Hons Neuroscience/Biomedical Sciences THE NEUROMUSCULAR JUNCTION IN HEALTH AND DISEASE Practical Exercises: Function and Structure of Neuromuscular Synapses Aim 1. To make recordings and explore the control of action potentials in your own motor units; 2. To investigate and measure the action, specificity, and efficacy of ions and drugs that affect synaptic transmission and muscle excitability at the neuromuscular junction. 3. To study and measure the structure and organization of neuromuscular synapses Skills you will develop - Recording action potentials (electromyogram, EMG) using surface electrodes applied to the skin overlaying your own muscles - Measuring twitch contractions in an accurate computer simulation of the classic, rat diaphragm nerve- muscle preparation - Constructing dose- response curves, in response to addition of ions and drugs - Appraising the morphology of healthy and diseased neuromuscular synapses - Making and keeping accurate records of your practical work - Answering questions about the experiments you have done and their theoretical background Background The exercises will be held in the Greenfield computer suite. However, all the software you will use is in the public domain and can be downloaded free of charge from the following websites: 1. Backyard Brains: https://backyardbrains.com/ 2. Strathclyde Institute of Pharmacy and Biomedical Sciences: http://spider.science.strath.ac.uk/sipbs/showpage.php?page=software_si ms 3. ImageJ: http://rsbweb.nih.gov/ij/ The exercises comprise a mixture of hands- on recording of motor unit activity in your own muscles; experiments using a computer simulation; an on- line tutorial on structure and function of neuromuscular junctions; and an exercise using image analysis techniques to obtain morphological data from images of neuromuscular junctions. 1
Exercise 1. Recording EMG using a Backyard Brains SpikerBox 1. Go to the website for Backyard Brains. You will find a youtube video there, which you can also find here: http://www.youtube.com/watch?v=e2dalnla8_g 2. Attach a pair of adhesive electrodes to your forearm and connect them to the spiker box input terminals. Attach the third (ground) electrode clip to an item of metal jewelry you are wearing or, to an adhesive electrode over your wrist. 3. From your Greenfield Suite computer, locate and run from the Start menu (bottom left, multicolour flags icon), the Backyard Brains Neuron Recorder program. Connect one output of the Spiker box to a set of earphones or headphones if you have them, and the other to the line- input of your computer (this will be demonstrated). (If you have an ipad, or a smartphone like an iphone or Android, you can download an App from the Backyard Brains website or Apple s AppStore and connect the Spiker box output to that, using one of the special cables provided. Ask a demonstrator. The Neuron Recorder program is also free so you could download and run it using your own laptop PC/Mac if you wish). 4. Start the Neuron recorder program and voluntarily contract your arm muscles, observing the spikes on the screen and the crackle and clicks you hear from the loudspeaker. How do the spikes change as you increase the strength of your muscle contraction? 5. Now semi- quantify this. Take the grip- strength dynamometer and exert forces of increasing magnitude that you can read of the digital display: try 1kg, 2kg, 5kg, 10kg, 20kg, 40kg. Note on a scale of 1-10, a) the frequency, b) the amplitude of the spikes as you apply increasing force. Kg force 0 1 2 5 10 20 40 Frequency Amplitude Summarise what you have learned, including making sketches of the responses you have elicited. How might you quantify the recordings? What do we need to know in order to understand what you have observed? Can you think of any other experiments you could do using the Spiker Box to help you find out? What other research and techniques might you use to take your knowledge and understanding of mechanisms and/or functions to a deeper level? What could go wrong, leading to dysfunction or disease? 2
Exercise 2. Simulation of neuromuscular function in the rat diaphragm The simulated preparation consists of the hemisected diaphragm of a rat, a classical skeletal muscle preparation in neuromuscular pharmacology. In a real experiment, the hemidiaphragm from one side (usually the left side, for technical reasons) is dissected complete with a long distal stump of the phrenic nerve. Stimulation of the nerve with single pulses, about 0.1 ms in duration and 1-5V in amplitude, triggers action potentials in the motor nerve fibres, which in turn cause release of acetylcholine (ACh) from synaptic vesicles in motor nerve endings. The ACh molecules diffuse across the narrow synaptic cleft and bind to specific, nicotinic receptors in the