Terminal Schwann cells guide the reinnervation of muscle after nerve injury

Schwann cells and axons labeled by transgene-encoded, fluorescent proteins can be repeatedly imaged in living mice to observe the reinnervation of neuromuscular junctions. Axons typically return to denervated junctions by growing along Schwann cells contained in the old nerve sheaths or “Schwann cell tubes”. These axons then commonly “escape” the synaptic sites by growing along the Schwann cell processes extended during the period of denervation. These “escaped fibers” grow to innervate adjacent synaptic sites along Schwann cells bridging these sites. Within the synaptic site, Schwann cells, originally positioned above the synaptic site continue to cover the acetylcholine receptors (AChRs) immediately following denervation, but gradually vacate portions of this site. When regenerating axons return, they first deploy along the Schwann cells and ignore sites of AChRs vacated by Schwann cells. In many cases these vacated sites are never reinnervated and are ultimately lost. Following partial denervation, Schwann cells grow in an apparently tropic fashion from denervated to nearby innervated synaptic sites and serve as the substrates for nerve sprouting. These experiments show that Schwann cells provide pathways that stimulate axon growth and insure the rapid reinnervation of denervated or partially denervated muscles.     

In vivo time-lapse imaging of synaptic takeover associated with naturally occurring synapse elimination

During development, competition between axons causes permanent removal of synaptic connections, but the dynamics have not been directly observed. Using transgenic mice that express two spectral variants of fluorescent proteins in motor axons, we imaged competing axons at developing neuromuscular junctions in vivo. Typically, one axon withdrew progressively from postsynaptic sites and the competing axon extended axonal processes to occupy those sites. In rare instances when the remaining axon did not reoccupy a site, the postsynaptic receptors rapidly disappeared. Interestingly, the progress and outcome of competition was unpredictable. Moreover, the relative areas occupied by the competitors shifted in favor of one axon and then the other. These results show synaptic competition is not always monotonic and that one axon's contraction in synaptic area is associated with another axon's expansion.     

Axon branch removal at developing synapses by axosome shedding

In many parts of the developing nervous system, the number of axonal inputs to each postsynaptic cell is dramatically reduced. This synapse elimination has been extensively studied at the neuromuscular junction, but how axons are lost is unknown. Here, we combine time-lapse imaging of fluorescently labeled axons and serial electron microscopy to show that axons at neuromuscular junctions are removed by an unusual cellular mechanism. As axons disappear, they shed numerous membrane bound remnants. These "axosomes" contain a high density of synaptic organelles and are formed by engulfment of axon tips by Schwann cells. After this engulfment, the axosome's contents mix with the cytoplasm of the glial cell. Axosome shedding might underlie other forms of axon loss and may provide a pathway for interactions between axons and glia.     

Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations

Synapse formation requires the coordination of pre- and postsynaptic differentiation. An unresolved question is which steps in the process require interactions between pre- and postsynaptic cells, and which proceed cell-autonomously. One current model is that factors released from presynaptic axons organize postsynaptic differentiation directly beneath the nerve terminal. Here, we used neuromuscular junctions (NMJs) of the zebrafish primary motor system to test this model. Clusters of neurotransmitter (acetylcholine) receptors (AChRs) formed in the central region of the myotome, destined to be synapse-rich, before axons extended and even when axon extension was prevented. Time-lapse imaging revealed that pre-existing clusters on early-born slow (adaxial) muscle fibers were incorporated into NMJs as axons advanced. Axons were, however, required for the subsequent remodeling and selective stabilization of synaptic clusters that precisely appose post- to presynaptic elements. Thus, motor axons are dispensable for the initial stages of postsynaptic differentiation but are required for later stages. Moreover, many AChR clusters on later-born fast muscle fibers formed at sites that had already been contacted by axons, suggesting heterogeneity in the signaling mechanisms leading to synapse formation by a single axon.     

