Induction of target-directed optic axon outgrowth: effect of retinae transplanted to anophthalmic mice

In previous work using neural transplants (Hankin and Lund, 1987) we demonstrated two basic components of optic axon outgrowth in the mammalian retinotectal system: one category of outgrowth utilizes the subpial margin of the rostral brain stem as a preferential substrate (as do normal retinotectal axons); the other type of outgrowth, from retinae embedded deep within the midbrain parenchyma, is distance-dependent and highly target-oriented, but shows little apparent substrate specificity. One explanation for this directed outgrowth is that it is in response to a diffusible factor emanating from cells in the superior colliculus. In the present study we use congenitally anophthalmic mice as recipients for retinal transplants to test whether prior optic innervation of the superior colliculus plays a role in establishing either component of outgrowth. We show that outgrowth along the subpial pathway from a graft placed on the surface of the brain stem can take place in the absence of prior innervation of the superior colliculus. The target-directed outgrowth exhibited by embedded grafts only occurs if the tectum is also innervated by a second graft placed on the surface of the brain stem. It is proposed that tectal cells produce a factor in response to optic innervation and that this directs the growth patterns of embedded grafts. This suggests that optic innervation is a necessary prerequisite for the superior colliculus to produce the proposed diffusible chemotropic signal. In normal development such a factor could function to improve the efficiency of target-finding by later growing optic axons, but it might serve a quite different role, encouraging branching and trophic maintenance of the optic pathway once it has reached the tectum.     

Regulation of axial patterning of the retina and its topographic mapping in the brain

Topographic maps are a fundamental organizational feature of axonal connections in the brain. A prominent model for studying axial polarity and topographic map development is the vertebrate retina and its projection to the optic tectum (or superior colliculus). Linked processes are controlled by molecules that are graded along the axes of the retina and its target fields. Recent studies indicate that ephrin-As control the temporal-nasal mapping of the retina in the optic tectum/superior colliculus by regulating the topographically-specific interstitial branching of retinal axons along the anterior-posterior tectal axis. This branching is mediated by relative levels of EphA receptor repellent signaling. A major recent advance is the demonstration that EphB receptor forward signaling and ephrin-B reverse signaling mediate axon attraction to control dorsal-ventral retinal mapping along the lateral-medial tectal axis. In addition, several classes of regulatory proteins have been implicated in the control of the axial patterning of the retina, and its ultimate readout of topographic mapping.     

Competitive interactions between retinal ganglion axons for tectal targets explain plasticity of retinotectal projection in the servomechanism model of retinotectal mapping

The mechanism of topographic mapping of retinal ganglion cells to the midbrain was previously elucidated by the servomechanism model, which is based on the fact that cells expressing Eph-receptors respond specifically to surface expressing membrane-bound ephrin-ligands at a critical level. The retina has increased nasal-to-temporal gradient of Eph receptor-density, and the optic tectum/superior colliculus has increased rostral-to-caudal gradient of membrane-bound ephrin-ligand. An axon from the retina has an identification tag of a certain level of Eph-receptor density depending on its retinal position, and adheres to the site on the tectum/superior colliculus expressing ephrin-ligands at a critical ligand-density level. The servomechanism model rigidly defines positions of axon terminals on the midbrain. However, optic nerve regeneration experiments combined with halved retina or tectum show a plastic or flexible mapping (expansion, compression and transposition of tectal projections). To reconcile the discrepancy between the rigid model and the plastic behavior, competition between retinal axon terminals for a target site was introduced to the servomechanism. The servomechanism/competition model succeeded in computer simulations of the plastic mapping of retinal axons on the tectum. Recent experiments of upregulated ligand-density on the tectum during nerve regeneration and the role of axonal competition are discussed.     

Pax genes in development and maturation of the vertebrate visual system: implications for optic nerve regeneration

