neuromuscular junction physiology

detailed description of neuromuscular junction , signal transmission, muscle contraction.
basics of NMJ function

1. Speakers Dr Gowri shankar B N-M junction and N-M blockade

2. Anatomy And Physiology Of Neuromuscular Junction Presented by Dr. Gowri shankar B Moderator Dr.Madhuri S kurdi

3. Introduction The neuromuscular junction (NMJ) is one of the most widely studied synapses. Electrical neurotransmission and the presence of chemical compounds with a critical function for the transmission of information from nerve to muscle was first described by Claude Bernard katz . Bernard Katz, was able to study the neuromuscular junction with intracellular electrodes, and the role of acetylcholine in this synapse was completely demonstrated.

4. Sir Henry Dale proved through series elegant experiments between 1929 and 1936, that acetylcholine is also a neurotransmitter in the neuromotor synapse. Eccles succeeded for the first time in inserting microelectrodes into nerve cells and also later instrumental in researching the ionic basis of membrane potentials in the synapse, studying sodium, potassium and calcium.

5. Sir John Carew Eccles Sir Bernard Katz Sir John Eccles and Sir Bernard Katz were both honoured with the Nobel Award of 1963 and 1970, respectively.

6. The neuromuscular junction is a chemical synapse at which a nerve impulse triggers the excitation of skeletal muscle. Motor neuron = presynaptic cell at the motor neuron: electrical signal  chemical signal skeletal muscle fiber = postsynaptic cell at the skeletal muscle fiber: chemical signal  electrical signal Neuromuscular Junction

7. Physiology Of Neuromuscular Transmission NMJ is specialised on the nerve side and on the muscle side to transmit and recieve chemical messages. Each motor neuron( α ) runs without interruption from the ventral horn of spinal cord to NMJ as a large myelinated axon. As it approaches muscle it branches to contact many muscle cells together into functional group known as Motor unit .

8. Motor unit- The Nerve-Muscle Functional Unit Each single motor neuron and the muscle fibers it innervates constitute a motor unit. In muscles concerned with fine, graded, precise movement (hand , eye), each motor unit innervates 3 to 6 muscle fibers. Large weight-bearing muscles (thighs, hips) have large motor units.

9. Motor Unit: Muscle fibers from a motor unit are spread throughout the muscle. Not confined to one fascicle. Therefore, contraction of a single motor unit causes weak contraction of the entire muscle. Stronger and stronger contractions of a muscle require more and more motor units being stimulated ( recruited )

10. Parts - The anatomy of NMJ consist of three parts: Pre-synaptic membrane. Synaptic cleft. Post-Synaptic membrane.

11. Presynaptic portion of neuromuscular junction As the axon of motor neuron approaches the muscle fiber, it loses its myelin sheath & divides extensively into several fine branches-axon terminal and it is covered by Schwann cells. Each terminal is expanded at its end to form a knob like structure- synaptic knob (terminal bouton ). Terminal bouton lies in the groove ( synaptic trough ) in the surface of muscle fiber. Vesicles are clustered around specific points- active zones .

12. Size – 40 to 50 nm . Formed by the Golgi apparatus in the cell body of the motor neuron in the spinal cord. Transported by axoplasm to the neuromuscular junction at the tips of the peripheral nerve fibers. About 300,000 of these small vesicles collect in the nerve terminals of a single skeletal muscle end plate. Synaptic Vesicles

13. MITOCHONDRIA Numerous. Supply ATP . Energy source for synthesis of excitatory neurotransmitter, acetylcholine . DENSE BARS Present on the inside surface of neural membrane. Terminal boutons also contain…

14. Ach (Synthesis, storage, release) Synthesized in the Presynaptic terminal from substrate Choline and Acetyl CoA. CAT CHOLINE + ACETYL CoA ACETYL CHOLINE COMT 50% Carrier Facilitated Transport Release CHOLINE + ACETYL CoA ACETYL CHOLINE Synaptic Cleft ACH is stored in vesicles in the nerve terminal.

