Hospital Practice
ISSN: 2154-8331 (Print) 2377-1003 (Online) Journal homepage: http://www.tandfonline.com/loi/ihop20
Principles of Neuromuscular Transmission A. Robert Martin To cite this article: A. Robert Martin (1992) Principles of Neuromuscular Transmission, Hospital Practice, 27:8, 147-158, DOI: 10.1080/21548331.1992.11705473 To link to this article: http://dx.doi.org/10.1080/21548331.1992.11705473
Published online: 17 May 2016.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ihop20 Download by: [RMIT University Library]
Date: 20 June 2016, At: 09:57
IPHYSllOlOCGY llN MIEDllCllNIE
Principles of Neuromuscular Transmission Downloaded by [RMIT University Library] at 09:57 20 June 2016
A. ROBERT MARTIN
University of Colorado
Activation of skeletal muscles, small and large alike, occurs when spinal cord neurons send action potentials to groups of fibers. The central role of acetylcholine and its receptors in this activation is well established. The mechanisms involved and the implications for understanding neuromuscular disorders and for rationalizing and improving therapy are discussed.
All acts of conscious movement-walking, throwing a ball, playing a piano, talking-involve activation of skeletal muscles, as motor neurons in the spinal cord send action potentials outward along motor nerves to groups of muscle fibers. An individual motor nerve may innervate only a few fibers in a small muscle or a thousand or more fibers in a large one. A large muscle contains many motor units, each comprising a motor neuron and the muscle fibers it supplies, and has a wide range of contractile strength, depending on how frequently each motor unit is activated and on progressive recruitment of motor units as the strength of a contraction increases. When action potentials in a motor nerve arrive at its terminal branches, the excitation moves through the chemical synapse ofthe neuromuscular junction into the muscle fibers. An action potential arriving in the motor nerve terminal releases acetyl-
Dr. Martin is Professor and Chairman, Department of Physiology, University of Colorado School of Medicine, Denver. This is the second article in a series on neuromuscular transmission disorders.
choline, which depolarizes the underlying muscle fiber, producing an action potential and contraction of the muscle.
The Neuromuscular Synapse The synapse consists of two main functional elements: the presynaptic nerve terminal and a postjunctional specialization in the muscle-the motor end plate (Figure 1 ). The terminal consists of numerous small unmyelinated branches partially embedded in the end-plate surface and covered with a layer of Schwann cells. The plasma membrane of the muscle end plate is thrown into regular folds under each terminal nerve branch. Piles of synaptic vesicles and associated thickenings in the presynaptic membrane, called active zones, lie over each postjunctional fold. Between the presynaptic and postsynaptic membranes is a glycoprotein layer, the basal lamina. Scanning electron microscopy reveals several important features: In the presynaptic membrane, regular rows of particles, believed to be calcium channels, line up along either side of the active zones; in the postsynaptic membrane, particles representing nicotinic acetylcholine receptors .are con-
In cooperation with the American Physiological Society Hospital Practice August 15,1992
147
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE centrated near the edges of the postjunctional folds. Acetylcholinesterase molecules are embedded in the postsynaptic membrane and the basal lamina. The general pharmacologic principles underlying neuromuscular transmission were worked out long before identification of the structural elements. By 1936, Henry Dale and his colleagues had determined that acetylcholine appears in muscle perfusates in response to motor nerve stimulation and, conversely, that acetylcholine injected into the artery supplying a muscle causes muscle contraction. In order to collect acetylcholine during nerve stimulation, they found that it was necessary to add a cholinesterase inhibitor, such as physostigmine or neostigmine, to the fluid perfusing the muscle. Furthermore, after addition of curare to the perfusate, acetylcholine could still be collected but the muscle did not contract. In other words, curare blocked the action of acetylcholine but not its release.
The End-Plate Potential Early electrical recordings from curarized muscle revealed that stimulation of the motor nerve resulted in a depolarization of the end-plate region. This depolarization was called the end-plate potential. The introduction of intracellular recording techniques using glass microelectrodes permitted detailed study ofthe end-plate potential, particularly by Bernard Katz and his colleagues. Such experiments involve placing a muscle (for example, a sartorius muscle dissected from the leg of a frog), with its attached motor nerve elevated on a pair of stimulating electrodes, in a chamber containing an isotonic salt solu148
Hospital Practice August 15, 1992
tion. As a glass electrode is advanced by a micromanipulator, penetration of the muscle fiber membrane at the end plate is signaled by the sudden appearance of a negative potential-the resting potential. In most muscle fibers, this is between -70 and -90 mV. Stimulation of the nerve fiber produces, after a short delay, an action potential in the muscle. (Usually the muscle contracts after such an action potential, dislodging the microelectrode.) The delay between the stimulation and the muscle action potential is due to two factors: 1) the time taken for the nerve action potential to travel from the stimulating electrode to the nerve terminals, and 2) a synaptic delay of about 1 msec, which is the time taken for the acetylcholine to be released from the terminal onto the end-plate membrane. There is no sign of an endplate potential in such a simple experiment because the endplate potential is of more than sufficient amplitude to trigger an action potential, which masks it. However, the end-plate potential is seen after the addition of curare because its amplitude no longer exceeds the threshold depolarization necessary to initiate an action potential; that is, nerve stimulation no longer produces muscle contraction, even though an end-plate potential is still present. If the curare concentration is increased, the end-plate potential becomes still smaller, disappearing entirely at sufficiently high concentrations. Thus, curare competes with acetylcholine for postsynaptic receptors but does not activate receptors. In other words, it is a competitive inhibitor of acetylcholine. The end-plate potential has two major electrical characteris-
tics: 1) After the initial depolarization, it decays passively at a rate determined by the electrical properties of the muscle fiber; 2) unlike an action potential, which is propagated along the whole length of the fiber, the endplate potential spreads passively away from the end-plate region and dies out over a short distance.
