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Introduction:
Skeletal muscle fibers contract, or twitch, when a stimulus is applied. The stimulus
is delivered by a nerve cell, or neuron. A muscle twitch is the response of a single muscle
fiber from a single nerve stimulus. (Widmaier, 2004, p. 284) The physiological location
where the communication between neurons and muscle fibers occur is the neuromuscular
junction. (Sherwood, 2010, p. 246) As the action potential sent by a neuron reaches the
neuromuscular junction, Ca2+ is able to enter the terminal axon. This causes acetylcholine
(Ach) to leave and bind to the motor end plate of the muscle fiber. (Sherwood, 2010, p.
246) The binding of ACh allows the muscle fiber to depolarize and initiate an action
potential.
The action potential travels to the transverse tubules that surround the muscle
fiber. This excites the sarcoplasmic reticulum, which then releases stored Ca2+ ions into the
muscle fiber. The Ca2+ then allows myosin and actin filaments, that make up the muscle
fiber, to bind and form a cross-‐bridge and contract the muscle. (Sherwood, 2010, p. 264)
This cross-‐bridge is formed by the myosin heads bonding to the actin filaments and
generating a pulling motion to initiate a muscle contraction. This pulling motion must be
repeated in rapid succession to provide maximal contraction. (A. Faller, 2004, p. 92)
Twitch strength, also known as twitch tension, can be increased by the summation
of the twitches or by recruiting more motor units. Many muscle fibers acting together by
stimulation of the motor neuron are known as a muscle unit. Muscle units can recruit more
muscle units when additional strength is required, this is known as muscle summation.
There are several sizes of motor units. The size recruited depends on the strength of
contraction and acuity required. (Sherwood, 2010, p. 270)
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With increased stimulation frequency there is no period of relaxation for the twitch.
This provides consistent and sustained contraction, known as tetanus. This occurs due to
the increase of Ca2+ concentration. This form of contraction is typically 300-‐400% stronger
than that of a single twitch. (Sherwood, 2010, p. 271)
The main process, which acts as the origination of a muscle stimulation, is the
binding of ACh to its receptor. The receptor used in skeletal muscle is called the nicotinic
acetylcholine receptor (nAChR). (Sherwood, 2010, p. 243) Muscle contraction can be
blocked, or inhibited through competitive binding of an antagonist known as tubocurare.
Tubocurare binds to the nAChR, blocking ACh from binding and reducing twitch tension.
Muscle neuron synapses can also be inhibited which will lower contraction response.
The goal of this experiment is to measure and understand how twitch tension differs
when the sciatic nerve is stimulated at varying voltages and frequencies. Additionally this
experiment will show neurotransmitter interactions with muscle twitch and evaluate
direct muscle stimulation using an electrode.
Tension is expected to increase as voltage is increased until a maximum tension is
observed. This is the point where the motor unit is being completely used and no more
force can be applied. With increased stimulation frequency it is expected that the muscle
twitches become closer together and at a higher tension, however, this will cause the
muscle to fatigue more quickly than with individual stimulation due to the inability to
replenish ATP. When tubocurare is injected into the frog’s leg, tension will decrease due to
the inhibition of the nAChR. However, direct stimulation by an electrical charge will
override the nerve and cause a contraction.
The subject used was a Leopard Frog.
3
Methods:
The procedure followed for the lab can be found in the NPB 101L Physiology Lab
Manual. (E. Bautista, 2009, pp. 9-‐18) Previous to the experiment the frogs were double
pithed by the lab TA’s. The brain and spinal cord were destroyed so the frog could not feel
pain or control it’s own movement. The frog was kept moist with a paper towel dampened
with deionized water, and once the muscle tissue was exposed the tissue was kept alive
with Ringer saline solution. The solution was high in electrolyte ions Ca2+, Na+, and K+.
