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The Properties of Skeletal Muscle in Frog Gastrocnemius

Wan Yi Wang

Group Members: Haiyan Wang, Justice Williams, Leona Wong

Section 062

Danielle Copper

July 13, 2014

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I. Introduction

Majority of body is built upon by three major muscles: skeletal, cardiac and smooth muscle

(Sherwood, 2010, 257). The contraction of the muscle involves many more structures

complementing each other. The first structure to notice is the smallest structural feature of the

skeletal muscle, myofibrils; it’s composed of myosin (thick filaments) and actin (thin filament).

To break it down even further, myofibrils have an A band, M line, Z line, H zone and sarcomere

to help the thick and thin filament interact to bind. Thus, as muscle fiber contracts, the Z line is

pulled in, the H band and I band reduce in width (Sherwood, 2010, 258). In order to initiate a

muscle contraction many factors play a role. This brings up the excitation-contraction coupling.

The purpose is to convert action potential (stimulus) into a response. During this process the

extracellular calcium influx into terminal button and brings acetylcholine vesicle merge into the

presynaptic membrane. In addition, the acetyl chlorine will bind to the nicotinic Ach receptors

which increase the sodium entry into the muscle fiber and cause a graded depolarization

(Sherwood, 2010, 250). The depolarization then can travel down to the muscle fiber by the

transverse tubules system (Sherwood, 2010, 266). As a result of opened ryanodine receptor,

sarcoplasmic reticulum releases the calcium into the cytoplasm. Consequently, it increase

intracellular calcium level and calcium binds to troponin C, removing the tropomyosin from the

blocking the actin-myosin binding site. This allows for the contraction of the muscle. A single

action potential or a direct stimulation elicits a twitch, small muscle contraction (Sherwood, 2010,

268). The accumulation and buildup of twitches upon each other is known as summation.

The goal of this lab is to explore the properties of skeletal muscle by isolating of the

gastrocnemius muscle and the sciatic nerve in a frog. In theory, the first part of lab illustrates

spatial summation. The sciatic nerve is stimulated with increasing levels of voltage. Thus, there

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should be linear relationships between increases in voltage and contractile force due to motor

unit recruitment increase. Second part of the lab displays the concept of temporal summation:

force increases as frequency increases. Indeed, it shows a fusion of twitches because the

sarcomere is stimulated again before it has time to fully relax. The fourth part of lab involves the

use of tubocurare, resulting in the decrease of muscle force (twitches) over time and no

depolarization. Lastly, theoretically, direct stimulation of a smaller area via a graded potential

requires a larger threshold because of higher resistance and thus higher voltage in order to create

a proportional amount of force as nerve stimulation.

II. Materials and Methods

The subject for this experiment was a frog. A more complete version of the methods can

be found in the NPB 101L: Systemic Physiology Lab Manual (Bautista, Korber 2009.9-17). The

sciatic nerve was isolated by blunt dissect of the seam apart then tie off the underlying tissue and

tendon. Next, the sciatic nerve was contacted by the electrode tips. Throughout the experiment a

MP35 stimulator unit was used. Hence, data values were collected for starting voltage threshold

and voltage maximum, for the test of stimulus intensity on muscle activity. Then, stimulus

frequency was raised on the muscle according to increments given in the lab manual. Next,

tubocurare was injected into the muscle. Lastly, data were collected on the effect of direct

electrical stimulation on muscle activity. One deviation from lab manual was the use of surgical

knot tie on the tendon instead of simple thread tie. To analyze the raw data and make

interpretation BioPac software was used.

