Animal Physiology lab 4B Date Performed: October 1, 2012 Date Submitted: October 12, 2012 Activity 4 Nerve Physiology

A. Sciatic Nerve Structure

Group 2 Benson, Jonah (Leader) Catayas, Keth Laurel Legarda, Criscire

In the experiment, frog sciatic nerve was used in order to study the relationship between nerve stimulation and skeletal muscle movement. As observed, the sciatic nerves near the spinal cord caused stronger leg twitches when stimulated with 1.5V. This pair of sciatic nerves was bigger than those found along the leg (Figure 1). The sciatic nerve on the leg was located underneath bundles of muscle tissue immediately below a blood vessel (Figure 2).

Spinal cord

Sciatic nerve

Blood vessel

Sciatic nerves

Dorsal view Figure 1 Sciatic nerve near the spinal cord Figure 2 Sciatic nerve on the leg As shown in Figure 3, the sciatic nerve is derived from three spinal nerves (7th, 8th, and 9th). It is the longest single nerve in the vertebrate body which starts from the lower back, runs through the buttocks, and terminates on the lower limbs (legs and

2 thighs). This nerve mainly innervates the skeletal muscles of the legs and thighs (McGill Physiology Virtual Lab, 2011).

Figure 3 Schematic diagram of the frog sciatic nerve A typical vertebrate nerve consists of several thousand axons. These axons are cylindrical structures that transmit neural messages in the form of electrical signals called action potentials. Axons that conduct action potentials toward the brain are called afferent fibers, while those that deliver signals away from the brain are efferent fibers. The cell bodies of the former are located outside the central nervous system, in a site called dorsal root ganglion, while the soma of the latter are located inside the spinal cord. Both efferent and afferent fibers are found in the sciatic nerve (Vizsolyi, 2004). Axons within the sciatic nerve differ in structure (myelinated or unmyelinated) and function (afferent or efferent). Fibers may be large (15-25 m) and myelinated, or small (around 0.2 m) and unmyelinated. In general, small (less than 25 m), myelinated and unmyelinated fibers are features of vertebrate nerves. Also, in vertebrates, the whole nerve is encased in a connective tissue sheath called the epineurium. The axons are further subgrouped into bundles called fasciculi (singular: fasciculus), each of which is also enclosed in a connective tissue sheath called the perineurium. Lastly, each individual fiber is further surrounded by a delicate connective tissue sheath called the endoneurium.

3 Figures 4 shows cross sections of a frog sciatic nerve depicting different connective tissues (McGill Physiology Virtual Lab, 2011).

Figure 4 Cross sections of frog sciatic nerve showing connective tissues B. Sciatic Nerve Response toward Electrical Stimulation (Untreated Ringers Solution) During the experiment, the sciatic nerves near the spinal cord were stimulated by 1.5-V electrical stimulus while being immersed in Ringers solution. This solution was used as control because it is approximately the same in ionic composition to the frogs extracellular fluids (Massachusetts Institute of Technology, 1999). As shown in Figure 5, the right and left sciatic nerves innervating the right and left legs, respectively, were stimulated individually with two successive stimuli separated by specific time intervals (1.75, 1.5, 1, and 0.5 second).

Figure 5 Stimulated left and right sciatic nerves The right sciatic nerve was first stimulated, horizontal and vertical displacements were recorded. The sum of the two distances reached by the leg in correspondence to the

4 two successive stimuli was used in the data. The stimulation was repeated three times, obtaining the following data: Table 1 Horizontal and vertical distances of right leg after stimulation 1.75 seconds H (mm) 20 53 68 47 V (mm) 15 5 10 10 1.5 seconds H (mm) 15 25 25 21.7 V (mm) 10 28 5 14.3 1 second H (mm) 0 5 0 1.7 V (mm) 0 10 0 3.3 0.5 second H (mm) 35 30 50 38.3 V (mm) 5 30 20 18.3