Acetylcholine and the Postsynaptic Membrane Why do we want to study the end-plate potential? One reason is to find out exactly how acetylcholine released from the nerve terminal produces depolarization of the muscle. Rapid depolarizations of this sort are always produced by changes in membrane permeability, usually an increase in sodium permeability. When permeability increases at the end plate, the relatively high extracellular concentration of sodium ions and the negative membrane potential drive sodium ions inward across the membrane. During inward sodium movement, the membrane becomes depolarized as the incoming positive ions accumulate on its inner surface. The intracellular concentration of sodium does not change significantly during the end-plate potential; the number of ions that accumulate on the inner surface of the membrane to change the membrane potential is infmitesimally small compared with the total number of sodium ions in the myoplasm. Voltage clamping makes it possible to record the end-plate current produced by acetylcholine rather than the change in membrane potential (Figure 2). The end-plate current, which is due mostly to sodium influx, reaches its peak during the ris-
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE
Figure 1. The two main functional components of the neuromuscular junction are the presynaptic nerve terminal and the postsynaptic end-plate region of skeletal muscle fiber. The presynaptic terminal originates from a myelin-sheathed motor neuron's axon; each terminal is overlaid by a Schwann
cell and has mitochondria and numerous acetylcholine-containing vesicles. Some vesicles are clustered over synaptic clefts in active zones; these are sites of release of acetylcholine, which crosses the synaptic cleft and basal lamina to activate specific receptors in postjunctional folds. Hospital Practice August 15, 1992
149
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE end-plate potential becomes smaller, as expected. However, increasing extracellular potassium or extracellular calcium concentrations also affects the endplate potential. It turns out that acetylcholine makes the endplate membrane permeable to a wide range of cations. The major cations normally present are sodium. potassium, and (to a much lesser extent) calcium. During the increase in permeability, sodium and a small amount of calcium move inward across the membrane to cause depolarization, which is limited to some extent by an accompanying efflux of potassium ions.
The Nicotinic Acetylcholine Receptor
Figure 2. Graphs contrast the amplitude and duration of the end-plate potential and end-plate current in the absence or presence of the anticholinesterase prostigmine. Without prostigmine (top), the end-plate current, whose duration reflects acetylcholine activity, peaks and decays relatively rapidly. Prostigmine, however, prolongs acetylcholine activity, enhancing the amplitude and duration of the end-plate current and end-plate potential (bottom).
ingphase of the end-plate potential and declines exponentially with a time constant of about 1 msec. After the current ceases, the excess positive charges accumulated on the inner face of the end-plate membrane gradually dissipate and the end-plate potential decays slowly to zero. Hydrolysis of acetylcholine by cholinesterase in the synaptic cleft normally limits both the amount of acetylcholine reaching the postsynaptic receptors and the 150
Hospital Practice August 15, 1992
duration of its action on thereceptors. When hydrolysis of acetylcholine is prevented, increases in both amplitude and duration of the end-plate current produce a corresponding increase in the end-plate potential. Anticholinesterases thus increase the effectiveness of transmission and so are important tools for treating neuromuscular defects. When extracellular sodium concentration is reduced, the
Acetylcholine increases postsynaptic cation permeability by acting on receptors in the endplate membrane. Activated acetylcholine receptors form aqueous channels, allowing cations to move through the membrane. The nicotinic acetylcholine receptor in skeletal muscle is a member of a family of channelforming proteins sensitive to acetylcholine, varieties of which are also found in eel electroplax, autonomic ganglia, and brain. (Muscarinic receptors, a different family of acetylcholine receptors, do not form channels when activated.) The nicotinic receptor family is part of a superfamily of channel-forming proteins, each activated by a specific ligand, such as y-aminobutyric acid (GABA) or glutamate. (The voltage-activated ion channels constitute another superfamily.) The nicotinic acetylcholine receptor in adult mammalian muscle is formed of five polypeptide subunits, two of them identical: a, a, ~. £, o. Each (continues)
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
(continued)
a subunit has an acetylcholine binding site, and the channel opens when both are occupied. (Receptors expressed in fetal muscles and in eel electroplaxes contain a y rather than an £ subunit and have somewhat different electrical properties.) The subunits are similar in amino acid composition; each contains several sequences of about 20 amino acids that form predominantly hydrophobic helical structures, which are embedded in the membrane lipid to form the transmembrane segments of the channel. Patch clamping. a remarkable technique developed by Erwin Neher. Bert Sakmann. and their colleagues. makes it possible to
study the responses of a single acetylcholine receptor (Figure 3). The tip of a glass pipette is brought up to the cell and by gentle suction is made to form a seal with the membrane. The exact nature of the seal between the glass and the membrane lipid is not well understood, but it is of very high resistance and effectively isolates the tiny circular patch of membrane within the pipette tip. When the fluid in the pipette is connected to a suitable amplifier, ion fluxes through a single channel can be recorded as it opens and closes. If the patch is made on an acetylcholine-sensitive membrane (such as the end plate) and the electrode contains acetylcholine, the technique can demonstrate the opening and closing of individual channels as
acetylcholine molecules bombard the receptors. Detailed observations of the behavior of acetylcholine receptors enabled investigators to make models of the steps involved in channel opening and closing. For our purposes only two observations are important: The number of channels opened determines the amplitude of the end-plate current, and the kinetics of the channels determines its time course. Under normal circumstances, each channel opens only once because acetylcholine in the synaptic cleft disappears rapidly after being released, some hydrolyzed by cholinesterase and the rest rapidly diffusing away. When an anticholinesterase is added, the acetylcholine concen-
Figure 3. Patch clamping permits individual acetylcholine receptor channels to be studied. A glass microelectrode (patch electrode) containing acetylcholine solution is pressed against the membrane of an isolated or cultured cell, and gentle suction is applied to form a seal (A). When fluid in the electrode is connected to an appropriate amplifier, ion fluxes through a single channel can be recorded as the channel opens and closes (B). Recordings typically show current pulses a few pA in amplitude of varying duration (downward deflections signify currents flowing into the cell).
Hospital Practice August 15. 1992
151
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
tration in the cleft remains relatively high for a longer period and the molecules can interact more than once with the channels, thus increasing and prolonging the end-plate current.
Quanta/ Release of Acetylcholine
Figure 4. Miniature end-plate potentials (A), each produced by release of a quan-
tum of acetylcholine at the neuromuscular junction, are the building blocks of the end-plate potential. Studies of acetylcholine release under conditions of low extracellular calcium concentrations demonstrate that end-plate potentials fluctuate according to the number of acetylcholine quanta released. In this example, the number of quanta ranges from three to none at all (B).
15 2
Hospital Practice August 15. 1992
In the early 1950s, Paul Fatt and Katz observed small depolarizing changes at irregular intervals in the end-plate region of frog muscle fibers, even when the motor nerve was not being stimulated. These potential changes had time courses similar to that of the end-plate potential but an amplitude of only 0.5 to 1.0 mV. Subsequent experiments showed that they occurred more frequently when the nerve terminals were depolarized-for example, by increasing the extracellular potassium concentration. The addition of curare to the bathing solution diminished the amplitude of the spontaneous potentials; prostigmine made them larger and longer. Thus, the small potential changes were due to the spontaneous release of small quantities of acetylcholine from the motor nerve terminals. The small potential changes became known as miniature end-plate potentials; the package of acetylcholine responsible for each miniature end-plate potential is the quantum (Figure 4). When an action potential arrives in the motor nerve termi-
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE nal, is acetylcholine released as one large package of molecules or as a large number of quanta? To approach this problem, Fatt and Katz took advantage of the fact that release of acetylcholine is diminished under conditions of low extracellular calcium. They found that the end-plate potential fluctuated in magnitude under such conditions: Sometimes there was no response at all, the smallest responses were identical in size and shape to the spontaneously occurring miniature end-plate potentials, and larger responses were multiples of this unit size. Thus, the miniature end-plate potential, produced by one quantum of acetylcholine, is the basic building block for the endplate potential. At low calcium concentrations, nerve stimulation causes the release of only a few quanta, the number varying from one trial to the next. At physiologic calcium concentration, release of 100 to 200 quanta produces a full-sized endplate potential. This large release provides a large safety factor for muscle activation, since fewer than 50 quanta are required to depolarize the muscle to threshold for the production of an action potential.
