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

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.