Skin was removed and the frog’s gastrocnemius was tied off and separated from the
leg at the lower tendon. The muscle hung at a 90° angle to the force transducer. The
tension was set to approximately 20g +/-‐ 5g at all times during the lab. The sciatic nerve
was identified and an electrode was placed under the nerve, with parafilm separating the
nerve from the tissue.
To find the threshold and maximum voltage, the voltage was increased at
increments of approximately 0.10V until the observed force no longer increased. To
observe the graded response an equation was used to calculate ΔV, taking the difference of
the Vmax and the Vthreshold and dividing by a factor of 4 to give a ΔV of 0.10V. The stimulator
was set to threshold voltage of 0.30V and voltage was increased at 10-‐second intervals by
our ΔV of 0.10V until Vmax of 1.0V was reached. To measure muscle twitch summation, the
voltage was set to the Vmax of 1.00V and frequencies were increased. To examine muscle
paralysis, the muscle was stimulated at 0.50 pps at 1.0V for about 60 seconds to establish a
baseline. The tubocurare was injected and observed for 5 minutes. To observe direct
electrical muscle stimulation, needle electrodes were inserted into the frog leg and voltages
were increased until a stimulus appeared.
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Results:
Part 1: Voltage Threshold and Maximum
Stimulation was delivered to the muscle at increasing increments of approximately
0.1V as shown in Table 1. The initial tension from the frog leg muscle was roughly 21
grams. The first twitch was recorded at 0.3V which is the Vthreshold and a force of 23.52g was
recorded. At 1.0V Vmax was recorded and a force of 124.75g was recorded as shown in
figure 1. The change in voltage needed to reach Vmax was 0.7V as shown in table 2.
Figure 1: Graph of voltage (V) vs. twitch tension (g). As the V increases, the force, also
known as the twitch tension increases until the voltage maximum which is at 1.0V and
124.75g of force. The voltages were recorded in gradually increasing increments.
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Table 1: The twitch tension (g) recorded and voltages associated with each change when
sciatic nerve of the frog was stimulated by the electrode.
Table 2: Threshold voltage, maximum voltage and the change in voltage for twitch tension.
To find the change in voltage needed to elicit a maximum response, the Vthreshold was
subtracted from the Vmax.
ΔV = Vmax – Vthreshold = 1.00-‐0.30 = 0.70V
Part 2: Graded muscle Responses
When the voltage increases, the tension increases as well, however the response is
graded. These data present the tension as minimally increasing from Vthreshold to Vmax as
seen in Figure 2. Individual measurements are indicated in Table 3.
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Figure 2: The tension (g) at specific voltages applied to the frog’s gastrocnemius. The
maximum tension was recorded at 8 specific points from Vthreshold to Vmax.
Table 3: The tension (g) at specific voltages (V) between Vthreshold to Vmax.
Part 3: Frequency and Twitch Summation
When the frequency of the electrode stimulation increased, the waves appeared
closer together. There were 7 variances in frequency shown in Table 4 and represented in
Figure 3. As frequency increased the waves became closer together leading to tetanus.
When the nerve was stimulated the frog leg would visibly twitch. As frequency increased
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and the waves fused together the twitches appeared to become a single contraction. The
tension decreased towards the end of each series of stimulation.
Figure 3: Raw data of muscle twitches at increasing frequencies for the frog gastrocnemius
muscle. Frequencies are indicated at the top of the graph and are measured in pps. Time is
indicated at the bottom along the X-‐axis and is measured in seconds. Twitch tension in
grams is measured along the Y-‐axis.
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Table 4: Maximum tension in grams as the frequency (pps) increases and the highest
measured twitch tension at that frequency.
Part 4: Tubocurare Injection and Muscle Paralysis
Prior to the tubocurare injection the control tension of the muscle at a frequency of
0.5 pps and measured Vmax of 1.0V was recorded for 60 seconds. 0.25mL of tubocurare was
injected in the frog muscle and tensions were monitored for approximately 5 minutes. The
injection caused a change in tension due to external force. Tension was recorded at 60-‐
second intervals and can be seen in Table 5. Tension visibly decreased over the 5-‐minute
period, which can be observed in Figure 4.