15. 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.

16. 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 be Spontaneous or In response to a nerve impulse.

17. c) Quantum: Smallest amount of ACH released. Probably the amount of ACH in a “standard” presynaptic vesicle is: Quantum = 2,000 to 10,000 ACH molecules.

18. 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 from 50-100 vesicles. 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.

19. 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 receptors. vesicle from the immediately releasable stores aligned al0ng the active zones.

20. Once the contents have been discharged, they are rapidly refilled from the reserve stores. The reserve vesicles are anchored to actin fibrils in the cytoskeleton, by vesicular proteins called synapsins Some calcium that enters the axoplasm , on the arrival of the nerve impulse binds to calmodulin , which activates protein kinase-2 which phosphorylates synapsins , which, in turn dissociates the vesicle from the actin fibrils allowing it to move forward to the release site/ active zones.

21. Docking of the vesicle and subsequent discharge of acetylcholine by exocytosis, involves several other proteins. Membrane protein called SNAREs ( Soluble N- ethylmatrimide sensitive attachment proteins) are involved in fusion, docking, and release of acetylcholine at the active zone. SNARE includes – synaptic vesicle protein synaptobrevin , synataxin and SNAP-25.

22. Synaptotagmin is the protein on the vesicular membrane acts as a calcium sensor and localizes the synaptic vesicle to synaptic zones rich in calcium channels, stabilizing the vesicles in docked state. Synaptophysin is a glycoprotein component of the vesicle membrane.

23. Phosphorlation of another membrane protein - synapsin facilitates vesicular trafficking to release site. Synaptobrevin is a vesicle associated membrane protein (VAMP). During depolarisation & entry of Ca it unfolds & forms a ternary complex with syntaxin / SNAP-25 .

24. Assembly of the ternary complex forces the vesicle in close apposition to the nerve membrane at the active zone with release of its contents, acetylcholine. The fusion is disassembled, and the vesicle is recycled.

25. Applied Botulinum toxin impairs neuromuscular transmission by binding to the presynaptic terminal and entering the presynaptic terminal through endocytosis , cleaves the membrane docking proteins and prevents acetylcholine exocytosis. Botulinum toxin A cleaves SNAP 25 while Botulinum toxin B cleaves synaptobrevin to limit exocytosis.

26. 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. 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

27. 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 antiport system The sodium is eventually removed from the cell by ATPase

28. Exocytosis at the Terminal Boutons Resting nerve with abundant vesicles Stimulate nerve and observe fusion of vesicles with membrane. Vesicle exocytosis releases ACh into synaptic space Synaptic cleft is 50 to 100 nm Time for diffusion of ACh is ~0.5 ms

29. Synaptic cleft: gap between the terminal bottom & the muscle fiber, which is about 50-100 nm wide. Muscle fiber is covered by basement membrane or basal lamina, consisting of collagen, glycoproteins & other extracellular matrix proteins ( neurexins ). Also contain acetylcholinesterase anchored to sarcolemma by colQ protein.

30. Post synaptic membrane Acetylcholine receptors: At the post synaptic membrane the area overlying the nerve terminal is called muscle end plate . The membrane here is thrown into primary and secondary clefts. The clefts greatly increase the membrane surface area. Shoulder of these clefts contain numerous Ach receptors arranged in pairs .

31. Motor End Plate Presynaptic terminal, with many small vesicles containing ACh Electron micrograph of nerve terminal Postsynaptic region of the skeletal muscle, with mitochondria and contractile filaments apparent in the cytoplasm 500 nm

32. Motor End Plate (continued) Each vesicle contains ~5000 ACh molecules. 1 vesicle=1 quanta Each vesicle is ~50 nm diameter Postsynaptic membrane: Clusters of nicotinic ACh receptors in the junctional folds Synaptic cleft between nerve and muscle cells Size 50 to 100 nm gap.