membranes of the motor endplate region of the muscle fibre. These ionotropic receptors mediate a current (the endplate current, EPC) that depolarizes the muscle fibre (the endplate potential, EPP). An EPP evoked in this way is normally large enough to trigger an action potential in the muscle fibre. The simultaneous firing of action potentials in all the muscle fibres can be detected and monitored by EMG recording (see part 1) and also by the twitch contraction of the muscle. These contractions can be measured, for example by attaching the muscle to a force transducer that changes its electrical resistance in proportion to the force applied to it. Incorporation of the transducer into a Wheatstone bridge circuit and recording the output through a analogue- to- digital interface allows the change in resistance (hence, force) to be digitized, measured and displayed on a computer screen. When we change the ionic environment, or add drugs to a nerve- muscle preparation, these ions or drugs may affect conduction of nerve impulses; synthesis, storage, release, action and inactivation of the ACh neurotransmitter; action potentials in muscle fibres; or muscle contractions. However, in this exercise what we are measuring in all cases is the end result: muscle contractile force. Thus, preparations like the rat phrenic nerve- hemidiaphragm have proved useful in giving us a quantitative but rough indication of the effects of drugs. The effects must be interpreted with caution, however. More sophisticated investigations are normally required, using electrophysiological techniques or ligand binding assays, to establish the precise mechanisms of action the ions or drugs, and on which specific components of the neuromuscular system they are acting. Accessing the Virtual Twitch Program 1. Log- in to an open- access computer. 2. From the Start menu (lowest left four- colour flag icon) select 'All Programs' then select '_School Applications_' then click on 'Medicine and Veterinary Medicine' and select the 'MedCAL' option. (You may find there is a MedCAL shortcut already on your Desktop display.) From the menu that appears, select Virtual Twitch. This program can also be downloaded to your own computer from here: http://spider.science.strath.ac.uk/sipbs/showpage.php?page=software_sims 3
Simulated twitch contractions of the diaphragm muscle are produced, as in a real experiment, either using a stimulating electrode attached to the phrenic Nerve ( indirect ); or by an electrode attached directly to the Muscle ( direct ) stimulation. (Note: It is important to appreciate that direct stimulation will also normally also stimulate the motor nerves that ramify throughout the muscle. However, if neuromuscular transmission is blocked, then direct stimulation will excite only the muscle fibres. ) Important Notes: 1. Ionic and drug concentrations in the program are given in exponent form: e.g. 2x10-6 M (or 2 µm), is entered as 2.0E- 006 M. 2. Drug additions are cumulative. This means that if you add the same dose twice, then the concentration is doubled in the bath. 3. When you apply Muscle (direct) stimulation, take care to return to Nerve (indirect) stimulation before giving the next drug Varying ion concentrations in the bathing medium and addition of drugs can be simulated in this program, to illustrate various presynaptic and postsynaptic effects. Experiment A: Changing Ions As you should have learned from lectures on excitable cells physiology, resting potentials and action potentials in nerve axons are very sensitive to changes in Na + and K + ions in the extracellular fluid. Synaptic transmission is, in addition, very sensitive to the concentration of Ca 2+ ions. This is because the probability of exocytosis of a synaptic vesicle and release of neurotransmitter (acetylcholine, ACh, in this case) is strongly influenced by Ca 2+ ions, which interact co- operatively with a Ca sensor proteins, including synaptotagmin, found in the SNARE complexes that control exocytosis. The main source of the Ca 2+ for exocytosis is from the extracellular fluid. Ca 2+ ions flow into the nerve terminal through voltage- gated Ca- channels in the nerve terminal membrane when it is depolarized by an action potential. The amount of Ca 2+ entering is very small (less than 1 pm per cm 2 of membrane), but the volume of the nerve terminal is also very small. Thus, following an action potential, intracellular Ca 2+ concentration increases from about 1 nm to about 0.1 µm. Thus, there is about a 100- fold increase in intracellular Ca 2+ concentration. Mg 2+ ions compete with Ca 2+ for entry through the Ca- channels, and possibly for binding sites on the Ca 2+ sensors. By contrast, Mg 2+ ions do not promote exocytosis: they inhibit it. 4
