Growth-cone attraction to netrin-1 is converted to repulsion by laminin-1.

Growing axons are guided by both diffusible and substrate-bound factors. Growth cones of retinal neurons exhibit chemoattractive turning towards the diffusible factor netrin-1 in vitro and are guided into the optic nerve head (ONH) by localized netrin-1. Here we report that, in Xenopus, laminin-1 from the extracellular matrix (ECM), converts netrin-mediated attraction into repulsion. A soluble peptide fragment of laminin-1 (YIGSR) mimics this laminin-induced conversion. Low levels of cyclic AMP in growth cones also lead to the conversion of netrin-induced attraction into repulsion, and we show that the amount of cAMP decreases in the presence of laminin-1 or YIGSR, suggesting a possible mechanism for laminin's effect. At the netrin-1-rich ONH, where axons turn sharply to leave the eye, laminin-1 is confined to the retinal surface. Repulsion from the region in which laminin and netrin are coexpressed may help to drive axons into the region where only netrin is present, providing a mechanism for their escape from the retinal surface. Consistent with this idea, YIGSR peptides applied to the developing retina cause axons to be misdirected at the ONH. These findings indicate that ECM molecules not only promote axon outgrowth, but also modify the behaviour of growth cones in response to diffusible guidance cues.     

Homing behaviour of axons in the embryonic vertebrate brain

In embryonic nervous systems, growing axons must often travel long distances through diverse extracellular terrains to reach their postsynaptic partners. In most embryos, axons grow to their appropriate targets along particular tracts or nerves, as though they were following guidance cues confined to specific pathways. For example, in all vertebrates, axons from the retina invariably grow to the tectum along the well-defined optic tract. Yet, transplant experiments demonstrate that retinal axons make tectal projections even though they enter the brain at locations which are distinctly off the optic tract. Only recently has it become possible to label discreet growing projections in the embryonic vertebrate brain. Thus, it is not yet known whether displaced retinal axons grow directly towards the tectum or find it accidently, through random extension. To resolve this question, pioneering axons from normal and transplanted eyes in embryonic Xenopus were labelled using a short-survival horseradish peroxidase (HRP) method, and their orientation during growth was quantitatively assessed. The finding that the ectopic fibres head towards their distant targets implies that guidance cues are not restricted to specific pathways but are distributed throughout the embryonic brain. The significance of this result is discussed with respect to the ontogeny and evolution of the visual pathway.     

旋转脉冲偏场下哑铃畴中VBL的特性

实验研究了顺时针、逆时针转动的哑铃畴(ID,IID)畴长与缩灭场的关系.结果显示,顺时针转动和逆时针转动的哑铃畴的缩灭场明显不同.对于同样畴长的ID,IID,顺时针转动的缩灭场高,较稳定,对应负的VBL;逆时针转动的缩灭场低,不稳定,对应正的VBL.实验首次验证了正、负布洛赫线模型是合理的.     

Position-dependent properties of retinal axons and their growth cones

The formation of the very orderly neuronal projection from the retina to the optic tectum is not yet understood, but several mechanisms are thought to be involved in a coordinated fashion. These mechanisms may include mechanical or chemical guidance in channels, guidance by spatial gradients of positional markers, gradients of temporal (maturation) markers or specific inter-axon interactions (see ref. 1 for review). The last-mentioned mechanism could explain the fibre order found in optic nerve and tract. It requires that some or all growing retinal axons can distinguish between retinal axons of various origins and grow preferentially along retinal axons originating from the same area as themselves. The in vitro experiments described here show that growth cones from the temporal half of the chick retina grow preferentially along temporal axons, whereas growth cones from nasal retina do not distinguish between nasal and temporal axons.     

