Aber Lenken Sie Auch? (But do they also guide?)

One of the most intriguing problems in developmental neurobiology is that of how growing axons find the correct way to their proper target cells. Often axonal connections are organized in topographic maps, where neighboring cells of the projecting area are connected to neighboring cells in the target area, thus allowing a faithful transfer of positionally stored information from one area to another. The retinotectal projection is the classical model system for studying topographic projections.     

Competitive and positional cues in the patterning of nerve connections

The visual system of lower vertebrates has served as an important testing ground for the mechanisms that generate topographic neuronal connections. During both the outgrowth and the regeneration of the optic nerve, a smoothly ordered map of the retina is formed on its major target, the optic tectum (the retinotectal projection). Experiments performed on this projection have offered support for a variety of mechanisms, including the matching of positional cues in the retina and tectum, the guidance of nerve fibers by interactions between fibers, competition for synaptic space, and the refinement of connections based on neuronal activity. Unfortunately, individual experiments that support any one of these mechanisms have been taken at times as evidence against the involvement of any other mechanism; for example, experiments demonstrating the importance of positional cues have been thought mistakenly to indicate that activity-based interactions are unimportant. Computer simulations, in which multiple, somewhat opposed, mechanisms are allowed to operate in concert demonstrate that such a hybrid model is able to generate a full range of experimental results. More importantly, the elimination of any one of the mechanisms renders the model unable to fit entire classes of findings. Thus, the patterning of the retinotectal projection is best viewed as a process in which the optic nerve terminals attempt to satisfy multiple constraints in selecting their target sites.     

Fiber optic mapping of the Xenopus visual system: shift in the retinotectal projection during development

Two new techniques for assaying the retina to tectum connections in the lower vertebrate visual system are presented. These techniques allow defined regions of the retina to be stimulated, thus circumventing some of the difficulties of the more conventional retinotectal mapping techniques. Applying these techniques to the Xenopus visual system demonstrates that the retina-to-tectum projection shifts during development. The central part of the retinotectal projection moves medially and caudally about 150 microns (10% of the size of the tectum) in two weeks. The presence of such plasticity in a normal developing animal indicates that the plasticity previously observed in experimentally altered animals probably reflects a normal developmental process.     

Assembly of lamina-specific neuronal connections by slit bound to type IV collagen

The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons.     

Pattern regulation in the eyebud of Xenopus studied with a vital-dye fiber-tracing technique

Evidence for pattern regulation in the developing Xenopus visual system has previously been obtained after surgical manipulations of the eyebud early in development. In one experimental paradigm, a "compound" eye is produced by combining a nasal (anterior) half-eyebud with normal dorsoventral polarity and a temporal (posterior) half-eyebud with inverted dorsoventral polarity. The adult retinotectal projection from such compound eyes, as assayed by electrophysiological mapping techniques, shows normal dorsoventral polarity in both halves, indicating an apparent reversal in the polarity of the surgically-inverted half. We have utilized a fluorescent vital-dye fiber-tracing technique to investigate the early events in this regulatory process. The results show that the change in dorsoventral polarity is not due to cell movements in the eyebud after surgery. Interestingly, the experiments also demonstrate that the pattern of connections initially formed by the developing eye does not reflect the pattern regulation observed in the adult retinotectal map; instead, the temporal half of the eye projects to the tectum with inverted dorsoventral order. Thus, the regulation observed in the adult does not become evident in the pattern of the projection until after early larval development.     

Adhesive Interactions and the Patterning of Nerve Connections: An Experimental and Theoretical Approach

Adhesive interactions have long been proposed to play a centralrole in the patterning of neural structures and their interconnections.Many of these ideas are based upon experiments on the projectionfrom the eye to the optic tectum (the retinotectal projection)in lowervertebrates. In order to test the feasibility of suchproposals, a detailed model, based largely on adhesive interactionsbetween cells, has been developed. Computer simulations of themodelshow that simple adhesive interactions are sufficient toexplain much of the literature on the patterning of the amphibianretinotectal projection. Aspects of the model have been experimetallytested through the use of antibodies to known adhesive molecules.The results of these experiments appear consistent with therole of adhesion in the patterning of the connections and withthe predictions of the model. Although such experiments demonstratethe power of adhesive cell interactions in the patterning ofnerve connections, additional experiments and simulations demonstratethat some other non-adhesive processes may play a role. In particular,the addition of a process that is dependent on the activityof the neurons allows the model to better fit the literature.An activity-dependent competition between neurons for adhesivesites on the target cells appears to be sufficient to play thisrole.     

