Microelectrode-based approaches for studying cholinergic neuromuscular transmission

Despite more than a century of research into the neuromuscular junction, it still remains a widely used experimental model for studying molecular mechanisms of synaptic transmission in various physiological and pathological conditions, as well as for testing different pharmacological agents. One of the generally accepted methodologies for achieving these goals is electrophysiological recordings using microelectrode techniques. Application of extracellular microelectrodes makes it possible to precisely evaluate the parameters of presynaptic action potential and its coupling with evoked exocytotic event. The use of intracellular microelectrodes allows the measurements of a resting membrane potential, amplitude-temporal parameters of evoked and spontaneous postsynaptic potentials, which depend on both neurotransmitter release and the postsynaptic membrane sensitivity to neurotransmitter. A voltage-clamp technique with two intracellular microelectrodes is the best option to access postsynaptic currents for characterization of both quantal content and functioning receptor-channel complexes within the end-plate region. To apply all of the above microelectrode approaches under conditions of motor nerve stimulation, an immobilization of neuromuscular preparations is necessary. Skeletal muscle paralysis can be accomplished by decreasing neurotransmitter release (via lowering extracellular Ca²⁺ and elevating external Mg²⁺ levels), a partial blockage of nicotinic acetylcholine receptors, a selective inhibition of skeletal muscle-specific voltage-gated sodium (Na_v1.4) channels, a reduction of membrane potential below the excitation threshold (due to a transverse dissection of the muscle fibers), uncoupling excitation-contraction (by hyperosmotic shock), and inhibiting skeletal muscle myosin ATPase. All of the listed methods have their advantages and disadvantages, which are discussed in this review.