The Muscle and the Neuromuscular Junction: Endplate, Motor Unit and Reserve

01Calcium and quantal release

The neuromuscular junction turns a nerve impulse into transmitter release, and calcium is the trigger

Calcium entry is what converts a nerve impulse into transmitter release. The action potential reaches the presynaptic terminal and opens voltage-gated calcium channels, and the calcium that enters triggers release of acetylcholine into the cleft. The transmitter crosses a gap tens of nanometers wide and lands on the motor endplate, a specialized region of sarcolemma dense with nicotinic acetylcholine receptors.

Release depends steeply on how long that calcium flows. In tetrodotoxin-treated frog nerve terminals, lengthening a focal depolarization from 1 to 2 milliseconds raised transmitter release more than fiftyfold Katz 1968. One extra millisecond of calcium entry multiplies output by a factor no later step in the pathway can match. Anything that touches presynaptic calcium handling therefore moves the whole junction, which is why the presynaptic disease of this synapse is a calcium channel disease.

Quanta leave from active zones, and the count barely varies

Transmitter leaves the terminal in packets. Each quantum holds on the order of 10,000 acetylcholine molecules and saturates a critical patch of receptor membrane covering about 0.3 square micrometers in the frog Matthews-Bellinger 1978. Quanta are released from discrete active zones, and release probability differs from one active zone to the next Slater 2015.

The number of quanta released by successive nerve impulses shows low statistical variability, well described by a binomial distribution Slater 2015. Reliability at this synapse is a property of many independent release sites reporting together rather than of one large event that has to work every time.

02Findings

What the research shows

03The endplate and its folds

The motor endplate answers with a receptor field so dense that most of it goes unused

Acetylcholine receptors are packed at the endplate and almost absent a short distance away. At frog junctions the density reaches 26,000 plus or minus 6,000 sites per square micrometer on the thickened postjunctional membrane Matthews-Bellinger 1978. Fifteen micrometers away from the axon terminal it has fallen to roughly 50 sites per square micrometer. The receptive surface of one frog endplate totals about 1,500 square micrometers, and an endplate potential built from 300 quanta activates less than 10 percent of it.

Mammalian endplates are built the same way. Bat diaphragm labeled to saturation with tritiated alpha-bungarotoxin carries 20,000 to 25,000 receptor sites per square micrometer at the crests of its junctional folds Porter 1975. Mouse endplates measure close to the same. Averaged over the whole postsynaptic membrane the figure is 8,800 sites per square micrometer. Cholinesterase sites are distributed uniformly over the folds while receptors crowd the crests nearest the nerve terminal. Geometry sorts the two molecules, so the transmitter meets its receptor before it meets the enzyme that ends it.

The enzyme is anchored rather than dissolved. Endplate acetylcholinesterase is an asymmetric molecule built from three collagenic ColQ strands, each carrying a tetramer of catalytic subunits, and the tail subunit holds the whole assembly in the synaptic basal lamina Ohno 2000. Hydrolysis the moment the transmitter dissociates from the receptor prevents rebinding, so each command is cut into a discrete event.

The safety factor is the margin the junction is built with

In isolated rat nerve-muscle preparations, nerve-evoked responses carry a safety factor of 3.5 in soleus and 5.0 in extensor digitorum longus Wood 1997. Quantal content was 61.8 and 79.4 in those muscles, while only about 13 to 23 quanta are needed to fire the fiber. The junctional folds double that margin, because the sodium channels in their depths convert a given endplate potential into an action potential more readily than flat membrane does. In the extrajunctional region, where the folds are absent, currents injected through an intracellular electrode give 1.7 in soleus and 2.5 in extensor digitorum longus.

One acetylcholine channel opens with a conductance near 24 picosiemens and stays open about 0.8 milliseconds in rat muscle at room temperature Sterz 1983. Thousands of them open at once, delivering three and a half to five times the depolarization that firing the fiber requires.

04Excitation and contraction

The endplate potential reaches the sarcomere through two proteins facing each other across a gap

Depolarization of the sarcolemma is carried into the interior of the muscle fiber by the transverse T tubules. Depolarizing the T tubule membrane changes the conformation of the dihydropyridine receptor, and that change opens the ryanodine receptor anchored in the membrane of the sarcoplasmic reticulum Allard 2018. Calcium floods out of the reticulum and activates the contractile proteins. Relaxation is not passive. The sarcoplasmic reticulum calcium ATPase pumps the ion back in, and mutations along this chain produce myotonia, weakness, periodic paralysis and wasting.