Procedure 1. Drag the screen dimensions for a large display or click the full screen button at the top right of the window. 2. Make sure the Stimulator is set to Nerve (indirect). Click the Start button. Observe successive twitch contractions as the trace scrolls across the screen. (You may notice periodic changes in the thickness of the vertical lines. This is a digital display artifact called aliasing ). Note how the peaks of the twitch contractions vary slightly. 3. Click the Stop button. Measure the average of ten successive twitch contractions by hovering the cursor over their peaks and recording the Twitch amplitude in grams. Enter the values into a Microscoft Excel spreadsheet (run from the Start flag All programs.. Microsoft Office..). Calculate the mean and standard deviation using the inbuilt AVERAGE and STDEV functions. Convert the mean value to Newtons of force by multiplying the value in kg by the gravitational constant, 9.81ms - 2 (note the measurements are in grams so you must convert to kg). Save the file with an appropriate name on your area of the server. 4. Open a Microsoft Powerpoint file. Make the Experiment window the top window by clicking it again to show the twitches. Now press <ALT>- PrintScreen together. This copies the window to the Clipboard. Click on your Powerpoint file and Paste (or <CTRL>- V) the image into the slide. Add text to describe what it is. Save the file with an appropriate name on your area of the server Effect of Calcium ions Change solution to one with zero calcium by selecting the Low Ca Kreb s from the Wash menu at the top of the window. After the trace has settled press the Stop button. Scroll to the region of interest and copy (<ALT>- PrintScreen) and paste the image into another Powerpoint slide. 1. What happens to the twitch contractions over time? 2. How rapidly does this happen? 3. Why do you think it takes so long? 5
Select Muscle (direct) stimulation and click Start. The contraction is back! After a few twitches have been recorded, press Stop. Click Nerve (indirect) stimulation. Select Wash from the top menu and select Normal Ca Kreb s solution. Click Start. Observe the twitch contractions gradually return. When the contractions have recovered, press Stop. 1. Why does the muscle still respond to direct but not indirect stimulation in low Calcium Krebs? 2. Why does contraction slowly return when Ca 2+ ions are returned? 3. Why do you think it takes so long? Now do an experiment to measure the effect of increasing Ca 2+ over a range from 100 µm to 1 mm. First, select Wash Low- Ca Krebs and press Start. Once the contractions have stopped, Select Ions, Calcium and from the dropdown menu 1.0E- 004 M. This means you are adding Ca 2+ to the bathing medium to a final concentration of 100 µm. Suppose the bath volume was 10 ml and you had a stock solution of 0.1M CaCl2. What volume of stock would you add to the bath to obtain a final concentration Ca 2+ concentration of 100 µm? Make sure you have Nerve (indirect) stimulation selected and press Start. You should see little or no response. We must therefore conclude that 100 µm Ca 2+ is not sufficient to restore or maintain synaptic transmission at any of the neuromuscular synapses. Now add another dose of Ca 2+ to increase the concentration to 200µM (by adding another 1.0E- 004 M ). Note that the accumulated concentration in the bath is indicated below the trace at the point in time when the solution was added. 6
Once the trace has stabilized, add successive doses of 100 µm Ca 2+ until you reach a bath concentration of 1 mm (that is, until the note under the trace 1.0E- 003 M). The normal twitch is almost restored. Press Stop. Use the scrollbar to display the result of your experiment and copy and paste it to your Powerpoint file. Enter text description (adding arrows, if you wish) and save the file. Return to the traces. Measure the average amplitude of three twitches in the region where the trace has stabilized, and record this against the bath concentration of Ca 2+ in your Excel Spreadsheet. Plot a graph of the amplitude of the twitch, as percentage of the maximum, against the logarithm of the Ca 2+ concentration in the bath. Copy and paste this graph into your Powerpoint file. Comment on the nature and steepness of the relationship between force of contraction and Ca 2+ concentration: Effect of Magnesium ions. Without washing the preparation, press Start and select Magnesium from the Ions menu. Increase the ionic concentration of Mg 2+ ions by 1 mm (1.0E- 003 M). Allow the trace to stabilize. Now progressively increase Mg 2+ in 1 mm steps, allowing the trace to stabilize each time, until the concentration of Mg 2+ in the bath has reached 10 mm. Press Stop; scroll so your region of interest fills the screen and copy and paste it into a new slide in your Powerpoint file. Add arrows and make notes as appropriate. Return to your traces, measure the average of three contractions at each concentration of Mg 2+, transfer the data to your Excel file and plot twitch contraction directly against Mg 2+ concentration. 7
