THE INFLUENCE OF IONS ON THE CONTRACTION OF ISOLATED EARTHWORM SMOOTH MUSCLE

Transcription

1 THE INFLUENCE OF IONS ON THE CONTRACTION OF ISOLATED EARTHWORM SMOOTH MUSCLE BACKGROUND READING Animal Physiology by Hill, Wyse & Anderson, 2004: pp ANIMALS & EQUIPMENT Living material Large earthworms (Lumbricus terrestrius). Equipment Scissors; pins; forceps; thread; ring stands and clamps; force transducer; bioamplifier; earthworm ringer; immersion bath; plastic waste beakers; known weights; Macintosh computer; PowerLab virtual chart recorder; BNC cables. (Include a sketch of the equipment set up in your lab book that is of sufficient quality that you could use it to set up the same experiment at a later date) A. Preparation of the equipment STANDARD PROCEDURE 1. Study the controls on the Bioamplifier. Set the amplifier to Bridge Mode, 100X amplification, and 50K filter. 2. Open the chart application and set up to record from Channel 1 only. 3. Set the controls to collect data at a slow speed... about 2 samples/sec is a good place to start. 4. Turn on the 50 Hz LP filter within the Input Amplifier. * You are probably expecting us to say: "Now you must calibrate the force transducer". Surprise!!! You don't have to! The forces that you measure today will be examined RELATIVE to each other. For the actual force to be really meaningful, you would need to determine the cross-section of the active muscle and the angle at which the muscle is pulling. Both of these measurements are beyond the scope of the lab. In other words, we're not letting you off easy, we're just not making you do something that won't really add anything of value to your analysis. B. Dissection of Earthworm (from Hoar and Hickman, 1975) **THIS IS A DIFFICULT DISSECTION! TAKE CARE BUT BE BOLD** * IF AVAILABLE PLEASE LOOK AT DEMONSTRATION DISSECTION BEFORE BEGINNING* *KEEP THE PREPARATION MOIST WITH RINGERS* 1

2 1. Place an earthworm in the dissecting tray and pin down the anterior end. Place another pin just anterior to the clitellum (Figure 1). DO NOT PLACE PINS DIRECTLY ON THE MIDLINE OF THE ANIMAL OR YOU MIGHT PUNCTURE THE ORGANS. Anterior Clitellum Posterior Figure 1. External view of Earthworm (from: 2. Make a mid-dorsal incision just anterior to the clitellum and proceed carefully forward, cutting just through the skin and body wall. Cut through septa and pin body wall open to reveal the internal organs. Identify the anatomical features in Figure 2 below to get your bearings. Figure 2. Illustration of the internal organs in an earthworm opened by a mid-dorsal incision. (From: 3. Loosen the esophagus, crop, gizzard and anterior portion of the intestine from the underlying tissues by carefully cutting the septa. 4. Carefully pass a long piece of thread under the esophagus and tie off. Do the same for the intestinal end, tying the intestine off very close to the end of the gizzard. 2

3 5. Cut the intestine to free the anterior end and then cut the intestine to free the posterior end. 6. Carefully mount the preparation in the muscle bath according to instructions given in the lab. You should see slow rhythmic contractions within a few minutes. If you do not, carefully adjust the tension on the preparation by lowering the muscle bath SLIGHTLY. If you do not see contractions within a few minutes, you'll have to do another dissection. C. Normal Activity and the Effects of Cation Concentration 1. After the preparation has recovered from its initial trauma, record normal activity of the gut preparation for around 5 min. You'll want to note the magnitude and frequency of the beat pattern. 2. Drain the normal ringer solution and replace with one containing either no CaCl2 or no KCl. Record for 5 min. 3. Rinse the preparation with at least 3 changes of normal ringer. Following the washes, immerse the preparation in normal ringer and record the response for 5 min. 4. Drain the normal ringer solution and replace with one containing either 2X CaCl2 or 2X KCl. Record for 5 min. or until you see a clear response. 5. Rinse the preparation with at least 3 changes of normal ringer. Following the washes, immerse the preparation in normal ringer and record the response for 5 min. PRE-LAB (Due at the start of lab) 1. List two ways in which the appearance of smooth muscle cells differ from skeletal muscle cells. List three major sub-cellular differences between smooth muscle cells and skeletal muscle cells. 2. Relative to skeletal muscle contractions, would you expect to use a faster, slower, or the same chart recorder speed to measure smooth muscle contractions? Why? 3. In general, how does the regulation of smooth muscle contraction differ from that of skeletal muscle? 4. In the experiment that you will do today, how will you induce the muscle to contract? (Hint: this is sort-of a trick question) 3

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5 POST-LAB (Due next week in lab) 1. Write a short paragraph that verbally describes your results. 2. Construct at least two figures and/or tables to back-up your statement(s) above. The figures should clearly show your main result. The figures can be raw or processed data, depending on what you believe best illustrates your main result(s). 5