Cholinergic Neuromuscular Junctions Research Articles

New advances in clinical application of neostigmine: no longer focusing solely on increasing skeletal muscle strength.

Neostigmine is a clinical cholinesterase inhibitor, that is, commonly used to enhance the function of the cholinergic neuromuscular junction. Recent studies have shown that neostigmine regulates the immune-inflammatory response through the cholinergic anti-inflammatory pathway, affecting perioperative neurocognitive function. This article reviews the relevant research evidence over the past 20years, intending to provide new perspectives and strategies for the clinical application of neostigmine.

Postsynaptic Calcium Extrusion at the Mouse Neuromuscular Junction Alkalinizes the Synaptic Cleft.

Neurotransmission is shaped by extracellular pH. Alkalization enhances pH-sensitive transmitter release and receptor activation, whereas acidification inhibits these processes and can activate acid-sensitive conductances in the synaptic cleft. Previous work has shown that the synaptic cleft can either acidify because of synaptic vesicular release and/or alkalize because of Ca2+ extrusion by the plasma membrane ATPase (PMCA). The direction of change differs across synapse types. At the mammalian neuromuscular junction (NMJ), the direction and magnitude of pH transients in the synaptic cleft during transmission remain ambiguous. We set out to elucidate the extracellular pH transients that occur at this cholinergic synapse under near-physiological conditions and identify their sources. We monitored pH-dependent changes in the synaptic cleft of the mouse levator auris longus using viral expression of the pseudoratiometric probe pHusion-Ex in the muscle. Using mice from both sexes, a significant and prolonged alkalization occurred when stimulating the connected nerve for 5 s at 50 Hz, which was dependent on postsynaptic intracellular Ca2+ release. Sustained stimulation for a longer duration (20 s at 50 Hz) caused additional prolonged net acidification at the cleft. To investigate the mechanism underlying cleft alkalization, we used muscle-expressed GCaMP3 to monitor the contribution of postsynaptic Ca2+ Activity-induced liberation of intracellular Ca2+ in muscle positively correlated with alkalization of the synaptic cleft, whereas inhibiting PMCA significantly decreased the extent of cleft alkalization. Thus, cholinergic synapses of the mouse NMJ typically alkalize because of cytosolic Ca2+ liberated in muscle during activity, unless under highly strenuous conditions where acidification predominates.SIGNIFICANCE STATEMENT Changes in synaptic cleft pH alter neurotransmission, acting on receptors and channels on both sides of the synapse. Synaptic acidification has been associated with a myriad of diseases in the central and peripheral nervous system. Here, we report that in near-physiological recording conditions the cholinergic neuromuscular junction shows use-dependent bidirectional changes in synaptic cleft pH-immediate alkalinization and a long-lasting acidification under prolonged stimulation. These results provide further insight into physiologically relevant changes at cholinergic synapses that have not been defined previously. Understanding and identifying synaptic pH transients during and after neuronal activity provides insight into short-term synaptic plasticity synapses and may identify therapeutic targets for diseases.

CASK and FARP localize two classes of post-synaptic ACh receptors thereby promoting cholinergic transmission.

Changes in neurotransmitter receptor abundance at post-synaptic elements play a pivotal role in regulating synaptic strength. For this reason, there is significant interest in identifying and characterizing the scaffolds required for receptor localization at different synapses. Here we analyze the role of two C. elegans post-synaptic scaffolding proteins (LIN-2/CASK and FRM-3/FARP) at cholinergic neuromuscular junctions. Constitutive knockouts or muscle specific inactivation of lin-2 and frm-3 dramatically reduced spontaneous and evoked post-synaptic currents. These synaptic defects resulted from the decreased abundance of two classes of post-synaptic ionotropic acetylcholine receptors (ACR-16/CHRNA7 and levamisole-activated AChRs). LIN-2's AChR scaffolding function is mediated by its SH3 and PDZ domains, which interact with AChRs and FRM-3/FARP, respectively. Thus, our findings show that post-synaptic LIN-2/FRM-3 complexes promote cholinergic synaptic transmission by recruiting AChRs to post-synaptic elements.

