BMPs as mediators of roof plate repulsion of commissural neurons

During spinal cord development, commissural (C) neurons, located near the dorsal midline, send axons ventrally and across the floor plate (FP). The trajectory of these axons toward the FP is guided in part by netrins. The mechanisms that guide the early phase of C axon extension, however, have not been resolved. We show that the roof plate (RP) expresses a diffusible activity that repels C axons and orients their growth within the dorsal spinal cord. Bone morphogenetic proteins (BMPs) appear to act as RP-derived chemorepellents that guide the early trajectory of the axons of C neurons in the developing spinal cord: BMP7 mimics the RP repellent activity for C axons in vitro, can act directly to collapse C growth cones, and appears to serve an essential function in RP repulsion of C axons.     

Representation of the glomerular olfactory map in the Drosophila brain

We explored how the odor map in the Drosophila antennal lobe is represented in higher olfactory centers, the mushroom body and lateral horn. Systematic single-cell tracing of projection neurons (PNs) that send dendrites to specific glomeruli in the antennal lobe revealed their stereotypical axon branching patterns and terminal fields in the lateral horn. PNs with similar axon terminal fields tend to receive input from neighboring glomeruli. The glomerular classes of individual PNs could be accurately predicted based solely on their axon projection patterns. The sum of these patterns defines an "axon map" in higher olfactory centers reflecting which olfactory receptors provide input. This map is characterized by spatial convergence and divergence of PN axons, allowing integration of olfactory information.     

Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord

Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.     

The role of floor plate contact in the elaboration of contralateral commissural projections within the embryonic mouse spinal cord

In vertebrate embryos, commissural axons extend toward and across the floor plate (FP), an intermediate target at the ventral midline (VM) of the spinal cord. After decussating, many commissural axons turn into the longitudinal plane and elaborate diverse projections. FP contact is thought to alter the responsiveness of these axons so that they can exit the FP and adopt new trajectories. However, a requirement for the FP in shaping contralateral commissural projections has not been established in higher vertebrates. Here we further analyze to what extent FP contact is necessary for the elaboration of decussated commissural projections both in cultured, FP-excised spinal cord preparations and in gli2-deficient mice, which lack a FP. In FP-lacking spinal cords, we observe a large number of appropriately projecting contralateral commissural projections in vivo and in vitro. Surprisingly, even though gli2 mutants lack a FP, slit1-3 mRNA and their receptors (Robo1/2) are expressed in a wild-type-like manner. In addition, blocking Robo-Slit interactions in FP-lacking spinal cord explants prevents commissural axons from leaving the VM and turning longitudinally. Thus, compared to FP contact, Slit-Robo interactions are more critical for driving commissural axons out of the VM and facilitating the elaboration of a subset of contralateral commissural projections.     

Axon regeneration in young adult mice lacking Nogo-A/B

After injury, axons of the adult mammalian brain and spinal cord exhibit little regeneration. It has been suggested that axon growth inhibitors, such as myelin-derived Nogo, prevent CNS axon repair. To investigate this hypothesis, we analyzed mice with a nogo mutation that eliminates Nogo-A/B expression. These mice are viable and exhibit normal locomotion. Corticospinal tract tracing reveals no abnormality in uninjured nogo-A/B(-/-) mice. After spinal cord injury, corticospinal axons of young adult nogo-A/B(-/-) mice sprout extensively rostral to a transection. Numerous fibers regenerate into distal cord segments of nogo-A/B(-/-) mice. Recovery of locomotor function is improved in these mice. Thus, Nogo-A plays a role in restricting axonal sprouting in the young adult CNS after injury.     