1st Trial 2nd Trial 3rd Trial Mean

Legend: H= horizontal distance; V= vertical distance Table 1 shows the large mean values of the horizontal distances (47, 21.7, and 38.3 mm) compared to their corresponding vertical distances (10, 14.3, and 18.3 mm, respectively), except in the stimulation with one-second interval having a horizontal distance of 1.7 mm and a vertical distance of 3.3 mm. These large values for horizontal distances were caused by the kind of leg movement. The right leg was folded inward instead of being stretched upon stimulation. Due to this, the horizontal distance was more observable than the vertical distance. Similar to what was done in the right sciatic nerve, the left nerve was also stimulated using the same amplitude of electrical stimulus. There were two successive stimuli applied following different time intervals. The procedure was repeated three times, having the following data: Table 2 Horizontal and vertical distances of left leg after stimulation 1.75 seconds H (mm) 15 10 0 8.3 V (mm) 0 0 0 0 1.5 seconds H (mm) 10 3 0 4.3 V (mm) 0 0 0 0 1 second H (mm) 15 10 3 9.3 V (mm) 0 0 0 0 0.5 second H (mm) 0 0 0 0 V (mm) 0 0 0 0

1st Trial 2nd Trial 3rd Trial Mean

Legend: H= horizontal distance; V= vertical distance Table 2 shows that majority of the stimulations in the left leg did not result to leg jerking that covered great vertical distances. However, horizontal distances were recorded from the three stimulations (1.75, 1.5 and 1 second). This was due to the fact that upon

5 the application of stimulus, the left leg in general did not move, instead, the muscles just twitched. Some factors may have affected the experiment thus coming up with the obtained data. The left leg was not able to produce distance values compared to the other leg because the left sciatic nerve may have been destroyed by the metal tools used. It must have been grazed by the scissors or probe during the dissection. In the study done by Massachusetts Institute of Technology (1999), glass and blunt tools were used to handle the sciatic nerve because it is very sensitive and easily destroyed. In addition, as discussed in their study, metal tend to attract the excess ions present in the nerve, thus affecting its excitability. Another factor that may have caused the great differences in the obtained measurements is the duration of the stimulation. Figure 1 shows the schematic diagram of how the nerves were stimulated. The intensity of electrical signals was equal all throughout the experiment, however the time intervals of stimulation differed. In the experiment conducted by McGill Physiology Virtual Lab (2011), the duration of stimulus was also considered. The large values for horizontal or vertical distance may have been caused by longer duration of stimulus applied instead of being affected by the duration of time intervals.

Figure 6. Schematic diagram of the stimulation process When a nerve bundle is stimulated, individual axons produce many action potentials simultaneously, however, other action potentials are not conducted the same time as the others. A nerve is composed of different axons with different structures and diameter, thus different conduction velocities. Conduction velocity depends on two fiber characteristics: fiber diameter and myelination. Myelinated fibers conduct action potentials faster than the unmyelinated types. Also, axons with small diameter tend to conduct action potentials more slowly than large diameter axons. This is related to the

6 duration of stimulus, once the nerve is stimulated, small diameter fibers are immediately excited, however, if the stimulus is present for a longer period of time, then the large diameter fibers are also stimulated leading to a stronger reaction or leg movement (Vizsolyi, 2004). C. Sciatic Nerve Response toward Electrical Stimulation (Caffeine Solution) Similar to the Ringers solution, the sciatic nerves found in the spinal cord were treated with a caffeine solution (36.7 mg/ 100 ml of distilled water) within 10 minutes and stimulated by 1.5V electrical stimulus. In Figure 7, the right and left sciatic nerves innervating the right and left legs, respectively, were stimulated individually with two sequential stimuli separated by different time intervals (1.75, 1.5, 1, and 0.5 second).

Figure 7 Stimulated left and right sciatic nerves Horizontal and vertical displacements of the right leg were recorded after it was first stimulated. Two distances influenced by the leg in relation to the two repetitive stimuli were summed up and used in the data. The stimulation was repeated three times, attaining the following data: Table 3 Horizontal and vertical distances of right leg after stimulation 1.75 seconds H (mm) 0 3 0 1 V (mm) 5 10 15 10 1.5 seconds H (mm) 0 0 0 0 V (mm) 10 5 5 6.7 1 second H (mm) 0 0 1 0.3 V (mm) 10 10 10 10 0.5 second H (mm) 0 0 0 0 V (mm) 5 0 5 3.3

