Physio-compilation.docx

PowerLab: Physiology of the Nerve

T. Pineda, P. M. Santiago, J. R. Serrano, A. E. Sutingco and M. T. Tuazon Keywords:

Summary The nervous system is made up of the brain andspinal cord, together with the nerves to conduct impulses to and from the CNS. In this experiment, the Compound Action Potential (CAP) was determined using the PowerLab. The data obtained yielded the following values: Threshold voltage= 0.3008mV; Maximum CAP potential= 3.3505; Conduction velocity= 3.33 m/s.

Introduction In this experiment, we stimulated the frog's sciatic nerve, activating a large number of individual nerve fibres simultaneously, and record the resulting aggregate electrical activity. The objective of this experiment is to determine the compound action potential (CAP) of nerves that are not exposed and exposed to anesthetics. We also studied the characteristics of the frog's sciatic nerve CAP, and also to construct a strength or duration curve of the nerve. The sciatic nerve is the main nerve trunk from the spinal cord to the leg. It consists of a bundle of nerve fibers, each of which is the axon of a neuron, whose cell body is near the spinal cord. Axons are long, cylindrical processes that project from the cell body of a neuron and that act as a conduit for neural messages called action potentials. Some axons conduct action potentials toward the brain: they are called afferent fibers. Others, called efferent fibers, conduct action potentials away from the brain. The sciatic nerve contains both efferent and afferent fibers. Axons within the sciatic nerve differ in

important structural and functional ways (Fig. I-1). Myelinated axons are surrounded by myelin, which looks in cross-section like tightly-packed, concentric rings around the perimeter of the axon. Other axons lack myelin. Axons that innervate internal organs and glands tend to be smaller in diameter than those that innervate skeletal muscles. Smaller diameter axons tend to conduct action potentials more slowly than larger diameter axons. The thresholds, refractory periods, and the durations of action potentials differ across types of axons. Interpretation of the properties of compound action potential involves thinking of the sciatic nerve as a heterogenous population of nerve fibers.

Fig.I-1: Cross section of the sciatic nerve of a frog consists of only a single bundle of fibers, surroundede by the perinium and loose epineurium

The origin of the action potential is based on some amazing properties of the cell membrane. All cells develop a membrane potential (Vm). A microelectrode with a very small tip can be carefully introduced into the cell through its membrane. Much research has been done to ascertain the origin of the membrane potential (Vm). Fundamentally, a cell develops a membrane voltage due to the separation of charged ions across the cell membrane. Because of differences in concentration and the selective permeability (or conductance) of the membrane, only certain ions can cross the barrier. The separation of charge across the membrane eventually achieves “electro-chemical” equilibrium and at this point the resting membrane potential is established. While all cells develop a resting membrane potential, only nerve and muscle cells have the ability to change it dramatically. If the Vm changes very quickly in response to particular stimuli, this property is called excitability. The response is called an action potential. As action potentials propagate along an axon, they produce electric potentials that can be recorded from the surface of the nerve. When a nerve bundle is stimulated, many axons produce action potentials synchronously. The resulting electric responses recorded from the surface of the nerve are called compound action potentials to distinguish them from the action potentials generated by individual axons. Stimulus electrodes are applied to one end of the nerve; recording electrodes are located along the nerve. If the nerve is stimulated with a current pulse of sufficient amplitude, the action potentials produced in the fibers propagate toward the recording electrodes.

The aggregate effect of the many action potentials is an extracellular wave of negative potential, moving along the surface of the nerve. If the recording electrodes are widely spaced, the wave of negative potential produces a negative pulse in recorded voltage as it passes the (+) recording electrode. At a later time, the negative wave of extracellular potential passes the (-) recording electrode, where it contributes a positive pulse to the recorded voltage. If the electrodes are more closely spaced, the negative and positive parts of the recorded voltage merge, and the resulting waveform is called a diphasic compound action potential. The propagation can be blocked by a number of methods including mechanical methods (pressure applied to the nerve or crushing the nerve with forceps), electrical methods (passing a blocking level of current through the nerve), or chemical methods (applying local anesthetics to the nerve). Materials and Methods The PowerLab was utilized to determine the physiology of the nerve. The equipment was first setup and calibrated. For the setup, the red and black alligator clips from the stimulator electrodes were connected to two of the metal rungs on the opposite sides of MLT012/B Nerve Bath. Next, the red and black BNC connector from the stimulator electrode was connected to the positive and negative analog output connector on the powerlab, respectively. The red and black leads from the first recording electrode were connected to two of the metal rungs of the MLT012/B Nerve Bath. Afterwards, the 8-pin pod connector was connected to the pod port on input 1 of the PowerLab. For the

