BC1002 Module 1 Biol BC1002, Spring semester 2005, Module 1, Lab 1-1
MODULE 1: LAB 1 MUSCLE PHYSIOLOGY Spring semester 2005, Week of January 24-28. Learning objectives to learn the microscopic structure of muscles to learn how to record and to interpret electromyography (EMG) recordings from your own muscles and/or those of your labmates to understand the causal connections between motor unit recruitment, the magnitude of the EMG signal, and force generated during a skeletal muscle contraction to learn how antagonistic muscles control the movement of bones around joints Laboratory overview Hour 1: Study and label drawings of cardiac, skeletal, and smooth muscle (Exercise 1). Hour 2: EMG muscle experiment 1 (Exercise 2). Hour 3: EMG muscle experiment 2 (Exercise 3). Readings: Asking about Life, 3 nd edition, pp. 689-702. Anatomy and Physiology for Dummies, pp. 91-100. Biol BC1002, Spring semester 2005, Module 1, Lab 1-2
Exercise 1: Muscle Histology (work individually) This week we will be examining the three types of muscle tissue that occur in our bodies: skeletal muscle, cardiac muscle and smooth muscle. Remember that muscle tissue is one of the four basic tissue types occurring in animals. (The other three types are epithelial, connective, and nervous tissue.) Skeletal muscle Smooth muscle Cardiac muscle Figure 1. Photomicrographs of three types of muscle tissue. Muscles are composed of many muscle cells that are also called muscle fibers. Skeletal and cardiac muscles are classified as striated muscles because their muscle fibers have the appearance of altering light and dark bands under the light microscope (these are most obvious in the skeletal photo above). Most skeletal muscles are attached to the bones of the skeleton, enabling them to control body movement. Note the very prominent striations in the photograph above. These fibers are large, multinucleate cells (several uninucleate cells fuse during formation of a skeletal muscle cell). The plasma membrane that surrounds these large, fused cells is called the sarcolemma. Cardiac muscle is found only in the heart and is responsible for moving blood through the circulatory system. Cardiac muscle is similar to skeletal muscle, but its cells are uninucleate and branched and it has intercalated discs (see arrow in photograph above) that facilitate the coordinated contraction of cardiac muscle cells. Smooth muscle is the primary muscle of surrounding internal organs and tubes such as the stomach, urinary bladder, and blood vessels. Smooth muscle fibers are small uninucleate cells without obvious banding patterns (smooth muscles still have actin and myosin, but they are not arranged in the same manner as in striated muscle). The nuclei of several smooth muscle fibers are labeled with N in the photograph above. Smooth muscle contracts with less tension, but over a greater range of lengths than skeletal muscle. In addition, it has slow contractions, but with more control over contraction strength than with skeletal muscle. Biol BC1002, Spring semester 2005, Module 1, Lab 1-3
Name Lab day/time/instructor BIOL BC1002 Spring semester 2005 Module 1, Lab 1, Worksheet 1: Muscle Histology Individually, observe each of the three slides available. Observe and draw longitudinal sections of each of the three types of muscle tissue under low, medium, and high magnification. Draw what you see under the highest magnification (using the 10X ocular and 40X objective). You can find labeled photos on pages 126-127 in the Photo Atlas for Biology (please share these books with your labmates during lab, and do not remove them from the lab room). A. Smooth muscle: label muscle fiber & nucleus B. Skeletal muscle: label muscle fiber, sarcolemma, nucleus, and striations Magnification 10X ocular x 40X objective = 400X total Uni- or multi-nucleate? Magnification Uni- or multi-nucleate? Striated or not? Striated or not? C. Cardiac muscle: label muscle fiber, nucleus, striations, and intercalated disc Magnification Uni- or multi-nucleate? Striated or not? D. How is cardiac muscle like skeletal muscle? E. How is cardiac muscle like smooth muscle? F. What special feature is present only in cardiac muscle? What is the function of these structures? Biol BC1002, Spring semester 2005, Module 1, Lab 1-4
EXERCISES 2 and 3 Introduction to the muscular system through electromyography (EMG) Up to this point, we have focused on the microscopic structures. In this second exercise, we will study the contractile properties of the tissues that move the skeleton i.e., the skeletal muscles. To this end, you will use a noninvasive technique, called electromyography (EMG), to monitor neural activity of these muscles. We will use EMG to study how the brain (a) generates graded amounts of force in the muscles, and (b) causes bones to move around a joint. Before discussing the methodological details of the exercises, we need to review three features of muscle anatomy and physiology. Please refer to your textbook for more details. Each muscle consists of thousands of cylindrically shaped cells (called muscle fibers) that are bound together by connective tissue. A muscle fiber contracts when it receives the appropriate amount of electrical