THE NEUROMUSCULAR PHYSIOLOGY

PRESENTER - DR. SOURAV MONDALMODERATOR – DR. HANSRAJ BAGHEL, MD

DEPT OF ANAESTHESIOLOGY , SSMC, REWA, MP, INDIA

PARTS OF NMJ

The anatomy of NMJ consist of following parts:

Pre-synaptic membrane

Synaptic cleft

Post-Synaptic membrane

STEPS IN NORMAL NM TRANSMISSION

ROLE OF CALCIUM• The concentration of calcium and the length of time during which it

flows into the nerve ending, determines the number of quanta release.

• If Ca+2 is not present , then depolarisation of the nerve , even by electrical stimulation will not produce the release of transmitter whereas doubling the extracellular Ca+2 results in 16-fold increase in the quantal content of an end-plate potential

• Calcium current is normally stopped by the out flow of potassium.

• Calcium channels are specialized proteins, which are opened by voltage change accompanying action potentials

• Part of calcium is captured by proteins in the endoplasmic reticulum & are sequestrated.

• Remaining part is removed out of the nerve by the Na+/Ca+2 antiport system

• The sodium is eventually removed from the cell by ATPase

THE ACETYLCHOLINE • Synthesized in the Presynaptic terminal from substrate

Choline and Acetyl CoA. Choline Acetyltransferase

CHOLINE + ACETYL CoA ACETYL CHOLINE Carrier Facilitated Transport Release

CHOLINE + ACETYL CoA ACETYL CHOLINE Acetylcholinesterase

Synaptic Cleft

SYNAPTIC VESICLE AND RECYCLING

• Different pools of acetylcholine in the nerve terminal have variable availability for release :The immediately releasable stores, VP2: Responsible for the

maintainance of transmitter release under conditions of low nerve activity. 1% of vesicles.

The reserve pool, VP1: Released in response to nerve impulses. 80% of vesicles.

The stationary store: The remainder of the vesicles.

• The vesicles in the VP2 pool are bit smaller and limited to an area very close to the nerve membrane , where they are bound to the active zones.

• Majority of the synaptic vesicles (VP1) are sequestered in the reserve pool and tethered to cytoplasmic skeleton in a filamentous network made up of primarily actin , synapsin , synaptotagmin and spectrin.

• Each vesicle contains approx 12,000 molecules of acetylcholine, which are loaded into the vesicles by an active transport process in the vesicle membrane involving a magnesium dependent H+ pump ATPase.

• Contents of a single vesicle constitute a quantum of acetylcholine.

• Release of acetylcholine may either be Spontaneous , or In response to a nerve impulse.

• When a nerve impulse invades the nerve terminal, calcium channels in the nerve terminal membrane are opened up.

• Calcium enters the nerve terminal and there is calcium dependant synchronous release of the contents .

• The number of quanta released by each nerve impulse is very sensitive to extracellular ionized calcium concentrations. Increased calcium concentration results in increased quanta released

• To enable this, vesicle must be docked at special release sites (active zones) in that part of the terminal where the axonal membrane faces the postjunctional acetylcholine receptor.

• The soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) protein are involved in fusion , docking and release of Acetylcholine at the active sites. The SNARE proteins involve the synaptic –vesicle protein, synaptobrevin; the plasmalemma-associated protein, syntaxin; and the synaptosome-associated protein of 25-kd (SNAP-25).

• These are vesicle from the immediately releasable stores

• Once the contents have been discharged, they are rapidly refilled from the reserve stores. Repeated stimulation requires the nerve ending to replenish its store of vesicles filled with transmitter , a process known as mobilization.

• Uptake of Choline and the activity of Choline acetyltransferase are probably the rate limiting steps.

PROCESS OF EXOCYTOSIS

• When there is an action potential and calcium ions enter , synapsin becomes phosphorylated , which frees the vesicle from its attachment to the cytoskeleton.

• Syntaxin and SNAP-25 are complexes attached to the plasma membrane . After an initial contact ,the synaptobrevin on the vesicle forms a ternary complex with syntaxin and SNAP-25.

• Synaptotagmin is the protein on 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.

• Assembly of the ternary complex forces the vesicles to move close to the underlying nerve terminal membrane and the vesicle is then ready for release.

ACETYLCHOLINESTERASE

• Acetylcholinesterase is a type B carboxylesterase enzyme.

• The protein enzyme is secreted from the muscle, but remain attached to it by thin stalks of collagen, attached to the basement membrane.

• Acetylcholine molecules that do not immediately react with a receptor or those released after binding to the receptor are almost instantly destroyed by acetylcholinesterase, in <1 ms, after its release into the junctional cleft.

CHOLINERGIC RECEPTORS (CHOLINOCEPTORS)

Two families of cholinoceptors, designated:

• MUSCARINIC receptors

• NICOTINIC receptors,

Types of cholinergic receptors

MUSCARINIC RECEPTORS• There are five subclasses of

muscarinic receptors: M1, M2, M3, M4, and M5.

