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INTRODUCTION To study muscle physiology, a simple experiment can be carried out by using the frog gastrocnemius muscle/sciatic nerve. The principles of muscle excitation, contraction and work performed in the frog are similar to all vertebrates including man. Using frog tissue has the practical advantage that it will function at room temperature without a blood supply; its oxygen requirements are met by diffusion from the air into the solution bathing the preparation. In this class, you will learn to use the Kymograph, which is used to study muscle physiology. The apparatus consists of a drum, which rotates at a pre-set speed, and traces are produced on the paper by means of an ink writing pointer. The stimulator within the kymograph delivers small electric pulses to the muscle or nerve tissue via a pair of platinum electrodes. If an electric current of suitable size and duration is passed through the tissue, the nerve fibres will initiate an impulse and the muscle cells will contract. The pulses are of fixed duration of 2msec each. The voltage strength may be varied upto 25 volts in small steps. The voltage used depends on the type of tissue. At the start of the experiment, it is wise to begin with a very low stimulus intensity and increase gradually to the threshold stimulus, i.e. the smallest voltage that will produce a full response. If too large a voltage is used the tissue will get damaged. Set the stimulator electrodes plugged into the black and red 2.5V sockets. (If the muscle does not twitch at all, check the electrodes before changing red electrode plug to 25V socket). The stimulus can deliver single pulses or repeated stimulation upto a frequency of 100 per sec. An audible click is produced when a pulse is delivered. The stimulator may also be triggered using the trigger switch attached to the kymograph spindle. MATERIALS & METHOD 1. The frog’s head is cut off and the spinal cord destroyed by pushing a metal pin down the vertebral canal with rotary movements. This is called pithing. During the dissection do not allow the tissues to dry; exposed nerve and muscle should be flooded with Ringers solution. Try not to touch the nerve tissue at all, pick it up by holding the adjacent connective tissue, do not stretch it and of course, never compress it with forceps. A glass rod can be used to lift a nerve from beneath. 2. Remove the skin from the lower part of the frog, either with scissors or pulling it off from the legs in one piece. 3. Lay the frog on its ventral surface on a corkboard. Identify the gastrocnemius (calf muscle) and the Achilles tendon which passes around the ankle joint (see Figure 3). Remember at all stages to keep the preparation moist by flooding with Ringer Solution. 4. Anchor the dissection by pinning the foot to the board. Cut through the Achilles tendon below the ankle joint and use forceps to pull away the attachment from the

ankle. Once the tendon is free, the gastrocnemius muscle can be lifted away from the lower leg leaving it still attached to the knee. (Figure 3.1) Figure 3: Initial Stage of the Dissection

Figure 3.1: Components of the Gastrocnemius/Sciatic Nerve Preparation

5. Trace the path of the sciatic nerve between the thigh muscles. Use forceps to separate the two major thigh muscles and identify the cream coloured sciatic nerve, which is accompanied by a darker and much thinner vessel. 6. Separate the sciatic nerve from the surrounding connective tissue, using forceps to pull on adjacent tissues and the hooked glass rod to gently lift the nerve. Keep it moist. 7. Now trace the path of the nerve from the lower pelvis to the vertebral column as follows. Grasping the tip of the urostyle with the forceps cut the muscles attached to its lower tip and continue cutting the muscles on either side until the whole structure can be removed. Take care, as the sciatic nerves are close beneath the surface, so keep the tips of the scissors pointing up. 8. With strong scissors bisect the vertebral column (Figure 3.2) Figure 3.2: The Bisection of the Pelvic Girdle

9. If the preparation is good, using forceps you should be able to lift out a portion of the vertebral column with the root of the sciatic nerve attached. 10. Trace the path of the nerve through the pelvic region. Lift the nerve from the vertebral column end and gently separate the connective tissue using forceps or the hooked glass rod. 11. The final stage is to isolate the preparation from the frog. Using strong scissors cut through the thigh muscles and femur close to the knee joint, carefully avoiding the sciatic nerve. Similarly cut through the tibia/fibula just below the knee. Take care not to stretch the nerve. (Figure 3.3)