Number of Acetylcholine Molecules in a Quantum Important information about synaptic transmission has emerged from studies based on ionophoresis, the application of tiny amounts of neurotransmitter to the postsynaptic membrane from a micropipette. A nerve muscle preparation is mounted in a bath with arrangements for intracellular recording, and a micropipette filled with a solution of acetylcholine is
placed close to the postsynaptic membrane. When a positive voltage pulse is applied to the micropipette, acetylcholine (a positively charged ion) is ejected from the pipette tip. The result is an acetylcholine potential, which is like an end-plate potential except that the acetylcholine comes from the pipette rather than from the presynaptic nerve terminal. The acetylcholine potential increases in amplitude with the size of the voltage pulse. Like the end-plate potential, it is blocked by curare and increased in amplitude and duration by neostigmine. When the pipette tip is close to the postsynaptic membrane, a brief pulse can produce an acetylcholine potential that is almost indistinguishable from a spontaneous miniature end-plate potential. By calibrating the amount of acetylcholine ejected by the voltage pulse, Steve Kuffler and Doju Yoshikami deduced that the miniature end-plate potential was produced by a few thousand acetylcholine molecules. The calculated number of acetylcholine molecules in a quantum agrees well with the calculated number of acetylcholine channels activated during the miniature end-plate potential. At the muscle resting potential, the current through a single 30-picosiemen channel is about 2.5 picoamperes (2.5 X I0- 12 A). The end-plate current associated with a miniature end-plate potential is about 4 nA (4x I0-9 A), or about 1,600 times larger. So a miniature end-plate potential involves activation of about 1,600 channels, each requiring two acetylcholine molecules for activation. In round numbers, then, about 5,000 acetylcholine molecules are released in each quantum, and 3,000 or so activate
about 1,500 acetylcholine receptors to produce the miniature end-plate potential.
Vesicle Recycling It is generally believed that a quantum of acetylcholine represents the contents of one synaptic vesicle. Quantitatively, 5,000 acetylcholine molecules inside a sphere 50 nm in diameter is equivalent to a concentration of about 130 mmol/L. With an equal number of negative counterions, the vesicle contents would be nearly isotonic. Acetylcholine is transported actively into the vesicles from the cytoplasm, and 130 mmol!L is well within the capabilities we might expect from such a transport system. An alternative view is that a quantum represents the contents of several vesicles released simultaneously. In any case, vesicles release their contents by exocytosis, fusing with the nerve terminal membrane and opening into the synaptic cleft. In doing so, the vesicle membrane becomes part of the plasma membrane of the presynaptic terminal, with its inner surface facing the synaptic cleft. Some form of membrane recovery must accompany exocytosis; otherwise, the nerve terminal would have to synthesize lipid membrane continually to form new vesicles and would expand continually with each endplate potential. Without membrane recovery, a nerve terminal supplying a postural muscle of the limbs could increase its surface area by as much as 30,000 times in a single day. After the quantum is released, the vesicle membrane migrates laterally and is recovered into the nerve terminal cytoplasm near the edge of the synaptic cleft. The reHospital Practice August 15. 1992
153
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE
Figure 5. When a motor neuron is stimulated, nerve terminal vesicles filled with acetylcholine (A) migrate to the plasma membrane, form fusion pores (B), and release their con-
covered vesicle is loaded with acetylcholine and is again ready for fusion (Figure 5).
Action ofCalcium Curare and calcium block neuromuscular transmission by different mechanisms: The action of curare is postsynaptic. competing with acetylcholine for receptor sites; that of calcium is presynaptic, reducing quantal release. The differences are mirrored in different types of neuromuscular disease: myasthenia gravis, caused by a reduction in postsynaptic sensitivity to acetylcholine; and the myasthenic (or Eaton-Lambert) syndrome, resulting from reduced acetylcholine release from the motor nerve terminals. 154
Hospital Practice August 15. 1992
tents into the synaptic cleft (C). Vesicles then recover their membranes in coated form (D), rejoin the vesicle pool minus their coats (E), and refill with acetylcholine (F).
Ionophoresis experiments by Katz and Ricardo Miledi and others demonstrated that release requires the presence of calcium when the presynaptic action potential invades the nerve terminal, and that calcium enters the nerve terminal during the action potential. Furthermore, elevated extracellular magnesium, which interferes with calcium entry into the presynaptic terminal. blocks acetylcholine release. Other experiments on the neuromuscular junction showed that artificial depolarizing pulses can trigger acetylcholine release from the motor nerve terminal, even when the presynaptic action potential is blocked with tetrodotoxin. In other words, any brief depolarization will do. The effect of the depolar-
ization is to open voltage-activated calcium channels in the active zones, allowing calcium to enter the terminals. Calcium influx through the activated channels then causes exocytosis of the acetylcholine-containing synaptic vesicles by a process that is still not well understood. Other than providing the necessary depolarization, the action potential itself has no special role in therelease process. The process of synaptic transmission at the neuromuscular junction is summarized in Figure 6. It is useful to review the actions of ions and drugs as well. Presynaptically. low serum calcium concentrations inhibit acetylcholine release, and high serum magnesium inhibits calci(continuesJ
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
(continued)
urn entry into the nerve terminal. Postsynaptically, curare blocks the end-plate potential by competingwith acetylcholine for receptors. Various quaternary ammonium compounds have similar effects, and some are used as muscle relaxants in company with anesthetics. Anticholinesterases prevent hydrolysis of acetylcholine in the synaptic space, thereby enhancing and prolonging the action of the transmitter. In neuromuscular diseases, anticholinesterase can be useful in restoring synaptic transmission regardless of whether the defect is presynaptic or postsynaptic. One point sometimes overlooked is that neostigmine and other anticholinesterases, because of their structural similarity to acetylcholine, also bind to and block postsynaptic acetylcholine receptors, even at moderate concentrations ( > 10 )lmol/L); that is, they are curarizing. Care must be taken, therefore, to use appropriate therapeutic doses.