Table 5: Tension (g) before, during, and after tubocurare injection into the muscle of the
leg.
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Figure 4: Graph of the tension (g) at times (seconds) before, during, and after the injection
of tubocurare. Initial data point is the tension prior to injection. 162 seconds is the initial
data point post injection. All data points are at a constant voltage (Vmax) and a constant
frequency (0.50pps).
Table 6: Average tension (g) of 5 twitches within each 60-‐second time point.
Time (seconds) Average Tension (g) 62.00 43.69 92.00 39.72 162.00 33.87 222.00 29.56 282.00 25.87 342.00 23.42 388.00 23.14
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00
Tension (g)
Time (seconds)
Tension (g) with Tubocurare
Tension (g)
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Figure 5: The average tension (g) of the time (seconds) that the twitch prolonged for. Five
twitches were averaged for the 10 seconds prior to the recorded time interval. Voltage was
set to Vmax.
Part 5: Direct Muscle Stimulation:
When an electrode directly stimulates the muscle, specific muscle fibers are
innervated. At the Vthreshold of 2.8V the muscle tension was 23.28g. This was the lowest
voltage where a twitch was visible. Voltage was then increased to V10xMax, which was 10V.
At this voltage tension was 34.82g, which can be seen in table 7.
Table 7: Tension (g) of the frog gastrocnemius with direct muscle stimulation via
electrode.
Voltage (V) Tension (g)
Vthreshold 2.8 23.28
V10xMax 10 34.82
0
10
20
30
40
50
0.00 100.00 200.00 300.00 400.00 500.00
Tension (g)
Time (seconds)
Average Tension (g)
Average Tension (g)
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Table 8: Tension (g) of the frog gastrocnemius with direct muscle stimulation at Vthreshold of
2.8V and V10xMax of 10V versus stimulation of the sciatic nerve at Vthreshold of 0.30V and Vmax
of 1.0V.
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Discussion:
Effects of Stimulus Intensity on Muscle Activity: Graded Response, Threshold and
Maximum
The membrane potential of the sciatic nerve is changed with stimulation from an
electrode. This allows an action potential to be sent to the frog’s gastrocnemius and cause
a muscle twitch. Considering a muscle twitch is an all-‐or-‐none response, the intensity of
the stimulation must meet a minimum voltage, or a threshold. The voltage threshold
(Vthreshold) is the lowest voltage where the muscle is able to twitch. This is the amount of
stimulation required to stimulate the nerve to send the action potential to the
neuromuscular junction. The lowest threshold that was observed was 0.3V with a force of
23.52g.
When the maximum voltage is reached (Vmax), the twitch tension is at its highest
point and no longer will increase with added intensity. The tension will no longer increase
due to all motor units being recruited. Motor units are composed of the motor neuron and
all of the muscle fibers they stimulate. (Sherwood, 2010, p. 270)
There was possibly error in the graded response experiment due to incorrect
stimulation settings. The multiplier on the voltage setting may have been incorrect
because the data did not follow the expected results. The expected tension at Vthreshold
should have compared to the established tension recorded of 23.52g, however it was
recorded at 122.51g.
By looking at the data in table 1, muscle recruitment can be seen. At 0.3V the
twitch tension is 23.52g and at 0.4V the tension is 31.87g. This shows that as voltage
increased, more motor units are recruited. Although twitches occur in an all-‐or-‐none
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fashion, the twitches can be graded in size due to the formation of more cross-‐bridges and
the recruitment of more motor units. (Sherwood, 2010, p. 270)
The data for increasing the voltage and recruiting more motor units to increase
tension supports the hypothesis according to table 1, however according to table 3 these
data were not repeatable.
