Introduction

Neuromuscular junction (NMJ) is responsible for the chemical transmission of the electrical impulse from a nerve to the muscle( skeletal/ smooth/ cardiac) in order to produce an appropriate muscle contraction.  Diseases of NMJ such as myasthenia gravis, Lambert-Eaton syndrome, and botulism affects neuromuscular impulse transmission and result in muscle weakness and paralysis. Many drugs and anesthetic agents also affect neuromuscular junction and impulse transmission to elicit their effects. In order to understand the pathophysiology and basis of treatment of the diseases that affect neuromuscular transmission, it is important to have a thorough knowledge of the structure of NMJ and the physiology of neuromuscular transmission.[1][2][3]

Issues of Concern

Failure of or a defect in neuromuscular transmission may occur in a number of disease states. These disease states produce weakness of muscles of eyes, face, limbs, respiration, etc. Though the clinical presentation of these diseases may appear similar, there are important differences in the etiology and the basis of treatment.[4].

Cellular Level

Physiological Anatomy of Neuromuscular Junction

The structure of NMJ of a skeletal, smooth, or cardiac muscle vary a little from each other, but all have three main parts; a motor nerve ending, also termed the presynaptic part; postsynaptic part, the motor endplate, which is a part of the muscle membrane; the synaptic cleft; an area between the motor nerve ending and the motor endplate.

Neuromuscular Junction of Skeletal Muscle

A skeletal NMJ is formed between the nerve endings of a motor neuron that arise either from the ventral horn of the spinal cord or from the medulla and the part of plasma membrane skeletal muscle called motor endplate. On reaching the target muscle, the myelinated motor neuron loses its myelin sheath to form a complex of 100-200 branching nerve endings called nerve terminal/ terminal boutons. Each nerve terminal lies against the motor endplate and is covered by Schwann cells. The nerve terminal is the presynaptic part of skeletal muscle NMJ. Its structure is quite different from that of the rest of the axon. It has voltage-gated calcium channels and potassium channels on its membrane and contains mitochondria, endoplasmic reticulum, and synaptic vesicles (SVs) in its cytoplasm.  The membrane of SVs has synaptotagmin and synaptobrevin proteins. SVs store acetylcholine (ACh), the neurotransmitter at skeletal NMJ. Each SV has around 5000-10000 molecules of Ach. The amount of neurotransmitter stored in each vesicle is sometimes referred to as “quanta. The SVs are concentrated around the active zones, the areas of membrane thickening at the nerve terminal that contains many proteins and rows of voltage-gated calcium (Ca) channels. The nerve terminal membrane also contains a family of SNAP proteins, syntaxin, and synaptosomal-associated protein 25 (SNAP-25). These proteins(syntaxin and SNAP-25) along with synaptic-vesicle protein synaptobrevin are referred to as SNARE (soluble N-ethylmaleimide-sensitive) proteins and are essential for docking and fusion of SVs to active zones and resulting exocytosis of ACh in the synaptic cleft. The space between the nerve terminal and the motor endplate on the plasma membrane of muscle is called the synaptic/junctional cleft and measures ∼50 nm. It is the site where presynaptic neurotransmitters, ACh is released before it interacts with nicotinic ACh receptors on the motor endplate. The synaptic cleft of NMJ contains acetylcholinesterase enzyme, responsible for the catabolism of released ACh so that its effect on the post-synaptic receptors is not prolonged. Motor endplate forms the postsynaptic part of NMJ. It is the thickened portion of the muscle plasma membrane (sarcolemma) that is folded to form depressions called junctional folds. The nerve terminal does not penetrate the motor endplate but fits into the junctional folds. Junctional folds have nicotinic ACh receptors concentrated at the top. The binding of ACh to these receptors opens the ion channels allowing the influx of sodium ions from the extracellular fluid into the muscle membrane. This creates endplate potential and generation and transmission of AP to muscle membrane.[2][3][5]

