Chapter 5: Practicum Guide: Frog Wet Lab & PhysioEx Dry Lab

The muscle practicum has three parts:

1. Wet laboratory: recording contractions of a real frog gastrocnemius on a kymograph.
2. Dry laboratory: the same experiments, plus a few that are hard to do on tissue, in the PhysioEx 9.1 simulation.
3. Body reflexes: examining reflexes on each other (Chapter 4).

This chapter walks through the first two step by step and explains what each result means, so that you know what to expect at the bench and can explain it in your report and at the responsi.

Wet lab: the frog nerve–muscle preparation

Aims

By the end of the wet lab you should be able to:

• explain the mechanism of skeletal muscle contraction, the mechanical events of a contraction, and the mechanism of muscle fatigue;
• explain the factors that affect how skeletal muscle works and contracts;
• prepare and carry out a neuromuscular experiment using a frog (Rana sp.) preparation.

Background

• Muscle is an excitable tissue: it receives and responds to stimuli by contracting.
• A single fiber follows the all-or-none principle: it contracts fully once the stimulus reaches threshold, and not at all below it. The whole muscle, however, can grade its force by recruitment and summation (Chapter 3).
• At the molecular level, contraction is the coordinated interaction of actin, tropomyosin, troponin and myosin, which shortens the fiber (Chapter 2).

Why a frog?

• It is poikilothermic (cold-blooded), so the tissue does not need to be kept at body temperature during the experiment.
• Its gastrocnemius is relatively large and has a long tendon, which makes dissection, stimulation and observation easy.
• It is easy to obtain and suitable for studying neuromuscular responses experimentally.

Materials and equipment

The kymograph

A kymograph ("wave writer") records movement against time. Its parts are:

• a rotating drum covered with graph (or smoked) paper, turning at a constant, known speed, so horizontal distance on the paper equals time;
• a muscle clamp on a stand, from which the muscle hangs;
• a writing lever (an arm with a pen) attached to the tendon, pivoted so that the muscle's shortening is magnified and traced on the drum;
• a weight on the lever that provides the load (and the resting stretch);
• an electrical stimulator with electrodes to the nerve or muscle.

Each time the muscle shortens, the lever rises and the pen draws a peak. The height of the peak shows the strength of contraction and its width shows its duration. When the drum turns fast, a single twitch shows its latent, contraction and relaxation phases; when it turns slowly, a series of twitches shows treppe, summation, tetanus and fatigue.

Step 1: pithing the frog

The slide calls this step "anesthesia" (anestesi katak), but what is actually done is pithing: the brain and spinal cord are destroyed so that the frog is dead and feels nothing, and so that no reflexes from the CNS interfere with the recording. Following the slides:

1. Rinse the frog and hold it firmly in the left hand to keep it steady.
2. Insert the scissors into the frog's mouth, from the right corner of the mouth to the left.
3. Tilt the scissors so that one blade lies over the back of the neck, then cut to kill the frog (decapitation, removing the brain; the slides show this as decerebration then decapitation).
4. Check that the hind legs no longer respond before continuing.
5. Insert the needle into the vertebral canal, pointing caudally, and move it back and forth to destroy the spinal cord.
6. Make sure both hind legs are limp. This shows the pithing is complete.

The hind legs may twitch briefly as the needle passes down the cord. That is expected, and flaccid legs afterwards confirm success.

Handling animals. Use the animals provided only for the purpose of the practicum, work quickly and confidently so the procedure is humane, and follow the assistants' instructions and the lab's ethics rules. The PhysioEx dry lab exists partly to reduce the number of animals needed.

Step 2: the nerve–muscle preparation

The goal is an isolated gastrocnemius with its sciatic nerve still attached, hanging from the knee with a thread on the Achilles tendon.

1. Cut the skin around the thigh in a ring.
2. Peel the skin down like a stocking until the Achilles tendon is visible.
3. Free the gastrocnemius from its neighbors and tie a thread around the Achilles tendon, then cut the tendon below the knot.
4. Cut through the leg below the knee (the tibiofibula), taking care not to cut the gastrocnemius itself.
5. Cut the tissue above the knee, but keep the nerve intact.
6. Find the sciatic nerve (n. ischiadicus) in the back of the thigh, tie a thread around it and cut it at its proximal end, so that a length of nerve comes away with the muscle.

