NEUROMUSCULAR PHYSIOLOGY Dr Elizabeth Joseph Govt Medical College

Neuromuscular junction is the region of approximation between a motor neuron and a muscle cell at which an electrical impulse is converted into muscle action potential and contraction by chemical transmitter. Skeletal muscle is innervated by nerve fibres that have their cell bodies in the anterior horn of the spinal cord or in the brain stem. Near its ending, each nerve fibre branches and each branch ends in apposition to a muscle fibre at the neuromuscular junction. The nerve fibre together with the fascicule of muscle fibre it innervates is called a motor unit. The myelin sheath surrounding the motor axon ends near the surface of the muscle fibre. The nerve is separated from the surface of the muscle by a gap of approximately 20nm called the junctional or synaptic cleft. The muscle surface is heavily corrugated with deep invaginations of the junctional cleft, primary and secondary clefts, thus increasing the total surface area. The shoulders of the folds are densely populated with acetylcholine receptors and the depths contain sodium channels. All muscle cells in a unit are excited by a single neuron, thus stimulation of a nerve causes all muscle cells in the motor unit to contract synchronously. Most adult human muscles have only one neuromuscular junction per cell with the exception of some cells in the extraocular muscles. The ocular muscles contract and relax slowly and they maintain a steady contracture holding the eye steadily in position. The depolarizing muscle relaxants cause a long lasting contracture that pulls the eye against the orbit and could increase intraocular tension. The perijunctional zone is the area of muscle immediately beyond the junctional area. It contains a smaller density of acetyl choline receptors and a high density of sodium channels. The perijunctional receptors respond to the depolarization produced by acetyl choline receptors and transduce it into a wave of depolarization that travel along the muscle to initiate muscle contraction. In the nerve endings vesicles are congregated towards the junctional surface, microtubules, mitochondria and other support structures are located towards the opposite side. The vesicles are present as clusters in the active zones, alongside which are the voltage gated calcium channels which allow calcium to enter the nerve and cause release of vesicles. When electrical potentials are measured across the endplate, occasional spontaneous depolarizing potentials occur, these are miniature end plate potentials (MEPP) and correlate with release of acetylcholine from single vesicles. The sum of many quanta of acetylcholine released simultaneously by a nerve impulse creates the end plate potential. Acetyl choline is synthesized from choline and acetate brought about by the enzyme choline acetyltransferase. Choline is obtained from the extracellular fluid and acetyl coenzyme A from the mitochondria. During a nerve action potential, sodium from outside flows across the membrane, and the resulting depolarizing voltage opens calcium channels, which allows entry of calcium ions into the nerve and

causes acetylcholine to be released. Calcium channels include faster P channels and slower L channels. P channels are found only in the nerve terminals and are located immediately adjacent to the active zones. They are voltage dependent and are responsible for normal release of transmitter. The slower L channels are located mainly in the cardiovascular system. The number of quanta released by a stimulated nerve is influenced by the concentration of ionized calcium in extracellular fluid. Outward flux of potassium returns the membrane potential to normal. Voltage gated and calcium activated potassium channels lie along the nerve terminal alongside the calcium channels. There are two pools of vesicles that release acetylcholine, a readily releasable pool and a reserve pool. Calcium ions enter the nerve through P channels and move a very short distance to encounter a vesicle and activate proteins in the vesicle wall. The activated protein reacts with the nerve membrane to form a pore through which acetylcholine is discharged into the junctional cleft. The larger reserve vesicles are firmly tethered to the cytoskeleton by actin, synapsin, synaptotagmin and spectrin. They replace worn out vesicles and may also participate in transmission when nerve is stimulated at high frequencies or for a long time. Here, calcium may enter nerve through the L channels. Acetyl choline is released by a process of exocytosis into the synaptic cleft. SNARE proteins (soluble N-ethylmaleimidesensitive attachment protein receptors) proteins are involved in fusion, docking, and release of acetylcholine at the active zone. They include synaptobrevin, syntaxin and SNAP 25. Syntaxin and SNAP 25 are attached to the plasma membranes, synaptobrevin is present on the vesicle. Synaptotagmin is the protein on the vesicular membrane that acts as a calcium sensor, localizes the synaptic vesicles to synaptic zones rich in calcium channels, and stabilizes the vesicles in the docked state. When calcium enters the nerve synaptobrevin forms a tertiary complex with syntaxin and SNAP25. The tertiary complex forces the vesicle in close apposition to the nerve membrane at the active zone with release of its contents, acetylcholine. The acetylcholine released from the nerve diffuses across the junctional cleft and reacts with specialized receptor proteins in the end plate to initiate muscle contraction. Transmitter molecules that do not react immediately with a receptor or those released after binding to the receptor are destroyed almost instantly by acetyl cholinesterase in the junctional cleft. It is a type B carboxyl esterase enzyme which is secreted from the muscle. It destroys acetylcholine in less than 1millisecond after it is released. Three isoforms of postjunctional nicotinic acetylcholine receptors have been described, junctional or mature receptor, extrajunctional (immature/foetal) receptors and neuronal 7 receptor. Acetylcholine Receptors are synthesized in muscle cells and are anchored to the end-plate membrane by a special 43-kd protein known as rapsyn. This cytoplasmic protein is associated with the receptors in a 1:1 ratio.[31] The receptors, formed of five subunit proteins, are arranged like the staves of a barrel into a cylindrical receptor with a central pore for ion channeling. Each receptor has five subunits. The mature receptor present in the innervated, adult neuromuscular junction consists of , , , and subunits. The foetal (immature, extrajunctional) receptor are seen in the foetus before innervations and after LMN/UMN injury, burns or sepsis consists of , , , and subunits. There are two subunits of and one of each of the others. The neuronal 7 Acetylcholine consists of five 7-subunits and they have been found in skeletal muscle during development and denervation. The agonists, Acetylcholine, succinylcholine, nicotine and choline antagonists, pancuronium, cobra toxin, -bungarotixin bind to the receptor.

