Exp 2 Physiology of Nerve a D

Physiology of the Nerve Astrid Amador, Mark Joseph Amancio, Mariah Jemm Amurao, and Rex Augustus Artuz 4Biology4 Group 1 Department of Biological Sciences College of Science University of Santo Tomas

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Abstract

The nervous system is a complex collection of nerves and specialized cells known as neurons that transmit signals between different parts of the body. It is essentially the body’s electrical wiring. Four activities were done to understand Keywords: the physiology of the nerve. Twitching was only observed through electrical Nerve function, effect among others in testing the function of nerves. Applying anesthesia and signal blocking, blocking the pressure increases the threshold voltage needed to elicit response reflex action as time goes by and had a minimum stimulus of 3 volts. The damaged brain and spinal cord of the frog showed minimal to no response in comparison to the nonpithed frog. For the action reflex and reflex time, it was observed that the 10% HCl gave the fastest reflex time averaging 0.56 sec. Introduction The nervous system consists of the brain, the spinal cord and the nerves that conduct impulses to and from the Central Nervous System (CNS). The nervous system consists of billions of neurons and neuroglial cells. These neurons are able to be stimulated by touch, light, sound and other stimuli, they also conduct impulses and communicate with each other. When the neurons receive electrical or chemical stimulation they produce an action potential. This study aims to determine the effect of mechanical, thermal, osmotic, and other factors on nerve conduction, the effect of single and double pithing on the different reflexes of a frog, to determine the response of the nerves that are exposed and not exposed to anesthetics, and to test the time of reflex action of the frog in different concentrations of acid. Materials and Methods Double-Pithing The frog was grasped using the left hand and the head was bended over the middle finger with the index finger. The foramen magnum was then located using dissection needle. The

dissection needle was pushed cranially into the foramen magnum to destroy brain. The dissection needle was then pushed caudally to destroy the spinal cord. Nerve Dissection The skin from legs and abdomen of the double-pithed frog was removed by cutting around the abdomen and peeling the skin downward and off the animal. The frog was kept moist using Ringer’s solution. The urostyle was grasped using forceps and was cut. The sciatic nerve was then located and lifted using a glass hook. The nerve from the spinal cord was cut and was deflected back onto the animal’s leg. A piece of thread was tied around the free end of the nerve. The nerve was severed from the gastrocnemius muscle. The nerve was placed in a petri dish containing frog Ringer’s solution. A. Nerve Function A muscle nerve preparation was made. The nerve was stimulated by pinching (mechanical), touching it with a hot glass rod (thermal), applying a few crystals of NaCl to

the fresh cut (osmotic), dipping the free end of the nerve in HCL (chemical), and applying a mild electrical shock using stimulator (electrical) B. Signal Blocking B1. Anesthetic solution The minimum stimulus that will elicit a response on the gastrocnemius muscle was determined by touching one end of the nerve with a stimulating electrode. Two cottons with anesthetic were placed between the middle portions of the nerve. This was covered with filter paper moistened with ringer’s solution. The free end of the nerve was then stimulated using the minimal stimulus previously determined. This was repeated with two-minute interval and the threshold stimulus was determined at each stage. The cotton was moistened with anesthesia periodically. The anesthetic was washed away with amphibian ringers’ solution and the minimum stimulus was determined at two-minute intervals. B2. Pressure Blocking The minimum stimulus that will elicit a response was determined. Pressure was applied gradually using prongs of forceps with parafilm on the middle portion of the nerve, which was then released immediately. The minimal stimulus was again determined and the same procedure was repeated at 1minute intervals. The results were tabulated. C. Functions of the Central Nervous System Nine physiological tests were performed for a non-pithed, single-pithed and double-pithed frog. It was first placed in a dissecting pan, the position of the head, eyes and legs were observed. The pulsation in the throat area was noted. Next, in the dissecting pan, the animal was placed on its back and the righting position of the frog as the pan is revolved horizontally was observed. The frog was then placed in a pail of water and the pulsation in

