1

Neeka Tabatabaei
Systems Physiology Laboratory
Section 08
23 April 2015

Frog Nerve-Muscle
Abstract: Muscle contraction is a very important part of body function and has many different
variables that can affect it. The main determiner though, is whether the stimulation is directly on
the muscle or indirectly on the nerve. Variables such as twitch response, latency, and effect of
fatigue were measured for both the nerve and the muscle on the gastrocnemius muscle and the
sciatic nerve of a Rana pipienspiens frog leg. Results showed that the twitch response in the
muscle required a stronger stimulus and generated a higher force. The onset and peak latencies
of the muscle, on the other hand, were slightly lower than those of the nerve. The muscle
exerted a higher force than the nerve under fatigue, but both decreased in force as time passed.

Introduction: Skeletal muscle fibers, like in nerve tissue, respond to a stimulus in an all-or-none
fashion called a twitch. In this experiment, we worked with the gastrocnemius muscle and
sciatic nerve of a Rana pipienspiens frog. In a study done on the interaction between
gastrocnemius muscle weakness and moderate exercise in a rat knee, it was discovered that
moderate exercise has a positive effect on joint homeostasis, but ankle muscle weakness may
change the mechanical environment of the knee and impair the integrity of joint cartilage with
moderate exercise (1).

2
A motor unit is a compilation of a single motor neuron and all the muscle fibers that it
innervates. Depending on the number of muscle fibers innervated, there are small motor units
and there are large motor units. The smaller the motor unit, the more control that muscle has
over its movement. The strength of muscle contraction depends on the intensity and frequency
of stimulation and can be increased by either recruitment, which is increasing the number of
active motor units, and/or by stimulating existing active units more frequently. In a study done
on former polio patients, it was determined that patients with post-polio syndrome (PPS) lost
both strength and motor unit (MU) size over ten years, and the rate of decline in strength was
related to the rate of decline in both MU size and number of active MUs (2).
The number of muscle fibers also affects the force that a muscle can generate.
Additionally, the force, mechanical work and power produced by muscle fibers are profoundly
affected by the length changes they undergo during a contraction (6). A factor to consider
during muscle contraction is fatigue, which is caused when there is a depletion of energy. Action
potentials are a main part of muscle contraction and are started when the membrane gets excited
and releases neurotransmitters, such as acetylcholine, into the synapse. These then diffuse across
the cleft, binding to post-synaptic receptors, depolarizing the membrane. The conduction of
sodium ions is increased during depolarization, and the result of depolarization is the release of
calcium ions. These calcium ions bind to contractile proteins, activating the formation of cross
bridges and ultimately leading to contraction and a twitch. The time before a contraction is
known as the latent period and is necessary so that the acetylcholine can be released indirectly
and that the detector can be reached. An action potential will form faster and sooner when the
resting potential is more negative, because it will be more sensitive.

3
The purpose of this experiment is to illustrate the importance of motor nerves for
activating muscle contraction. We will aim to determine the morphology and physiology of the
neuromuscular junction and explain the differences in conduction and synaptic transmission. It
is also important to determine the difference between stimulating the nerve (indirect stimulation)
or stimulating the muscle (direct stimulation) in terms of eliciting a muscle twitch and how that
affects the threshold and plateau. Illustrating the influence of time on the nerve/muscle
interaction is also stressed.

Methods: The leg of a Rana pipienspiens frog was dissected, examined, and manipulated in a
series of steps. The frogs were placed in a glass jar of isoflurane in order to anesthetize them.
They were then pithed, meaning their spinal chords were severed, and they were weighed. The
legs were then separated in order to begin the rest of the surgery.
The surgery of the leg began by peeling off the skin from the muscle, using one piece of
gauze to hold the bone and another piece of gauze to pull the skin. The muscle was then
loosened from the fibula bone using a glass rod and moistened with Ringers solution to super
fuse it. The Achilles tendon was loosened with scissors and a suture was passed and tied
through it. The length of the gastrocnemius was measured and found to be 3.3 centimeters. The
Achilles tendon was then cut below the knot of the suture, and the fibula bone was cut below the
knee. The next step was to locate and expose the sciatic nerve, which was done using a glass rod
and our hands. Once exposed, the sciatic nerve was moistened with Ringers solution a suture
was tied through it. The nerve was then separated from all other tissues, and the thigh was cut
above the knee.

