Today’s Objective: Neuromuscular Transmission

1. Today’s Objective:Neuromuscular Transmission • Describe the sequence of electrical and chemical events at the neuromuscular junction during transmission of an action potential. • Provide some of the evidence that acetylcholine (ACh) is released, and that the release is quantal in nature. • Describe the ionic mechanisms of the endplate potential (EPP), including the types of ion channels involved. • Consider the disorder Myasthenia Gravis, and the rationale for treatment with inhibitors of acetylcholinesterase.

2. Review: Main Components of Neurons • Soma, axon, myelin sheath, nodes of Ranvier and terminal boutons. Nodes of Ranvier Terminal Boutons: Sites of transmitter release

3. The Motor End Plate • A myelinated nerve fiber is seen reaching the surface of muscle cells that have been teased apart. • The terminal region branches extensively to form the neuromuscular junction. Striated muscle fibers Motor End Plate Axon From R.A. Bergman, A.K. Afifi and P.M. Heidger Virtual Hospital: Atlas of Microscopic Anatomy University of Iowa

4. Sequence of Events in Neuromuscular Transmission (1) Presynaptic Events • Action potential (AP) is initiated in the presynaptic motor neuron (MN) and invades the endplate region • Depolarization of MN terminal boutons, resulting in the opening of voltage-dependent calcium channels • Influx of Ca2+, down its concentration gradient • Rise of intracellular free [Ca2+] initiates fusion of vesicles containing acetylcholine (ACh) to the membrane of the terminal boutons, resulting in exocytosis of ACh • Diffusion of ACh across synaptic cleft to the muscle cell

5. Sequence of Events in Neuromuscular Transmission (2) Postsynaptic Events • Binding of ACh to nicotinic ACh-receptors at endplate • Receptor binding causes opening of cation channels, leading to influx of Na+ (ACh is then degraded by acetylcholinesterase present in the synaptic cleft) • The resulting depolarization of muscle cell membrane at the endplate is referred to as the endplate potential (EPP) • The local depolarization causes adjacent regions to be depolarized, causing an AP in the muscle cell membrane • AP spreads out in all directions from the endplate, propagates along muscle cell, initiating contraction

6. Motor End Plate Electron micrograph of nerve terminal Presynaptic terminal, with many small vesicles containing ACh Postsynaptic region of the skeletal muscle, with mitochondria and contractile filaments apparent in the cytoplasm From Alberts, Bray, Lewis, Raff, Roberts and Watson, Molecular Biology of the Cell, 2nd edition 500 nm

7. 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? A 50 to 100 nm gap.

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

9. Record Membrane potential of muscle Stimulate nerve Vm 0.5 mV Vm 5 mV Vm 5 mV Stimulate nerve Miniature EPP (spontaneous, 1 vesicle aka 1 quantum) EPP brings membrane to threshold and initiates action potential EPP (evoked, ~200 quanta) End Plate Potentials • From mini-EPP, to summation and EPP to Action Potential Schematic courtesy of T. Stavraky Stimulate nerve

10. Evidence that ACh is the neurotransmitter at the NMJ • ACh is synthesized and released by the nerve terminal; synthesis of ACh is blocked by hemicholinium (which inhibits choline uptake) • Small amounts of ACh applied to the end plate cause depolarization of the muscle. The ACh-induced depolarization and EPP have the same reversal potential and permeability to Na+ and K+. • The pharmacology of the ACh induced depolarization and EPP are the same, including: • (a) activation by ACh agonist (nicotine, carbachol, e.g.) • (b) blocking by ACh receptor antagonist (curare) • (c) enhancement and prolongation the EPP by anticholinesterase (e.g., neostigmine, which prevents the hydrolysis of ACh)

11. Nicotinic ACh Receptor • Receptor is a pentameric complex of 2 a, b, g and d subunits • Binding of 2 ACh molecules causes opening of the channel, which is a channel permeable to all cations, including Na+, K+ and Ca2+. • 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

12. Action potential invades presynaptic terminal Opening of Ca2+ channels leads to influx of Ca2+ (extracellular Ca2+ is essential) Vesicles fuse and release ACh into cleft + + + + ACh diffuses across synaptic cleft ACh activates cation channels to cause depolarization of the endplate. Summary of Neuromuscular Transmission ACh-activated channel is permeable to both Na+ and K+, so the reversal potential is a mixture of the two

13. + + + + Depolarization of the end plate initiates an action potential that spreads over muscle cell Summary of Neuromuscular Transmission ACh is destroyed by acetylcholinesterase enzymes in the synaptic cleft In healthy muscle, an AP in the motor neuron ALWAYS activates an AP in the muscle, leading to contraction (safety factor is ~2).

14. Pharmacology of the End Plate Potential And now for something completely different

15. Ancient Chinese Proverb I hear and I forget. I see and I remember. I DO and I understand. In the doing is the learning!

16. Control EPP + Neostigmine Vm 5 mV Vm 5 mV Control EPP + Curare Stimulate nerve Stimulate nerve Pharmacology of the End Plate Potential • Curare • Blocker of nicotinic receptors • Plant extract used to induce paralysis • Must be careful with patients, as they may feel pain but cannot show it • Neostigmine • Blocker of cholinesterases • Component of nerve gases and insecticides

17. Clinical Case: • A patient presents with muscle weakness, and fatigues easily Findings: • No muscle atrophy • Normal nerve conduction • muscle response to repeated stimulation is decreased • muscle response with repeated direct stimulation of the muscle is normal • When muscle biopsy is studied, small MEPPs and EPPs are observed. • Muscle weakness improves with neostigmine (tensilon has briefer duration, so is better in the clinical setting) From Principles of Neural science, 3rd edition, by E. Kandel, J. Schwartz and T. Jessel.

18. Tentative Diagnosis: Myasthenia Gravis • Myasthenia Gravis is an autoimmune disease in which patients develop antibodies against nicotinic ACh receptors. • Thus, the amplitudes of MEPPs and EPPs are reduced (since there will be less depolarization for the same amount of released ACh) and the muscle membrane may not be depolarized sufficiently to fire an action potential. • Treatment is to give an acetylcholinesterase inhibitor to prolong and increase the action of ACh at the available receptors and to restore the muscle action potential.