Effects of Stimulus Frequency on Muscle Activity: Summation
The action potential can be sent at various rates to initiate a response. The rate an
action potential is sent is referred to as the frequency. When the frequency of stimulation
to the nerve increases, the nerve sends action potentials at a faster rate thus increasing
frequency. When the action potentials are sent close together, they are not able to
completely depolarize due to the refractory period. This causes the twitches to summate.
(Sherwood, 2010, p. 270) When the cell is depolarized, Ca2+ ions enter into the fiber where
the myosin heads and actin filaments are located from the sarcoplasmic reticulum. When
Ca2+ is present, it binds to troponin, which pulls away tropomyosin and allows the myosin
heads to bind to actin, pulling the filament, leading to an increase in tension.
With an increase in stimulation frequency, action potentials are sent at a faster
rate, the Ca2+ concentration stays high enough that the cells are not able to return to
threshold forcing the myosin actin cross-‐bridge to stay intact. (Sherwood, 2010, p. 271)
When the action potentials are sent at a high enough frequency, they summate and cause
tetanus. Tetanus occurs when muscle fibers receive constant stimulation and the Ca2+
concentration cannot decrease so a constant contraction occurs at maximal strength.
(Sherwood, 2010, p. 271)
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The tension of a tetanus contraction is stronger than a twitch summation. In table
4, a single twitch where no summation occurred had a tension of 134.99g. At a frequency
of 25pps, where tetanus occurred, there was a tension of 146.88g. This shows how tetanus
contraction is stronger than twitch summation.
The tetanus lasts for a shorter amount of time as the frequency increases due to
fatigue, which can be observed in figure 3. Since the motor units are innervating all of the
muscle fibers available, there is no time to replenish Ca2+ and fatigue sets in. When muscles
are not in tetanus they exhibit asynchronous recruitment of motor neurons, allowing the
muscle fibers to relax. (Sherwood, 2010, p. 270) The higher the frequency, the faster
fatigue sets in, which is observed in figure 3.
Studies have shown that various types of muscle fatigue at various rates. In a
study on a rat gastrocnemius the fast fatigue motor units had a larger twitch tension and a
higher tetanus peak compared to the fast resistant and slow motor units (Lochynski, 2007,
pp. 23, 26). The motor units being innervated in the frog gastrocnemius varied with
frequency change, which is why the muscle fatigued more rapidly at the highest frequency,
25pps, without increasing tension substantially.
When frequency increased, the twitch tension also increased but twitch response
lasted a shorter amount of time. At 0.5pps the tension was 134.99g and remained fairly
steady. As the frequency increased and twitch moved towards tetanus from summation
twitch response decreased as well as twitch tension leading to fatigue as shown in figure 3.
When comparing the higher frequencies of 15 and 25pps, the decrease in tension after
tetanus was steeper. This can be due to tetanic depression, which occurs when a high
frequency stimulus, such as 25pps follows a low frequency stimulus and the force of the
15
motor unit decreases. (Celichowski, 2011, p. 19) In a study researching tetanic depression,
the medial gastrocnemius muscle in a cat and a rat was stimulated at a low frequency first
and subsequently at a high frequency. It was observed that when a low-‐frequency stimulus
and a high frequency stimulus occurred in succession, the higher frequency stimulus
exhibits lower twitch tension post tetanus. (Celichowski, 2011, p. 19)
Effects of Tubocurare on Muscle Activity: Paralysis
When an action potential is sent down the nerve to the neuromuscular junction, a
rise in Ca2+ signals vesicles in the terminal button of the nerve axon to release
acetylcholine, a neurotransmitter that signals the muscle fibers to allow Na+ to enter and
cause depolarization which leads to muscle contraction. (Sherwood, 2010, pp. 247-‐248)
Tubocurare is an antagonist to Ach meaning it has a similar conformation and will
competitively bind to the same receptors. This competitive binding allows it to bind to the
nAChR so that Ach cannot bind, preventing a muscle twitch. (Sherwood, 2010, p. 246)
As shown in figure 4, post injection of tubocurare, the tension of the muscle
twitches decreases as time progresses. The binding of tubocurare to the nAChR causes this,
leaing ACh. As time progresses the tension decreases ultimately stabilizing at the end of
the time sample.
