RGC) axon pathfinding and also participates in the branching and synaptic differentiation of mature RGC axons at their target. To investigate whether netrin also serves as an early target recognition signal in the brain, we examined the dynamic behavior of Xenopus RGC axons soon after they innervate the optic tectum. Time-lapse confocal microscopy imaging of RGC axons expressing enhanced yellow fluorescent protein demonstrated that netrin-1 is involved in early axon branching, as recombinant netrin-1 halted further advancement of growth cones into the tectum and induced back branching. RGC growth cones exhibited differential responses to netrin-1 that depended on the degree of differentiation of the axon and the developmental stage of the tadpole. Netrin-1 decreased the total number of branches on newly arrived RGC growth cones at the target, but increased the dynamic branching of more mature arbors at the later developmental stage. To further explore the response of axonal growth cones to netrin, Xenopus RGC axons were followed in culture by time-lapse imaging. Exposure to netrin-1 rapidly increased the forward advancement of the axon and decreased the size and expanse of the growth cone, while also inducing back branching. Taken together, the differential in vivo and in vitro responses to netrin-1 suggest that netrin alone is not sufficient to induce the cessation of growth cone advancement in the absence of a target but can independently modulate axon branching. Collectively, our findings reveal a novel role for netrin on RGC axon branch initiation as growth cones innervate their target.     

Mechanisms in the development of retinotectal projections in the chick embryo studied by surgical deflection of the retinal pathway

Retinotopic analysis of the pathways of normal and aberrant retinal axons within the tectum of developing chick embryos was performed by selective labeling of retinal axons with a fluorescent dye, rhodamine-B isothiocyanate. To produce aberrant retinal axons, the presumptive optic chiasma was surgically disorganized at the 3rd day of incubation. At the 11th and 13th days of incubation, more than half of the operated embryos exhibited several aberrant retinal axons which reached ectopic parts of the tectum. The pathways of these aberrant axons within the tectum depended on the position of their initial invasion into the tectum at the diencephalotectal junction, and not on their position of origin within the retina. The aberrant retinal axons did not show any sign of correction of their pathways toward their normal sites of innervation within the tectum. As development proceeded, elimination of the aberrant retinal axons occurred. By the 16th day of incubation, almost all operated embryos lacked aberrant retinal axons and although the total number of axons often appeared reduced, a nearly normal topography of retinotectal projections was established. These findings indicate that the initial invasion of the retinal axons into the tectum is conducted predominantly by nonspecific mechanisms and, thereafter, a selective maintenance of appropriate retinal axons occurs.