The nervous system acts like a highway along which your brain sends and receives information about bodily function and its external surroundings. This highway is made up of billions of nerve cells, or neurons that join together to make nerves. A neuron is made up of three components: a cell body, an axon, and dendrites. Dendrites gather impulses from adjacent cells and direct these electrical messages to the neuron cell body, or soma. The primary function of a neuron’s cell body is to gather and relay this synaptic information to other cells via the nerve cell’s long threadlike axon. An axon carries the impulses away from the cell body to dendrites of another cell or to an effector cell (Sherwood 2014). Dendrites propagate the electrochemical stimulation across the cell body as a graded potential with decreasing energy over time and space. As the energy reaches the axon hillock, the region connecting the soma to the axon, specialized voltage-sensitive membrane sodium channels are activated at -50 mV or higher. Upon this electrical or chemical stimulation, neurons and other excitable cells produce action potentials. Sodium ions rush into the cell, resulting in a membrane potential of +30 mV. Subsequently, the sodium channels close and the potassium channels open to allow potassium to rush into the cell. This results in a large increase in sodium permeability and membrane depolarization. Repolarization follows due to sodium permeability returning to its baseline value and potassium permeability transiently increasing as potassium channels open. The membrane channel reaches -80 mV until these channels close and the membrane returns to -70 mV once again (Sherwood 2014).
The rest period occurring from action potential initiation to resting membrane potential restoration is known as the refractory period. A refractory period gives the cell time to generate an ideal ion balance across the cell membrane. The refractory period is divided into two phases: absolute refractory period and relative refractory period. The absolute refractory period immediately follows the firing of a nerve fiber. Second action potentials are unable to be initiated because of sodium channels’ incapability to open due to their arrangement. The relative refractory period occurs shortly after the firing of a nerve fiber when partial repolarization has occurred and a greater than normal stimulus can elicit a second response. Although some sodium channels’ arrangement facilitates action potential, a very strong stimulus must be set to carry out an action potential (Brodal 2010).
Action potentials are “all-or-none” events in which the cell’s electrical membrane potential rapidly rises and falls. The action potential begins its course propagating down the axon, ultimately releasing a neurotransmitter into the synapse when it reaches the axon’s end. The velocity of an action potential’s path down an axon correlates with the fiber’s myelination and the fiber’s diameter (Sherwood 2014). Schwann cells forms a myelin sheath around the axon, keeping it from losing energy during impulse conduction. Found solely in the peripheral nervous system, these cells electrically insulate portions of the axon. Axons with this sheath are termed myelinated, while axons without are termed unmyelinated. Nerves coated with a myelin sheath of Schwann cells display a white pigment (Brodal 2010). Contiguous conduction occurs in unmyelinated fibers, while saltatory conduction takes place in myelinated fibers. Saltatory conduction propagates action potentials more rapidly than contiguous conduction because the action potential does not have to be regenerated at myelinated regions; it regenerates within every region of an unmyelinated axon (Sherwood 2014). The axon’s diameter also directly affects the required threshold voltage for an action potential. Hence, axons with large diameters are stimulated at lower voltages than smaller diameter axons (Sherwood 2014).
This study aimed to record action potentials from an isolated frog sciatic nerve. The thousands of axons that make up this peripheral nerve include afferent (sensory) and efferent (motor and autonomic) nerves. Their variations in diameter, myelination, excitability, threshold and conduction velocities make them individually distinct. CAPs are the summed “all-or-none” action potentials that occur from excited axons at a stimulus voltage. Increasing stimulus voltage progressively excites axons until the nerve’s axons are entirely excited. Consequently, the magnitude of the CAP increases as the strength of the stimulus increased. A resulting maximal response develops when supramaximal stimuli can no longer affect the magnitude of the CAP. CAPs are produced when the nerve is extracellularly stimulated; they are recorded with extracellular electrodes. The shape of the CAP reflects the difference in potential between two extracellular electrodes. An absent stimulus would yield a baseline recording since there is no difference. A stimulus, however, incites a wave of depolarization that passes down the nerve. Once the depolarization wave transverses the first electrode, the electrode becomes negative to its distal electrode and a positive uptake is recorded. As the wave reaches the second electrode, the electrode becomes negative to its proximal electrode and a negative recording is generated (McGill 2015).
Compound action potentials are very useful tools in examining nerve physiology. Nonetheless, they are also used in clinical settings to explore peripheral nerve lesions and diseases in patients. In this study, compound action potentials (CAPs) were measured from an isolated frog sciatic nerve to investigate the nerve impulses’ basic physiological features. The compound action potential and the refractory period graphs were hypothesized to represent the standard graphs depicting these physiologic properties.