stimulation. This electrical stimulation is usually provided by a motor neuron, which relays electrical signals from the brain and the spinal cord to each muscle fiber. Upon reaching the muscle, each motor neuron branches and innervates several different muscle fibers. The combination of a single motor neuron and all of the muscle fibers that it innervates is called a motor unit. Each muscle consists of many motor units. With numerous motor units, the brain, through motor neurons, can tell muscles how much force they should generate. When the brain wants a particular muscle to contract weakly, it causes a limited number of small motor units to contract. Upon receiving a stimulus form a neuron, a muscle fiber contracts for about 100 msec and then relaxes. If the repeated neuronal stimuli are separated by long intervals of time, the muscle fibers have time to relax completely between stimuli. When the brain wants a muscle to contract more forcefully, it can increase the frequency of electrical stimulation to each motor unit. If the interval of time between stimuli is shortened, the muscle fiber will not have relaxed completely at the time of the second stimulus, resulting in a more forceful contraction. This process is known as summation. The brain can also cause a greater number of motor units to contract. This process, called motor unit recruitment, also enables muscle to generate graded amounts of force. When the muscle fibers within a motor unit are stimulated by a motor neuron, they respond by generating their own electrical signal. These signals propagate across the surface of the muscle fiber, and then down into the center of the muscle fiber (via the transverse-tubules), where they let in Ca 2+ ions, which in turn activate the contractile machinery. This combination of electrical and mechanical events in a muscle fiber is called excitationcontraction coupling. You can record the muscle electrical activity with surface electrodes (i.e., electrodes placed on the surface of the skin) coupled to recording equipment. You should realize, however, that your EMG recordings will contain electrical activity from several motor units, and in some cases, from several muscles. Biol BC1002, Spring semester 2005, Module 1, Lab 1-5
The anatomical arrangement of muscles and bones in the body is directly related to how muscles work. When the bones attached to a muscle are connected by a flexible joint, contraction of the muscle results in movement of the skeleton. If the centers of the connected bones are brought closer together when the muscle contracts, the muscle is called a flexor. If the bones move away from each other when the muscle contracts, the muscle is called an extensor. Flexor-extensor pairs are called antagonist muscle groups because they exert opposite effects. Figure 2. Typical EMG results: the greater magnitude on the right is the result of higher motor unit recruitment. The x-axis is time (seconds), and the y-axis is magnitude of EMG response (mv). EMG (mv) time (seconds) EMG ACTIVITY 1 (work in groups of 4) Relationship between the size of the EMG signal and force output This exercise examines the relationship between the magnitude of the EMG signal from your forearm flexors and the amount of force generated while clenching an object. The forearm flexors are the muscles on the inside of your forearm. Contraction of these muscles causes your fingers to curl and, hence, clench an object in your hand. To release your grip on the same object, you would relax your forearm and contract your forearm extensor (i.e., the muscles located on the outside of your forearm). Hypothesis generation At this point, your group should generate a hypothesis about the relationship between the magnitude of the EMG signal and the force output of your forearm flexors (see worksheet 2). Your prediction should be stated graphically that is, construct a bivariate (x-y) plot with the magnitude of the EMG signal on the Y-axis and the amount of force exerted on the X-axis. Indicate your prediction by drawing a line on this graph. Biol BC1002, Spring semester 2005, Module 1, Lab 1-6
If you predict a direct linear relationship between the two variables, then draw a straight line with a positive slope. If you predict an inverse linear relationship, then draw a straight line with a negative slope. If you predict an asymptotic relationship, then draw such a line. If you predict no relationship, then draw a horizontal line with no slope. In addition to stating your hypothesis graphically, briefly write out your hypothesis. After you have completed these steps, test your hypothesis by performing an experiment. Procedure Electrode placement on dominant forearm (right forearm for right-handed person, left for left-handed) Your first task is to attach 3 surface electrodes, and their associated leads, to the subject. NOTE THAT ELECTRICTY WILL BE GOING FROM THE BODY TO THE COMPUTER, NOT THE OTHER WAY AROUND. THIS PROCEDURE IS VERY SAFE AND CARRIES NO RISK OF INJURY FROM ELECTRICITY. Figure 3. Where to position the 3 surface electrodes. Figure 4. Where to connect each of the 3 leads. Ask the subject to sit down on a chair at the lab bench nearest the recording equipment. Ask the