Neuromuscular function and transmission

PHYSIOLOGY

Neuromuscular function and transmission Anthony C Wareham

The initiation of contraction of skeletal muscle fibres requires the stimulus of an action potential from a motor neuron. One motor neuron connects to several muscle fibres to form a motor unit. A motor unit in a large limb muscle may contain 1000 fibres connected to one neuron. In contrast, the motor unit of an extra-ocular muscle may be composed of less than 10 muscle fibres. This correlates with the fineness of movement required from a muscle. The smallest movement possible is that produced by the simultaneous activation of all fibres in one motor unit. The axon running to the motor unit is invisible to the naked eye. It may travel as far as 90 cm or more from the spinal cord to its muscle fibres. As it enters a muscle, the axon of the motor neuron divides into fine branches called telodendria, the endings of which each make close approximation with a specialized region of the sarcolemma of one muscle fibre. The specialized region is the motor end-plate, which covers an area of muscle surface of about 3000 µm2. The term neuromuscular junction (NMJ) refers to the axon terminal of the motor neuron plus the motor end-plate. In humans, after maturity, only one NMJ occurs per skeletal muscle fibre. During development, multiple NMJs are formed by several different axons on each muscle fibre but all except one are eliminated during maturation. The role of the NMJ is to ensure that a motor neuron action potential results in the generation of a muscle action potential on every fibre in the motor unit, and ultimately leads to contraction of the whole unit. Unlike CNS synapses, the NMJ does not modify signals but is a simple effector system carrying out the demands of the motor output. All of the physiology and pharmacology of the NMJ is directed towards achieving this aim. The role of the NMJ is so important that everything is done to excess, for example excess transmitter is released, there is an excess of postsynaptic receptors and the postsynaptic potential is several times greater than the minimum required to exceed the action potential threshold. In order for the action potential in the motor axon to activate the muscle fibre by exciting the electrically excitable sarcolemma, several complex steps have to be completed. These include the release of the chemical transmitter acetylcholine (ACh) from the axon terminal by exocytosis, activation by transmitter of nicotinic ACh receptors at the end-plate, generation of an end-plate potential

Anthony C Wareham was Senior Lecturer in Physiological Sciences at the University of Manchester until taking early retirement in April 2001. He obtained his BSc in Zoology and his PhD from Durham University. His research interests focus on the electrophysical properties of skeletal muscle during development, denervation and disease, particularly muscular dystrophy.

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PHYSIOLOGY

Neuromuscular junction

Acetylcholine receptor at the neuromuscular junction

Myelin

Axon

ε subunit

Axonal end bulb

Synaptic vesicles containing acetylcholine

ACh

Dense body

α subunit δ subunit β subunit

Sarcolemma

50 nm synaptic gap

ACh

α subunit Synaptic cleft Agrin

Postsynaptic fold Voltage-gated Ca2+ channels Voltage-gated Na+ channels Acetylcholine receptor Acetylcholinesterase 2+

Rapsyn

Sarcoplasm Central pore allows movement of Na+ and K+

–6

Presynaptic Ca concentration is actively maintained below 10 M at rest. Activation of voltage-gated Ca2+ channels by an action potential invading the axonal end bulb increases Ca2+ concentration and initiates excitation–secretion coupling to release acetylcholine

When activated by two molecules of acetylcholine (ACh) the receptor acts as an ionopore to allow the passage of monovalent cations. The ε subunit is unique to the junctional receptor

1

2

and finally the production of the sarcolemmal action potential. All of these events must be completed rapidly because some skeletal muscle fibres are able to respond to a frequency of motor neuron action potentials greater than 100 Hz for short periods. The process of chemical transmission at the NMJ induces a delay of about 0.5 ms between the time of arrival of an action potential presynaptically and the appearance of a muscle action potential.

steep concentration gradient driving the diffusion of Ca2+ into the axon terminal because of the low intracellular concentration at rest. It is this subsequent influx of Ca2+, increasing intracellular concentration above 10-6 M that is directly responsible for releasing vesicular ACh. Vesicles are associated with specialized presynaptic areas called dense bodies, which occur opposite postsynaptic clefts and appear to be the sites of transmitter release (Figure 1). The vesicular membrane contains a variety of specialized proteins. One, synaptotagmin, has the ability to combine, in the presence of Ca2+, with others including neurexin and SNAP-25 present in the axolemma of the terminal region. When they combine they form relatively large channels that effectively link the interior of the vesicle with the synaptic cleft between presynaptic and postsynaptic structures. The formation of numerous such channels permits the unloading, by simple diffusion, of ACh from the vesicle into the synaptic cleft. Within 1 ms the depolarizing phase of the presynaptic action potential is over, the Ca2+ channels close, Ca2+ influx ceases and the free intracellular Ca2+ concentration is rapidly returned to below 10-6 M. In the absence of Ca2+ the now empty vesicles are recycled by a mechanism which is not yet understood. The way in which the vesicles touch and bind to the presynaptic terminal under the influence of Ca2+ and are then released to be recycled has been called a ‘kiss and run’ mechanism. Every evoked response at the neuromuscular junction requires the synchronous release of acetylcholine (ACh) from up to 100 or more vesicles. The process of vesicle recycling and ACh production therefore has to be very active and efficient to keep up with high stimulation rates. In very active NMJs, released ACh feeds back to populations of ligand-gated ACh receptors on the axon terminals and acts via a second messenger system to increase transmitter production in the terminal.

