Measuring neuromuscular junction functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle. Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1)

A synaptic nidogen: developmental regulation and role of nidogen-2 at the neuromuscular junction

Background: The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.Results: In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen- 2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.Conclusion: All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction

Congenital myasthenic syndromes and the therapeutic modulation of the neuromuscular junction

PhD ThesisImpairment of neuromuscular transmission at the neuromuscular junction is a primary pathomechanism of many human conditions. The congenital myasthenic syndromes (CMS) are caused by primary genetic defects which reduce the efficacy of neuromuscular transmission. CMS are increasingly diverse, both phenotypically and genetically, and the study of this group of disorders has improved our understanding of the role of neuromuscular junction proteins in health and disease. For most subtypes of CMS, symptomatic treatments are available. However, these are poorly understood and often limited by systemic side effects. In many CMS, sympathomimetics such as salbutamol and ephedrine lead to clinical benefit. However, the reason for this clinical benefit is unknown. Using animal models, this research aimed to explore the mechanisms underlying improved muscle strength from sympathomimetics in CMS. Experiments in zebrafish models revealed that salbutamol alters many aspects of neuromuscular junction development. Follow-up studies in the mouse model of end-plate acetylcholinesterase deficiency revealed that salbutamol leads to structural neuromuscular junction alterations which are primarily postsynaptic. The identification of novel CMS genes has been accelerated by the use of next generation sequencing. Genetic sequencing of undiagnosed patient cohorts lead to the identification of a novel presynaptic CMS subtype caused by mutations in SLC5A7, encoding the presynaptic choline transporter. Mutations in SLC5A7 were previously associated with an inherited motor neuropathy, and this finding expanded the overlap between disorders of the neuromuscular junction and of the motor nerve. In addition, through analysis of a patient cohort with CMS and episodic apnoea, I identified genetic, phenotypic and neurophysiological characteristics which provide mechanistic insights into this phenomenon. Understanding the mechanisms of neuromuscular junction dysfunction and of its therapeutic modulation are essential to facilitate earlier diagnosis and the development of targeted therapies for the wide range of disorders in which the neuromuscular junction is implicated

CXCL12/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axonterminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by -latrotoxin. CXCL12 acts via binding to the neuronal CXCR4 receptor. A CXCL12-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration invivo. Recombinant CXCL12 invivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons invitro. These findings indicate that the CXCL12-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage

Mathematical modeling of the Drosophila neuromuscular junction

Poster presentation: An important challenge in neuroscience is understanding how networks of neurons go about processing information. Synapses are thought to play an essential role in cellular information processing however quantitative and mathematical models of the underlying physiologic processes that occur at synaptic active zones are lacking. We are generating mathematical models of synaptic vesicle dynamics at a well-characterized model synapse, the Drosophila larval neuromuscular junction. This synapse's simplicity, accessibility to various electrophysiological recording and imaging techniques, and the genetic malleability intrinsic to Drosophila system make it ideal for computational and mathematical studies. We have employed a reductionist approach and started by modeling single presynaptic boutons. Synaptic vesicles can be divided into different pools; however, a quantitative understanding of their dynamics at the Drosophila neuromuscular junction is lacking [4]. We performed biologically realistic simulations of high and low release probability boutons [3] using partial differential equations (PDE) taking into account not only the evolution in time but also the spatial structure in two dimensions (the extension to three dimensions will be implemented soon). PDEs are solved using UG, a program library for the calculation of multi-dimensional PDEs solved using a finite volume approach and implicit time stepping methods leading to extended linear equation systems be solvedwith multi-grid methods [3,4]. Numerical calculations are done on multi-processor computers for fast calculations using different parameters in order to asses the biological feasibility of different models. In preliminary simulations, we modeled vesicle dynamics as a diffusion process describing exocytosis as Neumann streams at synaptic active zones. The initial results obtained with these models are consistent with experimental data. However, this should be regarded as a work in progress. Further refinements will be implemented, including simulations using morphologically realistic geometries which were generated from confocal scans of the neuromuscular junction using NeuRA (a Neuron Reconstruction Algorithm). Other parameters such as glutamate diffusion and reuptake dynamics, as well as postsynaptic receptor kinetics will be incorporated as well

The Wnt and BMP Families of Signaling Morphogens at the Vertebrate Neuromuscular Junction

The neuromuscular junction has been extensively employed in order to identify crucial determinants of synaptogenesis. At the vertebrate neuromuscular synapse, extracellular matrix and signaling proteins play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in invertebrate species have revealed crucial functions of early morphogens during the assembly and maturation of the neuromuscular junction. Here, we discuss growing evidence addressing the function of Wnt and Bone morphogenetic protein (BMP) signaling pathways at the vertebrate neuromuscular synapse. We focus on the emerging role of Wnt proteins as positive and negative regulators of postsynaptic differentiation. We also address the possible involvement of BMP pathways on motor neuron behavior for the assembly and/or regeneration of the neuromuscular junction

