Frogs are keystone species, an essential organism to aquatic ecosystems. They have both terrestrial and aquatic niches as predators and prey and serve as indicator species to assess the response of ecosystems to environmental change. To execute daily locomotion patterns, frogs use skeletal muscles. We wanted to determine the relationship between the strength of the stimulus and the response of the muscle. We also wanted to measure the amplitude of contraction produced in a muscle that is stimulated with repeated pulses delivered at progressively higher frequencies. We hypothesized that increasing stimulus voltage in the gastrocnemius muscle of a frog will result in an increase in stimulation amplitude and that an increase in stimulation frequency at a constant voltage will result in an increase in force generated by the muscle up until a point where it plateaus. We found that our hypotheses were supported and that muscle regulation was via temporal and spatial recruitment. This study is important because it serves as a model for understanding skeletal muscle mechanisms in other organisms including humans.

Frogs are widely distributed terrestrial amphibians that inhabit upland and wetland regions, found on all continents of the world except Antarctica. Many frog species, in both larval and adult stages serve as important prey for larger predators including fish, raccoons, snakes and birds of prey (Chalcraft and Resetarits 2003; Auniola and Kauhala 2001). Additionally frogs serve an important role as indicators of environmental stress (King 2010). A review of complex systems in temporary ponds by Wilbur (1997) makes the argument that frogs have two distinct niches, one terrestrial and one aquatic. Wilbur states “all frogs with free-living larvae change at metamorphosis from aquatic omnivorous tadpoles to amphibious carnivorous adults. The role of such connections among food webs is a fruitful area for both theoretical and empirical research because the foraging of animals across ectones may be an important biological mechanism linking elements of the mosaics of habitats that form landscapes.”

One trait frogs are most known for is locomotion. Frogs typically display two type of locomotion: jumping and swimming. Though frogs are traditionally presented as “jump specialists” most species also swim (Navas et al. 1999). Frogs exhibit these locomotive behaviors for a variety of reasons including escaping predators, often times by a short set of quick and powerful jumps (Carvalho, Gomes and Navas 2007). Frog locomotion is dependent on muscles, particularly skeletal muscles, which are muscles connected to the skeleton (Marsh and Olson 1998). Skeletal muscles are organized beginning with units called sarcomeres. A sarcomere consists of two opposing vertical Z-line discs each with actin filaments attached. A myosin filament floats between each horizontal actin section. Sarcomeres are connected to each other by Z-lines. One mechanism of muscle contraction begins with the sliding of the actin and myosin filaments. Parts of the myosin, known as myosin heads, bind to the free end of the actin, the end not attached to the Z-line, and pull it one way toward the center of the mysosin, in an accordian-like mechanism. The muscle shortens or contracts because the sarcomeres shorten.

The process by which the myosin binds to the actin is called the Cross-Bridge cycle. The binding of the myosin to actin is the trigger for the myosin head to tilt and release an ADP and a P as well as a powerstroke. ATP binds to the myosin head and the myosin releases the actin, in a “softening” effect. The ATP is hydrolyzed and delivers energy to moved the mysosin head back and it is ready for the next powerstroke. The sarcomeres move closer together by many of these powerstrokes occurring one after the other. In the Cross-Bridge cycle myosin is normally prevented from binding to the actin. Another protein called tropomyosin, which is wrapped around the actin, is in the way to block the actin-myosin binding site. Another protein, troponin, is attached to the tropomyosin and when triggered, moves the troponin away to allow the binding to occur. But what triggers the tropomyosin to move the troponin? The simple answer is calcium and this occurs in a process called excitation-contraction coupling. In excitation-contraction coupling an action potential or electrical stimulus, runs down a T-tubule in the muscle fiber. The stimulus reaches a ryanodyne receptor which opens ion channels in the sarcoplasmic reticulum, a storage space for calcium in the muscle fibers. Once the ion channels are opened, calcium runs out into the cell.

For the muscle to relax or return to its original resting position, calcium must be moved back into the sarcoplasmic reticulum by a SERCA pump. Because calcium is being moved against a concentration gradient, this relaxation requires ATP. The SERCA pump lowers calcium levels in the cytosol or cell and when the calcium is taken up again the muscle relaxes.

Since muscles are not contracting all the time muscle contraction must be regulated. Regulating the muscles allows frogs to change aspects of locomotive behavior, such as how far a frog is able to jump. Muscle contraction force can be regulated by calcium in three mechanisms: temporal recruitment, in which the firing rate at which individual motor neurons fire is changed; spatial recruitment, in which the number of active motor units is changed; and the length-tension relationship, in which the sarcomere length is changed to generate tension. This study focuses on the force of muscle contraction via temporal and spatial recruitment.

In temporal recruitment, the frequency of the action potential is changed, usually increased, so that more calcium is released into the muscle cell. More calcium in the cell results in more tension generated. Another mechanism for the regulation of muscle contraction force is motor unit recruitment, also known as spatial recruitment. A motor unit is comprised of muscle fibers and a motor neuron. There are different amounts of fibers per motor unit. In spatial recruitment the number of active motor units is increased to increase the strength of muscle contraction. More motor units means that more muscle fibers can be stimulated. If only half of the muscle fibers are stimulated, only half the amount of force will be generated. If all of the muscle fibers are stimulated, the maximum amount of force will be generated.

We hypothesized that if we increase voltage of an electrical stimulus in a frog muscle we will see an increase in stimulation amplitude and if we increase stimulation frequency at a constant voltage, we will see an increase in force generated by the muscle up until a point where it plateaus.