III. Results

1. The Effect of Stimulus Intensity on Muscle Activity: Graded Response

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The effect of stimulus intensity on muscle activity can be observed by gradually

increasing the stimulus strength with the same setup as in Part 1. The gastrocnemius was

stretched and stabilized at a baseline force of 10.8 gram; then the baseline force is subtracted

from the measured force to produce the reported force. The voltage was increased in increments

of .006V, starting at 0.15V and ending at 0.18V. The minimum value for the force of contraction

happened at 0.15V corresponding to a minimum force of 0.428 grams. The force at 0.156V was

1.94 grams, at 1.162V was 18.60 grams, at 0.168V was 9.51 grams, and at 0.174V was 68.13

grams. The maximum value 94.69 grams for the force of contraction occur as stimulation reach

0.180V. These data values are depicted in a graphical form in figure 1. Overall it followed a

trend of increased muscle tension as voltage was increased. An outlier data value occurred from

muscle tension of 9.51 grams to 94.69 grams. Result in a incomplete S-shaped curve. The muscle

tension value of 68.13 grams and 94.69 grams, these area should not show increased instead, a

slow decrease would help created expected result.

Figure 1 The Effect of Stimulus Intensity on Muscle Activity: Graded Response

0.43 1.94

18.6 9.51

68.13

94.69

0102030405060708090

100

0.14 0.15 0.16 0.17 0.18 0.19

Intn

esi

ty (

gram

)

Voltage (V)

The Effects of Stimulus Intensity on Mucle Activity: Grade Response

muscle tension

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Figure 1: Data was collected by using a MP35 stimulator unit on the sciatic nerve of a double-pithed frog.

The data were analyzed by the use of p-p value measurements through BioPac software. The stimulus

was increased in increments of .006 V. The force of contractions increased as the stimulus increase. The

smallest value of force achieved was .428 grams, and the highest value was 94.69 grams. There is a trend

in which the muscle tension increases as stimulus increased. From data value 9.51 gram on, yields an

unexpected result.

2. The Effect of Stimulus Frequency on Muscle Activity: Summation

The next part of lab is organized to demonstrate the effect of different frequencies of

stimulation with the same voltage on contractile force. The contractions were recorded at

frequencies of 1 pulse per second (pps), 2pps, 4pps, 8pps, 15pps, and 25 pps for 30 seconds each.

The muscle tension decreased starting from frequency of 0.5pps and ended at 2pps. Then,

starting at frequency of 4pps until 25 pps, the muscle tension increased. The result was not as

expected in the beginning of the frequency response curve. But the rest of the graph shows the

linearity relationship of the tension and frequency.

Figure 2 The Effect of Stimulus Frequency on Muscle Activity

Figure 2: Data was collected by using of a MP35 stimulator unit on the sciatic nerve of a double-pithed

frog. Then, p-p values were used to analyze the raw data to data points in the graph through BioPac

software. At the beginning of the graph, tension started decreasing from 96.18 gram, at 0.5 pps to 2 pps.

Then starting from 4 pps to 25 pps shows a linearity relationship of the tension and frequency.

3. The Effect of Tubocurare on Muscle Activity: Paralysis

96.18

50.96

16.72 19.72

35.81 45.66

88.89

0

20

40

60

80

100

120

0 5 10 15 20 25

Ten

sio

n (

gram

)

Frequency (PPS)

Frequency Response

muscle tension

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This part is designed to give an insight into the effect of the competitive inhibitor,

tubocurane on nAChR, on the resulting muscle contraction. Thus, the gastrocnemius was

stimulated at a frequency of 0.5PPs and at a voltage of 0.180V for one minute. Next, a 0.25

milliliter of tubocurare was injected into the gastrocnemius muscle, and then the data was

recorded for 10 minute with the stimulation still running. The maximum force of 90.06 grams was

recorded at 30 seconds after the injection. At 3.52 minute to 5.02 minute, the muscle tension

remained unchanged. Then, starting at 5.52 minute the muscle tension started to decreased. The

lowest muscle tension recorded was 7.37 gram at the end of the ten minute period, as shown in

figure 3. The trend followed was muscle activity decreased after the initial injection of tubocurare

starting after 1.50 minute. Lists of all recorded values for force and their corresponding times can

further illustrate the trend of decreasing twitches as time progress. (Appendix, Raw Data)

Figure 3 the Effect of Tubocurare on Muscle Activity

Figure 3: The graph shows the data collected when a 0.25ml of tubocurare into the gastrocnemius muscle.