Allotransplanted nerves regenerate inhibitory synapses on a crayfish muscle: Possible postsynaptic specification

Donor nerves of different origins, when transplanted onto a previously denervated adult crayfish abdominal superficial flexor muscle (SFM), regenerate excitatory synaptic connections. Here we report that an inhibitory axon in these nerves also regenerates synaptic connections based on observation of nerve terminals with irregular to elliptically shaped synaptic vesicles characteristic of the inhibitory axon in aldehyde fixed tissue. Inhibitory terminals were found at reinnervated sites in all 12 allotransplanted-SFMs, underscoring the fact that the inhibitory axon regenerates just as reliably as the excitatory axons. At sites with degenerating nerve terminals and at sparsely reinnervated sites, we observe densely stained membranes, reminiscent of postsynaptic membranes, but occurring as paired, opposing membranes, extending between extracellular channels of the subsynaptic reticulum. These structures are not found at richly innervated sites in allotransplanted SFMs, in control SFMs, or at several other crustacean muscles. Although their identity is unknown, they are likely to be remnant postsynaptic membranes that become paired with collapse of degenerated nerve terminals of excitatory and inhibitory axons. Because these two axons have uniquely different receptor channels and intramembrane structure, their remnant postsynaptic membranes may therefore attract regenerating nerve terminals to form synaptic contacts selectively by excitatory or inhibitory axons, resulting in postsynaptic specification.     

Coordinated motor neuron axon growth and neuromuscular synaptogenesis are promoted by CPG15 in vivo

We have used in vivo time-lapse two-photon imaging of single motor neuron axons labeled with GFP combined with labeling of presynaptic vesicle clusters and postsynaptic acetylcholine receptors in Xenopus laevis tadpoles to determine the dynamic rearrangement of individual axon branches and synaptogenesis during motor axon arbor development. Control GFP-labeled axons are highly dynamic during the period when axon arbors are elaborating. Axon branches emerge from sites of synaptic vesicle clusters. These data indicate that motor neuron axon elaboration and synaptogenesis are concurrent and iterative. We tested the role of Candidate Plasticity Gene 15 (CPG15, also known as Neuritin), an activity-regulated gene that is expressed in the developing motor neurons in this process. CPG15 expression enhances the development of motor neuron axon arbors by promoting neuromuscular synaptogenesis and by increasing the addition of new axon branches.     

Seeking long-term relationship: axon and target communicate to organize synaptic differentiation

Synapses form after growing axons recognize their appropriate targets. The subsequent assembly of aligned pre and postsynaptic specializations is critical for synaptic function. This highly precise apposition of presynaptic elements (i.e. active zones) to postsynaptic specializations (i.e. neurotransmitter receptor clusters) strongly suggests that communication between the axon and target is required for synaptic differentiation. What trans‐synaptic factors drive such differentiation at vertebrate synapses? First insights into the answers to this question came from studies at the neuromuscular junction (NMJ), where axon‐derived agrin and muscle‐derived laminin β2 induce post and presynaptic differentiation, respectively. Recent work has suggested that axon‐ and target‐derived factors similarly drive synaptic differentiation at central synapses. Specifically, WNT‐7a, neuroligin, synaptic cell adhesion molecule (SynCAM) and fibroblast growth factor‐22 (FGF‐22) have all been identified as target‐derived presynaptic organizers, whereas axon‐derived neuronal activity regulated pentraxin (Narp), ephrinB and neurexin reciprocally co‐ordinate postsynaptic differentiation. In addition to these axon‐ and target‐derived inducers of synaptic differentiation, factors released from glial cells have also been implicated in regulating synapse assembly. Together, these recent findings have profoundly advanced our understanding of how precise appositions are established during vertebrate nervous system development.     

