INTRODUCTION:
The skeletal muscle is an important organ in the human body that allows for movement and support of the skeleton, helping the body execute various tasks from precise control movements (writing a letter or picking up a chalk) to more abrupt, powerful movements (lifting weights or running). The skeletal muscle is one of the main types of muscle in the body (the other two being cardiac and smooth muscles) that allows movement by contraction of the muscles. When a motor unit, a single nerve that innervates all the muscle fibers, activates these muscles fiber, it causes a muscle contraction. These muscle contraction occurs when motor neurons from the spinal cord leaves and activates multiple muscle fibers, the number of muscle fibers depend on the type of muscle. According to the Hennemen size principle, muscles that react rapidly and exert fine control have smaller motor units than those with larger, weight-involving muscles, whose movements are less precise, will require larger motor units.
The means of movement and force generation is based on the skeletal muscles contraction. Contractile units of the skeletal muscles include thick myosin and thin actin myofillaments or fillaments. Sarcomere is the smallest contractile unit of a muscle fiber. The repetition of the sarcomere structure make up myofibrils and hundreds to thousands of these myofibrils make up a single muscle fiber (Sherwood 258). Contraction is initiated by an increased in the intracellular calcium concentration which triggers an action potential in the plasma membrane by the depolarization of voltage gated calcium channels. When the muscles contract, the skeletal muscle fibers actively shortens, the thin filaments are propelled toward the center of their sarcomere by movements of the myosin cross-bridges that bind to actin (Sherwood 271).
In the first part of the experiment, we examined and analyzed the effects of stimulus intensity and stimulus frequency on contraction of frog’s gastrocnemius. Contraction of the gastrocnemius was induced by applying electrical stimulus in the sciatic nerve of the frog to elicit muscle activity. The muscle twitches observed by the frog should have been a graded responses (an increase in stimulus voltage should cause an increased in muscle tension). To get a plot of stimulus intensity, the threshold voltage (lowest voltage that produces an observable twitch) and the maximum stimulus voltage (lowest stimulus voltage that produce maximum twitch tension) were recorded to calculate the ∆V as a measurement of the graded responses (Bautista 13). When testing stimulus frequency on the gastrocnemius of the frog, there was an increase in muscle tension with increasing stimulus frequency. This effect was due to the occurrence of temporal summation. Temporal summation is the phenomenon when the second contraction occurs before the muscle has completely relaxed, which causes a fusion of wave (summation) at a high enough frequency. However, at lower frequency, the waves would not be fused.
In the third part of the experiment, tubocarare was injected into the gastrocnemius of the frog decreasing tension when stimulating the sciatic nerve. Tubocurare is a competitive inhibitor of the Nicotinic Acetylcholine receptor since it competes with acetylcholine for receptor binding sites. Tubocurare is also a paralysis and the injection of tubocurare to the frog’s gastrocnemius will decrease muscle contraction The paralysis will only be temporarily because the acetylcholine receptor still has influx of calcium ions coming from the skeletal muscle. Lastly, we will be performing direct stimulation to the frog gastrocnemius to see whether neuronal input is absolutely needed to elicit muscle activity, and comparing direct and indirect stimulation to check which would give the most muscle contraction.
MATERIALS & METHODS:
Specific details of laboratory procedures can be found in the NPB101L Physiology Lab Manual. (Bautista, et al., 2009)
Transducer Calibration
Transducer was calibrated from grams to voltage by setting up the channels and changing the parameters on the computer.
Gastrocnemius and Sciatic Nerve Isolation
A double-pithed frog was acquired from the TA. Using the tools available for set-up, the gastrocnemius was isolated and the lower tendon from the heel was cut to position the muscles on to the transducer. By dissecting the seam between the lateral side of thigh between the quadriceps and hamstring muscles, the sciatic nerve was isolated and position on the electrode for measurement.
Effect of Stimulus Intensity of Muscle Activity
To begin the experiment, the transducers should have muscle tension value approximately around 20 grams. This value (baseline tension) is used as references to measure the force of increasing stimulus intensity by electrical impulse to the sciatic nerve. The stimulus voltage was gradually increased to check for the first observable twitch, which would be the threshold voltage. Then, stimulus voltage was steadily increased to find the lowest stimulus voltage that produce maximum twitch tension, which would be the maximum voltage. To calculate for the change in voltage, we simply follow the equation from NPB101 Lab Manual: ∆V = maximum voltage – threshold voltage/5

