Project description:Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis. DOI:http://dx.doi.org/10.7554/eLife.00723.001.
Project description:The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.
Project description:The focus of this review is on Duchenne muscular dystrophy (DMD), which is caused by the absence of the protein dystrophin and is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but data show that both human patients and animal models for DMD present with fragmented neuromuscular junction (NMJ) morphology. In addition to pre- and post-synaptic abnormalities, studies indicate increased susceptibility of the NMJ to contraction-induced injury, with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyographic activity. Such findings suggest that alterations in the NMJ of dystrophic muscle may play a role in muscle weakness via impairment of neuromuscular transmission. Further work is needed to fully understand the role of the NMJ in the weakness, susceptibility to injury, and progressive wasting associated with DMD.
Project description:ObjectiveElectromyography (EMG) is used routinely to diagnose neuromuscular dysfunction in a wide range of peripheral neuropathies, myopathies, and neuromuscular degenerative diseases including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Definitive neurological diagnosis may also be indicated by the analysis of pathological neuromuscular innervation in motor-point biopsies. Our objective in this study was to preempt motor-point biopsy by combining live imaging with electrophysiological analysis of slow degeneration of neuromuscular junctions (NMJs) in vivo.MethodsWe combined conventional needle electromyography with fiber-optic confocal endomicroscopy (CEM), using an integrated hand-held, 1.5-mm-diameter probe. We utilized as a test bed, various axotomized muscles in the hind limbs of anaesthetized, double-homozygous thy1.2YFP16: Wld (S) mice, which coexpress the Wallerian-degeneration Slow (Wld(S)) protein and yellow fluorescent protein (YFP) in motor neurons. We also tested exogenous vital stains, including Alexa488-α-bungarotoxin; the styryl pyridinium dye 4-Di-2-Asp; and a GFP conjugate of botulinum toxin Type A heavy chain (GFP-HcBoNT/A).ResultsWe show that an integrated EMG/CEM probe is effective in longitudinal evaluation of functional and morphological changes that take place over a 7-day period during axotomy-induced, slow neuromuscular synaptic degeneration. EMG amplitude declined in parallel with overt degeneration of motor nerve terminals. EMG/CEM was safe and effective when nerve terminals and motor endplates were selectively stained with vital dyes.InterpretationOur findings constitute proof-of-concept, based on live imaging in an animal model, that combining EMG/CEM may be useful as a minimally invasive precursor or alternative to motor-point biopsy in neurological diagnosis and for monitoring local administration of potential therapeutics.
Project description:Schwann cells (SCs), the glial cells of the peripheral nervous system, cover synaptic terminals, allowing them to monitor and modulate neurotransmission. Disruption of glial coverage leads to axon degeneration and synapse loss. The cellular mechanisms that establish and maintain this coverage remain largely unknown. To address this, we labeled single SCs and performed time-lapse imaging experiments. Adult terminal SCs are arranged in static tile patterns, whereas young SCs dynamically intermingle. The mechanism of developmental glial segregation appears to be spatial competition, in which glial-glial and axonal-glial contacts constrain the territory of single SCs, as shown by four types of experiments: (1) laser ablation of single SCs, which led to immediate territory expansion of neighboring SCs; (2) axon removal by transection, resulting in adult SCs intermingling dynamically; (3) axotomy in mutant mice with blocked axon fragmentation in which intermingling was delayed; and (4) activity blockade, which had no immediate effects. In summary, we conclude that glial cells partition synapses by competing for perisynaptic space.
Project description:Cancer cachexia is a major cause of patient morbidity and mortality, with no efficacious treatment or management strategy. Despite cachexia sharing pathophysiological features with a number of neuromuscular wasting conditions, including age-related sarcopenia, the mechanisms underlying cachexia remain poorly understood. Studies of related conditions suggest that pathological targeting of the neuromuscular junction (NMJ) may play a key role in cachexia, but this has yet to be investigated in human patients. Here, high-resolution morphological analyses were undertaken on NMJs of rectus abdominis obtained from patients undergoing upper GI cancer surgery compared with controls (N = 30; n = 1,165 NMJs). Cancer patients included those with cachexia and weight-stable disease. Despite the low skeletal muscle index and significant muscle fiber atrophy (P < 0.0001) in patients with cachexia, NMJ morphology was fully conserved. No significant differences were observed in any of the pre- and postsynaptic variables measured. We conclude that NMJs remain structurally intact in rectus abdominis in both cancer and cachexia, suggesting that denervation of skeletal muscle is not a major driver of pathogenesis. The absence of NMJ pathology is in stark contrast to what is found in related conditions, such as age-related sarcopenia, and supports the hypothesis that intrinsic changes within skeletal muscle, independent of any changes in motor neurons, represent the primary locus of neuromuscular pathology in cancer cachexia.