Tubocurare is a paralytic agent and is lethal to animals such as the frog used. It
lowers twitch response, inhibiting proper function of the muscles. Since land animals
require muscle function for movement and getting food, impairing muscle twitch can be
lethal. (Widmaier, 2004, p. 283) Although the frog was not alive for the experiment, muscle
tension did return to threshold tension while inhibited at 342 seconds as shown in table 5.
Effects of Direct Electrical Stimulation on Muscle Activity
16
When directly stimulating a muscle with an electrode, the neuromuscular junction
is bypassed. The electrode causes the muscle to contract, even while the nACHr is
competitively inhibited with tubocurare. Direct stimulation only innervates the muscle
fibers it is touching, however. This leads to a low response in tension at much higher
voltages. In a study on frog muscle fibers, direct stimulation led to a tension of 1/6th of that
observed by stimulation of the nerve. (Lehmann, 1979, p. 43) With a lower number of
muscle fibers being stimulated, the tension response is lower.
When comparing the twitch tension in part 1 and part 5 of the lab, there is a
noticeable difference in the twitch tension at Vmax, as shown in table 8. Vmax for sciatic
stimulation (1.0V) provided a tension of 124.75g of force. This is largely different than the
force of 34.82g measured with a direct stimulation of V10xMax (10.0V). This difference in
tension shows the properties of motor unit recruitment. When the sciatic nerve is
stimulated, it can innervate many muscle fibers, and recruit more motor units. With direct
stimulation, however, a single muscle fiber is stimulated and a lower tension response is
observed.
The use of Ringers solution maintained the presence of ions in the tissue. The
solution contains required ions such as Ca2+, Na+, and K+ that are needed for action
potentials, Ach, and myosin-‐actin cross-‐bridges to occur. Regardless of stimulation type,
there is a requirement for these ions. A lack of Ringers solution applied could lead to error
in the results due to atrophy of the muscle tissue.
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Conclusion:
Several components affect muscle tension and twitch. By altering these
components, such as voltage and frequency, motor unit and muscle fiber interactions can
be observed. If one variable is altered, the response changes. Each individual alteration
had a different impact and was observed.
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References
A. Faller, M. S. (2004). The Human Body: An Introduction to Structure and Function (13 ed.). (O. French, Trans.) Stuttgart, Germany: Georg Thieme Verlag, 92. Celichowski, D. L. (2011). The tetanic depression in fast motor units of mammalian skeletal muscle can be evoked by lengthening of one initial interpulse interval. Experimental Brain Research (241), 19. E. Bautista, J. K. (2009). NPB 101L Physiology Lab Manual (2 ed.). Mason: Cengage Learning. 9-‐18 Lehmann, S. (1979). Contractile Responses to Direct Stimulation of Frog Slow Muscle Fibres Before and After Denervation. European Journal of Physiology (382), 43. Lochynski, C. K. (2007). Changes of motor unit contractile output during repeated activity. Acta Neurobiologiae Experimentalis (67), 23-‐26. Sherwood, L. (2010). Human Physiology: From Cells to Systems (7 ed.). Belmont: Brooks/Cole, 243, 246, 247-‐248, 264, 270-‐271. Widmaier, R. S. (2004). Cander, Sherman and Luciano's Human Physiology (9th ed.). New York: McGraw-‐Hill 283-‐284.
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Raw Data:
Threshold Voltage 0.3V
Maximum Voltage 1.0V
Frequency Change Graph
20
Tubocurare
Tubocurare baseline 0-‐62 seconds
Tubocurare at 92 seconds (injection start)
21
Tubocurare at 162 seconds (post injection)
Tubocurare at 222 seconds
Tubocurare at 282 seconds
22
Tubocurare at 342 seconds
Tubocurare at 388 seconds