Neuromuscular Junction - Lecture 10 PDF

Neuromuscular Junction Dr. Oana Birceanu [email protected]  2 Our goal for today > - guides L /...

Neuromuscular Junction Dr. Oana Birceanu [email protected]  2 Our goal for today > - guides L / movement voluntary info leads t o action ers Neurotransmitt - using talks to synapses On Skeletalmuscular cells  3 Our goal for today The neuromuscular junction (NMJ)  4 Intended learning outcomes The neuromuscular junction Organization – main (NMJ) components of NMJ Biochemistry – how does the neuron communicate with the muscle cell? Signaling at the NMJ Modulation of the NMJ  5 The neuromuscular junction (NMJ) Propagation of action potential Voltage-gated calcium channel - synaptic cleft  6 The neuromuscular junction (NMJ) 1. Action potential (AP) travels down motor neuron, to presynaptic terminal causing depolaration , inside cell becomes m o re the Propagation of action potential 1 Voltage-gated calcium channel  7 The neuromuscular junction (NMJ) 1. Action potential (AP) travels down motor neuron, to presynaptic terminal rushes into cell , stimulates Propagation of resicles to m ove toward action potential ~ - Synaptic cleft 1 2. Activation and opening of the voltage-gated Ca2+ channels 2 Voltage-gated calcium channel  8 The neuromuscular junction (NMJ) 1. Action potential (AP) travels down motor neuron, to presynaptic terminal Propagation of action potential 1 2. Activation and opening of the voltage-gated Ca2+ channels 3. Ach-containing vesicles fuse with the post- 2 synaptic membrane and release ACh Voltage-gated calcium channel 3  9 The neuromuscular junction (NMJ) 1. Action potential (AP) travels down motor neuron, to presynaptic terminal Propagation of action potential 1 2. Activation and opening of the voltage-gated Ca2+ channels 3. Ach-containing vesicles fuse with the post- 2 synaptic membrane and release ACh Voltage-gated 4. Binding of neurotransmitter to ligand-gated ion calcium channel channels on muscle change in membrane permeability 3 opening of ion channels 4 ↳ a : c.. a Nicotinic receptors ↳ can also bind nicotine causing depolarization  10 The neuromuscular junction (NMJ) 1. Action potential (AP) travels down motor neuron, to presynaptic terminal Propagation of action potential 1 2. Activation and opening of the voltage-gated Ca2+ channels 3. Ach-containing vesicles fuse with the post- 2 synaptic membrane and release ACh Voltage-gated 4. Binding of neurotransmitter to ligand-gated ion calcium 5 channel channels on muscle recycled pre-synaptic - acetylcholine m a ke back to m o re to acetylcholine change in membrane permeability 3 L can diffuse out of synaptic cleft opening of ion channels by going↓ i ts [I grad. 4 5. ACh broken down by acetylcholinesterase (AChE)  11 Acetylcholine acts via fast transmission RE-LISTEN TO THIS SLIDE On the post-ganglionic Nicotinic acetylcholine receptors Transmembrane protein complex composed of 5 subunits Fast response - Acetylcholine binds to the 2 alpha subunit – 1 molecule per subunit Opens channel, potassium rushes with a little bit of sodium, causing depolarization Located at NMJ and at postganglionic cells of vertebrate autonomic nervous system  12 Acetylcholine acts via slow transmission GPCR is metaboltropic not ionotropic. Bind to GPCR causes opening of an ion channel Muscarinic receptors G-protein coupled receptors Acetylcholine (ACh) binds to G- protein coupled receptor Activation of G-protein complex on cytoplasmic side Alpha-subunit activation - can act directly on the ion channels or effector protein and intracellular messengers  13 Measuring activity of the nAChR Voltage clamp of skeletal muscle cell Presynaptic neuron synapses on the post synaptic neuron Purves et al., Neuroscience  14 Measuring activity of the nAChR Voltage clamp of skeletal muscle cell 1. Voltage clamp muscle using 2 electrodes, and simultaneously electrically stimulate the presynaptic motor neuron Purves et al., Neuroscience  15 Measuring