Physiology, Neuromuscular Transmission

Issues of Concern

Defects or failures in neuromuscular transmission can occur in various disease states, leading to weakness in muscles such as those of the eyes, face, limbs, and respiration. Although the clinical presentations of these diseases may appear similar, important differences exist in their etiology and treatment approaches.[4].

Cellular Level

Physiological Anatomy of Neuromuscular Junction

The structure of the NMJ varies slightly among skeletal, smooth, and cardiac muscles, but all share 3 main components, as mentioned below.

• Presynaptic part: The motor nerve ending, also known as the presynaptic terminal.
• Synaptic cleft: The area between the motor nerve ending and the motor endplate.
• Postsynaptic part: The motor endplate, which is a part of the muscle membrane.

Neuromuscular Junctions of Skeletal Muscle

A skeletal NMJ is formed between the nerve endings of a motor neuron and the motor endplate, which is a specialized region of the skeletal muscle plasma membrane. Motor neurons that contribute to NMJ formation originate from either the ventral horn of the spinal cord or the medulla. Upon reaching the target muscle, the myelinated motor neuron loses its myelin sheath to form a complex of 100 to 200 branching nerve endings called nerve terminal or terminal boutons. Each nerve terminal lies adjacent to the motor endplate and is covered by Schwann cells. The nerve terminal serves as the presynaptic component of the skeletal muscle NMJ, with a structure distinct from the rest of the axon. The terminal membrane contains voltage-gated calcium and potassium channels on its membrane, and the cytoplasm includes mitochondria, the endoplasmic reticulum, and synaptic vesicles (SVs).

The membrane of SVs contains synaptotagmin and synaptobrevin proteins. SVs store acetylcholine (ACh), the primary neurotransmitter at the skeletal NMJ, with each vesicle containing approximately 5000 to 10,000 molecules of ACh. The amount of neurotransmitter stored in each vesicle is sometimes referred to as a "quanta." SVs are concentrated around the active zones, regions of membrane thickening at the nerve terminal that house various proteins and rows of voltage-gated calcium (Ca²⁺) channels. The nerve terminal membrane also contains a family of synaptosomal-associated proteins (SNAP), including syntaxin and SNAP-25. Together with the SV protein synaptobrevin, these proteins form the SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) complex, which is essential for the docking and fusion of SVs at active zones, leading to the exocytosis of ACh into the synaptic cleft.

The space between the nerve terminal and the motor endplate on the muscle plasma membrane is called the synaptic or junctional cleft, which measures approximately 50 nm. This is the site where ACh, the presynaptic neurotransmitter, is released before interacting with nicotinic ACh receptors on the motor endplate. The synaptic cleft at the NMJ contains the enzyme acetylcholinesterase (AChE), which is responsible for the catabolism of released ACh to prevent prolonged activation of postsynaptic receptors. The motor endplate forms the postsynaptic part of the NMJ, and it is a thickened portion of the muscle plasma membrane (sarcolemma) with deep folds called junctional folds. The nerve terminal does not penetrate the motor endplate but fits into these junctional folds, where nicotinic ACh receptors are concentrated at the top. The binding of ACh to these receptors opens ion channels, allowing sodium ions to flow from the extracellular fluid into the muscle membrane. This influx generates an endplate potential, leading to the initiation and transmission of an action potential across the muscle membrane.[2][3][5]

The NMJ in smooth muscle is less structurally organized compared to that in skeletal muscle. In smooth muscle, the NMJ is formed by autonomic nerve fibers that branch diffusely, creating diffuse junctions. Unlike the typical nerve terminals seen in skeletal muscle NMJ, the autonomic nerve fibers in smooth muscle feature multiple varicosities distributed along its axis. While skeletal NMJ always uses ACh as a neurotransmitter, the SVs in the varicosities may contain ACh, norepinephrine, or other neurotransmitters. The Schwann cells are interrupted at the points where varicosities occur, allowing neurotransmitters to diffuse to the muscle cells. Smooth muscles consist of multiple layers of muscle cells, but the nerve fibers typically innervate only the outermost layer. Muscle excitation then spreads from the outer layer to the inner layers either through action potential conduction within the muscle mass or through further diffusion of the neurotransmitter. A single neuron controls many muscle fibers in areas where smooth muscle activity is relatively slow, such as the intestines. In contrast, in regions where activity is rapid, such as the iris, the autonomic nerve branches less extensively, innervating fewer muscle fibers.[2][3]

