Characterization of neuromuscular synapse function abnormalities in multiple Duchenne muscular dystrophy mouse models

Duchenne muscular dystrophy (DMD) is an X‐linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin‐associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction (NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn^+/? and mdx/utrn^?/? mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested in vivo, with mdx/utrn^?/? mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~ 40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation‐evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high‐rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn^?/? mice. Overall, we showed mild ‘myasthenia‐like’ neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration.     

Developmental consequences of neuromuscular junctions with reduced presynaptic calcium channel function

Evoked neurotransmitter release at the Drosophila neuromuscular junction (NMJ) is regulated by the amount of calcium influx at the presynaptic nerve terminal, as for most chemical synapses. Calcium entry occurs via voltage-gated calcium channels. The temperature-sensitive Drosophila mutant, cac(TS2), has a reduced amount of calcium entry during evoked stimulation. We have used this mutation to examine homeostatic regulatory mechanisms during development of the NMJ on muscle 6 within the developing larva. The amplitude of the excitatory postsynaptic potentials are reduced for both the Ib and Is motor neurons in 3rd instar larvae which have been raised at 33 degrees C from the 1st instar stage. Larvae raised at 25 degrees C and larvae pulsed at 33 degrees C from the late 2nd instar for various lengths of time show a reduced synaptic efficacy as a 3rd instar. The results indicate that the nerve terminal cannot fully compensate physiologically in the regulation of synaptic transmission during larval life for a reduced amount of evoked calcium entry. Morphological comparisons of Ib and Is terminals in relation to length and numbers of varicosities are significantly reduced in cac(TS2), which also suggests a lack in homeostatic ability. These findings are relevant since many deficits in synaptic transmission in various systems are compensated for either physiologically or structural over development, but not in this case for reduced calcium entry during evoked transmission.     

Immunohistological and electrophysiological evidence that N‐acetylaspartylglutamate is a co‐transmitter at the vertebrate neuromuscular junction

Immunohistochemical studies previously revealed the presence of the peptide transmitter N‐acetylaspartylglutamate (NAAG) in spinal motor neurons, axons and presumptive neuromuscular junctions (NMJs). At synapses in the central nervous system, NAAG has been shown to activate the type 3 metabotropic glutamate receptor (mGluR3) and is inactivated by an extracellular peptidase, glutamate carboxypeptidase II. The present study tested the hypothesis that NAAG meets the criteria for classification as a co‐transmitter at the vertebrate NMJ. Confocal microscopy confirmed the presence of NAAG immunoreactivity and extended the resolution of the peptide's location in the lizard (Anolis carolinensis) NMJ. NAAG was localised to a presynaptic region immediately adjacent to postsynaptic acetylcholine receptors. NAAG was depleted by potassium‐induced depolarisation and by electrical stimulation of motor axons. The NAAG receptor, mGluR3, was localised to the presynaptic terminal consistent with NAAG's demonstrated role as a regulator of synaptic release at central synapses. In contrast, glutamate receptors, type 2 metabotropic glutamate receptor (mGluR2) and N‐methyl‐d ‐aspartate, were closely associated with acetylcholine receptors in the postsynaptic membrane. Glutamate carboxypeptidase II, the NAAG‐inactivating enzyme, was identified exclusively in perisynaptic glial cells. This localisation was confirmed by the loss of immunoreactivity when these cells were selectively eliminated. Finally, electrophysiological studies showed that exogenous NAAG inhibited evoked neurotransmitter release by activating a group II metabotropic glutamate receptor (mGluR2 or mGluR3). Collectively, these data support the conclusion that NAAG is a co‐transmitter at the vertebrate NMJ.     

