Neuromuscular Junction - Research Methods

Research Methods

del Castillo and Katz used ionophoresis to determine the location and density of nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction. With this technique, a microelectrode is placed inside the motor end-plate of the muscle fiber, and a micropipette filled with acetylcholine (ACh) is placed directly in front of the endplate in the synaptic cleft. A positive voltage is applied to the tip of the micropipette, which causes a burst of positively charged ACh molecules to be released from the pipette. These ligands flow into the space representing the synaptic cleft and bind to AChRs. The intracellular microelectrode monitors the amplitude of the depolarization of the motor end plate in response to ACh binding to nicotinic (ionotropic) receptors. Katz and de Castillo showed that the amplitude of the depolarization (excitatory postsynaptic potential) depended on the proximity of the micropipette releasing the ACh ions to the endplate. The farther the micropipette was from the motor endplate, the smaller the depolarization was in the muscle fiber. This allowed the researchers to determine that the nicotinic receptors were localized to the motor end-plate in high density.

Toxins are also used to determine the location of acetylcholine receptors at the neuromuscular junction. α-bungarotoxin is a toxin found in the snake species Bungarus multicinctus that acts as an ACh antagonist and binds to AChRs irreversibly. By coupling assayable enzymes such as horseradish peroxidase (HRP) or fluorescent proteins to the α-bungarotoxin, AChRs on the motor end plate can be visualized and quantified.