Comment on the sensitivity and the relationship of the muscle contractions to Ca 2+ ions compared with the relationship and sensitivity to Mg 2+ : Use a web browser and google Pubmed. Locate the classic paper by Dodge and Rahamimoff (1967) PMID 6065887. Download the paper. Read the Summary at the front of the paper then locate Figure 3. Comment on the relationship between the amplitude of the End- plate potential (EPP) in a single muscle fibre as Ca 2+ ionic concentration is increased, in progressively increasing concentrations of Mg 2+ ions. How might the analysis in Dodge & Rahamimoff s research paper help you to understand the effects of Ca 2+ and Mg 2+ on muscle twitch contractions? Finally, go to this website: https://en.wikipedia.org/wiki/reference_ranges_for_blood_tests..and examine the ionic composition of human blood plasma from the link to 3.1: Ions and Trace Metals. Summarise what you have learned from this part of the practical and note the file names of your Excel and Powerpoint files for future reference. Print out the spreadsheet and graphs, and the representative traces you have saved and attach them to this workbook. Make sure your name and the date are noted on each page. This is good laboratory practice. 8
1. What are the normal concentrations of Ca 2+ and Mg 2+ ions in human blood plasma? 2. What are the normal concentrations of Ca 2+ and Mg 2+ ions in Krebs mammalian physiological saline? 3. What concentration of Ca 2+ ions was required to maintain indirect muscle twitch contractions of the rat diaphragm at half the maximum value? 4. In the presence of 1 mm Ca 2+, what concentration of Mg 2+ ions reduces the twitch contractions to half the initial amplitude? 5. Mathematically, what is the relationship between the amplitude of an endplate potential (EPP) and Ca 2+ concentration? 6. Mathematically, what is the relationship between the amplitude of an EPP and the Mg 2+ concentration? 9
Experiment B : Adding Drugs Drugs can act presynaptically, affecting neurotransmitter release; or postsynaptically, affecting sensitivity to neurotransmitter. In addition, some drugs interfere with the inactivation of neurotransmitter, by inhibiting or enhancing either its breakdown; or by inhibiting or enhancing the uptake of neurotransmitter or its breakdown products. At mammalian neuromuscular junctions, acetylcholine is released by exocytosis from synaptic vesicles; and the molecules bind to nicotinic acetylcholine receptors in the muscle membrane, embedded in the membranes of the junctional folds, at the motor endplate. Acetylcholine is normally broken down to acetate ions and choline, catalyzed by the enzyme acetylcholinesterase. Here you will investigate the effects of a drug that enhances neurotransmitter release (4- aminopyridine); drugs that inhibit nicotinic acetylcholine receptors (d- tubocurarine and suxamethonium); a drug inhibit acetylcholinesterase (neostigmine); and a drug that inhibits the sodium channels that cause action potentials (tetrodotoxin). 4- aminopyridine is used clinically in the treatment of Lambert- Eaton Myasthenic Syndrome (LEMS), an autoimmune disease that attacks voltage- sensitive Ca- channels in presynaptic motor nerve terminals, reducing neurotransmitter release. It is also used in the treatment of the demyelinating disease, multiple sclerosis, in which action potentials in nerve axons fail, causing loss of sensation and paralysis of movement, due to the loss of their insulating myelin sheath. Analogues of d- tubocurarine, such as atracurium or rocuronium, are used clinically to produce muscle relaxation, for example during surgery. Suxamethonium is also used as a muscle relaxant during certain, short surgical procedures. Neostigmine is used clinically in the treatment of the disease myasthenia gravis (MG), an autoimmune disease that attacks nicotinic ACh receptors at neuromuscular junctions, reducing end- plate sensitivity to acetylcholine. Another, short- acting anticholinesterase called edrophinium is used in a diagnostic test for MG: the tensilon test: patients with MG show sudden (temporary) recovery from muscle weakness when this drug is administered. Drugs that block sodium channels are commonly used as local anaesthetics, although tetrodotoxin is not used in this way because it is too potent and not metabolized, and therefore too dangerous. One of the most common local anaesthetics is lignocaine, widely used in dentistry, for example. It is safe, relatively short acting and swiftly metabolized. 10