Low dystrophin levels are insufficient to normalize the neuromuscular synaptic abnormalities of mdx mice

Dystrophin is a sub-sarcolemmal component of skeletal muscle fibres and is enriched at the postsynaptic membrane of the neuromuscular junction (NMJ). In the mdx mouse, dystrophin absence not only causes muscle damage but also mild synaptic dysfunctions and clear morphological aberrations at NMJs. In particular, reduction of postsynaptic sensitivity for the neurotransmitter acetylcholine and extra exhaustion of presynaptic acetylcholine release during intense synaptic activity exists. Current experimental therapeutic approaches in Duchenne muscular dystrophy aim to restore dystrophin expression. An important question is what dystrophin levels are needed to improve muscle function. Recent experimental and clinical studies suggested that levels as low as a few percent of normal can be beneficial. Similarly, it is of interest to know how dystrophin levels relate to NMJ function and morphology. We investigated NMJs of a series of mdx-Xist^Δhs mice, which expressed dystrophin between ~2% and 19% of normal. Most functional and morphological NMJ parameters of these mice remained comparable to mdx. On the other hand, mdx^+/- mice (expressing ~50% dystrophin) showed normal NMJ features. Thus, the minimal dystrophin level required for normal NMJ function and morphology lies between 19% and 50% of normal when expression of dystrophin is not uniform.     

Structural and Functional Synaptic Plasticity Induced by Convergent Synapse Loss in the Drosophila Neuromuscular Circuit

Throughout the nervous system, the convergence of two or more presynaptic inputs on a target cell is commonly observed. The question we ask here is to what extent converging inputs influence each other''s structural and functional synaptic plasticity. In complex circuits, isolating individual inputs is difficult because postsynaptic cells can receive thousands of inputs. An ideal model to address this question is the Drosophila larval neuromuscular junction (NMJ) where each postsynaptic muscle cell receives inputs from two glutamatergic types of motor neurons (MNs), known as 1b and 1s MNs. Notably, each muscle is unique and receives input from a different combination of 1b and 1s MNs; we surveyed multiple muscles for this reason. Here, we identified a cell-specific promoter that allows ablation of 1s MNs postinnervation and measured structural and functional responses of convergent 1b NMJs using microscopy and electrophysiology. For all muscles examined in both sexes, ablation of 1s MNs resulted in NMJ expansion and increased spontaneous neurotransmitter release at corresponding 1b NMJs. This demonstrates that 1b NMJs can compensate for the loss of convergent 1s MNs. However, only a subset of 1b NMJs showed compensatory evoked neurotransmission, suggesting target-specific plasticity. Silencing 1s MNs led to similar plasticity at 1b NMJs, suggesting that evoked neurotransmission from 1s MNs contributes to 1b synaptic plasticity. Finally, we genetically blocked 1s innervation in male larvae and robust 1b synaptic plasticity was eliminated, raising the possibility that 1s NMJ formation is required to set up a reference for subsequent synaptic perturbations.SIGNIFICANCE STATEMENT In complex neural circuits, multiple convergent inputs contribute to the activity of the target cell, but whether synaptic plasticity exists among these inputs has not been thoroughly explored. In this study, we examined synaptic plasticity in the structurally and functionally tractable Drosophila larval neuromuscular system. In this convergent circuit, each muscle is innervated by a unique pair of motor neurons. Removal of one neuron after innervation causes the adjacent neuron to increase neuromuscular junction outgrowth and functional output. However, this is not a general feature as each motor neuron differentially compensates. Further, robust compensation requires initial coinnervation by both neurons. Understanding how neurons respond to perturbations in adjacent neurons will provide insight into nervous system plasticity in both healthy and disease states.     

Is taurine a hypothalamic neurotransmitter?: a model of the differential uptake and compartmentalization of taurine by neuronal and glial cell particles from the rat hypothalamus

Although taurine has been postulated to be a neurotransmitter or neuromodulator in the mammalian CNS, little is known concerning its role in brain function. Evidence suggesting that taurine may influence endocrine and homeostatic mechanisms via the hypothalamus resulted in our investigations into its function in this brain region.The main objectives of the research were to characterize the specific binding, uptake, and release of taurine in the hypothalamus. A specific aim was to examine the proposed neurotransmitter role for taurine in the hypothalamus. This was accomplished by comparing the characteristics and properties of the binding, uptake, and release of taurine with those for the classical neurotransmitters which satisfy the criteria for a neurotransmitter. On such a comparative basis, the characteristics of taurine uptake satisfy the neurotransmitter criterion of inactivation of taurine in the hypothalamus. However, the observed characteristics of taurine binding and release in the hypothalamus do not satisfy the respective neurotransmitter criteria of specific receptors and Ca²⁺-dependent evoked release. Therefore, solely on the basis of the experimental observations reported herein, we must conclude that taurine apparently does not function as a neurotransmitter in the hypothalamus.Two uptake systems were found in the P₂ fraction, a high affinity uptake system and a low affinity uptake system. Uptake systems for taurine have previously been reported in glial and nerve cell homogenates, and therefore, because of the known contamination of crude synaptosomal preparations with glial particles, we sought to determine the cellular origin of the two taurine uptake systems in our crude preparation. Using a variety of diverse biochemical techniques such as hypo-osmotic shock, release experiments and Arrhenius plots, we determined that physical changes of the media or depolarizing stimuli which would influence neuronal and glial cell particles differently, also had differing effects on high and low affinity taurine uptake or its release from the respective uptake compartments. We conclude that the high affinity taurine uptake system/compartment is located on/in neuronal membranes/particles and that the low affinity taurine uptake system/compartment is located on/in glial membranes/particles. Such a model for the differential cellular transport and compartmentalization of taurine into neuronal and glial cells has important implications concerning its possible role in the CNS.     

