A connection between two neurons is referred to as a synapse. A connection between a neuron and a muscle whether it be skeletal, cardiac, or smooth is referred to as a neuromuscular junction. This is a special junction between an axon terminal and a muscle fiber however there is no direct contact between the axon and the muscle. The synapses between spinal motor neurons and skeletal muscle cells occur at specializations called end plates. The synaptic cleft is very narrow being roughly 50-100nm in width (Squire, 2008). All over the synaptic cleft are
Figure 1: Outline of Neuromuscular Junction→displays the motor end plate which is the differentiated version of membrane of muscle fiber which has a number of junctional folds to increase its surface area

numerous acetylcholine receptors which will utilize the chemical messenger released from the synaptic vesicle to open ion channels. The neurotransmitter released at the neuromuscular junction is acetylcholine which binds with the nicotinic cholinergic receptor at the post synaptic membrane. To completely understand the binding of the acetylcholine with the ACh receptors, the synaptic cleft can be viewed in terms of the “saturated disk” model (Adams, 2015). This model suggest the cleft be divided into subsections of where the acetylcholine travels to upon release from the synaptic vesicle. The diameter of the vesicle itself is labeled as the initial center of the numerous circles. This area is where the acetylcholine is initially released as a focused cloud of approximately 10^4 molecules and where it will begin to diffuse outwards (Adams 2015). Throughout its journey it will find and also bind to all available acetylcholine receptors as it makes its way through the vesicle diameter of the cleft. Most of the acetylcholine will make it past this area as there are far fewer binding sites for the acetylcholine than there is
Figure 2: “Saturated Disk” Model for Acetylcholine Transmission→ The Central Circle identifies the vesicle diameter (0.05um), going towards the next radius it encounters the saturated disk diameter (0.7 um) followed by the monoliganded fringe and lastly encountering the virgin receptors which extend to the edge of the synaptic cleft. (Adams 2015)

ACh despite the fact each receptor can be biliganded. As the acetylcholine diffuses outward in high concentrations, the next area will be filled with saturated and biliganded receptors known as the “saturated disk” zone. This zone contains 5000 receptors and it is here that the ACh concentration begins to deplete through the binding of ACh receptors and it will approach a concentration that no longer exceeds the equilibrium dissociation constant of the binding to the acetylcholine receptors. After approaching the monoliganded fringe, the concentration of the acetylcholine becomes similar to that of the dissociation constant. The virgin receptors begin beyond the monoliganded fringe followed by the edges of the synaptic cleft. A mere 10% of the initial acetylcholine makes it to the monliganded fringe while another 20% are taken by acetylcholinesterase (present at 20% of receptor concentration) immediately upon release into the synaptic cleft (vesicle diameter zone).
Acetylcholine has the same binding speed to acetylcholinesterase as it does to an acetylcholine receptor, however the higher concentration of receptors allows it to bind to more acetylcholine. But to activate the receptor, it must bind to 2 acetylcholines as it is biliganded. Paul Adams states that the reason why it takes 2 molecules to bind “is that the free energy change that occurs on binding must exceed the free energy change of the ‘conformational’ change that corresponds to pore opening” (Adams 2015). Essentially, when binding only one ligand, there is less of an energy change than when the ion channel is opening up, so it requires two of the ligands to bind to create enough energy to allow the channel to open. The nearly simultaneous activation of 2000 of these channels causes a slight depolarization of 0.5mV. Hundreds of these slight depolarizations occur after a postsynaptic spike with high reliability is triggered by a spike in nerve terminals. These are referred to as miniature synaptic potentials.