Throughout, keep the preparation moist with Ringer lactate. Handle the nerve only by its thread; pinching or stretching it with forceps damages it and the muscle will not respond to nerve stimulation.

Step 3: recording the contraction

1. Mount the preparation on the kymograph: fix the knee end in the clamp and tie the Achilles thread to the writing lever.
2. Stimulate it electrically, through the nerve or directly on the muscle.
3. Observe and label the tracing that forms on the drum.

Typical protocol and what you should see:

Common problems: a dry preparation (no response, keep it moist); a crushed or overstretched nerve (responds to direct muscle stimulation but not to nerve stimulation); a lever that is too heavy or rubs on the paper (flattened, distorted tracings); and a slack thread (the latent period looks too long).

Dry lab: PhysioEx Exercise 2

PhysioEx 9.1 is a laboratory simulation published by Pearson. In Exercise 2, Skeletal Muscle Physiology, a simulated muscle hangs in a holder attached to a force transducer. You control the stimulus voltage, the muscle length and, in later activities, the stimulus frequency and the load. The screen shows the force trace (an oscilloscope display) and the active, passive and total force, and Record Data saves each run in a table for your report.

Aims

• Explain the mechanism of skeletal muscle contraction.
• Carry out a practicum using the dry lab method.
• Explain the effects of stimulus strength, number of motor units and frequency on contraction.
• Explain the effects of muscle length and load on contraction.

Topics

1. Muscle tension and the latent period
2. The effect of stimulus voltage on contraction
3. The effect of stimulus frequency on contraction
4. Tetanus
5. Fatigue
6. Muscle length and force (length–tension)
7. Isotonic contraction and velocity (load–velocity)

The numbers below come from the practicum slides. Your version of the simulation may differ slightly, so always record what you actually measure.

Activity 1: the muscle twitch and the latent period

Background. In the body, a motor neuron's action potential releases ACh at the neuromuscular junction; ACh diffuses to the sarcolemma, binds receptors at the motor end plate, changes ion permeability, depolarizes the membrane, and the muscle contracts. In this experiment, electrical voltage replaces ACh as the trigger. The latent period is the time between the action potential and the start of contraction. Nothing visible happens yet, but Ca²⁺ is being released from the SR. Once Ca²⁺ is out, the contraction phase begins and lasts until tension peaks. The relaxation phase then lasts until the muscle returns to its resting state, as Ca²⁺ is pumped back into the SR.

Procedure:

1. Open PhysioEx.
2. Set the voltage to 0 V, click Stimulate, then Record Data.
3. Raise the voltage to 4 V, stimulate and record.
4. Click Measure and drag the yellow line to the point where force starts to rise, to read the latent period.
5. Increase the voltage in 2 V steps up to the maximum, stimulating and recording each time.
6. Print the results and compare them.

Expected result: no response at 0 V. Above threshold, force increases with voltage, but the latent period stays roughly the same (a few milliseconds), because it reflects the time needed for excitation–contraction coupling, not the strength of the stimulus.

Activity 2: the effect of stimulus voltage

Background. Electrical stimulation can trigger skeletal muscle contraction. A motor unit is one motor neuron and the fibers it controls, and the more motor units are active (motor unit recruitment), the greater the force. The threshold is the smallest stimulus that produces a response. As the stimulus increases, more fibers are activated and force increases, until maximal tension is reached when every fiber that can be activated has been recruited. After that, increasing the stimulus does not increase force.

Procedure:

1. Open PhysioEx.
2. Set 0 V, click Stimulate, then Record Data.
3. Raise the voltage to 0.2 V, stimulate and record.
4. Keep raising the voltage in 0.1 V steps, stimulating and recording, until a force trace appears. This is the threshold.
5. Click Clear Tracings.
6. Keep raising the voltage up to the maximum.

Expected result: force stays at zero until the threshold (about 0.8 V in the standard simulation), then rises steeply and levels off at the maximal stimulus (about 8.5 V). Going to 10 V gives no further increase. This is why 8.5 V is used in all later activities: it guarantees that every fiber is recruited, so any further change in force must come from frequency, length or load.