Presynaptic transmitter release The axon ending is typically enlarged and divided at the NMJ to form end bulbs arranged in the end-plate region like the fingers of a hand. The end bulbs contain ACh packaged into membrane vesicles (visible only under the electron microscope) at a high concentration of 5000–10,000 molecules per vesicle. Each end bulb contains thousands of these vesicles, a proportion of which have to release their contents synchronously to activate the endplate region. This process, called excitation–secretion coupling, requires Ca2+. At rest, the free Ca2+ concentration in presynaptic endings is kept below 10-6 M by the low membrane permeability to Ca2+, sequestration of Ca2+ by mitochondria and an active Na+/Ca2+ exchange pump in the axolemma. Consequently, at rest, most ACh is kept sealed in the presynaptic vesicles. There is a low level of resting release, thought to represent the random collision of vesicles with the presynaptic membrane leading to exocytosis, manifested as very small postsynaptic depolarizations of about 0.5 mV, termed miniature end-plate potentials. Their function, if any, is unknown but at any one end-plate region they occur at intervals of 1/s or less. The arrival of an action potential at the presynaptic region opens voltage-gated Ca2+ channels, which occur at high density in this region of the axolemma. Although the absolute concentrations of Ca2+ on either side of the axolemma are low, there is a

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PHYSIOLOGY

Comparison of the properties of an action potential and an end-plate potential Action potential

End-plate potential

Initiation

By depolarization

By acetylcholine

Rising phase

Selective increase in Na permeability

Simultaneous increase in Na+ and K+ permeabilities

Falling phase

Selective increase in K+ permeability

Passive decline in permeabilities due to acetylcholinesterase action

Potential change

Reverses polarity

Does not exceed –10 mV

Additional

Regenerative ascent followed by refractory period

No regenerative action and no refractory period

Pharmacology

Blocked by tetrodotoxin, not influenced by curare

Blocked by curare, not influenced by tetrodotoxin

+

3

Postsynaptic transmitter action The postsynaptic region of the end-plate is separated from the axon by a 50 nm gap. Typically the end-plate region forms a series of folds that increase the contact area significantly (Figure 1). At a typical NMJ, the synaptic area is about 3000–6000 µm2. This contrasts with about 1 µm2 of contact at a CNS synapse where subsynaptic folding is absent and underlines the differences in their functions. While the postsynaptic response to most stimuli to a CNS synapse is small and could not reach threshold for an action potential in the absence of summation, the postsynaptic response at the NMJ is always large and is always suprathreshold for a muscle action potential. The ACh released presynaptically diffuses rapidly across the synaptic cleft. At the crest of each subsynaptic fold there is a high density of nicotinic ACh receptors, which are held in place by two types of protein, agrin and rapsyn (Figure 2). At each NMJ there are about 3 × 107 ACh receptors, which equates to 104 ACh receptors per µm2. Each ACh receptor is composed of five protein subunits, two of which, the α units, react with ACh. The ACh receptor at the NMJ, which is blocked by curare, is similar to the nicotinic ACh receptor that occurs widely in the nervous system except that one subunit, the γ unit, is replaced in the NMJ receptor by an ε subunit. The receptor present on fetal muscle fibres does not have this ε subunit, therefore it is considered to occur as part of the maturation process of muscle. The extrajunctional receptors that typically appear all over muscle fibres after denervation, leading to denervation hypersensitivity, do not have the ε subunit either. The ACh receptor is also an ionopore and when the active sites of both units are each combined with a molecule of ACh the receptor changes its conformation to open a central pore through the receptor, which behaves as a non-specific cation channel (Figure 2). The receptor spans the postsynaptic membrane completely, which allows cations, mainly Na+ and K+, to move freely across the membrane. The net result is that the combined inward current flowing through all the activated ACh receptors at the endplate results in depolarization of the postsynaptic membrane to about –10 mV. ACh is rapidly broken down by the enzyme acetylcholinesterase, located close to the ACh receptors at the crests of the subsynaptic folds (Figure 1). Therefore, the inward current is transient and is followed by repolarization of the end-plate region to the resting state. Unlike voltage-gated Na+ channels,

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ACh receptors are not refractory and therefore there is no relative or absolute refractory period to the postsynaptic response. The transient depolarization and repolarization, termed the end-plate potential, is responsible for activating the sarcolemma to produce a muscle action potential. Activation of the sarcolemma The end-plate potential is a large depolarization of about 70–80 mV. However, it does not take the membrane potential above 0 mV (i.e. it does not overshoot zero potential as does the action potential). It is also slower than an action potential, lasting 10–15 ms, mainly owing to its slow repolarization. In the case of the end-plate potential, repolarization depends on the chemical breakdown of ACh by acetylcholinesterase to prevent further reactions with ACh receptors and to allow the channels to close. Thus, the properties of an end-plate potential differ in many respects from those of a normal action potential (Figure 3). Nevertheless, because the fast depolarizing phase of the end-plate potential is associated with a relatively intense current it is always strong enough to activate voltage-gated Na+ channels on the sarcolemma. These Na+ channels occur in high concentrations at the base of the subsynaptic folds and around the edges of the end-plate region (Figure 1). The endplate potential activates Na+ channels in the subsynaptic folds and the combination of the end-plate potential and the depolarization resulting from activated Na+ subsynaptic channels, is sufficient to overcome the considerable membrane capacitance of the end-plate region and to excite the population of Na+ channels outside the end-plate. Once these Na+ channels open, an all-or-none action potential is generated and a muscle action potential propagates in all directions from the end-plate across the sarcolemma. This action potential is responsible for activating muscle contraction via the process known as excitation–contraction coupling. 

FURTHER READING Levitan I B, Kaczmarek L K. The neuron. 2nd ed. Oxford: Oxford University Press, 1997. Silverthorn D U. Human physiology. An integrated approach. 2nd ed. Upper Saddle River, NJ: Prentice Hall, 2001.

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