Activation of apoptotic pathways at muscle fiber synapses is circumscribed and reversible in a slow-channel syndrome model

In the slow-channel syndrome (SCS) mutant acetylcholine receptors elicit calcium overload and myonuclear degeneration at the neuromuscular junction (NMJ), without muscle fiber death. Activated caspases are present at SCS motor endplates. We hypothesized that SCS represents a limited form of apoptosis. We found condensed chromatin and occasional single-strand DNA nicks in degenerating synaptic nuclei. Cleaved forms of caspases-3 and -9 were present in mouse SCS muscle homogenates and were specifically localized to NMJs. Finally, interruption of cholinergic activity by axotomy markedly reduced NMJ caspase activity and improved the morphological features of apoptosis at NMJs. These results demonstrate that in SCS processes leading to apoptosis may remain compartmentalized and reversible. Use of cysteine protease inhibitors may aid in treatment of this and other dystrophic muscle and excitotoxic disorders. Identification of extrasynaptic factors that prevent the spread of apoptosis in SCS muscle fibers may aid in developing treatments for neurological disorders characterized by excitotoxicity or apoptosis.     

Glutamatergic modulation of synaptic plasticity at a PNS vertebrate cholinergic synapse

The presence and the functionality of a glutamatergic regulation was studied at the frog neuromuscular junction (NMJ), a singly innervated cholinergic synapse. Bath application of glutamate reduced transmitter release without affecting nerve-evoked presynaptic Ca2+ entry and handling. (1S,3R)-aminocyclopentanedicarboxylic acid (ACPD), a metabotropic glutamate receptor (mGluR) agonist, mimicked the effects of glutamate while (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), a mGluR antagonist, blocked glutamate effects. MCPG had no effect on transmitter release evoked at low frequency (0.2 Hz) but significantly reduced synaptic depression (10 Hz, 80 s). This suggests that a frequency-dependent endogenous glutamatergic modulation is present at the frog NMJ and is mediated through mGluRs. Immunohistochemical labelling revealed the presence of mGluRs at the end plate area, primarily on muscle fibers. Functional glutamate uptake machinery was also found at the NMJ as blockade of glutamate transport by the inhibitor dl-threo-beta-benzyloxyaspartate (DL-TBOA) increased high frequency-induced depression, suggesting that the transporters system is used to eliminate glutamate from the extracellular space. Moreover, immunohistochemical labelling revealed that glutamate-aspartate transporters (GLASTs) are predominantly present on perisynaptic Schwann cells (PSCs). However, local application of glutamate on PSCs unreliability evoked small Ca2+ responses. Hence, these data suggest that functional glutamatergic interactions at a purely cholinergic synapse, shape synaptic efficacy and short-term plasticity in a frequency-dependent fashion.     

Postsynaptic IP3 receptor-mediated Ca2+ release modulates synaptic transmission in hippocampal neurons

Ca(2+)-dependent mechanisms are important in regulating synaptic transmission. The results herein indicate that whole-cell perfusion of inositol 1,4,5-trisphosphate receptor (IP(3)R) agonists greatly enhanced excitatory postsynaptic current (EPSC) amplitudes in postsynaptic hippocampal CA1 neurons. IP(3)R agonist-mediated increases in synaptic transmission changed during development and paralleled age-dependent increases in hippocampal type-1 IP(3)Rs. IP(3)R agonist-mediated increases in EPSC amplitudes were inhibited by postsynaptic perfusion of inhibitors of Ca(2+)/calmodulin, PKC and Ca(2+)/calmodulin-dependent protein kinase II. Postsynaptic perfusion of inhibitors of smooth endoplasmic reticulum (SER) Ca(2+)-ATPases, which deplete intracellular Ca(2+) stores, also enhanced EPSC amplitudes. Postsynaptic perfusion of the IP(3)R agonist adenophostin (AdA) during subthreshold stimulation appeared to convert silent to active synapses; synaptic transmission at these active synapses was completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Postsynaptic IP(3)R-mediated Ca(2+) release also produced a significant increase in spontaneous EPSC frequency. These results indicate that Ca(2+) release from intracellular stores play a key role in regulating the function of postsynaptic AMPARs.     