Six different twitches should have been recorded (one representing the threshold voltage, one for the maximum voltage, and the other four are in between these two values). These points were plot out to analyze the relationship between muscle tension and stimulus intensity.
Effect of Stimulus Frequency of Muscle Activity
In this experiment, the frog’s gastrocnemius was stimulated at different frequency, testing for muscle activity responses and observable twitches. Seven data sets were collected at fifteen seconds interval by varying stimulus frequency (0.5pps, 1pps, 2pps, 4pps, 8pps, 15pps, and 25pps). Event markers were made for each frequency and evaluated at each particular frequency values.
Effect of Tubocurare on Muscle Activity
Before the administration of the competitive antagonist of acetylcholine receptor, a control was required to compare the after effect of the tubocarare injection. The stimulator mode was switched to repeat for 60 seconds and was evaluated. After the control period, needle injection of 0.25ml of tubocurare was inserted to the gastrocnemius. Event markers for the start and end of injection were made to measure the effect of tubocarare on muscle activity.
Effect of Direct Electrical Stimulation on Muscle Activity
Direct stimulation was applied to the gastrocnemius of the frog after the tubocurare injection to observe if muscle activity is still operative. Two needle electrodes were hooked onto the ends of the gastrocnemius muscle and connected to the stimulator. Voltage was steadily increased until twitching of the muscle occurred. Voltage was then increased to 10X the maximum voltage to check if twitching would occur at ten times the maximum voltage. Data was recorded using the “maximum” function to find the muscle tension.
RESULTS:
Transducer Calibration
The calibration for the transducer when first set up gave a value of 0.49mV for CAL1 and 2.36mV for CAL2.
Effect of Stimulus Intensity on Muscle Activity
In order to examine how electrical stimulus would trigger muscle responses in the gastrocnemius, values for the threshold and maximum voltage was recorded. The muscle responses in the frog’s gastrocnemius gave a threshold voltage (Vthreshold) value of 1.60 Volts and a maximum voltage (Vmaximum) value of 2.85Volts. Using this information, the change in voltage (∆V) was calculated to be 0.25V. By taking a set of six data points, starting from Vthreshold and stopping at Vmaximum, a graph was plotted out to find the relationship between stimulus intensity vs. muscle activity. Each data point was measured by using the “maximum” value to figure out the muscle tension at that particular ∆V point. As seen in Fig. 1, the correlation between stimulus intensity and muscle activity responses seems to increase linearly.
Table 1: Maximum and Threshold Voltages and Corresponding Tensions. Variables | Voltage (V) | Tension (g) | Vmax | 1.6 | 18.3 | Vthreshold | 2.85 | 109.8 | ∆V | 0.25 | N/A | | | |
V = Volts; g = gram
Raw data (1) of increasing stimulus intensity on the frog’s gastrocnemius.
Figure 1. Graded response of muscle tension by gastrocnemius of the frog to increasing stimulus intensity. Horizontal data sets represent stimulus intensity increased each time by ∆V (2.65V). Graph shows a linear trend between stimulus intensity and muscle tension at successive step.
Effect of Stimulus Frequency on Muscle Activity
Next, we examine the effect of stimulus frequency on muscle activity by taking seven different stimulus frequencies (.5pps, 1pps, 2pps, 4pps, 8pps, 15pps, and 25pps). Each cluster of peak represents muscle tension at different stimulus frequencies. In Figure 2, the relationship between muscle responses and stimulus frequency decreases with each increasing frequency stimulus, which then plateau and remain fairly constant after 2pps. The degree of summation starts to become greater after 4pps since the waves starts to fuse into a sustained contraction plateau. Maximum muscle responses occurs at 0.5pps giving a value of 81.7 grams and minimum muscle tension occurs at 4pps giving a value of 38.45grams.

Raw data (2) Effect of increasing stimulus frequency on muscle activity.

Figure 2. Muscle responses to increase in stimulus frequency. The graph shows higher muscle contraction at lower frequency (0.5pps and 1pps), but did not show summation at these frequency. The maximum twitch tension stayed relatively constant after 2pps and showed summation after 4pps.
Effect of Tubocurare on Muscle Activity
To examine the effect of Tubocurare effect on muscle activity, a control was made from 73 seconds to 110 seconds before the intramuscular injection was inserted to the gastrocnemius of the frog. Once the tubocurare injection was started around 120 seconds, recording was made after the injection had ended at around 151 seconds. At the 151 seconds mark, this point is essentially time zero for the tubocurare trials. For the two minute interval, a 30seconds window was highlighted and added on to the 151 second mark. This means 30 second was added to the 151 mark ending at 181 seconds. Time calculations were done in similar manner at 30 second intervals all the way to the two minute mark. In Figure 3, plot was made to compare the control versus the tubocurare’s injection after three minutes. After the injection of tubocurare to the muscle, muscle tension starts to decline as seen in Figure 4.