Neuromuscular junction disorders mimicking myopathy

Introduction: Small‐amplitude, short‐duration motor unit action potentials are non‐specific findings seen in myopathies and neuromuscular junction (NMJ) disorders. NMJ studies (repetitive nerve stimulation and single‐fiber electromyography) can determine if such findings are related to NMJ abnormalities but are not considered routinely in atypical cases. Methods: Medical records of 338 patients with confirmed NMJ disorders were reviewed to identify cases with a clinical or electrodiagnostic impression of myopathy during initial evaluation. A history of muscle biopsy with findings that did not support a myopathic process was required for inclusion. Results: Four patients met the inclusion criteria. NMJ studies were abnormal in all cases. One patient had elevated acetylcholine receptor antibodies. Three patients were antibody negative: 2 demonstrated immunotherapy responsiveness, and 1 had a Rapsyn mutation. Conclusions: NMJ disorders may mimic myopathies, and NMJ studies should be performed to clarify so‐called “myopathic” electromyographic findings to avoid unnecessary testing and delayed diagnosis. Muscle Nerve 50 : 854–856, 2014     

Interrelation between MEPP amplitude and MEPP frequency in different regions along the frog neuromuscular junction

Experiments were done to study the relationship between miniature endplate potential (MEPP) frequency and MEPP amplitude in different regions along the frog neuromuscular junction (NMJ). The position of the release site producing a MEPP and the amplitude of a MEPP at its release site were evaluated by using the spatial decay method after simultaneous intracellular recordings at each distal end of the NMJ. The NMJ was divided in 10 regions of equal length. It was found that MEPP amplitude and MEPP frequency are smaller in distal regions than in proximal ones. Moreover, we observed a logarithmic relationship between MEPP frequency of a given region and the mean MEPP amplitude of the same region. These results suggest that the probability to produce a MEPP and the quantity of neurotransmitter liberated are two synaptic functions controlled or affected by common mechanisms.     

Effects of intravesicular loading of a Ca2+ chelator and depolymerization of actin fibers on neurotransmitter release in frog motor nerve terminals

Ca²⁺‐induced Ca²⁺ release (CICR) via type‐3 ryanodine receptor enhances neurotransmitter release in frog motor nerve terminals. To test a possible role of synaptic vesicle in CICR, we examined the effects of loading of EGTA, a Ca²⁺ chelator, into synaptic vesicles and depolymerization of actin fibers. Intravesicular EGTA loading via endocytosis inhibited the ryanodine sensitive enhancement of transmitter release induced by tetanic stimulation and the associated rises in intracellular‐free Ca²⁺ ([Ca²⁺]_i: Ca²⁺ transients). Latrunculin A, a depolymerizer of actin fibers, enhanced both spontaneous and stimulation‐induced transmitter release, but inhibited the enhancement of transmitter release elicited by successive tetanic stimulation. The results suggest a possibility that the activation of CICR from mobilized synaptic vesicles caused the enhancement of neurotransmitter release.     

