This document summarizes an experiment that examined the effects of direct and indirect stimulation on a frog gastrocnemius muscle. It found that: 1) Direct stimulation of the muscle required a higher threshold voltage than indirect stimulation of the nerve to induce contraction. 2) Onset and peak latencies were shorter when stimulating the muscle directly compared to stimulating the nerve indirectly. 3) Both direct and indirect prolonged stimulation induced muscle fatigue, with an 83-85% reduction in contractile force over 30 seconds.

1. Effects of Direct Stimulation, Indirect Stimulation, and Fatigue in Frog Nerve and
Muscle
By
Anastasia O. Belyakova
Systems Physiology Lab 357
Section 5

2. Abstract
Studying physiological effects of the body with animal experimentation has
progressed the knowledge and understanding humans have on bodily functions. The
purpose of this experiment was to examine different kinds of muscle stimulation in the
Northern Leopard Frog, and compare muscle activation characteristics by direct muscular
stimulation, indirect nerve stimulation, latency period and fatigue effects in the
gastrocnemius muscle. The frogs were anesthetized and dissected leaving the leg,
followed by a surgical procedure to isolate and remove the gastrocnemius muscle –
careful not to damage the sciatic nerve. Once the muscle was removed, it was maintained
in room temperature Ringer’s solution and then positioned onto the force transducer
followed by series of tests with varying electrical stimuli.
In the first part of the experiment, the threshold voltage to induce action potential
by direct stimulation of the muscle was found to be approximately 450mV with a peak at
1800mV, as compared to a much lower threshold during indirect stimulation of the nerve
– at about 35mV, with a peak at 160mV. The plateau voltages were found to be 675mV
for the muscle and 52.5mV in the nerve. In the second part of the experiment, the onset
and peak twitch latency in the muscle and nerve were compared. In the nerve, the onset
latency was at 13.5ms while the peak reached 54ms, while in the muscle they were
12.5ms and 50ms, respectively. In the third experiment, fatigue of the muscle was
examined, and it was found that maximum contraction of muscle by direct super
excitation decreased from 25.847mV to 4.461mV (exertion force loss of 83 per cent),
while in the nerve it decreased from 32.279mV to 4.821mV (force loss of 85 per cent).

3. The results of these experiments determined that excitation of the muscle yields
different results when an electrode is stimulating the muscle directly and the nerve
indirectly. The first experiment determined that the threshold, peak, and overall voltage
range of stimulation is a lot higher for direct stimulation than for the indirect stimulation,
while the second experiment showed that the onset and peak latencies were higher in the
nerve than in the muscle. The final experiment illustrated the effects of prolonged
stimulation of the muscle and compared the force exertions throughout the duration of
direct and indirect stimuli. From these experiments, a comprehensive understanding of
the way that muscles and the nervous system work together to produce movement was
achieved; such experiments would further understanding in what may prevent the
physical functioning of the body, and push medicine further in treating physically
limiting conditions.
Introduction
While the study of physiology is not a young science, the understanding of the
morphology and functionality of systems within the organism is. It was not until
technological advancements within the last century did scientists begin to see the inside
of a living organism, as opposed to exploring the body post-mortem. In 1980,
neuromuscular morphology was studied in Japan, and early imaging techniques by
scanning electron microscopy allowed scientists to visualize the structure of the
neuromuscular junction – as well as establish comparisons among various animal species
(Desaki & Uehara, 1980). It was proved that structure between species differed, but the
reactions to stimuli essentially were the same. From this point forward, methods of
muscle stimulation and studies continued to progress.

4. The understanding of the neuromuscular junction advanced from imaging to
stimulation techniques. By the twenty-first century, nerve-clamping electrodes that place
the nerve within the tube set up were created – which is useful in stimulating nerves of
various lengths and examining the effects of toxins that may affect contraction by
inhibiting acetylcholine (Hilmas et al., 2010). Indirect muscle stimulation allows testing
the functionality of the nerve and muscle response, which would be useful in
comprehension of factors affecting physically compromised patients.
In order to determine issues in physical injuries, studies in healthy subjects have
been conducted. Experimentation with electrical impulses has gained advancements in
neuromuscular knowledge by comparing voluntary muscular contractions to electrically
induced ones (Ward & Shkuratova, 2002). This study explored the increase of muscle
force in young Russian athletes as well as older individuals: to test whether muscular
output gains were better affected by muscular growth, electrical stimuli, or both. The
finding that a combination of the two yielded the best results supports the benefits of
electrical stimuli in producing stronger muscular output, which in turn could also
challenge the effects of muscle fatigue during prolonged exercise.
In these experiments, basic neuromuscular functions were explored. Using
electrodes to induce direct and indirect stimulation of the muscle showed the differences
between stimulation voltages required to activate the nerve, and the muscle itself, for a
contraction. Contrasting values for latency as well as muscular reaction to fatigue
explains what is happening within the neuromuscular junction, which leads to the
conclusion that establishing the differences in nerve and muscle stimulation is the key to
aiding physically impaired individuals with future advancements in technology.