Materials & Methods:

We used the gastrocnemius muscle of a frog in two experiments. In the first experiment we used a single stimulus, changing the voltage of the stimulus from 0 volts to 2.0 volts. The force of the muscle was recorded. In the second experiment we stimulated the muscle in series of ten using a constant voltage identified in the first experiment. The frequency of the stimuli was progressively increased starting at 0.5 and ending at 30 Hz.

Results:

Our results showed that as the stimulus increases the amplitude of the muscle twitches increases up until a point where it plateaus. Our results also showed that as the stimulation frequency increases the passive tension of the muscle increase up until a point where it plateaus.

Figure 1 shows a normalized graph for the effects of increasing stimulus on the amplitude of muscle twitches in the gastrocnemius muscle of a frog. The x-axis is the recorded stimulus in volts and the y-axis is the amplitude of the twitches (displayed as a percentage of the maximum). The graph shows that as the stimulus increases the amplitude of the muscle twitches increases up until a point where it plateaus.

Table 1 shows a set of group data from the first experiment, in which amplitude and times of muscle twitches were generated by stimulus pulses of different amplitudes. As in Figure 1, Table 1 shows that as the stimulus increases the amplitude of the muscle twitches increases up until a point where it plateaus. The contraction time and latency period remains largely unchanged with changing stimulus amplitude.

Figure 2 shows a normalized graph for the effects of increasing stimulation frequency on the passive tension in the gastrocnemius muscle of a frog. The x-axis is the stimulus frequency in hertz and the y-axis is the passive tension in the muscle (displayed as a percentage of the maximum). The graph shows that as the stimulation frequency increases the passive tension of the muscle increase up until a point where it plateaus.

Table 2 shows a set of group data from the second experiment, in which the strength of muscle contraction was examined during mechanical summation and tetanus. As in Figure 2, Table 2 shows that as the stimulus increases the amplitude of the muscle twitches increases up until a point where it plateaus. The amplitude of the first twitch remains largely unchanged with changing stimulus amplitude.

Discussion:

The data shows that the direct electrical stimulation produces contraction of the muscle via motor units. A little bit of force is generated when a few of these motor units are being used and a lot of force is generated when lots of motor units are being used. The muscle does not respond to the low stimulus voltages because the electrical stimulus is not directly touching the muscle, it is touching the surrounding connective tissue. The low stimulus voltages are not strong enough to penetrate the tissue. As noted in Figure 1 and Table 1 the amplitude of the muscle response increases with increasing stimulus voltages. This is so because more and more of the muscle mass is stimulated as the voltages increase. At high stimulus voltages, the muscle response reaches maximum amplitude. The muscle response does not continue to increase with increasing stimulus voltages because the muscle is already functioning at the best of its ability. The muscle cells have reached the point where all the troponins are activated by calcium. Releasing more calcium into the cell will not result in any more tension generated, as the system is already working at its maximum capacity.

Latency is the interval between stimulus and a response to the stimulus, here meaning muscle contraction. Over this period, the action potential sweeps across the cell membrane of the muscle cell and the sarcoplasmic reticulum releases calcium ions. The muscle fiber does not produce tension during the latent period, because the contraction cycle has yet to begin. The latency period in this study was constant at 0.025 seconds.This result been found by anyone else and it seems does not vary among other species, since it is roughly the same for humans (Hamilton and Osborn 1977).

Since contraction amplitude is dependent upon the increases in concentration and persistence of intracellular calcium, the question of why the contraction amplitudes of single twitches are the same is raised. This can be explained because the same amount of calcium is being put in for the same repeated event. As noted in Table 2, the amplitude of the first twitch seems to be constant (value). This can be explained because the muscle is utilizing the same amount of calcium and is thus generating the same amount of force. Tetanus is the complete contraction of a muscle. Tetanus requires high stimulus frequencies. This tells us that the calcium re-uptake by the sarcoplasmic reticulum is slower than the original release. The rate of muscle relaxation is much slower after tetanus than after a single twitch because more calcium needs to be re-taken up and it takes longer to get all the extra added calcium back into the sarcoplasmic reticulum.

A study on jumping bullfrogs by Marsh and Roberts reveals two points of interest: first, frogs jump farther than they should, considering only the force their muscles are able to generate. Second, muscles are able to do the most work when they contract slowly, however frog jumping involves a very rapid movement. They explain that by “separating the performance of muscular work from the application of mechanical work to the body,” a catapult-like mechanism, which works by loading elastic elements into the limbs prior to initiating a jump, overcomes the constraints of skeletal muscle function” (Marsh and Roberts 2003).

Another study by Aerts and Nauwelaerts (2006) indicates that by taking more small jumps as opposed to fewer larger jumps, frogs can increase their flexibility in movement because they would be able to change direction during the forward movement part jumping. Theoretically this means they would spend less time in the same spot during landing and recovery of the jumping cycle, which makes them more likely to be snatched by a predator.

Frogs have physiological mechanisms that have enabled their muscles to generate enough force for jumping and swimming locomotion including changing the frequency of the action potential and increasing the number of active motor units. As mentioned before, frogs are a keystone species, meaning other organisms rely on it and not always directly in a predator-prey relationship. Without frogs, food webs would collapse and lead to the demise of many other species and potentially entire ecosystems. This study is important because it serves as a model for understanding skeletal muscle mechanisms in other organisms including humans.