4
Before beginning the data-collecting segment of the experiment, the force transducer was
zeroed in order to convert from mV to the more meaningful units of Newtons (N). Once zeroed,
the transducer was calibrated using a known weight of 5 grams. The nerve-muscle preparation
was placed into the metal tab of the force transducer with a C-shaped hook. The nerve suture
was then passed through the two nerve-stimulating electrodes. Another suture was used to hold
the knee joint firmly in place. The negative, black alligator lead was attached closest to the
muscle and the positive, red loop was attached to the adjacent loop. The muscle-nerve
preparation was super fused with Ringers solution in order to keep it moist.
The first procedure conducted measured the threshold voltage during nerve stimulation
by delivering a single stimulus after an initial delay. Once the threshold voltage was reached,
increasing stimulus amplitudes were applied until there were three successive stimuli that did not
produce any further increase in the force of the muscle contraction. This is called the peak
amplitude.
The next procedure was to compare the time it took for the muscle to respond to either
nerve stimulation or direct muscle stimulation, which is called latency. The time delay between
the application of the stimulus and the muscle contraction was observed and recorded in order to
determine both onset and peak latencies. Lastly, the effects of prolonged stimulation on force
were tested, ultimately testing fatigue. The frogs were disposed based on Rutgers Environmental
Health Services policies.

5
Results:

Figure 1: The threshold voltage for both the muscle (direct) and the nerve (indirect) were measured during
nerve stimulus. As the stimulus strength increases, the force also increases until the threshold is reached.
Then there is a plateau, and the strength starts to decrease.

Table 1: Twitch Latency

Onset (ms)

Peak (ms)

Nerve

10

57

Muscle

8.6

56

Table 1: The onset and peak latencies were measured both for the nerve and the muscle. The results are
similar, with the nerve having slightly higher values.

Figure 2: The effects of prolonged stimulation on force were tested on both the muscle and the nerve. The
nerve values follow about the same trend as that of the muscle, but are slightly lower at all times.

The threshold voltage was measured both directly from the muscle and indirectly from
the nerve. The muscle required a higher voltage than the nerve to be stimulated. The muscle
also generated greater force than the nerve after being stimulated. On the other hand, the
opposite trend can be seen in the twitch latency. Here, the nerve has slightly higher values for
both onset and peak latencies. The fatigue follows the same trend as the twitch responses. The
force decreases with time as the muscle and nerve become more and more fatigued. The force of
the nerve is always slightly lower than that of the muscle.

Discussion: The results of the frog nerve-muscle experiment show correlations between the
nerve and muscle through the measurements of muscle twitch, twitch latency, and fatigue. It was
determined that the muscle needs a greater stimulus to generate a force, as compared to the

7
nerve. This can be due to two different reasons. The nerve has a more negative resting potential,
making the membrane more sensitive, and therefore causing a sharper and sooner action
potential. The other reason is that the stimulation of the nerve leads to the recruitment of muscle
fibers to create a stimulus, while in the muscle, more voltage is needed to go deeper to stimulate
more. In a study done on the knee extensor muscle, during maximal isometric contractions,
neither direct nor indirect superimposed twitch techniques produce force increases (3).
When a muscle fiber is depolarized, by an action potential or a voltage clamped pulse, a
clear latency is seen between the onset of depolarization and the first detectable rise in
intracellular calcium (4). The twitch latency results showed the opposite trend, with both the
onset and peak latencies of the nerve being higher than that of the muscle. This is due to the
time it takes for the neurotransmitter, acetylcholine, to be released in indirect stimulation of the
nerve. This takes time and is why the latent period of the nerve is essentially longer than that of
the muscle.
The nerve and the muscle both showed the same trend when fatigued, with the nerves
values staying lower than the muscles the whole time. In a study done on previously fatigued
muscle, it was concluded that acute passive stretching, when applied to a previously
fatigued muscle, further depresses the maximum force-generating capacity (5). The force of
contraction constantly decreased as both the muscle and the nerve were stimulated for a
prolonged time. This is due to the nerve and muscle becoming fatigued.

8
References:
1. Ozawa J, Tanaka R, Matsuura N, et al. Interaction between gastrocnemius muscle weakness
and moderate exercise deteriorates joint integrity in rat knee. Scandinavian Journal Of Medicine
And Science In Sports [serial online]. 2015; 25(1): e11-e19.

2. Bickerstaffe A, van Dijk J, Beelen A, Zwarts M, Nollet F. Loss of motor unit size and
quadriceps strength over 10years in post-polio syndrome. Clinical Neurophysiology [serial
online]. 2014; 125: 1255-1260.

3. Pfeifer K, Vogt L, Obermller R, Banzer W. Direct and indirect interpolated electric muscle
stimulation to discover incomplete muscle activation. Physikalische Medizin
Rehabilitationsmedizin Kurortmedizin. 2001; 11(3): 87-93.

4. Zhu P, Parker I, Miledi R. Minimal Latency of Calcium Release in Frog Twitch Muscle
Fibres. Proceedings of the Royal Society of London. Series B, Biological Sciences. 1986:39.
5. Esposito F, Ce E, Rampichini S, Veicsteinas A. Acute passive stretching in a previously
fatigued muscle: Electrical and mechanical response during tetanic stimulation. Journal Of
Sports Sciences. 2009; 27(12): 1347.

6. Azizi E, Deslauriers A. Regional heterogeneity in muscle fiber strain: the role of fiber
architecture. Frontiers In Physiology [serial online]. 2014; 5: 1-5.