5. Understanding the effects of voltages on the muscle or the nerve has resulted in the field
of creation of robotic limbs, which could dramatically improve with more studies into the
future.
Methods
The Northern Leopard Frog was used in this experiment, placed in a jar of
isoflurane to anesthetize it. After 10-15 minutes the frog achieved deep anesthesia,
evidenced by lack of pain response to pulling its toes. The frog was removed from the jar,
pithed at the neck through the spinal cord using standard techniques approved by the
Rutgers University Laboratory Animal Science veterinarians (RU, LAS). Then the leg
was removed and pinned down to the board for surgery.
First, the skin was detached from the muscular tissues of the leg. While keeping
the muscle moist with room temperature Ringer’s solution, a hole was punctured between
the Achilles tendon and the tibia with a glass rod in order to prevent any overstimulation
of the muscle and nerve to be exposed. A surgical silk suture of approximate length 10-
15cm and a hook at one end was tied tightly around the bottom of the tendon to prepare it
for direct stimulation experiments. Then a cut with surgical scissors was made at the
bottom of the Achilles tendon, detaching it from the bone. Next, the optimal muscle
length was measured and found to be 3.0cm long, and then surgical scissors were used to
cut the tibia below the kneecap. After the muscle was freed from the lower leg, a glass
rod was used to expose the sciatic nerve, and further separate it from upper leg muscle
tissues so that another suture could be tied around the end of the nerve for indirect
stimulation experimentation. Finally, the thigh muscle and femur were cut off the leg
above the kneecap, leaving the knee along with the gastrocnemius muscle and sciatic

6. nerve extracted. While the frog leg rested in room temperature Ringer’s solution, the
force transducer was zeroed and calibrated.
The force transducer was zeroed and calibrated before attaching the frog muscle
and running tests. This was done by recording 5 seconds of data by the ECG with no
weight attached. Then the transducer was calibrated with a 1.0g weight, and the software
measured a 0.010N force. The weight was taken off the force transducer, the muscle was
placed to have two electrodes stimulating it from the top of the apparatus, the bottom two
electrodes were stimulating the sciatic nerve. All results were recorded by Lab Tutor
software.
In the first experiment, the resting potential was recorded. The threshold of the
nerve was determined by increasing the administered voltage by increments of +5mV,
until a response occurred. From there, additional increments of +25mV were
administered until the peak was reached. A similar format of stimulation was done for the
muscle, starting at 300mV stimulation additional increments of +50mV lead to the
threshold and peak voltages.
From the recorded data in the first experiment, the onset and peak latency of
direct and indirect stimulation were measured. To find the onset latency, a marker was
positioned at the baseline of the action potential at the moment the shock was
administered, and ranged to the point where contraction began and an action potential
occurred. Similarly, the peak latency was measured from the same initial marker at the
administration of the stimulus, to the peak of the action potential.

7. All throughout experimentation the muscle specimen was washed with room
temperature Ringer’s solution. In the third experiment, the ECG shock was administered
with constant voltage duration of 30 seconds to stimulate the muscle and test its capacity
for fatigue. The first part of the experiment tested the indirect prolonged stimulation of
the nerve, followed by a two-minute rest to let the muscle recover, and then direct
stimulation of fatigue was administered. After all testing was completed, the muscle
specimen was untied and detached from the force transducer apparatus, the surgical silk
sutures were cut off and washed, and the frog leg was disposed of following the REHS
guidelines into a brown bag.
Results
Figure 1: Twitch Response
This graph shows the threshold and peak voltages of the nerve and the muscle, thereby
showing direct and indirect stimulation ranges that produce muscle contractions.
Figure 1 illustrates direct and indirect stimulation of the muscle: from the threshold
voltage when action potential is first experienced, to the plateau – when the contractile

8. force no longer increases. When stimulating the nerve, the threshold voltage is
determined to be at 35mV, and contractile force rises until approximately 8.92 – 9.114N
at 160mV administration, where the plateau is reached. This is a much smaller voltage
range in comparison to direct muscle stimulation: the threshold being at 450mV, reaching
a plateau around 1650 – 1800mV, producing contractile forces at 12.782 – 13.033N.
Table 1: Latency Onset and Peak Latency in Nerve and Muscle
This table shows the comparison of time frames in milliseconds for the onset of latency,
as well as the peak in latency for the nerve and the muscle.
Onset (ms) Peak (ms)
Nerve 13.5 54.0
Muscle 12.5 50.0
Table 1 compares and contrasts the latency onset and peaks of direct and indirect
stimulation. It is evident that the onset of latency for the muscle (12.5ms) takes less time
than for the nerve (13.5ms), confirmed by the peak latencies: which were 50.0ms and
54.0ms respectively. This proves that stimulating the muscle directly causes a faster
contraction of the muscle than indirectly stimulating the nerve.
Figure 2: Fatigue in Nerve and Muscle
Figure 2 shows the change in force that is exerted during the administration of a 30
second prolonged stimulus to the muscle via direct and indirect stimulation.