Interaction of Nootropic Drugs with Neurotransmitter Enzyme Acetylcholinesterase: An Insilico Approach: A Review

Nootropic drugs are the class of drugs or supplements that are claimed to enhance cognitive functions, specifically executive functions, memory and creativity in healthy individual. They are sometime referred as cognitive enhancers or smart drugs as they are associated with memory improvement functioning. Some of them are well known drugs and clinically approved by the Food and Drug Administration (FDA). All metabolic reactions are purely dependent on enzymatic actions as they play a very important role in regulating and maintaining most of the biological responses and various processes. An enzyme Acetylcholinesterase (AChE) seems to play an essential role in the conduction of cholinergic brain synapses and neuromuscular junctions. There have been different nootropic drugs identified and approved for curing neurodegenerative disorders such as Alzheimer, Parkinson and Huntington's disease. Their binding efficiency and energy have been well studied an established by using the in-silico docking tools. There are different docking tools available today for analysis of molecules such as PyRx, Auto dock and schrodinger suite. The advent of these tools is being widely used by the pharmaceutical industries for the virtual screening of the formulated drugs against the desired target molecule. It has made the drug formulation process more time efficient and cost effective. Thus, an in-silico approach has been widely accepted for drug discovery and its design.

The HSPG syndecan is a core organizer of cholinergic synapses.

The extracellular matrix has emerged as an active component of chemical synapses regulating synaptic formation, maintenance, and homeostasis. The heparan sulfate proteoglycan (HSPG) syndecans are known to regulate cellular and axonal migration in the brain. They are also enriched at synapses, but their synaptic functions remain more elusive. Here, we show that SDN-1, the sole orthologue of syndecan in C. elegans, is absolutely required for the synaptic clustering of homomeric α7-like acetylcholine receptors (AChRs) and regulates the synaptic content of heteromeric AChRs. SDN-1 is concentrated at neuromuscular junctions (NMJs) by the neurally secreted synaptic organizer Ce-Punctin/MADD-4, which also activates the transmembrane netrin receptor DCC. Those cooperatively recruit the FARP and CASK orthologues that localize α7-like-AChRs at cholinergic NMJs through physical interactions. Therefore, SDN-1 stands at the core of the cholinergic synapse organization by bridging the extracellular synaptic determinants to the intracellular synaptic scaffold that controls the postsynaptic receptor content.

The Snail transcription factor CES-1 regulates glutamatergic behavior in C. elegans.

Regulation of AMPA-type glutamate receptor (AMPAR) expression and function alters synaptic strength and is a major mechanism underlying synaptic plasticity. Although transcription is required for some forms of synaptic plasticity, the transcription factors that regulate AMPA receptor expression and signaling are incompletely understood. Here, we identify the Snail family transcription factor ces-1 in an RNAi screen for conserved transcription factors that regulate glutamatergic behavior in C. elegans. ces-1 was originally discovered as a selective cell death regulator of neuro-secretory motor neuron (NSM) and I2 interneuron sister cells in C. elegans, and has almost exclusively been studied in the NSM cell lineage. We found that ces-1 loss-of-function mutants have defects in two glutamatergic behaviors dependent on the C. elegans AMPA receptor GLR-1, the mechanosensory nose-touch response and spontaneous locomotion reversals. In contrast, ces-1 gain-of-function mutants exhibit increased spontaneous reversals, and these are dependent on glr-1 consistent with these genes acting in the same pathway. ces-1 mutants have wild type cholinergic neuromuscular junction function, suggesting that they do not have a general defect in synaptic transmission or muscle function. The effect of ces-1 mutation on glutamatergic behaviors is not due to ectopic cell death of ASH sensory neurons or GLR-1-expressing neurons that mediate one or both of these behaviors, nor due to an indirect effect on NSM sister cell deaths. Rescue experiments suggest that ces-1 may act, in part, in GLR-1-expressing neurons to regulate glutamatergic behaviors. Interestingly, ces-1 mutants suppress the increased reversal frequencies stimulated by a constitutively-active form of GLR-1. However, expression of glr-1 mRNA or GFP-tagged GLR-1 was not decreased in ces-1 mutants suggesting that ces-1 likely promotes GLR-1 function. This study identifies a novel role for ces-1 in regulating glutamatergic behavior that appears to be independent of its canonical role in regulating cell death in the NSM cell lineage.