Cross-species axial signaling, with realignment of retinal axes, in embryonic Xenopus eyes

The locus specificities which enable retinal ganglion cells to assemble a topographic retinotectal map are patterned about a pair of (anteroposterior and dorsoventral) retinal axes set down in the early eye bud. We have transplanted a Xenopus laevis eye bud, at stage $\text{23}\text{24}$ when the retinal field is still responsive to the axial signals from the surrounding tissues, into the enucleated eye socket of a comparable stage Ambystoma maculatum embryo. Three days later, when the Xenopus eye had reached early larval stages and was no longer responsive to extraocular signals, the eye was retransplanted into the socket of the Xenopus final carrier embryo. The pattern of retinotectal connections between the eye and the carrier's optic tectum was examined by electrophysiological analysis of the visuotectal projections. The results indicated that many of the retinae had patterned locus specificities about axes derived from the salamander intermediate host. We infer that axial signaling involves fundamental cellular processes which have been highly conserved during evolution.     

The application of the Gierer-Meinhardt equations to the development of the retinotectal projection

Model calculations are presented for the several properties of the development of the retinotectal projection in amphibians and fishes, using the Gierer-Meinhardt equations. One of these properties is the maintenance of topographic mapping between the retina and the tectum during their development despite the fact that the two tissues grow in morphologically different ways. Another is the existence of a critical period, at which the coordinates of the retina with respect to the tectum are irrevocably determined. It is assumed that the connections between the retinal and the tectal cells are made on the correspondence of positional markers which are given as a form of the distribution of a specific activator, the dynamics of which is described by the Gierer-Meinhardt equations. The monotonic distributions of the activator and the existence of the critical period are shown by a computer simulation of the proliferating retina. Several changes of the retinotectal projection after surgical operations on the retina or the tectum are also explained.Some of the results in this paper were presented at the poster session of the 6th International Biophysics Congress in Kyoto 1978     

Patterning of retinotectal connections in the vertebrate visual system.

Recent work on the retinotectal projection clearly establishes the roles of neuronal activity and position-based cues in the patterning of nerve connections. In some species, the high degree of spatial order has been shown to emerge from a continued process of terminal growth and refinement. The future challenge is now to determine how multiple cues work together to guide the sculpting of the final pattern.     

Formation of retinotopic connections: Selective stabilization by an activity-dependent mechanism

During regeneration of the optic nerve in goldfish, the ingrowing retinal fibers successfully seek out their correct places in the overall retinotopic projection on the tectum. Chemospecific cell-surface interactions appear to be sufficient to organize only a crude retinotopic map on the tectum during regeneration. Precise retinotopic ordering appears to be achieved via an activity-dependent stabilization of appropriate synapses and is based upon the correlated activity of neighboring ganglion cells of the same receptive-field type in the retina. Four treatments have been found to block the sharpening process: (a) blocking the activity of the ganglion cells with intraocular tetrodotoxin (TTX), (b) rearing in total darkness, (c) correlating the activation of all ganglion cells via stroboscopic illumination and (d) blocking retinotectal synaptic transmission with alpha-bungarotoxin (alphaBTX). These experiments support a role for correlated visually driven activity in sharpening the diffuse projection and suggest that this correlated activity interacts within the postsynaptic cells, probably through the summation of excitatory postsynaptic potentials (EPSPs). Other experiments support the concept that effective synapses are stabilized: a local postsynaptic block of transmission causes a local disruption in the retinotectal map. The changes that occur during this disruption suggest that each arbor can move to maximize its synaptic efficacy. In development, initial retinotectal projections are often diffuse and may undergo a similar activity-dependent sharpening. Indirect retinotectal maps, as well as auditory maps, appear to be brought into register with the direct retinotopic projections by promoting the convergence of contacts with correlated activity. A similar mechanism may drive both the formation of ocular dominance patches in fish tectum and kitten visual cortex and the segregation of different receptive-field types in the lateral geniculate nucleus. Activity-dependent synaptic stabilization may therefore be a general mechanism whereby the diffuse projections of early development are brought to the precise, mature level of organization.     