1st Trial 2 Trial 3 Trial Mean

rd nd

Legend: H= horizontal distance; V= vertical distance

7 As shown in the table 3, the vertical distances in different time intervals obtained the large mean values (10, 6.7, 10, and 3.3 mm) compared to corresponding horizontal distances (0 mm), except in the stimulation with 1.75 and 1 second intervals having vertical distances of 1 mm and 0.3 mm, respectively. The large values for vertical distances were affected by the movement produced by the leg. The right leg was being stretched upon stimulation, compared to ringers solution in which the leg folded inward instead. After measuring the vertical and horizontal distances produced by the right leg, the left nerve was also stimulated using the same amplitude of electrical stimulus. Two consecutive stimuli were applied in different time intervals. The obtained data for left leg after three times of treatment is shown below. Table 4 Horizontal and vertical distances of left leg after stimulation 1.75 seconds H (mm) 0 0 0 0 V (mm) 15 5 15 11.7 1.5 seconds H (mm) 0 0 0 0 V (mm) 10 15 15 13.3 1 second H (mm) 0 0 0 0 V (mm) 5 5 10 6.7 0.5 second H (mm) 0 0 0 0 V (mm) 0 0 3 1

1st Trial 2nd Trial 3rd Trial Mean

Legend: H= horizontal distance; V= vertical distance As shown in table 4, vertical distances (11.7, 13.3, 6.7, and 1 mm) obtained the large mean values in contrast to the zero mean values of horizontal distances. This was due to the reaction manifested by the left leg after being treated with stimuli in different time intervals. The left leg just stretched upon stimulation and not folded inward, consequently, only the vertical distances were evident than horizontal distances. In the study of Hussain and Thai (2002), the sciatic nerve of a female bull frog (Rana catesbeiana) was placed in the Ringers solution with caffeine for five minutes. The amplitude and velocity of the bullfrogs sciatic nerve compound action potential in response to the caffeine treatment were recorded and analyzed. Results showed that the average speed of the control measurements was 3.6 + 0.2 cm/ms, while average speed of the caffeine-treated nerve was 3.7 + 0.2 cm/ms. The mean amplitude for the control experiments was -0.68 + 0.05 mV, while the mean amplitude for the caffeine-treated nerve was -1.68 + 0.32 mV. It showed that the velocity of the compound action potential was unaffected by caffeine treatment, while the amplitude was increase in absolute value. It can be explained by the anatomy of the sciatic nerve. The sciatic nerve is actually a bundle of individual nerve fibers. Each nerve fiber consists of many nerve cells and each cell can have a different threshold potential. The

8 speed of the action potential for each cell is determined by the diameter of the nerve cell axons, and the presence of myelin sheath surrounding the axon. The speed of the compound action potential is a weighted average of the speeds of the individual nerve cells. Caffeine can have no effect on either the diameter of the axon or the presence of the myelin sheath, and thus can have no effect on the speed of the propagation of the action potential (Hussain and Thai, 2002). Caffeine is a stimulant of the central nervous system (CNS) by inhibiting the enzyme phosphodiesterase and acting as a competitive inhibitor of adenosine. Adenosine slows down nerve cell activity by binding to the adenosine receptors on the outside of nerve cell axon. This receptor has been linked with the inhibition of adenylyl cyclase activity. The receptor is thought to mediate the inhibition of transmitter release and the reduction in neuronal activity. Because caffeine is similar to adenosine in shape, caffeine binds to the same receptor, blocks the adenosine, and increases nerve activity. Caffeine is known to increase the excitability of nerve cells by lowering the threshold voltage, an effect known as facilitation. By lowering the voltage of individual nerve cells, caffeine increases the fraction of cells that are activated at a given stimulating voltage, and hence the amplitude of the compound action potential (Hussain and Thai, 2002). D. Sciatic Nerve Response toward Electrical Stimulation (Analgesic Solution) The sciatic nerves found in the spinal cord were treated with an analgesic solution (1 tablet / 20 ml of distilled water) using a 500 mg paracetamol within 10 minutes and stimulated by 1.5V electrical stimulus. The right and left sciatic nerves innervating the right and left legs, respectively, were stimulated individually with two sequential stimuli separated by different time intervals (1.75, 1.5, 1, and 0.5 second) (Figure 8).