Pharyngeal Pumping Variability Assessment Reveals Subtle Mutant and Treatment Effects in the Nematode C. elegans

In C. elegans, feeding is achieved through a well characterized behavior, pharyngeal pumping. This pumping is controlled by two pairs of pharyngeal motor neurons (MC and M3). Each pump cycle corresponds to the propagation of a single pharyngeal muscle action potential, initiated by MC and transmitted across a single cholinergic neuromuscular junction. The eat‐2 gene is required for nicotinic neurotransmission at the pharynx. We assessed pumping patterns exhibited by wild type (N2) and a series of eat‐2 mutant strains, each identified as possessing deficits in pumping, including: (Strain: genotype) DA465: eat‐2(ad465) II; DA1113: eat‐2(ad1113) II; DA1116: eat‐2(ad1116) II. We also assessed pumping patterns in N2 strain animals incubated in the anthelmintic and acetylcholine receptor antagonist Levamisole ((−)‐Tetramisole hydrochloride; 0 – 50 uM). All studies we done in animals incubated in 10mM serotonin in M9 buffer. We recorded electropharyngeograms (EPG) using the recently developed ScreenChip system (NemaMetrix) to detect individual pump events, and analyzed recordings using an algorithm (WormBeat) to distinguish and quantify neuromuscular fidelity of the pharynx and pharyngeal pumping pace variation. As expected, mutant strains and Levamisole‐exposed N2 worms each illustrated pump pattern defects. Results show that the WormBeat algorithm can identify subtle treatment effects and distinguish phenotypic variation between mutant strains. Pump pattern assessment distinguished individual strains and allowed comparison of defect to the Levamisole dose‐responses of N2s.Support or Funding InformationWork reported in this publication was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers 1R15HL126105, 1SC2GM112570, UL1GM118979, TL4GM118980, and RL5GM118978. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.

Endophilin A and B Join Forces With Clathrin to Mediate Synaptic Vesicle Recycling in Caenorhabditis elegans.

Synaptic vesicle (SV) recycling enables ongoing transmitter release, even during prolonged activity. SV membrane and proteins are retrieved by ultrafast endocytosis and new SVs are formed from synaptic endosomes (large vesicles—LVs). Many proteins contribute to SV recycling, e.g., endophilin, synaptojanin, dynamin and clathrin, while the site of action of these proteins (at the plasma membrane (PM) vs. at the endosomal membrane) is only partially understood. Here, we investigated the roles of endophilin A (UNC-57), endophilin-related protein (ERP-1, homologous to human endophilin B1) and of clathrin, in SV recycling at the cholinergic neuromuscular junction (NMJ) of C. elegans. erp-1 mutants exhibited reduced transmission and a progressive reduction in optogenetically evoked muscle contraction, indicative of impaired SV recycling. This was confirmed by electrophysiology, where particularly endophilin A (UNC-57), but also endophilin B (ERP-1) mutants exhibited reduced transmission. By optogenetic and electrophysiological analysis, phenotypes in the unc-57; erp-1 double mutant are largely dominated by the unc-57 mutation, arguing for partially redundant functions of endophilins A and B, but also hinting at a back-up mechanism for neuronal endocytosis. By electron microscopy (EM), we observed that unc-57 and erp-1; unc-57 double mutants showed increased numbers of synaptic endosomes of large size, assigning a role for both proteins at the endosome, because endosomal disintegration into new SVs, but not formation of endosomes were hampered. Accordingly, only low amounts of SVs were present. Also erp-1 mutants show reduced SV numbers (but no increase in LVs), thus ERP-1 contributes to SV formation. We analyzed temperature-sensitive mutants of clathrin heavy chain (chc-1), as well as erp-1; chc-1 and unc-57; chc-1 double mutants. SV recycling phenotypes were obvious from optogenetic stimulation experiments. By EM, chc-1 mutants showed formation of numerous and large endosomes, arguing that clathrin, as shown for mammalian synapses, acts at the endosome in formation of new SVs. Without endophilins, clathrin formed endosomes at the PM, while endophilins A and B compensated for the loss of clathrin at the PM, under conditions of high SV turnover.