Chemotropic guidance of developing axons in the mammalian central nervous system

In the developing nervous system, axons project considerable distances along stereotyped pathways to reach their targets. Axon guidance depends partly on the recognition of cell-surface and extracellular matrix cues derived from cells along the pathways. It has also been proposed that neuronal growth cones are guided by gradients of chemoattractant molecules emanating from their intermediate or final cellular targets. Although there is evidence that the axons of some peripheral neurons in vertebrates are guided by chemotropism and the directed growth of some central axons to their targets is consistent with such a mechanism, it remains to be determined whether chemotropism operates in the central nervous system. During development of the spinal cord, commissural axons are deflected towards a specialized set of midline neural epithelial cells, termed the floor plate, which could reflect guidance by substrate cues or by diffusible chemoattractant molecules. Here we provide evidence in support of chemotropic guidance by demonstrating that the rat floor-plate cells secrete a diffusible factor(s) that influences the pattern and orientation of commissural axon growth in vitro without affecting other embryonic spinal cord axons. These findings support the hypothesis that chemotropic mechanisms guide developing axons to their intermediate targets in the vertebrate CNS.     

Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit

Retinal ganglion cells are the projection neurons that link the retina to the brain. Peptide immunoreactive cells in the ganglion cell layer (GCL) of the mammalian retina have been noted but their identity has not been determined. We now report that, in the rabbit, 25-35% of all retinal ganglion cells contain substance P-like (SP) immunoreactivity. They were identified by either retrograde transport of fluorescent tracers injected into the superior colliculus, or by retrograde degeneration after optic nerve section. SP immunoreactive cells are present in all parts of the retina and have medium to large cell bodies with dendrites that ramify extensively in the proximal inner plexiform layer. Their axons terminate in the dorsal lateral geniculate nucleus, superior colliculus and accessory optic nuclei, and these terminals disappear completely after contralateral optic nerve section and/or eye enucleation. In the dorsal lateral geniculate nucleus large, beaded, immunoreactive axons and varicosities make up a narrow plexus just below the optic tract, where they define a new geniculate lamina. The varicosities make multiple synaptic contacts with dendrites of dorsal lateral geniculate nucleus projection neurons and presumptive interneurons in complex glomerular neuropil. This is direct evidence that some mammalian retinal ganglion cells contain substance P-like peptides and strongly suggests that, in the rabbit, substance P (or related tachykinins) may be a transmitter or modulator in a specific population or populations of retinal ganglion cells.     

Order in the initial retinotectal map in Xenopus: a new technique for labelling growing nerve fibres

Retinal nerve fibres form an orderly map of visual space in several centres in the vertebrate brain. Such topographic maps are a common feature of central nervous system organization, yet the way in which they develop is poorly understood. Early nerve projections in the fetal and neonatal mammalian brain have been found in several cases to be less restricted than those in the adult, suggesting that nerve fibres may initially form a diffuse set of connections in their target structure from which the adult map is sculpted by the elimination of terminals. Indeed, previous electrophysiological data indicate that the retinotectal map in Xenopus laevis might be initially disorganized. We report here, however, that the retinotectal projection is ordered from the beginning of tectal innervation (stage 39/40). We demonstrate this first autoradiographically by tracing groups of growing ganglion cell axons which we labelled by incubating sectors of eye rudiments, before axonal outgrowth, in 3H-proline and replacing them orthotopically. Separate labelling of dorsal and ventral parts of the initial projection showed that retinal fibres are organized topographically, as in the adult, in the tectal rudiment and throughout much of the pathway. Second, we show that visual responses are ordered in the tectum from the first stage that they can be mapped (stage 40). We conclude that the topographic ordering of retinotectal connections develops as a result of directed axonal outgrowth.     

Disruption of retinogeniculate afferent segregation by antagonists to NMDA receptors.

Afferent activity has an important role in the formation of connections in the developing mammalian visual system. But the extent to which the activity of target neurons shapes patterns of afferent termination and synaptic contact is not known. In the ferret's visual pathway, retinal ganglion cell axons from each eye segregate early in development into eye-specific laminae in the lateral geniculate nucleus (LGN). The dorsal laminae (termed laminae A and A1) then segregate further into inner and outer sublaminae that retain input from on-centre and off-centre retinal axons, respectively. Thus, individual retinogeniculate axons form terminal arbors within laminae A and A1 that are restricted to one inner or outer sublamina. We report here that blockade of N-methyl-D-aspartate (NMDA) receptors on LGN cells with specific antagonists during the period of sublamina formation prevents retinal afferents from segregating into 'On' and 'Off' sublaminae. Retinogeniculate axons have arbors that are not restricted appropriately, or are restricted in size but inappropriately positioned within the eye-specific laminae. NMDA receptor antagonists may specifically disrupt a mechanism by which LGN neurons detect correlated afferent and target activity, and have been shown to reduce retinogeniculate transmission more generally, causing LGN cells to have markedly reduced levels of activity. These results therefore indicate that the activity of postsynaptic cells can significantly influence the patterning of inputs and the structure of presynaptic afferents during development.     