The subject is a double-pithed frog. Then, the data was analyzed by finding the p-p value through BioPac

software. Then data points were inputted into excels. The maximum value of force occurred at 90.06

grams and minimum was at 7.37 grams. The trend is the muscle twitches decreased as time progressed

after tubocurare was injected.

4. The Effect of indirect Electrical Stimulation versus Direct Stimulation on Muscle Activity

89.57 90.02 90.06 89.96 85.11

75.4

64.91 61.87 55.9

52.45

40.3 33.22

26.35 22.4 20.22 17.29 15.71 13.25

9.89 8.1 7.37

0

10

20

30

40

50

60

70

80

90

100

3.504.004.505.005.506.006.507.007.508.008.509.009.5010.0010.5011.0011.5012.0012.5013.0013.5014.0014.5015.00

Inte

nsi

ty (

gram

)

Time ( minute)

The Effect of Tubocurare Injection

muscle tension aftertubocurare

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2.27 4.19 22.18

97.16

0

50

100

150

0 0.5 1 1.5 2 2.5

ten

sio

n (

gram

)

Voltage (V)

Indirect stimulation Vs. Direct stimulation

direct stimulation

indirect stimulation

The last part of the lab demonstrates indirect stimulation of sciatic nerve shows a

different force of contraction compared to direct stimulation of gastrocnemius muscle. At a point

value of 0.15 voltages, the first twitch was observed for indirect stimulation of sciatic nerve.

When compare the initial threshold for both direct and indirect stimulation, direct required a

higher voltage. Indirect stimulation has a higher muscle tension value compare to direct

stimulation of muscle. Observation of higher threshold was needed to elicit a threshold and thus

a higher V maximum in direct stimulation of muscle.

Table 1: Indirect Stimulation versus Direct Stimulation

Table 1: Data for direct stimulation was collected by using two needle electrodes and inserting them into

about 5 mm from each end of the muscle. The subject being studied is a double-pithed frog. Then, raw

data was analyzed by using the P-P values measurement tool through BioPac software. In this table,

indirect stimulation requires a smaller voltage to elicit threshold and have a lower data value for

Vmaximum.

Figure 4 Comparison of indirect stimulation to direct stimulation in terms of intensity

Figure 4: Data was collected by using a MP35 stimulator unit on the sciatic nerve of a double-pithed

frog. For the direct stimulation, two needle electrodes were inserted about 5 mm from each end of the

muscle. The subject being studied is a double-pithed frog. Then, raw data was analyzed by use the P-P

Indirect

stimulation

(voltage )

Indirect stimulation

Tension (gram)

Direct

stimulation

voltage

Direct stimulation

Tension

(gram)

Vthreshold 0.15 22.18 2.2 2.27

V maximum 0.18 97.16 1.8 4.19

Delta V .006 N/A N/A N/A

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value measurement tool through BioPac software. The indirect stimulation revealed a trend of increases

in muscle tension from 22.18 grams to 97.16 grams as the voltage increased from .15 V to .18 V. The

direct stimulation on the muscle shows a decrease in muscle tension 4.19 grams to 2.27 grams as voltage

increased from 2.2 V to 1.8 V.

IV. Discussion

The muscle contraction has been referred to with the law called, all-or-none law. This law

explains the logic that when a muscle is stimulated with a certain force or intensity, the muscle

will contract accordingly (Barabarb, 2012, 10). Whole muscle contraction involves motor units

which are made of motor neuron that connects to different muscle fibers. As contraction

occurred, motor unit recruitment happens. The number of motor unit recruitment depends on the

strength of contraction. This can be tied to the Henneman size principal. It states that increasing

the number of motor units depends on the strength of contraction and how hard a muscle works

(Sherwood, 2010, 270).