BDNF increases synapse density in dendrites of developing tectal neurons in vivo

Neuronal connections are established through a series of developmental events that involve close communication between pre- and postsynaptic neurons. In the visual system, BDNF modulates the development of neuronal connectivity by influencing presynaptic retinal ganglion cell (RGC) axons. Increasing BDNF levels in the optic tectum of Xenopus tadpoles significantly increases both axon arborization and synapse density per axon terminal within a few hours of treatment. Here, we have further explored the mechanisms by which BDNF shapes synaptic connectivity by imaging tectal neurons, the postsynaptic partners of RGCs. Individual neurons were co-labeled with DsRed2 and a GFP-tagged postsynaptic density protein (PSD95-GFP) to visualize dendritic morphology and postsynaptic specializations simultaneously in vivo. Immunoelectron microscopy confirmed that PSD95-GFP predominantly localized to ultrastructurally identified synapses. Time-lapse confocal microscopy of individual, double-labeled neurons revealed a coincident, activity-dependent mechanism of synaptogenesis and axon and dendritic arbor growth, which is differentially modulated by BDNF. Microinjection of BDNF into the optic tectum significantly increased synapse number in tectal neuron dendritic arbors within 24 hours, without significantly influencing arbor morphology. BDNF function-blocking antibodies had opposite effects. The BDNF-elicited increase in synapse number complements the previously observed increase in presynaptic sites on RGC axons. These results, together with the timescale of the response by tectal neurons, suggest that the effects of BDNF on dendritic synaptic connectivity are secondary to its effects on presynaptic RGCs. Thus, BDNF influences synaptic connectivity in multiple ways: it enhances axon arbor complexity expanding the synaptic territory of the axon, while simultaneously coordinating synapse formation and stabilization with individual postsynaptic cells.     

Neuromuscular synaptogenesis in wild-type and mutant zebrafish

Genetic screens for synaptogenesis mutants have been performed in many organisms, but few if any have simultaneously screened for defects in pre- and postsynaptic specializations. Here, we report the results of a small-scale genetic screen, the first in vertebrates, for defects in synaptogenesis. Using zebrafish as a model system, we identified seven mutants that affect different aspects of neuromuscular synapse formation. Many of these mutant phenotypes have not been previously reported in zebrafish and are distinct from those described in other organisms. Characterization of mutant and wild-type zebrafish, from the time that motor axons first arrive at target muscles through adulthood, has provided the new information about the cellular events that occur during neuromuscular synaptogenesis. These include insights into the formation and dispersal of prepatterned AChR clusters, the relationship between motor axon elongation and synapse size, and the development of precise appositions between presynaptic clusters of synaptic vesicles in nerve terminals and postsynaptic receptor clusters. In addition, we show that the mechanisms underlying synapse formation within the myotomal muscle itself are largely independent of those that underlie synapse formation at myotendinous junctions and that the outgrowth of secondary motor axons requires at least one cue not necessary for the outgrowth of primary motor axons, while other cues are required for both. One-third of the mutants identified in this screen did not have impaired motility, suggesting that many genes involved in neuromuscular synaptogenesis were missed in large scale motility-based screens. Identification of the underlying genetic defects in these mutants will extend our understanding of the cellular and molecular mechanisms that underlie the formation and function of neuromuscular and other synapses.     

Increased neuromuscular activity causes axonal defects and muscular degeneration

Before establishing terminal synapses with their final muscle targets, migrating motor axons form en passant synaptic contacts with myotomal muscle. Whereas signaling through terminal synapses has been shown to play important roles in pre- and postsynaptic development, little is known about the function of these early en passant synaptic contacts. Here, we show that increased neuromuscular activity through en passant synaptic contacts affects pre- and postsynaptic development. We demonstrate that in zebrafish twister mutants, prolonged neuromuscular transmission causes motor axonal extension and muscular degeneration in a dose-dependent manner. Cloning of twister reveals a novel, dominant gain-of-function mutation in the muscle-specific nicotinic acetylcholine receptor alpha-subunit, CHRNA1. Moreover, electrophysiological analysis demonstrates that the mutant subunit increases synaptic decay times, thereby prolonging postsynaptic activity. We show that as the first en passant synaptic contacts form, excessive postsynaptic activity in homozygous embryos severely impedes pre- and postsynaptic development, leading to degenerative defects characteristic of the human slow-channel congenital myasthenic syndrome. By contrast, in heterozygous embryos, transient and mild increase in postsynaptic activity does not overtly affect postsynaptic morphology but causes transient axonal defects, suggesting bi-directional communication between motor axons and myotomal muscle. Together, our results provide compelling evidence that during pathfinding, myotomal muscle cells communicate extensively with extending motor axons through en passant synaptic contacts.     