Effect of 4- aminopyridine In Experiment A you varied the Ca 2+ /Mg 2+ ionic concentration ratio and found this affected twitch contractions evoked by nerve stimulation. This is most simply explained by the requirement of Ca 2+ ions for neurotransmitter release. In the present experiment you will examine the effect of 4- aminopyridine when the twitch contractions are partially inhibited by a reduced concentration of Ca 2+ ions. Begin by starting a New Rat from the File menu. Ensure you have Nerve (indirect) stimulation. Wash with low- Ca Krebs so that the contractions fall to zero. Now add back Ca 2+ to a final bath concentration of 500µM (5.0E- 004 M). Once the contractions have stabilized, select Drugs, 4- aminopyridine and add 1 µm (1.0E- 006 M). Once the trace has stabilized, press Stop; adjust the control to bring the complete trace into the window, and copy and paste the record into your Powerpoint file. 1. What effect did 4- aminopyridine have on the muscle twitch contractions? 2. 4- aminopyridine is a drug that blocks the voltage- gated potassium channels that are present in nerve terminal membranes. These channels are normally responsible for repolarization of the membrane during the action potential. How would you therefore expect 4- amino pyridine to affect the action potential? 3. Ca 2+ ions enter motor nerve terminals through voltage- sensitive Ca- channels but 4- aminopyridine has no direct effect on these channels. So, why is Ca 2+ entry prolonged, leading to enhanced neurotransmitter release, in the presence of 4- aminopyridine? 4. How do you think you might test your hypothesis/explanation? 11
Effect of d- tubocurarine. This drug is a competitive nicotinic cholinergic receptor antagonist and, thus, a blocker of neuromuscular transmission in skeletal muscle, where ligand- gated, nicotinic acetylcholine receptors are found at motor end- plates. Start a new experiment by selecting New Rat from the File menu. Ensure you are using indirect stimulation. Press Start. Record a few twitches, then add d- tubocurarine to a concentration of 0.2µM (2.0E- 007 M). Suppose the bath volume was 10 ml and you had a stock solution of 10 mg/ml d- tubocurarine (MW 625). What volume would you need to add to the bath to achieve a concentration of 0.2 µm? Now progressively increase the dose of d- tubocurarine in steps of 0.2 µm. Allow the twitches to stabilize at their new level before adding each additional dose. Continue adding doses until the final concentration is 1 µm. Once your reach this concentration, progressively increase the bath concentration by 1µM until it reaches 5 µm. Switch to Muscle (direct) stimulation. The contraction comes back!! Press Stop. Transfer the record to your Powerpoint File. Measure the average response of three twitches from the stable regions at each concentration of d- tubocurarine and transfer the data to your Excel spreadsheet. Plot a log dose- response curve (twitch amplitude as percentage of maximum on the y- axis (ordinate) against logarithm of the d- tubocurarine concentration and calculate the EC50. Return to your experiments. Switch back to Nerve (indirect) stimulation. Press Start. From the Drugs menu select Neostigmine and a concentration of 1µM. You will observe a substantial recovery of the twitch. Copy the trace to your Powerpoint file and label it appropriately. 12
1. What effect did d- tubocurarine have on the muscle twitch contractions? 2. What was the EC50 of d- tubocurarine (concentration required to reduce the twitch contraction in normal Krebs solution by 50%)? 3. d- tubocurarine acts postsynaptically: so, how come direct muscle stimulation produce a maximal twitch contraction? 4. Why does administration of neostigmine counteract the blocking effect of d- tubocurarine? 5. d- tubocurarine is a drug that blocks nicotinic Ach receptors. These ligand gated channels are normally responsible for depolarization of the muscle at the motor endplate following a nerve stimulus: the endplate potential (EPP). EPP s are normally twice the size they need to be to trigger a muscle action potential. Each EPP is made up of quantal steps about 1 mv in amplitude (miniature EPP s). How would you expect d- tubocurarine to affect EPPs and mepps? 13