Rapid activity-dependent sprouting of optic fibers into a local area denervated by application of beta-bungarotoxin in goldfish tectum

The retinotectal projection is known to be capable of extensive long-term expansion of connections, but it is not known how fast such changes can occur or what triggers sprouting of terminals. We studied sprouting of optic fibers into an area denervated by local microinjection of beta-bungarotoxin (β-BTX), a specific presynaptic neurotoxin with phospholipase A₂ activity that destroys nerve terminals at the neuromuscular junction. After injection of 0.1 pmol of β-BTX, the optic terminals fired spontaneously with decreasing amplitude and became silent within 1 to 2 h. Outside the injection zone, the retinotectal map was normal, so the silent zone was associated with a scotoma in the visual field. Horseradish peroxidase (HRP) staining of the entire optic nerve showed a denervated region at the injection site with beaded, degenerating fibers at its edge. Between 3 and 9 days later, optic units were recorded within the injection zone whose receptive fields lay just outside the scotoma in the visual field, indicating that intact surrounding terminals had sprouted into the area. These sprouts made functional connections, as indicated by field potential recordings and current source-density analysis. At this time, HRP staining also demonstrated retinal innervation within the injection zone. By 12 days, normal maps with no scotoma were recorded and HRP staining was normal at the injection site, indicating that the β-BTX-damaged fibers had regenerated to reclaim their tectal sites. The results show that the retinotectal projection of goldfish is very dynamic, since intact optic fibers can sprout into adjacent vacant postsynaptic territory within 2 to 3 days, much faster than previously reported. In a final experiment, we showed that this sprouting is activity-dependent, since it could be prevented by blocking retinal activity with intraocular tetrodotoxin (TTX) during the first 2 days postinjection, even though TTX block of activity does not block regeneration in this system. One possible mechanism for this rapidly triggered sprouting is that arachidonic acid liberated by β-BTX acts as a sprouting factor to attract surrounding healthy fibers into the denervated region but requires activity at the terminals to be effective. © 1996 John Wiley & Sons, Inc.     

Pathways of Retinotectal Projection in Developing Xenopus Tadpoles Revealed by Selectively Labeling of Retinal Axons with Horseradish Peroxidase (HRP)

Anatomical mapping was made of the retinal central pathways from the chiasm to the targets within the tectum in the developing Xenopus tadpoles, after labeling a specific regional population of retinal axons with horseradish peroxidase (HRP). In the tadpoles at stage 50, pathway sorting of retinal axons within the optic tract was clear for the dorsoventral axis of the retina, but not for the nasotemporal axis. Most nasal retinal axons and some dorsal and ventral retinal axons invaded the tectum directly at the diencephalotectal junction, and arrived at their correct sites of innervation after running through ectopic parts of the tectum. These findings indicate that the pathway orientation before targets is not a prerequisite factor for establishment of the orderly map of the retinotectal projection. Rather, a direct interaction between ingrowing retinal axons and tectal cells seems to be a predominant factor for specification of retinal central connections.     

Development and regeneration of the retinotectal map in goldfish: a computational study

We present a simple computational model to study the interplay of activity-dependent and intrinsic processes thought to be involved in the formation of topographic neural projections. Our model consists of two input layers which project to one target layer. The connections between layers are described by a set of synaptic weights. These weights develop according to three interacting developmental rules: (i) an intrinsic fibre-target interaction which generates chemospecific adhesion between afferent fibres and target cells; (ii) an intrinsic fibre-fibre interaction which generates mutual selective adhesion between the afferent fibres; and (iii) an activity-dependent fibre-fibre interaction which implements Hebbian learning. Additionally, constraints are imposed to keep synaptic weights finite. The model is applied to a set of eleven experiments on the regeneration of the retinotectal projection in goldfish. We find that the model is able to reproduce the outcome of an unprecedented range of experiments with the same set of model parameters, including details of the size of receptive and projective fields. We expect this mathematical framework to be a useful tool for the analysis of developmental processes in general. <br>     

Synaptic organization of retinotectal connections of the frog application of pulse triggered averaging

Pulse-triggered averaging technique was applied to retinotectal connections of the frog. An extracellular single unit was first isolated from the terminals of retinal fibers, and then intracellular responses were recorded from a tectal neuron in the vicinity of the extracellular recording electrode. Intracellular potentials in response to a moving stimulus were averaged by triggering with the isolated presynaptic impulses. The results show that "on-off" retinal fibers monosynaptically excite E-E type (EPSP at "on" and "off" of light) and EI-EI type (EPSP-IPSP at "on" and "off" of light). One of the E-E type neurons was identified as a large ganglionic neuron in layer 8.     