33. The released acetylcholine diffuses to the muscle type nicotinic acetylcholine receptors which are concentrated at the tops of junctional folds of membrane of the motor end plate. Binding of acetylcholine to these receptors increases Na and K conductance of membrane and resultant influx of Na produces a depolarising potential -EPP. The current created by the local potential depolarise the adjacent muscle membrane to firing level.

34. Acetylcholine is then removed by acetylcholinesterase from synaptic cleft, which is present in high concentration at NMJ. Action potential is generated due to opening of the voltage gated Na channels on either side of end plate and are conducted away from end plate in both directions along muscle fiber. The muscle action potential in turn initiates muscle contraction.

35. Acetylcholine Receptors Acetylcholine-gated ion channels, molecular weight -275,000 Types – Junctional or mature. Located almost entirely near the mouths of the sub neural clefts lying immediately below the dense bar areas. Extra junctional or immature - tend to be concentrated around the end plate, where they mix with post junctional receptors but may be found anywhere on the muscle membrane. The adult ε subunit is replaced by the fetal γ subunit.

36. Extrajunctional Receptor Not found in normal active muscle, but appear very rapidly after injury or whenever muscle activity has ended. Can appear within 18hrs of injury and an altered response to neuromuscular blocking drugs can be detected in 24hrs of the insult.

37. 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.

38. The immature isoform containing the γ-subunit has long open times and low-amplitude channel currents. The mature isoform containing the ε-subunit has shorter open times and high-amplitude channel currents during depolarization. Application of acetylcholine to the α7 AchR also results in a fast, rapidly decaying inward current.

39. Nicotinic ACh Receptor( post junctional ) Receptor is a pentameric complex of 2 a , b ,  and d subunits. Ligand gated ionic channel. Binding of 2 ACh molecules causes opening of the channel, which is a channel permeable to all cations , including Na + , K + and Ca 2+ . The evoked currents reverse direction close to 0 mV. Thus, at the resting potential, the primary effect is influx of Na + , accounting for the depolarization.

40. The muscle nicotinic ACh receptor is a typical ionotropic receptor The receptor is made up of five subunits; 2  and 3 non   Binding of 2 ACh molecules opens the channel through a conformational change

41. ACh receptor subunits have conserved domains 4 hydrophobic transmembrane domains of 20 amino acids form 4 alpha helixes Subunits are 50% conserved suggesting similar structure M2 domains line the channel pore

42. Aligned negatively charged amino acids (purple) flanking M2 of each subunit form rings that contribute to Ion selectivity (repulsing anions) Conformational changes are thought to reorientate the residues within the pore, allowing Ions to flow. Ring of hyrdophobic leucine residues occlude the pore where the M2 is kinked inward Functional model of the nicotinic ACh receptor channel threonine/serine ring contribute ion selectivity filter

43. Opened Ach Channel Diameter- 0.65 nanometer. Allows important positive ions —SODIUM, potassium, and calcium to move easily through the opening. Disallows negative ions, such as chloride to pass through because of strong negative charges in the mouth of the channel that repel these negative ions.

44. Sodium Ions Far more sodium ions flow through the acetylcholine channels to the inside than any other ions . The very negative potential on the inside of the muscle membrane, –80 to –90 mili volts, pulls the positively charged sodium ions to the inside of the fiber. Simultaneously prevents efflux of the positively charged potassium ions when they attempt to pass outward.

45. The ACh receptor is permeable to Na + and K + Na + K + Na + K + At resting potential the inward driving force for Na + is large and the outward driving force for K + is small I EPSP =g EPSP X (V m - E EPSP ) Na + K + Na + K + At reversal potential the driving forces for Na + and K + are equal and opposite so the net ion flow is zero

46. End plate potential Binding of ACh with the ACh receptors in the post synaptic membrane of the motor end plate causes opening of ligand gated Na+ & K+ channels which cause greater influx of Na+ than efflux of K+ leading to localized depolarization of motor end plate to about + 50-75mv called as the End Plate Potential . EPP is not an action potential but it is simply depolarization of specialized motor end plate.