Neuromuscular Junctions of Cardiac Muscle

Cardiac muscle fibers are interconnected by multiple gap junctions, allowing for the rapid spread of contraction within the muscle. Each cardiac muscle fiber is innervated by postganglionic parasympathetic and sympathetic nerve endings, which lose their myelin sheath closer to the individual muscle fibers. This proximity allows for the free diffusion of neurotransmitters from the innervating nerve axon to the muscle fiber. The parasympathetic and sympathetic fibers terminate at the sinoatrial node, atrioventricular node, and the bundle of His to form the NMJ. Sympathetic fibers also innervate the ventricular muscle. The exact nature of the nerve endings on nodal tissue remains unclear. In the ventricle, the contacts between sympathetic fibers and cardiac muscle fibers resemble those found in smooth muscle.[1][2][3]

Organ Systems Involved

Any organ with neurological innervation is involved in neuromuscular transmission. That includes skeletal muscles, smooth muscles, and cardiac muscles.[6]

Function

The nervous system can exert excitatory control over muscle contraction or inhibit reflex contractions and hyperreflexia. This control is achieved through the transfer of action potentials from the nerve to the target muscle via neurotransmitters, allowing the nerve impulse to be converted into the appropriate muscle contraction in skeletal, smooth, or cardiac muscles. All vital functions requiring voluntary or involuntary muscle contractions—such as circulation, respiration, digestion, urination, and locomotion—depend on an intact NMJ and proper neuromuscular transmission for efficient execution.

Mechanism

When a nerve impulse from the peripheral or central nervous system reaches the presynaptic membrane (nerve terminal) of the NMJ as an action potential, it causes the voltage-gated Ca²⁺ channels at the active zones of the nerve terminal to open, allowing Ca²⁺ ions to enter the nerve terminal from the extracellular space. The increased intracellular calcium interacts with SNARE proteins, stimulating synaptic vesicles (SVs) to fuse with the active zones and release their contents—ACh—into the synaptic cleft. This process is called exocytosis. This process is known as exocytosis. Increased intracellular calcium in nerve terminals triggers the simultaneous simultaneous release of multiple quanta of ACh. The total number of quanta released by a stimulated nerve is highly dependent on the extracellular Ca²⁺ concentration. In the absence of Ca²⁺ ions, even electrical stimulation of the nerve fails to induce neurotransmitter release. A 2-fold increase in extracellular calcium can result in a 16-fold increase in the quantal content of an endplate potential.

Released ACh crosses the synaptic cleft and binds to nicotinic ACh receptors on the motor endplate, thereby opening ACh-gated ion channels. This process increases the permeability of the muscle membrane to Na⁺ ions, causing the membrane potential to shift from −90 mV to −45 mV. This reduction in membrane potential is known as the endplate potential. In the skeletal NMJ, the endplate potential is strong enough to initiate an action potential across the surface of the muscle membrane. This action potential travels along the muscle fiber through the T-tubule system, triggering the release of Ca²⁺ ions from the sarcoplasmic reticulum into the muscle's sarcoplasm, leading to muscle contraction. The remaining ACh in the synaptic cleft is then hydrolyzed by the enzyme AChE.

Interestingly, the presynaptic part of the NMJ, the nerve terminal, also contains nicotinic ACh receptors. These receptors detect ACh in the synaptic cleft and regulate ACh release through a feedback system. When the concentration of ACh in the synaptic cleft increases sufficiently, the presynaptic ACh receptors sense this change, leading the nerve terminal to reduce further release of ACh. The key difference between the presynaptic and postsynaptic ACh receptors lies in their response to various ACh receptor agonists and antagonists.[5][7]

Related Testing

NMJ function in skeletal muscle is assessed through a specific nerve conduction study known as repetitive nerve stimulation (RNS). Neuromuscular transmission failure during RNS is characteristic of NMJ disorders, such as myasthenia gravis and LES.

During RNS testing, a sequence of supramaximal intensity impulses is applied to the nerve, and the response is recorded from the corresponding muscle. A decrement or increment in response indicates a specific NMJ pathology. A decremental response to RNS is commonly seen in clinical muscle fatigue and weakness. Abnormal RNS results are observed in more than 50% to 70% of generalized myasthenia gravis patients, but it is often normal in those with only ocular myasthenia gravis. Usually, a 10% decrement on slow RNS (2-3 Hz) is typically observed in patients with myasthenia gravis. In those with ocular myasthenia gravis, electromyographic (EMG) abnormalities may only be detectable in the facial muscles.