the throat area was observed. The frog was placed on the table and a loud noise was made. The reaction of the frog was observed. After that, the firmness and flaccidness of the frog’s legs was observed. Also, the hind leg of the frog was pulled and was observed if a resistance is present. Next, the toe of the frog was pinched and its reaction was noted. Then, the eyes of the frog were touched with a piece of cotton or tissue paper. Lastly, the toes of the right foot of the frog were soaked in heated water in a beaker. All the procedures were repeated and results were recorded. D. Reflex Action in the Frog The frog was single-pithed following the pithing procedure. The frog was suspended by the jaw with a wire hook attached to a clamp on a stand. The long toe of the frog was immersed with 10% sodium bicarbonate then tap water and it was subsequently immersed in 10% HCl. The time before the reflex action obtained without the toe touching any part of the beaker was noted. This procedure was repeated after 2 minutes but using different concentration of the acids. Results and Discussions Before examining the physiology of the nerve, double-pithing procedure was done. Double-pithing procedure is a known method to immobilized amphibians for experimental purposes. This is used to reduce the pain and bleeding of the frog (Suckow, Murthy & Silverman, 2007). After the double pithing, brain was destroyed therefore perception of sensory phenomena is impossible. Also, spinal cord was destroyed which makes the frog’s legs limp. However, the frog remained alive due to cutaneous respiration (Biopac, 2001). After the pithing procedure, nerve dissection procedure was performed. During this procedure, the frog should be kept moist by Ringer’s solution. Ringers solution is used to create isotonic solution relative to bodily

fluids of animal. It is a solution that typically contains sodium chloride, potassium chloride, calcium chloride dissolved in water (Adams, 2016). It is important to keep the frog moist because the frog is still breathing through the skin. After the nerve was dissected, metal tools or fingers must not touch it because this will cause the depolarization of membrane, which can affect future experiments. The nerve must also be kept moist by ringer’s solution so the nerve stays flexible. In addition, if it is dry, the nerve becomes brittle and hence, will break easily. (Biopac, 2001) Table 2.1: Nerve Function

Stimulus Mechanical Thermal Osmotic Chemical Electrical

Observation No twitching No twitching No twitching No twitching Twitching was observed

Expected Twitching of muscle

After dissection of the nerve, its function was examined through mechanical, thermal, osmotic, chemical and electrical stimulation. Most of the stimulus given to the frog elicited no response except for electrical stimuli as opposed to the expected observation (Table 2.1). There should have been a response for all the stimuli because frogs have sensory receptors i.e. mechanoreceptors, thermoreceptors, nociceptors and photoreceptors that recognizes stimuli and initiates sensory transduction. Sensory transduction is a process that converts the stimuli recognized by the sensory receptors to neural signals so that an electrical response in the cell membrane is elicited (McMillan, 2014). Initially, a neuron is at its resting membrane potential. i.e, the inside is negative relative to the inside due to the presence of protein ions with negative charge. In addition, during

resting membrane potential, most ion channels are closed. However, some potassium channels are open therefore potassium can freely diffuse out of the cell. The resting membrane potential of a neuron is about -70 mV which means that the inside of a neuron is 70 mV less than the outside. When a nerve is stimulated, the sodium channels are opened causing an influx of sodium ions into the cell membrane making the cell membrane more positive relative to the outside. This is called the depolarization state. When depolarization reaches about -55 mV, a neuron will fire an action potential (threshold). If the neuron does not reach this threshold level, no action potential is fired. After a cell has been depolarized, sodium ion channel begins to close. Meanwhile, the sodium potassium pump also pumps three sodium ions outside and two potassium ions inside. This will re establish the resting potential membrane of the nerve (Purves, 2001). The probable reason behind the observed result is that the threshold potential was not reached creating no response and the nerve was not kept moist. Table 2.2: Signal and Pressure Blocking