Pax genes play a pivotal role in development of the vertebrate visual system. Pax6 is the master control gene for eye development: ectopic expression of Pax6 in Xenopus laevis and Drosphila melanogaster leads to the formation of differentiated eyes on the legs or wings. Pax6 is involved in formation of ganglion cells of the retina, as well as cells of the lens, iris and cornea. In addition Pax6 may play a role in axon guidance in the visual system. Pax2 regulates differentiation of the optic disk through which retinal ganglion cell axons exit the eye. Furthermore, Pax2 plays a critical role in development of the optic chiasm and in the guidance of axons along the contralateral or ipsilateral tracts of the optic nerve to visual targets in the brain. During development Pax7 is expressed in neuronal cells of one of the major visual targets in the brain, the optic tectum/superior colliculus. Neurons expressing Pax7 migrate towards the pia and concentrate in the stratum griseum superficiale (SGFS), the target site for retinal axons. Together, expression of Pax2, 6 and 7 may guide axons during formation of functional retinotectal/collicular projections. Highly regulated Pax gene expression is also observed in mature animals. Moreover, evidence suggests that Pax genes are important for regeneration of the visual system. We are currently investigating Pax gene expression in species that display a range of outcomes of optic nerve regeneration. We predict that such information will provide valuable insights for the induction of successful regeneration of the optic nerve and of other regions of the central nervous system in mammals including man.     

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

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

Retinal neural progenitors express topographic map markers

Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Retinal ganglion cell axons project to visual centers of the brain forming circuits in precise topographic order. In chick, dorsal retinal neurons project to ventral optic tectum, ventral neurons to dorsal tectum, anterior neurons to posterior tectum and posterior neurons to anterior tectum; forming a continuous point-to-point map of retinal cell position in the tectal projection. We found that when stem cells derived from ventral retina were implanted in dorsal host retina, the stem cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors exhibit topographic markers of cell position in retina. Indeed, retinal neural progenitors express topographic markers: dorsal stem cells expressed more Ephrin B2 than ventral stem cells and, conversely, ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells. The fact that neural progenitors express topographic markers has pertinent implications in using neural stem cells in cell replacement therapy for replacing projecting neurons that express topographic order, e.g., analogous neurons of the visual, auditory, somatosensory and motor systems.     

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments

Summary Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose severed axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.     

Two homeobox genes define the domain of EphA3 expression in the developing chick retina

Graded expression of the Eph receptor EphA3 in the retina and its two ligands, ephrin A2 and ephrin A5 in the optic tectum, the primary target of retinal axons, have been implicated in the formation of the retinotectal projection map. Two homeobox containing genes, SOHo1 and GH6, are expressed in a nasal-high, temporal-low pattern during early retinal development, and thus in opposing gradients to EphA3. Retroviral misexpression of SOHo1 or GH6 completely and specifically repressed EphA3 expression in the neural retina, but not in other parts of the central nervous system, such as the optic tectum. Under these conditions, some temporal ganglion cell axons overshot their expected termination zones in the rostral optic tectum, terminating aberrantly at more posterior locations. However, the majority of ganglion cell axons mapped to the appropriate rostrocaudal locations, although they formed somewhat more diffuse termination zones. These findings indicate that other mechanisms, in addition to differential EphA3 expression in the neural retina, are required for retinal ganglion axons to map to the appropriate rostrocaudal locations in the optic tectum. They further suggest that the control of topographic specificity along the retinal nasal-temporal axis is split into several independent pathways already at a very early time in development.     

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.     

Aberrant axonal projections in mice lacking EphA8 (Eek) tyrosine protein kinase receptors.

We have generated mice homozygous for a mutation that disrupts the gene encoding EphA8, a member of the Eph family of tyrosine protein kinase receptors, previously known as Eek. These mice develop to term, are fertile and do not display obvious anatomical or physiological defects. The mouse ephA8/eek gene is expressed primarily in a rostral to caudal gradient in the developing tectum. Axonal tracing experiments have revealed that in these mutant mice, axons from a subpopulation of tectal neurons located in the superficial layers of the superior colliculus do not reach targets located in the contralateral inferior colliculus. Moreover, ephA8/eek null animals display an aberrant ipsilateral axonal tract that projects to the ventral region of the cervical spinal cord. Retrograde labeling revealed that these abnormal projections originate from a small subpopulation of superior colliculus neurons that normally express the ephA8/eek gene. These results suggest that EphA8/Eek receptors play a role in axonal pathfinding during development of the mammalian nervous system.     

Proteoglycans as cues for axonal guidance in formation of retinotectal or retinocollicular projections

Understanding the formation of neuronal circuits has long been one of the basic problems in developmental neurobiology. Projections from the retina to their higher center, the optic tectum in nonmammalian vertebrates and the superior colliculus in mammals, are most amenable to experimental approaches; thus, much information has been accumulated about the mechanisms of axonal guidance. The retinal axons navigate along the appropriate pathway with the help of a series of guidance cues. Although much of the work has focused on proteinaceous factors, proteoglycans have been identified as playing important roles in retinal axon guidance. Chondroitin sulfate proteoglycans and heparan sulfate proteoglycans are involved in essential decisions of axon steering along the pathway. However, it has not been determined whether diversity of the carbohydrate chains results in differential effects and how their diversity is recognized by growth cones, which represent an important area of future research.     

