Neuromuscular Junction

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Chapter 22 Neuromuscular Junction

Skeletal Muscle

The most intensively studied effector endings are those that innervate muscle, particularly skeletal muscle. All neuromuscular (myoneural) junctions are axon terminals of somatic motor neurones. They are specialized for the release of neurotransmitter onto the sarcolemma of skeletal muscle fibres, causing a change in their electrical state that leads to contraction. Each axon branches near its terminal and subsequently innervates from several to hundreds of muscle fibres, depending on the precision of motor control required. The detailed structure of a motor terminal varies with the type of muscle innervated. Two major endings are recognized: those typical of extrafusal muscle fibres, and endings on the intrafusal fibres of neuromuscular spindles. In the former, each axon terminal usually ends midway along a muscle fibre in a discoidal motor end-plate (Figs 22.1–22.3). This type usually initiates action potentials, which are rapidly conducted to all parts of the muscle fibre. In the latter, the axon has numerous subsidiary branches that form a cluster of small expansions extending along the muscle fibre. In the absence of propagated muscle excitation, these excite the fibre at several points. Both types are associated with a specialized receptive region of the muscle fibre, the sole plate, where a number of muscle cell nuclei are grouped within the granular sarcoplasm.

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Fig. 22.1 Neuromuscular junction in a whole-mount preparation of teased skeletal muscle fibres (pale, faintly striated, diagonally oriented structures). The terminal part of the axon (silver-stained, brown) branches to form motor end-plates on adjacent muscle fibres. The sole plate recesses in the sarcolemma, into which the motor end-plates fit, are demonstrated by the presence of AChE (shown by enzyme histochemistry, blue).

(Courtesy of Dr. Norman Gregson, Division of Neurology, GKT School of Medicine, London. Photograph by Sarah-Jane Smith.)

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Fig. 22.2 Neuromuscular junction. Note the axonal motor end-plate and the deeply infolded sarcolemma.

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Fig. 22.3 Neuromuscular junction in skeletal muscle. The expanded motor end-plate of the axon is filled with vesicles containing synaptic transmitter (ACh; above), and the deep infoldings of the sarcolemmal sole plate (below) form subsynaptic gutters.

(Courtesy of D. N. Landon, Institute of Neurology, University College London.)

The sole plate contains numerous mitochondria, endoplasmic reticulum and Golgi complexes (see Figs 22.2, 22.3). The neuronal terminal branches are plugged into shallow grooves in the surface of the sole plate (primary clefts), from which numerous pleats extend for a short distance into the underlying sarcoplasm (secondary clefts). The axon terminal contains mitochondria and many clear 60-nm spherical vesicles, similar to those in presynaptic boutons, clustered over the zone of membrane apposition. The motor terminal is ensheathed by Schwann cells whose cytoplasmic projections extend into the synaptic cleft. The plasma membranes of the nerve terminal and the muscle cell are separated by a 30- to 50-nm gap, with a basal lamina interposed. The basal lamina follows the surface folding of the sole plate membrane into the secondary clefts. It contains specialized components, including specific isoforms of type IV collagen and laminin and agrin, a heparan sulphate proteoglycan. Endings of fast and slow twitch muscle fibres differ in detail: the sarcolemmal grooves are deeper, and the presynaptic vesicles more numerous, in the fast fibres.

Junctions with skeletal muscle are cholinergic, and the release of acetylcholine (ACh) changes the ionic permeability of the muscle fibre. Clustering of ACh receptors at the neuromuscular junction depends in part on the presence of agrin, synthesized by the motor neurone. Agrin affects muscle cytoskeletal attachments to the ACh receptor cytoplasmic domain and prevents their lateral diffusion out of the junction. When the depolarization of the sarcolemma reaches a particular threshold, it initiates an all-or-none action potential in the sarcolemma, which is then propagated rapidly over the whole cell surface and also deep within the fibre via the invaginations (T-tubules) of the sarcolemma, causing contraction. The amount of ACh released by the arrival of a single nerve impulse is sufficient to trigger an action potential. However, because ACh is very rapidly hydrolysed by the enzyme acetylcholinesterase (AChE), present at the sarcolemmal surface of the sole plate, a single nerve impulse gives rise to only one muscle action potential—that is, there is a one-to-one relationship between neural and muscle action potentials. Thus, the contraction of a muscle fibre is controlled by the firing frequency of its motor neurone. Neuromuscular junctions are partially blocked by high concentrations of lactic acid, as in some types of muscle fatigue.