Self-organizing mechanism for the formation of ordered neural mappings

A model for the formation of ordered neural mappings in general, and of retinotectal connections, in particular is given. The main point came from the theory of noise induced transitions, i.e. order may be the result of the interplay between deterministic and random interactions. An activity-dependent self-organizing mechanism is presented in terms of modifiable synapses. Simulation experiments were done not only for the normal ontogenetic development but also for the plastic behaviour of the retinotopic connections.     

Ganglioside patterns and cholera toxin--peroxidase labeling of aggregating cells from the chick optic tectum.

2The ganglioside compositions of the chick optic tectum and aggregating tectal cell cultures were examined. Both showed similar trends in changes in ganglioside patterns during development. GD3 and GD1b were the predominant gangliosides early in development, while GD1a and several other multisialogangliosides increased in relative amounts with increasing age in vivo and in vitro. Four gangliosides were present early in development which have not previously been reported. These gangliosides are not present at later developmental times suggesting a possible role for them during the critical early stages of nervous tissue differentiation. Some differences were noted when comparing in vivo versus in vitro ganglioside patterns; these differences may possibly be due to the lack of normal retinotectal connections in the cultures. Cytochemical studies on the localization of the presumed cholera toxin--peroxidase binding site GM1 showed conjugate binding correlates with increasing levels of GM1 in the cultures. In older cultures, the conjugate was uniformly localized on all cells and processes in the aggregates. The conjugate also bound to synaptic membranes and intensely stained the synaptic cleft. This latter observation suggests an enrichment of GM1 in the synaptic cleft region.     

The Tumor Suppressor Gene Retinoblastoma-1 Is Required for Retinotectal Development and Visual Function in Zebrafish

Mutations in the retinoblastoma tumor suppressor gene (rb1) cause both sporadic and familial forms of childhood retinoblastoma. Despite its clinical relevance, the roles of rb1 during normal retinotectal development and function are not well understood. We have identified mutations in the zebrafish space cadet locus that lead to a premature truncation of the rb1 gene, identical to known mutations in sporadic and familial forms of retinoblastoma. In wild-type embryos, axons of early born retinal ganglion cells (RGC) pioneer the retinotectal tract to guide later born RGC axons. In rb1 deficient embryos, these early born RGCs show a delay in cell cycle exit, causing a transient deficit of differentiated RGCs. As a result, later born mutant RGC axons initially fail to exit the retina, resulting in optic nerve hypoplasia. A significant fraction of mutant RGC axons eventually exit the retina, but then frequently project to the incorrect optic tectum. Although rb1 mutants eventually establish basic retinotectal connectivity, behavioral analysis reveals that mutants exhibit deficits in distinct, visually guided behaviors. Thus, our analysis of zebrafish rb1 mutants reveals a previously unknown yet critical role for rb1 during retinotectal tract development and visual function.     

The Caenorhabditis elegans Six/sine oculis class homeobox gene ceh-32 is required for head morphogenesis

Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. We have analyzed the distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon during the period when visual connections are being established. We have found that both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor-ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph-ephrin molecules was observed. Finally, we also show that a similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway.     

Emergence of input specificity of ltp during development of retinotectal connections in vivo.

Input specificity of activity-induced synaptic modification was examined in the developing Xenopus retinotectal connections. Early in development, long-term potentiation (LTP) induced by theta burst stimulation (TBS) at one retinal input spreads to other unstimulated converging inputs on the same tectal neuron. As the animal develops, LTP induced by the same TBS becomes input specific, a change that correlates with the increased complexity of tectal dendrites and more restricted distribution of dendritic Ca(2+) evoked by each retinal input. In contrast, LTP induced by 1 Hz correlated pre- and postsynaptic spiking is input specific throughout the same developmental period. Thus, input specificity of LTP emerges with neural development and depends on the pattern of synaptic activity.