kymograph

MEMBRANE ACTION POTENTIALS INTRODUCTION To study muscle physiology, a simple experiment can be carried out by using the frog gastrocnemius gastrocnemius muscle/sciat muscle/sciatic ic nerve. The principles principles of muscle muscle excitation, contraction contraction and work performed performed in the frog are similar to all vertebrates including including man. Using frog tissue 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 this class class,, you you will will lear learn n to use use the Kymogr Kymograp aph, h, whic which h is used used to study study musc muscle le physiology. physiology. The apparatus 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. pointer. The stimulator stimulator within the kymogra kymograph ph deliver deliverss small small electri electricc pulses pulses to the muscle muscle or nerve nerve tissue tissue via a pair pair of platinum platinum electrodes. electrodes. If an electric electric current of suitable suitable size and duration duration is passed through through the tissue, the nerve fibres fibres will initiate initiate an impulse and the muscle muscle cells will contract. contract. The pulses pulses are of fixed fixed duration duration of 2msec 2msec each. each. The voltage strength may be varied upto 25 volts in small steps. steps. The voltage used depends on the type of tissue. tissue. At the start of the experime experiment, nt, it is wise to begin with 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 used the tissue will get damaged. Set the stimulator stimulator electrodes 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 audible click is produced produced when when a pulse is delivere delivered. d. The stimulator stimulator may may also be triggered using the trigger switch attached to the kymograph spindle.

MATERIALS & METHOD 1. The frog’s frog’s head head is cut off and the spinal spinal cord cord destroy destroyed ed by pushing pushing a metal pin pin down the verte vertebral bral canal canal with with rotary rotary movem movements ents.. This is is called called pithing pithing.. During 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 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 rod can be used to to lift a nerve from beneath. 2. Remove Remove the skin skin from the lower lower part of the the frog, either either with with scissors scissors or pullin pulling g it off from the legs in one piece. 3. Lay the the frog on on its ventra ventrall surface surface on on a corkboar corkboard. d. Identi Identify fy the gastr gastrocne ocnemius mius (calf (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. Ancho Anchorr the disse dissect ction ion by pinnin pinning g the foot foot to the board. board. Cut Cut throug through h the Achill Achilles es tendon below the ankle joint and use forceps to pull away the attachment from the

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ankle. Once the tendon is free, free, the gastrocnemius gastrocnemius muscle 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 Dissection

Figure 3.1: Components of the Gastrocnemius/ Gastrocnemius/Sciatic Sciatic Nerve Preparation





5. Trace Trace the path path of the sciati sciaticc nerve betwe between en the thigh thigh muscle muscles. s. Use force forceps ps to separa separate te 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 Now trace trace the path path of the nerve nerve from from the lower lower pelvis pelvis to the vertebr vertebral al column column as follows. Grasping the tip of the urostyle with the 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 beneath the surface, so keep the tips of the scissors pointing up. 8. With strong scissors scissors bisect bisect the the vertebral vertebral column (Figure (Figure 3.2) Figure 3.2: The Bisection of the Pelvic Girdle

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





Figure 3.3: The Gastrocnemius/Sciatic Gastrocnemius/Sci atic Nerve Preparation

12. Tie a piece of thread about 20cm long to the Achilles Achilles tendon close to the muscle. 13. Transfe Transferr the preparat preparation ion to the muscle muscle bath, bath, which which is filled with Ringer Solution. Solution. Secure the preparation in the appropriate position by pinning through the knee joint. 14. Connect the thread thread from the the tendon to the lever. lever. Finally, Finally, drape the sciatic sciatic nerve across 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 tissues are easily damaged damaged and great great care is needed needed in the the dissection. dissection. 2. Do not gras grasp p the the gast gastro rocne cnemiu miuss musc muscle le and espe especia ciall lly y the the nerve nerve with finger fingerss or forceps. 3. Avoid stretc stretching hing the nerve, only only lifting lifting it it with with the hooked hooked glass glass rods. rods. 4. Tissues Tissues taken taken from the body body need to be bathed in tissue tissue fluid. fluid. An artificia artificiall tissue tissue fluid fluid ‘Frog ‘Frog Ringe Ringerr Solut Solution ion’’ is provi provide ded d whic which h rese resemb mble less the the blood blood plas plasma ma in ionic ionic composition. Always keep your preparation moist with Frog Ringer. 5. Physiol Physiology ogy equipm equipment ent is expens expensive ive and often often fragil fragile. e. Please Please make make every every attemp attemptt to treat it with care and respect. 6. Lift Lift the the kymog kymograp raph h by its base base only. only. 7. Rotate the the drum manually manually when when the speed speed stimulat stimulator or is switch switch to a neutral neutral (indicate (indicated d by a DOT) position. 8. Make sure sure that that the equipme equipment nt is packed away away carefully carefully and in clean condition. condition. 9. If there there is any any defective defective equipment equipment,, report report the matter matter immediat immediately ely