47. Miniature End Plate Potentials- MEPP Small quanta (packets) of Ach are released randomly from nerve cell at rest spontaneously, each producing smallest possible change in membrane potential of motor end plate, the MINIATURE EPP. Usually about 0. 5mV. Significance of MEPP not known, probably responsible for the tone of muscle.

48. Quantal theory Proposed by Bernard katz to explain MEPP. Because MEPPs are too big to be produce by a single molecule of Ach, it was deduced that they are produced by uniformly sized packages or quanta of transmitter released from the nerve( in the absence of stimulation). The stimulus evoked end plate potential is the additive depolarization produced by the synchronous discharge of quanta from several hundred vesicles.

49. Na Channel Cylindrical, Membrane protein. Has two gates- voltage dependent and time dependent. Both should be open to allow passage of ions . Voltage dependent gate is closed in resting state and opens only on application of a depolarising voltage, remains open as long as the voltage persists.

50. voltage dependent time dependent

51. The time dependent gate is normally open at rest closing a few milliseconds after the voltage gate opens and remains closed as long as the voltage gate is open It reopens after the voltage gate closes. The channel is patent, allowing sodium ions only when the gates are open.

52. 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

53. Summary of Neuromuscular Transmission Action potential invades presynaptic terminal + + Opening of Ca 2+ channels leads to influx of Ca 2+ (extracellular Ca 2+ is essential ) Vesicles fuse and release ACh into cleft ACh diffuses across synaptic cleft + + ACh activates cation channels to cause depolarization of the endplate. ACh -activated channel is permeable to both Na + and K + , so the reversal potential is a mixture of the two

54. Summary of Neuromuscular Transmission + + + + Depolarization of the end plate initiates an action potential that spreads over muscle cell ACh is destroyed by acetylcholinesterase enzymes in the synaptic cleft

55. Contractile Apparatus It is formed by thin actin , thick myosin filaments tropomyosin & troponin. The shortening of this apparatus causes the contraction of the muscle.

56. Physical Channel Blockade Various drugs can block the neuromuscular junction and prevent depolarisation . Blockade can occur in two modes Blocked when open Blocked when closed

57. Open Channel Block In this, the drug molecule enters a channel which has been opened by acetylcholine. This is use dependent block Physical blockade by a molecule of an open channel relies on the open configuration of the channel and the development of this is proportional to the frequency of channel opening.

58. This mechanism may explain the synergy that occurs with certain drugs such as local anaesthetic, antibiotics and muscle relaxants. In addition, the difficulty in antagonizing profound neuromuscular blockade may be due to open channel block by the muscle relaxants

59. Closed Channel Block The drugs occupy the mouth of the channel and prevents ions from passing through the channel to depolarise the end plate. Tricyclic drugs and naloxone may cause physical blockade of a closed by impending interaction of acetylcholine with the receptor. For drugs interfering with the function of the acetylcholine receptor, without acting as an agonist or antagonist, the receptor lipid membrane interface may also be another site of action. Eg : Volatile agents, Local anaesthetic and Ketamine

60. REFERENCES: Millers text book of anesthesia,7 th ed. Clinical anesthesia, Barasch 5 th ed. Morgan`s principles of anesthesia 5 th ed Textbook of physiology, Ganong 23 rd ed. Guyton & Hall Textbook of physiology. Neurons and Synapses: The History of Its Discovery By Renato M.E. Sabbatini , PhDBrain & Mind Magazine, 17, April-July 2003 Basic principles of neuromuscular transmission J. A. J. Martyn,1,2 M. Jonsson Fagerlund3 and L. I. Eriksson4 ----- THANK YOU -----