Kismet Positively Regulates Glutamate Receptor Localization and Synaptic Transmission at the Drosophila Neuromuscular Junction

The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is structurally and functionally similar to mammalian glutamatergic synapses. These synapses can, as a result of changes in activity, alter the strength of their connections via processes that require chromatin remodeling and changes in gene expression. The chromodomain helicase DNA binding (CHD) protein, Kismet (Kis), is expressed in both motor neuron nuclei and postsynaptic muscle nuclei of the Drosophila larvae. Here, we show that Kis is important for motor neuron synaptic morphology, the localization and clustering of postsynaptic glutamate receptors, larval motor behavior, and synaptic transmission. Our data suggest that Kis is part of the machinery that modulates the development and function of the NMJ. Kis is the homolog to human CHD7, which is mutated in CHARGE syndrome. Thus, our data suggest novel avenues of investigation for synaptic defects associated with CHARGE syndrome.     

Neuron-glia interactions: the roles of Schwann cells in neuromuscular synapse formation and function

The NMJ (neuromuscular junction) serves as the ultimate output of the motor neurons. The NMJ is composed of a presynaptic nerve terminal, a postsynaptic muscle and perisynaptic glial cells. Emerging evidence has also demonstrated an existence of perisynaptic fibroblast-like cells at the NMJ. In this review, we discuss the importance of Schwann cells, the glial component of the NMJ, in the formation and function of the NMJ. During development, Schwann cells are closely associated with presynaptic nerve terminals and are required for the maintenance of the developing NMJ. After the establishment of the NMJ, Schwann cells actively modulate synaptic activity. Schwann cells also play critical roles in regeneration of the NMJ after nerve injury. Thus, Schwann cells are indispensable for formation and function of the NMJ. Further examination of the interplay among Schwann cells, the nerve and the muscle will provide insights into a better understanding of mechanisms underlying neuromuscular synapse formation and function.     

Prolonged effects of a post-synaptic blocking fraction of Naja siamensis venom of skeletal muscle of the mouse.

A sublethal dose of a post-synaptic blocking fraction of Naja siamensis venom was injected into the soleus muscle of the mouse inhibiting neuromuscular transmission for 2-3 days. The paralysed soleus muscle behaved as if denervated, developing extra-junctional sensitivity to acetylcholine and accepting innervation by an implanted foreign nerve. Since the only known action of the post-synaptic blocking fraction of this venom is due to its affinity to acetylcholine receptors, the results suggest that the spread in the sensitivity of muscle fibres to acetylcholine and their ability to accept a foreign nerve is a consequence of neuromuscular blockade.     

Muscle specific kinase: organiser of synaptic membrane domains

Muscle Specific Kinase (MuSK) is a transmembrane tyrosine kinase vital for forming and maintaining the mammalian neuromuscular junction (NMJ: the synapse between motor nerve and skeletal muscle). MuSK expression switches on during skeletal muscle differentiation. MuSK then becomes restricted to the postsynaptic membrane of the NMJ, where it functions to cluster acetylcholine receptors (AChRs). The expression, activation and turnover of MuSK are each regulated by signals from the motor nerve terminal. MuSK forms the core of an emerging signalling complex that can be acutely activated by neural agrin (N-agrin), a heparin sulfate proteoglycan secreted from the nerve terminal. MuSK activation initiates complex intracellular signalling events that coordinate the local synthesis and assembly of synaptic proteins. The importance of MuSK as a synapse organiser is highlighted by cases of autoimmune myasthenia gravis in which MuSK autoantibodies can deplete MuSK from the postsynaptic membrane, leading to complete disassembly of the adult NMJ.     

Investigating the safety factor at an invertebrate neuromuscular junction

Fidelity of synaptic transmission is essential at the neuromuscular junction (NMJ). To ensure that transmission does not fail, vertebrate motoneurons often release more neurotransmitter than is required for muscle contraction. This safety factor allows some loss of synaptic function without failure of muscle contraction. It is not known whether a similar mechanism operates at the invertebrate neuromuscular junction. In our study of the Drosophila NMJ, we find that glutamate receptor mutants can exhibit a substantial decrease in synaptic function while maintaining muscle contraction. The persistence of neuromuscular function in these mutants is not explained by synaptic facilitation, temporal summation of high frequency stimuli, or a hyperpolarizing shift in the activation range of muscle calcium channels. Instead, the attenuated synaptic response is sufficient to drive muscle contraction. Quantitative analysis of the decrease in synaptic transmission in these mutants implies that at the wild-type NMJ there is an approximately five- to ninefold excess in released transmitter. Hence, the presence of a synaptic safety factor is a conserved feature of neuromuscular organization in both invertebrates and vertebrates.     