The sarcomere is the repeating unit of interdigitating actin and myosin filaments, and force depends on how much of the thick filament the thin filaments overlap. In single frog fibers, isometric tension holds a plateau between sarcomere lengths of 2.05 and 2.2 micrometers and falls steeply below about 1.67 micrometers Gordon 1966. Each thin filament's tension is the sum of equal contributions from every cross-bridge it overlaps.

Sarcomere length sets how much force a nerve command produces

The plateau is narrow. A sarcomere held short or stretched long produces less force from the same nerve command, because fewer cross-bridges can form Gordon 1966. Resting length is set by joint position, and joint position is held by the muscles around it. The model reads sarcomere length as the point where a postural state becomes a mechanical one. A segment held short runs its sarcomeres off the plateau, and the force lost there is force no extra transmitter restores.

05The motor unit

One motor neuron and its fibers act as one unit, and unit size sets how finely a muscle can be graded

In cat medial gastrocnemius an average motor unit contains 400 to 800 muscle fibers, and the count holds across physiological types Burke 1973. A motor unit is one motor neuron in the spinal cord or brainstem plus every fiber it innervates. Units differ in force because their fibers differ in size and in specific tension. In that muscle, specific tension ran about 0.6 kg per square centimeter in slow units, 1.5 to 2.0 in fast fatigable units and 2.6 to 2.9 in fast fatigue-resistant units. Fiber density implies that a single region of the muscle is shared by as many as fifty motor units.

Intrinsic hand muscles run small units and large limb muscles run large ones, because few fibers per neuron buys precision. Cat soleus motor neurons innervate from under 50 to over 400 fibers, with an estimated average of 140 to 190 fibers per unit Burke 1974. Every soleus unit studied was slow and extremely resistant to fatigue, with twitch contraction times of 64 to 131 milliseconds and a mean near 97 milliseconds. The fibers of one unit scatter widely through the muscle instead of clustering, so postural force builds smoothly rather than in lumps.

Skeletal muscle carries three fiber types in proportions set by function. Type I fibers are small, red, and rich in mitochondria and capillaries. They resist fatigue and predominate in postural muscle. Type IIb fibers are large, mitochondria poor, white, and built for brief powerful contractions. Type IIa fibers sit between the two. Single fibers were taken from twelve healthy young adults. Those expressing type II myosin produced 24 to 42 percent higher specific force than type I fibers at every cross-sectional area Miller 2015. The difference traces to the number and stiffness of bound cross-bridges rather than to fiber size.

Fiber type is written into the endplate itself

Functional acetylcholine receptor density is 62 percent lower at slow soleus endplates than at fast extensor digitorum longus endplates in the rat Sterz 1983. The total number of functional receptors is 60 percent lower. Single channel conductance is nearly identical in the two, at 24.6 and 23.9 picosiemens. The difference sits in how many channels the endplate owns, and it lines up with the lower safety factor measured in the same muscle Wood 1997. Fatigable weakness therefore does not strike all muscles equally, because they do not start with equal margins.

The motor unit pool thins before weakness is felt

Physically active men near 71 years of age carry between 50 and 60 percent fewer motor units in vastus lateralis than men near 25 Piasecki 2016. The count takes into account the 30 percent smaller cross-sectional area of the older muscle. The units that survive have grown, with motor unit potentials 25 percent larger and firing rates 8 percent lower. Near-fiber jiggle rose 11 percent, a direct index of unstable neuromuscular transmission. The pool is remodeled and transmission is already less stable before muscle function has measurably declined.

06Muscle energy metabolism

Muscle runs its own fuel supply, and it changes settings within seconds

Energy metabolism supplies the ATP that powers every cross-bridge cycle, and the standing supply is tiny. During near-maximal exercise in man the muscle store of ATP is depleted in under one second, so ATP must be resynthesized continuously for contraction to continue Hultman 1991. Phosphocreatine and glycogen carry the first 30 seconds. Fatigue in short maximal work follows type II fibers depleting phosphocreatine faster than glycogenolysis can compensate, which makes early fatigue a supply problem inside the fiber.

The pathways behind that supply are the ones every cell uses. Glycolysis is an oxygen-independent source of ATP and pyruvate. Pyruvate is either reduced in the cytosol to lactate or oxidized in the mitochondrial matrix to acetyl-CoA, which feeds the TCA cycle. Beta oxidation of fatty acids and the TCA cycle produce NADH and FADH2, which the electron transport chain oxidizes to pump hydrogen ions and drive oxidative phosphorylation of ADP. The machinery is taught in full at mitochondria and ATP.