The neuromuscular junction of the smooth muscle is not as highly structured as skeletal muscle NMJ. Smooth muscle NMJ is formed between the autonomic nerve fibers that branch diffusely on smooth muscle to form diffuse junctions. The autonomic nerve in smooth muscle NMJ does not have typical nerve terminals as seen in skeletal NMJ, but instead, it has multiple varicosities distributed along its axis. Unlike skeletal NMJ that always has Ach as a neurotransmitter, the SVs present in the varicosities may contain ACh or norepinephrine, or another transmitter. The Schwann cells are interrupted at the points where varicosities are present so that neurotransmitters can diffuse to the cells. The smooth muscles have many layers of muscle cells, but the nerve fibers often innervate only the outer layer. Muscle excitation travels from this outer layer to the inner layers by action potential conduction in the muscle mass or additional diffusion of the transmitter substance. In body parts where smooth muscle activity is relatively slow, like intestines, one neuron controls a large number of muscle fibers, but parts where the activity is fast, such as iris, the autonomic nerve branch less extensively and controls fewer muscle fibers.[2][3]

Neuromuscular Junctions of Cardiac Muscle

Cardiac muscle fibers are connected by multiple gap junctions that provide a rapid spread of contraction within the muscle. Each cardiac muscle fiber is innervated by postganglionic parasympathetic and sympathetic nerve endings, which lose the myelin sheath closer to the individual muscle fiber; this allows free diffusion of the neurotransmitter from the innervated nerve axon to the muscle fiber. The parasympathetic and sympathetic nerve fibers end on the sinoatrial node, the atrioventricular node, and the bundle of His to form NMJ. Sympathetic fibers also innervate the ventricular muscle. The exact nature of the endings on nodal tissue is not known. In the ventricle, the contacts between the sympathetic fibers and the 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 can have an inhibitory influence on reflex contractions and hyperreflexia. This control is achieved through the transfer of action potential from the nerve to the target muscle with the help of a neurotransmitter so that nerve impulse can be converted to appropriate muscle contraction of the skeletal, smooth, or cardiac muscle. All vital functions that need voluntary or involuntary contraction of skeletal, smooth, or cardiac muscles, such as circulation, respiration, digestion, urination, locomotion, etc., could be carried out efficiently due to intact NMJ and proper neuromuscular transmission.

Mechanism

When the nerve impulse from the peripheral or central nervous system reaches the presynaptic membrane (nerve terminal) of the neuromuscular junction in the form of the action potential, it triggers voltage-gated Ca2+ channels at the active zones of the nerve terminal to open, and Ca2+ ions enter the nerve terminal from the extracellular space. Increased intracellular calcium interact with SNARE proteins; this stimulates synaptic vesicles to fuse with active zones and release their content – ACh into the synaptic cleft. This process is named exocytosis. Increased intracellular calcium in nerve terminals triggers the simultaneous release of a number of ACh quanta. The total number of quanta of ACh released by a stimulated nerve markedly depends on the concentration of Ca2+ ions in the extracellular fluid. If Ca2+ ions are not present, even electrical stimulation of the nerve will not produce the release of transmitter. A 2-fold increase in the extracellular calcium will cause a 16-fold increase in the quantal content of an endplate potential.

Released ACh travels across the synaptic cleft towards the motor endplate and binds with the nicotinic ACh receptors, triggering ACh-gated channels to open. The motor endplate on the muscle membrane becomes more permeable to Na+ ions. This changes the membrane potential at the muscle membrane from -90 mV to -45 mV. This decrease in membrane potential is called endplate potential. In the skeletal NMJ, the endplate potential is strong enough to propagate action potential over the surface of the skeletal muscle membrane. This potential is carried along the muscle fiber through the system of T tubules and triggers to release Ca2+ ions from the sarcoplasmic reticulum into the sarcoplasm of muscle, which results in the contraction of the muscle. The remaining ACh in the synaptic cleft gets hydrolyzed by the enzyme acetylcholinesterase (AChE). Interestingly, the presynaptic part of NMJ, the nerve terminal, also has nicotinic ACh receptors. These receptors sense ACh in the synaptic cleft and, via a feedback system, controls the release of ACh. If the concentration of ACh in the synaptic cleft has increased appropriately, the presynaptic ACH receptors will sense, and the nerve terminal will shut down more release.  The difference between pre-and postsynaptic ACh receptors is the response of these receptors to different ACh receptor agonists and antagonists.[5][7]

Related Testing

Neuromuscular junction testing in skeletal muscle is done by a specific nerve conduction study called repetitive nerve stimulation (RNS). Neuromuscular transmission failure during RNS is a trademark of NMJ diseases such as Myasthenia Gravis (MG) and Lambert-Eaton syndrome (LES).  