The receptor-protein complex passes entirely through the membrane and protrudes beyond the extracellular surface of the membrane and into the cytoplasm. The binding site for acetylcholine is on each of the -subunits. Agonists and antagonists are attracted to the binding site, and either may occupy the site.

In the conventional Acetyl choline Receptors, binding of even one of the 1-subunits by an antagonist results in inactivation of that receptor because acetylcholine needs both 1-subunits of the Acetyl choline Receptors for its activation. In the 7 A Acetyl choline Receptors, however, even when three subunits are bound by an antagonist (e.g., muscle relaxant), two other subunits are still available for binding to agonist and causing depolarization. This feature may account for the resistance of 7 Acetyl choline Receptors as opposed to conventional Acetyl choline Receptors, to the blocking effects of drugs such as pancuronium. The depolarizing effects of succinylcholine and choline on the upregulated 7 Acetyl Choline Receptors can result in continued leakage of intracellular potassium and flooding of extracellular fluid, including plasma, thereby leading to hyperkalemia

BASIC ELECTROPHYSIOLOGY OF NEUROTRANSMISSION

Normally, the pore of the channel is closed by approximation of the cylinders (i.e., subunits). When an agonist occupies both -subunit sites, the protein molecule undergoes a conformational change in which a channel is formed in the centre through which ions can flow along a concentration gradient. When the channel is open, sodium and calcium flow from the outside of the cell to the inside and potassium flows from the inside to the outside. The channel in the tube is large enough to accommodate many cations and electrically neutral molecules, but it excludes anions (e.g., chloride). The current carried by the ions depolarizes the adjacent membrane. The net current is depolarizing and creates the end-plate potential that stimulates the muscle to contract. The pulse stops when the channel closes and one or both agonist molecules detach from the receptor.

DRUG EFFECTS ON POSTJUNCTIONAL RECEPTORS

Classic Actions of Nondepolarizing Muscle Relaxants: Neurotransmission occurs when acetylcholine released by the nerve action potential binds to Acetyl choline Receptors. All NDMRs impair or block neurotransmission by competitively preventing the binding of acetylcholine to its receptor. Normally acetyl cholinesterase destroys acetyl choline, cholinesterase inhibitors like neostigmine inhibit the enzyme resulting in a high concentration of the agonist in the junctional cleft, thus competitively inhibiting non depolarising muscle relaxants Classic Actions of Depolarizing Muscle Relaxants: succinyl choline is structurally two molecules of acetyl choline bound together. It binds to the receptor and depolarizes the end plate, however it is not metabolised by acetyl cholinesterase and the drug remains in the junctional cleft. Excitation of muscle contraction is followed by blockade of transmission by depolarizing relaxants as the end plate is continuously depolarized. This is due to the presence of a sodium channel which does not respond to chemicals but opens when exposed to a transmembrane voltage change. The sodium

channel is a transmembrane protein through which sodium ions flow. Two parts of its structure act as gates that allow or stop the flow of sodium ions. Both gates must be open if sodium is to flow through the channel; closing of either cuts off the flow. Because these two gates act sequentially, a sodium channel has three functional conformational states and can move progressively from one state to another.