• Only M1, M2 and M3, receptors have been functionally characterized.

• These receptors, in addition to binding acetylcholine, also recognize muscarine.

• Muscarine is an alkaloid that is present in certain poisonous mushrooms.

LOCATIONS OF MUSCARINIC RECEPTORS

Although all five subtypes have been found on neurons, M1 receptors are also found on gastric parietal cells, M2 receptors on cardiac cells and smooth muscle, and M3 receptors on the bladder, exocrine glands, and smooth muscle.

When M1 and M3 receptors are activated, the receptor undergoes a conformational change and interacts with a G protein, designated Gq , which in turn activates phospholipase C (PC). This leads to the hydrolysis of phosphatidylinositol-(4,5)-bisphosphate- P2 to yield diacylglycerol and inositol (1,4,5)-trisphosphate , which cause an increase in intracellular Ca2+ .

This action can then interact to stimulate or inhibit enzymes, or cause hyperpolarization, secretion, or contraction.

MECHANISMS OF ACETYLCHOLINE SIGNAL TRANSDUCTION ON M1 & M3 RECEPTORS

MECHANISMS OF ACETYLCHOLINE SIGNALTRANSDUCTION ON M2 RECEPTOR

M2 subtype on the cardiac muscle stimulates a G protein, designated Gi , that inhibits adenylyl cyclase

and increases K+ conductance, to which the heart responds with a decrease in rate and force of contraction.

NICOTINIC RECEPTORSThe nicotinic receptor is composed of five subunits, and it functions as a ligand-gated ion channel.

Binding of two acetylcholine molecules on α subunits elicits a conformational change that allows the entry of sodium ions, resulting in the depolarization of the effector cell.

Another isoform of Ach contains a γ subunit instead of the ε subunit known as fetal or immature receptor, because this form initially expressed in fetal muscle, often referred to as extrajunctional receptors.

LOCATION OF NICOTINIC RECEPTORS

Nicotinic receptors are located in the CNS, adrenal medulla, autonomic ganglia, and the neuromuscular junction. Those at the neuromuscular junction are sometimes designated NM and the others NN.

THE ACETYLCHOLINE RECEPTORS

POST JUNCTIONAL RECEPTORS Three isoforms of

postjunctional niconitinic AChRs exist :

A junctional or mature receptors

an extrajunctional or immature (fetal) receptor

the recently described neuronal α7 receptor

JUNCTIONAL OR MATURE RECEPTORS

• Present in the post junctional membrane of the motor end plate & are of nicotinic type. These receptors exist in pairs.

• It consists of protein made up of 1000 amino acids, made up of 5 protein subunits designated as alpha (α), beta (β) , delta (δ) and epsilon (ε) joined to form a channel that penetrates through and projects on each side of the membrane.

• Each receptor has central funnel shaped core which is an ion channel, 4 nm in diameter at entrance narrowing to less than 0.7nm within the membrane.

• The receptor is 11 nm in length and extends 2nm into the cytoplasm of the muscle cell.

• The receptor has 2 gates, an Upper voltage- dependent and a Lower time-dependent.

When acetylcholine receptors bind to the pentameric complex, they induce a conformational change in the proteins of the alpha (α) subunits which opens the channel and it occurs only if it binds to both the alpha (α) binding sites.

For ions to pass through the channel both the gates should be open.

Cations flow through the open channel, Na+ and Ca+2 in and K+ out, thus generating end plate potential.

Na+ ions are attracted to the inside of the cell which induces depolarisation.

THE SODIUM CHANNELS• Sodium channels are present in muscle membrane.

• Perijunctional areas of muscle membrane have a higher density of these sodium channels than other parts of the membrane.

• These sodium channels have two types of gate - voltage dependent - time dependent

• Sodium ions pass only when both gates are open.

The channel therefore possesses three functional states.

A...At rest, the lower gate is open but the upper gate is closed

B...reaches threshold voltage depolarization, the upper gate opens and sodium can pass

C...Shortly after the upper gate opens the

time dependent lower gate closes

POSSIBLE CONFIGURATION OF Na CHANNELS

• Resting state: Voltage gate closed Time gate open Channel closed

• Depolarization: Voltage gate open Time gate open Channel open

• With in a few milliseconds: Voltage gate open Time gate closed Channel closed

• End of depolarization: Voltage gate closed Time gate open Channel closed

EXTRAJUNCTIONAL RECEPTOR• These tend to be concentrated around the end plate,

where they mix with post junctional receptors but may be found anywhere on the muscle membrane. In them, the adult epsilon (ε) subunit is replaced by the fetal gamma (γ) subunit.

• They are not found in normal active muscle, but appear very rapidly after injury or whenever muscle activity has ended.

• They can appear within 18 hrs of injury and an altered response to neuromuscular blocking drugs can be detected in 24 hrs of the insult.

