‘Calcium bombs' as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions

Excitotoxicity is thought to be an important factor in the onset and progression of amyotrophic lateral sclerosis (ALS). Evidence from human and animal studies also indicates that early signs of ALS include degeneration of motor nerve terminals at neuromuscular junctions (NMJs), before degeneration of motor neuron cell bodies. Here we used a model of excitotoxicity at NMJs in isolated mouse muscle, utilizing the organophosphorus (OP) compound omethoate, which inhibits acetylcholinesterase activity. Acute exposure to omethoate (100 μM) induced prolonged motor endplate contractures in response to brief tetanic nerve stimulation at 20–50 Hz. In some muscle fibers, Fluo-4 fluorescence showed association of these contractures with explosive increases in Ca(2+) (“calcium bombs”) localized to motor endplates. Calcium bombs were strongly and selectively mitigated by increasing Mg(2+) concentration in the bathing medium from 1 to 5 mM. Overnight culture of nerve-muscle preparations from Wld(S) mice in omethoate or other OP insecticide components and their metabolites (dimethoate, cyclohexanone, and cyclohexanol) induced degeneration of NMJs. This degeneration was also strongly mitigated by increasing [Mg(2+)] from 1 to 5 mM. Thus, equivalent increases in extracellular [Mg(2+)] mitigated both post-synaptic calcium bombs and degeneration of NMJs. The data support a link between Ca(2+) and excitotoxicity at NMJs and suggest that elevating extracellular [Mg(2+)] could be an effective intervention in treatment of synaptic pathology induced by excitotoxic triggers

Donepezil inhibits neuromuscular junctional acetylcholinesterase and enhances synaptic transmission and function in isolated skeletal muscle

BACKGROUND AND PURPOSE: Donepezil, a piperidine inhibitor of acetylcholinesterase (AChE) prescribed for treatment of Alzheimer's disease, has adverse neuromuscular effects in humans, including requirement for higher concentrations of non‐depolarising neuromuscular blockers during surgery. Here, we examined the effects of donepezil on synaptic transmission at neuromuscular junctions (NMJs) in isolated nerve‐muscle preparations from mice. EXPERIMENTAL APPROACH: We measured effects of therapeutic concentrations of donepezil (10 nM to 1 μM) on AChE enzymic activity, muscle force responses to repetitive stimulation, and spontaneous and evoked endplate potentials (EPPs) recorded intracellularly from flexor digitorum brevis muscles from CD01 or C57BlWld(S) mice. KEY RESULTS: Donepezil inhibited muscle AChE with an approximate IC(50) of 30 nM. Tetanic stimulation in sub‐micromolar concentrations of donepezil prolonged post‐tetanic muscle contractions. Preliminary Fluo4‐imaging indicated an association of these contractions with an increase and slow decay of intracellular Ca(2+) transients at motor endplates. Donepezil prolonged spontaneous miniature EPP (MEPP) decay time constants by about 65% and extended evoked EPP duration almost threefold. The mean frequency of spontaneous MEPPs was unaffected but the incidence of ‘giant’ MEPPs (gMEPPs), some exceeding 10 mV in amplitude, was increased. Neither mean MEPP amplitude (excluding gMEPPs), mean EPP amplitude, quantal content or synaptic depression during repetitive stimulation were significantly altered by concentrations of donepezil up to 1 μM. CONCLUSION AND IMPLICATIONS: Adverse neuromuscular signs associated with donepezil therapy, including relative insensitivity to neuromuscular blockers, are probably due to inhibition of AChE at NMJs, prolonging the action of ACh on postsynaptic nicotinic acetylcholine receptors but without substantively impairing evoked ACh release

“Calcium bombs” as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions

Excitotoxicity is thought to be an important factor in the onset and progression of amyotrophic lateral sclerosis (ALS). Evidence from human and animal studies also indicates that early signs of ALS include degeneration of motor nerve terminals at neuromuscular junctions (NMJs), before degeneration of motor neuron cell bodies. Here we used a model of excitotoxicity at NMJs in isolated mouse muscle, utilizing the organophosphorus (OP) compound omethoate, which inhibits acetylcholinesterase activity. Acute exposure to omethoate (100 μM) induced prolonged motor endplate contractures in response to brief tetanic nerve stimulation at 20–50 Hz. In some muscle fibers, Fluo-4 fluorescence showed association of these contractures with explosive increases in Ca2+ (“calcium bombs”) localized to motor endplates. Calcium bombs were strongly and selectively mitigated by increasing Mg2+ concentration in the bathing medium from 1 to 5 mM. Overnight culture of nerve-muscle preparations from WldS mice in omethoate or other OP insecticide components and their metabolites (dimethoate, cyclohexanone, and cyclohexanol) induced degeneration of NMJs. This degeneration was also strongly mitigated by increasing [Mg2+] from 1 to 5 mM. Thus, equivalent increases in extracellular [Mg2+] mitigated both post-synaptic calcium bombs and degeneration of NMJs. The data support a link between Ca2+ and excitotoxicity at NMJs and suggest that elevating extracellular [Mg2+] could be an effective intervention in treatment of synaptic pathology induced by excitotoxic triggers

The progressive nature of Wallerian degeneration in wild-type and slow Wallerian degeneration (Wld(S)) nerves

