Neurophysiology Of Neuromuscular Transmission 11 Anatomy of the Terminal End Plate Region

The physiology of the neuromuscular junction (NMJ) derives from the anatomy of the terminal axon and motor end plate, also referred to as the presynaptic and postsynaptic regions (Fig. 1). Motor nerve fibers end in an arborization of fine intramuscular twigs ending at the terminal bouton. The motor twigs are myelinated until the very terminus, with a Schwann cell covering all but the synaptic interface. The terminal axon is separated from the motor end-plate region of the muscle fiber by an extracellular space approx 70-nm wide, called the primary synaptic cleft. On the far side of this cleft, in the motor end-plate region, are a series of invaginations of the muscle membrane, which are called the secondary synaptic clefts. These junc-tional folds are unique to NMJs.

The terminal axon membrane contains voltage-gated calcium channels (VGCC) of the P/Q type, which are arranged in active zones in a semigeometric array. Within the terminal axon, synaptic vesicles containing acetylcholine collect at the active zones (Fig. 1). Acetylcholine is synthesized from choline and acetate in the cytoplasm by the enzyme choline acetyltrans-ferase. Acetylcholine enters the synaptic vesicles using the vesicular acetylcholine transporter, which also exports the acetylcholine during exocytosis. Choline is actively transported by an energy-dependent reuptake mechanism.

From: The Clinical Neurophysiology Primer Edited by: A. S. Blum and S. B. Rutkove © Humana Press Inc., Totowa, NJ

Fig. 1. A cartoon of a neuromuscular junction (NMJ). The synaptic vesicles contain acetylcholine. The active zones contain voltage-gated calcium channels, and part of the apparatus necessary for exocytosis. The basal lamina in the primary synaptic cleft contains acetylcholinesterase. Voltage-gated sodium channels necessary for propagation of the action potential generated at the neuromuscular junction are in the muscle fiber membrane, including the depths of the secondary synaptic clefts.

Fig. 1. A cartoon of a neuromuscular junction (NMJ). The synaptic vesicles contain acetylcholine. The active zones contain voltage-gated calcium channels, and part of the apparatus necessary for exocytosis. The basal lamina in the primary synaptic cleft contains acetylcholinesterase. Voltage-gated sodium channels necessary for propagation of the action potential generated at the neuromuscular junction are in the muscle fiber membrane, including the depths of the secondary synaptic clefts.

The primary synaptic cleft is divided by a basal lamina, a loosely organized, porous boundary containing acetylcholinesterase, which catabolizes acetylcholine as it diffuses across the primary synaptic cleft. Approximately 50% of the acetylcholine released from the presynap-tic membrane is catabolized before it reaches the postsynaptic membrane.

The most important components of the postsynaptic membrane are the acetylcholine receptors. These receptors are of the nicotinic type, and contain a ligand-activated cation channel. At the base of the secondary clefts are voltage-gated sodium channels, essential for transmitting any action potential along the muscle membrane. Acetylcholine receptors are manufactured by membrane-bound ribosomes in the cytoplasm and then inserted in the post-synaptic membrane. There are approx 10,000 receptors per square micrometer at the terminal and upper areas of the secondary clefts. Each adult acetylcholine receptor (Fig. 2) is a tetramer containing 2 a-subunits, and one each of the P-, 5-, and e-subunits. Fetal acetylcholine receptors substitute a y-subunit for the e-subunit. Of note, ocular muscles, which differ from other skeletal muscles in a number of ways, have an enriched population of fetal-type receptors. The half-life for adult acetylcholine receptors is 8 to 11 d. Ligand sites for acetylcholine are located on each of the a-subunits, and both most be engaged to activate the receptor channel. The main immunogenic region (MIR) is also located on each a-subunit, but separate from the ligand-binding site. After acetylcholine dissociates from the receptor, it is catabolized by acetylcholinesterase. Application of agents that inhibit the activity of acetyl-cholinesterase will prolong the activity of acetylcholine at the postsynaptic receptors.

Fig. 2. A cartoon of the acetylcholine receptor, showing a transverse section, a top view, and a deconstructed view of the a-subunit. MIR, the main immunogenic region of the receptor; ACh, the ligand-binding site for acetylcholine. From Engel AG, 1999 with permission.

1.2. Presynaptic Physiology of Neuromuscular Transmission

The motor nerve action potential generated in the cell body is transmitted to the terminal axon membrane. As it traverses the presynaptic membrane, it activates the VGCCs, which open, allowing the movement of calcium into the terminal axon. The influx of calcium activates calmodulin-dependent protein kinase II, which binds with synapsin I, resulting in the docking of the synaptic vesicle with the terminal membrane. Release of the acetylcholine by exocytosis involves a complicated interaction of many proteins, including synaptobrevin, syntaxin, and SNAP-25. The synaptic membrane remains part of the axonal membrane and is recycled.