The experiment begins with demonstration of the effects of motor unit recruitment as the

sciatic nerve was externally stimulated by increasing voltage. The expected outcome of

increasing tension with increasing voltage was an S-shaped curve. The sciatic nerve was being

stimulated with increasing voltage from .006 voltages increments until it reached the maximum

voltage of 0.18. Referring to figure 1, the tension increased as the stimulus voltage increases in

which bought upsurge in motor unit recruitment because of the spatial summation. Overall, the

muscle tension increases from a small tension value of 0.43 grams to 94.69 grams. This finding

agreed with the research in the cat medial gastrocnemius muscle, which follows the trends of the

Henneman Size Principal. The motor units are recruiting from small to largest as the force

increased (Gordon, Thomas, Munson, and Stein, 2004, 656). During the data values of 9.51

grams to 68.13 gram, a random error happened contributing to an outlier in the curve and gave

an incomplete S-shape curve. The cause could be the muscle of the subject (frog) was over active,

in which the muscle tension continued to went up.

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During contraction and relaxation, the sarcomere plays a vital role in the skeletal muscle,

allowing the sliding of filaments. Indeed, to initiate a muscle contraction in the next component

involves a cross bridge which is tied to troponin and tropomyosin. During relaxation,

tropomyosin sits in the actin-myosin binding site. It inhibits myosin from having contact with

actin, thus prohibiting the cross bridge process (Sherwood, 2010, 261). Calcium was released by

the stimulation from the sarcoplasmic reticulum due to depolarization. Once calcium is present in

the cytoplasm, it will bind to the tropomyosin. This allows the myosin to bind to actin and

supported by the consumption of ATP. ATP is converted to useful energy, ADP + Pi, which puts

cross bridge in binding position. When ATP is attached to myosin, the cross bridge is in a sort of

resting state, not binding to actin. Without calcium removing tropomyosin from the actin-myosin

binding site, the cross bridge will stop and the muscle will relax (Sherwood, 2010, p 261).

The next part of experiment involved the stimulation of the sciatic with increasing

frequencies (pulse per seconds). A research on the characteristic of tetanic force produced by the

sternomastoid muscle of the rat demonstrates a similar result, which was, muscle contraction

(twitches) are joined and merged, and then at given frequency, it reached tetanic summation

(Sobotka and Mu, 2010, 6). Refer to figure 3, an notable sharp decrease went from 96.18 gram at

frequency 0.15 to 16.72 gram at 2pps. Then there was a trend of steady increase from the tension

of 16.72 gram at 2 pps until it ends at tension of 88.89 gram at 25 pps. Frequency increases

indicated action potential are sent out, therefore, calcium release into the cytoplasm are greater in

compare to return of calcium to sarcoplasmic reticulum (Sherwood, 2010, 261).When large

amount of calcium present in cytoplasm, the cross-bridge and muscle contraction occurs nonstop

until it reach temporal summation and then end at tetanus (Sherwood, 2010, 264). Temporal

summation occurs with a pattern of increase in frequency cause twitches to combine. Then

tetanic takes place as frequencies increases, the muscle tension has recruited all available motor

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unit, so, the muscle tension shown no increase (Sherwood, 2010, 271). A similar research on rat

illustrates the relationship of muscle tension as response to increase in frequency. At 50Hz, the

muscle contractions are at temporal summation and reached tetanic contraction at 150 Hz.

(Sobotka S and Mu L. 2010, 6). The data obtained from lab gave unexpected result for the

beginning part of the curve. The data value of the curve started with the highest muscle tension

value of 96.18 gram corresponding to the lowest frequency started with 0.5 pps. Incorrect

experimental data can be caused by errors or taking inaccurate measurement such as missing the

Ringer’s solution. Another reason could also be due to a high baseline tension.

