ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.     

Activity-dependent elimination of neuromuscular synapses

At developing neuromuscular synapses in vertebrates, different motor axon inputs to muscle fibers compete for maintenance of their synapses. Competition results in progressive changes in synaptic structure and strength that lead to the weakening and loss of some inputs, a process that has been called synapse elimination. At the same time, a single input is strengthened and maintained throughout adult life, consistently recruiting muscle fibers to contract even at rapid firing rates. Work over the last decade has led to an understanding of some of the cell biological mechanisms that underlie competition and how these culminate in synapse elimination. We discuss current ideas about how activity modulates neuromuscular synaptic competition, how competition leads to synapse loss, and how these processes are modulated by cell-cell signaling. A common feature of competition at neuromuscular as well as CNS synapses is that temporally correlated activity seems to slow or prevent competition, while uncorrelated activity seems to trigger or enhance competition. Important questions that remain to be addressed include how patterns of motor neuron activity affect synaptic strength, what is the temporal relationship between changes in synaptic strength and structure, and what cellular signals mediate synapse loss. Answers to these questions will expand our understanding of the mechanisms by which activity edits synaptic structure and function, writing permanent changes in neural circuitry.     

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.     

Neuromuscular synapses mediate motor axon branching and motoneuron survival during the embryonic period of programmed cell death

The embryonic period of motoneuron programmed cell death (PCD) is marked by transient motor axon branching, but the role of neuromuscular synapses in regulating motoneuron number and axonal branching is not known. Here, we test whether neuromuscular synapses are required for the quantitative association between reduced skeletal muscle contraction, increased motor neurite branching, and increased motoneuron survival. We achieved this by comparing agrin and rapsyn mutant mice that lack acetylcholine receptor (AChR) clusters. There were significant reductions in nerve-evoked skeletal muscle contraction, increases in intramuscular axonal branching, and increases in spinal motoneuron survival in agrin and rapsyn mutant mice compared with their wild-type littermates at embryonic day 18.5 (E18.5). The maximum nerve-evoked skeletal muscle contraction was reduced a further 17% in agrin mutants than in rapsyn mutants. This correlated to an increase in motor axon branch extension and number that was 38% more in agrin mutants than in rapsyn mutants. This suggests that specializations of the neuromuscular synapse that ensure efficient synaptic transmission and muscle contraction are also vital mediators of motor axon branching. However, these increases in motor axon branching did not correlate with increases in motoneuron survival when comparing agrin and rapsyn mutants. Thus, agrin-induced synaptic specializations are required for skeletal muscle to effectively control motoneuron numbers during embryonic development.     

Gonadal steroid preservation of central synaptic input to hamster facial motoneurons following peripheral axotomy

In recent work, we have demonstrated that testosterone propionate accelerates recovery from facial nerve injury in the adult male hamster. Central synaptic stripping following peripheral motor neuron damage is a well-established component of the injury response. Gonadal steroids regulate synaptogenesis in the normal nervous system. In this study, we tested the hypothesis that testosterone propionate administration at the time of facial nerve transection alters the synaptic connectivity of injured facial motoneurons. Adult hamsters were subjected to right facial nerve transection at the level of the stylomastoid foramen. Half the animals received subcutaneous implants of testosterone propionate; the other half were sham implanted. At 5 days postoperative, the animals were killed by intracardiac perfusion-fixation, and the control and axotomized facial nuclear groups from the brainstems of nonhormone- and testosterone propionate-treated animals processed for routine transmission electron microscopy. Quantiative analysis of the synaptic ratio (percent somal membrane covered by synaptic profiles) and the average length of axosomatic synapses was accomplished. The results indicate that axotomy alone resulted in an 81% reduction in the synaptic ratio and a 26% decrease in the average synaptic length of axosomatic synapses. Exposure to testosterone propionate from the time of facial nerve transection resulted in only a 48% reduction in the synaptic ratio and a 16% decrease in the average synaptic length of axosomatic synapses following injury. Thus, testosterone propionate significantly attenuated the amount of synaptic stripping that occurred at 5 days postoperative and the decrease in average length of the remaining synapses as well. It is concluded that gonadal steroids modulate central synaptic plasticity following peripheral nerve injury. The results are discussed in light of our recent findings of steroidal effects on the central astrocyctic response to facial nerve injury as well.     

Ultrastructural evidence for two-cell and three-cell neural pathways in the tentacle epidermis of the sea anemone Aiptasia pallida.