Maintaining a skin temperature between 32 ºC and 34 ºC before starting RNS is essential, as cold extremities can lead to false-negative results. Lower temperatures may reduce ACh release during the initial stimuli, leaving more quanta available for subsequent stimuli. In LES, high-frequency RNS (at rates 30-50 Hz) typically produces a significant increase in response, often exceeding 50% to 200% of the baseline value. Similarly, botulism also demonstrates an incremental response on fast RNS.

Before conducting RNS testing, it is recommended to first test at least one motor and one sensory nerve in both an upper and lower extremity. This helps rule out other potential causes related to the nerve or muscle, such as peripheral neuropathy or motor neuron disease. Needle EMG testing is conducted using single fiber studies, which measure a response called "jitter." Jitter is typically stable, but if it becomes unstable, it suggests the presence of an NMJ disorder.[8][9]

The detection of specific antibodies in a patient's serum aids in diagnosing myasthenia gravis and LES, both of which are autoimmune diseases. Approximately 85% of patients with generalized myasthenia gravis and 50% of those with only ocular myasthenia gravis have ACh receptor antibodies in their serum. For patients with ocular myasthenia gravis who lack ACh receptor antibodies in their serum, detecting muscle-specific kinase (MuSK) antibodies can be helpful for diagnosis. A positive serum voltage-gated calcium channel antibody test, along with characteristic EMG findings, can assist in diagnosing LES.[4]  

Clinically, intravenous edrophonium is used to assist in diagnosing myasthenia gravis. Edrophonium is a short-acting cholinesterase inhibitor that transiently improves weakness associated with myasthenia gravis. However, because edrophonium administration can cause severe cholinergic reactions, including syncope, it is essential to have atropine and appropriate resuscitative facilities available before conducting this test.[4]

Pathophysiology

Myasthenia Gravis

Myasthenia gravis is one of the most common diseases affecting the skeletal NMJ. In over 60% of cases, hyperplasia of the thymus gland is present, where excessive T-cell activity may contribute to an autoimmune response. Myasthenia gravis is an autoimmune disorder where the body produces antibodies against its own ACh receptors on the postsynaptic membrane. These antibodies bind to the ACh receptors, blocking the interaction between ACh and the receptors, resulting in impaired NMJ transmission, muscle weakness, and paralysis. ACh receptor antibodies are present in approximately 85% of patients with generalized symptoms but only in about 50% of patients with purely ocular involvement.

Myasthenia gravis typically presents with double vision (diplopia), drooping of the upper eyelids (ptosis), difficulty speaking (dysarthria), difficulty swallowing (dysphagia), and generalized muscle fatigue. Symptoms are usually mildest in the morning and worsen throughout the day as ACh availability at the postsynaptic membrane decreases with continued muscle activity. In progressive cases, muscle weakness may worsen over time, potentially leading to a myasthenic crisis and, in severe cases, death. A decrement of more than 10% on RNS testing is a diagnostic criterion for myasthenia gravis, indicating depletion of functional ACh in the synaptic cleft.[6][4]

Muscle weakness in myasthenia gravis can be temporarily relieved with AChE inhibitors, such as physostigmine or neostigmine, which increase the amount of ACh in the synaptic cleft and enhance conduction through the NMJ. Corticosteroids and steroid-sparing agents, including azathioprine, cyclophosphamide, tacrolimus, and mycophenolate, are used to suppress the immune response in myasthenia gravis. Rituximab, an anti-CD20 B-cell monoclonal antibody, may be used in refractory patients, especially those with MuSK-related myasthenia gravis. Plasmapheresis is used to remove antibodies from the body, while both plasmapheresis and IV immunoglobulins are used to manage complications, including acute flare-ups, myasthenic crises, and respiratory involvement. Patients experiencing a respiratory crisis may also require ventilatory support.[10]

Lambert-Eaton Myasthenic Syndrome

In LEMS, autoantibodies are produced against presynaptic membrane Ca²⁺ channels. The blockage of these Ca²⁺ channels reduces calcium entry into the nerve terminal, leading to a decrease in ACh release. This results in skeletal muscle weakness and fatigue, which typically improves after physical activity. The exact cause of LEMS is unknown, but it is often associated with lung tumors, particularly small-cell lung cancer. The symptoms of LEMS are similar to those of myasthenia gravis, including muscle weakness and fatigue. However, what distinguishes LEMS from myasthenia gravis is the involvement of proximal limb muscles, the presence of depressed tendon reflexes, and the improvement of weakness with use. This improvement occurs because repeated attempts at muscle contraction gradually build up a calcium gradient outside the presynaptic Ca²⁺ channels. Eventually, this allows the endogenous calcium to outcompete the autoantibodies, triggering the release of ACh in the synaptic cleft.