When the sodium channel is in its resting state, the lower gate (i.e., the time-dependent or inactivation gate) is open, but the upper gate (i.e., the voltage-dependent gate) is closed, and sodium ions cannot pass. When the molecule is subjected to a sudden change in voltage by depolarization of the adjacent membrane, the top gate opens, and because the bottom (timedependent) gate is still open, sodium flows through the channel. The voltage-dependent gate stays open as long as the molecule is experiencing a depolarizing influence from the membrane around it; it will not close until the depolarization disappears. However, shortly after the voltage-dependent gate opens, the bottom gate closes and again cuts off the flow of ions. It cannot open again until the voltage-dependent gate closes. When depolarization of the end plate stops, the voltage-dependent gate closes, the time-dependent gate opens, and the sodium channel returns to its resting state. This whole process is short lived when depolarization occurs with acetylcholine. The initial response of a depolarizing muscle relaxant resembles that of acetylcholine, but because the relaxant is not hydrolyzed rapidly, depolarization of the end plate is not brief. Depolarization of the end plate by the depolarizing relaxant initially causes the voltage gate in adjacent sodium channels to open, thereby producing a wave of depolarization that sweeps along the muscle and generates a muscle contraction. Shortly after the voltage-dependent gate opens, the time-dependent inactivation gate closes. The end plate continues to be depolarised as muscle relaxant is not removed from the cleft and the voltage dependent gate remains open. As sodium cannot flow through this channel in this state, the perijunctional membrane does not depolarize. The channels downstream the perijunctional membrane are freed from the depolarising influence. The muscle membrane is thus in three zones: the end plate, which is depolarized by succinyl choline; the perijunctional muscle membrane, in which the sodium channels are frozen in an inactivated state; and the rest of the muscle membrane, in which the sodium channels are in the resting state. This phenomenon is known as accommodation. Since the extraocular muscles are multiply innervated and the presence of both mature and foetal receptors, they undergo a sustained contracture in the presence of succinyl choline. The tension thus developed forces the eye against the orbit and accounts for part of the increase in intraocular pressure produced by depolarizing relaxants.

Nonclassic and Noncompetitive Actions of Neuromuscular Drugs: Several drugs react with the neuromuscular receptor to change its function and impair transmission but do not act through the acetylcholine binding site. These reactions cause drug-induced changes in the dynamics of the receptor, and instead of opening and closing sharply, the modified channels are sluggish. Two clinically important reactions: receptor desensitization and channel blockade are seen. The former occurs in the receptor molecule, whereas the latter occurs in the ion channel. Desensitization Block; During normal neuromuscular transmission receptors may be free of agonist i.e., resting with channel closed or may have two molecules of agonist attached to subunit resulting in opening up of the ion channel. However, some receptors that bind to agonists do not undergo the conformational change to open the channel. The receptors are not sensitive to the channel opening actions of agonists i.e. they are desensitised. Some evidence suggests that desensitization is accompanied by phosphorylation of a tyrosine unit in the receptor protein. Desensitized receptors decreases the efficacy of neuromuscular transmission Channel Block; the normal flow of ions through the receptor is impaired, thereby resulting in prevention of depolarization of the end plate and a weaker or blocked neuromuscular transmission. It may be an open channel or a closed channel block. Channel block is believed to play a role in some of the antibiotic, cocaine, quinidine, piperocaine, tricyclic antidepressant, naltrexone, naloxone, and histrionicotoxin-induced alterations in neuromuscular function. Phase II Block; A phase II block is a complex phenomenon that occurs slowly at junctions continuously exposed to depolarizing agents. The junction is depolarized by the initial application of a depolarizing relaxant, but then the membrane potential gradually recovers toward normal, even though the junction is still exposed to drug. Neuromuscular transmission usually remains blocked throughout the exposure. The repeated opening of channels allows a continuous efflux of potassium and influx of sodium, and the resulting abnormal electrolyte balance distorts the function of the junctional membrane. Calcium entering the muscle through the opened channels can cause disruption of receptors and the subend-plate elements themselves. The activity of the sodiumpotassium adenosine triphosphatase pump in the membrane increases with increasing intracellular sodium and, by pumping sodium out of the cell and potassium into it, works to restore the ionic balance and membrane potential toward normal. As long as the depolarizing drug is present, the receptor channels remain open and ion flux through them remains high. Antagonism of Neuromuscular Blockade The NDMRs block neuromuscular transmission predominantly by competitive antagonism of acetylcholine at the postjunctional receptor. Two factors are important, concentration of acetylcholine and the length of time that acetylcholine is in the cleft. Increasing the number of molecules of acetylcholine in the junctional cleft changes the agonist-to-antagonist ratio and increases the probability that agonist molecules will occupy recognition sites of the receptor. It also increases the probability that an unoccupied receptor will become occupied. The second factor important to the competitive position of acetylcholine is Acetylcholine must wait for the antagonist to dissociate spontaneously before it can compete for the freed site. NDMRs bind to the receptor for slightly less than 1 millisecond, which is longer than the normal lifetime of acetylcholine. The destruction of acetylcholine normally takes place so quickly that most of it is destroyed before any significant number of antagonist molecules have dissociated from the receptor. Prolonging the time

during which acetylcholine is in the junction allows time for the available acetylcholine to bind to receptor when the antagonist dissociates from the receptors. Used Three classes of drugs, potassium channelblocking drugs, acetylcholinesterase inhibitors, and -cyclodextrin derivatives, can be used clinically to reverse nondepolarizer-induced paralysis. The commonly used antagonists of neuromuscular block inhibit acetylcholinesterase. Cholinesterase inhibitors act preferentially at the neuromuscular junction but also act at other synapses thet use the same transmitter including muscarinic receptors, hence atropine like drug is co administered to counter effects of acetylcholine on muscarinic recptors. Cyclodextrins bind steroidal muscle relaxants resulting in inactive muscle relaxants, the kidney thenremoves these complexes.