UNC-6/netrin and its receptor UNC-5 locally exclude presynaptic components from dendrites

Polarity is an essential feature of many cell types, including neurons that receive information from local inputs within their dendrites and propagate nerve impulses to distant targets through a single axon. It is generally believed that intrinsic structural differences between axons and dendrites dictate the polarized localization of axonal and dendritic proteins. However, whether extracellular cues also instruct this process in vivo has not been explored. Here we show that the axon guidance cue UNC-6/netrin and its receptor UNC-5 act throughout development to exclude synaptic vesicle and active zone proteins from the dendrite of the Caenorhabditis elegans motor neuron DA9, which is proximal to a source of UNC-6/netrin. In unc-6/netrin and unc-5 loss-of-function mutants, presynaptic components mislocalize to the DA9 dendrite. In addition, ectopically expressed UNC-6/netrin, acting through UNC-5, is sufficient to exclude endogenous synapses from adjacent subcellular domains within the DA9 axon. Furthermore, this anti-synaptogenic activity is interchangeable with that of LIN-44/Wnt despite being transduced through different receptors, suggesting that extracellular cues such as netrin and Wnts not only guide axon navigation but also regulate the polarized accumulation of presynaptic components through local exclusion.     

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.     

Netrin-1-mediated axon outgrowth requires deleted in colorectal cancer-dependent MAPK activation

Neuronal growth cones are guided to their targets by attractive and repulsive guidance cues. In mammals, netrin-1 is a bifunctional cue, attracting some axons and repelling others. Deleted in colorectal cancer (Dcc) is a receptor for netrin-1 that mediates its chemoattractive effect on commissural axons, but the signalling mechanisms that transduce this effect are poorly understood. Here we show that Dcc activates mitogen-activated protein kinase (MAPK) signalling, by means of extracellular signal-regulated kinase (ERK)-1 and -2, on netrin-1 binding in both transfected cells and commissural neurons. This activation is associated with recruitment of ERK-1/2 to a Dcc receptor complex. Inhibition of ERK-1/2 antagonizes netrin-dependent axon outgrowth and orientation. Thus, activation of MAPK signalling through Dcc contributes to netrin signalling in axon growth and guidance.     

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.     

Semaphorin 7A promotes axon outgrowth through integrins and MAPKs

Striking parallels exist between immune and nervous system cellular signalling mechanisms. Molecules originally shown to be critical for immune responses also serve neuronal functions, and similarly neural guidance cues can modulate immune function. We show here that semaphorin 7A (Sema7A), a membrane-anchored member of the semaphorin family of guidance proteins previously known for its immunomodulatory effects, can also mediate neuronal functions. Unlike many other semaphorins, which act as repulsive guidance cues, Sema7A enhances central and peripheral axon growth and is required for proper axon tract formation during embryonic development. Unexpectedly, Sema7A enhancement of axon outgrowth requires integrin receptors and activation of MAPK signalling pathways. These findings define a previously unknown biological function for semaphorins, identify an unexpected role for integrins and integrin-dependent intracellular signalling in mediating semaphorin responses, and provide a framework for understanding and interfering with Sema7A function in both immune and nervous systems.     

Relationship between prospective memory and vigilance: Evidence from ERP

Event-related potentials (ERPs) were used in this study to investigate the neural correlates of prospective memory (PM) and vigilance. Twenty college or graduate students participated in this study. They were administered a PM and a vigilance task and physiological data were collected at the same time. Behavioral results showed that the RT associated with PM cues was longer than those associated with vigilance targets. ERP results showed that PM cues and vigilance targets did not show significant difference in the N2 but PM cues evoked greater N300 than vigilance targets, and vigilance targets evoked greater parietal positivity/P3 than PM cues, suggesting vigilance and PM have similar but also distinctive neural basis.     

RGM is a repulsive guidance molecule for retinal axons

Axons rely on guidance cues to reach remote targets during nervous system development. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.     