Perisynaptic Schwann cells at neuromuscular junctions revealed by a novel monoclonal antibody

This study aimed to generate a probe for perisynaptic Schwann cells (PSCs) to investigate the emerging role of these synapse-associated glial cells in the formation and maintenance of the neuromuscular junction (NMJ). We have obtained a novel monoclonal antibody, 2A12, which labels the external surface of PSC membranes at the frog NMJ. The antibody reveals PSC fine processes or “fingers” that are interposed between nerve terminal and muscle membrane, interdigitating with bands of acetylcholine receptors. This antibody also labels PSCs at the avian neuromuscular junction and recognizes a 200 kDa protein in Torpedo electric organs. In frog muscles, axotomy induces sprouting of PSC processes beyond clusters of acetylcholine receptors and acetylcholinesterase at denervated junctional branches. PSC branches often extend across several muscle fibers. At some junctions, PSC sprouts join the tips of neighboring branches. The average length of PSC sprouts is approximately 156 µ at 3-week denervated NMJs. PSC sprouting is accompanied by a significant increase in the number of Schwann cell bodies per NMJ. Following nerve regeneration, nerve terminals reinnervate the junction along the PSC processes. In vivo observations of normal frog muscles also show PSC processes longer than nerve terminals at some junctional branches. The results suggest that nerve injury induces profuse PSC sprouting that may play a role in guiding nerve terminal regeneration at frog NMJs. In addition, antibody 2A12 reveals the fine morphology of PSCs in relation to other synaptic elements and is a useful probe in elucidating the function of these synapse-associated glial cells in vivo.     

Synaptogenetic mechanisms controlling postsynaptic differentiation of the neuromuscular junction are nerve-dependent in human and nerve-independent in mouse C2C12 muscle cultures

Acetylcholinesterase (EC 3.1.1.7, AChE) is one of the components of the neuromuscular junction (NMJ). Its expression and targeting in the skeletal muscle fiber is therefore under the control of the mechanisms responsible for the formation of the highly complex structure of this synapse. Recently, it has been demonstrated that myotubes of the C2C12 mouse muscle cell line form highly differentiated pretzel-like postsynaptic accumulations of acetylcholine receptors (AChRs) in the complete absence of the nerve if they are cultured on the laminin coating. This finding questions previously stressed importance of the nerve-derived factors in NMJ synaptogenesis and therefore deserves additional testing. The aim of this paper was to test whether the reported nerve-independency can be demonstrated also in the cultured human muscle meaning that the findings on C2C12 cultures can be extrapolated also to the human muscle. In our experiments aneurally cultured human myotubes failed to form AChR clusters on its surface, no matter if they were grown on normal gelatine or laminin coating. However, when innervated by neurons extending from the rat embryonic spinal cord, human myotubes formed AChR clusters with elaborate topography but strictly on the areas contacted by the nerve. One can hypothesize that higher nerve dependency of the NMJ synaptogenesis in humans in comparison to other species reflects species-specific differences in the organization of movement. Humans have the highest "fractionation of movement" capacity which probably requests different, more nerve-controlled development of the motor system including nerve-restricted development of the neuromuscular contacts.     

Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice.

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron-neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin-deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin-deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age-matched wild-type neurons during the first 3 weeks in culture. These results demonstrate that neuron-specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction.     

Motor function recovery: deciphering a regenerative niche at the neuromuscular synapse

The coordinated movement of many organisms relies on efficient nerve–muscle communication at the neuromuscular junction (NMJ), a peripheral synapse composed of a presynaptic motor axon terminal, a postsynaptic muscle specialization, and non-myelinating terminal Schwann cells. NMJ dysfunctions are caused by traumatic spinal cord or peripheral nerve injuries as well as by severe motor pathologies. Compared to the central nervous system, the peripheral nervous system displays remarkable regenerating abilities; however, this capacity is limited by the denervation time frame and depends on the establishment of permissive regenerative niches. At the injury site, detailed information is available regarding the cells, molecules, and mechanisms involved in nerve regeneration and repair. However, a regenerative niche at the final functional step of peripheral motor innervation, i.e. at the mature neuromuscular synapse, has not been deciphered. In this review, we integrate classic and recent evidence describing the cells and molecules that could orchestrate a dynamic ecosystem to accomplish successful NMJ regeneration. We propose that such a regenerative niche must ensure at least two fundamental steps for successful NMJ regeneration: the proper arrival of incoming regenerating axons to denervated postsynaptic muscle domains, and the resilience of those postsynaptic domains, in morphological and functional terms. We here describe and combine the main cellular and molecular responses involved in each of these steps as potential targets to help successful NMJ regeneration.     