subject to rotate the forearm of her dominant arm so that the palm of her hand is facing up and her arm is resting on the lab bench. Clean the subject s dominant wrist and forearm with an alcohol pad where the electrodes will be attached. This will ensure good contact between the electrode and the skin. If your subject has used a lot of moisturizing lotion recently, be sure to clean extra-well. Allow the alcohol to evaporate before attaching the electrodes. Attach one electrode to the forearm (see Fig. 2 for details). Attach two electrodes to the wrist (again, see Fig. 2). It is important to get good contact between the gel on the electrodes and the skin in order for the electrical activity of the underlying muscles to be recorded. Clip the 3 leads (via the pinch connectors) FROM CHANNEL 3 onto each of the surface electrodes as shown in Fig. 4. The pinch connectors on the leads work like a small clothespin, but will only latch onto the nipple of the electrode from one side of the connector. Biol BC1002, Spring semester 2005, Module 1, Lab 1-7
Equipment set-up Click on the desktop icon labeled EMG Activity 1. A menu will appear asking you to choose among several lessons. Select the lesson titled L01-EMG-1. If you wish, type in the name of the subject in the box that appears (this is not necessary for the program to work). If someone with the same name has already used the program, click to reuse the name. Next, a new screen should appear containing an empty window and a button labeled CALIBRATE in the upper left corner. Click on this button and follow the instructions carefully (we have no headphones, so ignore those instructions). This calibration procedure lasts 8 seconds and is critical for optimum performance. The computer needs to take an EMG reading while you clench your fist (i.e., activate your forearm flexors) maximally to set internal parameters of the program. Once the OK button is clicked, the subject should relax her fist for two seconds, squeeze her fist tightly for two seconds, relax her fist for two seconds, and squeeze her fist for two seconds. For best results, follow the timing on the screen. You should see an EMG signal appear as soon as the subject begins clenching her fist it should look like a cluster of vertical lines. Once this calibration procedure has been completed, your group will be ready to run the experiment. Follow these instructions, not the instructions on the computer screen. Experimental procedure You should have four group members: divide the tasks among yourselves: subject dynamometer reader computer operator recorder and overseer Before beginning the experiment, your subject should hold the dynamometer in her hand and place her forearm flat on the lab bench. Have the subject practice clenching her fist to varying degrees of intensity. She should become proficient at generating 7 different levels of clench intensity (make sure the lowest clench intensity is low enough to allow for 6 greater intensities). One member of your group should become proficient at reading the hand dynamometer to quantify the amount of force she generates with each clench. After becoming proficient with these skills, your group will be ready to collect data. Begin by having the computer operator click on the RECORD button. At this point, the subject should make the weakest fist clench for approximately 1.0 second. After the clench has been released, click on the SUSPEND button and record the amount of force (in kg) from the dynamometer in the appropriate place on Worksheet 1. Remember the first clench should be weak. Next, click RESUME and record a second EMG response, but have the subject clench her fist for approximately 1.0 second a bit more forcefully this time. Click on the SUSPEND button immediately after the clench has been released, and record the amount of force from the hand dynamometer to the worksheet. Continue in this manner until you have recorded the EMG response and force output during an ascending series of successively more forceful clenches (7 clenches total). You will notice that there are two windows on the data collection screen. The upper window contains the raw EMG signal, and the lower window contains the integrated EMG signal (i.e., a moving average of the EMG signal over time). The vertical axis of each window represents the magnitude of the EMG signal (in millivolts, mv), and the horizontal axis represents real time (in seconds). Finally, remove the leads from your subject, peel off the electrodes (and dispose of them), and then wash the electrode residue off the subject s skin with soap and water. Biol BC1002, Spring semester 2005, Module 1, Lab 1-8