THE FINE STRUCTURE OF MOTOR ENDPLATE MORPHOGENESIS

The fine structure of the developing neuromuscular junction of rat intercostal muscle has been studied from 16 days in utero to 10 days postpartum. At 16 days, neuromuscular relations consist of close membrane apposition between clusters of axons and groups of myotubes. Focal electron-opaque membrane specializations more intimately connect axon and myotube membranes to each other. What relation these focal contacts bear to future motor endplates is undetermined. The presence of a group of axons lying within a depression in a myotube wall and local thickening of myotube membranes with some overlying basal lamina indicates primitive motor endplate differentiation. At 18 days, large myotubes surrounded by new generations of small muscle cells occur in groups. Clusters of terminal axon sprouts mutually innervate large myotubes and adjacent small muscle cells within the groups. Nerve is separated from muscle plasma membranes by synaptic gaps partially filled by basal lamina. The plasma membranes of large myotubes, where innervated, simulate postsynaptic membranes. At birth, intercostal muscle is composed of separate myofibers. Soleplate nuclei arise coincident with the peripheral migration of myofiber nuclei. A possible source of soleplate nuclei from lateral fusion of small cells' neighboring areas of innervation is suspected but not proven. Adjacent large and small myofibers are mutually innervated by terminal axon networks contained within single Schwann cells. Primary and secondary synaptic clefts are rudimentary. By 10 days, some differentiating motor endplates simulate endplates of mature muscle. Processes of Schwann cells cover primary synaptic clefts. Axon sprouts lie within the primary clefts and are separated from each other. Specific neural control over individual myofibers may occur after neural processes are segregated in this manner.     

Neural Organization of the Median Ocellus of the Dragonfly : II. Synaptic structure

Two types of presumed synaptic contacts have been recognized by electron microscopy in the synaptic plexus of the median ocellus of the dragonfly. The first type is characterized by an electron-opaque, button-like organelle in the presynaptic cytoplasm, surrounded by a cluster of synaptic vesicles. Two postsynaptic elements are associated with these junctions, which we have termed button synapses. The second synaptic type is characterized by a dense cluster of synaptic vesicles adjacent to the presumed presynaptic membrane. One postsynaptic element is observed at these junctions. The overwhelming majority of synapses seen in the plexus are button synapses. They are found most commonly in the receptor cell axons where they synaptically contact ocellar nerve dendrites and adjacent receptor cell axons. Button synapses are also seen in the ocellar nerve dendrites where they appear to make synapses back onto receptor axon terminals as well as onto adjacent ocellar nerve dendrites. Reciprocal and serial synaptic arrangements between receptor cell axon terminals, and between receptor cell axon terminals and ocellar nerve dendrites are occasionally seen. It is suggested that the lateral and feedback synapses in the median ocellus of the dragonfly play a role in enhancing transients in the postsynaptic responses.     

Asynchronous synapse elimination in neonatal motor units: studies using GFP transgenic mice

In developing muscle, synapse elimination reduces the number of motor axons that innervate each postsynaptic cell. This loss of connections is thought to be a consequence of axon branch trimming. However, branch retraction has not been observed directly, and many questions remain, such as: do all motor axons retract branches, are eliminated branches withdrawn synchronously, and are withdrawing branches localized to particular regions? To address these questions, we used transgenic mice that express fluorescent proteins in small subsets of motor axons, providing a unique opportunity to reconstruct complete axonal arbors and identify all the postsynaptic targets. We found that, during early postnatal development, each motor axon loses terminal branches, but retracting branches withdraw asynchronously and without obvious spatial bias, suggesting that local interactions at each neuromuscular junction regulate synapse elimination.     