In a single fiber, the all-or-none law holds. The graded response here is possible only because the simulated whole muscle contains fibers with different thresholds.

Activity 3: the effect of stimulus frequency

Background. Treppe is a stepwise increase in force when the muscle is stimulated repeatedly at short intervals, each after complete relaxation. Summation occurs when the next stimulus comes before the muscle has finished relaxing, so the new contraction builds on the previous one. The higher the frequency, the greater the tension. If stimuli come so fast that the muscle cannot relax at all, the contraction becomes a tetanus.

Procedure:

1. Open PhysioEx and set the voltage to 8.5 V. Click Single Stimulus and Record Data.
2. Click Single Stimulus again as soon as the first twitch has returned to baseline, and record.
3. Click Clear Tracings. Give a single stimulus, then a second one before the trace has returned to baseline, and record.
4. Click Single Stimulus several times in quick succession and record.
5. Click Clear Tracings, raise the voltage to 10 V, give a single stimulus and record.
6. Click Clear Tracings, return the voltage to 8.5 V and give a final single stimulus.

Expected result:

• Stimuli after full relaxation give separate twitches of equal height (the simulation usually does not reproduce treppe).
• A second stimulus during relaxation gives a higher second peak: wave summation.
• Rapid repeated stimuli give even more summation.
• Raising the voltage to 10 V gives a single twitch no stronger than at 8.5 V, because every fiber is already recruited. The lesson: once all motor units are recruited, frequency, not voltage, increases force.

Activity 4: tetanus

Background. Increasing the stimulus frequency increases the force of skeletal muscle. Summation waves come in two forms:

• Unfused (incomplete) tetanus: stimuli come before relaxation is complete, but there is still partial relaxation between them. The record is a wavy plateau.
• Fused (complete) tetanus: stimuli come so fast that the peaks and valleys merge into a straight line.
• Maximal tetanic tension: the frequency beyond which force no longer increases.

The practicum slide on tetanus swaps the definitions of fused and unfused; the pairing above is the correct one (see Chapter 3).

Procedure:

1. Open PhysioEx.
2. Set the voltage to 8.5 V and the stimuli per second to 50. Click Multiple Stimulus, then Record Data.
3. Increase to 130 stimuli/s, click Multiple Stimulus and record.
4. Click Clear Tracings.
5. Increase to 140 stimuli/s, stimulate and record.
6. Increase by 2 stimuli/s at a time from 140 to 150, stimulating and recording each time.

Expected result: 50 stimuli/s gives unfused tetanus (a wavy plateau). By 130 stimuli/s the trace is almost smooth, fused tetanus. Between 140 and 150 stimuli/s, force rises only slightly and then stops rising: maximal tetanic tension (graph b above). This is several times the force of a single twitch.

Activity 5: fatigue

Background. Complete (fused) tetanus is followed by fatigue: a fall in muscle force, or a failure to produce contractions, after prolonged or repeated activity. In the practicum it is explained by the accumulation of lactic acid, ADP and Pi after high-intensity activity; Chapter 3 discusses the other mechanisms.

Procedure:

1. Open PhysioEx.
2. Set the voltage to 8.5 V and the stimuli per second to 120, then click Multiple Stimulus.
3. Let the tetanic contraction continue while force declines. Click Stop Stimulus once the trace has fallen (the slides say to wait until the force reaches 0), then Record Data.

Expected result: force rises to a tetanic plateau, then falls steadily despite continued stimulation. The full PhysioEx exercise also lets you rest the muscle for different intervals before restimulating: the longer the rest, the more force recovers, because the muscle has time to restore ATP and creatine phosphate, remove Pi and H⁺, and reload the SR with Ca²⁺.

Activity 6: muscle length and force

Background. Skeletal muscle contractions are isotonic or isometric. Here the muscle contracts isometrically, at a fixed length, and the factor varied is the length of the muscle before stimulation.

• Passive force comes from stretching the muscle, the elastic recoil of the tissue, caused mainly by the protein titin. The more the muscle is stretched, the greater the passive force.
• Active force comes from crossbridge cycling, powered by ATP. The more the muscle is stretched beyond its optimal length, the lower the active force.
• Total force = active force + passive force.

