Wnt Signaling in Neuromuscular Junction Development

Wnt proteins are best known for their profound roles in cell patterning, because they are required for the embryonic development of all animal species studied to date. Besides regulating cell fate, Wnt proteins are gaining increasing recognition for their roles in nervous system development and function. New studies indicate that multiple positive and negative Wnt signaling pathways take place simultaneously during the formation of vertebrate and invertebrate neuromuscular junctions. Although some Wnts are essential for the formation of NMJs, others appear to play a more modulatory role as part of multiple signaling pathways. Here we review the most recent findings regarding the function of Wnts at the NMJ from both vertebrate and invertebrate model systems.Wnt proteins are evolutionarily conserved, secreted lipo-glycoproteins involved in a wide range of developmental processes in all metazoan organisms examined to date. In addition to governing many embryonic developmental processes, Wnt signaling is also involved in nervous system maintenance and function, and deregulation of Wnt signaling pathways occurs in many neurodegenerative and psychiatric diseases (De Ferrari and Inestrosa 2000; Caricasole et al. 2005; Okerlund and Cheyette 2011). The first link between Wnt signaling and synapse development was established by Salinas and colleagues in the vertebrate nervous system (Lucas and Salinas 1997; Hall et al. 2000) and by Budnik and colleagues at the invertebrate neuromuscular junction (NMJ) (Packard et al. 2002). Since then, Wnt signaling has emerged as an essential regulator of synaptic development and function in both central and peripheral synapses. Although important roles for Wnt signaling have become known from studies in both the central and peripheral nervous system, this article is concerned with the role of Wnts at the NMJ.     

Acetylcholine Synthesis at the Rat Neuromuscular Junction

Acetylcholine (ACh) synthesis in homogenates of rat soleus muscles had two components. One component, specifically inhibited by bromoacetylcholine (BrACh), had a Km for choline of 0.26 mM; the other, resistant to BrACh, had a Km for choline of 45 mM. The component with a low Km was absent from denervated muscle, and was identical in kinetic terms to ACh synthesising activity in homogenates of sciatic nerve. It is therefore considered choline acetyltransferase (ChAT)-specific. The use of BrACh as a specific inhibitor of ChAT activity allowed the calculation of ACh synthesis at individual motor end-plates in the soleus muscle of the rat: 2.1 X 10(-3) nmol h-1. Since the number of muscle fibres and the number of motor units are known for this muscle, ACh synthesis per motor unit could be calculated: 0.15 nmol h-1. It is concluded that BrACh can be used as a specific inhibitor of ChAT activity in homogenates of skeletal muscle and that its use will obviate the necessity of dividing biopsied muscle or small rodent muscles into neural and aneural segments.     

Electron Microscope Study of the Human Neuromuscular Junction

A preliminary electron microscope study of human neuromuscular junction is presented. The biopsy material was taken from the palmarus longus, and fixed routinely in osmium tetroxide and embedded in methacrylate. The structure of the motor endings and the relationship of the synaptic vesicles to the axolemmal membrane are described. The synaptic clefts are filled with an homogeneous material in continuity with the basement membrane covering the muscle fiber. The subneural apparatus is described, and special attention is paid to a vesicular component present in the sarcoplasm of the junctional area, which differs from synaptic vesicles and is presumed to be a derivate of the sarcoplasmic reticulum.     

Caenorhabditis elegans Neuromuscular Junction: GABA Receptors and Ivermectin Action