Macroscopic properties of spontaneous mutations in slow-channel syndrome: correlation by domain and disease severity

The slow-channel syndrome (SCS) is a neuromuscular disorder characterized by fatigability, progressive weakness, and degeneration of the neuromuscular junction. The SCS is caused by missense mutations in the four subunits of the skeletal muscle acetylcholine receptor (AChR), which leads to altered channel gating, prolonged neuromuscular postsynaptic currents, and impaired neuromuscular transmission. Although a diverse set of mutations in different functional domains of the AChR appear to be associated with symptoms of widely ranging severity, there is as yet no mutant channel property or combination that explains the variations in disease severity. By observing the recovery time of AChR from desensitization, the authors determined that this process is significantly enhanced in SCS channels. In addition, as expected, the authors found that SCS macroscopic decay currents in transfected HEK293 cells are slower than wild type currents. While slight differences in relative Ca(2+) permeability between some SCS mutations were identified, they did not correlate with apparent disease severity. These results suggest that of the different AChR kinetic features studied, only recovery from desensitization and slow postsynaptic currents correlate with the severity observed in the different mutations of this syndrome.     

Postsynaptic TRPC1 function contributes to BDNF-induced synaptic potentiation at the developing neuromuscular junction

Brain-derived neurotrophic factor (BDNF) induces synaptic potentiation at both neuromuscular junctions (NMJs) and synapses of the CNS through a Ca2+ -dependent pathway. The molecular mechanism underlying BDNF-induced synaptic potentiation, especially the regulation of Ca2+ dynamics, is not well understood. Using the Xenopus NMJ in culture as a model system, we show that pharmacological inhibition or morpholino-mediated knockdown of Xenopus TRPC1 (XTRPC1) significantly attenuated the BDNF-induced potentiation of the frequency of spontaneous synaptic responses at the NMJ. Functionally, XTRPC1 was required specifically in postsynaptic myocytes for BDNF-induced Ca2+ elevation and full synaptic potentiation at the NMJ, suggesting a previously underappreciated postsynaptic function of Ca2+ signaling in neurotrophin-induced synaptic plasticity, in addition to its well established role at presynaptic sites. Mechanistically, blockade of the p75 neurotrophin receptor abolished BDNF-induced postsynaptic Ca2+ elevation and restricted BDNF-induced synaptic potentiation, while knockdown of the TrkB receptor in postsynaptic myocytes had no effect. Our study suggests that BDNF-induced synaptic potentiation involves coordinated presynaptic and postsynaptic responses and identifies TRPC1 as a molecular mediator for postsynaptic Ca2+ elevation required for BDNF-induced synaptic plasticity.     

Localization of inositol 1,4,5-trisphosphate receptors in mouse retinal ganglion cells

Inositol 1,4,5-trisphosphate receptors (IP(3)R) are ligand-gated intracellular Ca(2+)channels that mediate release of Ca(2+) from intracellular stores into the cytosol on activation by second messenger IP(3.). Similarly, IP(3)R mediated changes in cytosolic Ca(2+) concentrations control neuronal functions ranging from synaptic transmission to differentiation and apoptosis. IP(3)R-generated cytosolic Ca(2+) transients also control intracellular Ca(2+) release and subsequent retinal ganglion cell (RGC) physiology and pathophysiology. The distribution of IP(3)R isotypes in primary adult mouse RGC cultures was determined to identify molecular substrates of IP(3)R mediated signaling in these neurons. Immunocytochemical labeling of IP(3)Rs in retinal sections and cultured RGCs was carried out using isoform specific antibodies and was detected with fluorescence microscopy. RGCs were identified by the use of morphologic criteria and RGC-specific immunocytochemical markers, neurofilament 68 kDa, Thy 1.1, and Thy 1.2. RGC morphology and immunoreactivity to neurofilament 68 kDa and Thy 1.1 or Thy 1.2 were identified in both RGC primary cultures and tissue cryosections. RGCs showed localization on intracellular membranes with a differential distribution of IP(3)R isoforms 1, 2, and 3. IP(3)R Types 1 and 3 were detected intracellularly throughout the cell whereas Type 2 was expressed predominantly in soma. Expression of all three IP(3)Rs by RGCs indicates that all IP(3)R types potentially play a role in Ca(2+) homeostasis and Ca(2+) signaling in these cells. Differential localization of IP(3) receptor subtypes in combination with biophysical properties of IP(3)R types may be an important molecular mechanism by which RGCs organize their cytosolic Ca(2+) signals.     