Facilitation and Depression So far, we have discussed events at the neuromuscular junction that occur when the motor nerve is stimulated one shock at a time. In real life. muscles are activated by bursts of activity in motor neurons or by continuous discharges at relatively low frequencies. Experimentally, end-plate potentials produced by repetitive stimulation of the motor nerve are not of constant amplitude but instead are altered by previous activity. End-plate potentials recorded from a curarized end plate during repetitive stimulation of the motor nerve increase, then de-
crease in amplitude (Figure 7). When a steady state is reached near the end ofthe train of stimuli, the amplitude of the endplate potential is less than half that of the first response. The initial increase is called facilitation, the later decrease depression. Both are due to changes in the release of acetylcholine, not to changes in sensitivity of the postsynaptic receptors. Facilitation results from a more effective release process. Somehow the calcium entering the terminal with each stimulus becomes progressively more effective in causing vesicle fusion and exocytosis. It is generally believed that this increased effectiveness is due to accumulation of calcium in the terminal; that is, each incoming bolus of calcium sums with residual calcium from the previous stimuli to augment vesicle fusion. Depression is less complicated. It occurs, for the most part, as the active zones begin to run out of vesicles. Thus, even though the release process remains effective, the end-plate potential amplitude falls. A steady state is reached when the rate at which vesicles are released falls to the rate at which they can be replaced in the active regions. To summarize, during repetitive stimulation the processes of facilitation and depression interact. Facilitation causes an increase in the ability of the nerve terminal to release vesicles, increasing the end-plate potential amplitude. This increased ability persists but is soon overridden by depletion of vesicles in the release zones, causing a depression of end-plate potential amplitude. During a sustained contraction in a human limb muscle, the end-plate potential amplitudes may drop to 30% of their initial level. If the neuroHospital Practice August 15, 1992
155
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
Figure 7. When a motor nerve to a curarized muscle fiber is exposed to a brief train of stimuli, end-plate potential amplitudes first increase and then decrease (A). The increase, called facilitation, is due to an increase in the number of vesicles that release acetylcholine; the decrease reflects a depressed vesicle pool. After a longer series of stimuli (B), single shocks produce end-plate potentials that are less than (at 10 seconds), equivalent to (at 30 seconds), or greater than (at 2 to 10 minutes) the control amplitude. This prolonged increase in amplitude is called potentiation. Both facilitation and potentiation are believed to involve calcium accumulation in the nerve terminal.
muscular synapses are normal, the end-plate potential amplitudes are still well above threshold for initiation of action potentials in the muscle fibers, and muscle contraction is maintained. In neuromuscular disease-for example, myasthenia gravis-the end-plate potential amplitudes may fall below threshold, and neuromuscular block will ensue.
Post-Tetanic Potentiation Facilitation and depression are examples of synaptic modulation. A more dramatic example is post-tetanic potentiation, which is an increase in synaptic potential amplitude after a peri(continuesJ
156
Hospital Practice August 15. 1992
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
(continued)
od of repetitive stimulation. Normally, this increase lasts for several minutes (Figure 7). Like facilitation, post-tetanic potentiation involves accumulation of calcium in the presynaptic nerve terminal. If calcium is removed from the bathing solution during the tetanus and then quickly replaced, post-tetanic potentiation does not occur. If the motor nerve terminals are depolarized repetitively at high extracellular calcium concentrations, the resulting post-tetanic potentiation can last for several hours. The detailed mechanisms underlying this process are not understood. The neuromuscular junction is typical of excitatory chemical synapses, in which a neurotransmitter released from the presynaptic nerve terminal produces a postsynaptic depolarization-in muscle, the end-plate potential. The transmitter. in this case acetylcholine, is packaged in presynaptic vesicles
gathered over release zones in the presynaptic membrane. On activation, the vesicles release their contents by exocytosis. and the fused vesicle membrane is recycled back into the presynaptic vesicle pool. Vesicle fusion is caused by an increase in presynaptic calcium concentration in the release zones when an action potential depolarizes the motor nerve terminal. opening voltageactivated calcium channels. Exocytosis of a single synaptic vesicle releases about 5,000 molecules of acetylcholine (the quantum of transmitter). Acetylcholine released from the presynaptic terminal acts on nicotinic receptors in the postsynaptic membrane and is hydrolyzed by cholinesterase in the synaptic cleft. The postsynaptic receptors are transmembrane proteins that. when activated, open to form aqueous pores in the membrane that are permeable to small cations. 1\vo acetylcholine molecules are required to open a single channel. Under
Selected Reading Nicholls JG, Martin AR, Wallace GB: From Neuron to Brain: A Molecular and Cellular Approach to the Function ofthe Nervous System. Sinauer, Sunderland, Mass, 1992, chap 2, 7, 9 Boyd lA, Martin AR: The end plate potential in mammalian muscle. J Physlol132:74, 1956 Takeuchi A, Takeuchi N: On the permeability of the end-plate membrane during the action of transmitter. J Physlol 154: 52, 1960 Kuffler SW, Yoshikami D: The number of acetylcholine molecules In a quantum: An estimate from iontophoretic application of acetylcholine at the neuromuscular junction. J Phys1ol251 : 465, 1975 Hamill OP et al: Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch391 :85. 1981 Unwin N: The structure of ion channels In excitable cells. Neuron 3 : 665, 1989 Dan! JA: SUe-directed mutagenesis and single channel currents define the tonic channel of the nicotinic acetylcholine receptor. Trends Neurosci 12: 127, 1989
158
Hospital Practice August 15, 1992
normal ionic conditions, channel activation results in entry of sodium across the end-plate membrane, causing depolarization. A single quantum of acetylcholine activates about 1,500 receptors to produce the miniature end-plate potential. The endplate potential in vertebrate skeletal muscle results from the simultaneous release of 100 to 200 quanta of acetylcholine. Quanta! release of transmitter from the presynaptic nerve terminal varies during repetitive activation, as would occur with limb movement or postural adjustments. During such activity, an increase in the ability of the terminal to release transmitter (facilitation) coincides with depletion of the vesicle supply at the release zones, eventually producing a reduction in release (depression). After prolonged repetitive activity, recovery from depression is followed by posttetanic potentiation, which lasts for several minutes. Various changes in extracellular fluid ion composition and a number of pharmacologic agents affect transmission at the neuromuscular junction. Reduced extracellular calcium or elevated serum magnesium reduces quantal release. In clinical conditions, such changes usually have no significant effect on neuromuscular synapses if they are otherwise normal, as the nerve terminal releases many more quanta than are necessary to produce muscle contraction. On the postsynaptic side of the junction, curare-like agents are commonly used to produce muscle relaxation during anesthesia. In states of neuromuscular impairment, anticholinesterases can improve transmission considerably by protecting acetylcholine from hydrolysis. D
ISSN: 2154-8331 (Print) 2377-1003 (Online) Journal homepage: http://www.tandfonline.com/loi/ihop20
Principles of Neuromuscular Transmission A. Robert Martin To cite this article: A. Robert Martin (1992) Principles of Neuromuscular Transmission, Hospital Practice, 27:8, 147-158, DOI: 10.1080/21548331.1992.11705473 To link to this article: http://dx.doi.org/10.1080/21548331.1992.11705473
Published online: 17 May 2016.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ihop20 Download by: [RMIT University Library]
Date: 20 June 2016, At: 09:57
IPHYSllOlOCGY llN MIEDllCllNIE
Principles of Neuromuscular Transmission Downloaded by [RMIT University Library] at 09:57 20 June 2016
A. ROBERT MARTIN
University of Colorado
Activation of skeletal muscles, small and large alike, occurs when spinal cord neurons send action potentials to groups of fibers. The central role of acetylcholine and its receptors in this activation is well established. The mechanisms involved and the implications for understanding neuromuscular disorders and for rationalizing and improving therapy are discussed.