Interval 1 2 3 4 5 6 7 8 9 10

w/

w/o

Anesthetic

Anesthetic

3 6 9 9 12 9 12 -

3 3 4.5 4.5 4.5 4.5 3 6 4.5 4.5

Pressure Block 3 3 4.5 9 12 12 -

*Minimum Stimulus: 3 volts

After examining nerve function, the group also examined the effects of blocking the

signals by applying anesthetic solution and pressure. Local anesthesia inhibits depolarization of the nerve membrane by interfering with Na+ and K+ currents. Based on the results, applying anesthesia increases the threshold voltage needed to elicit response as time goes by. This is because the sciatic nerve is composed of three types of neurons based on diameter. Type A being the largest, Type B moderate, and Type C, the smallest. As a result, anesthesia block type C fibers easily than type A fibers (Gymrek, 2015). In the early time interval, the effect of anesthesia is not yet apparent because only type C fibers are blocked. However, as the time goes by, the Type A fibers are also blocked therefore no response was elicited. Meanwhile, when no anesthetic solution is applied, the threshold voltage also increases but very minimal compared to that with anesthetic solution because the signals are not blocked. However, the myelin sheath degrades with time. As a result the impulses are slowed down because the charges are not insulated. On the other hand, when pressure is applied, the nerve diameter was reduced until such time that no more signals are able to pass. In addition, the influence of pressure in blocking conduction is entirely dependent upon deformation of the tissue due to its unbalanced action (Causey, 1949). Table 2.3: Reaction of the single-pithed frog

Position of the head Righting In Water Reaction to Noise Firmness of Legs Eye Response

Lying position, eyes blinking, slow pulsation of throat are No reaction No Reaction No Reaction Flaccid Closes eye for a short period of time

Table 2.4: Reaction of the double-pithed frog

Position of the head Righting In Water Reaction to Noise Firmness of Legs Eye Response

Lying position, eyes are not blinking, slow pulsation of throat No reaction Does not do its righting position No movement Very flaccid No reaction

Table 2.5: Reaction of the non-pithed frog

Position of the head Righting In Water Reaction to Noise Firmness of Legs Eye Response

Normal position, blinking eyes, fast rhythmic pulsation of throat The frog immediately went to righting position The frog immediately went to righting position The frog jumped away from the source of the noise Firm hind limbs Closes eye for a long period of time

The non-pithed frog responded normally to the physiological tests that were given while the double-pithed frog has no reflex actions at all since the brain and spinal cord are fully damaged. However, the single-pithed frog still has little response action in comparison to the non-pithed frog due to the intact spinal cord. The CNS consists of the brain and the spinal cord. Its main focus is to coordinate incoming and outgoing neural impulses, integrating data, and forming motor output. When CNS receives a sensory input, it produces motor responses through nerves. The central nervous system is divided into the central, peripheral, somatic and autonomic nervous

Table 2.6: Reflex Action of the frog in HCl

Conc. 2% 4% 6% 8% 10%

1st trial 2.88 1.09 1.00 0.61 0.43

Time (sec) 2nd trial Mean 2.92 2.9 1.88 2.97 1.01 1.005 0.9 0.755 0.69 0.56

In this activity, we determined whether the reaction time to a stimulus is influenced by the strength of the stimulus. In this case, the stimulus is the hydrochloric acid (HCl). The skin of frogs and toads are more sensitive to drought and chemicals in water because it can absorb water and breathe through the skin as blood vessels are found underneath it. Acidic solutions can therefore be felt more by the amphibian skin, especially in areas where there is only a thin covering, like the toes, which is also convenient for determining the withdrawing reflex action as it only exposes a minimal surface area, can be easily dipped, and washed without affecting most of the body. Using 10% concentration HCl gave the fastest reflex