Conduction of the Nervous Impulse

All cells generate a steady electrochemical potential across their plasma membranes (a membrane potential) because of the different ionic concentrations inside and outside the cell (Fig. 22.4). Neurones use minute fluctuations in this potential to receive, conduct and transmit information across their surfaces.

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Fig. 22.4 Types of changes in electrical potential that can be recorded across the cell membrane of a motor neurone at the points indicated by the arrows. Excitatory and inhibitory synapses on the surfaces of the dendrites and somata cause local graded changes of potential that summate at the axon hillock and may initiate a series of all-or-none action potentials, which in turn are conducted along the axon to the effector terminals.

The membrane potential of a neurone, known as the resting potential, is similar to that of non-excitable cells. In most neurones it is approximately 80 mV, being negative inside. The entry into neurones of sodium or, in some sites, calcium ions causes depolarization of the cell, whereas an increased chloride influx or an increased potassium efflux results in hyperpolarization. Plasma membrane permeability to these ions is altered by the opening or closing of ion-specific transmembrane channels, triggered by chemical or electrical stimuli. Chemically triggered ionic fluxes occur at synapses and may be either direct, whereby the chemical agent (neurotransmitter) binds to the channel itself to cause it to open, or indirect, whereby the neurotransmitter is bound by a transmembrane receptor molecule that is not itself a channel but that activates a complex second messenger system within the cell to open separate transmembrane channels. Electrically induced changes in membrane potential depend on the presence of voltage-sensitive ion channels that, when the transmembrane potential reaches a critical level, open to allow the influx or efflux of specific ions. In all cases, the channels remain open only transiently, and the numbers that open and close determine the total flux of ions across the membrane.

The types and concentrations of transmembrane channels and related proteins, and therefore the electrical activity of the membranes, vary in different parts of the cell. Dendrites and neuronal somata depend mainly on neurotransmitter action and show graded potentials, whereas axons have voltage-gated channels that give rise to action potentials.

In graded potentials, a flow of current from or into adjacent areas of the cell occurs when a synapse is activated, and this contributes to the total degree of polarization of the membrane covering the cell body. However, the influence of an individual synapse on neighbouring regions decreases with distance, so that, for instance, synapses on the distal tips of dendrites may, on their own, have relatively little effect. The electrical state of a neurone therefore depends on many factors, including the number and position of thousands of excitatory and inhibitory synapses, their degree of activation, the branching pattern of the dendritic tree and the geometry of the cell body. The target of these integrated factors is a small part of the neurone surface, the axon hillock, where voltage-sensitive channels are concentrated (unlike the dendrites or somata). The axon hillock is the site where action potentials are generated before being conducted along the axon.

CASE 1 Botulism

A 22-year-old woman presents in the early morning with double vision, slurred speech and swallowing difficulties, preceded the night before by dry mouth, blurred vision, abdominal cramping and constipation. During the next several hours, she develops limb weakness and finally shortness of breath requiring intubation. She has otherwise been well. However, she participated in a covered-dish dinner at a family reunion on the day the symptoms began. Several other family members who attended also complained of abdominal cramps and diarrhea or constipation, and later the same day her mother experienced double vision and slurred speech.

On examination, she exhibits eyelid ptosis and complete bilateral ophthalmoplegia. Her pupils fail to react to light. She has marked facial and tongue weakness. Strength is reduced throughout, with absent reflexes.

Discussion: Botulinum toxin ingestion in adults initially causes gastrointestinal symptoms, followed by weakness, especially of ocular and bulbar muscles. Because the toxin’s site of action is presynaptic, decreased or lack of ACh release occurs at all peripheral cholinergic nerve endings, both nicotinic (neuromuscular junction) and muscarinic (autonomic) receptors. The toxin is taken up into the nerve terminal by endocytosis, then cleaves proteins required for the docking and fusing of vesicles to the presynaptic member, thereby blocking exocytosis and release of ACh. The binding of the toxin is permanent and results in destruction of the axon terminal. Regrowth of the nerve terminal must occur for function to return. Those affected early after ingestion usually have a more devastating course than those affected later. Clustering of cases frequently occurs.