All acts of conscious movement-walking, throwing a ball, playing a piano, talking-involve activation of skeletal muscles, as motor neurons in the spinal cord send action potentials outward along motor nerves to groups of muscle fibers. An individual motor nerve may innervate only a few fibers in a small muscle or a thousand or more fibers in a large one. A large muscle contains many motor units, each comprising a motor neuron and the muscle fibers it supplies, and has a wide range of contractile strength, depending on how frequently each motor unit is activated and on progressive recruitment of motor units as the strength of a contraction increases. When action potentials in a motor nerve arrive at its terminal branches, the excitation moves through the chemical synapse ofthe neuromuscular junction into the muscle fibers. An action potential arriving in the motor nerve terminal releases acetyl-
Dr. Martin is Professor and Chairman, Department of Physiology, University of Colorado School of Medicine, Denver. This is the second article in a series on neuromuscular transmission disorders.
choline, which depolarizes the underlying muscle fiber, producing an action potential and contraction of the muscle.
The Neuromuscular Synapse The synapse consists of two main functional elements: the presynaptic nerve terminal and a postjunctional specialization in the muscle-the motor end plate (Figure 1 ). The terminal consists of numerous small unmyelinated branches partially embedded in the end-plate surface and covered with a layer of Schwann cells. The plasma membrane of the muscle end plate is thrown into regular folds under each terminal nerve branch. Piles of synaptic vesicles and associated thickenings in the presynaptic membrane, called active zones, lie over each postjunctional fold. Between the presynaptic and postsynaptic membranes is a glycoprotein layer, the basal lamina. Scanning electron microscopy reveals several important features: In the presynaptic membrane, regular rows of particles, believed to be calcium channels, line up along either side of the active zones; in the postsynaptic membrane, particles representing nicotinic acetylcholine receptors .are con-
In cooperation with the American Physiological Society Hospital Practice August 15,1992
147
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE centrated near the edges of the postjunctional folds. Acetylcholinesterase molecules are embedded in the postsynaptic membrane and the basal lamina. The general pharmacologic principles underlying neuromuscular transmission were worked out long before identification of the structural elements. By 1936, Henry Dale and his colleagues had determined that acetylcholine appears in muscle perfusates in response to motor nerve stimulation and, conversely, that acetylcholine injected into the artery supplying a muscle causes muscle contraction. In order to collect acetylcholine during nerve stimulation, they found that it was necessary to add a cholinesterase inhibitor, such as physostigmine or neostigmine, to the fluid perfusing the muscle. Furthermore, after addition of curare to the perfusate, acetylcholine could still be collected but the muscle did not contract. In other words, curare blocked the action of acetylcholine but not its release.
The End-Plate Potential Early electrical recordings from curarized muscle revealed that stimulation of the motor nerve resulted in a depolarization of the end-plate region. This depolarization was called the end-plate potential. The introduction of intracellular recording techniques using glass microelectrodes permitted detailed study ofthe end-plate potential, particularly by Bernard Katz and his colleagues. Such experiments involve placing a muscle (for example, a sartorius muscle dissected from the leg of a frog), with its attached motor nerve elevated on a pair of stimulating electrodes, in a chamber containing an isotonic salt solu148
Hospital Practice August 15, 1992
tion. As a glass electrode is advanced by a micromanipulator, penetration of the muscle fiber membrane at the end plate is signaled by the sudden appearance of a negative potential-the resting potential. In most muscle fibers, this is between -70 and -90 mV. Stimulation of the nerve fiber produces, after a short delay, an action potential in the muscle. (Usually the muscle contracts after such an action potential, dislodging the microelectrode.) The delay between the stimulation and the muscle action potential is due to two factors: 1) the time taken for the nerve action potential to travel from the stimulating electrode to the nerve terminals, and 2) a synaptic delay of about 1 msec, which is the time taken for the acetylcholine to be released from the terminal onto the end-plate membrane. There is no sign of an endplate potential in such a simple experiment because the endplate potential is of more than sufficient amplitude to trigger an action potential, which masks it. However, the end-plate potential is seen after the addition of curare because its amplitude no longer exceeds the threshold depolarization necessary to initiate an action potential; that is, nerve stimulation no longer produces muscle contraction, even though an end-plate potential is still present. If the curare concentration is increased, the end-plate potential becomes still smaller, disappearing entirely at sufficiently high concentrations. Thus, curare competes with acetylcholine for postsynaptic receptors but does not activate receptors. In other words, it is a competitive inhibitor of acetylcholine. The end-plate potential has two major electrical characteris-
tics: 1) After the initial depolarization, it decays passively at a rate determined by the electrical properties of the muscle fiber; 2) unlike an action potential, which is propagated along the whole length of the fiber, the endplate potential spreads passively away from the end-plate region and dies out over a short distance.
Acetylcholine and the Postsynaptic Membrane Why do we want to study the end-plate potential? One reason is to find out exactly how acetylcholine released from the nerve terminal produces depolarization of the muscle. Rapid depolarizations of this sort are always produced by changes in membrane permeability, usually an increase in sodium permeability. When permeability increases at the end plate, the relatively high extracellular concentration of sodium ions and the negative membrane potential drive sodium ions inward across the membrane. During inward sodium movement, the membrane becomes depolarized as the incoming positive ions accumulate on its inner surface. The intracellular concentration of sodium does not change significantly during the end-plate potential; the number of ions that accumulate on the inner surface of the membrane to change the membrane potential is infmitesimally small compared with the total number of sodium ions in the myoplasm. Voltage clamping makes it possible to record the end-plate current produced by acetylcholine rather than the change in membrane potential (Figure 2). The end-plate current, which is due mostly to sodium influx, reaches its peak during the ris-
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE
Figure 1. The two main functional components of the neuromuscular junction are the presynaptic nerve terminal and the postsynaptic end-plate region of skeletal muscle fiber. The presynaptic terminal originates from a myelin-sheathed motor neuron's axon; each terminal is overlaid by a Schwann
cell and has mitochondria and numerous acetylcholine-containing vesicles. Some vesicles are clustered over synaptic clefts in active zones; these are sites of release of acetylcholine, which crosses the synaptic cleft and basal lamina to activate specific receptors in postjunctional folds. Hospital Practice August 15, 1992
149
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE end-plate potential becomes smaller, as expected. However, increasing extracellular potassium or extracellular calcium concentrations also affects the endplate potential. It turns out that acetylcholine makes the endplate membrane permeable to a wide range of cations. The major cations normally present are sodium. potassium, and (to a much lesser extent) calcium. During the increase in permeability, sodium and a small amount of calcium move inward across the membrane to cause depolarization, which is limited to some extent by an accompanying efflux of potassium ions.