time, averaging 0.56 seconds, which is due to the higher proton (H+) available of all the concentrations. The acid disrupts the pH of the frog skin demanding water and other buffering agents from the skin as it is needed to balance out the disruption. Therefore, excess acid, like any other ions promotes a feeling of dehydration (Doratt, 2008) to the toad. 4 TIME!(SEC)

system. The central nervous system is made up of the brain, spinal cord and nerves while the peripheral nervous system consists of sensory neurons, ganglia and nerves that connect to one another and to the central nervous system. Nerves in the peripheral nervous system contain both sensory and motor neurons. The somatic nervous system is a division of the PNS that includes all of the voluntary efferent neurons. The SNS is the only consciously controlled part of the PNS and is responsible for stimulating skeletal muscles in the body while the autonomic nervous system is a division of the PNS that includes all of the involuntary efferent neurons. The ANS controls subconscious effectors such as visceral muscle tissue, cardiac muscle tissue, and glandular tissue

3 2 1 0 0%

5%

10%

15%

CONCENTRATION

Figure 2.1. Graph of the concentration of the acid vs time in seconds showing that as the concentration of the acid increases, the reaction time decreases.

The time 1.005 s and 0.755 s for 6% and 8% respectively agrees that the lower the acid concentration the less effect there is to the frog giving longer reflex time. Exposure to the acid is done from the lowest concentration to the highest concentration because the sense might get used to the acid sensation in higher concentration and will then tolerate the succeeding lower concentration acids. Washing the frog with 10% sodium bicarbonate and water after every exposure to acid is important to stabilize and ideally get the previous condition of the frog, and it is done together with keeping the frog moist. Human errors contributed might be not being able to wipe properly the toe after washing with alkali and water. Drying it too much might have increased or could have diminished the sensitivity of the toe compared to the natural conditions or the time when the experiment began when nothing has been done to the toe prior to the experiment. The experiment

shows the susceptibility of the amphibian skin to slight changes in acidity. Conclusion The observations say otherwise but stimuli of different kinds (mechanical, thermal, chemical, osmotic, and electrical) to the nerve will elicit twitching of the muscle. In signal blocking, by applying pressure to the nerve, the diameter of the nerve becomes smaller and there will come a time that the signal will not be able to pass. Applying anesthetic to the nerve increases the threshold voltage needed to elicit a response. A nonpithed frog would react normally to physiological tests, while a single pithed frog will only react when it’s eyes is touched and the eyes are still blinking. While a double pithed frog would not react at all to the physiological tests. The reflex action of the frog in response to stimulus is faster in a high pH condition. References Adams, M. P. (2016). Pharmacology: Connections to Nursing Practice (Second ed.). Content Technologies. Biopac. (2001). Frog Pith & Preparation. Retrieved February 21, 2016, from http://www.biopac.com/wpcontent/uploads/a01.pdf Causey, G., & Palmer, E. (1949). The effect of pressure on nerve conduction and nervefibre size. The Journal of Physiology, 109(12), 22--231. Retrieved February 21, 2016, fromhttp://www.ncbi.nlm.nih.gov/pmc/articl es/PMC1392600/?page=11 Doratt, RE. 2008. The Effects of Skin and Body Hydration on theSusceptibility of the Frog, Eleutherodactylus coqui, to Citric Acidas a Control Agent. Accessed from:

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Gymrek, R. (2015). Local and Regional Anesthesia. Medscape. Retrieved February 21, 2016, from http://emedicine.medscape.com/article/1831 870-overview McMillan, R. (2014). Biology: The Dynamic Science (Third ed.). Cengage Learning. Purves D, Augustine GJ, Fitzpatrick D, et al., editors. Neuroscience. 2nd edition. Sunderland (MA): Sinauer Associates; 2001. Available from: http://www.ncbi.nlm.nih.gov/books/NBK10 799/ Suckow, M., & Murthy, S. (2007). The IACUC Handbook (Second ed.) (J. Silverman, Ed.). New York: CRC Press.