THREE-DIMENSIONAL ULTRASTRUCTURE OF THE CRAYFISH NEUROMUSCULAR APPARATUS

The synapse-bearing nerve terminals of the opener muscle of the crayfish Procambarus were reconstructed using electron micrographs of regions which had been serially sectioned. The branching patterns of the terminals of excitatory and inhibitory axons and the locations and sizes of neuromuscular and axo-axonal synapses were studied. Excitatory and inhibitory synapses could be distinguished not only on the basis of differences in synaptic vesicles, but also by a difference in density of pre- and postsynaptic membranes. Synapses of both axons usually had one or more sharply localized presynaptic "dense bodies" around which synaptic vesicles appeared to cluster. Some synapses did not have the dense bodies. These structures may be involved in the physiological activity of the synapse. Excitatory axon terminals had more synapses, and a larger percentage of terminal surface area devoted to synaptic contacts, than inhibitory axon terminals. However, the largest synapses of the inhibitory axon exceeded in surface area those of the excitatory axon. Both axons had many side branches coming from the main terminal; often, the side branches were joined to the main terminal by narrow necks. A greater percentage of surface area was devoted to synapses in side branches than in the main terminal. Only a small fraction of total surface area was devoted to axo-axonal synapses, but these were often located at narrow necks or constrictions of the excitatory axon. This arrangement would result in effective blockage of spike invasion of regions of the terminal distal to the synapse, and would allow relatively few synapses to exert a powerful effect on transmitter release from the excitatory axon. A hypothesis to account for the development of the neuromuscular apparatus is presented, in which it is suggested that production of new synapses is more important than enlargement of old ones as a mechanism for allowing the axon to adjust transmitter output to the functional needs of the muscle.     

Pervasive synaptic branch removal in the mammalian neuromuscular system at birth

Using light and serial electron microscopy, we show profound refinements in motor axonal branching and synaptic connectivity before and after birth. Embryonic axons become maximally connected just before birth when they innervate ～10-fold more muscle fibers than in maturity. In some developing muscles, axons innervate almost every muscle fiber. At birth, each neuromuscular junction is coinnervated by approximately ten highly intermingled axons (versus one in adults). Extensive die off of terminal branches occurs during the first several postnatal days, leading to much sparser arbors that still span the same territory. Despite the extensive pruning, total axoplasm per neuron increases as axons elongate, thicken, and add more synaptic release sites on their remaining targets. Motor axons therefore initially establish weak connections with nearly all available postsynaptic targets but, beginning at birth, massively redistribute synaptic resources, concentrating many more synaptic sites on many fewer muscle fibers. Analogous changes in connectivity may occur in the CNS. VIDEO ABSTRACT:     

In vivo imaging of presynaptic terminals and postsynaptic sites in the mouse submandibular ganglion

Much of what is currently known about the behavior of synapses in vivo has been learned at the mammalian neuromuscular junction, because it is large and accessible and also its postsynaptic acetylcholine receptors (AChRs) are readily labeled with a specific, high-affinity probe, alpha-bungarotoxin (BTX). Neuron-neuron synapses have thus far been much less accessible. We therefore developed techniques for imaging interneuronal synapses in an accessible ganglion in the peripheral nervous system. In the submandibular ganglion, individual preganglionic axons establish large numbers of axo-somatic synapses with postganglionic neurons. To visualize these sites of synaptic contact, presynaptic axons were imaged by using transgenic mice that express fluorescent protein in preganglionic neurons. The postsynaptic sites were visualized by labeling the acetylcholine receptor (AChR) alpha7 subunit with fluorescently tagged BTX. We developed in vivo methods to acquire three-dimensional image stacks of the axons and postsynaptic sites and then follow them over time. The submandibular ganglion is an ideal site to study the formation, elimination, and maintenance of synaptic connections between neurons in vivo.     

The function of p120 catenin in filopodial growth and synaptic vesicle clustering in neurons