Raw Data (3) showing a control period, start of Tubocurare injection, and end of injection

Raw Data (4) showing muscle tension (post-injection) from the intervals of 151-181 seconds

Raw Data (4) showing muscle tension (post-injection) from the intervals of 181-211 seconds

Raw Data (4) showing muscle tension (post-injection) from the intervals of 211-241seconds

Figure 3. Comparison of Muscle Responses before and after Tubocurare injection. The graph showed tubocurare decreasing muscle tension even when stimulated with electrical impulse to the sciatic nerve.

Figure 4. Muscle Responses to Injection of Tubocurare on Frog’s Gastrocnemius. Each set of data points on the horizontal axis represents 30 second windows within at two minute intervals. Twitch tension within these windows were averaged and plotted as a function of time.

Effect of Direct Electrical Stimulation on Muscle Activity
Finally, we examined the effect of direct electrical stimulation on muscle activity by taking two different trials: direct stimulation before and after tubocurare injection and direct stimulation after tubocurare injection. At threshold voltage before tubocurare injection, the muscle tension gave a voltage threshold of 17.4 grams compared to after injection which gave a voltage threshold of 17.8 grams. For 10XMaximum voltage, the pre-injection gave a V10Xmax of 97.1 grams and post injection gave a V10X max of 20.3 grams. Looking at threshold voltage, there is little discrepancy between control and post direct stimulation (Figure 6). However, looking at the V10Xmaximum control and post direct stimulation showed there was a big difference in muscle tension values. Raw Data (5) showing muscle tension at maximum voltage and when increased to 10X maximum voltage for direct stimulation of the gastrocnemius before tubocurare injection.

Max voltage, 3:08:32 PM
10x max voltage, 3:10:30 PM
60.00
120.00 seconds 0.00
10.00
20.00
30.00
grams
Force
Raw Data (6) graph showing muscle tension at threshold and when increased to 10X maximum voltage for direct stimulation of the gastrocnemius after tubocurare injection

Figure 5. Muscle responses to direct stimulation of gastrocnemius before and after Tubocurare injection. Data sets show little differences between control (17.8 g) and post tubocurare injection (17.4 g) in Vthreshold. However, there was a major difference in muscle contraction when voltage was increased to V10Xmax between control (20.3 g) and post-direct stimulation (97.1 g).

DISCUSSION: Skeletal muscle composed about 40 percent of the human body weight, both in males and females, and it is one of the most important tissues that allows for movement and protection (Sherwood 257). Without a doubt, they are very important for everyday movements such as walking, running, biking, etc., and are absolutely necessary for animal survival via fight or flight response. For example, without muscle contraction, in face of a predator, there is hardly any way for the prey to escape from danger. As a result, understanding the structure and mechanism of how skeletal muscles contract, how its contraction is affected by various stimulation factors such as intensity and frequency, and how contraction can be affected by chemical agents is essential.
A skeletal muscle contracts with varying force and for different periods of time in response to stimuli of varying frequencies and intensities. In the first and second part of the experiment, varying stimulus intensities and frequencies were generated causing muscle contraction in the gastrocnemius of the frog. As seen in Figure 1, muscle tension increases along with increasing stimulus intensity, showing a constant rate of change from Vthreshold to Vmaximum. However, even if we try to increase stimulus intensity beyond Vmax, muscle tension does not increase but stays constant. These results is what we expected from our data because when the motor neuron fires (transmit an electrical impulse), all the muscle fiber contracts. Even if increased beyond Vmaximum, the muscle tension would not increase because the numbers of muscle fibers are already stimulated by the motor units. More forceful muscle contraction requires more muscle fibers which then require more motor units (Sherwood 270). This implies manipulating the number of motor units recruited is possible and it is. According to the Henneman Size Principle, smaller, slower motor units would be recruited first and then larger, faster motor units (McCarthy and Haradeem, 2006). Since this an in vivo experiment, it does not recruit in the same manner as the Henneman Size Principle when the muscle is directly stimulated, because there is less resistance to voltage in the axons of larger motor units and so larger motor units can conduct action potentials faster than smaller motor units (McCarthy and Haradeem, 2006). In other words, even though motor neurons and muscles usually respond in all or none fashion, the twitches in Figure 1 occur by a graded muscle responses. Despite the fact this experiment shows the reversal of the Henneman Size Principle, McCarthy and Haradeem does not agree with this. They believe that motor unit recruitment is not selective and that there is no particular order in which different types of fibers is recruited. They proposed works of other researchers as evidence (McCarthy and Haradeem). Differing views on the same principle indicates more experiments and research will be needed to know which side is more plausible.
Stimulus frequency can also affect the magnitude of muscle contraction and we expect summation to occur once a high enough frequency is reached. Our data was not what was expected since the magnitude of muscle tension starting off at 0.5 pps and 1.0 pps was much greater than those at higher frequency. However, summation did occur after 4pps (Raw Data 2). The force of muscle contraction did not increased with increased in stimulus frequency as seen in Figure 2, but decreased down to 38.5 grams at 4pps, then starts to steadily increase again after each successive frequency.
One of the possibilities as to why the muscle tension magnitude decreased was due to prolonged and strong contraction of the frog’s muscles that leads to the well known state of muscle fatigue. Studies in paraplegic have shown that muscle fatigue increases in almost direct proportion to the rate of depletion of muscle glycogen. In normal skeletal muscle fibers, action potentials trigger force production by initiating a complex sequence of events (Giat et. al 2006). However, this mechanism of recruitment of muscle fibers is more likely compliant to fatigue because the fibers are continually contracting at not only at higher frequency but also maximum stimulus intensity (Vmax). Therefore, fatigue results mainly from inability of the contractile and metabolic processes of the muscle fibers to continue supplying the same work output.
Twitch summation happens when there is an increase in the firing rate of motor neurons, causing greater muscular force contraction to occur (Sherwood 271). For example, if two identical stimuli (electrical shocks or nerve impulses) are delivered to a muscle in rapid succession, the second twitch will be stronger than the first. Once the second contraction occurs, the muscle has not had the time completely relaxed and the muscle would be partially contracted. When the next stimulus arrives and more calcium is being released to replace that being reclaimed by the sarcoplasmic reticulum (SR), muscle tension produced during the second contraction causes more shortening than the first. In other words, the contractions are summed. Finally, as the stimulation frequency continues to increase, muscle tension increases until a maximal tension is reached. At this point all evidence of muscle relaxation disappears and the contractions fuse into a smooth, sustained contraction plateau called fused or complete tetanus.