Editorial introduction: from the structure and functions of the neuromuscular junction to the diseases

The neuromuscular junction has been recognized as a site for autoimmune and genetic disorders. Myasthenia gravis (MG) is mainly caused by postsynaptic nicotinic acetylcholine receptor (AChR) IgG1 antibodies that are directed against α-subunit 67-76 and 125-147 and activate complement. Thymic abnormalities are present in the autoimmune background. A proportion of MG patients without conformation-dependent AChR antibodies assayed by the cell-based method have muscle-specific tyrosine kinase (MuSK) antibodies which are largely IgG4 and partially IgG1. MuSK is activated by Dok-7 and Lrp4 (agrin receptor) and contributes to AChR clustering at the postsynaptic membrane via various kinase cascades in collaboration with Wnt-MuSK/Frizzled-Dishevelled signaling. Rapsyn interacts with MuSK-linked chaperones to stabilize postsynaptic architecture and also contributes to AChR phosphorylation. MG-associated thymomas express antigens that trigger antibody responses which play a part in disease generation and modification. Among these, ryanodine receptor-1 (RyR1; acts on sarcoplasmic Ca2+ release) antibodies cause muscle contractile weakness. Transient receptor potential canonical-3 (TRPC3) antibodies are also detected in thymoma-associated MG patients; they may participate in muscle contractile weakness because TRPC3 acts on RyR1, and may also impair the refill of sarcoplasmic Ca2+ stores since TRPCs contribute to the receptor-operated Ca2+ influx via the phospholipase C (PLC)-diacylglycerol (DAG) pathway in cooperation with the store-operated, STIM1/Orai1-mediated Ca2+ influx and TRPCs-Homerl-IP3R interaction. Lambert-Eaton myasthenic syndrome (LEMS) is caused by reduced ACh quantal release that occurs mainly because of presynaptic P/Q-type voltage-gated Ca2+ channel (VGCC) antibodies. Physicians should be vigilant for LEMS because it may predict an underlying malignancy, particularly small-cell lung carcinoma; SOX-1 antibodies are usually present in these patients and are absent in those who do not have caucer. Some patients with LEMS have antibodies against synaptotagmin-1, which associates with SNARE complex and functions as Ca2+ sensor for exocytosis. The stimulation of the M1-type presynaptic muscarinic AChR (mAChR)(G-proterin-coupled receptor) can compensate for the deficiency of Ca2+-mediated ACh quantal release via the PLC/DAG-mediated mechanism; This acts in a manner similar to the BDNF/NT4-TrkB interaction. The detection of M1 mAChR antibodies in LEMS suggests an impaired compensatory mechanism and corresponds, at least in part, to autonomic symptoms. Congenital myasthenic syndromes are classified into presynaptic, synaptic basal lamina and postsynaptic defects.     

Changes in acetylcholinesterase in experimental autoimmune myasthenia gravis and in response to treatment with a specific antisense

Controlled regulation of synaptic nicotinic acetylcholine receptors (AChRs) and acetylcholinesterase (AChE), together with maintenance of a dynamic balance between them, is a requirement for proper function of cholinergic synapses. In the present study we assessed whether pathological changes in AChR perturb this balance, and whether such changes can be corrected. We studied the influence of AChR loss, caused by experimental autoimmune myasthenia gravis (EAMG), on muscle AChE, as well as the reciprocal effect of an antisense targeted towards AChE on both AChR and AChE at the neuromuscular synapse. The extensor digitorum longus (EDL) muscles of EAMG Lewis rats were isolated, and AChE levels and isoform compositions were examined. Although AChE levels in the muscles of healthy and EAMG rats were similar, marked changes were observed in isoform composition. Healthy EDL muscles contained globular (G_1,2, G₄) and asymmetric (primarily A₁₂) isoforms. G_1,2‐AChE was significantly reduced in EAMG muscles, whereas both G₄‐ and A₁₂‐AChE remained unchanged. Treatment of EAMG rats with the antisense EN101 resulted in decreased total muscle AChE, with recovery in G_1,2 and reduction in A₁₂‐AChE. AChE/AChR ratios were determined at the neuromuscular junctions (NMJ). The decrease in AChR levels that occurred as the disease progressed resulted in a dramatic increase in this ratio, and a significant recovery towards normal ratios occurred after EN101 treatment. This improvement was primarily due to increased synaptic AChR content. Our findings emphasise the tight connection between AChR and AChE at the myasthenic NMJ, and the importance of the AChE/AChR ratio in maintaining the required cholinergic balance.     