The Nicotinic Acetylcholine Receptor
Figure 2. Graphs contrast the amplitude and duration of the end-plate potential and end-plate current in the absence or presence of the anticholinesterase prostigmine. Without prostigmine (top), the end-plate current, whose duration reflects acetylcholine activity, peaks and decays relatively rapidly. Prostigmine, however, prolongs acetylcholine activity, enhancing the amplitude and duration of the end-plate current and end-plate potential (bottom).
ingphase of the end-plate potential and declines exponentially with a time constant of about 1 msec. After the current ceases, the excess positive charges accumulated on the inner face of the end-plate membrane gradually dissipate and the end-plate potential decays slowly to zero. Hydrolysis of acetylcholine by cholinesterase in the synaptic cleft normally limits both the amount of acetylcholine reaching the postsynaptic receptors and the 150
Hospital Practice August 15, 1992
duration of its action on thereceptors. When hydrolysis of acetylcholine is prevented, increases in both amplitude and duration of the end-plate current produce a corresponding increase in the end-plate potential. Anticholinesterases thus increase the effectiveness of transmission and so are important tools for treating neuromuscular defects. When extracellular sodium concentration is reduced, the
Acetylcholine increases postsynaptic cation permeability by acting on receptors in the endplate membrane. Activated acetylcholine receptors form aqueous channels, allowing cations to move through the membrane. The nicotinic acetylcholine receptor in skeletal muscle is a member of a family of channelforming proteins sensitive to acetylcholine, varieties of which are also found in eel electroplax, autonomic ganglia, and brain. (Muscarinic receptors, a different family of acetylcholine receptors, do not form channels when activated.) The nicotinic receptor family is part of a superfamily of channel-forming proteins, each activated by a specific ligand, such as y-aminobutyric acid (GABA) or glutamate. (The voltage-activated ion channels constitute another superfamily.) The nicotinic acetylcholine receptor in adult mammalian muscle is formed of five polypeptide subunits, two of them identical: a, a, ~. £, o. Each (continues)
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
(continued)
a subunit has an acetylcholine binding site, and the channel opens when both are occupied. (Receptors expressed in fetal muscles and in eel electroplaxes contain a y rather than an £ subunit and have somewhat different electrical properties.) The subunits are similar in amino acid composition; each contains several sequences of about 20 amino acids that form predominantly hydrophobic helical structures, which are embedded in the membrane lipid to form the transmembrane segments of the channel. Patch clamping. a remarkable technique developed by Erwin Neher. Bert Sakmann. and their colleagues. makes it possible to
study the responses of a single acetylcholine receptor (Figure 3). The tip of a glass pipette is brought up to the cell and by gentle suction is made to form a seal with the membrane. The exact nature of the seal between the glass and the membrane lipid is not well understood, but it is of very high resistance and effectively isolates the tiny circular patch of membrane within the pipette tip. When the fluid in the pipette is connected to a suitable amplifier, ion fluxes through a single channel can be recorded as it opens and closes. If the patch is made on an acetylcholine-sensitive membrane (such as the end plate) and the electrode contains acetylcholine, the technique can demonstrate the opening and closing of individual channels as
acetylcholine molecules bombard the receptors. Detailed observations of the behavior of acetylcholine receptors enabled investigators to make models of the steps involved in channel opening and closing. For our purposes only two observations are important: The number of channels opened determines the amplitude of the end-plate current, and the kinetics of the channels determines its time course. Under normal circumstances, each channel opens only once because acetylcholine in the synaptic cleft disappears rapidly after being released, some hydrolyzed by cholinesterase and the rest rapidly diffusing away. When an anticholinesterase is added, the acetylcholine concen-
Figure 3. Patch clamping permits individual acetylcholine receptor channels to be studied. A glass microelectrode (patch electrode) containing acetylcholine solution is pressed against the membrane of an isolated or cultured cell, and gentle suction is applied to form a seal (A). When fluid in the electrode is connected to an appropriate amplifier, ion fluxes through a single channel can be recorded as the channel opens and closes (B). Recordings typically show current pulses a few pA in amplitude of varying duration (downward deflections signify currents flowing into the cell).
Hospital Practice August 15. 1992
151
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
tration in the cleft remains relatively high for a longer period and the molecules can interact more than once with the channels, thus increasing and prolonging the end-plate current.
Quanta/ Release of Acetylcholine
Figure 4. Miniature end-plate potentials (A), each produced by release of a quan-
tum of acetylcholine at the neuromuscular junction, are the building blocks of the end-plate potential. Studies of acetylcholine release under conditions of low extracellular calcium concentrations demonstrate that end-plate potentials fluctuate according to the number of acetylcholine quanta released. In this example, the number of quanta ranges from three to none at all (B).
15 2
Hospital Practice August 15. 1992
In the early 1950s, Paul Fatt and Katz observed small depolarizing changes at irregular intervals in the end-plate region of frog muscle fibers, even when the motor nerve was not being stimulated. These potential changes had time courses similar to that of the end-plate potential but an amplitude of only 0.5 to 1.0 mV. Subsequent experiments showed that they occurred more frequently when the nerve terminals were depolarized-for example, by increasing the extracellular potassium concentration. The addition of curare to the bathing solution diminished the amplitude of the spontaneous potentials; prostigmine made them larger and longer. Thus, the small potential changes were due to the spontaneous release of small quantities of acetylcholine from the motor nerve terminals. The small potential changes became known as miniature end-plate potentials; the package of acetylcholine responsible for each miniature end-plate potential is the quantum (Figure 4). When an action potential arrives in the motor nerve termi-
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE nal, is acetylcholine released as one large package of molecules or as a large number of quanta? To approach this problem, Fatt and Katz took advantage of the fact that release of acetylcholine is diminished under conditions of low extracellular calcium. They found that the end-plate potential fluctuated in magnitude under such conditions: Sometimes there was no response at all, the smallest responses were identical in size and shape to the spontaneously occurring miniature end-plate potentials, and larger responses were multiples of this unit size. Thus, the miniature end-plate potential, produced by one quantum of acetylcholine, is the basic building block for the endplate potential. At low calcium concentrations, nerve stimulation causes the release of only a few quanta, the number varying from one trial to the next. At physiologic calcium concentration, release of 100 to 200 quanta produces a full-sized endplate potential. This large release provides a large safety factor for muscle activation, since fewer than 50 quanta are required to depolarize the muscle to threshold for the production of an action potential.