Experimentally induced alteration in the polarity of developing neurons

Despite the great diversity of shapes exhibited by different classes of nerve cells, nearly all neurons share one feature in that they have a single axon and several dendrites. The two types of processes differ in their morphology, in their rate of growth, in the macromolecular composition of their cytoskeletons and surface membranes, and in their synaptic polarity. When hippocampal neurons are dissociated from the embryonic brain and cultured, they reproducibly establish this basic form with a single axon and several dendrites, despite the absence of any spatially organized environmental cues, and without the need for cell to cell contact. We have cut the axons of young hippocampal neurons within a day of their development: in some cases the initial axon regenerated, but more frequently one of the other processes, which if undisturbed would have become a dendrite, instead became the axon. Frequently the stump of the original axon persisted following the transection and subsequently became a dendrite. Evidently the neuronal processes that first develop in culture have the capacity to form either axons or dendrites. The acquisition of axonal characteristics by one neuronal process apparently inhibits the others from becoming axons, so they subsequently become dendrites.     

The Drosophila Netrin receptor Frazzled guides axons by controlling Netrin distribution

Netrin is a secreted protein that can act as a chemotropic axon guidance cue. Two classes of Netrin receptor, DCC and UNC-5 (refs 6-9), are required for axon guidance and are thought to mediate Netrin signals in growth cones through their cytoplasmic domains. However, in the guidance of Drosophila photoreceptor axons, the DCC orthologue Frazzled is required not in the photoreceptor neurons but instead in their targets, indicating that Frazzled also has a non-cell-autonomous function. Here we show that Frazzled can capture Netrin and 'present' it for recognition by other receptors. Moreover, Frazzled itself is actively localized within the axon through its cytoplasmic domain, and thereby rearranges Netrin protein into a spatial pattern completely different from the pattern of Netrin gene expression. Frazzled-dependent guidance of one pioneer neuron in the central nervous system can be accounted for solely on the basis of this ability of Frazzled to control Netrin distribution, and not by Frazzled signalling. We propose a model of patterning mechanism in which a receptor rearranges secreted ligand molecules, thereby creating positional information for other receptors.     

Genetic and epigenetic mechanisms contribute to motor neuron pathfinding

Many lines of evidence indicate that genetically distinct subtypes of motor neurons are specified during development, with each type having characteristic properties of axon guidance and cell-body migration. Motor neuron subtypes express unique combinations of LIM-type homeodomain factors that may act as intrinsic genetic regulators of the cytoskeletal events that mediate cell migration, axon navigation or both. Although experimentally displaced motor neurons can pioneer new routes to their targets, in many cases the axons of motor neurons in complete isolation from their normal territories passively follow stereotypical pathways dictated by the environment. To investigate the nonspecific versus genetically controlled regulation of motor connectivity we forced all motor neurons to express ectopically a LIM gene combination appropriate for the subgroup that innervates axial muscles. Here we show that this genetic alteration is sufficient to convert the cell body settling pattern, gene-expression profile and axonal projections of all motor neurons to that of the axial subclass. Nevertheless, elevated occupancy of the axial pathway can override their genetic program, causing some axons to project to alternative targets.     

The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system

Blood vessels and nerves are complex, branched structures that share a high degree of anatomical similarity. Guidance of vessels and nerves has to be exquisitely regulated to ensure proper wiring of both systems. Several regulators of axon guidance have been identified and some of these are also expressed in endothelial cells; however, the extent to which their guidance functions are conserved in the vascular system is still incompletely understood. We show here that the repulsive netrin receptor UNC5B is expressed by endothelial tip cells of the vascular system. Disruption of the Unc5b gene in mice, or of Unc5b or netrin-1a in zebrafish, leads to aberrant extension of endothelial tip cell filopodia, excessive vessel branching and abnormal navigation. Netrin-1 causes endothelial filopodial retraction, but only when UNC5B is present. Thus, UNC5B functions as a repulsive netrin receptor in endothelial cells controlling morphogenesis of the vascular system.     