Figure 8 Stimulated left and right sciatic nerves In our results, the treatment of sciatic nerve with analgesic has the least mean based on the responses of the right and left legs when stimulated by an electric current from a 1.5-volt battery. Repeated stimulation would result in receptor fatigue or sensitization wherein it became more sensitive to any stimulation. In the tables 5 and 6, the mean which determines the movement of the right and left legs is higher as shown in

9 1 sec and 0.5 sec compared to 2 sec and 1.5 sec because at this time of stimulation, it had experienced sensitization (Nielsen et al., 1991). Table 5 Horizontal and vertical distances of right leg after stimulation 1.75 seconds H (mm) 0 0 1 0.3 V (mm) 5 5 5 5 1.5 seconds H (mm) 0 0 1 0.3 V (mm) 5 20 5 10 1 second H (mm) 1 1 0 0.7 V (mm) 15 15 5 11.7 0.5 second H (mm) 0 0 0 0 V (mm) 10 3 20 11

1st Trial 2 Trial 3 Trial Mean

rd nd

Legend: H= horizontal distance; V= vertical distance Analgesic is a drug used to relieve pain. It actions in blocking pain signals thus changing how the brain interprets the signal. It slows down the central nervous system. It primarily relieves unpleasant sensory stimuli associated with tissue damage. Paracetamol is usually used as an analgesic. It inhibits the release of prostaglandins in the central nervous system. These chemicals are involved in the recognition of pain by the brain. This is the reason why treatment of the sciatic nerve with analgesic had the least response from electrical stimulation because it basically restrict the brain to recognize pain or stimulation (Nielsen et al., 1991). Table 6 Horizontal and vertical distances of left leg after stimulation 1.75 seconds H (mm) 0 0 10 3.3 V (mm) 0 3 15 6 1.5 seconds H (mm) 0 0 2 0.7 V (mm) 3 3 3 3 1 second H (mm) 10 0 1.5 3.8 V (mm) 3 0 7 3.3 0.5 second H (mm) 10 5 0 5 V (mm) 10 0 3 4.3

1st Trial 2 Trial 3 Trial Mean

rd nd

Legend: H= horizontal distance; V= vertical distance In general, the stimulations and time intervals of stimulation used in this experiment should be able to produce horizontal and vertical distances of leg movements, unless other variables (e.g. application of analgesic) or factors (e.g. destruction of nerve) were added in the experimental set-up. This is due to the fact that muscle action potential roughly lasts for 2 to 3 ms and conduction velocity is approximately 5 m/s. Refractory periods are also not reasons for the delayed movement of the leg since an absolute

10 refratory period only lasts for about 1 to 3 ms (Frankenhaeuser & Wilden, 1956). The values used for characterizing signal conduction in nerves are measured in terms of milliseconds and in this experiment, time intervals were applied in seconds. This only implicate that any stimulation applied in the sciatic nerve in this experiment should result to leg movement.

11 REFERENCES Frankenhaeuser, B. & Widen, L. 1956. Anode break excitation in desheathed frog nerve. Journal of Physiology, 131, 243-247. Hussain, D. & Thai, C. 2002. Caffeine increases the amplitude of the compound action potential of the frog sciatic nerve. Department of Electrical Engineering and Computer Science, 3-13. Massachusetts Institute of Technology. 1999. The compound action potential of the frog sciatic nerve. Department of Electrical Engineering and Computer Science, Harvard-MIT, 1-26. McGill Physiology Virtual Lab. 2011. Compound action potential. Retrieved from: http://www.medicine.mcgill.ca/physio/vlab/CAP/refract.htm Nielsen, J. C. & Bjerring, P., & Arendt-Nielsen, L.1991. A comparison of the hypoalgesic effect of Paracetamol in slow-release and plain tablets on laserinduced pain. British Journal of Pharmacology, 31, 267-270. Vizsolyi, E. 2004. Nerve conduction in frogs and humans. Department of Zoology, University of British Columbia, Vancouver, British Columbia.