Retinotopic order in the absence of axon competition

The retinotectal projection has long been studied experimentally and theoretically, as a model for the formation of topographic brain maps. Neighbouring retinal ganglion cells (RGCs) project their axons to neighbouring positions in the optic tectum, thus re-establishing a continuous neural representation of visual space. Mapping along this axis requires chemorepellent signalling from tectal cells, expressing ephrin-A ligands, to retinal growth cones, expressing EphA receptors. High concentrations of ephrin A, increasing from anterior to posterior, prevent temporal axons from invading the posterior tectum. However, the force that drives nasal axons to extend past the anterior tectum and terminate in posterior regions remains to be identified. We tested whether axon-axon interactions, such as competition, are required for posterior tectum innervation. By transplanting blastomeres from a wild-type (WT) zebrafish into a lakritz (lak) mutant, which lacks all RGCs, we created chimaeras with eyes that contained single RGCs. These solitary RGCs often extended axons into the tectum, where they branched to form a terminal arbor. Here we show that the distal tips of these arbors were positioned at retinotopically appropriate positions, ruling out an essential role for competition in innervation of the ephrin-A-rich posterior tectum. However, solitary arbors were larger and more complex than under normal, crowded conditions, owing to a lack of pruning of proximal branches during refinement of the retinotectal projection. We conclude that dense innervation is not required for targeting of retinal axons within the zebrafish tectum but serves to restrict arbor size and shape.     

Retinal ganglion cells lose response to laminin with maturation

The decisive role played by adhesive interactions between neuronal processes and the culture substrate in determining the form and extent of neurite outgrowth in vitro has greatly influenced ideas about the mechanisms of axonal growth and guidance in the vertebrate nervous system. These studies have also helped to identify adhesive molecules that might be involved in guiding axonal growth in vivo. One candidate molecule is laminin, a major glycoprotein of basal laminae which has been shown to induce a wide variety of embryonic neurones to extend neurites in culture. Moreover, laminin is found in large amounts in injured nerves that can successfully regenerate but is absent from nerves where regeneration fails. However, it is unclear to what extent the mechanisms that regulate axonal regeneration also operate in the embryo when axon outgrowth is initiated. Here we have examined the substrate requirements for neurite outgrowth in vitro by chick embryo retinal ganglion cells, the only cells in the retina to send axons to the brain. We show that while retinal ganglion cells from embryonic day 6 (E6) chicks extend profuse neurites on laminin, those from E11 do not, although they retain the ability to extend neurites on astrocytes via a laminin-independent mechanism. This represents the first evidence that central nervous system neurones may undergo a change in their substrate requirements for neurite outgrowth as they mature.     

Axon guidance in cultured epithelial fragments of Drosophila wing

Growing axons can be guided by a number of different cues: adhesive substrates, diffusible factors, electrical fields and even factors intrinsic to the neurone itself have all been shown to affect axon orientation and outgrowth in vitro. However, in most intact systems it has proved difficult to test directly the role played by these putative guidance cues. Here, we describe a system, the developing wing of the fruitfly, in which we have tested simultaneously two putative guidance mechanisms, physical constraints to axon growth (channels) and the position of neuronal somata (guideposts), using surgical techniques. We show that pioneer sensory axons can navigate correctly and form their normal stereotyped pattern of axon bundles in wing fragments that apparently lack both physical and neural cues. This technique allows access to the surface along which neuronal pathfinding takes place, making possible a wide range of experimental manipulations on the developing system.     

En passant neurotrophic action of an intermediate axonal target in the developing mammalian CNS.