During RNS testing, a sequence of supramaximal intensity impulses are applied to the nerve, and the response is recorded from the corresponding muscle. Decrement or increment of response indicates a specific NMJ pathology. A decremental response to RNS is seen in clinical muscle fatigue and weakness. Abnormal RNS is seen in more than 50% to 70% of generalized MG patients, but in patients having only ocular MG, it is often normal. A 10% decrement on slow RNS (2-3 Hz) is typically seen in patients with MG. In patients with ocular MG, electromyographic abnormalities may be detectable only in facial muscles. It is important to maintain a skin temperature between 32ºC – 34ºC before beginning RNS because cold extremities may give false-negative results. If the skin is cold, there may be a reduced release of Ach with the first stimuli, and this will leave more quanta for subsequent stimuli. In the LES, repetitive nerve stimulation at high rates (30-50 Hz) will cause considerable increments, usually exceeding 50% to 200% of the baseline value. Botulism also shows an incremental response on fast RNS. Before conducting RNS testing, it is recommended to at least test one motor and one sensory nerve in an upper and lower extremity first. The idea is to rule out any other underlying cause that involves nerve or muscle, such as peripheral neuropathy or motor neuron disease. Needle EMG test is done utilizing singer fiber studies.  These studies utilize a response called "jitter." Jitter is usually a stable action, but when unstable, it suggests NMJ disorder is present.[8][9]

Detection of specific antibodies in a patient's serum also aids the diagnosis of MG and LES. Both MG and LES are autoimmune diseases. Around 85% of generalized MG and 50% of only ocular MG patients have ACh receptor antibodies in their serum. For patients with ocular MG, who do not have ACh receptor antibodies in their serum, the detection of muscle-specific kinase (MuSK) antibodies can be helpful in making the diagnosis. A positive serum voltage-gated calcium channel antibody test and characteristic EMG findings may help the diagnosis of LES.[4]  

Clinically, intravenous edrophonium is used to help make a diagnosis of MG. Edrophonium is a short-acting cholinesterase inhibitor; its administration transiently improves weakness due to MG. Since edrophonium administration may lead to severe cholinergic reactions, including syncope, it is important to have atropine and proper resuscitative facilities available before conducting this test.[4]

Pathophysiology

Myasthenia Gravis

Among the most common diseases of the skeletal neuromuscular junction is myasthenia gravis (MG). In over 60% of cases, hyperplasia of the thymus gland is present when excessive T cells may predispose to an autoimmune response. MG is an autoimmune disorder in which the body produces antibodies against its own ACh receptors in the postsynaptic membrane. These antibodies bind to the ACh receptors and block the interaction of ACh with them, resulting in the blockage of NMJ transmission, muscle weakness, and paralysis. Acetylcholine receptor antibodies are present in about 85% of patients with generalized symptoms, but only 50% of patients with purely ocular involvement MG characteristically presents with double vision (diplopia), drooping of the upper eyelids (ptosis), difficulty in speaking (dysarthria), difficulty in swallowing (dysphagia) and general muscle fatigue. The symptoms are typically least expressed in the morning and are worse in the evening as the amount of ACh bound to the postsynaptic membrane receptor decreases due to various muscle activities during the day. In the progressive form of the disease, the weakness may become steadily worse, may cause myasthenic crisis and death. Decrement of over 10% observed following repetitive nerve stimulation is a diagnostic criterion for MG due to depletion of functional ACh in the synaptic cleft.[6][4]

Muscle weakness can be temporarily relieved by ACh-esterase drugs such as physostigmine or neostigmine since they increase the amount of ACh in the synaptic cleft and enhance conduction through the NMJ. Corticosteroids and steroid-sparing agents (azathioprine, cyclophosphamide, tacrolimus, or mycophenolate) are used to inhibit the immune response in MG. Rituximab, an anti-CD20 B cell monoclonal antibody, may be used in refractory patients, especially with MuSK. Plasmapheresis is used to remove antibodies from the body. Plasmapheresis and IV immunoglobulins are used to treat complications of MG that include acute flare-ups of myasthenic crises and respiratory involvement. Patients with a respiratory crisis may also require ventilatory support.[10]