At the developing neuromuscular junction (NMJ), physical contact between motor axons and muscle cells initiates presynaptic and postsynaptic differentiation. Using Xenopus nerve-muscle cocultures, we previously showed that innervating axons induced muscle filopodia (myopodia), which facilitated interactions between the synaptic partners and promoted NMJ formation. The myopodia were generated by nerve-released signals through muscle p120 catenin (p120ctn), a protein of the cadherin complex that modulates the activity of Rho GTPases. Because axons also extend filopodia that mediate early nerve-muscle interactions, here we test p120ctn's function in the assembly of these presynaptic processes. Overexpression of wild-type p120ctn in Xenopus spinal neurons leads to an increase in filopodial growth and synaptic vesicle (SV) clustering along axons, whereas the development of these specializations is inhibited following the expression of a p120ctn mutant lacking sequences important for regulating Rho GTPases. The p120ctn mutant also inhibits the induction of axonal filopodia and SV clusters by basic fibroblast growth factor, a muscle-derived molecule that triggers presynaptic differentiation. Of importance, introduction of the p120ctn mutant into neurons hinders NMJ formation, which is observed as a reduction in the accumulation of acetylcholine receptors at innervation sites in muscle. Our results suggest that p120ctn signaling in motor neurons promotes nerve-muscle interaction and NMJ assembly.     

The synaptic organization of visual interneurons in the lobula complex of flies

Summary The synaptic organization of three classes of cobalt-filled and silver-intensified visual interneurons in the lobula complex of the blowfly Calliphora (Col A cells, horizontal cells and vertical cells) was studied electron microscopically. The Col A cells are regularly spaced, columnar, small field neurons of the lobula, which constitute a plexus of arborizations at the posterior surface of the neuropil and the axons of which terminate in the ventrolateral protocerebrum. They show postsynaptic specializations in the distal layer of their lobula-arborizations and additional presynaptic sites in a more proximal layer; their axon terminals are presynaptic to large descending neurons projecting into the thoracic ganglion. The horizontal and vertical cells are giant tangential neurons, the arborizations of which cover the anterior and posterior surface of the lobula plate, respectively, and which terminate in the perioesophageal region of the protocerebrum. Both classes of these giant neurons were found to be postsynaptic in the lobula plate and pre- and postsynaptic at their axon terminals and axon collaterals. The significance of these findings with respect to the functional properties of the neurons investigated is discussed.     

A large-scale RNAi screen identifies functional classes of genes shaping synaptic development and maintenance

Neuronal circuit development and function require proper synapse formation and maintenance. Genetic screens are one powerful method to identify the mechanisms shaping synaptic development and stability. However, genes with essential roles in non-neural tissues may be missed in traditional loss-of-function screens. In an effort to circumvent this limitation, we used neuron-specific RNAi knock down in Drosophila and assayed the formation, growth, and maintenance of the neuromuscular junction (NMJ). We examined 1970 Drosophila genes, each of which has a conserved ortholog in mammalian genomes. Knock down of 158 genes in post-mitotic neurons led to abnormalities in the neuromuscular system, including misapposition of active zone components opposite postsynaptic glutamate receptors, synaptic terminal overgrowth and undergrowth, abnormal accumulation of synaptic material within the axon, and retraction of synaptic terminals from their postsynaptic targets. Bioinformatics analysis demonstrates that genes with overlapping annotated function are enriched within the hits for each phenotype, suggesting that the shared biological function is important for that aspect of synaptic development. For example, genes for proteasome subunits and mitotic spindle organizers are enriched among the genes whose knock down leads to defects in synaptic apposition and NMJ stability. Such genes play essential roles in all cells, however the use of tissue- and temporally-restricted RNAi indicates that the proteasome and mitotic spindle organizers participate in discrete aspects of synaptic development. In addition to identifying functional classes of genes shaping synaptic development, this screen also identifies candidate genes whose role at the synapse can be validated by traditional loss-of-function analysis. We present one such example, the dynein-interacting protein NudE, and demonstrate that it is required for proper axonal transport and synaptic maintenance. Thus, this screen has identified both functional classes of genes as well as individual candidate genes that are critical for synaptic development and will be a useful resource for subsequent mechanistic analysis of synapse formation and maintenance.     

Cell adhesion molecules: signalling functions at the synapse

Many cell adhesion molecules are localized at synaptic sites in neuronal axons and dendrites. These molecules bridge pre- and postsynaptic specializations but do far more than simply provide a mechanical link between cells. In this review, we will discuss the roles these proteins have during development and at mature synapses. Synaptic adhesion proteins participate in the formation, maturation, function and plasticity of synaptic connections. Together with conventional synaptic transmission mechanisms, these molecules are an important element in the trans-cellular communication mediated by synapses.