Project description:Desmin, the major intermediate filament (IF) protein in muscle cells, interlinks neighboring myofibrils and connects the whole myofibrillar apparatus to myonuclei, mitochondria, and the sarcolemma. However, desmin is also known to be enriched at postsynaptic membranes of neuromuscular junctions (NMJs). The pivotal role of the desmin IF cytoskeletal network is underscored by the fact that over 100 mutations of the human DES gene cause hereditary and sporadic myopathies and cardiomyopathies. A subgroup of human desminopathies comprises autosomal recessive cases resulting in complete abolition of desmin protein. In these patients, who display a more severe phenotype than the autosomal dominant cases, it has been reported that some individuals also suffer from a myasthenic syndrome in addition to the classical occurrence of myopathy and cardiomyopathy. Since further studies on the NMJ pathology is hampered by the lack of available human striated muscle biopsy specimens, we exploited homozygous desmin knock-out mice which closely mirror the striated muscle pathology of human patients lacking desmin protein. Here, we report on the impact of the lack of desmin on the structure and function of NMJs and on the transcription of genes coding for postsynaptic proteins. Desmin knock-out mice display a fragmentation of NMJs in soleus, but not in extensor digitorum longus muscle. Moreover, soleus muscle fibers show larger NMJs. Further, transcription levels of acetylcholine receptor (AChR) genes are increased in muscles from desmin knock-out mice, especially of the AChR subunit, which is known as a marker of muscle fiber regeneration. Electrophysiological recordings depicted a pathological decrement of nerve-dependent endplate potentials and a faster rise time of the nerve-independent miniature endplate potentials. The latter is indicating an enhanced opening time of the AChR channels. Our study highlights the essential role of desmin for the structural and functional integrity of mammalian neuromuscular junctions.
Project description:Topological materials and their unusual transport properties are now at the focus of modern experimental and theoretical research. Their topological properties arise from the bandstructure determined by the atomic composition of a material and as such are difficult to tune and naturally restricted to ≤3 dimensions. Here we demonstrate that n-terminal Josephson junctions with conventional superconductors may provide novel realizations of topology in n-1 dimensions, which have similarities, but also marked differences with existing 2D or 3D topological materials. For n≥4, the Andreev subgap spectrum of the junction can accommodate Weyl singularities in the space of the n-1 independent superconducting phases, which play the role of bandstructure quasimomenta. The presence of these Weyl singularities enables topological transitions that are manifested experimentally as changes of the quantized transconductance between two voltage-biased leads, the quantization unit being 4e(2)/h, where e is the electric charge and h is the Planck constant.
Project description:Mice deficient in the glycosyltransferase Large are characterized by severe muscle and central nervous system abnormalities. In this study, we show that the formation and maintenance of neuromuscular junctions in Large(myd) mice are greatly compromised. Neuromuscular junctions are not confined to the muscle endplate zone but are widely spread and are frequently accompanied by exuberant nerve sprouting. Nerve terminals are highly fragmented and binding of alpha-bungarotoxin to postsynaptic acetylcholine receptors (AChRs) is greatly reduced. In vitro, Large(myd) myotubes are responsive to agrin but produce aberrant AChR clusters, which are larger in area and less densely packed with AChRs. In addition, AChR expression on the cell surface is diminished suggesting that AChR assembly or transport is defective. These results together with the finding that O-linked glycosylation at neuromuscular junctions of Large(myd) mice is compromised indicate that the action of Large is necessary for proper neuromuscular junction development.
Project description:The physiological processes regulating neuromuscular transmission are highly dependent on the structural features of the motor neuron and motor endplate, and detailing the structure of neuromuscular junctions (NMJs) in muscle biopsies is a powerful method for research and diagnostics. The observation of NMJ ultrastructure, however, is complicated by the difficulty in locating NMJs for analysis by electron microscopy. Consequently, a correlative confocal-transmission electron microscopy method was developed. Fixed muscle samples were cryo-protected in sucrose, sectioned on a cryostat, and stained with fluorescent alpha-bungarotoxin for confocal microscopy. Sections containing junctions were mapped and then processed for transmission electron microscopy (TEM). Cryostat sections allowed large expanses of muscle tissue to be rapidly screened and enabled specific junctions to be targeted for TEM. The morphology of the junctions was well preserved with all essential features of the pre- and postsynaptic elements readily identifiable without freeze damage. Unlike NMJ correlative methods using histochemical stains and DAB photo-oxidation, no electron dense precipitate was deposited over the NMJ, enabling an unobstructed view of the pre- and postsynaptic structures.