Developmental and adult-specific processes contribute to de novo neuromuscular regeneration in the lizard tail

Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response.     

The signaling pathways mediated by P2Y nucleotide receptors in the formation and maintenance of the skeletal neuromuscular junction

The motor neuron, the Schwann cell and the muscle cell are highly specialized at the vertebrate skeletal neuromuscular junction (NMJ). The muscle cell surface contains a high local density of acetylcholine (ACh) receptors (AChRs), acetylcholinesterase (AChE) and their interacting macromolecules at the NMJ, forming the postsynaptic specializations. During the early stages of development, the incoming nerve terminal induces the formation of these postsynaptic specializations; the nerve secretes agrin and neuregulin (NRG), which are known to aggregate existing AChRs and to increase the expression of AChR at the synaptic region, respectively. In addition, adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is coreleased with ACh during muscle contraction. Recent evidence suggests that ATP can play a role in forming and maintaining the postsynaptic specializations by activating its corresponding receptors. In particular, one of the nucleotide receptor subtypes, the P2Y(1) receptor, is specifically localized at the NMJs. The gene expression of AChR and AChE is upregulated after the activation of P2Y(1) receptors. Thus, the synaptic ATP together with agrin and NRG can act as a synapse-organizing factor to induce the expression of postsynaptic functional effectors.     

Hypothalamic Sirt1 protects terminal Schwann cells and neuromuscular junctions from age‐related morphological changes

Neuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age‐related changes. Aging effects on the NMJ components—motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)—have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild‐type mice, all NMJ components showed age‐associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain‐specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic‐specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age‐related changes via sympathetic innervation.     

The ultrastructure of neuromuscular and neural-neural relationships in in vitro maintained Dirofilaria Immitis

Relationships of neuromuscular junctions of the somatic musculature and associated neural-neural synapses in the ventral nerve trunk of the canine adult heartworm, Dirofilaria immitis, were studied by transmission electron microscopy. The heartworms were maintained in vitro prior to study. Nerve fibres in the trunk were highly invaginated into the cytoplasm of hypodermal cells and connected through the intercellular spaces via mesaxons. The nerve fibres contained neurotubules, neurofilaments and ribosomes. The nerve trunk and the muscle arms were separated by an epineurium averaging 250 nm in width. At the junctional site, a marked reduction in width of the epineurium was noted at the synaptic cleft. Often when two adjacent nerve fibres had adjacent neuromuscular junctions, an axo-axonal synapse and common mesaxon between the adjacent fibres were present. Varicosities were evident on some cross-sections through nerve fibres and ranged from a simple outward swelling against the muscle arm mass to exaggerated outgrowths measuring several micrometers in length.     

Glutamatergic synapses of Drosophila neuromuscular junctions: a high-resolution model for the analysis of experience-dependent potentiation

The glutamatergic synapses of developing neuromuscular junctions (NMJ) of Drosophila larvae are readily accessible, morphologically simple, and physiologically well-characterized. They therefore have a long and highly successful tradition as a model system for the discovery of genetic and molecular mechanisms of target recognition, synaptogenesis, NMJ development, and synaptic plasticity. However, since the development and the activity-dependent refinement of NMJs are concurrent processes, they cannot easily be separated by the widely applied genetic manipulations that mostly have chronic effects. Recent studies have therefore begun systematically to incorporate larval foraging behavior into the physiological and genetic analysis of NMJ function in order to analyze potential experience-dependent changes of glutamatergic transmission. These studies have revealed that recent crawling experience is a potent modulator of glutamatergic transmission at NMJs, because high crawling activities result after an initial lag-phase in several subsequent phases of experience-dependent synaptic potentiation. Depending on the time window of occurrence, four distinct phases of experience-dependent potentiation have been defined. These phases of potentiation can be followed from their initial induction (phase-I) up to the morphological consolidation (phase-III/IV) of previously established functional changes (phase-II). This therefore establishes, for the first time, a temporal hierarchy of mechanisms involved in the use-dependent modification of glutamatergic synapses.     