Effects of Atropine, Suxamethonium and Tetrodotoxin Atropine is a drug that blocks a different type of ACh receptor: the so- called muscarinic type of receptor, found for example in cardiac and smooth muscle. By contrast, d- tubocurarine has no effect on muscarinic receptors. We can therefore test a hypothesis that the ACh receptors in a type of muscle tissue are muscarinic or nicotinic, by comparing the effects of atropine and d- tubocurarine. The test is not definitive, but it is a good start. Start with a New Rat from the File menu. Ensure Nerve (indirect) stimulation is selected. Press Start and collect a few twitch contractions. Select Drug Atropine Add 1 mm (1.0E- 003 M) to the bath. Wait about a minute, then add 1 µm (1.0E- 006 M) of d- tubocurarine Wash with Normal Krebs solution. Press Stop when the contractions are fully recovered. Suxamethonium is a dimer of acetylcholine. It has complex effects on ACh receptors, initially activating them then desensitizing them and inactivating depolarization. For this reason it is referred to as a depolarizing blocker. It is metabolized readily and is used in some forms of surgery requiring brief muscle relaxation. Ensure you are stimulating via the Nerve (indirect). Press Start and collect a few baseline twitches. Select Drug Suxamethonium.Add 20µM to the bath (2.0E- 005 M). Observe that the twitch contractions are substantively reduced. Now add neostigmine at 10 times the dose that was effective when d- tubocurarine was inhibiting the contractions (ie 10µM; 1.0E- 005 M). Not only does neostigmine not counteract the effect of suxamethonium, it adds to the latter s inhibitory effect!!. Switch to Muscle (direct) stimulation. Collect a few twitches, then press Stop. Copy the trace to your Powerpoint file and label it appropriately 14
Describe the effect of atropine, how it differs from the effect of d- tubocurarine, and why: Describe the effect of suxamethonium. How does the effect of neostigmine differ from the effect when d- tubocurarine is present instead of suxamethonium? Suxamethonium is a depolarising blocker of ACh receptors, whereas d- tubocurarine is a competitive antagonist of the nicotinic receptors. Why does the effect of neostigmine in the presence of suxmethonium differ from its effect in the prescence of d- tubocurarine? Tetrodotoxin is a non- selective blocker of voltage- gated sodium ion channels (NaV 1 type). It therefore blocks action potentials and, hence, muscle contractions. Wash the preparation with normal Krebs solution. Ensure that the Stimulator is set for Nerve (indirect) stimulation. Press Start and collect a few twitches. When the contractions have stabilized, add tetrodotoxin (1 µm; 1.0E- 006 M) to the bathing medium. Observe the rapid and almost complete blocking effect. Remember how when we switched to direct stimulation in either low Ca- Krebs or in the presence of d- tubocurarine, how the contractions were restored with Muscle (direct) stimulation? Why might you expect it to be different this time? To test your hypothesis, switch to Muscle (direct) stimulation. After about a minute, Wash with normal Krebs solution and observe the recovery. Switch back to Nerve (indirect) stimulation and observe that the nerve- evoked response has recovered as well. Press Stop and copy your records to your Powerpoint file and label it. Save the Powerpoint file and your Excel file. 15
Final Note: The effects of TTX are utilized by Japanese gourmet chefs: fugu is a fish dish prepared by cooking puffer fish sufficient to reduce the toxicity of TTX, but retaining sufficient levels to produce tingling and numbness in the lips of diners. Needless to say, there are a few deaths each year caused by inadequate preparation of fugu.. Describe the effects of tetrodotoxin on Nerve (indirect) and Muscle (direct) stimulation: Tetrodotoxin is a non- specific, reversible blocker of voltage- gated sodium channels. How does this explain the effects you have observed and described? µ- conotoxin is a selective sodium- channel antagonist that blocks voltage- gated sodium ion channels of the Nav1.5 subtype, found in skeletal muscle fibre membranes. A µ- conotoxin insensitive type of Nav channel is found in nerve axons. In light of this, how would twitch responses in µ- conotoxin differ from the effect of TTX? Suppose instead you were making a microelectrode recording in a muscle fibre rather than muscle twitches. If the preparation was then treated with µ- conotoxin, how would the response differ from EPPs recorded in the presence of d- tubocurarine? If you are interested in testing your explanation, go back to the MedCAL menu and try out the intracellular EPP simulator program: Virtual NMJ. Block the muscle action potential with 10µM µ- conotoxin and note or measure the EPP. Then add 1 µm d- tubocurarine and observe and measure the effect. You can also investigate the effect of reduced Ca 2+ and/or increasing Mg 2+ in this simulation. 16