Serial human biopsies during three 30-second maximal bouts show the handover happening. Glycogen phosphorylase switches on within 6 seconds and switches off again. Pyruvate dehydrogenase rises from 14 percent activation at rest to 48 percent at 6 seconds and 95 percent by 15 seconds, then stays on Parolin 1999. Muscle lactate climbed from 2.7 to 76.1 millimoles per kilogram dry weight in the first bout and rose no further by the third. With each successive bout the fiber leans further on oxidative phosphorylation.

Endurance training enlarges existing mitochondria rather than making new ones

Six weeks of endurance training raised mitochondrial volume density by 55 plus or minus 9 percent and citrate synthase activity by 44 plus or minus 12 percent in twenty-one healthy men Meinild Lundby 2018. The number of mitochondrial profiles per area of muscle stayed unchanged. Existing organelles grew.

Spacing decides what that capacity is worth. Ten men trained through fourteen high-intensity cycling sessions, half of them daily for two weeks and half with two days of rest after each session Parra 2000. The daily group raised phosphofructokinase 107 percent, citrate synthase 38 percent and 3-hydroxyacyl-CoA dehydrogenase 60 percent. The group given two days between sessions raised the same enzymes 68, 28.4 and 38.7 percent. The daily group failed to improve performance and the rested group improved it, which is what the model expects: the same work delivered into a different recovery state writes a different adaptation.

Aging oxidizes the calcium release channel of the sarcoplasmic reticulum

Age-related skeletal muscle dysfunction reaches up to half the population aged 80 or older, and the calcium release channel is one of the places it starts. Mitochondrial free radicals oxidize the ryanodine receptor and strip away its stabilizing subunit calstabin1, leaving the channel leaking calcium out of the sarcoplasmic reticulum between contractions Umanskaya 2014. Aged transgenic mice overexpressing human catalase in their mitochondria showed larger tetanic calcium transients, less intracellular leak and higher specific force than wild-type littermates. Mitochondrial output and the calcium release channel are one system, so a metabolic drift in the fiber shows up as a mechanical one.

07Diseases of the junction

Diseases of the neuromuscular junction break it one station at a time

Myasthenia gravis destroys the receiving membrane. Antibodies against the acetylcholine receptor are specific for the disease and present in up to 85 percent of patients Andersen 2014. The same Norwegian national cohort put prevalence at 131 to 145 per million and annual incidence at 8.8 to 16.0 per million. Morphometry at the endplate shows the loss is graded. How much receptor-bearing postsynaptic surface survives predicts the miniature endplate potential amplitude linearly, and it predicts the patient's clinical state Engel 1977. The regions with the greatest receptor loss were the simplest and most degenerated folds, so the disease erases the architecture and not only the receptors.

Transferring the immunoglobulin fraction of patient serum into mice reproduced the disease. After daily injections for up to fourteen days, miniature endplate potential amplitudes fell by 65 percent Toyka 1976. Receptors available for bungarotoxin binding fell 38 percent in extensor digitorum longus and 54 percent in soleus. The animals showed decrement on repetitive stimulation and visible weakness, while animals given control serum did not. The antibody is the lesion.

Ocular complaints open the illness. Ptosis and diplopia are the usual first symptoms, and weakness then spreads in a cranial to caudal direction. Only 15 percent of patients stay purely ocular for the whole course, and generalization is most frequent in the first two years Evoli 2001. The edrophonium test, a brief dose of a short-acting acetylcholinesterase inhibitor, transiently restores strength by sparing the transmitter that is still being released. Longer-acting neostigmine gives a separate confirmatory test.

Lambert-Eaton syndrome attacks the presynaptic calcium channel

Lambert-Eaton myasthenic syndrome attacks the sending terminal. Autoantibodies bind the presynaptic P/Q-type voltage-gated calcium channel and interfere with the calcium-dependent release of acetylcholine, which is why weakness begins in the proximal legs and spreads caudal to cranial Sakai 2013. The compound muscle action potential increments rather than decrements with repetitive nerve stimulation. Between 50 and 60 percent of these patients carry a tumor, most often a small cell lung carcinoma, so the electrodiagnostic finding sets off a search outside the nervous system.