Differential expression of calretinin in the developing and regenerating zebrafish visual system

Calretinin is a calcium-binding protein which participates in a variety of functions including calcium buffering and neuronal protection. It also serves as a developmental marker of retinal ganglion cells (RGCs). In order to study the role of calretinin in the development and regeneration of RGCs, we have studied its pattern of expression in the retina at different developmental stages, as well as during optic nerve regeneration by means of immunohistochemistry. During development, calretinin is found for the first time in RGCs when they connect with the optic tectum. Optic nerves from adult zebrafish were crushed and after different survival times, calretinin expression in the retina, optic nerve tract and optic tectum was studied. From the day of crushing to 10 days later, calretinin expression was found to be downregulated within RGCs and their axons, as was also observed during the early developmental stages of RGCs, when they are not committed to a definite cell phenotype. Moreover, 13 days after lesion, when the regenerating axons arrived at the optic tectum, a recovery of calretinin immunoreactivity within the RGCs was observed. These results indicate that calretinin may play an important role during optic nerve regeneration, Thus, the down-regulation of Calretinin during the growth of the RGC axons towards the target during development as well as during their regeneration after injury, indicates that an increase the availability of cytosolic calcium is integral to axon outgrowth thus recapitulating the pattern observed during development.     

GABA and development of the Xenopus optic projection

In the developing visual system of Xenopus laevis retinal ganglion cell (RGC) axons extend through the brain towards their major target in the midbrain, the optic tectum. Enroute, the axons are guided along their pathway by cues in the environment. In vitro, neurotransmitters have been shown to act chemotropically to influence the trajectory of extending axons and regulate the outgrowth of developing neurites, suggesting that they may act to guide or modulate the growth of axons in vivo. Previous work by Roberts and colleagues (1987) showed that populations of cells within the developing Xenopus diencephalon and mid-brain express the neurotransmitter gamma amino butyric acid (GABA). Here we show that Xenopus RGC axons in the midoptic tract grow alongside the GABAergic cells and cross their GABA immunopositive nerve processes. Moreover, RGC axons and growth cones express GABA-A and GABA-B receptors, and GABA and the GABA-B receptor agonist baclofen both stimulate RGC neurite outgrowth in culture. Finally, the GABA-B receptor antagonist CGP54626 applied to the developing optic projection in vivo causes a dose-dependent shortening of the optic projection. These data indicate that GABA may act in vivo to stimulate the outgrowth of Xenopus RGC axons along the optic tract.     

Visual map development: bidirectional signaling, bifunctional guidance molecules, and competition

Topographic maps are a two-dimensional representation of one neural structure within another and serve as the main strategy to organize sensory information. The retina's projection via axons of retinal ganglion cells to midbrain visual centers, the optic tectum/superior colliculus, is the leading model to elucidate mechanisms of topographic map formation. Each axis of the retina is mapped independently using different mechanisms and sets of axon guidance molecules expressed in gradients to achieve the goal of representing a point in the retina onto a point within the target. An axon's termination along the temporal-nasal mapping axis is determined by opposing gradients of EphAs and ephrin-As that act through their forward and reverse signaling, respectively, within the projecting axons, each of which inhibits interstitial branching, cooperating with a branch-promoting activity, to generate topographic specific branching along the shaft of the parent axons that overshoot their correct termination zone along the anterior-posterior axis of the target. The dorsal-ventral termination position is then determined using a gradient of ephrin-B that can act as a repellent or attractant depending on the ephrin-B concentration relative to EphB levels on the interstitial branches to guide them along the medial-lateral axis of the target to their correct termination zone, where they arborize. In both cases, axon-axon competition results in axon mapping based on relative rather than absolute levels of repellent or attractant activity. The map is subsequently refined through large-scale pruning driven in large part by patterned retinal activity.     