Neuroligin-2 controls the establishment of fast GABAergic transmission in adult-born granule cells

GABAergic inhibition is critical for the precision of neuronal spiking and the homeostatic regulation of network activity in the brain. Adult neurogenesis challenges network homeostasis because new granule cells (GCs) integrate continuously in the functional dentate gyrus. While developing, adult-born GCs undergo a transient state of enhanced excitability due to the delayed maturation of perisomatic GABAergic inhibition by parvalbumin interneurons (PV-INs). The mechanisms underlying this delayed synaptic maturation remain unknown. We examined the morphology and function of synapses formed by PV-INs onto new GCs over a 2-month interval in young adult mice, and investigated the influence of the synaptic adhesion molecule neuroligin-2 (NL2). Perisomatic appositions of PV-IN terminals onto new GCs were conspicuous at 2 weeks and continued to grow in size to reach a plateau over the fourth week. Postsynaptic knockdown of NL2 by expression of a short-hairpin RNA (shNL2) in new GCs resulted in smaller size of synaptic contacts, reduced area of perisomatic appositions of the vesicular GABA transporter VGAT, and the number of presynaptic active sites. GCs expressing shNL2 displayed spontaneous GABAergic responses with decreased frequency and amplitude, as well as slower kinetics compared to control GCs. In addition, postsynaptic responses evoked by optogenetic stimulation of PV-INs exhibited slow kinetics, increased paired-pulse ratio and coefficient of variation in GCs with NL2 knockdown, suggesting a reduction in the number of active synapses as well as in the probability of neurotransmitter release (P_r). Our results demonstrate that synapses formed by PV-INs on adult-born GCs continue to develop beyond the point of anatomical growth, and require NL2 for the structural and functional maturation that accompanies the conversion into fast GABAergic transmission.     

Targeting therapy to the neuromuscular junction: Proof of concept

Introduction: The site of pathology in myasthenia gravis (MG) is the neuromuscular junction (NMJ). Our goal was to determine the ability to direct complement inhibition to the NMJ. Methods: A single‐chain antibody directed against the alpha subunit of the acetylcholine receptor was synthesized (scFv‐35) and coupled to decay‐accelerating factor (DAF, scFv‐35‐DAF). scFv‐35‐DAF was tested in a passive model of experimentally acquired MG. Results: Administration of scFv‐35‐DAF to mice deficient in intrinsic complement inhibitors produced no weakness despite confirmation of its localization to the NMJ and no evidence of tissue destruction related to complement activation. Rats with experimentally acquired MG treated with scFV‐35‐DAF showed less weakness and a reduction of complement deposition. Conclusions: We demonstrate a method to effectively target a therapeutic agent to the NMJ. Muscle Nerve 49 : 749–756, 2014     

Glutamate released spontaneously from astrocytes sets the threshold for synaptic plasticity

Astrocytes exhibit spontaneous calcium oscillations that could induce the release of glutamate as gliotransmitter in rat hippocampal slices. However, it is unknown whether this spontaneous release of astrocytic glutamate may contribute to determining the basal neurotransmitter release probability in central synapses. Using whole‐cell recordings and Ca²⁺ imaging, we investigated the effects of the spontaneous astrocytic activity on neurotransmission and synaptic plasticity at CA3–CA1 hippocampal synapses. We show here that the metabolic gliotoxin fluorocitrate (FC) reduces the amplitude of evoked excitatory postsynaptic currents and increases the paired‐pulse facilitation, mainly due to the reduction of the neurotransmitter release probability and the synaptic potency. FC also decreased intracellular Ca²⁺ signalling and Ca²⁺‐dependent glutamate release from astrocytes. The addition of glutamine rescued the effects of FC over the synaptic potency; however, the probability of neurotransmitter release remained diminished. The blockage of group I metabotropic glutamate receptors mimicked the effects of FC on the frequency of miniature synaptic responses. In the presence of FC, the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N ′,N ′‐tetra‐acetate or group I metabotropic glutamate receptor antagonists, the excitatory postsynaptic current potentiation induced by the spike‐timing‐dependent plasticity protocol was blocked, and it was rescued by delivering a stronger spike‐timing‐dependent plasticity protocol. Taken together, these results suggest that spontaneous glutamate release from astrocytes contributes to setting the basal probability of neurotransmitter release via metabotropic glutamate receptor activation, which could be operating as a gain control mechanism that regulates the threshold of long‐term potentiation. Therefore, endogenous astrocyte activity provides a novel non‐neuronal mechanism that could be critical for transferring information in the central nervous system.     