Materials and Methods
The northern leopard frog, Rana pipiens, was initially subjected to decapitation with scissors. A metal rod was then used to push down the frog’s spinal column, providing instant death. The skin was peeled off with tweezers and scissors were used to open its body cavity. Its pelvic region muscles were then cut to expose and isolate the sciatic nerves extending down its hind legs. The sciatic nerves were extracted with a glass hooked probe and placed into separate beakers containing Frog Ringer’s solution (formula (g/L): NaCl 6.5, KCl 0.41, CaCl 0.12, NaHCO3 0.2) (ADInstruments 2016).
Stimulation probes were connected to one end of an axon stimulation box. Starting with the stimulation end, the recording probes were then attached along the sides. The probe ends were finally connected to each appropriate socket on the PowerLab box. The lower reservoir of the nerve bath was then filled with Frog Ringer's solution, confirming that the Ringer's solution was not touching any of the wires. The extracted nerve was placed in the chamber and the lid of the chamber was placed firmly on top to preserve the bath’s humidified air (ADInstruments 2016).
The PowerLab box was set to 10 mV and the nerve was excited with a stimulus, as Lab Tutor plotted the effect of the stimulus on muscle contractile response. The response output by the nerve generated a curve on the graph. The magnitude of the stimulation was then gradually increased by 10 mV until the graph curve plateaued, indicating the CAP amplitude. The purpose of this exercise was to determine the threshold of the nerve and the maximum compound action potential, CAP, amplitude in millivolts (mV) (ADInstruments 2016).
The frog’s sciatic nerve’s relative and absolute refractory periods were also examined using the CAP amplitude. Paired stimuli were applied to the nerve at intervals of 4.0 milliseconds (ms). The interval time was decreased by 0.5 ms until 2.0 ms, and then by 0.1 ms until 1.0 ms. The refractory periods were identified as the second stimulates peak initially decreased in amplitude and approached 0 mV (ADInstruments 2016).
The sciatic nerve’s velocity was determined by measuring and recording the length (cm) of the nerve region between the two recording probes. As the magnitude of the CAP amplitude was applied, the signal’s travel time in seconds between the probes was recorded. Lab Tutor used both data values to calculate the value for the conduction velocities of several groups of fibers in the nerve in meters per second (ADInstruments 2016).
Results
This figure displayed CAP (mV) increasing in size and duration with increasing stimulus strength. As the stimulus strength increased, more action potentials collectively produced a larger bell-shaped curve. The stimulus showed no recorded response until ~50 mv, exhibiting the threshold. It significantly increased until ~150 mV, indicating a positive correlation between the applied stimulus voltage and the sciatic nerve’s response (mV). A third set of data points followed with a plateau towards 200 mV. The peak amplitude of the CAP was 200 mV, which was the voltage value of the peak of the CAP response.
Figure 1. The electrical stimulus voltage (10-200 mV) versus recorded CAP response through a frog sciatic nerve.
This figure displayed the time interval between the two stimuli and the amplitude of the second CAP plotted against each other. The nerve was stimulated twice with a maximal stimulus of 90 mV and the interval between the stimuli was progressively reduced; the resulting observation was the effect of reducing the stimulus interval on the recorded second CAP response through the sciatic nerve. As the time intervals between stimuli shortened, the graph displayed a downward slope until a plateau at ~2.5 mV.
Figure 2. The recorded electrical second CAP response of a frog’s sciatic nerve to twin maximal stimuli as the stimulus interval varied from 4-1.5 ms.
This table measured the conduction velocity of several groups of fibers in the nerve. The variation in conduction velocity in the various fibers in the nerve highly regulates the shape of the Compound Action Potential (CAP) because each fiber’s conduction velocity determines the latency of its contribution to the CAP.