Exercise 3: Morphology of Neuromuscular Junctions Do this part of the practical in your own time. Go to the following web page: http://www.dns.ed.ac.uk/rrrweb/nmjtutorial.htm Follow the sequence of images you find there and answer the following questions: What techniques can be used to visualize a) motor nerve terminals b) motor endplates c) synaptic basal lamina How many neuromuscular junctions are there in a typical mouse muscle motor unit? What four cell types are found at a mammalian neuromuscular junctions? What is the approximate length of a motor endplate? What is the approximate area of a motor end- plate? How does the shape of the motor nerve terminal (presynaptic) relate to the structure of the motor endplate (postsynaptic)? Where is acetylcholinesterase located? Whereabouts on the motor endplate are acetylcholine receptors located? Whereabouts on the motor endplate are voltage- gated sodium channels located? How many active zones are present in one synaptic bouton? What are the approximate dimensions of the following: - Synaptic vesicle : - Synaptic cleft : - Junctional fold : - Muscle sarcomere : Comment on the innervation of immature neuromuscular junctions compared with adults: Comment on the structure of degenerated neuromuscular junctions after nerve injury: Comment on the structure of reinnervated neuromuscular junctions after nerve regeneration: Comment on the abnormal structure of neuromuscular junctions in the series of images taken at differen stages of the motor neurone disease, ALS: 17
Exercise 4: Measurement and analysis of EPP s A logical extension of measurement of the effects of ions and drugs on nerve- evoked muscle contractions is to record the effects of these treatments on the electrical responses of individual muscle fibres: endplate potentials or endplate currents. From the MedCAL menu select Virtual NMJ. This program can also be downloaded to your own computer from here: http://spider.science.strath.ac.uk/sipbs/showpage.php?page=software_sims Record the responses to nerve stimuli. Investigate the effects of drugs. Copy and paste illustrative records and annotate them in a Powerpoint file. Exercises 1. Compare the effects of µ- conotoxin and d- tubocurarine on the generation of action potentials and on the amplitude of the EPP. 2. Measure the amplitude, rise time and time- to- half- decay of a series of EPPs in low Ca- High Mg. Note how they fluctuate from stimulus to stimulus: this is due to the probabilistic nature of exocytosis and transmitter release, despite the relative constant amplitude trigger of an action potential 3. Calculate the quantal content using the Direct Method by dividing the mean amplitude of a series of EPPs by the mean amplitude of spontaneous MEPP s that you will occasionally observe on some of the traces. 4. Reduce the Ca- ion concentration until several of the nerve stimuli fail to evoke an EPP. Count the number of failures in a series of 100 stimuli. Use the failures method to calculate the mean quantal content of the EPPs (including zeroes). Do the estimates agree with the Direct Method? 5. Measure the mean amplitude (including zeroes, from failures) in the series of 100 EPP s and calculate the variance and coefficient of variation (CV=standard deviation/mean). The variance method estimates quantal content from 1/CV 2. How does the estimate compare with the Direct and Failures methods? Note: this Virtual NMJ simulation is based on a sophisticated and versatile synaptic recording and analysis program called WinWCP, that can be downloaded from here: http://spider.science.strath.ac.uk/sipbs/software_ses.htm 18
Exercise 5. Measurement of neuromuscular junction morphology ImageJ can also be freely downloaded to your own home computer from here: http://rsbweb.nih.gov/ij/ Download and follow the tutorial Using ImageJ to measure NMJ from: http://www.dns.ed.ac.uk/rrrweb/nmjtutorial.htm Go to the following URL: http://www.dns.ed.ac.uk/rrrweb/yfp/yfpwld01.htm The image you observe there is a montage of an axotomised lumbrical muscle from a Wld S mutant mouse, in which degeneration of axons and motor nerve terminals occurs about ten times more slowly than in normal, wild- type mice. The preparation was made and imaged using a confocal microscope, 5 days after transecting the sciatic nerve under anaesthesia. The green fluorescence is due to transgenic expression of Yellow Fluorescent Protein (YFP). A single motor unit is labeled, due to mosaic expression of YFP in a small percentage of motor neurons in this transgenic line (thy1.2- YFPH), which was crossbred into homozygous Wld S mice. Each of the red patches is a motor endplate whose Ach receptors were labeled with a fluorescent rhodamine (TRITC) conjugate of α- bungarotoxin. Clicking your mouse pointer over any of the innervated NMJ s in this image will bring up a magnified version of that endplate, which you can copy and paste into ImageJ Questions: 1. What is the motor unit size in this unit? 2. What are the average dimensions of neuromuscular junctions in the labeled motor unit? 3. What is the distribution of fractional endplate occupancies in the labeled motor unit? Generate a hypothesis and an experimental test to explain what you have observed and measured. 19