Sensory and ganglion cells in the tentacle epidermis of the sea anemone Aiptasia pallida were traced in serial transmission electron micrographs to their synaptic contacts on other cells. Sensory cell synapses were found on spirocytes, muscle cells, and ganglion cells. Ganglion cells, in turn, synapsed on sensory cells, spirocytes, muscle cells, and other neurons and formed en passant axo-axonal synapses. Axonal synapses on nematocytes and gland cells were not traced to their cells of origin, i.e., identified sensory or ganglion cells. Direct synaptic contacts of sensory cells with spirocytes and sensory cells with muscle cells suggest a local two-cell pathway for spirocyst discharge and muscle cell contraction, whereas interjection of a ganglion cell between the sensory and effector cells creates a local three-cell pathway. The network of ganglion cells and their processes allows for a through-conduction system that is interconnected by chemical synapses. Although the sea anemone nervous system is more complex than that of Hydra, it has similar two-cell and three-cell effector pathways that may function in local responses to tentacle contact with food.     

The nerve-muscle synapse of the garter snake

Snake nerve-muscle preparations are well-suited for study of both motor innervation patterns at the systems level and NMJ function at the cellular level. Their small size (～100 myofibers) and thinness (one fiber) allows access to all NMJs in one muscle. Snake NMJs are of three types, two twitch subtypes and a single tonic type. Properties of the NMJs supplied by a particular motor neuron, and of the motor unit fibers they innervate, are precisely regulated by the motor neuron in a manner consistent with the Henneman Size Principle. Unlike its amphibian or mammalian cousins, the snake NMJ comprises ～50 (twitch) or ～20 (tonic) individual one-bouton synapses, similar to synapses found in the central nervous system. Each bouton releases a few quanta per stimulus. Larger fibers, which require more synaptic current to initiate contraction, receive nerve terminals that contain more boutons and express receptor patches with higher sensitivity to transmitter. Quantal analysis suggests that transmitter release sites in one bouton do not behave independently; rather, they may cooperate to reduce fluctuations and enhance reliability. After release, two mechanisms coexist for retrieval and reprocessing of spent vesicles–one involving clathrin-mediated endocytosis, the other macropinocytosis. Unanswered questions include how each mechanism is regulated in a use-dependent manner.     

Lrp4 is a receptor for Agrin and forms a complex with MuSK

Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin.     

Kismet Positively Regulates Glutamate Receptor Localization and Synaptic Transmission at the Drosophila Neuromuscular Junction

The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is structurally and functionally similar to mammalian glutamatergic synapses. These synapses can, as a result of changes in activity, alter the strength of their connections via processes that require chromatin remodeling and changes in gene expression. The chromodomain helicase DNA binding (CHD) protein, Kismet (Kis), is expressed in both motor neuron nuclei and postsynaptic muscle nuclei of the Drosophila larvae. Here, we show that Kis is important for motor neuron synaptic morphology, the localization and clustering of postsynaptic glutamate receptors, larval motor behavior, and synaptic transmission. Our data suggest that Kis is part of the machinery that modulates the development and function of the NMJ. Kis is the homolog to human CHD7, which is mutated in CHARGE syndrome. Thus, our data suggest novel avenues of investigation for synaptic defects associated with CHARGE syndrome.     

Neuroanatomical and immunocytochemical studies of the head retractor muscle innervation in the pond snail, Lymnaea stagnalis L

Axonal tracing and immunocytochemical techniques were used to study the innervation of the head retractor muscle (HRM) in the pond snail Lymnaea stagnalis L. Fibers of both the superior and inferior cervical nerves which innervate the HRM form endings that comply with the structure of chemical synapses. The somata of neurons with axons in these nerves are located in all except the buccal ganglia of the central nervous system, and this seems to be a special feature of the HRM motor system. By staining the filamentous actin with Oregon-green conjugated phalloidin, we demonstrated that the HRM has a multiterminal innervation and one muscle fiber can contain several synaptic endings which appear to be both morphologically and physiologically different. The morphological diversity of synaptic vesicles suggests a multiplicity of neurotransmitters acting on these nerve-muscle junctions. Immunocytochemical evidence was found for a strong serotonergic and FMRFamidergic innervation of muscle fibers through axons of the inferior cervical nerve. The thin fibers of the inferior cervical nerve possess immunoreactivity to glutamate, gamma-aminobutyric acid (GABA) and choline-acetyltransferase, and form sparser innervation patterns in the muscle. Our results indicate that several neurotransmitters are present in the nerves innervating the Lymnaea HRM and may therefore participate in the control of this muscle. The possible behavioral significance of such different neurotransmitter sets involved in the regulation of contractions is discussed.     