Number of Acetylcholine Molecules in a Quantum Important information about synaptic transmission has emerged from studies based on ionophoresis, the application of tiny amounts of neurotransmitter to the postsynaptic membrane from a micropipette. A nerve muscle preparation is mounted in a bath with arrangements for intracellular recording, and a micropipette filled with a solution of acetylcholine is
placed close to the postsynaptic membrane. When a positive voltage pulse is applied to the micropipette, acetylcholine (a positively charged ion) is ejected from the pipette tip. The result is an acetylcholine potential, which is like an end-plate potential except that the acetylcholine comes from the pipette rather than from the presynaptic nerve terminal. The acetylcholine potential increases in amplitude with the size of the voltage pulse. Like the end-plate potential, it is blocked by curare and increased in amplitude and duration by neostigmine. When the pipette tip is close to the postsynaptic membrane, a brief pulse can produce an acetylcholine potential that is almost indistinguishable from a spontaneous miniature end-plate potential. By calibrating the amount of acetylcholine ejected by the voltage pulse, Steve Kuffler and Doju Yoshikami deduced that the miniature end-plate potential was produced by a few thousand acetylcholine molecules. The calculated number of acetylcholine molecules in a quantum agrees well with the calculated number of acetylcholine channels activated during the miniature end-plate potential. At the muscle resting potential, the current through a single 30-picosiemen channel is about 2.5 picoamperes (2.5 X I0- 12 A). The end-plate current associated with a miniature end-plate potential is about 4 nA (4x I0-9 A), or about 1,600 times larger. So a miniature end-plate potential involves activation of about 1,600 channels, each requiring two acetylcholine molecules for activation. In round numbers, then, about 5,000 acetylcholine molecules are released in each quantum, and 3,000 or so activate
about 1,500 acetylcholine receptors to produce the miniature end-plate potential.
Vesicle Recycling It is generally believed that a quantum of acetylcholine represents the contents of one synaptic vesicle. Quantitatively, 5,000 acetylcholine molecules inside a sphere 50 nm in diameter is equivalent to a concentration of about 130 mmol/L. With an equal number of negative counterions, the vesicle contents would be nearly isotonic. Acetylcholine is transported actively into the vesicles from the cytoplasm, and 130 mmol!L is well within the capabilities we might expect from such a transport system. An alternative view is that a quantum represents the contents of several vesicles released simultaneously. In any case, vesicles release their contents by exocytosis, fusing with the nerve terminal membrane and opening into the synaptic cleft. In doing so, the vesicle membrane becomes part of the plasma membrane of the presynaptic terminal, with its inner surface facing the synaptic cleft. Some form of membrane recovery must accompany exocytosis; otherwise, the nerve terminal would have to synthesize lipid membrane continually to form new vesicles and would expand continually with each endplate potential. Without membrane recovery, a nerve terminal supplying a postural muscle of the limbs could increase its surface area by as much as 30,000 times in a single day. After the quantum is released, the vesicle membrane migrates laterally and is recovered into the nerve terminal cytoplasm near the edge of the synaptic cleft. The reHospital Practice August 15. 1992
153
Downloaded by [RMIT University Library] at 09:57 20 June 2016
PHYSIOLOGY IN MEDICINE
Figure 5. When a motor neuron is stimulated, nerve terminal vesicles filled with acetylcholine (A) migrate to the plasma membrane, form fusion pores (B), and release their con-
covered vesicle is loaded with acetylcholine and is again ready for fusion (Figure 5).
Action ofCalcium Curare and calcium block neuromuscular transmission by different mechanisms: The action of curare is postsynaptic. competing with acetylcholine for receptor sites; that of calcium is presynaptic, reducing quantal release. The differences are mirrored in different types of neuromuscular disease: myasthenia gravis, caused by a reduction in postsynaptic sensitivity to acetylcholine; and the myasthenic (or Eaton-Lambert) syndrome, resulting from reduced acetylcholine release from the motor nerve terminals. 154
Hospital Practice August 15. 1992
tents into the synaptic cleft (C). Vesicles then recover their membranes in coated form (D), rejoin the vesicle pool minus their coats (E), and refill with acetylcholine (F).
Ionophoresis experiments by Katz and Ricardo Miledi and others demonstrated that release requires the presence of calcium when the presynaptic action potential invades the nerve terminal, and that calcium enters the nerve terminal during the action potential. Furthermore, elevated extracellular magnesium, which interferes with calcium entry into the presynaptic terminal. blocks acetylcholine release. Other experiments on the neuromuscular junction showed that artificial depolarizing pulses can trigger acetylcholine release from the motor nerve terminal, even when the presynaptic action potential is blocked with tetrodotoxin. In other words, any brief depolarization will do. The effect of the depolar-
ization is to open voltage-activated calcium channels in the active zones, allowing calcium to enter the terminals. Calcium influx through the activated channels then causes exocytosis of the acetylcholine-containing synaptic vesicles by a process that is still not well understood. Other than providing the necessary depolarization, the action potential itself has no special role in therelease process. The process of synaptic transmission at the neuromuscular junction is summarized in Figure 6. It is useful to review the actions of ions and drugs as well. Presynaptically. low serum calcium concentrations inhibit acetylcholine release, and high serum magnesium inhibits calci(continuesJ
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
(continued)
urn entry into the nerve terminal. Postsynaptically, curare blocks the end-plate potential by competingwith acetylcholine for receptors. Various quaternary ammonium compounds have similar effects, and some are used as muscle relaxants in company with anesthetics. Anticholinesterases prevent hydrolysis of acetylcholine in the synaptic space, thereby enhancing and prolonging the action of the transmitter. In neuromuscular diseases, anticholinesterase can be useful in restoring synaptic transmission regardless of whether the defect is presynaptic or postsynaptic. One point sometimes overlooked is that neostigmine and other anticholinesterases, because of their structural similarity to acetylcholine, also bind to and block postsynaptic acetylcholine receptors, even at moderate concentrations ( > 10 )lmol/L); that is, they are curarizing. Care must be taken, therefore, to use appropriate therapeutic doses.
