Justice Ross
Physiology Lab
Dr. Xu
2 March 2016
Frog Skeletal Muscle Lab
Introduction
A biologist by the name Jan Swammerdam performed the first experiments on muscle physiology between 1661 and 1665. Swammerdam demonstrated that an isolated frog muscle can contract if the sciatic nerve is irritated with a metal object. Almost a century later, a physician by the name Luigi Galvani demonstrated that frog muscle responds to electrical currents. In the late 1840s, either Carlo Matteucci or Carl Ludwig attributed to the invention of the kymograph, which is illustrated as a rotating drum powered by a clockwork motor. The invention of the kymograph changed experimental physiology by allowing muscle contractions, and other actions, to be recorded and analyzed for the first time ever. Muscle cells, or muscle fibers, are defined as the basic unit of a muscle. Therefore, complete muscles are constructed from bundles of muscle fibers, but there are no gap junctions between adjacent cells so each fiber performs independently. Myofibrils establish an ordered structure for single muscle fibers. Every myofibril is comprised of actin and myosin. Actin and myosin are contractile proteins that can slide past each other when Ca2+ and ATP are present. A motor unit is described as a single motor neuron and all of its innervating muscle fibers. Like nerve tissue, muscle fibers produce an all-or-none response to a stimulus, generating a twitch. A single motor neuron supplies numerous muscle fibers to make up a motor unit. Motor units can range from a few muscle fibers innervated by a single neuron to thousands innervated. Although most muscles consist of a variety of different motor unit sizes, smaller motor units have finer control of movement. Since the muscles that control movement in the fingers and eyes have smaller motor units, muscles controlling larger limb movements have larger motor units. More fibers can be activated depending on the intensity and frequency of stimulation, however, higher recruitment results in an increase in the strength of a muscle contraction. Therefore, the strength of a muscle contraction can either be increased by increasing the number of active motor units, or by stimulating existing active motor units more frequently. The total force that a muscle can produce depends on the total amount of muscle fibers. For example, muscles with larger cross-sectional areas produce larger forces, and muscles with smaller cross-sectional areas produce smaller forces. The neurotransmitter acetylcholine is released from the motor end plates by motor nerves. When the acetylcholine is released into the junctional cleft it binds to receptors on the muscle membrane. These receptors are directly coupled to cation-selective channels. When these cation-selective channels open, the muscle fiber goes through depolarization and intracellular calcium is released from the sarcoplasmic reticulum, which is a modification of smooth endoplasmic reticulum. The biochemical actions that trigger a contraction are prepared to begin when the cytosolic calcium is increased. In this same area acetylcholine esterase hydrolyzes the acetylcholine on the membrane. Because this is occurring, the acetylcholine does not gather in the junctional cleft. This shows that skeletal muscles can undergo isometric or isotonic conditions.
The muscle twitch is known as the mechanical response of the muscle, and they last considerably longer than action potentials. For instance, in skeletal muscles, the action potentials only last a few milliseconds. In a process known as summation, if a second stimulus arrives before the muscle can relax again a second twitch will occur on top of the first creating greater peak tension. Tetanus can be seen with increasing frequency of stimulation. When the frequency of stimulation increases, the time for the muscle fiber to relax between stimuli is reduced. In time, the contractions will fuse into a smooth powerful contraction known as tetanus. The degree of stretch also influences the strength of muscle contraction so it is important to separate the passive and active forces. The passive forces redirect the contributions of variable components, and the active force is generated by the contractile machinery when the fibers are stimulated. The total force during nerve stimulation and contraction and the passive force in the absence of verve stimulation is measured at different degrees of stretch. The active force is the difference between the two at any muscle length. Metabolic energy is required for a muscle contraction. Results are stored in fatigue when there is a reduction of energy. The muscle fibers that are more resistant to fatigue have a greater capacity for oxidative metabolism. Instead of a result of reduced energy, fatigue mainly occurs because the motor drive in the brain is reduced.
***ADD HYPOTHESES***
Materials and Methods During lab, we will investigate the physiological properties of skeletal muscle using the isolated frog gastrocnemius. Concepts we will explore include the single twitch, graded response and the relationship between muscle length and tension generated. We will also explore tetanus and muscle fatigue.
Five exercises: 1. The graded response→ 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→ increase the stretch of the muscle by raising a micro-positioner. 3. Effect of pulse frequency on contraction force→ stimulate the muscle with twin pulses at different pulse intervals and observe their effect on muscle contractions. 4. Tetanus→ examine the muscle’s response to a continuous stimulus at different frequencies. 5. Muscle fatigue→ stimulate the muscle constantly for a long duration to examine the effects of muscle fatigue.
15 steps from LabTutor: 1. Introduction→ a. We will investigate the physiological properties of skeletal muscle using the isolated frog gastrocnemius. Concepts we will explore include the single twitch, graded response and the relationship between muscle length and tension generated. We will also explore tetanus and muscle fatigue b. By the end of lab we should be able to describe the relationship between the intensity of muscle stimulation and contractile force, and describe and explain the effects of muscle stretch, summation, tetanus and fatigue on the strength of contraction 2. Dissection→ c. Using a scalpel or sharp scissors, cut the skin of the frog around its abdomen d. Peel the skin down and off the legs of the frog 3. Equipment Setup→ e. PowerLab i. Connect the bridge pod to input 1 of the PowerLab ii. Connect the positive and negative BNC connectors of the stimulating electrode leads to the analog Outputs on the PowerLab 4. Force Transducer Setup→ f. Force transducer setup iii. Raw output from the force transducer is in millivolts. It needs to be calibrated to give the more meaningful units of Newtons (N). Force transducers also often have some residual offset voltage that needs to be corrected for. g. Zeroing Procedure iv. Leave the force transducer undisturbed and click start. v. The baseline value will be displayed in the LabTutor panel. Use the knob on the front of the Bridge Pod to adjust the baseline value to zero, then click stop. h. Calibrating Procedure vi. Once the force transducer has been zeroed, click start and record for about 5 seconds, then click stop. vii. Now hang a known weight (between 5 and 50 grams) from the force transducer and record for another 5 seconds, then click stop viii. Enter weight in the Unit Conversion table and LabTutor will calculate the N value ix. Use the Waveform Cursor to select a stable section of the record before you added the weight, then click the arrow next to ‘Point 1’ in the Calibration Panel and, if it is not already entered, type ‘0’ in the right-hand box x. Now select a stable section of the record after adding the weight, click the arrow next to ‘Point 2’ and enter the calculated N value in the right-hand box xi. Click the ‘Apply’ button to implement the calibration 5. Exercise 1: Twitch Recruitment→ 6. Analysis: Twitch Recruitment→ 7. Exercise 2: Effects of Stretch→ 8. Analysis: Effects of Stretch→ 9. Exercise 3: Summation→ 10. Analysis: Summation→ 11. Exercise 4: Tetanus→ 12. Analysis: Tetanus→ 13. Exercise 5: Fatigue→ 14. Analysis: Fatigue→ 15. Questions/Results
Results and Discussion

Questions Conclusions