No abstract

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.

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 nondepolarising 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 posttetanic 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.