Axons of sacral preganglionic neurons in the cat: I. Origin,initial segment,and myelination

Parasympathetic preganglionic neurons in the cat sacral spinal cord innervate intraspinal neurons and pelvic target organs. Retrograde tracing studies have revealed little of the morphology of their axons including their origin, initial segments, or their myelin, due to methodological limitations. Intracellular labeling of single neurons with neurobiotin or HRP has overcome these problems. Axons were studied in 24 preganglionic neurons. In 21 neurons the axon originated as a branch of a dendrite, without a detectable axon hillock, at distances from the soma ranging from 10 to 110 μm (average 34.1 μm ). In 3 neurons the axon was derived from the soma. Initial segments, present in all cells, ranged from 15 to 40 μm (average 26.8 μm). Nearly all axons followed the initial segment with unmyelinated segments that varied between 59 to 630 μm, followed by myelin and nodes of Ranvier. Internodal distances were variable and relatively short (average 93 μm). Axonal diameters measured over the intraspinal course in 18 axons averaged 1.3 μm (range 0.6–2.4 μm) and were relatively constant compared with other neurons. Spine-like protrusions were observed on the initial segments of 12 cells. Axon collaterals originated from unmyelinated sections and nodes of Ranvier. Antidromic action potentials showing initial segment, soma-dendritic inflections, did not differentiate between soma-derived and dendrite-derived axons. The data suggest that axons originating from a dendrite are the normal structure of preganglionic neurons in the lateral sacral parasympathetic nucleus. It is proposed that the particular structure of these axons may be part of a timing mechanism that coordinates preganglionic neurons with other spinal neurons involved in target organ reflexes.     

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.     

An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe

In the developing Drosophila visual system, glia migrate into stereotyped positions within the photoreceptor axon target fields and provide positional information for photoreceptor axon guidance. Glial migration conversely depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. Our results support the view that this requirement for retinal innervation reflects a role of photoreceptor axons in the establishment of an axonal scaffold that guides glial cell migration. Optic lobe cortical axons extend from dorsal and ventral positions towards incoming photoreceptor axons and establish at least four separate pathways that direct glia to proper destinations in the optic lobe neuropiles. Photoreceptor axons induce the outgrowth of these scaffold axons. Most glia do not migrate when the scaffold axons are missing. Moreover, glia follow the aberrant pathways of scaffold axons that project aberrantly, as occurs in the mutant dachsous. The local absence of glia is accompanied by extensive apoptosis of optic lobe cortical neurons. These observations reveal a mechanism for coordinating photoreceptor axon arrival in the brain with the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival.     

Regeneration of retinal axons in the lizard Gallotia galloti is not linked to generation of new retinal ganglion cells

Using anterograde tracing with HRP and antibodies (ABs) against neurofilaments, we show that regrowth of retinal ganglion cell (RGC) axons in the lizard Gallotia galloti commences only 2 months after optic nerve transection (ONS) and continues over at least 9 months. This is unusually long when compared to RGC axon regeneration in fish or amphibians. Following ONS, lizard RGCs up-regulate the immediate early gene C-JUN for 9 months or longer, indicating their reactive state. In keeping with the in vivo data, axon outgrowth from lizard retinal explants is increased above control levels from 6 weeks, reaches its maximum as late as 3 months, and remains elevated for at least 1 year after ONS. By means of BrdU incorporation assays and antiproliferating cell nuclear antigen immunohistochemistry, we show that the late axon outgrowth is not derived from new RGCs that might have arisen in reaction to ONS: no labeled cells were detected in lizard retinas at 0.5, 1, 1.5, 3, 6, and 12 months after ONS. Conversely, numbers of RGCs undergoing apoptosis were too low to be detectable in TUNEL assays at any time after ONS. These results demonstrate that retinal axon regeneration in G. galloti is due to axon regrowth from the resident population of RGCs, which remain in a reactive state over an extended time interval. Neurogenesis does not appear to be involved in RGC axon regrowth in G. galloti.     

A little nip and tuck: axon refinement during development and axonal injury

While building the nervous system, regions of some developing axons are eliminated; this can also happen as a result of axonal injury. During development, many axon branches that are formed in excess of an organism's needs are fated for removal in a process called axon pruning. By contrast, when axons are injured the axon segment distal to the injury site is compartmentalized and eliminated. In both cases, the end result is similar -- a region of an axon is selected for removal. Recent evidence suggests that there are some similarities in the cellular and molecular mechanisms that regulate axon elimination in development and during axonal injury.     