activity of the nAChR Voltage clamp of skeletal muscle cell 1. Voltage clamp muscle using 2 electrodes, and simultaneously electrically stimulate the presynaptic motor neuron 2. Stimulation of motor neuron will cause the release of acetylcholine from the presynaptic motor neuron If stimulate the middle of the axon with a current, it can go either way Purves et al., Neuroscience  16 Measuring activity of the nAChR Voltage clamp of skeletal muscle cell 1. Voltage clamp muscle using 2 electrodes, and simultaneously electrically stimulate the presynaptic motor neuron 2. Stimulation of motor neuron will cause the release of acetylcholine from the presynaptic motor neuron 3. This will enable you to record macroscopic end plate currents (EPCs) Because the region where the neuromuscular region is called the neuromotor end plate Purves et al., Neuroscience  20 Measuring activity of the nAChR Voltage clamp of skeletal muscle cell 1. Individual nAChr open briefly 2. Measures activity of multiple channels opening at the synapse Purves et al., Neuroscience  21 Measuring activity of the nAChR Measuring EPC with voltage clamp Voltage clamp of skeletal muscle cell 1. Individual nAChr open briefly 2. Measures activity of multiple channels opening at the synapse 3. Brief opening of a large number of channels is synchronized, such that a macroscopic current can be recorded (summed End-Plate Current – EPC) Current thats being generated from multiple nicotinic channels open from acetylcholine binding the receptors Purves et al., Neuroscience  22 The end plate potential Voltage clamp of skeletal muscle cell  23 The end plate potential Voltage clamp of skeletal muscle cell nAchR opening creates an excitatory postsynaptic potential in the muscle, triggering the opening of voltage-gated Na+ and K+ channels along a muscle fiber Creating an AP Resting membrane potential is -90 mV in the muscles because it has more leaky channels than a neuron where it’s -70 mV RMP  24 The end plate potential This membrane depolarization of ~+30- Voltage clamp of skeletal muscle cell 40mV is the threshold for action potential in the muscle! nAchR opening creates an excitatory postsynaptic potential in the muscle, triggering the opening of voltage-gated Na+ and K+ channels along a muscle fiber  25 The end plate potential EPPs are NOT APs because EPP can be summed to reach AP This membrane depolarization of ~+30- Voltage clamp of skeletal muscle cell 40mV is the threshold for action potential in the muscle! nAchR opening creates an excitatory postsynaptic potential in the muscle, triggering the opening of voltage-gated Na+ and K+ channels along a muscle fiber Leads to a suprathreshold depolarization and therefore an action potential in the skeletal muscle (and therefore muscle contraction!) It could be a graded potential to be summed to create an AP  26 The end plate potential This membrane depolarization of ~+30- Voltage clamp of skeletal muscle cell 40mV is the threshold for action potential in the muscle! nAchR opening creates an excitatory postsynaptic potential in the muscle, triggering the opening of voltage-gated Na+ and K+ channels along a muscle fiber Leads to a suprathreshold depolarization and therefore an action potential in the skeletal muscle (and therefore muscle contraction!) EPPs are not APs – they trigger them!  27 EPP causes opening of voltage-gated Na+ channels ACh-induced depolarization Indentations on membrane to increase surface area so that there’s causes End Plate Potential more nicotinic receptors only at motor end plate of neuromuscular (EPP) junction acetylcholine doesn’t go into muscular cells, it only binds to nicotinic receptors at the neuromuscular junction Acetylcholine binding allows sodium to go into the cell, there is some sodium outflow at the same time 27 Kandel, Principles of Neural Science  28 EPP causes opening of voltage-gated Na+ channels ACh-induced depolarization causes End Plate Potential (EPP) If threshold is not