Lambert Eaton Myasthenic Syndrome

LEMS is another autoimmune disease affecting skeletal muscle NMJ when the body produces antibodies against its own presynaptic membrane Ca2+ channels. Blocking Ca2+ channels in the presynaptic membrane leads to less calcium entry into the nerve terminal and less ACh being released, resulting in weakness of skeletal muscles and fatigue that usually improves after physical activity. The exact cause of LEMS is unknown but correlates with tumors of the lung, usually small cell lung cancer. The symptoms of LES are similar to MG, such as muscle weakness and fatigue; however, what separates LES from MG is that in LES, proximal limb muscles are involved in the presence of depressed tendon reflexes a weakness improves with use. This is because with repeated attempts at muscle contraction, a calcium gradient builds up outside the presynaptic Ca channel, eventually allowing the endogenous calcium to outcompete the auto-antibodies to trigger the release of ACh in the synaptic cleft. 80% of LES patients complain of proximal muscle weakness in both 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 may be seen. The triad of proximal muscle weakness, areflexia, and autonomic dysfunction aids in making the diagnosis. The increased response following repetitive nerve stimulation is a diagnostic criterion for LEMS due to increased Ca2+ after supra-maximal stimulation, resulting in increased functional ACh in the synaptic cleft.[4]

Botulism

Botulism is a potentially fatal syndrome of diffuse, flaccid paralysis caused by neurotoxins produced caused by Clostridium botulinum, an anaerobic gram-positive, spore-forming bacterium. Of the several types of neurotoxins produced by C. botulinum toxins B, D, F, and G selectively affect one or all of these SNARE proteins and block docking, a fusion of SVs and exocytosis of Ach, resulting in muscle weakness or reversible flaccid paralysis. Botulism can be foodborne. In adults, ingestion of food contaminated with spores or toxins (home-canned food) of clostridium botulism result in nausea, vomiting, blurred vision, diplopia, and descending flaccid paralysis. In severe cases, bulbar paralysis and respiratory failure may occur rapidly. Infant botulism can occur due to ingestion of honey contaminated with spores of C. botulism. Toxins are produced within the GI tract of infants, and infants present with constipation, weak feeding, weak crying, and flaccid paralysis. History, clinical presentation, and the detection of botulinum toxin and the presence of C. botulinum in serum, stool, gastrointestinal content, or wound exudates form the basis of diagnosis. Treatment is done by administering antitoxin. Early administration of antitoxin decreases mortality and shortens the disease course. Patients may require prolonged periods of ventilation. Botulism can also occur due to contamination of a wound with dust containing C. botulism spores. Such infection particularly common after traumatic injuries and among drug abusers. Therapeutically, botulinum toxin is used to treat spasticity in conditions such as blepharospasm, torticollis, anal sphincter spasm, etc. It is also used to treat axillary hyperhidrosis and cosmetically used to correct wrinkles.[4][11]

Clinical Significance

Various Drugs Affecting NMJ

There are few drugs like nicotine and carbamylcholine that mimic acetylcholine's action because of their similar chemical structure. A direct agonist of ACh that directly binds to the ACh receptors includes bethanechol (which treats post-operative ileus, urinary retention), carbachol, and pilocarpine (both used to treat glaucoma by constriction of the pupillary muscle), and methacholine (challenge test to diagnose asthma in a patient who presents asymptomatically).

NMJ blockers are used to induce muscle relaxation and paralysis. These can be divided into depolarizing (succinylcholine) and non-depolarizing (tubocurarine, atracurium, mivacurium, pancuronium, vecuronium, rocuronium). Drugs such as d-tubocurarine compete with ACh and bind to the ACh receptor on the postsynaptic membrane, thus causing the skeletal muscle to relax instead of contraction after locally produced ACh. They are called competitive blocking agents.

Drugs like succinylcholine also paralyze the skeletal muscle but through continued depolarization that prevents repolarization of the motor endplate, resulting in ACh receptors becoming desensitized and inactivated. These drugs are used in general anesthesia to help avoid large doses of general anesthetics. Administration of neuromuscular blocking agents and drugs can also result in bulbar and respiratory muscle failure. ACh esterase inhibitors like physostigmine neostigmine, pyridostigmine, and edrophonium increase the levels of ACh in the synaptic cleft. Physostigmine and neostigmine are used for the treatment of Myasthenia Gravis (MG). Irreversible inhibitors of ACh esterase include the organophosphates commonly used as an insecticide, which include malathion and parathion. Exposure to these may cause organophosphate toxicity syndrome that includes diarrhea, urination, myosis, bronchospasm, excessive lacrimation, and salivation. The effects of irreversible inhibitors of ACh esterase can be reversed by using a competitive inhibitor such as atropine and/or pralidoxime, which regenerates ACh esterase if given early enough before enzyme aging occurs.[7][12]