Kinetic Scheme of Acetylcholine Binding
Each of the open channels contributes a current of 2pA summating to a “mini” synaptic current of 4nA. There is a brief (100 usec) rise time and an exponential decay with a 1 msec time constant. Once bound to the receptor, the channels open on average for 1 msec. However, these openings have been seen to have momentary “blinks” of sorts. The overall event is considered a burst of openings momentarily broken up by periods of these “blinks”. The kinetic scheme of the binding of the acetylcholine to the ACh receptor illustrates that the blinks are in fact transitions from the biliganded receptor including the rate of channel opening, which is the only open phase of the binding transitions, and the biliganded receptor phase. This is a special characteristic at the neuromuscular junction as some channels do not reopen after a blink due to the loss of an acetylcholine molecule and the unlikelihood of regaining it. Using the reciprocal of the rate of leaving the blink state (40 1/msec), by opening of the channel or by losing an acetylcholine molecule, gives the mean blink duration of 25 usec. Upon calculation of the rate of binding of ACh to the receptor (10^8 1/Msec), it is visible that this is approaching the upper limit of a diffusion controlled reaction. This demonstrates how unlikely it would be for an ACh molecule to reach a binding site and be unable to bind it. If this rate of binding had been at a significantly lower value, the concentration of ACh molecules would have to be largely increased in the synaptic vesicle during the initial synapse. This also suggests that of the 3500 biliganded receptors from the saturated disk, only around 1700 will open while the other 1700 lose an ACh which is immediately taken up by acetylcholinesterase.
Miniature End Plate Potentials
The opening of the channels will come as two bursts of opening events separated by a blink in most cases while other events could contain anywhere between 1-4 bursts. The distance of each receptor from the synaptic vesicle affects the rise time of the miniature synaptic potential Figure 3: The experimental synaptic transmission at the neuromuscular junction→ B. There is a depolarization of the motor end plate at an end plate potential with a graded potential whose magnitude depends on the amount and duration of ACh at the end plate. When this EPP reaches its threshold, it causes an action potential in the adjacent muscle membrane. C. The random movement of vesciles at rest spontaneously releases ACh through exocytosis which causes a small depolarization of the membrane known as the miniature end plate potentials. These are no followed by an action potential. D. If the neuromuscular junction is at a low concentration of calcium ions, the stimulation of the motor neurons causes the amplitude of the EPP to be similar to that of a MEPP. caused by the bursts as the ones closer to the vesicle are bound by their ACh ligand sooner than the receptors at the monoliganded fringe. The transient depolarization of the postsynaptic muscle fiber causes a change in membrane potential known as the end plate potential (Purves 2004). An end plate potential response is created when there is stimulation of a motor neuron that is normally above threshold thus causing an action potential in the post synaptic cell. However, in the absence of presynaptic stimulation there are still spontaneously occurring depolarizations known as miniature end plate potentials caused by the release of ACh from the resting synaptic vesicle which go through this process (Purves 2004).
Effect of Curare, Open Channel Blockers, and Anticholinesterase on MEPPs There are numerous drugs that can affect the ability to create a miniature end plate potential or to the degree of which the MEPP is present. Curare for example is a potent antagonist that is incapable of leaving the ACh receptor during the actions of a bound ACh causing essentially a non-competitive situation. By taking up these binding sites for itself, it is taking away the available number of binding sites for the ACh. Although this does reduce the amplitude of the mini, it is not by much as the ACh carries further down the “saturated disk” model towards the monoliganded fringe with some molecules being intercepted by the available acetylcolinesterase. The impact of the curare comes through the wasted binding of available ACh to its unbound receptor with the bound curare. Seeing as with the curare bound the receptor cannot function properly, binding the available ACh reduces the concentration available to those receptors to which ACh can properly activate in their biliganded form. The effect of an open channel blocker would be similar to that of the curare drug but to a greater extent. Whereas the curare drug was affecting the number of available receptors to which ACh can effectively bind to, the open channel blocker is binding inside the pore of the ion channel and blocks the flow of the ions. This ion flow is the direct cause of the MEPP so blocking it effectively negates the mini in its entirety. Although the open channel blocker also may just cause the ACh to flow further down the “saturated disk”, the imprint left by the open channel blocker would be significantly greater than that of the curare in a negative way. Anticholinesterases included a variety of reversible and irreversible drugs along with two types of nerve gas. As opposed to the previous two drugs that inhibit the amplitude of the MEPP, these drugs slightly increase the size of the mini and prolong decay time by a factor of two (Adams 2015). They accomplish this by allowing 20% more ACh to initially survive because instead of just leaving the receptor and being taken up by acetylcolinesterase, it diffuses a bit and then rebinds causing the receptor to once again open the channel. After this diffusion however, it soon reaches the edge of the synaptic cleft that causes the ACh to escape resulting in only the doubling of the decay time. This illustrates the effectiveness of using diffusion to empty the synaptic cleft of the transmitter to make the mini faster.