Glial modulation of synaptic transmission at the neuromuscular junction

The neuromuscular junction (NMJ) is a cholinergic synapse that controls muscle contraction. Glial cells, called perisynaptic Schwann cells, surround nerve terminals at the NMJ. Transmitter release induced by repetitive nerve stimulation, elicit a frequency-dependent activation of G-protein-coupled receptors on perisynaptic Schwann cells and the release of calcium from internal stores. In return, perisynaptic Schwann cells modulate synaptic activity during and following high-frequency stimulation through short-term plasticity. In the present review, we discuss evidence of glial involvement in the short-term plasticity at the NMJ and the potential impact of such modulation on synaptic efficacy.     

Severely impaired neuromuscular synaptic transmission causes muscle weakness in the Cacna1a-mutant mouse rolling Nagoya

The ataxic mouse rolling Nagoya (RN) carries a missense mutation in the Cacna1a gene, encoding the pore-forming subunit of neuronal Ca(v)2.1 (P/Q-type) Ca2+ channels. Besides being the predominant type of Ca(v) channel in the cerebellum, Ca(v)2.1 channels mediate acetylcholine (ACh) release at the peripheral neuromuscular junction (NMJ). Therefore, Ca(v)2.1 dysfunction induced by the RN mutation may disturb ACh release at the NMJ. The dysfunction may resemble the situation in Lambert-Eaton myasthenic syndrome (LEMS), in which autoantibodies target Ca(v)2.1 channels at NMJs, inducing severely reduced ACh release and resulting in muscle weakness. We tested neuromuscular function of RN mice and characterized transmitter release properties at their NMJs in diaphragm, soleus and flexor digitorum brevis muscles. Clinical muscle weakness and fatigue were demonstrated using repetitive nerve-stimulation electromyography, grip strength testing and an inverted grid hanging test. Muscle contraction experiments showed a compromised safety factor of neuromuscular transmission. In ex vivo electrophysiological experiments we found severely impaired ACh release. Compared to wild-type, RN NMJs had 50-75% lower nerve stimulation-evoked transmitter release, explaining the observed muscle weakness. Surprisingly, the reduction in evoked release was accompanied by an approximately 3-fold increase in spontaneous ACh release. This synaptic phenotype suggests a complex effect of the RN mutation on different functional Ca(v)2.1 channel parameters, presumably with a positive shift in activation potential as a prevailing feature. Taken together, our studies indicate that the gait abnormality of RN mice is due to a combination of ataxia and muscle weakness and that RN models aspects of the NMJ dysfunction in LEMS.     

Mechanisms of postsynaptic plasticity: remodeling of the junctional acetylcholine receptor cluster induced by motor nerve terminal outgrowth

Motor nerve terminal outgrowth (NTO) at neuromuscular junctions (NMJs) occurs rapidly in response to denervation changes in muscle. We have previously found that NTO can produce an elongation of the synaptic area of the NMJ as defined by cholinesterase-silver staining. In the present study, we examined the effects of NTO on a postsynaptic muscle membrane component, the usually stable cluster of acetylcholine receptors (AChRs) at the NMJ. NTO was evoked in rat soleus muscles using botulinum toxin. AChRs were demonstrated using immunocytochemistry or autoradiography of alpha-bungarotoxin binding. Our results show that NTO induces rapid elongation of the cluster of AChRs at the NMJ within 7 d of treatment with botulinum toxin. The growth in the size of the AChR clusters was accompanied by an increase in the number of AChRs/NMJ. No elongation of AChR clusters was seen following surgical denervation, suggesting that cluster growth is related to NTO and not to denervation changes in muscle per se. Growth of NMJ-AChR clusters appeared to result primarily from 2 processes: insertion of new AChRs into the NMJ membrane and, surprisingly, redistribution of preexisting NMJ-AChRs. These results show that NTO can cause rapid changes in the normally stable cluster of AChRs at the NMJ. Motor nerve terminals provide a strong and anatomically precise control of AChRs at the NMJ.     