Elements of molecular machinery of GABAergic signaling in the vertebrate cholinergic neuromuscular junction

It is generally accepted that gamma-aminobutyric acid (GABA) is a signaling molecule abundant in central synapses. In a number of studies though, it has been shown that GABA signaling functions in the peripheral nervous system as well, in particular, in the synapses of sympathetic ganglia. However, there exists no firm evidence on the presence of GABAergic signaling cascade in the intercellular junctions of the somatic nerve system.By the use of immunohistochemistry methods, in the synaptic area of cholinergic neuromuscular contact in rat diaphragm, we have detected glutamate decarboxylase, the enzyme involved in synthesis of GABA, molecules of GABA, and also GAT-2, a protein responsible for transmembrane transport of GABA. Earlier we have also shown that metabotropic GABAB receptors have overlapping localization in the same compartment. Moreover, activation of GABAB receptors affects the intensity of acetylcholine release. These data taken together, allows us to suggest that in the mammalian cholinergic neuromuscular junction, GABA is synthesized and performs certain synaptic signaling function.

Congenital Myasthenic Syndromes or Inherited Disorders of Neuromuscular Transmission: Recent Discoveries and Open Questions.

Congenital myasthenic syndromes (CMS) form a heterogeneous group of rare diseases characterized by fatigable muscle weakness. They are genetically-inherited and caused by defective synaptic transmission at the cholinergic neuromuscular junction (NMJ). The number of genes known to cause CMS when mutated is currently 30, and the relationship between fatigable muscle weakness and defective functions is quite well-understood for many of them. However, some of the most recent discoveries in individuals with CMS challenge our knowledge of the NMJ, where the basis of the pathology has mostly been investigated in animal models. Frontier forms between CMS and congenital myopathy, which have been genetically and clinically identified, underline the poorly understood interplay between the synaptic and extrasynaptic molecules in the neuromuscular system. In addition, precise electrophysiological and histopathological investigations of individuals with CMS suggest an important role of NMJ plasticity in the response to CMS pathogenesis. While efficient drug-based treatments are already available to improve neuromuscular transmission for most forms of CMS, others, as well as neurological and muscular comorbidities, remain resistant. Taken together, the available pathological data point to physiological issues which remain to be understood in order to achieve precision medicine with efficient therapeutics for all individuals suffering from CMS.

My research life: from synaptic transmission to behavior

I have studied signal transmission at synapses and the effects of drugs on it at the molecular and cellular levels. Specific areas of research interest are outlined here. 1) Electrophysiological experiments in cats and rabbits suggested that a new type of analgesic, the phenothiazine derivative levomepromazine, exerts analgesic effects by depressing emotional responses accompanying the sensation of pain. 2) It was hypothesized that motoneurons had long-term effects on muscle cell membrane properties, in addition to controlling moment-to-moment activities. The substance to recover the post-denervation changes in muscle properties in culture was partially purified from mouse nerve extract, which suggested that trophic influences were exerted by substances released from motoneurons. 3) Muscles innervated by adrenergic fibers had sites responsive to acetylcholine as well as to adrenaline in early life in chicks, but only the adrenaline-responsive sites remained during development. Acetylcholine receptor clusters on Xenopus muscles were concentrated at the cholinergic neuromuscular junctions by the movement of receptors from outside the junctions during development. The passive diffusion-trap mechanism explained the accumulation of synaptic receptors at synapses. 4) We found two endocytic pathways and pools of synaptic vesicles contributing to low- and high-frequency synaptic transmission at Drosophila nerve terminals. We then identified two Ca2+ channels designated for the low- and high-frequency endocytosis of synaptic vesicles, straightjacket Ca2+ channels in the active zone and La3+-sensitive Ca2+ channels in the inactive zone at the terminals, respectively. Recently, Drosophila melanogaster has been used as a model for studying the social brain, and the heat avoidance response of the flies was found to be socially enhanced. Future studies are expected to reveal mechanisms underlying social brain functions at the gene level.

Synaptic vesicle cycling is not impaired in a glutamatergic and a cholinergic synapse that exhibit deficits in acidification and filling

The purpose of the present work was to investigate synaptic vesicle trafficking when vesicles exhibit alterations in filling and acidification in two different synapses: a cholinergic frog neuromuscular junction and a glutamatergic ribbon-type nerve terminal in the retina. These synapses display remarkable structural and functional differences, and the mechanisms regulating synaptic vesicle cycling might also differ between them. The lipophilic styryl dye FM1-43 was used to monitor vesicle trafficking. Both preparations were exposed to pharmacological agents that collapse ΔpH (NH4Cl and methylamine) or the whole ΔµH+ (bafilomycin), a necessary situation to provide the driving force for neurotransmitter accumulation into synaptic vesicles. The results showed that FM1-43 loading and unloading in neuromuscular junctions did not differ statistically between control and experimental conditions (P > 0.05). Also, FM1-43 labeling in bipolar cell terminals proved highly similar under all conditions tested. Despite remarkable differences in both experimental models, the present findings show that acidification and filling are not required for normal vesicle trafficking in either synapse.