Facilitation and Depression So far, we have discussed events at the neuromuscular junction that occur when the motor nerve is stimulated one shock at a time. In real life. muscles are activated by bursts of activity in motor neurons or by continuous discharges at relatively low frequencies. Experimentally, end-plate potentials produced by repetitive stimulation of the motor nerve are not of constant amplitude but instead are altered by previous activity. End-plate potentials recorded from a curarized end plate during repetitive stimulation of the motor nerve increase, then de-
crease in amplitude (Figure 7). When a steady state is reached near the end ofthe train of stimuli, the amplitude of the endplate potential is less than half that of the first response. The initial increase is called facilitation, the later decrease depression. Both are due to changes in the release of acetylcholine, not to changes in sensitivity of the postsynaptic receptors. Facilitation results from a more effective release process. Somehow the calcium entering the terminal with each stimulus becomes progressively more effective in causing vesicle fusion and exocytosis. It is generally believed that this increased effectiveness is due to accumulation of calcium in the terminal; that is, each incoming bolus of calcium sums with residual calcium from the previous stimuli to augment vesicle fusion. Depression is less complicated. It occurs, for the most part, as the active zones begin to run out of vesicles. Thus, even though the release process remains effective, the end-plate potential amplitude falls. A steady state is reached when the rate at which vesicles are released falls to the rate at which they can be replaced in the active regions. To summarize, during repetitive stimulation the processes of facilitation and depression interact. Facilitation causes an increase in the ability of the nerve terminal to release vesicles, increasing the end-plate potential amplitude. This increased ability persists but is soon overridden by depletion of vesicles in the release zones, causing a depression of end-plate potential amplitude. During a sustained contraction in a human limb muscle, the end-plate potential amplitudes may drop to 30% of their initial level. If the neuroHospital Practice August 15, 1992
155
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
Figure 7. When a motor nerve to a curarized muscle fiber is exposed to a brief train of stimuli, end-plate potential amplitudes first increase and then decrease (A). The increase, called facilitation, is due to an increase in the number of vesicles that release acetylcholine; the decrease reflects a depressed vesicle pool. After a longer series of stimuli (B), single shocks produce end-plate potentials that are less than (at 10 seconds), equivalent to (at 30 seconds), or greater than (at 2 to 10 minutes) the control amplitude. This prolonged increase in amplitude is called potentiation. Both facilitation and potentiation are believed to involve calcium accumulation in the nerve terminal.
muscular synapses are normal, the end-plate potential amplitudes are still well above threshold for initiation of action potentials in the muscle fibers, and muscle contraction is maintained. In neuromuscular disease-for example, myasthenia gravis-the end-plate potential amplitudes may fall below threshold, and neuromuscular block will ensue.
Post-Tetanic Potentiation Facilitation and depression are examples of synaptic modulation. A more dramatic example is post-tetanic potentiation, which is an increase in synaptic potential amplitude after a peri(continuesJ
156
Hospital Practice August 15. 1992
PHYSIOLOGY IN MEDICINE
Downloaded by [RMIT University Library] at 09:57 20 June 2016
(continued)
od of repetitive stimulation. Normally, this increase lasts for several minutes (Figure 7). Like facilitation, post-tetanic potentiation involves accumulation of calcium in the presynaptic nerve terminal. If calcium is removed from the bathing solution during the tetanus and then quickly replaced, post-tetanic potentiation does not occur. If the motor nerve terminals are depolarized repetitively at high extracellular calcium concentrations, the resulting post-tetanic potentiation can last for several hours. The detailed mechanisms underlying this process are not understood. The neuromuscular junction is typical of excitatory chemical synapses, in which a neurotransmitter released from the presynaptic nerve terminal produces a postsynaptic depolarization-in muscle, the end-plate potential. The transmitter. in this case acetylcholine, is packaged in presynaptic vesicles
gathered over release zones in the presynaptic membrane. On activation, the vesicles release their contents by exocytosis. and the fused vesicle membrane is recycled back into the presynaptic vesicle pool. Vesicle fusion is caused by an increase in presynaptic calcium concentration in the release zones when an action potential depolarizes the motor nerve terminal. opening voltageactivated calcium channels. Exocytosis of a single synaptic vesicle releases about 5,000 molecules of acetylcholine (the quantum of transmitter). Acetylcholine released from the presynaptic terminal acts on nicotinic receptors in the postsynaptic membrane and is hydrolyzed by cholinesterase in the synaptic cleft. The postsynaptic receptors are transmembrane proteins that. when activated, open to form aqueous pores in the membrane that are permeable to small cations. 1\vo acetylcholine molecules are required to open a single channel. Under
Selected Reading Nicholls JG, Martin AR, Wallace GB: From Neuron to Brain: A Molecular and Cellular Approach to the Function ofthe Nervous System. Sinauer, Sunderland, Mass, 1992, chap 2, 7, 9 Boyd lA, Martin AR: The end plate potential in mammalian muscle. J Physlol132:74, 1956 Takeuchi A, Takeuchi N: On the permeability of the end-plate membrane during the action of transmitter. J Physlol 154: 52, 1960 Kuffler SW, Yoshikami D: The number of acetylcholine molecules In a quantum: An estimate from iontophoretic application of acetylcholine at the neuromuscular junction. J Phys1ol251 : 465, 1975 Hamill OP et al: Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch391 :85. 1981 Unwin N: The structure of ion channels In excitable cells. Neuron 3 : 665, 1989 Dan! JA: SUe-directed mutagenesis and single channel currents define the tonic channel of the nicotinic acetylcholine receptor. Trends Neurosci 12: 127, 1989
158
Hospital Practice August 15, 1992
normal ionic conditions, channel activation results in entry of sodium across the end-plate membrane, causing depolarization. A single quantum of acetylcholine activates about 1,500 receptors to produce the miniature end-plate potential. The endplate potential in vertebrate skeletal muscle results from the simultaneous release of 100 to 200 quanta of acetylcholine. Quanta! release of transmitter from the presynaptic nerve terminal varies during repetitive activation, as would occur with limb movement or postural adjustments. During such activity, an increase in the ability of the terminal to release transmitter (facilitation) coincides with depletion of the vesicle supply at the release zones, eventually producing a reduction in release (depression). After prolonged repetitive activity, recovery from depression is followed by posttetanic potentiation, which lasts for several minutes. Various changes in extracellular fluid ion composition and a number of pharmacologic agents affect transmission at the neuromuscular junction. Reduced extracellular calcium or elevated serum magnesium reduces quantal release. In clinical conditions, such changes usually have no significant effect on neuromuscular synapses if they are otherwise normal, as the nerve terminal releases many more quanta than are necessary to produce muscle contraction. On the postsynaptic side of the junction, curare-like agents are commonly used to produce muscle relaxation during anesthesia. In states of neuromuscular impairment, anticholinesterases can improve transmission considerably by protecting acetylcholine from hydrolysis. D