• When a large number of extrajunctional receptors are present, resistance to non-depolarising muscle relaxants develops, yet there is an increased sensitivity to depolarising muscle relaxants.

• In most extreme cases, increased sensitivity to succinylcholine results in lethal hyperkalemic receptors with an exaggerated efflux of intracellular potassium.

• The longer opening time of the ion channel on the extrajunctional receptor also results in larger efflux.

THE NEURONAL α7 RECEPTORS• The neuronal α7 AchRs consists of five α7 subunits. Each of all

receptor subunits consists of approximately 400 to 500 amino acids.

• The receptor protein complex pass entirely through the membrane and protudes beyond the extracellular surface of the membrane and into the cytoplasm.

• In α7 AChRs , however , even when three subunits are bound by an antagonist , the two other subunits are still available for binding by agonist and cause depolarisation . This feature may account for some of the resistance to muscle relaxants when α7 AChRs are expressed In muscle and in other tissues during pathologic states like sepsis, denervation , immobilisation , burns etc.

MAINTENANCE OF MATURE NEUROMUSULAR JUNCTIONS

• Multiple factors including electrical activity, growth factor signaling and the presence or absence of innervation , control the expression of the three type of receptor isoforms.

• The nerve releases several growth factors that influence the synaptic apparatus of nearby nuclei.

• Before innervation , as in the fetus , AChRs are present throughout the muscle membrane. After innervation , AChRs become more and more concentrated at the postsynaptic membrane and are virtually absent in the extrasynaptic area after birth.

• In the active adult, and normal innervated muscle , just the nuclei under and very near the end plate direct the synthesis of the receptor . Nuclei beyond the junctional area are not active , and therefore no receptors are expressed anywhere in the muscle cells beyond the perijunctional area.

PREJUNCTIONAL RECEPTORS

• These are nicotinic receptors that control ion channel specific for Ca+2 which is essential for synthesis and mobilization of acetylcholine and are composed of alpha (α) and beta (β) subunits only.

• They contain protein subunits that are blocked by non depolarising muscle relaxants resulting in tetanic fade and train-of-four fade.

NEUROMUSCULAR BLOCKING AGENTS –CLASSIFICATION ,MECHANISM & DURATION OF

ACTIONDEPOLARIZING

Short-acting

• Succinylcholine

NONDEPOLARIZING

Short-acting

• Gantacurium• Mivacurium

Intermediate-acting

• Atracurium • Cisatracurium • Vecuronium • Rocuronium

Long-acting

• Pancuronium• Pipecuronium• Doxacurium

MECHANISM OF ACTION of DEPOLARIZING NMBA

1 •Depolarizing muscle relaxants closely resemble ACh and readily bind to ACh receptors, generating a muscle action potential.

2 •Unlike ACh, however, these drugs are not metabolized by acetylcholinesterase, and their concentration in the synaptic cleft does not fall as rapidly, resulting in a prolonged depolarization of the muscle end-plate.

3 •Continuous end-plate depolarization causes muscle relaxation

4 • opening of perijunctional sodium channels is time limited (sodium channels rapidly “inactivate” with continuing depolarization)

PHASES OF BLOCK IN DEPOLARIZING NMBA

Phase I block

• Perijunctional sodium channel cannot reopen until the end-plate repolarizes.• The end-plate cannot repolarize as long as the depolarizing muscle relaxant continues to bind to

ACh receptors; this is called a phase I block.

Phase II Block

• After a period of time, prolonged end-plate depolarization can cause changes in the ACh receptor that result in a phase II block.

MECHANISM OF ACTION OF NON-DEPOLARIZING NMBA

Nondepolarizing muscle relaxants

function as competitive antagonists.

Nondepolarizing muscle relaxants bind ACh receptors but are incapable of inducing the

conformational change necessary for ion channel opening.

Because ACh is prevented from binding to its

receptors, no end-plate potential develops.

Neuromuscular blockade occurs even if only one α subunit is

blocked.

NEUROMUSCULAR JUNCTION AT EXTREMES OF AGE

• NEWBORN

Just before birth , the AChRs are all clustered around the junctional area, and minimal extrajunctional AChRs are present.

The newborn postsynaptic membrane ,itself , is not specialised , having almost no synaptic folds , a widened synaptic space , and a reduced number of AChRs.

The early postnatal AChR clusters appears as an oval plaque.

Within a few days simplified folds appear.

With continued maturation , the plaque is transformed to a multiperforated pretzel-like structure.

The polyinnervated end plate is converted to a singly innervated juntion because of a retraction of all but one terminal.

• OLD AGE

Anatomic changes involve increased preterminal and axonal branching within the individual neuromuscular junction, either with or without an increase in the junctional size.

The points of contact between the junctional and post-junctional membrane decrease , resulting in a decline in trophic interactions between nerve and muscle and stimulus transmission which in turn results in age-associated functional denervation, muscle wasting and weakness.

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