Impaired neuromuscular function by conjoint actions of organophosphorus insecticide metabolites omethoate and cyclohexanol with implications for treatment of respiratory failure, Clinical Toxicology,

Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Some survivors develop a severe, acute or delayed myasthenic syndrome. In animal models, similar myasthenia has been associated with increasing plasma concentration of one insecticide solvent metabolite, cyclohexanol. We investigated possible mechanisms using voltage and current recordings from mouse neuromuscular junctions (NMJs) and transfected human cell lines. Cyclohexanol (10–25 mM) reduced endplate potential (EPP) amplitudes by 10–40% and enhanced depression during repetitive (2–20 Hz) stimulation by up to 60%. EPP decay was prolonged more than twofold. Miniature EPPs were attenuated by more than 50%. Cyclohexanol inhibited whole‐cell currents recorded from CN21 cells expressing human postjunctional acetylcholine receptors (hnAChR) with an IC50 of 3.74 mM. Cyclohexanol (10–20 mM) also caused prolonged episodes of reduced‐current, multi‐channel bursting in outside‐out patch recordings from hnAChRs expressed in transfected HEK293T cells, reducing charge transfer by more than 50%. Molecular modelling indicated cyclohexanol binding (−6 kcal/mol) to a previously identified alcohol binding site on nicotinic AChR α‐subunits. Cyclohexanol also increased quantal content of evoked transmitter release by ∼50%. In perineurial recordings, cyclohexanol selectively inhibited presynaptic K+ currents. Modelling indicated cyclohexanol binding (−3.8 kcal/mol) to voltage‐sensitive K+ channels at the same site as tetraethylammonium (TEA). TEA (10 mM) blocked K+ channels more effectively than cyclohexanol but EPPs were more prolonged in 20 mM cyclohexanol. The results explain the pattern of neuromuscular dysfunction following ingestion of organophosphorus insecticides containing cyclohexanol precursors and suggest that cyclohexanol may facilitate investigation of mechanisms regulating synaptic strength at NMJs. Key points Intentional ingestion of agricultural organophosphorus insecticides is a significant public health issue in rural Asia, causing thousands of deaths annually. Survivors may develop a severe myasthenic syndrome or paralysis, associated with increased plasma levels of cyclohexanol, an insecticide solvent metabolite. Analysis of synaptic transmission at neuromuscular junctions in isolated mouse skeletal muscle, using isometric tension recording and microelectrode recording of endplate voltages and currents, showed that cyclohexanol reduced postsynaptic sensitivity to acetylcholine neurotransmitter (reduced quantal size) while simultaneously enhancing evoked transmitter release (increased quantal content). Patch recording from transfected cell lines, together with molecular modelling, indicated that cyclohexanol causes selective, allosteric antagonism of postsynaptic nicotinic acetylcholine receptors and block of presynaptic K+‐channel function. The data provide insight into the cellular and molecular mechanisms of neuromuscular weakness following int...

We present a model of excitability in larval Drosophila muscles. Our model was initially based on modified Hodgkin-Huxley equations, adapted to represent variable, regenerative depolarisations (action potentials) we have occasionally observed in intracellular recordings and that can be triggered by excitatory junction potentials at neuromuscular synapses. We modified several kinetic equations describing voltage sensitive Ca 2+ and K + ionic currents, previously used to predict excitability in muscle cells of the mammalian cardiac atrioventricular node. The resulting nonlinear differential equations had multiple unknown parameters. Thus, to fit the model to experimental observations of variable excitability, we developed a new implementation of particle swarm optimisation. This GPU-based implementation allows us to adopt an ensemble model approach in which each experimental observation is used to find a plausible parameterisation, resulting in a set of models accounting for cell-to-cell variability of muscle excitability in Drosophila larvae, and with potential applications to population-based modeling of other excitable cell types.