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San Beda College of Medicine
Course: Medical Physiology
Academic Year: 2014-2015





EXPERIMENT 2 - FROG MUSCLE PHYSIOLOGY
INTRODUCTION:

In this laboratory, you will investigate the physiological properties of skeletal muscle using
the isolated frog gastrocnemius. Concepts that you will explore include the single twitch,
graded response and the relationship between muscle length and tension generated. You
will also explore tetanus and muscle fatigue.
These experiments illustrate the collective understanding of muscle physiology gained
from over 400 years of research.

BACKGROUND:

The frog muscle preparation that you will use in the laboratory is the earliest isolated
tissue preparation. The first experiments on muscle physiology appear to have been
performed between 1661 and 1665 by Jan Swammerdam, who demonstrated that an
isolated frog muscle could be made to contract when the sciatic nerve was irritated with a
metal object. Later, Luigi Galvani (17371798) demonstrated that frog muscle responded
to electrical currents. Here we focus on the mechanical properties of skeletal muscle. The
invention of the kymograph (a rotating drum powered by a clockwork motor) in the late
1840s, attributed to either Carlo Matteucci (18111868) or Carl Ludwig (18161895),
revolutionized experimental physiology for it enabled events such as muscle contractions
to be recorded and analyzed for the first time.

Today, the computer has taken the place of the kymograph but physiology students of the
late 1800s would recognize the experiments described here. These demonstrate some of
the important functional characteristics of skeletal muscle.






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The basic unit of a muscle is the muscle cell, or fiber (Figure 1).



Figure 1. Skeletal muscle structure.

Whole muscles are made up of bundles of these fibers. Unlike cardiac muscle cells, there
are no gap junctions between adjacent cells. This means that each fiber behaves
independently. A single muscle fiber has a very regular structure, defined by myofibrils.
Each myofibril consists of an arrangement of the contractile proteins actin and myosin,
which are able to slide past each other in the presence of Ca
2+
and ATP.
A single motor neuron, and all the muscle fibers that it innervates, is known as a motor unit
(Figure 2).



Figure 2. A motor unit.

Skeletal muscle is similar to nerve tissue in that the fiber responds to a stimulus in an all-
or-none fashion. This response is called a twitch.

One motor neuron supplies a number of muscle fibers to constitute a motor unit. Motor
units vary greatly in size, from just a few muscle fibers innervated by a single neuron (small

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motor unit) up to thousands (large motor unit). The smaller the motor unit, the finer the
control of movement in that muscle. Thus the muscles controlling the movements of the
fingers and eyes have small motor units whereas those controlling the large limb muscles
may have very large motor units. However, most muscle consists of a range of motor unit
sizes.

Depending on the intensity and frequency of stimulation, greater numbers of fibers are
activated. The strength of a muscle contraction, therefore, can be increased in two ways: by
increasing the number of active motor units (termed recruitment) and by stimulating
existing active motor units more frequently. The absolute force that a muscle can generate
is dependent on the total number of muscle fibers. So muscles with large cross-sectional
areas are able to generate larger forces than those with small cross-sectional areas.

Motor nerves release the neurotransmitter acetylcholine from their terminals, called motor
end plates.


Figure 3. A neuromuscular junction.

The acetylcholine released into the junctional cleft binds to receptors on the muscle
membrane that are directly coupled to cation-selective ion channels. Opening of these
channels depolarizes the muscle fiber and leads to the release of intracellular calcium from
the sarcoplasmic reticulum, a variant of smooth endoplasmic reticulum. The increased
cytosolic calcium sets in motion the biochemical events that underlie contraction. The

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acetylcholine is rapidly hydrolyzed by acetylcholine esterase on the skeletal muscle
membrane in this region and thus does not accumulate in the junctional cleft.
Skeletal muscle can be studied under isometric (constant length) or isotonic (constant
load) conditions. Here the force is measured isometrically.

Action potentials in skeletal muscle, like those in nerve, last for only a few milliseconds
(ms). In contrast the mechanical response of the muscle the muscle twitch last
significantly longer.




Figure 4. Temporal relationship between muscle action potential and consequent contraction.