Obtain the following Data A. Measurement Measurement of the threshold threshold and and maximal maximal stimulus stimulus intensities intensities 1. Arrange Arrange a nerve-muscle nerve-muscle preparation preparation as previously previously described described 2. Stimulate Stimulate the nerve nerve with a single single pulse pulse of 2msec 2msec duration duration at voltage voltage strength strength just above above zero. 3. Mark the trace trace at the point point of of stimulati stimulation on and indicate the voltage voltage intensity. intensity. Manually Manually move the drum for about 0.5cm, slightly increase the voltage strength and stimulate the preparation preparation again. Repeat this procedure procedure until the muscle contracts. contracts. The smallest smallest stimulus intensity that produces a muscular contraction is known as the THRESHOLD STIM STIMUL ULUS US.. A weak weaker er stim stimul ulus us that that fail failss to exci excite te the the nerv nervee is terme termed d as SUBTHRESHOLD STIMULI. 4. Increase Increase the voltage voltage strengt strength h and stimul stimulate ate the nerve nerve again to produce produce a larger larger muscle 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). Stimulus intensities from Threshold point to the Maximal point are are known as SUBMAXI SUBMAXIMAL MAL STIMUL STIMULII (Figure (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







period of time. Higher intensities intensities may alter or damage the electrical electrical properties properties of the excitable tissues. B. Summat Summation ion of Subthr Subthresho eshold ld Stimu Stimuli li fails to produce a contraction and stimulate the Use a stimulus intensity, which just fails preparation preparation with rapidly rapidly repeated repeated shocks. Find the minimum minimum frequency frequency of Subthreshold 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. The Singl Singlee Isotonic Isotonic Muscle Muscle Contra Contractio ction n 1. Stimulate at supramaximal strength. 2. Set the drum rotating rotating at its maximum maximum rate rate and record record a base line. line. 3. Record a single contraction contraction only by pressing pressing the ‘key switch’ switch’ long long enough enough for one click of the drum contacts to occur. 4. Stop Stop the drum drum.. 5. Rotat Rotatee the drum drum by hand and and pres presss the ‘key’ ‘key’ swit switch ch at the same same time time.. As the the drum drum contact contactss close close the switch, switch, the prepar preparatio ation n will will receive receive a stimulus. 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 Effect of Temperature Temperature on on the Isotonic Isotonic Frog Muscle Contrac Contraction tion With With refe refere rence nce to the proce procedur duree of expe experi rime ment nt C, reco record rd a singl singlee isot isotoni onicc musc muscle le 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. Summa Summatio tion n of Contr Contrac actio tion n 1. Arrange Arrange the preparation preparation and and stimulat stimulator or as for experiment experiment C. 2. Set the contacts on the drum for for two two successive successive stimuli stimuli (S1 and S2). Mark the points points of of stimulation on your record. 3. When When the interval interval betwee between n the two stimul stimulii is large, large, two separat separatee muscle muscle contracti contractions ons





Figure 3.8: Example Records showing Summation of Contraction

F. Measure Measuremen mentt of Refract Refractory ory Period Period 1. Set up the the nerve nerve muscle muscle prepa preparat ration ion as for for experi experiment ment E. E. 2. Find the maximu maximum m interval interval between between stimuli stimuli,, which show respo response nse only to the first 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 1 st; (d) single response to the 1st stimulus only –the 2 nd stimulus falls within the refractory period of the response due to the 1 st Stimulus





G. The Generation Generation of Tetanus Tetanus (the effect effect of increasing increasing the frequency frequency of stimulation) stimulation) 1. Set up the muscle-ner muscle-nerve ve preparati preparation on to record record simple simple isotonic isotonic contractions contractions 2. Set the drum drum speed speed at 25mm/s 25mm/sec ec and start start stimu stimulat lating ing at a frequenc frequency y of 1/sec. 1/sec. Record Record contractions for 1-2 seconds. 3. Then Then move move the drum drum onto a clea clean n part part of the paper paper and stimul stimulat atee at 5/sec 5/sec for 1-2 seconds. 4. Repeat at frequencies frequencies of 10, 10, 15, 20, 20, 25 /sec. /sec. It may may be necessar necessary y to allow allow the the muscle muscle to recover at points points during the experiment. experiment. At high frequencies frequencies of stimulation stimulation a single smooth smooth contract contraction ion is produced, produced, this is called a FUSED FUSED TETANUS TETANUS.. The lower lower freq freque uency ncy at whic which h the indiv individu idual al contr contract action ionss cease cease to be visib visible le is term termed ed 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. impulses. Even alcohol can can depress nerve nerve conducti Of the diff





Precautions 1. Ensure Ensure that that the print printing ing on the the chart chart paper paper is the righ rightt way up. up. This will will ensur ensuree the writing point will ride over the join and not come up against it. 2. When starting starting an an experiment, experiment, the join join should should be facing you-consider you-consider the positi position on of the join when carrying carrying out experiments; experiments; it is not advisable advisable to have an important important part of your record over a join. 3. Fill the the ink pen using using the syringe syringe supplied. supplied. Slowly Slowly inject inject ink until until a drop appears appears at the writing tip. 4. When When usin using g the the scia sciati ticc gast gastro rocn cnem emiu iuss prep prepar arat atio ion, n, the the writ writin ing g poin pointt shou should ld be positioned 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 pressur pressuree of the writing writing point point on the paper should should be the minimum minimum neces necessar sary y to produce a satisfactory satisfactory trace. If you increase the pressure pressure of the writing writing tip during an experiment experiment this will decrease decrease the height to which the lever moves. Be careful to avoid this this effe effect ct for for insta instanc ncee when when inves investi tiga gati ting ng heigh heightt of contr contrac acti tion on in rela relati tion on to stimulation intensity.

The use of the Kymograph will will be demonstrated to you. The choice of the drum speed speed will depend on what you are investigating. investigating. If you are interested interested in what is happening during during one contraction/relaxation cycle, then a fast speed should be used so that events are well spread spread out. If on the other hand, the change change in magnitu magnitude de of contractio contractions ns with time is required then a slow speed will be more satisfactory.

kymograph

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