Approximately 80% of LEMS patients report proximal muscle weakness in both the arms and legs. Oropharyngeal and ocular muscles are mildly affected, so eyelid ptosis and mild diplopia may also be present. Autonomic symptoms, such as dry mouth, constipation, impotence in males, and postural hypotension, can occur. The triad of proximal muscle weakness, areflexia, and autonomic dysfunction is key to making the diagnosis. An increased response following RNS serves as a diagnostic criterion for LEMS, as the enhanced Ca²⁺ influx after supramaximal stimulation results in increased functional ACh in the synaptic cleft.[4]

Botulism

Botulism is a potentially fatal syndrome characterized by diffuse, flaccid paralysis caused by neurotoxins produced by Clostridium botulinum—an anaerobic, gram-positive, spore-forming bacterium. Of the several neurotoxins produced by C botulinum, toxins B, D, F, and G selectively affect one or more SNARE proteins, blocking docking, fusion of SVs, and exocytosis of ACh. This results in muscle weakness or reversible flaccid paralysis. Botulism can be foodborne. 

In adults, ingestion of food contaminated with C botulinum spores or toxins, often from home-canned foods, can cause nausea, vomiting, blurred vision, diplopia, and descending flaccid paralysis. In severe cases, bulbar paralysis and respiratory failure may develop rapidly. Infant botulism occurs when infants ingest honey contaminated with C botulinum spores. The spores colonize the gastrointestinal tract and produce toxins, leading to symptoms such as constipation, weak feeding, weak crying, and flaccid paralysis.

Diagnosis of botulism is based on history, clinical presentation, and the detection of botulinum toxin or C botulinum in serum, stool, gastrointestinal content, or wound exudates. Treatment involves the administration of antitoxin. Early antitoxin administration reduces mortality and shortens the disease course. Patients may require prolonged ventilation. Botulism can also occur from wound contamination with dust containing C botulinum spores, which is particularly common following traumatic injuries or among drug abusers. Therapeutically, botulinum toxin is used to treat spasticity in conditions such as blepharospasm, torticollis, and anal sphincter spasm. The toxin is also used to manage axillary hyperhidrosis and for cosmetic purposes, such as wrinkle correction.[4][11]

Clinical Significance

Drugs Affecting Neuromuscular Junction

Certain drugs, such as nicotine and carbamylcholine, mimic ACh’s action due to their similar chemical structure. Direct ACh agonists, which bind directly to ACh receptors, include bethanechol (used to treat postoperative ileus and urinary retention), carbachol and pilocarpine (both used for treating glaucoma by inducing pupillary muscle constriction), and methacholine (used in challenge tests to diagnose asthma in asymptomatic patients).

NMJ blockers are used to induce muscle relaxation and paralysis. They are classified into depolarizing agents (eg, succinylcholine) and non-depolarizing agents (eg, tubocurarine, atracurium, mivacurium, pancuronium, vecuronium, and rocuronium). Drugs such as d-tubocurarine compete with ACh for binding to the ACh receptors on the postsynaptic membrane. This prevents ACh from inducing muscle contraction, resulting in skeletal muscle relaxation instead. These drugs are referred to as competitive blocking agents.

Drugs such as succinylcholine also paralyze skeletal muscle by causing continuous depolarization, which prevents repolarization of the motor endplate. This leads to ACh receptors becoming desensitized and inactivated. These drugs are commonly used during general anesthesia to reduce the need for large doses of anesthetics. However, the administration of neuromuscular blocking agents can also result in bulbar and respiratory muscle failure. AChE inhibitors such as physostigmine neostigmine, pyridostigmine, and edrophonium increase the levels of ACh in the synaptic cleft. Physostigmine and neostigmine are commonly used for the treatment of myasthenia gravis.

Irreversible inhibitors of AChE include organophosphates, such as malathion and parathion, which are commonly used as insecticides. Exposure to these compounds can lead to organophosphate toxicity syndrome, characterized by diarrhea, urination, miosis, bronchospasm, excessive lacrimation, and salivation. The effects of irreversible AChE inhibitors can be reversed with competitive inhibitors such as atropine and/or pralidoxime, which regenerate AChE if administered early enough, before the enzyme undergoes aging.[7][12]