The third part of this experiment explored the effect of tubocurare injection into the middle

of the muscle. The expected result with the present of tubocurare in the frog was notable for the

fast decreased in the depolarization of the skeletal muscle fiber due to the blockage of

neurotransmitter (Magleby, Pallotta, and Terrar 1981, 88). Tubocurare competes with the

acetylcholine (Ach) to bind with nicotinic acetylcholine receptors (nAChR) (Jenkinson, 1960,

309). The motor nerves within the muscle could be numbed by the tubocurare shot, decreasing

the amount of excited nerve; thus, less motor neuron are recruited. (Barabarb and Clarac 13).

This was consistent with research performed by Barabara and Clarac and figure 3. In Figure 3, as

a result of tubocurare injection, the contraction of muscle got banned then muscle tension

decreased. As observed in graph, the muscle contraction was level-off, with no decline at 3.53

minute to 5.02 minute with muscle tension around the range of approximately, 85-90 grams. As

time progressed to 5:52 minute, there was a sharp declined of muscle tension from initially at

89.57 grams to 75.40 grams, then, it was further declined until it reached 7.37 grams. So, the

initial muscle tension was 12X greater then force at the end of the ten minutes. In addition, the

result of decline muscle tension due to tubocurare is reversible. An increase of calcium influx

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into the axon terminal can cause an increased level of acetylcholine available to bind with

nAchR, thus, out-compete tubocurare (Barabarb and Clarac 2011, 13).

Going to direct stimulation, it’s notable that direct stimulation is different from indirect

stimulation of the sciatic nerve. Direct stimulation stimulates those muscles which located at the

motor endplate and creates graded potential. Graded potential has amplitude diminishment as it

travels away from the initial site (decremented). Thus, graded potentials have no refractory

period. Recruitment of the motor unit went from largest to smallest. Thus, the stimulation

avoided the neuromuscular junction and graded potential was created (Barbara, Griffith , and

Lam, 2012, 207). Refer to figure 4, the motor unit recruitment went from a high muscle tension

value of 4.19 grams to lower value 2.27 grams. In table 1 and figure 4, the direct stimulation of

the muscle took a higher voltage to generate the threshold at 2.2 voltages. It bought about a 2.05

voltage difference compared with the indirect stimulation. An inverse relationship observed

between the tension and voltage when compared direct stimulation with indirect stimulation. It

took a larger amount of voltage to elicit gastrocnemius muscle and it was responded with lower

tension than sciatic nerve stimulation. Thus, motor recruitment in direct stimulation is less

effective because of the bypassed pathway of neuromuscular junction.

Action potentials are triggered by membrane depolarization to threshold. It also has a

characteristic of all or none nature and refractory periods; so, summation is not possible with

action potentials (Sherwood, 2010, 90). In lab, indirect stimulation of the sciatic nerve with

increasing voltage generated action potentials, which propagated to the entire motor unit to

create a response afterwards. In figure 4, indirect stimulation increased sharply from muscle

tension of 22.18 grams at voltage of .15 and ended at muscle tension of 97.16 gram during

Vmaximum. Therefore, it indicated motor unit are recruited from smallest to largest, which

matched with the Henneman size principle. In contrast, direct stimulation of the gastrocnemius is

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the reverse of the Henneman size principle. The graphed curve was imperfect and without the

expected curve shape. The size of the frog, how the frog was positioned and contacted the

electrode tips might have played a role in the deviation of the curve. Yet, the curve was still

capable to illustrate the concept and the pattern projected.

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References

Barbara DM, Lam A, and Griffith L. Neuromuscular Electrical Stimulation for Skeletal Muscle

Function. Yale Journal of Biology and Medicine. 2012 June; 85(2): 207

Bautista Erwin; and Korber Julia. NPB 101L: Physiology Lab Manual. 2nd Ed. Ohio: Cengage

Learning, 2009.