During development, neurons extend axons to their targets, then become dependent for their survival on trophic substances secreted by their target cells. Competition for limiting amounts of these substances is thought to account for much of the extensive naturally-occurring cell death that is seen throughout the nervous system. Here we show that spinal commissural neurons, a group of long projection neurons in the central nervous system (CNS), are also dependent for their survival on trophic support from one of their intermediate targets, the floor plate of the spinal cord. This dependence occurs during a several-day-long period when their axons extend along the floor plate, following which they develop additional trophic requirements. A dependence of neurons on trophic support derived en passant from their intermediate axonal targets provides a mechanism for rapidly eliminating misprojecting neurons, which may help to prevent the formation of aberrant neuronal circuits during the development of the nervous system.     

Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor

Numerous inherited retinal degenerations exist in animals and humans, in which photoreceptors inexplicably degenerate and disappear. In RCS rats with inherited retinal dystrophy, the mutant gene is expressed in the retinal pigment epithelial (RPE) cell, and leads to the loss of photoreceptor cells. Photoreceptors can be rescued from degeneration if they are juxtaposed to wild-type RPE cells in experimental chimaeras or by the transplantation of RPE cells from normal rats. In both cases, the rescue effect extends beyond the immediate boundaries of the normal RPE cells, suggesting trophic action of a diffusible factor(s) from the normal RPE cells. We considered that the fibroblast growth factors, aFGF and bFGF, might have such a trophic role as they are found in the retina and RPE cells; bFGF acts as a neurotrophic agent after axonal injury in several regions of the central nervous system, and bFGF induces retinal regeneration from developing RPE cells. Here we report that subretinal injection of bFGF results in extensive rescue of photoreceptors in RCS rats for at least two months after the injection, and that intravitreal injection of bFGF results in even more widespread rescue, across almost the entire retina. The findings demonstrate for the first time that bFGF can act as a survival-promoting neurotrophic factor in a hereditary neuronal degeneration of the central nervous system.     

Target size regulates calibre and myelination of sympathetic axons

Axons in vertebrate peripheral nerves are ensheathed by Schwann cells. For some axons, this sheath consists of a single layer of glial cell cytoplasm and plasma membranes; for other axons, Schwann cells form multilayered myelin. Whether or not a Schwann cell makes myelin is determined by a signal from the axon, but the nature of this signal is not known. Here I show that sympathetic postganglionic axons, which are normally not myelinated, become myelinated when their calibre is increased as a result of increasing the size of the peripheral target they innervate. This result implies that axon calibre, which is known to be correlated with myelination, is in fact the crucial determinant of whether an axon becomes myelinated. Furthermore, the finding that increasing or decreasing target size causes corresponding increases or decreases in axon size indicates that axon calibre is itself regulated by retrograde signals from peripheral target tissues.     

A cell surface molecule distributed in a dorsoventral gradient in the perinatal rat retina

Brain topography may have its earliest expression as spatial gradients of molecules controlling the deposition of neurones and neuronal processes. In the vertebrate visual system there is evidence that the stereotyped alignment of central retinal projections relies on an initial spatially organized distribution of molecules in both the retina and its central target nuclei. We used an immunological approach to look for molecules that are so organized and produced a monoclonal antibody (JONES) which shows a pronounced dorsal to ventral gradient of binding in the rat retina throughout the period when retinal ganglion cell axons are forming topographically organized projections within the central nervous system (CNS). Binding is present throughout the radial thickness of the retinal epithelium in regions where postmitotic neurones are generated but is not associated with any consistent histological characteristic of the tissue. The antibody was shown to bind on the cell surface of freshly dissociated retinal cells, and dorsal retinal quadrants were found in vitro to have nearly twice as much antigen as ventral retinal quadrants. Initial biochemical characterization of the target epitope reveals that it is a lipid present in chloroform/methanol extracts from perinatal retina and is sensitive to neuraminidase digestion.     