A CHOLINERGIC COMPONENT IN THE INNERVATION OF THE LONGITUDINAL SMOOTH MUSCLE OF THE GUINEA PIG VAS DEFERENS : The Fine Structural Localization of Acetylcholinesterase

Acetylcholinesterase (AChE) has been detected on the plasma membrane of about 25% of the axons in the longitudinal smooth muscle tissue of guinea pig vas deferens. These axons are presumably cholinergic. No enzyme was detected in the remaining 75% of axons. These axons are presumably adrenergic. The plasma membrane of the Schwann cells associated with the cholinergic axons also stained for AChE. Some axon bundles contained only cholinergic or adrenergic axons while others contained both types of axon. When a cholinergic axon approached within 1100 A of a smooth muscle cell, there was a patch of AChE activity on the muscle membrane adjacent to the axon. It is suggested that these approaches are the points of effective transmission from cholinergic axons to smooth muscle cells. Butyrylcholinesterase activity was detected on the plasma membranes of all axons and smooth muscle cells in this tissue.     

Cortical axons branch to multiple subcortical targets by interstitial axon budding: implications for target recognition and "waiting periods"

We are studying how axons branch in vivo. Individual cortical neurons send axons to both the spinal cord and the basilar pons. Here we show that the corticopontine projection develops by an interstitial budding of collaterals from parent axons rather than a reported mechanism of axon branching, growth cone bifurcation. This mechanism is used regardless of whether the parent axon's postpontine segment, which forms the corticospinal projection, is permanent (motor cortex) or transient (visual cortex). Budding occurs days after the parent axons grow spinally past the pons, accounting for the "waiting period" reported in this system in contrast to an alternative explanation that the growth cones pause outside of their target. Timing and location of pontine collateral budding vary with cortical origin of the parent axon and are correlated with the temporal ordering of axon arrival.     

Lack of enhanced spinal regeneration in Nogo-deficient mice

The failure of regeneration of severed axons in the adult mammalian central nervous system is thought to be due partly to the presence of endogenous inhibitors of axon regeneration. The nogo gene encodes three proteins (Nogo-A, -B, and -C) that have been proposed to contribute to this inhibition. To determine whether deletion of nogo enhances regenerative ability, we generated two lines of mutant mice, one lacking Nogo-A and -B but not -C (Nogo-A/B mutant), and one deficient in all three isoforms (Nogo-A/B/C mutant). Although Nogo-A/B-deficient myelin has reduced inhibitory activity in a neurite outgrowth assay in vitro, tracing of corticospinal tract fibers after dorsal hemisection of the spinal cord did not reveal an obvious increase in regeneration or sprouting of these fibers in either mouse line, suggesting that elimination of Nogo alone is not sufficient to induce extensive axon regeneration.     

New views on retinal axon development: a navigation guide

The eye is a peripheral outpost of the central nervous system (CNS) where the retinal ganglion cells (RGCs) reside. RGC axons navigate to their targets in a remarkably stereotyped and error-free manner and it is this process of directed growth that underlies the complex organization of the adult brain. The RGCs are the only retinal neurons to project into the brain and their peripheral location makes them an unusually accessible population of projection neurons for experiments involving in vivo gene transfer, anatomical tracing, transplantation and in vitro culture. In this paper, we review recent findings that have contributed to our understanding of some of the guidance decisions that axons make in the developing visual system. We look at two choice points in the pathway, the optic nerve head (onh) and the midline chiasm, and discuss evidence that supports the idea that key molecules in guiding axon growth at these junctures are netrin-1 (onh) and ephrin-B (chiasm). In the optic tectum where RGC axon terminals are arrayed in topographic order, we present experimental evidence to suggest that in the dorso-ventral dimension, the B-type ephrins and Eph receptors are of prime importance, possibly through attractive interactions. This complements the anterior-posterior topographic mapping known to be mediated through A-type ephrin/Eph repulsive interactions. An emerging theme is that guidance molecules such as ephrin-B and netrin-1 have complex patterns of restricted expression in the pathway and play multiple and changing roles in axon guidance.     