CASE 2 Lambert–Eaton Syndrome

A 68-year-old man presents with generalized weakness of 3 months’ duration. He finds it most difficult to climb stairs or rise from a chair. He has a dry mouth most of the time but denies other symptoms, except for a recent 20-pound weight loss. He has a 50–pack-year smoking history. Examination shows a dry mouth; proximal muscle weakness, especially in the lower extremities; and decreased reflexes throughout. Following a brief full contraction of biceps, the biceps reflex is briefly normal but diminishes again within 1 minute. When asked to grip the examiner’s hand, his grip gets stronger as it is maintained.

Discussion: This man has Lambert–Eaton (“myasthenic”) syndrome. In this disorder, the presynaptic release of ACh at the neuromuscular junction and in autonomic nerves is abnormal. Antibodies to presynaptic calcium channels block calcium flow into the presynaptic nerve following its depolarization; with a decreased influx of calcium, synaptic vesicles containing ACh cannot bind to the presynaptic membrane, and ACh cannot be released. The result is weakness, often generalized, along with autonomic complaints such as dry mouth and impotence. Because this is a presynaptic issue, both nicotinic and muscarinic receptors are affected. Increasing strength and reflexes following muscle contraction occur by virtue of the fact that exercise forces more calcium into the presynaptic nerve terminal, resulting in increased release of ACh and a temporary increase in strength or reflex, as in this man. These pathophysiological events are also reflected in electrophysiological studies; the initial amplitude of a compound muscle action potential is very low, but following a brief period of exercise of the tested muscle or repetitive stimulation of a motor nerve, the compound muscle action potential amplitude increases in an incremental manner (opposite the response seen in true myasthenia gravis).

Lambert–Eaton syndrome is a paraneoplastic syndrome in more than 50% of affected individuals, most commonly associated with small cell carcinoma of the lung. In this man, his weight loss and smoking history underscore this possibility and should prompt appropriate laboratory and radiographic investigations.

CASE 3 Myasthenia Gravis

A 24-year-old woman complains of difficulty holding her arms over her head to wash her hair. For the last 6 months she has also noted bilateral eyelid ptosis, worse at the end of the day, and intermittent diplopia, especially when driving at night. She has occasional difficulty swallowing, and if she drinks water quickly, she may cough, resulting in nasal regurgitation of the liquid. She has no pain and otherwise feels well. On examination, she has mild bilateral ptosis, increasing after 60 seconds of up-gaze. There is no diplopia in primary gaze, but she develops diplopia after 20 seconds of sustained up-gaze. She has palatal weakness, especially after repeated phonation, and has a nasal voice. Routine testing of strength demonstrates only mild neck flexor weakness, but she cannot keep her arms extended for more than 30 seconds. She is able to rise from a chair six times before exhibiting difficulty. The remainder of the neurological examination is normal. A bedside edrophonium test results in a transient improvement in strength.

Discussion: Myasthenia gravis (MG) is a postsynaptic disorder of the neuromuscular junction, with antibodies to ACh receptors being demonstrated there. Ocular, bulbar and systemic muscles may all be affected to varying degrees. As noted, ACh is released from synaptic vesicles in the terminal twigs of motor axons, then diffuses across the discontinuous neuromuscular junction to find receptors in the synaptic cleft in the postsynaptic sarcolemma. Binding of ACh to the receptors causes end-plate depolarization by opening sodium channels, and an all-or-none action potential ensues, spreading rapidly over the cell surface and inducing muscle contraction. ACh is then rapidly hydrolysed by AChE at the sarcolemmal surface. In MG, muscle weakness worsens with repetition, as available stores of ACh released from the presynaptic nerve terminal are depleted. Administration of edrophonium, an AChE inhibitor, produces improvement by slowing the breakdown of ACh, allowing more effective binding of the neurotransmitter with more postsynaptic receptors, and transiently improving strength.