Ultrastructural organization of the synapses in ganglia of the hen intestinal nerve under various conditions of its activity

The interneuronal connections in ganglia of the caudal part of the hen intestinal nerve of Remak are presented as axodendritic and axosomatic synapses and symmetric axo-axonal, dendro-dendritic and axodendritic contacts, often forming complicated complexes. Under conditions of preliminary decentralization or under certain disturbances of nervous connections with the intestine, a part of synapses remains, and a part of them degenerates, this demonstrates participation of peripheral afferent neurons in formation of the synaptic apparatus of the ganglia mentioned. The axonal terminals differentiate by composition of the synaptic vesicles: some contain mainly light agranular vesicles, others--a large amount of granular ones. The characteristic peculiarities of the hen intestinal nerve ganglia, in contrast to analogous mammalian ganglia, are abundant axosomatic synapses in some neurons, and presynaptic terminals, containing a large number of granular vesicles.     

内源性大麻素系统对皮层下运动中枢的调控作用

以往研究已证明,内源性大麻素系统广泛存在于中枢和外周神经组织中,并作为逆向信号分子在突触信号传递中发挥重要调节作用。本文就内源性大麻素系统对皮层下运动中枢的调控作用及相关机制进行综述,以期系统地论述皮层下运动中枢在躯体运动、动作选择和运动技能学习等高级神经活动过程中的突触和神经环路机制,并为相关疾病的治疗和靶向药物开发提供理论依据。     

Patterning of skeletal muscle

Recent studies challenge the view that signals provided by motor neurons are required to activate subsynaptic nuclei and induce postsynaptic specializations in developing skeletal muscle. New findings show that acetylcholine receptor genes are expressed and that acetylcholine receptor clusters form preferentially in the prospective synaptic region of muscle independently of motor innervation. These results indicate that developing myotubes are patterned by mechanisms intrinsic to developing muscles and raise the possibility that patterning of muscles may influence the growth pattern of motor axons and the sites where synapses form.     

Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle

Acetylcholine receptors (AChRs) mediate synaptic transmission at the neuromuscular junction, and structural and functional analysis has assigned distinct functions to the fetal (alpha2beta(gamma)delta) and adult types of AChR (alpha2beta(epsilon)delta). Mice lacking the epsilon-subunit gene die prematurely, showing that the adult type is essential for maintenance of neuromuscular synapses in adult muscle. It has been suggested that the fetally and neonatally expressed AChRs are crucial for muscle differentiation and for the formation of the neuromuscular synapses. Here, we show that substitution of the fetal-type AChR with an adult-type AChR preserves myoblast fusion, muscle and end-plate differentiation, whereas it substantially alters the innervation pattern of muscle by the motor nerve. Mutant mice form functional neuromuscular synapses outside the central, narrow end-plate band region in the diaphragm, with synapses scattered over a wider muscle territory. We suggest that one function of the fetal type of AChR is to ensure an orderly innervation pattern of skeletal muscle.     

Synaptic specificity is generated by the synaptic guidepost protein SYG-2 and its receptor, SYG-1

Synaptic connections in the nervous system are directed onto specific cellular and subcellular targets. Synaptic guidepost cells in the C. elegans vulval epithelium drive synapses from the HSNL motor neuron onto adjacent target neurons and muscles. Here, we show that the transmembrane immunoglobulin superfamily protein SYG-2 is a central component of the synaptic guidepost signal. SYG-2 is expressed transiently by primary vulval epithelial cells during synapse formation. SYG-2 binds SYG-1, the receptor on HSNL, and directs SYG-1 accumulation and synapse formation to adjacent regions of HSNL. syg-1 and syg-2 mutants have defects in synaptic specificity; the HSNL neuron forms fewer synapses onto its normal targets and forms ectopic synapses onto inappropriate targets. Misexpression of SYG-2 in secondary epithelial cells causes aberrant accumulation of SYG-1 and synaptic markers in HSNL adjacent to the SYG-2-expressing cells. Our results indicate that local interactions between immunoglobulin superfamily proteins can determine specificity during synapse formation.     

