Neuromuscular transmission and its pharmacology

Nerve - Muscle Physiology

1. Neuromuscular Transmission and its Pharmacology

2. N euromuscular J unction S ynapses are the junctions where the axon or some other portion of one cell (the presynaptic cell ) terminates on the dendrites, soma, or axon of another neuron or in some cases a muscle or gland cell (the postsynaptic cell ) Synapses between axons of motor neurons and skeletal muscle fibers are called NEUROMUSCULAR JUNCTION or MYONEURAL JUNCTIONS

3. Motor Unit Motor unit One neuron Muscle cells stimulated by that neuron Motor neuron & all the muscle fibers it supplies

4. Motor Unit

5. Physiological Anatomy of NMJ PRESYNAPTIC MEMBRANE (TERMINAL BOUTONS/END FEET) : small, clear vesicles contain ing acetylcholine SYNAPTIC CLEFT/SPACE : space between pre and post-synaptic membrane, 20-40 nm, also contain enzyme ACETYLCHOLINESTERASE POSTSYNAPTIC MEMBRANE (MOTOR END PLATE): thickened portion of muscle membrane at the junction

6. Physiological Anatomy of NMJ Axon makes a single point of synaptic contact with a skeletal muscle fiber, midway along the length of the fiber As axon approaches its termination, it loses its myelin sheath and divides into a number of terminal buttons, or endfeet The endfeet contain many small, clear vesicles that contain acetylcholine (Ach) , the transmitter at these junctions

7. Physiological Anatomy of NMJ Vesicles – formed in the cell body and are transported by fast axonal transport via the microtubules ACh is synthesized in the nerve terminal – outside the vesicle – from choline and acetyl coenzyme A by the enzyme choline acetyltransferase The ACh moves into the synaptic vesicles via a specific ACh -H + exchanger

8. Release of Acetylcholine Nerve impulse reaching axon terminal increases permeability of pre-synaptic membrane to Ca 2+ Ca 2+ enters the nerve terminal through voltage-gated channels Ca 2+ causes fusion of synaptic vesicles with the pre-synaptic membrane Amount of neurotransmitter released is directly proportional to Ca 2+ influx Mg 2+ decreases this process

9. Physiological Anatomy of NMJ Synaptic vesicles fuse at differentiated regions of the presynaptic membrane called Active Zones and releases ACh E ndings fit into junctional folds, which are depressions in the motor end plate Synaptic basal lamina contains high concentration of enzyme AChE ( Acetylcholinesterase ) - terminates transmission by rapidly hydrolyzing free ACh to choline and acetate

10. Effect of ACh on Postsynaptic Membrane ACh binds to ACh Receptors which are ligand -gated ion channels in postsynaptic membrane ACh receptor is a heteropentamer with a subunit composition of α 2 βγδ Subunits are homologous to one another Each α subunit has 4 membrane spanning segments

11. Acetylcholine Receptor

12. Acetylcholine Receptor

13. Acetylcholine Receptor

14. Effect of ACh on Postsynaptic Membrane Binding of acetylcholine to these receptors increases the Na + and K + conductance Na + influx, creates a local positive potential change inside the muscle fiber membrane called the END PLATE POTENTIAL (EPP) ACh released into synaptic cleft is removed rapidly by enzyme Acetylcholinesterase , present in synaptic cleft. Removal is rapid to prevent re-excitation of the receptors after first action potential

15. Voltage gated calcium channels Voltage gated sodium channels Voltage gated potassium channels End of the nerve fiber ( Presynaptic )

16. Muscle fiber(Postsynaptic)

17. Entry of sodium ions ( Depolarization ) Depolarization causes opening of voltage gated calcium channels. Calcium entry Opening of potassium channels ( hyperpolarization ) Closure of calcium channels Entry of Calcium after the arrival of impulse (action potential) Events at the end of the nerve fiber

18. Release of vesicles Entry of Calcium Movement of vesicle towards membrane Release of Ach One impulse = 125 vesicles released At rest = 1 – 2 Hz release Events at the end of the nerve fiber

19. Binding to AChR Opening of Ligand gated channel Events on the muscle fiber

20. Binding of ACh to receptor increases the Na + and K + conductance of the membrane R esultant influx of Na + produces a depolarizing potential - end plate potential C urrent sink created by this local potential depolarizes the adjacent muscle membrane to its firing level Normally not recordable as action potential is almost always generated End Plate Potential (EPP)

21. End Plate Potential (EPP) Average human end plate contains about 15–40 million ACh receptors Each nerve impulse releases about 60 ACh vesicles, and each vesicle contains about 10,000 molecules of the neurotransmitter Can activate 10 times the number of acetylcholine receptors Therefore, a propagated response in the muscle is regularly produced