Autonomic Motor Terminations

Autonomic neuromuscular junctions differ in several important ways from the skeletal neuromuscular junction and from synapses in the central nervous system (CNS) and peripheral nervous system (PNS). There is no fixed junction with well-defined pre- and postjunctional specializations. Unmyelinated, highly branched postganglionic autonomic axons become beaded or varicose as they reach the effector smooth muscle. These varicosities are not static but are able to move along axons. They are packed with mitochondria and vesicles containing neurotransmitters, which are released from the varicosities during conduction of an impulse along the axon. The distance (cleft) between the varicosity and smooth muscle membrane varies considerably, depending on the tissue—from 20 nm in densely innervated structures, such as the vas deferens, to 1 to 2 µm in large elastic arteries. Unlike skeletal muscle, the effector tissue is a muscle bundle rather than a single cell. Gap junctions between individual smooth muscle cells are low-resistance pathways, allowing electronic coupling and the spread of activity within the effector bundle. They vary in size from punctate junctions to junctional areas of more than 1 µm in diameter.

Adrenergic sympathetic postganglionic terminals contain dense-core vesicles. Cholinergic terminals, which are typical of all parasympathetic and some sympathetic endings, contain clear spherical vesicles like those in motor end-plates of skeletal muscle. A third category of autonomic neurones has non-adrenergic, non-cholinergic endings that contain a wide variety of chemicals with transmitter properties. Conjugated purine (ATP, a nucleoside), is probably the neurotransmitter at these terminals, which are thus classed as purinergic. Typically, their axons contain large (80- to 200-nm), dense, opaque vesicles congregated in varicosities at intervals along axons. They are formed in many sites, including the external muscle layers and sphincters of the alimentary tract, lungs, vascular walls, urogenital tract and CNS. In the intestinal wall, neuronal somata lie in the myenteric plexus, and their axons spread caudally for a few millimetres, mainly to innervate circular muscle. Purinergic neurones are under cholinergic control from preganglionic sympathetic neurones. Their endings mainly hyperpolarize smooth muscle cells, causing relaxation (e.g. preceding peristaltic waves), opening sphincters and probably causing reflex distension in gastric filling.

Autonomic efferents also innervate glands, myoepithelial cells and adipose and lymphoid tissue.

Action Potential

The action potential is a brief complete reversal of polarity that propagates itself along membranes. It depends on an initial influx of sodium ions, which causes a reversal of polarity to about 40 mV (positive inside), followed by a rapid return to the resting potential as potassium ions flow out (the detailed mechanism differs somewhat between CNS and PNS). The whole process is completed in approximately 5 msec. For a particular neurone, the size and duration of action potentials are always the same (described as all or none), no matter how much a stimulus exceeds the threshold value.

Once initiated, an action potential spreads rapidly and at a constant velocity because it triggers the opening of neighbouring voltage-gated channels of the same sort. The velocity of conduction, ranging from 4 to 120 m/sec, depends on a number of factors related to the way the current spreads, including axonal cross-sectional area, membrane capacitance (influenced by the presence of myelin) and the number and position of ion channels. At the end of an action potential, there is an irreducible delay—the refractory period—during which another action potential cannot be triggered. This determines the maximum frequency at which action potentials can be conducted along a nerve fibre; its value differs in different neurones and affects the amount of information that can be carried by an individual fibre.

Myelinated fibres are electrically insulated along most of their lengths, except at nodes of Ranvier. Voltage-gated sodium channels are clustered at nodes, and the nodal membrane is the only place where an action potential can be propagated down the axon. The action potential thus jumps from node to node across internodal distances of 0.2 to 2.0 mm, depending on the axon diameter—a process known as saltatory conduction. This greatly speeds the rate of conduction. In demyelinating disease, the speed and security of conduction are severely compromised.

Axonal conduction is naturally unidirectional, from dendrites and somata to axon terminals. When an action potential reaches the axon terminals, it causes depolarization of the presynaptic membrane, and as a result, quanta of neurotransmitter (corresponding to the content of individual vesicles) are released to change the degree of excitation of the next neurone, muscle fibre or glandular cell (Kandel and Schwartz 2000).

References

Kandel E.R., Schwartz J.H. Principles of Neural Science, fourth ed. New York: McGraw-Hill; 2000.

Lehmann-Horn F., Jurkat-Rott K., Rudel R. Periodic paralysis: understanding channelopathies. Neurology. 2006;66(Suppl. 1):62-69.

Miller A.E., editor. Myasthenic disorders and ALS: CONTINUUM Lifelong learning in neurology. Philadelphia: Lippincott Williams & Wilkins, 2009.

Ohno K., Engel A.G. Congenital myasthenic syndromes: genetic defects of the neuromuscular junction. Neurology. 2006;66(Suppl. 1):77-88.

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