Convergence of multisensory inputs in Xenopus tadpole tectum

The integration of multisensory information takes place in the optic tectum where visual and auditory/mechanosensory inputs converge and regulate motor outputs. The circuits that integrate multisensory information are poorly understood. In an effort to identify the basic components of a multisensory integrative circuit, we determined the projections of the mechanosensory input from the periphery to the optic tectum and compared their distribution to the retinotectal inputs in Xenopus laevis tadpoles using dye‐labeling methods. The peripheral ganglia of the lateral line system project to the ipsilateral hindbrain and the axons representing mechanosensory inputs along the anterior/posterior body axis are mapped along the ventrodorsal axis in the axon tract in the dorsal column of the hindbrain. Hindbrain neurons project axons to the contralateral optic tectum. The neurons from anterior and posterior hindbrain regions project axons to the dorsal and ventral tectum, respectively. While the retinotectal axons project to a superficial lamina in the tectal neuropil, the hindbrain axons project to a deep neuropil layer. Calcium imaging showed that multimodal inputs converge on tectal neurons. The layer‐specific projections of the hindbrain and retinal axons suggest a functional segregation of sensory inputs to proximal and distal tectal cell dendrites, respectively. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

The multiple decisions made by growth cones of RGCs as they navigate from the retina to the tectum in Xenopus embryos

Retinal ganglion cells (RGCs) of Xenopus laevis send axons along a stereospecific pathway from the retina to their target the optic tectum. Viewed from the point of the growth cone, this journey is reflected by discrete processes of axon initiation, axon outgrowth, navigation, target recognition, and innervation. These processes are characterised by distinct signalling mechanisms that trigger dynamic changes in growth cone morphology and behavior. Here we review work primarily from our laboratory, examining these events from a cellular and molecular perspective, focusing on the roles of FGFs, netrins, receptors, and intracellular effectors.     

The directed growth of retinal axons towards surgically transposed tecta in Xenopus; an examination of homing behaviour by retinal ganglion cell axons

The growth of optic axons towards experimentally rotated tecta has been studied. In stage 24/25 embryos, a piece of the dorsal neural tube, containing the dorsal midbrain rudiment, was rotated through 180 degrees. At later stages of development, the pathways of growing optic axons were investigated by labelling with either horseradish peroxidase or fluorescent dye. It is shown that retinal ganglion cell axons followed well-defined pathways, in spite of the abnormal structure of the brain, and were able to locate displaced tecta. This directed outgrowth of retinal axons in the optic tracts appears to be related either to the tectum or to some other component included in the graft operations. In tadpoles in which the midbrain rudiment was removed, optic axons still followed the normal course of the optic tract. This observation argues against long-range target attraction as being essential in guiding growing retinal axons towards the tectum. An alternative axon guidance mechanism, selective fasciculation, is discussed as a possible alternative to explain the directed axon outgrowth which occurs in both the normal and in these experimentally manipulated tadpoles.     

Visual Stimuli Evoked Action Potentials Trigger Rapidly Propagating Dendritic Calcium Transients in the Frog Optic Tectum Layer 6 Neurons

The superior colliculus in mammals or the optic tectum in amphibians is a major visual information processing center responsible for generation of orientating responses such as saccades in monkeys or prey catching avoidance behavior in frogs. The conserved structure function of the superior colliculus the optic tectum across distant species such as frogs, birds monkeys permits to draw rather general conclusions after studying a single species. We chose the frog optic tectum because we are able to perform whole-cell voltage-clamp recordings fluorescence imaging of tectal neurons while they respond to a visual stimulus. In the optic tectum of amphibians most visual information is processed by pear-shaped neurons possessing long dendritic branches, which receive the majority of synapses originating from the retinal ganglion cells. Since the first step of the retinal input integration is performed on these dendrites, it is important to know whether this integration is enhanced by active dendritic properties. We demonstrate that rapid calcium transients coinciding with the visual stimulus evoked action potentials in the somatic recordings can be readily detected up to the fine branches of these dendrites. These transients were blocked by calcium channel blockers nifedipine CdCl₂ indicating that calcium entered dendrites via voltage-activated L-type calcium channels. The high speed of calcium transient propagation, >300 μm in <10 ms, is consistent with the notion that action potentials, actively propagating along dendrites, open voltage-gated L-type calcium channels causing rapid calcium concentration transients in the dendrites. We conclude that such activation by somatic action potentials of the dendritic voltage gated calcium channels in the close vicinity to the synapses formed by axons of the retinal ganglion cells may facilitate visual information processing in the principal neurons of the frog optic tectum.