Analysis of the EMG results Once you have recorded the EMG responses, click on the Stop button. You will be prompted, Are you finished with both forearm recordings? Click Yes. Click Done. Next, select Analyze current data file. At this point, you need to record the highest integrated EMG value (in millivolts) obtained for each fist clench (corresponding to dynamometer readings) and enter these values on Worksheet 1. To select the parameters you will measure, click on the little box that says none next to the first 3 in the upper left hand part of the screen. Select p-p from the drop-down menu. (What does p-p represent on the graph? It represents the difference between the maximum peak and the minimum trough, a measure of the magnitude of the EMG.) The box to the right of p-p will display these values. Click on the small icon that looks like an I (with curved tops and bottoms) on the lower right end of the graph. Using your cursor, trace each hand-squeeze on your graph, beginning at the far left side with fist clench 1. Using worksheet 1, record p-p for each of the 7 fist clenches (after each clench). This is the data you will use to test your hypotheses. To trace each cycle, hold the left mouse button down while you move the cursor over the desired part of the cycle. Release the button when you are finished. It is not crucial exactly where you begin and end your tracings, but you should be consistent. To print an EMG sample, use the scroll bar at the bottom of the window to center the part of the EMG that you would like to print. Next, go to print in the drop-down menu under File. Continue with the following printing instructions: Printing summary: Select Print from File menu. Select graph. Select set-up. Select landscape. Select OK. Select 4 copies to print. Select OK. Be sure to label your printouts clearly. When your group is finished: Select Quit from the File menu. You will be prompted to save changes, but DO NOT SAVE CHANGES TO THE PROGRAM!!!! You will need to plot your findings on the graph on your EMG worksheet, with EMG response on the x- axis and force output on the y-axis (don t forget to always label all axes and include units). Finally, you need to explain your results on EMG worksheet 1. Did your results support your hypothesis? Provide a brief physiological explanation for your results on your EMG worksheet. PLEASE note that you can NEVER PROVE a hypothesis. NEVER, EVER. Your data can support a hypothesis or not, but NEVER prove. Biol BC1002, Spring semester 2005, Module 1, Lab 1-9
EMG WORKSHEET 1 BC1002 Spring 2005 Name Name Name Name 1. Your group should generate a hypothesis about the relationship between the magnitude of the EMG signal and the force output of your forearm flexors. You should write out your hypothesis in words in the space below as well as graph your hypothesis on the graph to the left below. Written hypothesis: Graphed hypothesis Amount of force (kg) Magnitude of EMG signal (mv) 2. Collect data in the following table: (Record just two numbers after the decimal for the EMG values, e.g., 0.54.) Forearm contraction fist clench 1 (weakest) fist clench 2 fist clench 3 fist clench 4 fist clench 5 fist clench 6 fist clench 7 (strongest) clench force (in kg from dynamometer) EMG magnitude (mv) Biol BC1002, Spring semester 2005, Module 1, Lab 1-10
3. Plot the data from your table on the following graph (you ll need to label the axes with appropriate numbers): DATA FROM YOUR EXPERIMENT Amount of force (kg) Magnitude of EMG signal (mv) 4. Based on the graph of your data, was your hypothesis supported by your data? Briefly explain how your hypothesis was or was not supported by your data. Biol BC1002, Spring semester 2005, Module 1, Lab 1-11
EMG Activity 2 The Action of antagonist muscles The muscles you activate while performing a specific movement (e.g., lifting a cup of coffee up to your mouth) are referred to as the agonists. The muscles that oppose this movement are called the antagonists. Antagonist muscles are usually silent (i.e., not contracting) during a movement. These 2 sets of opposing muscles lie on opposite sides of joints and serve diametrically opposed functions flexion (closing) vs. extension (opening) of the joint. In this experiment, you will study the action of the muscles that flex and extend your elbow joint. Your biceps muscle flexes your elbow joint, and in so doing, brings your forearm towards your shoulder (see Fig. 5). Fig. 5. Antagonistic muscle groups. Muscle contraction can pull on a bone, but cannot push a bone away. Muscles in the body are therefore usually arranged in pairs or groups around a joint. In the diagram above, the biceps muscle flexes (closes) the elbow joint, and the triceps muscle extends (opens) the elbow joint. Biol BC1002, Spring semester 2005, Module 1, Lab 1-12
Procedure Electrode placement Ask the subject to flex her biceps muscle (e.g., lift something heavy). Palpate the biceps as she performs this movement to determine where the contraction is strongest (i.e., where the biceps feel the hardest). Attach one electrode at this location (before attaching electrodes, always clean with the alcohol pad and allow the alcohol to evaporate). Then, ask the subject to flex her triceps muscle (e.g., place her palm on the table and push downwards). Palpate the triceps as she performs this movement to determine where the muscle contraction is strongest. Attach one electrode at this location. Then, attach four electrodes to the ventral side of her wrist (it s fine if the sticky parts of the electrodes overlap slightly, but make sure the gel parts of the electrodes have good contact with the skin). Lead placement You will need to use 2 sets of leads for this exercise. (Note that each set of leads contains a white, red and black