Gap junctional communication among motor and other neurons shapes patterns of neural activity and synaptic connectivity during development

We are studying the functional roles of neuronal gap junctional coupling during development, using motor neurons and their synapses with muscle fibers as a model system. At neuromuscular synapses, several studies have shown that the relative pattern of activity among motor inputs competing for innervation of the same target muscle fiber determines how patterns of innervation are sculpted during the first weeks after birth. We asked whether gap junctional coupling among motor neurons modulates the relative timing of motor neuron activity in awake, behaving neonatal mice. We found that the activity of motor neurons innervating the same muscle is temporally correlated perinatally, during the same period that gap junction-mediated electrical and dye coupling are present. In vivo blockade of gap junctions abolished temporal correlations in motor neuron activity, without changing overall motor behavior, motor neuron activity patterns or firing frequency. Together with preliminary studies in mice lacking gap junction protein Cx40, our data suggest that developmentally regulated gap junctional coupling among motor and other neurons affects the activity in nascent neural circuits and thus in turn affects synaptic connectivity. Dynamic monitoring of dye coupling can be used to explore this possibility in normal mice and in mice lacking gap junction proteins during embryonic and neonatal development.     

Astroglial cradle in the life of the synapse

Astroglial perisynaptic sheath covers the majority of synapses in the central nervous system. This glial coverage evolved as a part of the synaptic structure in which elements directly responsible for neurotransmission (exocytotic machinery and appropriate receptors) concentrate in neuronal membranes, whereas multiple molecules imperative for homeostatic maintenance of the synapse (transporters for neurotransmitters, ions, amino acids, etc.) are shifted to glial membranes that have substantially larger surface area. The astrocytic perisynaptic processes act as an ‘astroglial cradle’ essential for synaptogenesis, maturation, isolation and maintenance of synapses, representing the fundamental mechanism contributing to synaptic connectivity, synaptic plasticity and information processing in the nervous system.     

Synaptic diversity and differentiation: Crustacean neuromuscular junctions

Crustacean motor neurons exhibit a wide range of synaptic responses. Tonically active neurons generally produce small excitatory postsynaptic potentials (EPSPs) at low impulse frequencies, and are able to release much more transmitter as the impulse frequency increases. Phasic neurons typically generate large EPSPs in their target cells, but have less capability for frequency facilitation, and undergo synaptic depression during maintained activity. These differences depend in part upon the neuron's ongoing levels of activity; phasic neurons acquire physiological and morphological features of tonic neurons when their activity level is altered. Molecules responsible for adaptation to activity can be sought in single identified phasic neurons with current techniques. The fact that both phasic and tonic neurons innervate the same target muscle fibers is evidence for presynaptic determination of synaptic properties, but there is also evidence for postsynaptic determination of specific properties of different endings of a single neuron. The occurrence of high- and low-output endings of the same tonic motor neurons on different muscle fibers suggests a target-specific influence on synaptic properties. Structural variation of synapses on individual terminal varicosities leads to the hypothesis that individual synapses have different probabilities for release of transmitter. We hypothesize that structurally complex synapses have a higher probability for release than the less complex synapses. This provides an explanation for the larger quantal contents of high-output terminals (where the proportion of complex synapses is higher), and also a mechanism for progressive recruitment of synapses during frequency facilitation.     

Localisation of the high-affinity choline transporter-1 in the rat skeletal motor unit

The rate-limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline via a high-affinity transporter. We have generated antisera against the recently identified transporter CHT1 to investigate its distribution in rat motor neurons and skeletal muscle and have used these antisera in combination with (1) antisera against the vesicular acetylcholine transporter (VAChT) to identify cholinergic synapses and (2) Alexa-488-labelled alpha-bungarotoxin to identify motor endplates. In the motor unit, immunohistochemistry and RT-PCR have demonstrated that CHT1 is restricted to motoneurons and absent from the non-neuronal ACh-synthesizing elements, e.g. skeletal muscle fibres. In addition, CHT1 is also present in parasympathetic neurons of the tongue, as evidenced by immunohistochemistry and RT-PCR. CHT1 immunoreativity is principally found at all segments (perikaryon, dendrites, axon) of the motoneuron but is enriched at neuro-neuronal and neuro-muscular synapses. This preferential localisation matches well with its anticipated pivotal role in synaptic transmitter recycling and synthesis.     

Synapse formation and plasticity: recent insights from the perspective of the ubiquitin proteasome system

The formation of synaptic connections during the development of the nervous system requires the precise targeting of presynaptic and postsynaptic compartments. Furthermore, synapses are continually modified in the brain by experience. Recently, the ubiquitin proteasome system has emerged as a key regulator of synaptic development and function. The modification of proteins by ubiquitin, and in many cases their subsequent proteasomal degradation, has proven to be an important mechanism to control protein stability, activity and localization at synapses. Recent work has highlighted key questions of the UPS during the development and remodeling of synaptic connections in the nervous system.