Molecular architecture of the neuromuscular junction

The neuromuscular junction (NMJ) is a complex structure that serves to efficiently communicate the electrical impulse from the motor neuron to the skeletal muscle to signal contraction. Over the last 200 years, technological advances in microscopy allowed visualization of the existence of a gap between the motor neuron and skeletal muscle that necessitated the existence of a messenger, which proved to be acetylcholine. Ultrastructural analysis identified vesicles in the presynaptic nerve terminal, which provided a beautiful structural correlate for the quantal nature of neuromuscular transmission, and the imaging of synaptic folds on the muscle surface demonstrated that specializations of the underlying protein scaffold were required. Molecular analysis in the last 20 years has confirmed the preferential expression of synaptic proteins, which is guided by a precise developmental program and maintained by signals from nerve. Although often overlooked, the Schwann cell that caps the NMJ and the basal lamina is proving to be critical in maintenance of the junction. Genetic and autoimmune disorders are known that compromise neuromuscular transmission and provide further insights into the complexities of NMJ function as well as the subtle differences that exist among NMJ that may underlie the differential susceptibility of muscle groups to neuromuscular transmission diseases. In this review we summarize the synaptic physiology, architecture, and variations in synaptic structure among muscle types. The important roles of specific signaling pathways involved in NMJ development and acetylcholine receptor (AChR) clustering are reviewed. Finally, genetic and autoimmune disorders and their effects on NMJ architecture and neuromuscular transmission are examined.     

Focal caspase activation underlies the endplate myopathy in slow-channel syndrome

Slow-channel syndrome (SCS) is a progressive neuromuscular disorder caused by abnormal gating of mutant acetylcholine receptors (AChRs) in the neuromuscular junction (NMJ). The pathological hallmark is selective degeneration of the NMJ termed endplate myopathy. Endplate myopathy consists of a combination of ultrastructural abnormalities, including degenerating subsynaptic nuclei, mitochondria, and postsynaptic folds, caused by localized cation overload through mutant AChRs. Because some of these changes resemble those seen in programmed cell death, we evaluated SCS muscle for evidence of focal activation of apoptotic pathways. Using antisera specific for the activated forms of caspases, the family of cysteine proteases that underlies apoptosis, we demonstrated that active forms of initiator and effector caspases are selectively localized at the NMJ in SCS. In comparison with an electron microscopic assessment of the abnormalities seen in endplate myopathy, we found that activated caspases were present at between 15 and 57% of endplates, similar to the proportion of endplates with degenerating mitochondria or vacuoles. This greatly exceeds the number of NMJs exhibiting nuclear degeneration. These findings provide the first evidence supporting the view that caspase activation in human disease can play a prominent role in localized cellular degenerative processes without causing nuclear or cell death.     