Chemotropic effect of specific target epithelium in the developing mammalian nervous system

Developing nerve fibres are guided to their targets by specific directional cues which are thought to be expressed in the tissues along the route and may involve the extracellular matrix. Another possibility, that directional cues emanate from the target itself, is consistent with the recent demonstration of homing behaviour by ectopic retinal ganglion axons and our previous demonstration that early trigeminal neurites grow directly to their virgin peripheral target in vitro. Here we show that this chemotropic effect is precisely limited to the trigeminal system; trigeminal ganglion neurites grow directly to their own target field but not to the adjoining field, normally innervated by the geniculate ganglion; furthermore, the trigeminal field does not influence the growth of geniculate neurites. Also, when trigeminal ganglia are co-cultured with isolated tissue layers of their target, neurites grow only towards the epithelial and not the mesenchymal component. These findings suggest that trigeminal epithelium is specified to attract correct innervation and that pathway mesenchyme, in which preformed guidance cues have been postulated, may provide favourable conditions for nerve fibre growth but not govern its direction.     

Transient expression of a surface antigen on a small subset of neurones during embryonic development

During development, neurones find and interconnect with their targets in a remarkably precise way. The unfolding of neuronal specificity involves a series of highly specific recognition events which are likely to be coordinated by the spatial and temporal expression of many different surface molecules. At early stages of development, neuronal recognition occurs most dramatically at the tips of growing axons, at growth cones and their filopodia. Previous studies on the grasshopper embryo suggest that specific filopodial contacts lead to the stereotyped patterns of selective axonal fasciculation; these results support the 'labelled pathways' hypothesis which predicts that the different neighbouring axon fascicles in the embryonic neuropil within filopodial grasp are differentially labelled. To uncover the molecular labels on fasciculating embryonic axons, we screened 2,000 monoclonal antibodies generated against the embryonic neuroepithelium. Here we describe three antibodies which reveal surface antigens whose temporal and spatial expression during embryogenesis correlate with the predictions of the model. In particular, the Mes-2 antibody recognizes an antigen which is transiently expressed on the surface of only 4 out of approximately 1,000 neurones in each metathoracic hemisegment during a short period of embryogenesis. The growth cones of two of these neurones fasciculate in the periphery and innervate the same target. Moreover, they transiently express the Mes-2 surface antigen while doing so.     

Requirement for subplate neurons in the formation of thalamocortical connections

The neurons of layer 4 in the adult cerebral cortex receive their major ascending inputs from the thalamus. In development, however, thalamic axons arrive at the appropriate cortical area long before their target layer 4 neurons have migrated into the cortical plate. The axons accumulate and wait in the zone below the cortical plate, the subplate, for several weeks before invading the cortical plate. The subplate is a transient zone that contains the first postmitotic neurons of the telencephalon. These neurons mature well before other cortical neurons, and disappear by cell death after the thalamic axons have grown into the overlying cortical plate. The close proximity of growing thalamocortical axons and subplate neurons suggests that they might be involved in interactions important for normal thalamocortical development. Here we show that early in development the deletion of subplate neurons located beneath visual cortex prevents axons from the lateral geniculate nucleus of the thalamus from recognizing and innervating visual cortex, their normal target. In the absence of subplate neurons, lateral geniculate nucleus axons continue to grow in the white matter past visual cortex despite the presence of their target layer 4 neurons. Thus the transient subplate neurons are necessary for appropriate cortical target selection by thalamocortical axons.     

Segregation of functionally distinct axons in the monkey's optic tract

The classical neuro-ophthalmologic literature describes the organization of the primate's optic tract as containing a single topographic representation of the complete contralateral visual hemifield. In contrast, cats have separate visual field representations for the optic axons of the functionally distinct retinal ganglion cell classes. As the line of decussation for each ganglion cell class in the cat occupies a different location on the retinal surface, whereas in primates they are all superimposed, such a species difference might be expected. We report that implants of horseradish peroxidase placed in either the deep or superficial extremes of the monkey's optic tract produce retrograde labelling of distinct retinal ganglion cell classes, and produce anterograde labelling confined to distinct laminae of the lateral geniculate nucleus. Hence, the optic tract of the primate cannot contain a single representation of the contralateral visual hemifield; rather, independent visual field representations for the functionally distinct optic axons must exist. Their anatomical segregation may account for the clinical observation of selective impairments of distinct visual abilities following partial interruption of the optic tract in man.