second recording electrode, the alligator clips were paced further Way from the stimulus electrode; and the pod connector was attached to pod port on input 2 on the PowerLab. For the setup for calibration, the lower reservoir of the Nerve Bath was filled with frog Ringer's solution carefully to avoid contact between the solution and the metal electrode rungs which can cause short circuit. A strip of paper was laid over the wires in the nerve bath so that it touches both stimulating electrodes and recording electrodes. The strip was moistened with frog Ringer's solution, and the cover was placed on the nerve bath. As for the calibration, the PowerLab was connected to the USB port in the computer and was switched on. LabChart was launched and the file named CAP Set Chart was opened. The Macro menu was selected and Test Connection was performed. A series of stimulus pulses were recorder; if there is none, check the setup. After the connections were tested confirmed to be working properly, the experiment proper was conducted. An isolated frog sciatic nerve was utilized for the experiment. The nerve was lifted out of the dish by grasping the thread tied to the nerve using forceps. The nerve was blotted with tissue paper to remove excess Ringer's solution. The filter paper from the nerve bath was removed and the nerve was laid across the wire electrodes making sure it is in contact with each of the active connections. The cover was placed on the nerve bath. The first experiment was to determine the threshold voltage and maximal CAP. A series of electrical stimuli, each with increasing amplitude, were give to the nerve. The stimulus

range was between 20 to 400 mV, and increases in 10 mV. The Macro: Threshold Voltage was selected from the Chart window to stimulate and record the nerve for 1.1 seconds. The table was filled and the data were analyzed using Waveform Cursor in order to measure CAP amplitude at each stimulus voltage. The data were recorded and maximum CAP altitude was recorded. The second experiment is the determination of the refractory period. In this part of the experiment, the PowerLab stimulated the nerve with a series if pulses. The pulse interval decreased in each block of data. Using the data from the first experiment, the minimum stimulus voltage required to elicit maximal CAP was determined. Next, Macro: Refractory mV was selected and one of the four versions of Refractory macro was chosen, that is, the voltage that is nearest the intensity determined beforehand. The chart then recorded 15 data block with each block having 10 milliseconds in duration. For the analysis, the first two CAPS's recorded in CAP1 were selected in each block of data recorded in part 2. The zoom window was opened and the data trace was examined using Waveform Cursor. The amplitude for the second CAP was recorded. The relative refractory period was determined as the first decrease in the amplitude of the second CAP. Lastly, the absolute refractory period was determined as complete disappearance of the second CAP. The last experiment is the determination of nerve conduction velocity. The first step was to measure in centimeters the distance between the black leads of each of the two recording electrodes. The Macro: Conduction Velocity was selected from the chart

window. A block of data in two channels was recorded in the chart. For the analysis, first, a selection of the first CAP in both channels was made. The time interval for the CAP to travel between the two recording electrodes was determined using the Marker and WaveformCursor. Channel 1 was selected and the marker was placed on the first peak. Then, channel 2 was selected and waveform cursor was placed over the second CAP peak. The value for the time differential was read and recorded in the data notebook. The following formula was used to determine the conduction velocity: Conduction Velocity (m/sec) = Distance between electrodes(cm) x Time interval between CAP′s(ms) 1m 100cm

x

1000ms 1sec

RESULTS AND DISCUSSION Table 1- CAP amplitude VS stimulus intensity; threshold stimulus voltage highlighted in yellow and maximum CAP amplitude highlighted in orange. Stimulu s amplitu de (mV) 20 30 40 50 60 70 80 90 100 110 120 130 140

CAP amplitu de (mV) 0.0000 0.0000 0.0000 0.0000 0.3008 0.9744 1.8235 2.2991 2.6495 2.9007 3.0293 3.1247 3.2735

Stimulu s amplitu de (mV) 220 230 240 250 260 270 280 290 300 310 320 330 340

CAP amplitu de (mV) 3.2239 3.1759 3.1743 3.0991 3.1503 3.0751 3.1247 2.9503 3.0239 2.9503 3.9503 2.9263 2.9007

150 160 170 180 190 200 210

2.2239 3.2751 3.3487 3.3503 3.3247 3.2751 3.2239

350 360 370 380 390 400 410

2.9247 2.8735 2.8495 2.7503 2.7759 2.8495 2.7759

At the start, with low initial stimulus amplitude of 20 mV no compound action potential (CAP) is visible. This is apparent until the 4th stimulus amplitude (50mV). However when 60 mV was reached a sign of CAP was apparent and the CAP amplitude equal to 0.3008 mV indicates this. The CAP is seen as a deflection after the stimulus artifact. Figure 1 is showing the first sign of CAP which is also denoted as the threshold stimulus voltage, which is where the first sign of CAP can be discerned. The threshold stimulus voltage signifies the voltage needed to generate at least on action potential from the sciatic nerve fiber. So at 60mV we can see that the sciatic nerve is capable of conduction action potentials.