A second stimulus arriving before the muscle has relaxed again, causes a second twitch on
top of the first so that greater peak tension is developed. This is called summation. With
increasing frequency of stimulation, there is less and less time for the muscle fiber to relax
between stimuli and eventually the contractions fuse and a smooth powerful contraction
tetanus is seen.



Figure 5. Effects of repeated frequency of stimulation.

Indeed, normally skeletal muscles are activated by volleys of action potentials and operate
in a state of fused contractions.
The strength of muscle contraction is also influenced by the degree of stretch of the muscle.
In considering the force of the response of a muscle to stimulation, it is necessary to
separate out the passive and active forces. The passive forces reflect the contributions of

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elastic elements in the muscle both extracellularly and within the fibers themselves. The
active force is that generated by the contractile machinery when the fibers are stimulated.



Figure 6. Skeletal muscle length - tension relationship.

Experimentally, what is measured at different degrees of stretch is the total force during
nerve stimulation and contraction, and the passive force in the absence of nerve
stimulation. The difference between the two is the active force at any muscle length.

Skeletal muscle contraction requires metabolic energy. A depletion of energy stores results
in fatigue. Some muscle fibers are more resistant to fatigue than others. These have a
greater capacity for oxidative metabolism (Note, however, that in the intact animal, fatigue
occurs primarily because the motor drive from the brain is reduced, rather than as a result
of an appreciable depletion of the muscle energy reserves).








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WHAT YOU WILL DO IN THE LABORATORY:

There are five exercises that you will complete during this Laboratory.

1. Twitch recruitment. In this experiment, you will give the muscle a series of stimuli
of increasing amplitude to examine the effects of stimulus amplitude on contraction
force.
2. Effect of stretch on contraction force. Here you will increase the stretch of the
muscle by raising a micropositioner.
3. Effect of stimulus frequency on contraction force. In this experiment, you will
stimulate the muscle with twin pulses at different pulse intervals and observe their
effect on muscle contractions.
4. Tetanus. In this part of the laboratory, you will examine the muscles response to a
repetitive stimulus at different frequencies.
5. Muscle fatigue. Here you will stimulate the muscle at a high frequency (50 Hz) for a
long duration to examine the effects of muscle fatigue.

GUIDE QUESTIONS:
Exercise 1: Twitch recruitment

As you increase voltage to the muscle describe how it responds to the increased stimulus:
_____________________________________________________________________________________

1. What was the smallest voltage required to produce a contraction (the threshold
voltage)? What proportion of the fibers in the muscle do you think were contracting to
produce this small response?
2. What was the smallest voltage required to produce the maximum (largest) contraction?
What proportion of the fibers in the muscle do you think were contracting to produce
this maximal response?
3. What do you conclude happened to the number of fibers contracting as the voltage was
raised from threshold to that required to produce a maximal contraction?

4. In light of the all or none law of muscle contraction, how can you explain the graded
response?


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Exercise 2: Effect of stretch on contraction force

Describe how the isolated muscle behaved as it was stretched progressively. ________
________________________________________________________________________________________________

1. What effect does stretching the muscle have on contraction strength? Is this effect
linear.

2. What stretch resulted in the highest contraction force? What happens to the muscle at
the highest stretch levels?

Exercise 3: Effect of stimulus frequency on contraction force

Describe how the isolated muscle behaved as the stimulus interval was decreased
progressively ________________________________________________________________________________

1. What effect does varying the stimulation frequency have on contraction force? Which
stimulus interval caused the greatest contraction force?
Exercise 4: Tetanus

Describe how the isolated muscle behaved as the stimulus interval was further decreased.
________________________________________________________________________________

1. Define tetanus. At which stimulus interval did you observe tetanus? Explain the
mechanism behind this phenomenon.
Exercise 5: Muscle fatigue

Describe how the isolated muscle behaved with continued high frequency stimulation.
________________________________________________________________________________

1. At what time point did your muscle begin to fatigue? Comment on the percent decrease
in contraction force by the end of the experiment..

2. Provide a possible mechanism for why the muscle was unable to maintain a prolonged
contraction.


3. Would your results have differed if you were measuring from smooth muscle tissue?
Why?