22. End Plate Potential (EPP) Only 6 vesicles are reqd for activation from -90 to -65 mv Each nerve impulse releases 60 ACh vesicles Every vesicle has 10,000 molecules of ACh 10-fold safety factor Action potential always generated

23. Depolarization due to net entry of cations threshold Time (ms) Membrane voltage (mv) End plate potential (graded potential ) Action potential Events on the Muscle Fiber

24. End Plate Potential (EPP) EPP can be seen if the tenfold safety factor is overcome A dministration of small doses of curare (competitive inhibitor of ACh ) is used for studying EPP The response is then recorded only at the end plate region and decreases exponentially away from it EPP undergo es temporal summation

25. Miniature End Plate Potential (MEPP) At rest - Random release of small packets / quanta of ACh - Quantal Release of Transmitter Small depolarising spike Amplitude = 0.5mv Amount released ∞ Ca 2+ concentration 1/∞ Mg ++ concentration When a nerve impulse comes , no. of quanta released increases result ing in large EPP that exceeds the firing level of the muscle fiber producing AP

26. MEPP

27. Muscle Nerve RMP -90mv -70mv Action Potential Duration 2-4msec varies Velocity 5 m/sec Varies with fiber Absolute Refractory period 1-3 msec 2-4 msec Ionic distribution is similar to that in nerve

28. Transmission of Nerve Impulse to Muscle Sodium rushes into the cell generates an action potential (AP) The action potential travels along the T-tubules to the SR to stimulate release of Calcium ions The Ca 2+ ions travels to the muscle tissue and bind to the ACTIN regulatory proteins ( Troponin C)

29. Transmission of Nerve Impulse to Muscle This UNCOVERS Myosin Head BINDING Sites on ACTIN so as to allow CROSS BRIDGING ( once myosin is powered by ATP) Activation by nerve causes myosin heads ( crossbridges ) to attach to binding sites on the thin filament Myosin heads then bind to the next site of the thin filament - Sliding Filament Theory of Muscle Contraction

30. Transmission of Nerve Impulse to Muscle

31. End of Neuromuscular Transmission Acetylcholine, the neurotransmitter is broken down by the enzyme acetylcholinesterase SO the stimulus to muscle ceases! – prevents continued muscle reexcitation Calcium ions are actively transported back to the SR by SERCA (calcium ATPase ) The actin and myosin cross bridges break RELAXES------S T R E T C H of the sarcomere

32. Drugs that Enhance or Block Transmission at N-M Junction 1 . STIMULATE THE MUSCLE FIBER BY ACh LIKE ACTION : methacholine , carbachol and nicotine : these drugs are not destroyed or are destroyed very slowly by cholinesterase, action persists for many minutes to several hours can cause muscle spasm

33. 2.STIMULATE THE NEURO-MUSCULAR JUNCTION BY INACTIVATING ACETYL-CHOLINESTERASE neostigmine , physostigmine : inactivate acetylcholinesterase for upto several hours diisopropyl flourophosphate : inactivate acetylcholinesterase for weeks, “nerve” gas poison Drugs that Enhance or Block Transmission at N-M Junction

34. 3. BLOCK TRANSMISSION AT NEURO-MUSCULAR JUNCTION Curariform drugs d-Tubocurarine blocks the action of ACh on the muscle fiber Acetylcholine receptors, thus preventing sufficient increase in permeability of the muscle membrane channels to initiate an action potential Drugs that Enhance or Block Transmission at N-M Junction

35. Neuromuscular blocking agents: Two types - neuromuscular blocking agents:- Depolarizing Non – depolarizing Depol . Type blockers- Agents act like Ach but resistant to Ach esterase Constant stimulation/contraction of muscles Persistent depol . leads to block due to inactivation of voltage gated Na + channels Non depol . Type blockers – Drugs complete with Ach for Ach receptors e.g. curare, gallamine or flaxedil

36. Neuromuscular blocking agents: Used in surgery because they relax muscle and abolish reflexes Reduces the dose of anesthetic agent necessary Patient who receive neuromuscular blockers, need artificial respiration, because respiratory muscles ( S.K.muscles /diaphragm) being paralysed , may lead to death within minutes

37. Drugs that Enhance or Block Transmission at N-M Junction

38. Botulinum and Tetanus toxin Decrease Ach release Tetanus toxin – markedly increases activity of motor neurons Causes spastic paralysis - Lockjaw Botulinum toxin( A-G ) Causes Flaccid paralysis

39. Myasthenia Gravis Eaton Lambert Syndrome Clinical disorders of NMJ

40. Myasthenia Gravis Afflicts 25-125 of every million people Can occur at any age but has a bimodal distribution with peak occurrences in 20s (mainly women) and 60s (mainly men) Antibodies against ACh receptors are present in blood of most patients with this disease Autoimmune disease Antibody detected in 50% of pts with pure ocular MG 90-95% of pts with generalized MG