A study performed by Li-Wei Chou, Samuel C. Lee, Therese E. Johnston, and Stuart A. Binder-Macleod on patients with spinal cord injuries discovered that a simultaneous increase in both stimulus intensity and frequency caused stronger contractions in the quadriceps femoris muscle opposed to stimulation of one of these variables. When only stimulus intensity or only stimulus frequency were introduced, they recorded low levels of muscle contraction. When intensity was increased, more muscle units were recruited, resulting in maximum force of contraction. However, when stimulus intensity was increased first, followed by an increase in stimulus frequency, muscle contraction reached their highest levels (Chou et al. 2008). This experiment expanded on our by taking it one step further, which was increasing stimulus intensity and frequency simultaneously to produce a force of greater muscle contraction.
Once tubocurare injection was applied to the gastrocnemius, it showed a steady decreased in muscle tension which was expected and seen in Figure 4. Significant drop was observed during the first time interval recorded and continued to decrease as time went on. This speedy decrease (approximately about three minutes) in muscle tension shows how quickly paralytic agent can kick into effect (Raw data 4). Muscle tension before the tubocurare injection gave a baseline tension of 21.4 and after the tubocurare injection gave muscle tension to 18.6 grams, which is what we expected (Figure 3).Tubocurare is a competitive inhibitor of the nicotinic acetylcholine receptor (nAChR) by competing with acetylcholine for space at receptor binding site. The mechanism that fits them best appears to be a combination of competitive block (or block of shut channels), with a strongly voltage-dependent block of open ion channels by tubocurarine (Colquhoun et al.). Opening of the nicotinic acetylcholine receptor is necessary for the depolarization of the muscle cell (Schlumberge and Sasak). If there was no depolarization of the voltage gated calcium channels, then there would be no influx of calcium that triggers the synaptic vesicles for the release of acetylcholine, which means there would be no muscle contraction.
Lastly, in the final experiment, direct stimulation was done to the gastrocnemius itself as oppose to the sciatic nerve being electrically innervated. It was expected that the muscle tension for Vthreshold and Vmax before and after the injection should be remain the same. However, this expectation was not the case, for there were significant differences in maximum muscle tension for both cases of control and post-injection (Figure 5). Direct stimulation of the muscle should have only cause depolarization along the membrane of the muscle cell. Tubocurare should have affected both muscle contraction of post-injection and pre-injection in the same manner since stimulating the muscle directly bypass the need for the opening the nicotine acetylcholine receptor. Thus there are many possibilities that could lead to this effect. It was mentioned that the baseline changed throughout the experiment and calibration values were fluctuated as well so this could have contributed to the differences observed (Figure 5).

CONCLUSION:

Our skeletal muscles are a marvel. They enable up to perform the remarkable task of moving and getting around. In this lab, the goal was to gain an understanding of how the physiological mechanism of our skeletal muscle works and function. With that concept, the goal was to look and analyze the effect of stimulus intensity and stimulus frequency on muscle contraction. Furthermore, by analyzing at how receptors and neurotransmitter work, paralyzing agents such as Tubocurare could be examine and classified. Together these experiments allowed a better understanding of how properties of skeletal muscle work in the human body.

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