Modification of retinal ganglion cell axon morphology by prenatal infusion of tetrodotoxin

The cellular mechanisms by which the axons of individual neurons achieve their precise terminal branching patterns are poorly understood. In the visual system of adult cats, retinal ganglion cell axons from each eye form narrow cylindrical terminal arborizations restricted to alternate non-overlapping layers within the lateral geniculate nucleus (LGN). During prenatal development, axon arborizations from the two eyes are initially simple in shape and are intermixed with each other; they then gradually segregate to form complex adult-like arborizations in separate eye-specific layers by birth. Here we report that ganglion cell axons exposed to tetrodotoxin (TTX) to block neuronal activity during fetal life fail to form the normal pattern of terminal arborization. Individual TTX-treated axon arborizations are not stunted in their growth, but instead produce abnormally widespread terminal arborizations which extend across the equivalent of approximately two eye-specific layers. These observations suggest that during fetal development of the central nervous system, the formation of morphologically appropriate and correctly located axon terminal arborizations within targets is brought about by an activity-dependent process.     

The role of different cues in the brain mechanism on visual spatial attention

The visual spatial attention mechanism in the brain was studied in 16 young subjects through the visual search paradigm of precue-target by the event-related potential (ERP) technique, with the attentive ranges cued by different scales of Chinese character and region cues. The results showed that the response time for Chinese character cues was much longer than that for region cues especially for small region cues. With the exterior interferences, the target stimuli recognition under region cues was much quicker than that under Chinese character cues. Compared with that under region cues, targets under Chinese character cues could lead to increase of the posterior P1, decrease of the N1 and increase of the P2. It should also be noted that the differences between region cues and Chinese character cues were affected by the interference types. Under exterior interferences, no significant difference was found between region cues and Chinese character cues; however, it was not the case under the interior interferences. Considering the difference between the exterior interferences and the interior interferences, we could conclude that with the increase of difficulty in target recognition there was obvious difference in the consumption of anterior frontal resources by target stimuli under the two kinds of cues.     

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.     

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.     

Defining brain wiring patterns and mechanisms through gene trapping in mice

The search to understand the mechanisms regulating brain wiring has relied on biochemical purification approaches in vertebrates and genetic approaches in invertebrates to identify molecular cues and receptors for axon guidance. Here we describe a phenotype-based gene-trap screen in mice designed for the large-scale identification of genes controlling the formation of the trillions of connections in the mammalian brain. The method incorporates an axonal marker, which helps to identify cell-autonomous mechanisms in axon guidance, and has generated a resource of mouse lines with striking patterns of axonal labelling, which facilitates analysis of the normal wiring diagram of the brain. Studies of two of these mouse lines have identified an in vivo guidance function for a vertebrate transmembrane semaphorin, Sema6A, and have helped re-evaluate that of the Eph receptor EphA4.     

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.     

Oligodendroglia metabolically support axons and contribute to neurodegeneration

Oligodendroglia support axon survival and function through mechanisms independent of myelination, and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been proposed. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and use. Here we show that the most abundant lactate transporter in the central nervous system, monocarboxylate transporter 1 (MCT1, also known as SLC16A1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and in mouse models of, amyotrophic lateral sclerosis, suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.     

Turnover of fluorescently labelled tubulin and actin in the axon.

The cytoskeleton has an important role in the generation and maintenance of the structure of the axon. Microtubules, neurofilaments and actin, together with various kinds of associated proteins, form highly organized dynamic cytoskeletal structures. Because tubulin and actin molecules are essential cytoskeletal components and are transported down the axon, it is important to understand their dynamic behaviour within the axon. Although previous pulse-labelling studies have indicated that the axonal cytoskeleton is a static complex travelling down the axon, this view has been challenged by the results of several recent experiments. We have now addressed this question by analysing the recovery of fluorescence after photobleaching fluorescent analogues of tubulin and actin in the axons of cultured neurons. We did not observe movement or spreading of bleached zones along the axon, both in neurons injected with fluorescein-labelled tubulin and actin. All bleached zones recovered their fluorescence gradually, however, indicating that microtubules and actin filaments are not static polymers moving forward within the axon, but are dynamic structures that continue to assemble along the length of the axon.