Barbaraa, JG, Clarac, F. Historical concepts on the relations between nerves and muscles. Brain

Res. 2011 Aug 29;1409:13

Gordon T, Thomas CK, Munson JB, and Stein RB. The resilience of the size principle in the

organization of motor unit properties in normal. Can. J. Physiol. Pharmacol. 2004; Vol.82: 646

Jenkinson DH. The antagonism between tubocruraine and substances which depolarize the motor

end-plate. J. Physiol. 1960; 152: 309

Magleby KL, Pallotta BS, and Terrar DA. The Effect of (+)-Tubocurarine on Neuromuscular

Transmission During Repetitive Stimulaiton in the Rat, Mouse, and Frog. J. Physiol. 1981; 99

Sherwood, Lauralee. Human Physiology: From Cells to Systems. 7th ed. Canada: Brooks/Cole,

2010. 90, 249,250, 257, 260-262, 264,267-271

Sobotka S and Mu L. Characteristics of Tetanic Force Produced by the Sternomastoid Muscle of

the Rat. Journal of Biomedicine and Biotechnology. 2010; Article ID 194984: 6.

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Appendix: Raw Data

The Voltage Threshold

The subject being studied is a double-pithed frog. Then, raw data was analyzed by using the P-P values

measurement tool. In graph, the threshold voltages were illustrated as the spikes. Threshold voltage refers

to when the first twitch observed. The trend in this graph shows a steady flow of twitches.

The Voltage Maximum

The subject being studied is a double-pithed frog. Then, raw data was analyzed by using the P-P values

measurement tool. In graph, the voltage maximums are shown represented by the spikes. Voltage

maximum are at a given voltage which the muscle twitch couldn’t go any higher. The trend in this graph

shows a steady flow of twitches at maximum.

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The Stimulus Intensity on Muscle Activity: Graded Response

Data was collected by using a MP35 stimulator unit on the sciatic nerve of a double-pithed frog. The data

were analyzed by the use of p-p value measurements through BioPac software. Illustrated in this graph

were all the muscle twitches in response to stimulus. The trend shown was the twitch was small as

stimulus was small then increased as stimulus given was increased.

Stimulus Frequency on Muscle Activity: Summation

Data was collected by using of a MP35 stimulator unit on the sciatic nerve of a double-pithed frog. Then,

p-p values were used to analyze the raw data to data points in the graph through BioPac software.

Illustrated in this graph were all the twitches in response to increased frequency with voltage at voltage

maximum. The trend shown was increase in twitches are frequency increased.

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Tubocurare Injection and Paralysis of Muscle Activity

The graph shows the data collected when a 0.25ml of tubocurare into the gastrocnemius muscle. The

subject is a double-pithed frog. Then, the data was analyzed by finding the p-p value. The graph

illustrated all the twitches in response to the injection of tubocurare. The trend shown was decreased

twitches as time progressed.

Direct Stimulation of Muscle: Voltage Threshold

Direct Stimulation of Muscle: Voltage Maximum

Data for direct stimulation was collected by using two needle electrodes and inserting them into about 5

mm from each end of the muscle. The subject being studied is a double-pithed frog. Then, raw data was

analyzed by using the P-P values measurement tool. In graphs above, it illustrated all the twitches in

response to direct stimulation of the muscle and reflected the twitches as the voltage was set to the 10x

the voltage maximum in indirect stimulation.

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Raw Data for Tubocurare Injection

Minute Tension (gram)

3.52 89.57

4.02 90.02

4.52 90.06

5.02 89.96

5.52 85.11

6.02 75.4

6.52 64.91

7.02 61.87

7.52 55.9

8.02 52.45

8.52 40.3

9.02 33.22

9.52 26.35

10.02 22.4

10.52 20.22

11.02 17.29

11.52 15.71

12.02 13.25

12.52 9.89

13.02 8.1

13.52 7.37

These were the data value plot in to the tubocurare injection curve displayed in figure 3. The

subject is a double-pithed frog. Then, the data was analyzed by finding the p-p value then putted into

excels. The graph illustrated all the twitches in response to the injection of tubocurare. The trend shown

was decreased twitches as time progressed.