41. Myasthenia Gravis Antibodies destroy some of the receptors and bind others to neighboring receptors, triggering their removal by endocytosis Normally, number of quanta released declines with successive repetitive stimuli Neuromuscular transmission fails at these low levels of quantal release Leads to the major clinical feature of the disease–muscle fatigue with sustained or repeated activity

42. Myasthenia Gravis

43. Myasthenia Gravis Two major forms of the disease – Involves weakness of only the extraocular muscles Results in generalized weakness of all skeletal muscles In severe cases, paralysis of respiratory muscles can lead to death

44. Clinical Manifestation of MG Symptoms worsen with exercise, end of day (Fatigue) Ocular Droopy eyelids ( ptosis ) Double vision ( diplopia ) Extremity weakness Arms > legs Dysarthria & Dysphagia Respiratory Shortness of breath

45. Muscle Weakness of MG EASY FATIGABILITY

46. Myasthenia Gravis Treatment: neostigmine or some other anticholinesterase drug – alone or combined with thymectomy or immunosuppression Cholinesterase inhibitors prevent metabolism of ACh compensating for the normal decline in released neurotransmitters during repeated stimulation Removal of Thymoma leads to clinical improvement in 75% of cases

47. Clinical Problem A 18-year-old college woman comes to the student health service complaining of progressive weakness. She reports that occasionally her eyelids “droop” she tires easily, even when completing ordinary daily tasks such as brushing her hair. She has fallen several times while climbing a flight of stairs. These symptoms improve with rest.

48. Lambert- eaton Myasthenic Syndrome (LEMS) Antibodies against Ca 2+ channels in the motor nerve terminals no. of Ca 2+ channels decrease less calcium enters the nerve terminal and less neurotransmitter is released Symptoms - muscular weakness & diminished stretch reflexes Muscle strength increases with prolonged contraction as more Ca becomes available

49. Lambert- eaton Myasthenic Syndrome (LEMS) The major clinical finding is progressive weakness that does not usually involve the respiratory muscles and the muscles of face In contrast to MG, symptoms of LEMS tend to be worse in the morning and improve with exercise The proximal parts of the legs and arms are predominantly affected Many patients have autonomic symptoms like dry mouth or impotence. Reflexes are usually reduced or absent

50. Differences between MG LEMS Antibodies are formed against the ACh Receptors on the Post synaptic membrane Primarily attacks the ocular and bulbar muscles Repeated muscle stimulation leads to decrease in contractile strength Antibodies are formed against the presynaptic Calcium channels Primarily attacks the limb muscles Repeated muscle stimulation leads to increasing contractile strength

51. N-M Junction in Smooth & Cardiac Muscle

52. Autonomic nerve fibers that innervate smooth muscle branch diffusely on top of sheet of muscle fibers These fibers do not make direct contact with smooth muscle fiber cell membranes but form diffuse junctions Vesicles may contain ACh in some & NE in other autonomic nerve fiber endings N-M Junction in Smooth & Cardiac Muscle

53. N-M Junction in Smooth & Cardiac Muscle No typical end feet as seen in skeletal muscle, axons have multiple varicosities distributed along their axes V aricosities are about 5 μ m apart, with up to 20,000 varicosities per neuron Transmitter is liberated at each varicosity, ie, at many locations along each axon This arrangement permits one neuron to innervate many effector cells The type of contact in which a neuron forms a synapse on the surface of another neuron or a smooth muscle cell and then passes on to make similar contacts with other cells is called a synapse en passant

54. NMJ: Smooth muscles

55. Denervation Hypersensitivity When the motor nerve to skeletal muscle is cut and allowed to degenerate muscle gradually becomes extremely sensitive to acetylcholine - denervation hypersensitivity or supersensitivity due to an upregulation of its receptors Muscle atrophies Also seen in smooth muscle Does not atrophy hyperresponsive to the chemical mediator that normally activates it

56. Summary

57. Thank you References: Guyton- Textbook of Medical Physiology Ganong’s - Review of Medical Physiology Boron-Medical Physiology Kandel -Principles of Neural Science Silbernagl -Color atlas of Physiology Ira Fox- Medical Physiology

58. Neuromuscular Junction Types of myoneural junction En plaque (extra fusal) : The end plate En grappe (intra fusal)

59. Safety factor of transmission Minature end plate potential(MEPP) = 0.4 mV About +20 to + 30 mV of depolarization is sufficient EPP is about + 50 to + 70 mV .Thus, a safety of 2 – 3 times Plus, Ach receptors are stimulated 3 times that required for action potential Leading to a safety factor of around 10 times