Tissue-type plasminogen activator triggers the synaptic vesicle cycle in cerebral cortical neurons

The active zone (AZ) is a thickening of the presynaptic membrane where exocytosis takes place. Chemical synapses contain neurotransmitter-loaded synaptic vesicles (SVs) that at rest are tethered away from the synaptic release site, but after the presynaptic inflow of Ca⁺² elicited by an action potential translocate to the AZ to release their neurotransmitter load. We report that tissue-type plasminogen activator (tPA) is stored outside the AZ of cerebral cortical neurons, either intermixed with small clear-core vesicles or in direct contact with the presynaptic membrane. We found that cerebral ischemia-induced release of neuronal tPA, or treatment with recombinant tPA, recruits the cytoskeletal protein βII-spectrin to the AZ and promotes the binding of SVs to βII-spectrin, enlarging the population of SVs in proximity to the synaptic release site. This effect does not require the generation of plasmin and is followed by the recruitment of voltage gated calcium channels (VGCC) to the presynaptic terminal that leads to Ca⁺²-dependent synapsin I phosphorylation, freeing SVs to translocate to the AZ to deliver their neurotransmitter load. Our studies indicate that tPA activates the SV cycle and induces the structural and functional changes in the synapse that are required for successful neurotransmission.     

Axonal domain disorganization in Caspr1 and Caspr2 mutant myelinated axons affects neuromuscular junction integrity,leading to muscle atrophy

Bidirectional interactions between neurons and myelinating glial cells result in formation of axonal domains along myelinated fibers. Loss of axonal domains leads to detrimental consequences on nerve structure and function, resulting in reduced conductive properties and the diminished ability to reliably transmit signals to the targets they innervate. Thus, impairment of peripheral myelinated axons that project to the surface of muscle fibers and form neuromuscular junction (NMJ) synapses leads to muscle dysfunction. The goal of our studies was to determine how altered electrophysiological properties due to axonal domain disorganization lead to muscle pathology, which is relevant to a variety of peripheral neuropathies, demyelinating diseases, and neurodegenerative disorders. Using conventional Contactin‐Associated Protein 1 (Caspr1 ) and Caspr2 single or double mutants with disrupted paranodal, juxtaparanodal, or both regions, respectively, in peripheral myelinated axons, we correlated defects in NMJ integrity and muscle pathology. Our data show that loss of axonal domains in Caspr1 and Caspr2 single and double mutants primarily alters distal myelinated fibers together with presynaptic terminals, eventually leading to NMJ denervation and reduction in postsynaptic endplate areas. Moreover, reduction in conductive properties of peripheral myelinated fibers together with NMJ disintegration leads to muscle atrophy in Caspr1 mutants or muscle fiber degeneration accompanied by mitochondrial dysfunction in Caspr1 /Caspr2 double mutants. Together, our data indicate that proper organization of axonal domains in myelinated fibers is critical for optimal propagation of electrical signals, NMJ integrity, and muscle health, and provide insights into a wide range of pathologies that result in reduced nerve conduction leading to muscle atrophy. © 2017 Wiley Periodicals, Inc.     

Sensing and expressing homeostatic synaptic plasticity

Chronic changes in the level of neuronal activity (over a period of days) trigger compensatory changes in synaptic function that seem to contribute to the homeostatic restoration of neuronal activity. Changes in both quantal amplitude and vesicle release contribute to homeostatic synaptic plasticity, but they are often considered as the same phenomenon. In this review, we propose a new approach to studying how neuronal activity is sensed and changes in synaptic function are expressed during synaptic compensation. Changes in quantal amplitude and vesicle release should be considered separately in an attempt to identify the sensors that trigger homeostatic synaptic plasticity. Although data are limited, current evidence suggests that the sensors triggering changes in the quantal amplitude and vesicle release exist at different locations. Furthermore, it is important to recognize that at least two different mechanisms underlie changes in quantal amplitude during homeostatic synaptic plasticity: changes in both the number of postsynaptic receptors and loading of synaptic vesicles with neurotransmitter. Finally, modulation of the probability of neurotransmitter release contributes to the changes in vesicle release associated with homeostatic synaptic plasticity. An improved understanding of where and how neuronal activity is sensed, in addition to the types of changes in synaptic function that are induced, will be needed both to design future experiments and to understand the consequences of synaptic compensation following injury to the nervous system.     