connector). Channel 1 leads from Biopac box. Attach: White connector to the electrode on the subject s biceps muscle Red and Black connectors to 2 of the electrodes on the subject s wrist. Channel 2 leads from Biopac box. Attach: White connector to the electrode on the subject s triceps muscle, Red and black connectors to the other 2 electrodes on the subject s wrist. Experimental Procedure Open the EMG Activity 2 program on the computer. Select Open from under the File menu and open EMG Activity 2 from the Lab 1 folder. You do not need to calibrate the hardware for this experiment (you also won t need to tell the program you are done collecting data and are ready to analyze the data). You should have your electrodes and leads attached, the computer program open, and have your subject ready to perform three exercises. A) and B) should be performed while seated. C) should be performed while standing next to the lab bench. A) lift a small weight (mortar) from the lab bench to your chin; B) lower the weight back down to the bench from your chin (with your elbow resting on the lab bench); and C) push down on the lab bench with your palm. Biol BC1002, Spring semester 2005, Module 1, Lab 1-13
Single click on the Start button in the lower right corner of the window to begin recording data. The program will ask if you want to overwrite existing data; say yes. Each data recording will proceed for a maximum of 30 seconds, so you should have the subject repeat each of the above exercises three times within the 30-second time period (the subject does not have to do all nine in the same 30-second period; she can do three in a first run, three in a second run, and three in a third run). As the subject is performing the actions, have one member of your team carefully watch the computer screen and make note of when (according to time on the x-axis) the actions are performed. Record the P-P values for each of the three repetitions on Worksheet 2. To do so, adapt the data analysis instructions from EMG activity 1 in an appropriate manner. Ask your instructor or TA if you have questions. Calculate average values for the three repetitions, and record these in Worksheet 2. Print only the EMG for your last trial. Using the scroll bar at the bottom of the window, center the part of the EMG that you would like to print in the window. Next, go to print in the drop-down menu under File. Choose print graph. Next, click on the setup button and change the page setup from portrait to landscape and click ok. Then, print 4 copies (one for each member of your group). Be sure to label your printouts clearly. When your group is finished, select Quit from the File Menu. DO NOT SAVE CHANGES. Analyze your data and draw conclusions about whether your data support your hypothesis. Complete Worksheet 2. Remember that you can NEVER PROVE a hypothesis. NEVER, EVER. Your data can support a hypothesis or not, but NEVER prove. Carefully coil the leads from each channel and place them neatly on the back lab bench. Be gently with the leads as they are quite expensive. Biol BC1002, Spring semester 2005, Module 1, Lab 1-14
EMG WORKSHEET 2 Module 1, Lab 1, BC1002, Spring 2005 Name Name Name Name Hypotheses: Which muscle(s) do you expect to be active during: (1) Lifting a small weight from the lab bench to your chin? (2) Lowering the weight back to the bench? (3) Pushing down on the lab bench with your hand? (4) How will you use EMG recordings to test your hypothesis? Specifically state your predictions of EMG activity in relation to muscle movement. Biol BC1002, Spring semester 2005, Module 1, Lab 1-15
Module 1, Lab 1, Worksheet 2 continued (5) Record the EMG readings during these movements in the chart below. Print out a copy of your EMG record for activity 3 (pushing hand on bench) and attach it to this worksheet. LIFTING Weight Action 1 Action 2 Action 3 Average BICEPTS (mv) TRICEPS (mv) LOWERING Weight Action 1 Action 2 Action 3 Average BICEPTS (mv) TRICEPS (mv) PUSHING HAND on bench BICEPTS (mv) TRICEPS (mv) Action 1 Action 2 Action 3 Average (6) Was your hypothesis supported or not supported by your EMG data? Explain in the space below. Biol BC1002, Spring semester 2005, Module 1, Lab 1-16
Assignments due at the beginning of lab next week. Individually: 1. labeled drawings of muscle histology slides and answers to questions on the worksheet As a group: (turn in one packet for the whole group; remember that all group members must contribute equally) 2. EMG worksheet 1 and labeled printout of your recording 3. EMG worksheet 2 and labeled printout of your recording for pushing hand on bench 4. Typed answers to the following questions (you may work together to answer these questions or break them up per group member, whichever your group prefers.): a) What is the source of the signal detected by the EMG electrodes? b) What physiological process(es) underlie the observed relationship between EMG response and force output? c) What does the term motor unit recruitment mean? d) When you were lowering a weight using your forearm (i.e., extension) why did the EMG machine indicate that both the biceps and triceps muscles were both active? Biol BC1002, Spring semester 2005, Module 1, Lab 1-17