Genetic defects and disorders at the neuromuscular junction

Genetic defects in molecules expressed at the neuromuscular junction (NMJ) cause congenital myasthenic syndromes (CMSs), which are characterized by muscle weakness, abnormal fatigability, amyotrophy, and minor facial anomalies. Muscle weakness mostly develops under 2 years but is also sometimes seen in adults. Mutations identified to date include (i) muscle nicotinic acetylcholine receptor (AChR) subunits, (ii) rapsyn that anchors and clusters AChRs at the neuromuscular junction, (iii) agrin that is released from the nerve terminal and induces AChR clustering by stimulating the downstream LRP4/MuSK/Dok-7/rapsyn/AChR pathway, (iv) muscle-specific kinase (MuSK) that transmits the AChR-clustering signal from agrin/LRP4 to rapsyn/AChR, (v) Dok-7 that transmits the AChR-clustering signal from agrin/LRP4/MuSK to rapsyn/AChR, (vi) skeletal muscle sodium channel type 1.4 (Nav1.4) that spreads the depolarization potential from the endplate throughout muscle fibers, (vii) collagen Q that anchors acetylcholinesterase to the synaptic basal lamina, and (viii) choline acetyltransferase that resynthesizes acetylcholine from recycled choline at the nerve terminal. In addition, mutations in the heparin sulfate proteoglycan perlecan, which binds to many molecules including collagen Q and dystroglycan, causes Schwartz-Jampel syndrome. Interestingly, mutations in LRP4 cause Cenani-Lenz syndactyly syndrome but not CMS. AChR, MuSK, and LRP4 are also targets of auto-antibodies in myasthenia gravis. In addition, molecules at the NMJ are targets of many other disease states AChRs are blocked by the snake toxin alpha-bungarotoxin and the plant poison curare. The presynaptic SNARE complex is attacked by botulinum toxin. Acetylcholinesterase is inhibited by the nerve gas sarin and by organophosphate pesticides. This review focuses on the molecular bases underlying defects of AChR, rapsyn, Nav1.4, collagen Q, and choline acetyltransferase.     

Cholinergic induction of input-specific late-phase LTP via localized Ca2+ release in the visual cortex

Acetylcholine facilitates long-term potentiation (LTP) and long-term depression (LTD), substrates of learning, memory, and sensory processing, in which acetylcholine also plays a crucial role. Ca(2+) ions serve as a canonical regulator of LTP/LTD but little is known about the effect of acetylcholine on intracellular Ca(2+) dynamics. Here, we investigated dendritic Ca(2+) dynamics evoked by synaptic stimulation and the resulting LTP/LTD in layer 2/3 pyramidal neurons of the rat visual cortex. Under muscarinic stimulation, single-shock electrical stimulation (SES) inducing ～20 mV EPSP, applied via a glass electrode located ～10 μm from the basal dendrite, evoked NMDA receptor-dependent fast Ca(2+) transients and the subsequent Ca(2+) release from the inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. These secondary dendritic Ca(2+) transients were highly localized within 10 μm from the center (SD = 5.0 μm). The dendritic release of Ca(2+) was a prerequisite for input-specific muscarinic LTP (LTPm). Without the secondary Ca(2+) release, only muscarinic LTD (LTDm) was induced. D(-)-2-amino-5-phosphopentanoic acid and intracellular heparin blocked LTPm as well as dendritic Ca(2+) release. A single burst consisting of 3 EPSPs with weak stimulus intensities instead of the SES also induced secondary Ca(2+) release and LTPm. LTPm and LTDm were protein synthesis-dependent. Furthermore, LTPm was confined to specific dendritic compartments and not inducible in distal apical dendrites. Thus, cholinergic activation facilitated selectively compartment-specific induction of late-phase LTP through IP(3)-dependent Ca(2+) release.     

Astrocyte calcium signaling transforms cholinergic modulation to cortical plasticity in vivo

Global brain state dynamics regulate plasticity in local cortical circuits, but the underlying cellular and molecular mechanisms are unclear. Here, we demonstrate that astrocyte Ca(2+) signaling provides a critical bridge between cholinergic activation, associated with attention and vigilance states, and somatosensory plasticity in mouse barrel cortex in vivo. We investigated first whether a combined stimulation of mouse whiskers and the nucleus basalis of Meynert (NBM), the principal source of cholinergic innervation to the cortex, leads to enhanced whisker-evoked local field potential. This plasticity is dependent on muscarinic acetylcholine receptors (mAChR) and N-methyl-d-aspartic acid receptors (NMDARs). During the induction of this synaptic plasticity, we find that astrocytic [Ca(2+)](i) is pronouncedly elevated, which is blocked by mAChR antagonists. The elevation of astrocytic [Ca(2+)](i) is crucial in this type of synaptic plasticity, as the plasticity could not be induced in inositol-1,4,5-trisphosphate receptor type 2 knock-out (IP(3)R2-KO) mice, in which astrocytic [Ca(2+)](i) surges are diminished. Moreover, NBM stimulation led to a significant increase in the extracellular concentration of the NMDAR coagonist d-serine in wild-type mice when compared to IP(3)R2-KO mice. Finally, plasticity in IP(3)R2-KO mice could be rescued by externally supplying d-serine. Our data present coherent lines of in vivo evidence for astrocytic involvement in cortical plasticity. These findings suggest an unexpected role of astrocytes as a gate for cholinergic plasticity in the cortex.     