Surprises from Drosophila: genetic mechanisms of synaptic development and plasticity

Drosophila are excellent models for the study of synaptic development and plasticity, thanks to the availability and applicability of a wide variety of powerful molecular, genetic, and cell-biology techniques. Three decades of study have led to an intimate understanding of the sequence of events leading to a functional and plastic synapse, yet many of the molecular mechanisms underlying these events are still poorly understood. Here, we provide a review of synaptogenesis at the Drosophila glutamatergic neuromuscular junction (NMJ). Next, we discuss the role of two proteins that forward genetic screens in Drosophila have revealed to play crucial-and completely unexpected-roles in NMJ development and plasticity: the origin of replication complex protein Latheo, and the enzyme glutamate decarboxylase. The requirement for these proteins at the NMJ highlights the fact that synaptic development and plasticity involves intense inter- and intracellular signaling about which we know almost nothing.     

Snapin is critical for presynaptic homeostatic plasticity

The molecular mechanisms underlying the homeostatic modulation of presynaptic neurotransmitter release are largely unknown. We have previously used an electrophysiology-based forward genetic screen to assess the function of >400 neuronally expressed genes for a role in the homeostatic control of synaptic transmission at the neuromuscular junction of Drosophila melanogaster. This screen identified a critical function for dysbindin, a gene linked to schizophrenia in humans (Dickman and Davis, 2009). Biochemical studies in other systems have shown that Snapin interacts with Dysbindin, prompting us to test whether Snapin might be involved in the mechanisms of synaptic homeostasis. Here, we demonstrate that loss of snapin blocks the homeostatic modulation of presynaptic vesicle release following inhibition of postsynaptic glutamate receptors. This is true for both the rapid induction of synaptic homeostasis induced by pharmacological inhibition of postsynaptic glutamate receptors, and the long-term expression of synaptic homeostasis induced by the genetic deletion of the muscle-specific GluRIIA glutamate receptor subunit. Loss of snapin does not alter baseline synaptic transmission, synapse morphology, synapse growth, or the number or density of active zones, indicating that the block of synaptic homeostasis is not a secondary consequence of impaired synapse development. Additional genetic evidence suggests that snapin functions in concert with dysbindin to modulate vesicle release and possibly homeostatic plasticity. Finally, we provide genetic evidence that the interaction of Snapin with SNAP25, a component of the SNARE complex, is also involved in synaptic homeostasis.     

Skeletal muscle IP3R1 receptors amplify physiological and pathological synaptic calcium signals

Ca(2+) release from internal stores is critical for mediating both normal and pathological intracellular Ca(2+) signaling. Recent studies suggest that the inositol 1,4,5-triphosphate (IP(3)) receptor mediates Ca(2+) release from internal stores upon cholinergic activation of the neuromuscular junction (NMJ) in both physiological and pathological conditions. Here, we report that the type I IP(3) receptor (IP(3)R(1))-mediated Ca(2+) release plays a crucial role in synaptic gene expression, development, and neuromuscular transmission, as well as mediating degeneration during excessive cholinergic activation. We found that IP(3)R(1)-mediated Ca(2+) release plays a key role in early development of the NMJ, homeostatic regulation of neuromuscular transmission, and synaptic gene expression. Reducing IP(3)R(1)-mediated Ca(2+) release via siRNA knockdown or IP(3)R blockers in C2C12 cells decreased calpain activity and prevented agonist-induced acetylcholine receptor (AChR) cluster dispersal. In fully developed NMJ in adult muscle, IP(3)R(1) knockdown or blockade effectively increased synaptic strength at presynaptic and postsynaptic sites by increasing both quantal release and expression of AChR subunits and other NMJ-specific genes in a pattern resembling muscle denervation. Moreover, in two mouse models of cholinergic overactivity and NMJ Ca(2+) overload, anti-cholinesterase toxicity and the slow-channel myasthenic syndrome (SCS), IP(3)R(1) knockdown eliminated NMJ Ca(2+) overload, pathological activation of calpain and caspase proteases, and markers of DNA damage at subsynaptic nuclei, and improved both neuromuscular transmission and clinical measures of motor function. Thus, blockade or genetic silencing of muscle IP(3)R(1) may be an effective and well tolerated therapeutic strategy in SCS and other conditions of excitotoxicity or Ca(2+) overload.