reached, no voltage gated sodium channels open 28 Kandel, Principles of Neural Science  29 EPP causes opening of voltage-gated Na+ channels ACh-induced depolarization causes End Plate Potential (EPP) 29 Kandel, Principles of Neural Science  30 BRAIN DRAIN!!! Time to review what you know on your own! NMJ Voltage clamping NMJ components signaling of a muscle cell List Draw the NMJ Describe the components Signaling Advantages and pathway – steps disadvantages 1-5 EPC, 30 EPP, AP  32 Pathophysiology of NJM Myasthenia gravis A chronic neuromuscular disease that is characterized by weakness in skeletal muscles  33 Pathophysiology of NJM Myasthenia gravis A chronic neuromuscular disease that is characterized by weakness in skeletal muscles Autoimmune disease, more common in women (2x the chance in men) Occurs due to autoantibodies that attack the nicotinic acetylcholine receptor If antibody attacks and binds the nicotinic receptors, it breaks down the receptors as they’re formed into the cell. Acetylcholine does not bind to the receptor, and it gets broken down by AChE  34 Pathophysiology of NJM Myasthenia gravis A chronic neuromuscular disease that is characterized by weakness in skeletal muscles Occurs due to autoantibodies that attack the nicotinic acetylcholine receptor ACh in the synaptic cleft cannot bind to receptors – broken down by acetylcholinesterase (AChE) Reduced EPP and muscle contraction  35 Pathophysiology of NJM Myasthenia gravis A chronic neuromuscular disease that is characterized by weakness in skeletal muscles Occurs due to autoantibodies that attack the nicotinic acetylcholine receptor ACh in the synaptic cleft cannot bind to receptors – broken down by acetylcholinesterase (AChE) Body will think it needs less nicotinic receptors so it also makes less of it. Indentations will be lost because we use The nicotinic receptor less frequently Reduced EPP and muscle contraction  36 Diseases of NJM and pharmacological modulations Healthy NJM Myasthenia gravis Acetylcholine stays in the synaptic cleft longer, increasing the chance of breaking down by AChE Endplate potential represents the difference In potential between the inside and outside of the cell Micro EPP occurs in the muscle Endplate potentials will not reach threshold so AP can not be fired. There will always be a little bit of acetylcholine released it becomes harder and harder to Because it’s not a perfectly enclosed system reach threshold With myasthenia gravis, AchR In a healthy muscle, autoantibodies lead to loss of have EPP and mEPPs AChR, smaller EPP and mEPP; EPP may not reach threshold Phillps & Vincent (2016) F1000Research  37 Cogan’s eyelid twitch – tool to diagnose MG Eyelid twitch response (Cogan’s eyelid twitch) – first symptom of MG Elicited by looking down and then straight 57yo woman – normal brain MRI, generalized weakness MG was confirmed by repetitive nerve stimulation and positive anti-nAChR binding antibody test https://n.neurology.org/content/87/5/e55.short  What do you think would be a 38 good therapeutic approach here? A drug that inhibits the aChE so that acetylcholine doesn’t degregate as quick and gives it a chance to bind nicotinic receptors  39 Therapeutic approaches Myasthenia gravis AChE inhibitor – allows ACh to be in the synaptic cleft for longer  40 Therapeutic approaches Myasthenia gravis AChE inhibitor – allows ACh to be in the synaptic cleft for longer Pharmacological agents: Neostigmine – preferred treatment course Endrophonium – short anti-AChE activity Physostigmime – not used – crosses the blood brain barrier, leading to secondary , undesired, effects Why is neostigmine the preferred pharmaceutical? It lasts longer, and you don’t want it to be in the BBB  Brain drain! 41 Write down as much as you can in 2 min The neuromuscular junction (NMJ) Organization – main Pathophysiology of the NJM components of NJM Signaling – how does the Pharmacological modulation of neuron communicate with the NJM the muscle cell?