BACKGROUND: The progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration (Wld(S)) mutant mice. RESULTS: In wild-type nerves, we directly observed partially fragmented axons (average 5.3%) among a majority of fully intact or degenerated axons 37–42 h after transection and 40–44 h after crush injury. Axons exist in this state only transiently, probably for less than one hour. Surprisingly, axons degenerated anterogradely after transection but retrogradely after a crush, but in both cases a sharp boundary separated intact and fragmented regions of individual axons, indicating that Wallerian degeneration progresses as a wave sequentially affecting adjacent regions of the axon. In contrast, most or all Wld(S )axons were partially fragmented 15–25 days after nerve lesion, Wld(S )axons degenerated anterogradely independent of lesion type, and signs of degeneration increased gradually along the nerve instead of abruptly. Furthermore, the first signs of degeneration were short constrictions, not complete breaks. CONCLUSIONS: We conclude that Wallerian degeneration progresses rapidly along individual wild-type axons after a heterogeneous latent phase. The speed of progression and its ability to travel in either direction challenges earlier models in which clearance of trophic or regulatory factors by axonal transport triggers degeneration. Wld(S )axons, once they finally degenerate, do so by a fundamentally different mechanism, indicated by differences in the rate, direction and abruptness of progression, and by different early morphological signs of degeneration. These observations suggest that Wld(S )axons undergo a slow anterograde decay as axonal components are gradually depleted, and do not simply follow the degeneration pathway of wild-type axons at a slower rate

Effect of Limb Lengthening on Internodal Length and Conduction Velocity of Peripheral Nerve

The influences of axon diameter, myelin thickness, and internodal length on the velocity of conduction of peripheral nerve action potentials are unclear. Previous studies have demonstrated a strong dependence of conduction velocity on internodal length. However, a theoretical analysis has suggested that this relationship may be lost above a nodal separation of ∼0.6 mm. Here we measured nerve conduction velocities in a rabbit model of limb lengthening that produced compensatory increases in peripheral nerve growth. Divided tibial bones in one hindlimb were gradually lengthened at 0.7 mm per day using an external frame attached to the bone. This was associated with a significant increase (33%) of internodal length (0.95–1.3 mm) in axons of the tibial nerve that varied in proportion to the mechanical strain in the nerve of the lengthened limb. Axonal diameter, myelin thickness, and g-ratios were not significantly altered by limb lengthening. Despite the substantial increase in internodal length, no significant change was detected in conduction velocity (∼43 m/s) measured either in vivo or in isolated tibial nerves. The results demonstrate that the internode remains plastic in the adult but that increases in internodal length of myelinated adult nerve axons do not result in either deficiency or proportionate increases in their conduction velocity and support the view that the internodal lengths of nerves reach a plateau beyond which their conduction velocities are no longer sensitive to increases in internodal length

Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wlds) gene

<p>Abstract</p> <p>Background</p> <p>Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (<it>Wld</it>^<it>s</it>) have a unique neuroprotective phenotype, where axonal and synaptic compartments of neurons are protected from degeneration following a wide variety of physical, toxic and inherited disease-inducing stimuli. This remarkable phenotype has been shown to delay onset and progression in several mouse models of neurodegenerative disease, suggesting that <it>Wld</it>^<it>s</it>-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of <it>Wld</it>^{<it>s </it>}mice with mouse models of neurodegenerative diseases is used increasingly to understand the roles of axon and synapse degeneration in disease. However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation. Since the <it>Wld</it>^{<it>s </it>}mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant <it>Wld</it>^{<it>s </it>}alleles is using copy number. Current approaches to genotype <it>Wld</it>^{<it>s </it>}mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR).</p> <p>Results</p> <p>We have developed a rapid, robust and efficient genotyping method for <it>Wld</it>^{<it>s </it>}using QPCR. This approach differentiates, based on copy number, homozygous and heterozygous <it>Wld</it>^{<it>s </it>}mice from wild-type mice and each other. We show that this approach can be used to genotype mice carrying the spontaneous <it>Wld</it>^{<it>s </it>}mutation as well as animals expressing the <it>Wld</it>^{<it>s </it>}transgene.</p> <p>Conclusion</p> <p>We have developed a QPCR genotyping method that permits rapid and effective genotyping of <it>Wld</it>^{<it>s </it>}copy number. This technique will be of particular benefit in studies where <it>Wld</it>^{<it>s </it>}mice are cross-bred with other mouse models of neurodegenerative disease in order to understand the neuroprotective processes conferred by the <it>Wld</it>^{<it>s </it>}mutation.</p

Segmentation of the mouse fourth deep lumbrical muscle connectome reveals concentric organization of motor units

Connectomic analysis of the nervous system aims to discover and establish principles that underpin normal and abnormal neural connectivity and function. Here we performed image analysis of motor unit connectivity in the fourth deep lumbrical muscle (4DL) of mice, using transgenic expression of fluorescent protein in motor neurones as a morphological reporter. We developed a method that accelerated segmentation of confocal image projections of 4DL motor units, by applying high resolution (63×, 1.4 NA objective) imaging or deconvolution only where either proved necessary, in order to resolve axon crossings that produced ambiguities in the correct assignment of axon terminals to identified motor units imaged at lower optical resolution (40×, 1.3 NA). The 4DL muscles contained between 4 and 9 motor units and motor unit sizes ranged in distribution from 3 to 111 motor nerve terminals per unit. Several structural properties of the motor units were consistent with those reported in other muscles, including suboptimal wiring length and distribution of motor unit size. Surprisingly, however, small motor units were confined to a region of the muscle near the nerve entry point, whereas their larger counterparts were progressively more widely dispersed, suggesting a previously unrecognised form of segregated motor innervation in this muscle. We also found small but significant differences in variance of motor endplate length in motor units, which correlated weakly with their motor unit size. Thus, our connectomic analysis has revealed a pattern of concentric innervation that may perhaps also exist in other, cylindrical muscles that have not previously been thought to show segregated motor unit organisation. This organisation may be the outcome of competition during postnatal development based on intrinsic neuronal differences in synaptic size or synaptic strength that generates a territorial hierarchy in motor unit size and disposition