The Presynaptic Dense Projection of theCaenorhabiditis elegansCholinergic Neuromuscular Junction Localizes Synaptic Vesicles at the Active Zone through SYD-2/Liprin and UNC-10/RIM-Dependent Interactions

The active zone (AZ) of chemical synapses is a specialized area of the presynaptic bouton in which vesicles fuse with the plasma membrane and release neurotransmitters. Efficient signaling requires synaptic vesicles (SVs) to be recruited, primed, and retained at the AZ, in close proximity to voltage-dependent calcium channels that are activated during presynaptic depolarization. The electron-dense specializations at the AZ might provide a molecular platform for the spatial coordination of these different processes. To investigate this hypothesis, we examined high-resolution three-dimensional models of Caenorhabditis elegans cholinergic neuromuscular junctions generated by electron tomography. First, we found that SVs are interconnected within the bouton by filaments similar to those described in vertebrates. Second, we resolved the three-dimensional structure of the dense projection centered in the AZ. The dense projection is a more complex structure than previously anticipated, with filaments radiating from a core structure that directly contact SVs in the interior of the bouton as well as SVs docked at the plasma membrane. Third, we investigated the functional correlate of these contacts by analyzing mutants disrupting two key AZ proteins: UNC-10/RIM and SYD-2/liprin. In both mutants, the number of contacts between SVs and the dense projection was significantly reduced. Similar to unc-10 mutants, the dependence of SV fusion on extracellular calcium concentration was exacerbated in syd-2 mutants when compared with the wild type. Hence, we propose that the dense projection ensures proper coupling of primed vesicles with calcium signaling by retaining them at the AZ via UNC-10/RIM and SYD-2/liprin-dependent mechanisms.

A secreted complement-control-related protein ensures acetylcholine receptor clustering

Efficient neurotransmission at chemical synapses relies on spatial congruence between the presynaptic active zone, where synaptic vesicles fuse, and the postsynaptic differentiation, where neurotransmitter receptors concentrate. Diverse molecular systems have evolved to localize receptors at synapses, but in most cases, they rely on scaffolding proteins localized below the plasma membrane. A few systems have been suggested to control the synaptic localization of neurotransmitter receptors through extracellular interactions, such as the pentraxins that bind AMPA receptors and trigger their aggregation. However, it is not yet clear whether these systems have a central role in the organization of postsynaptic domains in vivo or rather provide modulatory functions. Here we describe an extracellular scaffold that is necessary to cluster acetylcholine receptors at neuromuscular junctions in the nematode Caenorhabditis elegans. It involves the ectodomain of the previously identified transmembrane protein LEV-10 (ref. 6) and a novel extracellular protein, LEV-9. LEV-9 is secreted by the muscle cells and localizes at cholinergic neuromuscular junctions. Acetylcholine receptors, LEV-9 and LEV-10 are interdependent for proper synaptic localization and physically interact based on biochemical evidence. Notably, the function of LEV-9 relies on eight complement control protein (CCP) domains. These domains, also called 'sushi domains', are usually found in proteins regulating complement activity in the vertebrate immune system. Because the complement system does not exist in protostomes, our results suggest that some of the numerous uncharacterized CCP proteins expressed in the mammalian brain might be directly involved in the organization of the synapse, independently from immune functions.