Table 1. Estimated Conduction Velocity of Frog Sciatic Nerve Fibers
Group Calculated Conduction Velocity
α Nerve Fiber 25.2 m/s
β Nerve Fiber 19.6 m/s
γ Nerve Fiber 12.5 m/s
Discussion
A frog’s sciatic nerve’s extracellular, biphasic, compound action potential was assessed using an extracellular technique. The compound action potential, or CAP, is the total computation of the action potentials of fibers that are fired by a stimulus (ADInstruments 2016). An electrode’s position and the myelination of axons within the nerve affect the compound action potential’s magnitude. The CAP is biphasic in the sense that it has both positive and negative refractions. The negative phase is a result of the recording technique. After-hyperpolarization causes a negative phase in an intracellular action potential. The initial experiment reflected in Figure 1 displays the frog’s sciatic nerve’s response to increasing stimulus. Initially, there was no recorded response to the stimulus less than 50 mV. In order to trigger an action potential, a minimal threshold of 50 mV needed to be achieved. The graph plateaued towards 200 mV, indicating that the neuron’s nerves had overcame their threshold and that they were all conducting a stimulus. There was a graded potential due to the opening of voltage-gated sodium channels along the membrane in response to the stimulus. The membrane began to depolarize at the axon hillock as sodium ions flowed in. The current was leaked out of the leak channels and the response was then increased with an increased stimulus. More stimuli allowed for more fibers to achieve threshold and carry action potentials. As a result, the total response increased. The influx stops as potassium rushes out of the cell, hyperpolarizing the membrane potential. The positive and negative slopes in Figure 1 display the neuron’s membrane potential states of depolarization and hyperpolarization during action potential (Raymond 1979). The maximal CAP value of the American bullfrog (Rana catesbeiana) has been identified as ~100.7 mV (Tasaki and Frank 1955). This experiment’s maximal CAP value was determined to be 200 mV; this is a 99.3 mV difference with that of the American bullfrog. The observation that Rana pipiens had a higher value than that of the Rana catesbeiana may suggest that species within the same genus do not physiologically share the same sciatic nerve.
Refractory periods occur at de-polarization peaks when voltage-gated sodium channels are closed and incapable of being opened or inactivated until resting membrane potential is restored. Figure 2 displays the data collected from the refractory periods of the frog’s sciatic nerve. More fibers are becoming refractory as the time interval between two maximal stimuli was reduced. This caused a drop in the recorded 2nd CAP response through the frog sciatic nerve. Reading from right to left, the graph’s downward slope’s starting point indicated the relative refractory period. The end of the relative refractory period was displayed at 3.5 ms. As the graph approached the plateau at ~2.5 mV, the end of relative refractory period was determined to be greater than 2.5 ms long because of the test ran until the interval time was ~2.0 ms. The end of the absolute refractory period on the graph was displayed when the graph approached 0 mV on the y-axis. In "Nerve fibre velocity and refractory period distributions in nerve trunks," Betts displays a graph similar to Figure 2 that plots the second nerve action potential amplitude and the pulse interval. His graph correlates with the data value findings of this experiment (Betts 1976). The relative refractory period of the frog can also be compared to the relative refractory period of the Trembler mice’s sciatic nerve. A study determined that its relative refractory period value was ~9.7 ms, which is at least three times smaller than that of the frog’s sciatic nerve (Low and McLeod 1977). An exact time for the frog’s relative refractory period, however, could not be determined because the experiment ended at ~2.0 ms with a ~2.5 mV response.
Axonal diameter has an effect on conduction velocity. A wider diameter allows a greater number of ions to move through freely. Myelination insulates the membrane with a lipid-like substance that does not polarize. Myelination optimizes the propagation of action potentials along axons, resulting in an overall faster conduction. De-polarization happens faster with a myelinated axon because it decreases the current’s surface area as the action potential jumps from node to node along the axon (Sherwood 2014). The α nerve fiber was the fastest, generating a conduction velocity of 25.2 m/s. The β nerve fiber was the intermediate fiber, with a conduction velocity of 19.6 m/s. The slowest fiber was the γ nerve fiber, generating a 12.5 m/s conduction velocity. These conduction velocity values can be compared with that of a mature chicken. In "Excitation and conduction in immature nerve fibers of the developing chick," the mature chick’s sciatic nerve velocity was determined to be 50 m/s (Carpenter and Bergland 1957). This indicates that a mature chicken’s sciatic nerve conduction velocity is slower than a frog. Since a chicken is warm-blooded and a frog is cold-blooded, it is safe to hypothesize that cold-blooded organisms have a slower nerve conduction velocity than warm-blooded organisms. This may be because processes that are conducted at higher temperatures occur at faster rates. The fastest α nerve fiber of the frog was most likely myelinated. The β nerve fiber was less myelinated and the γ nerve fiber had the least myelination. Previous studies have revealed that the sciatic nerve’s amplified action wave on a cathode ray oscillograph separates during conduction into 4 components of alpha, beta, gamma, and delta. The alpha component is the quickest because of its motor feature that passes into the ventral spinal and dorsal roots. The beta and gamma waves are exclusively sensory (Erlenger 1926).
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