Figure 3.3: The Gastrocnemius/Sciatic Nerve Preparation

12. Tie a piece of thread about 20cm long to the Achilles tendon close to the muscle. 13. Transfer the preparation to the muscle bath, which is filled with Ringer Solution. Secure the preparation in the appropriate position by pinning through the knee joint. 14. Connect the thread from the tendon to the lever. Finally, drape the sciatic nerve across the two electrodes. (Figure 3.4) Figure 3.4: Frog Nerve Experimental Set up of Kymograph

CAUTION when preparing the Nerve-Muscle Experiment 1. Living tissues are easily damaged and great care is needed in the dissection. 2. Do not grasp the gastrocnemius muscle and especially the nerve with fingers or forceps. 3. Avoid stretching the nerve, only lifting it with the hooked glass rods. 4. Tissues taken from the body need to be bathed in tissue fluid. An artificial tissue fluid ‘Frog Ringer Solution’ is provided which resembles the blood plasma in ionic composition. Always keep your preparation moist with Frog Ringer. 5. Physiology equipment is expensive and often fragile. Please make every attempt to treat it with care and respect. 6. Lift the kymograph by its base only. 7. Rotate the drum manually when the speed stimulator is switch to a neutral (indicated by a DOT) position. 8. Make sure that the equipment is packed away carefully and in clean condition. 9. If there is any defective equipment, report the matter immediately

Obtain the following Data A. Measurement of the threshold and maximal stimulus intensities 1. Arrange a nerve-muscle preparation as previously described 2. Stimulate the nerve with a single pulse of 2msec duration at voltage strength just above zero. 3. Mark the trace at the point of stimulation and indicate the voltage intensity. Manually move the drum for about 0.5cm, slightly increase the voltage strength and stimulate the preparation again. Repeat this procedure until the muscle contracts. The smallest stimulus intensity that produces a muscular contraction is known as the THRESHOLD STIMULUS. A weaker stimulus that fails to excite the nerve is termed as SUBTHRESHOLD STIMULI. 4. Increase the voltage strength and stimulate the nerve again to produce a larger muscle contraction. Continue to increase the stimulus strength until you reach the point where further increases in stimulus intensity will not produce a larger muscle contraction (MAXIMAL STIMULUS). Stimulus intensities from Threshold point to the Maximal point are known as SUBMAXIMAL STIMULI (Figure 3.5)

Figure3.5: Response to Stimuli of Varying Strengths

NOTE: • The MAXIMAL stimuli should be found for each preparation EACH TIME you start the experiment as each sensitivity of the preparation depends on the frog and the tissue preparation. • It is then advisable to use a voltage intensity that is just above the maximal (i.e. in the lower range of supramaximal stimuli) for stimulating the preparation. This level of stimulation will give rise to the best performance of the preparation for a considerable

period of time. Higher intensities may alter or damage the electrical properties of the excitable tissues. B. Summation of Subthreshold Stimuli Use a stimulus intensity, which just fails to produce a contraction and stimulate the preparation with rapidly repeated shocks. Find the minimum frequency of Subthreshold stimuli that produce a single contraction. NOTE: To avoid muscle fatigue and possible damage to the preparation, do not apply prolonged repetitive stimulation and also allow the muscle to rest in between tests. C. 1. 2. 3. The Single Isotonic Muscle Contraction Stimulate at supramaximal strength. Set the drum rotating at its maximum rate and record a base line. Record a single contraction only by pressing the ‘key switch’ long enough for one click of the drum contacts to occur. 4. Stop the drum. 5. Rotate the drum by hand and press the ‘key’ switch at the same time. As the drum contacts close the switch, the preparation will receive a stimulus. If the drum is ALMOST STATIONARY at that moment, the muscle contracts will mark the moment of stimulation (Figure 3.6)

