<P> These findings were demonstrated in part by mouse "knockout" studies . In mice which are deficient for either agrin or MuSK, the neuromuscular junction does not form . Further, mice deficient in Dok - 7 did not form either acetylcholine receptor clusters or neuromuscular synapses . </P> <P> The development of neuromuscular junctions is mostly studied in model organisms, such as rodents . In addition, in 2015 an all - human neuromuscular junction has been created in vitro using human embryonic stem cells and somatic muscle stem cells . In this model presynaptic motor neurons are activated by optogenetics and in response synaptically connected muscle fibers twitch upon light stimulation . </P> <P> José del Castillo and Bernard Katz used ionophoresis to determine the location and density of nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction . With this technique, a microelectrode was placed inside the motor endplate 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 was applied to the tip of the micropipette, which caused a burst of positively charged ACh molecules to be released from the pipette . These ligands flowed into the space representing the synaptic cleft and bound to AChRs . The intracellular microelectrode monitored the amplitude of the depolarization of the motor endplate in response to ACh binding to nicotinic (ionotropic) receptors . Katz and del 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 endplate in high density . </P> <P> 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 such as green fluorescent protein (GFP) to the α - bungarotoxin, AChRs can be visualized and quantified . </P>

At the vertebrate neuromuscular junction how many nerves innervate each muscle fibre