9. Figure 2 demonstrates the effect of muscle stimulation for duration of 30 seconds. The
voltage was administrated, and the muscle exerted a contractile force demonstrating
fatigue. When stimulating the nerve, there is a consistent drop in the force the muscle
exerts throughout the duration of the stimulus. The initial contractile force via the nerve
was 32.279N, which dropped to 4.821N – losing 85% of muscle exertion. A similar
observation can be made for the muscle, which had an initial contractile force of 25.847N
and diminished to 4.461N – losing 83% of exertion power. The contractile force for
indirect stimulation was significantly lower than for indirect stimulation, due running a
direct stimulation after the first 30 second long duration, where the muscle was allowed
to rest for a few minutes. Upon the second 30-second direct stimulation, the muscle had
already been fatigued and therefore produced a lower initial muscle contractile force.
Discussion
In order to understand what is wrong with something, it is crucial to understand
the result when it functions properly. Any movement in the body is controlled by

10. electrical impulses that travel from the central nervous system and affect the muscle,
forcing it to contract. When the brain sends an electrical signal to initiate movement, the
signal travels down the axon of the terminal somatic motor neuron, which has several
branches that each attach to a muscle fiber. When the electrical impulse reaches threshold
voltage of the motor neuron, it causes the neuron depolarize and produce an action
potential. Acetylcholine is released into the synaptic cleft and attach to the postsynaptic
receptors of the muscle – which then also experiences an action potential, inducing a
contraction (Silverthorn, 2012). This is the sequence of activation of muscle contraction
via indirect stimulation of the nerve, however direct stimulation of the muscle yields
several changes.
In a direct stimulation, it is seen that there are voltage differences that will cause
an action potential sequence for contraction. The first experiment showed that it takes a
much higher voltage to directly stimulate contraction than to activate the nerve to
produce the same result. The typical resting potential of a motor neuron is -70mV, and if
the neuron is depolarized to -50mV an action potential results (Birkill et al.). However,
each presynaptic terminal of a motor neuron affects one fiber of muscle, so stimulation of
the entire muscle would require a high voltage in order to depolarize the muscle to cause
a contraction.
When testing latency in the second experiment, it was discovered that the onset
and peak of latencies were shorter for the muscle than for the nerve. This is due to the
mechanism behind eliciting a contraction. When stimulating the nerve, it takes a fraction
of a second longer because acetylcholine needs to be released into the synaptic cleft, and
then sodium ions need to depolarize the muscle membrane in order to activate the DHP

11. receptor to release calcium ions out of the sarcoplasmic reticulum. The calcium ions
induce contraction between the actin and myosin filaments (Silverthorn). When
stimulating the muscle directly, although it requires a higher voltage, the muscle itself
gets depolarized and contraction is immediately induced. The difference in eliciting the
contractions is the distance the electrical signal must travel – it takes longer for the action
potential to travel from the nerve to the muscle than from within the muscle itself.
In the third experiment, direct and indirect administration of prolonged voltage
stimulus induced muscle fatigue. Muscular fatigue is the decrease in the contractile force
of the muscle for the duration of a stimulus rather than the inability to perform a task
(Enoka & Duchateau, 2008) as was established in the experiment as well. Both direct and
indirect electrical prolonged forced contractions showed an 83 – 85 per cent loss of
contractile force within that time. It was also evident that the initial contractile force was
diminished by 20 per cent since the first experimentation of a prolonged stimulus, which
proves that muscle contractile force decreases with each round of exertion. This explains
why an individual gets more tired with each repeat of an exercise.
Citations:
1. Birkill, C., Van Rensburg, R., & Raath, R. (n.d.). Electrophysiology and Nerve
Stimulators. South African Journal of Regional Anaesthetics.
2. Desaki, J., & Uehara, Y. (1980). The overall morphology of neuromuscular
junctions as revealed by scanning electron microscopy. Journal of
Neurocytology, (10), 101-110.
3. Enoka, R., & Duchateau, J. (2007). Muscle Fatigue: What, Why And How It
Influences Muscle Function.The Journal of Physiology, (586), 11-23.

12. 4. Hilmas, C., Scherer, J., & Williams, P. (2010). A Nerve Clamp Electrode Design
For Indirect Stimulation Of Skeletal Muscle. Biotechniques, 49, 739-744.
5. Silverthorn, D. (2012). Chapter 12: Muscles. In Human Physiology: An Integrated
Approach (6th ed., pp. 410-413). Austin, TX: Benjamin-Cummings Publishing
Company.
6. Ward, A., & Shkuratova, N. (2002). Russian Electrical Stimulation: The Early
Experiments. Physical Therapy, 82(10), 1019-1030.