Contamination of bivalve haemolymph samples by adductor muscle components: implications for biomarker studies

Haemolymph samples and haemocytes collected via the adductor muscles of bivalve molluscs are extensively used in ecotoxicological studies. Withdrawal of haemolymph from mussels, Mytilus edulis, via the posterior adductor muscle, may lead to contamination with the intracellular contents of adductor myocytes. Lysopine dehydrogenase (LyDH) activity, an adductor myocyte marker, was used to investigate the impact of this potential contamination on levels of total glutathione, glutathione peroxidase (GPx) and acetylcholinesterase (AChE) measured in cell-free haemolymph. The mean glutathione content of cell-free haemolymph from 28 mussels was 3.2 +/- 1.8 microM (mean +/- SD). There was a linear relationship (slope = 0.28 +/- 0.03 min; mean +/- SE; P < 0.0001, n = 28) with haemolymph LyDH levels suggesting that at least some of the glutathione measured in cell-free haemolymph had arisen from contamination. Haemolymph LyDH activity was significantly higher in samples extracted using larger diameter needles, and also in samples where there had been some difficulty in the extraction. Exposure of mussels to oxidative stress using 40 microg l(-1) Cu for 5 days resulted in a 1.7 fold increase in glutathione (P = 0.033), but no increase (P = 0.810) in LyDH activity in adductor muscle. This was reflected in a similar increase in the slope of a plot of cell-free haemolymph glutathione versus LyDH activity (P = 0.011), consistent with both of these having originated from the adductor muscle. Cell-free haemolymph GPx and AChE activities also correlated with LyDH activity (Spearman rank correlation coefficients of 0.531 (P = 0.0068) and 0.537 (P = 0.0062), respectively, n = 27) suggesting that these also arise from contamination of the haemolymph. For GPx there was a significant linear relationship (P = 0.025) with haemolymph LyDH levels consistent with both enzymes originating from the myocytes. However, there was hyperbolic relationship (P = 0.0004) between haemolymph AChE and LyDH activities. It appears that this is because the AChE originates from a different compartment to the LyDH, i.e. cholinergic neuromuscular junctions in the adductor muscle. We conclude that it would be prudent, when considering the possibility of using a biomarker in cell-free haemolymph from bivalve molluscs, to check whether contamination could be an issue.

An RNAi Screen Identifies Genes that Regulate GABA Synapses

GABA synapses play a critical role in many aspects of circuit development and function. For example, conditions that perturb GABA transmission have been implicated in epilepsy. To identify genes that regulate GABA transmission, we performed an RNAi screen for genes whose inactivation increases the activity of C. elegans body muscles, which receive direct input from GABAergic motor neurons. We identified 90 genes, 21 of which were previously implicated in seizure syndromes, suggesting that this screen has effectively identified candidate genes for epilepsy. Electrophysiological recordings and imaging of excitatory and inhibitory synapses indicate that several genes alter muscle activity by selectively regulating GABA transmission. In particular, we identify two humoral pathways and several protein kinases that modulate GABA transmission but have little effect on excitatory transmission at cholinergic neuromuscular junctions. Our data suggest these conserved genes are components of signaling pathways that regulate GABA transmission and consequently may play a role in epilepsy and other cognitive or psychiatric disorders.

Aberrant morphology and residual transmitter release at the Munc13-deficient mouse neuromuscular synapse.

In cultured hippocampal neurons, synaptogenesis is largely independent of synaptic transmission, while several accounts in the literature indicate that synaptogenesis at cholinergic neuromuscular junctions in mammals appears to partially depend on synaptic activity. To systematically examine the role of synaptic activity in synaptogenesis at the neuromuscular junction, we investigated neuromuscular synaptogenesis and neurotransmitter release of mice lacking all synaptic vesicle priming proteins of the Munc13 family. Munc13-deficient mice are completely paralyzed at birth and die immediately, but form specialized neuromuscular endplates that display typical synaptic features. However, the distribution, number, size, and shape of these synapses, as well as the number of motor neurons they originate from and the maturation state of muscle cells, are profoundly altered. Surprisingly, Munc13-deficient synapses exhibit significantly increased spontaneous quantal acetylcholine release, although fewer fusion-competent synaptic vesicles are present and nerve stimulation-evoked secretion is hardly elicitable and strongly reduced in magnitude. We conclude that the residual transmitter release in Munc13-deficient mice is not sufficient to sustain normal synaptogenesis at the neuromuscular junction, essentially causing morphological aberrations that are also seen upon total blockade of neuromuscular transmission in other genetic models. Our data confirm the importance of Munc13 proteins in synaptic vesicle priming at the neuromuscular junction but indicate also that priming at this synapse may differ from priming at glutamatergic and gamma-aminobutyric acid-ergic synapses and is partly Munc13 independent. Thus, non-Munc13 priming proteins exist at this synapse or vesicle priming occurs in part spontaneously: i.e., without dedicated priming proteins in the release machinery.