Figure 3.6: Single Isotonic Muscle Contraction

D. The Effect of Temperature on the Isotonic Frog Muscle Contraction With reference to the procedure of experiment C, record a single isotonic muscle contraction of the frog muscle, which has been bathed in frog ringer at 5°C, 15°C, room temperature and 35°C. Your recordings should be similar to Figure 3.7. Figure 3.7: Example Records showing Effect of Temperature on Isotonic Muscle Contraction

E. Summation of Contraction 1. Arrange the preparation and stimulator as for experiment C. 2. Set the contacts on the drum for two successive stimuli (S1 and S2). Mark the points of stimulation on your record. 3. When the interval between the two stimuli is large, two separate muscle contractions are obtained. Gradually reduce the intervals between the stimuli until the two muscle contractions are partially fused (with two observable peaks Fig 3.8a) and finally completely fused into one (with only one observable peak Fig 3.8d). 4. Use a drum revolving at maximum speed and record each pair of contractions on a fresh place on the drum.

Figure 3.8: Example Records showing Summation of Contraction

F. Measurement of Refractory Period 1. Set up the nerve muscle preparation as for experiment E. 2. Find the maximum interval between stimuli, which show response only to the first stimulus (Figure 3.9) Figure 3.9: Example Records showing Different Responses due to Different Stimulus Intervals: (a) Single Response to one Stimulus; (b & c) summated response to two stimuli- the 2nd stimulus is outside the refractory period due to the 1st; (d) single response to the 1st stimulus only –the 2nd stimulus falls within the refractory period of the response due to the 1st Stimulus

G. The Generation of Tetanus (the effect of increasing the frequency of stimulation) 1. Set up the muscle-nerve preparation to record simple isotonic contractions 2. Set the drum speed at 25mm/sec and start stimulating at a frequency of 1/sec. Record contractions for 1-2 seconds. 3. Then move the drum onto a clean part of the paper and stimulate at 5/sec for 1-2 seconds. 4. Repeat at frequencies of 10, 15, 20, 25 /sec. It may be necessary to allow the muscle to recover at points during the experiment. At high frequencies of stimulation a single smooth contraction is produced, this is called a FUSED TETANUS. The lower frequency at which the individual contractions cease to be visible is termed the FUSION FREQUENCY (Figure 3.10) Figure 3.10: Effect of Increasing the Frequency of Stimuli

H. Drug Blockade Many agents such as general anesthetics ether and chloroform can block the propagation of nerve impulses. Even alcohol can depress nerve conduction. Of the different nerve types, the fibers that have relatively small diameters are the most sensitive to anesthetic action and larger fibers are the most resistant. 1. Dampen a small piece of cotton with 50% ethanol, squeeze out the excess fluid and place the cotton on the nerve. 2. Stimulate the nerve and observe the changes in the action potential over a period of time. 3. Remove the cotton and rinse with Ringers solution to bring about the recovery of the nerve.

Precautions 1. Ensure that the printing on the chart paper is the right way up. This will ensure the writing point will ride over the join and not come up against it. 2. When starting an experiment, the join should be facing you-consider the position of the join when carrying out experiments; it is not advisable to have an important part of your record over a join. 3. Fill the ink pen using the syringe supplied. Slowly inject ink until a drop appears at the writing tip. 4. When using the sciatic gastrocnemius preparation, the writing point should be positioned as shown in the diagram - slightly ahead of the mid-line of the drum and at right angles to the drum surface. 5. The pressure of the writing point on the paper should be the minimum necessary to produce a satisfactory trace. If you increase the pressure of the writing tip during an experiment this will decrease the height to which the lever moves. Be careful to avoid this effect for instance when investigating height of contraction in relation to stimulation intensity. The use of the Kymograph will be demonstrated to you. The choice of the drum speed will depend on what you are investigating. If you are interested in what is happening during one contraction/relaxation cycle, then a fast speed should be used so that events are well spread out. If on the other hand, the change in magnitude of contractions with time is required then a slow speed will be more satisfactory.