Figure 1 - CAP channel graph at 60mV showing the threshold stimulus voltage.

Figure 2 - CAP channel graph at 180mV showing the maximum CAP amplitude.

Finally after increasing the stimulus amplitude to 180mV we can see that it is at it’s maximum CAP potential (Figure 2). Afterwards we can observe a decline in the magnitude of the CAP amplitude despite raising its stimulus amplitude. The maximum CAP potential, which is at 3.3503mV, is the point at which a further increase in stimulus voltage generates further increase in the CAP amplitude. The size of the CAP will no longer increase since all the A-alpha fibers, which make up the nerve, are excited and are conducting action potentials.

E.2 Determination period

of

refractory

Figure 3 - Refractory period Once the action potential starts, voltage-gated sodium channels open and at the peak of the action potential, these channels will be inactive. After this point, the channels are not able to close right away; hence, a “recovery” period takes place, the refractory period. An absolute refractory period is defined as an interval in which a second action

potential is not produced regardless of increasing the intensity of stimulus. During this period, depolarization and repolarization takes place. An influx of sodium (Na+) ions and positive charge inside the membrance will take place during depolarization and otherwise during repolarization, which would entail increase of K+ ions and negative charge inside the membrane. On the other hand, relative refractory period is defined as an interval in which a second action potential may be produced if the intensity of stimulus is increased. Hyperpolarization takes place during this period, which prepares the membrane for other upcoming electrical signals. Voltage-gated sodium channels will be closed during the course of hyperpolarization. In addition, if the stimulus is kept constant and the interval decreases, the threshold voltage for the second action potential will be higher than the first. However, if a stimulus is constant and the interval increase, then a second action potential will occur. E.3 Calculating Velocity

the

Conduction

The variation in conduction velocity in the various fibres in the nerve is an important factor in determining the shape of the Compound Action Potential, since the conduction velocity of each fibre determines the latency of its contribution to the CAP. Conduction velocity is systematically related to fibre diameter in fibres of a given type.

The conduction velocity can be easily calculated by knowing both the distance the action potential travels or between CAP1 (𝑑1 ) and CAP2 (𝑑2 ) the amount of time it takes. Velocity has the units of distance per time or m/s. For the example given in the table below, the given value for the distance between recording electrodes is (0.1m) but since the table above is in cm, 0.1 is converted to 10cm. The time interval between CAP1 and CAP2 is 0.03s but since the table above is in ms, 0.03 is converted to 30ms. Then using the formula: Conduction

Velocity

(m/sec)

Distance between electrodes(cm) Time interval between CAP′s(ms) 1m 1000ms 100cm

x

= x

1sec

Distance 10 between recording electrodes Time 30 interval between CAP1 and CAP2 Conduction 3.33 Velocity

cm

ms

m/s

Substituting the values, the Conduction Velocity is 3.33m/s.

final

In summary, to calculate the conduction velocity by the Difference Method, we must take latency measurements using two different recording positions. The conduction distance is the distance between the first recording electrode for

each position, and the conduction time is the difference between the latencies at the first recording electrode for each position.

References: Characteristics of the Compound Action Potential. (n.d.). Retrieved February 22, 2016, from http://www.medicine.mcgill.ca/physio/vl ab/CAP/character.htm Goodman, B. P., Harper, C. M., & Boon, A. J. (2009). Prolonged compound muscle action potential duration in critical illness myopathy. Muscle & Nerve Muscle Nerve, 40(6), 1040-1042. Remington, L. A., & Remington, L. A. (2012). Clinical anatomy and physiology of the visual system. St. Louis, MO: Elsevier/Butterworth Heinemann.

Retrieved from: http://www.medicine.mcgill.ca/physio/vl ab/cap/methods.htm DeLisa, J. A., & Mackenzie, K. (1982). Manual of nerve conduction velocity techniques. New York: Raven Press. Retrieved from: http://retina.anatomy.upenn.edu/~rob/lan ce/conduction_velocity.html Neuronal Action Potential – Important features of the neuronal action potential. (2014). Retrieved from http://www.physiologyweb.com/l ecture_notes/neuronal_action_po tential/neuronal_action_potential _refractory_periods.html Compound Action Potential – Refractory period. (n.d.). Retrieved from http://www.medicine.mcgill.ca/p hysio/vlab/CAP/refract.htm