Formation of functional synaptic connections between cultured cortical neurons from agrin‐deficient mice

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron–neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin‐deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin‐deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma‐aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age‐matched wild‐type neurons during the first 3 weeks in culture. These results demonstrate that neuron‐specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 547–557, 1999     

神经营养因子对神经肌肉接头传递的调制作用

运动单位由运动神经元及其支配的肌纤维组成。神经肌肉接头(neuromuscular junction,NMJ)传递受到严密的调节,因而能和运动单位的活动协调一致。在NMJ,神经调制物质的释放与运动单位的活动有关,并能决定突触传递的效能。脑源性神经营养因子(brain—derived neurotrophic factor,BDNF)和神经营养因子4(neurotrophin-4,NT-4)由运动神经末梢和肌纤维产生。肌肉释放营养因子受肌肉活动调节。在NMJ,BDNF和NT-4通过激活酪氨酸激酶B受体(tyrosine kinase receptor B,TrkB),能加强自发性和诱导性的突触活动。突触前Ca^2 量的迅速增加或突触胞吐过程的易化,都能增加突触囊泡的释放,从而改善NMJ的突触传递。事实上,BDNF能促进突触前细胞内Ca^2 的释放,TrkB的激活也能通过有丝分裂活化蛋白激酶,引起突触素I(synapsinI)的磷酸化,进而增加可释放的突触囊泡的数量。在NMJ,神经营养因子还能通过影响神经调节素(neuregulin)或其他神经源性调制物质的局部释放,对接头传递进行调节。本文对近年来在NMJ突触传递的调节,运动单位的NMJ特性以及神经营养因子对突触传递效能的影响等方面的研究进展做一综述。     

Congenital myasthenic syndromes: spotlight on genetic defects of neuromuscular transmission

The neuromuscular junction (NMJ) is a complex structure that efficiently communicates the electrical impulse from the motor neuron to the skeletal muscle to induce muscle contraction. Genetic and autoimmune disorders known to compromise neuromuscular transmission are providing further insights into the complexities of NMJ function. Congenital myasthenic syndromes (CMSs) are a genetically and phenotypically heterogeneous group of rare hereditary disorders affecting neuromuscular transmission. The understanding of the molecular basis of the different types of CMSs has evolved rapidly in recent years. Mutations were first identified in the subunits of the nicotinic acetylcholine receptor (AChR), but now mutations in ten different genes - encoding post-, pre- or synaptic proteins - are known to cause CMSs. Pathogenic mechanisms leading to an impaired neuromuscular transmission modify AChRs or endplate structure or lead to decreased acetylcholine synthesis and release. However, the genetic background of many CMS forms is still unresolved. A precise molecular classification of CMS type is of paramount importance for the diagnosis, counselling and therapy of a patient, as different drugs may be beneficial or deleterious depending on the molecular background of the particular CMS.     

Long-term in vitro maintenance of neuromuscular junction activity of Drosophila larvae

The larval Drosophila neuromuscular junction (NMJ) has proven to be an excellent system to test fundamental aspects of synaptic transmission, such as relationships among ion channel function, subtypes of glutamate receptors, and the functions of synaptic proteins in the presynaptic compartment. Recent advances in understanding bi-directional communication between nerves and muscles of Drosophila are helping uncover developmental as well as maintenance cues that could be applicable to all chemical synapses. The development of HL3 medium makes it possible to record synaptic responses at NMJs for prolonged periods of time. We demonstrate that media commonly used to culture CNS neurons and imaginal disks of Drosophila such as Schneider's and M3 completely block glutamatergic synaptic transmission at the NMJ. The depressed postsynaptic excitatory junction potentials (EJPs) partially recover from exposure to such media shortly after switching to the HL3 medium. Preliminary results from NMJs of filleted 3rd instar larvae for 4 days in vitro bathed in a modified HL3 medium show great promise. The resting membrane potential and the EJP amplitudes after 4 days in vitro are normal. These results demonstrate the possibility for chronic studies of developmental regulation in culture, which in some cases are impractical in the whole animal.     

Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation In vitro

Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin‐1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin‐coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co‐localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 551–565, 2016     

Dedifferentiation and remodeling of motor endplates following experimental localized lesions of muscle fibers

Local anaesthetics, cardiotoxin and mechanical injuries may cause necrosis of muscle fibres while leaving the motor nerve fibres and their terminals intact. With local injuries to mouse muscles carried out by freezing or cutting we made a point of preserving both the nerve terminals and the muscle fibre portions on which these terminals were located. It was thus possible to follow the changes induced at endplates by these lesions. Within two or three days of the freezing or cutting, the muscle fibres underwent very different degrees of regression of the contractile material and T-system. The neuromuscular junctions also underwent changes, principally affecting their postsynaptic portion, in particular the folds of the subneural apparatus. After dedifferentiation of subsynaptic areas, we observed sprouting of the nerve terminal on muscle fibres which survived the amputation of one end and formed actively new myofibrils. This sprouting restored synaptic connections at the original sites, but with new structural features and correlative changes in the distribution of cholinergic receptors and cholinesterases. It is probable that after a phase of involution followed by a phase of recovery, the injured muscle fibres triggered off the nerve terminal sprouting which led to the remodelling of the endplates.