The formation of complex acetylcholine receptor clusters requires MuSK kinase activity and structural information from the MuSK extracellular domain

Efficient synaptic transmission at the neuromuscular junction (NMJ) requires the topological maturation of the postsynaptic apparatus from an oval acetylcholine receptor (AChR)-rich plaque into a complex pretzel-shaped array of branches. However, compared to NMJ formation very little is known about the mechanisms that regulate NMJ maturation. Recently the process of in vivo transformation from plaque into pretzel has been reproduced in vitro by culturing myotubes aneurally on laminin-coated substrate. It was proposed that the formation of complex AChR clusters is regulated by a MuSK-dependent muscle intrinsic program. To elucidate the structure-function role of MuSK in the aneural maturation of AChR pretzels, we used muscle cell lines expressing MuSK mutant and chimeric proteins. Here we report, that besides its role during agrin-induced AChR clustering, MuSK kinase activity is also necessary for substrate-dependent cluster formation. Constitutive-active MuSK induces larger AChR clusters, a faster cluster maturation on laminin and increases the anchorage of AChRs to the cytoskeleton compared to MuSK wild-type. In addition, we find that the juxtamembrane region of MuSK, which has previously been shown to regulate agrin-induced AChR clustering, is unable to induce complex AChR clusters on laminin substrate. Most interestingly, MuSK kinase activity is not sufficient for laminin-dependent AChR cluster formation since the MuSK ectodomain is also required suggesting a so far undiscovered instructive role for the extracellular domain of MuSK.     

Non-uniform release at the frog neuromuscular junction: evidence of morphological and physiological plasticity

The frog neuromuscular junction (NMJ) is a fusiform structure parallel to the muscle fiber with a few secondary and tertiary branches. Both sprouting and regression can occur on the same nerve terminal, suggesting a continuous on-going remodelling of the mature neuromuscular junction. Thus, the frog NMJ is a dynamic structure. Ultrastructural observations of the nerve terminal suggest that the active zones are distributed equally along the mature nerve terminal. Disorganized active zones have however been observed in distal regions. The density of synaptic vesicles is also uniform throughout the whole structure. However, mitochondria appear to be more abundant in the very distal regions of the nerve terminal. The postjunctional folds and the cholinergic receptors are also uniformly distributed along the NMJ. However, during remodelling periods, the distributions of postjunctional folds and of cholinergic receptors are not uniform in the degenerating and regenerating regions. Fig. 1 summarizes these morphological data. The frequency of spontaneous release (MEPPs) at the NMJ is higher in the proximal region than in the distal regions and recent evidence suggests that the mean MEPP amplitude is higher in the proximal than in the distal portions. Evoked transmitter release is also non-uniform along the frog NMJ. As for spontaneous release, it is higher in the proximal regions than in the distal regions. Failures of the active propagation of the PNAP at low safety points, such as the end of the myelinated axon and the branching points, may be one of the mechanisms responsible for unequal evoked release. It is also possible that the PNAP does not actively invade the whole extend of the nerve terminal since Na+ channels are absent from the distal regions. Fig. 2 summarizes these physiological data.     

Calcium as a Trigger for Cerebellar Long-Term Synaptic Depression

Cerebellar long-term depression (LTD) is a form of long-term synaptic plasticity that is triggered by calcium(Ca2+) signals in the postsynaptic Purkinje cell. This Ca2+comes both from IP3-mediated release from intracellular Ca2+ stores, as well as from Ca2+ influx through voltage-gated Ca2+ channels. The Ca2+ signal that triggers LTD occurs locally within dendritic spines and is due to supralinear summation of signals coming from these two Ca2+ sources. The properties of this postsynaptic Ca2+signal can explain several features of LTD, such as its associativity, synapse specificity, and dependence on thetiming of synaptic activity, and can account for the slow kinetics of LTD expression. Thus, from a Ca2+ signaling perspective, LTD is one of the best understood forms of synaptic plasticity.