A conserved postsynaptic transmembrane protein affecting neuromuscular signaling in Caenorhabditis elegans.

For a motor unit to function, neurons and muscle cells need to adopt their correct cell fate, form appropriate cellular contacts, and assemble a specific repertoire of signaling proteins into presynaptic and postsynaptic structures. In the nematode Caenorhabditis elegans, a disruption of any of these steps causes uncoordinated locomotory behavior (unc phenotype). We report here the positional cloning of a new unc gene, unc-122, which we show by mosaic analysis and tissue-specific rescue experiments to act in muscle to affect locomotory behavior. unc-122 codes for a phylogenetically conserved type II transmembrane protein with collagen repeats and a cysteine-rich olfactomedin domain. Together with uncharacterized proteins in C. elegans, Drosophila, and vertebrates, UNC-122 defines a novel family of proteins that we term "Colmedins." UNC-122 protein is expressed exclusively in muscle and coelomocytes and localizes to the postsynaptic surface of GABAergic and cholinergic neuromuscular junctions (NMJs). Presynaptic and postsynaptic structures are present and properly aligned in unc-122 mutant animals, yet the animals display neurotransmission defects characterized by an altered sensitivity toward drugs that interfere with cholinergic signaling. Moreover, unc-122 mutant animals display anatomical defects in motor axons that are likely a secondary consequence of neurotransmission defects. Both the neuroanatomical and locomotory defects worsen progressively during the life of an animal, consistent with a role of unc-122 in acute signaling at the NMJ. On the basis of motifs in the UNC-122 protein sequence that are characteristic of extracellular matrix proteins, we propose that UNC-122 is involved in maintaining a structural microenvironment that allows efficient neuromuscular signaling.

Botulism in foals less than 6 months of age: 30 cases (1989-2002).

Botulism has been recognized as a clinical entity in foals since the 1960s. Also known as "Shaker foal" disease, the toxicoinfectious form of botulism affects foals, with the highest incidence in the United States seen in Kentucky and the mid-Atlantic region. The disease is characterized by progressive muscular weakness caused by the action of botulism neurotoxin at cholinergic neuromuscular junctions. Increased number of episodes and duration of recumbency, muscular trembling, and dysphagia are seen in affected foals. Left untreated, the disease can be rapidly fatal, with death occuring secondary to respiratory muscle paralysis within 24 to 72 hours of the onset of clinical signs. Very mildly affected foals can survive with minimal treatment Despite advances made in treatment of these foals, including administration of botulism antitoxin early in the course of the disease, there is still an impression that the disease carries a high mortality rate. The purpose of this study was to evaluate outcome in 30 foals <6 months of age diagnosed with botulism between 1989 and 2002 at the George D. Widener Large Animal Hospital, New Bolton Center. Two foals were euthanized for economic reasons early in the disease course, and I died while being treated. Survival of treated cases was greater than 96%. Approximately 50% of the cases required oxygen therapy, whereas 30% required mechanical ventilation. All foals, excepting 1 mildly affected foal, received botulism antitoxin. Mean duration of hospitalization was 14 days. With appropriate treatment, foals with botulism have a high survival rate.

Association of Dystrophin-Related Protein 2 (DRP2) with Postsynaptic Densities in Rat Brain

The fundamental function of the membrane-associated cytoskeletal proteins dystrophin and utrophin remains unclear. To gain further insights into the dystrophin family of proteins, we have studied dystrophin-related protein 2 (DRP2), whose expression is largely confined to the vertebrate central nervous system. Both human and rat DRP2 are expressed from two alternative but neighboring transcriptional start sites and have simple transcript structures. Antibodies raised against DRP2 detect a characteristic quartet of bands (∼100–120 kDa) in Western blots of rat brain. The DRP2 protein is associated with brain membrane fractions and highly enriched in the postsynaptic density. Immunohistochemistry shows DRP2 to be widely distributed in a punctate pattern on neuronal dendrites and in neuropil, with particular concentration in regions of the brain involved in cholinergic synaptic transmission. Given the presence of utrophin in the cholinergic neuromuscular junction, and perturbations of cholinergic transmission in dystrophin-deficient nematodes, our findings may suggest a role for DRP2 in the organization of central cholinergic synapses.