Axon fasciculation and differences in midline kinetics between pioneer and follower axons within commissural fascicles

Early neuronal scaffold development studies suggest that initial neurons and their axons serve as guides for later neurons and their processes. Although this arrangement might aid axon navigation, the specific consequence(s) of such interactions are unknown in vivo. We follow forebrain commissure formation in living zebrafish embryos using timelapse fluorescence microscopy to examine quantitatively commissural axon kinetics at the midline: a place where axon interactions might be important. Although it is commonly accepted that commissural axons slow down at the midline, our data show this is only true for leader axons. Follower axons do not show this behavior. However, when the leading axon is ablated, follower axons change their midline kinetics and behave as leaders. Similarly, contralateral leader axons change their midline kinetics when they grow along the opposite leading axon across the midline. These data suggest a simple model where the level of growth cone exposure to midline cues and presence of other axons as a substrate shape the midline kinetics of commissural axons.     

Area specificity and topography of thalamocortical projections are controlled by ephrin/Eph genes

The mechanisms generating precise connections between specific thalamic nuclei and cortical areas remain poorly understood. Using axon tracing analysis of ephrin/Eph mutant mice, we provide in vivo evidence that Eph receptors in the thalamus and ephrins in the cortex control intra-areal topographic mapping of thalamocortical (TC) axons. In addition, we show that the same ephrin/Eph genes unexpectedly control the inter-areal specificity of TC projections through the early topographic sorting of TC axons in an intermediate target, the ventral telencephalon. Our results constitute the first identification of guidance cues involved in inter-areal specificity of TC projections and demonstrate that the same set of mapping labels is used differentially for the generation of topographic specificity of TC projections between and within individual cortical areas.     

Signals from the cerebellum guide the pathfinding of inferior olivary axons

During development, inferior olivary axons cross the floor plate and project from the caudal to the rostral hindbrain, whence they grow into the cerebellar plate. We have investigated the axon guidance signals involved in the formation of this projection in vitro. When the cerebellar plate was grafted ectopically along the margin of the hindbrain in organotypic cultures, inferior olivary axons could pathfind to the ectopic cerebellum, establishing a topographically normal projection. Following rostrocaudal reversal of a region of tissue in the axon pathway between the inferior olive and the cerebellum, olivary axons still navigated towards the cerebellum. Moreover, olivary axons could cross a bridging tissue explant (spinal cord) to reach a cerebellar explant. In collagen gel cultures of inferior olive explants, olivary axon outgrowth increased significantly in the presence of cerebellar explants and axons deflected towards the cerebellar tissue. These results show that the cerebellum is a source of diffusible axon guidance signals for olivary axons. We also found that, in organotypic cultures, olivary axons which had crossed the floor plate showed an increased tendency to respond to cerebellar cues. Taken together, these results indicate that the cerebellum is the source of cues that are chemoattractant and growth-promoting for inferior olivary axons; prior exposure to the floor plate increases responsiveness to these cues.     

Homing behaviour of regenerating axons in the amphibian limb

Following peripheral nerve deviation in the limbs of urodele amphibians axons regrow distally toward their previous target muscles (Holder et al. 1984; Proc. Roy. Soc. Lond. B 222, 477-489). This study describes analysis of this axon regeneration over time following deviation of the forearm flexor nerve in Triturus cristatus and the extensor cranialis nerve in the axolotl. Using horseradish peroxidase (HRP) axonal tracing, electrophysiology and electron microscopy, we describe the sequence of events leading to reestablishment of functional innervation. HRP fills reveal axons leaving the deviated nerve via a number of possible routes and they invariably grow distally. Many axons take a path close to that of the original nerve but others fasciculate forming parallel paths. Electrophysiology and electron microscopy show that axons in the deviated region of the nerve degenerate extensively compared with cut, but undeviated, controls. The results are discussed in terms of the possible axon-growth-promoting mechanisms that result in directed growth.