The prevalence of human and animal helminth infections remains staggeringly high, thus urging the need for concerted efforts towards this area of research. GABA receptors, encoded by the unc-49 gene, mediate body muscle inhibition in Caenorhabditis elegans and parasitic nematodes and are targets of anthelmintic drugs. Thus, the characterization of nematode GABA receptors provides a foundation for rational anti-parasitic drug design. We therefore explored UNC-49 channels from C. elegans muscle cultured cells of the first larval stage at the electrophysiological and behavioral levels. Whole-cell recordings reveal that GABA, muscimol and the anthelmintic piperazine elicit macroscopic currents from UNC-49 receptors that decay in their sustained presence, indicating full desensitization. Single-channel recordings show that all drugs elicit openings of ∼2.5 pA (+100 mV), which appear either as brief isolated events or in short bursts. The comparison of the lowest concentration required for detectable channel opening, the frequency of openings and the amplitude of macroscopic currents suggest that piperazine is the least efficacious of the three drugs. Macroscopic and single-channel GABA-activated currents are profoundly and apparently irreversibly inhibited by ivermectin. To gain further insight into ivermectin action at C. elegans muscle, we analyzed its effect on single-channel activity of the levamisol-sensitive nicotinic receptor (L-AChR), the excitatory receptor involved in neuromuscular transmission. Ivermectin produces a profound inhibition of the frequency of channel opening without significant changes in channel properties. By revealing that ivermectin inhibits C. elegans muscle GABA and L-AChR receptors, our study adds two receptors to the already known ivermectin targets, thus contributing to the elucidation of its pleiotropic effects. Behavioral assays in worms show that ivermectin potentiates piperazine-induced paralysis, thus suggesting that their combination is a good strategy to overcome the increasing resistance of parasites, an issue of global concern for human and animal health.     

Phenobarbital Increases Spontaneous Transmitter Release at the Frog Neuromuscular Junction

Phenobarbital (1-2 × 10^-4M) markedly increases the frequency of miniature end-plate potentials at the neuromuscular synapse of the frog. This effect was seen in calcium free media containing EGTA. The drug probably acts presynaptically at an intracellular locus to increase the presynaptic free calcium concentration.     

An Excess-Calcium-Binding-Site Model Predicts Neurotransmitter Release at the Neuromuscular Junction

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca²⁺-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20–40 independent Ca²⁺-binding sites on synaptic vesicles, only a fraction of which need to bind Ca²⁺ to trigger fusion, are sufficient to predict physiological release. Our excess-Ca²⁺-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca²⁺-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca²⁺ dynamics during synapse activation.     

An Excess-Calcium-Binding-Site Model Predicts Neurotransmitter Release at the Neuromuscular Junction

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca²⁺-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20–40 independent Ca²⁺-binding sites on synaptic vesicles, only a fraction of which need to bind Ca²⁺ to trigger fusion, are sufficient to predict physiological release. Our excess-Ca²⁺-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca²⁺-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca²⁺ dynamics during synapse activation.     

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses ¹. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs ^2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization ⁴ and hyperosmotic saline, all of which will be briefly described.Open in a separate windowClick here to view.^{(91M, flv)}     

Drosophila Neuroligin3 Regulates Neuromuscular Junction Development and Synaptic Differentiation

Neuroligins (Nlgs) are a family of cell adhesion molecules thought to be important for synapse maturation and function. Mammalian studies have shown that different Nlgs have different roles in synaptic maturation and function. In Drosophila melanogaster, the roles of Drosophila neuroligin1 (DNlg1), neuroligin2, and neuroligin4 have been examined. However, the roles of neuroligin3 (dnlg3) in synaptic development and function have not been determined. In this study, we used the Drosophila neuromuscular junctions (NMJs) as a model system to investigate the in vivo role of dnlg3. We showed that DNlg3 was expressed in both the CNS and NMJs where it was largely restricted to the postsynaptic site. We generated dnlg3 mutants and showed that these mutants exhibited an increased bouton number and reduced bouton size compared with the wild-type (WT) controls. Consistent with alterations in bouton properties, pre- and postsynaptic differentiations were affected in dnlg3 mutants. This included abnormal synaptic vesicle endocytosis, increased postsynaptic density length, and reduced GluRIIA recruitment. In addition to impaired synaptic development and differentiation, we found that synaptic transmission was reduced in dnlg3 mutants. Altogether, our data showed that DNlg3 was required for NMJ development, synaptic differentiation, and function.