An increment above 60 percent after brief exercise or one second of 50 hertz stimulation is the diagnostic criterion Komatsu 2013, and increments above 100 percent on repetitive stimulation are common Sakai 2013. In a retrospective series of eight patients, needle electromyography looked myopathic in six, and the motor unit potentials normalized only after sustained strong contraction. A presynaptic lesion can pass for muscle disease, and the way to tell is to make the terminal work before reading it.

COLQ mutations leave endplate acetylcholinesterase unanchored

Mutations in COLQ leave the catalytic subunits of acetylcholinesterase unanchored, and nine novel mutations were identified in seven patients with endplate acetylcholinesterase deficiency Ohno 2000. The enzyme is made and never reaches the basal lamina. These patients have myasthenia that anticholinesterase drugs do not relieve, which is the mirror-image proof that the enzyme's placement in the cleft is functional rather than incidental. Three diseases, three stations, one synapse.

08A junction tuned by use

The neuromuscular junction changes its output according to how the muscle is used

Overloading a muscle roughly doubles what its junctions release. In rat plantaris overloaded by cutting the tendons of its synergists, quantal content rose from 37.0 in controls to 74.3 Argaw 2004. Cumulative quantal release ran 139 and 153 percent higher at 25 and 50 hertz, and the safety factor of the junction rose with it. How much transmitter each impulse spends is a value the body writes from how the muscle is used.

The same setting moves the other way to defend force. In several mouse models of Duchenne muscular dystrophy, miniature endplate potential amplitudes ran about 40 percent smaller than wild type van der Pijl 2016. The nerve terminal answered by raising quantal content until evoked endplate potentials came out unchanged. Receptor clusters were fragmented and deficits appeared only at high firing rates. What the terminal holds constant is the endplate potential. Transmitter output is the adjustable term.

The Unified Model of Tone treats the safety factor as the tone value a muscle carries, written at its endplates. A junction carrying a margin of 5.0 has options. A soleus junction starting at 3.5 has fewer, and load, disease and age each spend that margin further down. The muscles holding the smallest margins fail first under a load that leaves their neighbors untouched. That is why the same antibody titer, the same dystrophin mutation and the same training week produce weakness in one person and none in another. The insult meets a margin that use has already set.

Descending drive decides whether the junction is asked to work

Excitability upstream decides whether the junction is ever asked to work. The V wave indexes descending drive reaching the motoneuron pool. It can move while nothing at the endplate has changed, so a change in voluntary force after a spinal input may sit in the cord rather than in the muscle. What a spinal input measurably changes in that drive, and in whom, is set out at tone as a measurable state. This page carries the machinery downstream of that drive: the terminal, the endplate, the calcium release channel and the cross-bridge.

The model predicts which way each link moves. Give a muscle more traffic and its terminals raise quantal content until the endplate potential clears threshold with room left over, as they did in overloaded plantaris. Take the receptors away and the terminals spend more per impulse to hold that same endplate potential. When there is nothing further to spend, rundown at high firing rates becomes weakness at ordinary ones. A junction that has spent its margin still fires at rest and still passes a first contraction. It fails partway through the load its neighbor absorbs, which is why the first sign of junction disease is a task that cannot be finished rather than one that cannot be started.

09Tone

How this system expresses tone

Tone at this synapse is a margin, and the margin is measurable. A rat junction spends 62 to 79 quanta on a command that 13 to 23 quanta would carry Wood 1997.

Prediction: the fiber commits its fuel setting before the work is over, and pyruvate dehydrogenase in human muscle rises from 14 percent activation at rest to 95 percent within 15 seconds. Time course: six weeks of endurance training raised mitochondrial volume density 55 percent in twenty-one healthy men by enlarging existing mitochondria rather than building new ones. Constraint: slow rat soleus endplates carry a functional receptor density 62 percent lower than fast ones, which caps what any quantity of transmitter can accomplish there. Coupling: a nerve command yields force only through filament overlap, and single frog fibers hold peak isometric tension across a sarcomere plateau of 2.05 to 2.2 micrometers. Input quality: fourteen high-intensity cycling sessions raised muscle enzymes in both groups of men, and only the group given two days between sessions improved performance. Oscillation: Lambert-Eaton weakness hides at rest and declares itself at frequency, with the compound muscle action potential incrementing above 60 percent after one second of 50 hertz stimulation.

10Across the library

How this page relates to the rest of the library

11Frequently asked

Questions about this topic

12The sources

References

Sources: primary literature, linked inline.