Procedure:

1. Open PhysioEx.
2. Set the voltage to 8.5 V and the muscle length to 75 mm, click Stimulate, then Record Data.
3. Shorten the muscle in 5 mm steps down to 50 mm, stimulating and recording at each length. (The slide says "55 mm"; it should be 5 mm steps.)
4. Click Clear Tracings.
5. Lengthen the muscle in 5 mm steps from 75 mm up to 100 mm, stimulating and recording each time.
6. Plot active, passive and total force against length.

Expected result: the graph reproduces the length–tension curve in Chapter 3. Active force is highest near 75 mm (the simulated muscle's optimal length) and falls at both shorter and longer lengths. Passive force is zero at 75 mm and below, and rises steeply as the muscle is stretched beyond it. Total force dips beyond the optimum, then climbs again because of passive force.

Activity 7: isotonic contraction and load

Background. An isotonic contraction changes muscle length. When lifting a load, the muscle first contracts isometrically, developing tension until it matches the load; only then does it shorten and move the weight. In the simulation, the latent period is measured as the time during which the muscle is already contracting but the load has not yet moved. The heavier the load, the more time is needed, and the lower the velocity of contraction. (The practicum slide heads this step "6. Muscle length and force" again; it is really the seventh activity.)

Procedure:

1. Open PhysioEx.
2. Set the voltage to 8.5 V and attach a 0.5 g weight. Click Stimulate.
3. Watch the muscle shorten, then Record Data.
4. Replace the weight with 1.0 g, 1.5 g and 2.0 g in turn, stimulating and recording each time.
5. Compare the results: latent period, distance shortened and shortening velocity.

Expected result: as the load increases, the latent period gets longer, the muscle shortens more slowly and over a shorter distance. If the load exceeds the maximum force the muscle can develop, it cannot lift it at all: velocity falls to zero and the contraction is isometric (graph c above). This is the load–velocity relationship: you can lift a pencil quickly, but a heavy suitcase only slowly.

Writing the practicum report

The Modules section has a place for practicum reports under "Practicum → Reports". A clear report usually has:

1. Title, date and group.
2. Aims: copy them from the practicum guide.
3. Background theory: a short summary of Chapters 1–3 or 4, in your own words.
4. Materials and methods: what you used and did, including any changes from the guide.
5. Results: tables of recorded data, labeled tracings or graphs with axes and units, and your measured values (threshold voltage, maximal voltage, latent periods, maximal tetanic tension, reflex grades and reaction times).
6. Discussion: explain each result with its mechanism, compare it with the expected result, and give reasons for any difference (for example tissue damage, fatigue from repeated stimulation, or timing errors).
7. Conclusion: one or two sentences per aim.
8. References.

Self-check before the pretest

1. Why is the frog pithed before the experiment? To kill it humanely and to destroy the brain and spinal cord so that reflexes cannot interfere with the recording.
2. What replaces acetylcholine in these experiments? The electrical stimulus, which depolarizes the membrane to threshold directly.
3. Why does force stop increasing above about 8.5 V? All fibers (motor units) are already recruited: the maximal stimulus.
4. How can force increase further once all units are recruited? By increasing stimulus frequency: summation and tetanus.
5. What is the difference between unfused and fused tetanus? Unfused: partial relaxation between stimuli, wavy trace. Fused: no relaxation, smooth trace at maximal tension.
6. What produces passive force? Elastic recoil of stretched titin and connective tissue.
7. Why does a heavier load lengthen the latent period? The muscle must first develop tension equal to the load (isometrically) before it can shorten.
8. What causes fatigue? Central factors plus peripheral ones: reduced ACh release, impaired E–C coupling, reduced Ca²⁺ release or sensitivity, depletion of creatine phosphate and glycogen, and accumulation of Pi and H⁺.

Key points

• Wet lab: pith the frog, prepare the gastrocnemius with its sciatic nerve, keep it moist with Ringer lactate, mount it on a kymograph, stimulate it and label the tracing.
• The kymograph turns time into horizontal distance and shortening into vertical deflection.
• PhysioEx activities show: a constant latent period; threshold then maximal stimulus with voltage; summation and tetanus with frequency; fatigue with sustained tetanus; the length–tension curve; and slower shortening with heavier loads.
• Always record what you measured, and explain any difference from the expected pattern with physiology.