Snake and spider toxins induce a rapid recovery of function of botulinum neurotoxin paralysed neuromuscular junction

Botulinum neurotoxins (BoNTs) and some animal neurotoxins (-Bungarotoxin, -Btx, from elapid snakes and -Latrotoxin, -Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis. This result provides a proof of principle that, by causing the complete degeneration, reabsorption, and regeneration of a paralysed nerve terminal, one could favour the recovery of function of a biochemically- or genetically-altered motor axon terminal. These observations might be relevant to dying-back neuropathies, where pathological changes first occur at the neuromuscular junction and then progress proximally toward the cell body

A Drosophila Mutant with a Temperature-Sensitive Block in Nerve Conduction

A mutant, napts (no action potential, temperature-sensitive), is described in which axonal conduction fails at high temperature. Synaptic transmission at the larval neuromuscular junction is unimpaired. Larvae and adults are rapidly paralyzed at restrictive temperatures; they recover rapidly when the temperature is decreased. The mutant gene is recessive and is located on the second chromosome at map position 56

Depolarization-activated potentiation of the T fiber synapse in the blue crab

The blue crab T fiber synapse, associated with the stretch receptor of the swimming leg, has a nonspiking presynaptic element that mediates tonic transmission. This synapse was isolated and a voltage clamp circuit was used to control the membrane potential at the release sites. The dependence of transmitter release on extracellular calcium, [Ca]o, was studied over a range of 2.5-40 mM. A power relationship of 2.7 was obtained between excitatory postsynaptic potential (EPSP) rate of rise and [Ca]o. Brief presynaptic depolarizing steps, 5-10 ms, presented at 0.5 Hz activated EPSP's of constant amplitude. Inserting a 300-ms pulse (conditioning pulse) between these test pulses potentiated the subsequent test EPSPs. This depolarization-activated potentiation (DAP) lasted for 10-20 s and decayed with a single exponential time course. The decay time course remained invariant with test pulse frequencies ranging from 0.11 to 1.1 Hz. The magnitude and decay time course of DAP were independent of the test pulse amplitudes. The magnitude of DAP was a function of conditioning pulse amplitudes. Large conditioning pulses activated large potentiations, whereas the decay time constants were not changed. The DAP is a Ca-dependent process. When the amplitude of conditioning pulses approached the Ca equilibrium potential, the magnitude of potentiation decreased. Repeated application of conditioning pulses, at 2-s intervals, did not produce additional potentiation beyond the level activated by the first conditioning pulse. Comparison of the conditioning EPSP waveforms activated repetitively indicated that potentiation lasted transiently, 100 ms, during a prolonged release. Possible mechanisms of the potentiation are discussed in light of these new findings.The blue crab T fiber synapse, associated with the stretch receptor of the swimming leg, has a nonspiking presynaptic element that mediates tonic transmission. This synapse was isolated and a voltage clamp circuit was used to control the membrane potential at the release sites. The dependence of transmitter release on extracellular calcium, [Ca]o, was studied over a range of 2.5-40 mM. A power relationship of 2.7 was obtained between excitatory postsynaptic potential (EPSP) rate of rise and [Ca]o. Brief presynaptic depolarizing steps, 5-10 ms, presented at 0.5 Hz activated EPSP's of constant amplitude. Inserting a 300-ms pulse (conditioning pulse) between these test pulses potentiated the subsequent test EPSPs. This depolarization-activated potentiation (DAP) lasted for 10-20 s and decayed with a single exponential time course. The decay time course remained invariant with test pulse frequencies ranging from 0.11 to 1.1 Hz. The magnitude and decay time course of DAP were independent of the test pulse amplitudes. The magnitude of DAP was a function of conditioning pulse amplitudes. Large conditioning pulses activated large potentiations, whereas the decay time constants were not changed. The DAP is a Ca-dependent process. When the amplitude of conditioning pulses approached the Ca equilibrium potential, the magnitude of potentiation decreased. Repeated application of conditioning pulses, at 2-s intervals, did not produce additional potentiation beyond the level activated by the first conditioning pulse. Comparison of the conditioning EPSP waveforms activated repetitively indicated that potentiation lasted transiently, 100 ms, during a prolonged release. Possible mechanisms of the potentiation are discussed in light of these new findings.NS-07942 - NINDS NIH HHS; NS-13742 - NINDS NIH HH