Acetylcholine-containing vesicles are organized into at least two pools. The immediately available pool is the smallest and consists of those vesicles actually lined up at the active zones for release. During a series of axonal depolarizations, such as when attempting to contract a muscle, the immediately available store will become depleted, and fewer synaptic vesicles will be released. The second pool is considerably larger than the immediately available store and is called the mobilization or reserve pool. During the course of a few seconds, it will replenish the immediately available pool.

The influx of calcium via the VGCCs is by passive diffusion. In contrast, calcium egress is by active transport and takes longer. During a sustained series of action potentials, the calcium concentration in the terminal axon will continue to increase and facilitate release of synaptic vesicles, partially countering the effects of depletion of the immediately available pool.

1.3. Postsynaptic Electrophysiology of Neuromuscular Transmission

The acetylcholine molecules passively diffuse across the primary synaptic cleft. Those that escape catabolism bind with acetylcholine receptors on the postsynaptic membrane. The activated receptors undergo a conformational change, allowing sodium to enter the cell and potassium to leave. This causes a small depolarization of the immediately adjacent muscle membrane. Release of single synaptic vesicles from the presynaptic membrane causes a reproducible level of depolarization, approx 1 mV, called a miniature end-plate potential (MEPP). This is the basis for the quantal theory of neuromuscular transmission (NMT). Because many synaptic vesicles are released with each depolarization of the terminal axon membrane, many MEPPs are produced. The MEPPs summate temporally and spatially to form an end-plate potential (EPP). If this EPP is sufficient to depolarize the membrane to threshold, an action potential is generated, which is then propagated along the muscle membrane by voltage-gated sodium channels in the sarcolemmal membrane, eventually resulting in muscle fiber contraction, the defining purpose of NMT.

An important concept is that of the safety factor. The EPP normally produced after each action potential activation of the presynaptic membrane is approximately four times that necessary to reach threshold. It is calculated by the formula m = n x p, where m is the quantal of the EPP, and n is the percentage of vesicles released from the immediately available pool, which is determined by p, the probability of release. This excessive EPP derives from the abundance of acetylcholine released presynaptically and of acetylcholine receptors postsynapti-cally. Thus, even with the normally encountered depletion of the immediately available pool of acetylcholine-containing synaptic vesicles during a rapid train of motor nerve action potentials, the safety factor always provides successful NMT in the healthy NMJ. When healthy muscle fatigues, it is not because of failure of NMT but, rather, because of muscle metabolic issues, such as lactic acid buildup and failure of energy pathways. However, in the abnormal NMJ, if either the amount of acetylcholine released or the number of acetylcholine receptors declines, the safety factor will begin to fall as the size of the EPP falls. If the EPP safety factor falls below 1, that is, that needed to just reach threshold for action potential generation, then NMT will fail. This is the basis for fatigable weakness in disorders of postsynaptic NMT, such as myasthenia gravis (MG). As the number of receptors falls, the EPP also decreases. In many NMJs, the safety factor will fall below 1, and that muscle fiber will be, in essence, denervated. In many other fibers, the safety factor will hover just at or above 1, and initially, there will be successful NMT. However, during a train of motor nerve action potentials, the normal depletion of the immediately available store of acetylcholine-containing synaptic vesicles will result in a decrease in acetylcholine reaching the decreased number of receptors on the postsynaptic membrane. This will cause the safety factor to fall below 1, and NMT will fail, the muscle fiber will not contract, and the muscle will weaken.

The safety factor is also important in understanding incremental strength during sustained effort in disorders of presynaptic NMJ. In these disorders, there are abundant postsynaptic acetylcholine receptors, but release of the acetylcholine-containing synaptic vesicles is impaired. In a weak muscle, the fibers do not contract because the lack of acetylcholine release causes the safety factor to fall below 1, with resultant failure of NMT. In the case of Lambert-Eaton syndrome (LEMS), this is caused by loss of VGCCs and decreased influx of calcium to start the process of synaptic vesicle release. In botulism, it is impairment of the release of vesicles themselves. In both cases, with rapid repetitive activation of the presynap-tic membrane, such as during strong effort, calcium will build up in the terminal axon, potentially increasing to levels that may approximate that observed during normal presynaptic function. At this point, acetylcholine release will improve toward normal levels, as will the EPP. As the safety factor increases and surpasses 1, NMT will successfully resume, though it will quickly fail as soon as the rapid train of motor action potentials ceases. This effect is much more evident in LEMS than botulism, but, if observed, is diagnostic of a presynaptic disorder.

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