Nerve Physiology Experiment #2 PDF

2-1 NERVE PHYSIOLOGY Experiment #2 PHYSIOLOGICAL PRINCIPLES he body is constantly being barraged with changes in both the internal and the external T environment. Sensory re...

2-1 NERVE PHYSIOLOGY Experiment #2 PHYSIOLOGICAL PRINCIPLES he body is constantly being barraged with changes in both the internal and the external T environment. Sensory receptors are responsible for detecting these changes and conveying the information to the central nervous system for integration. This information is transmitted to the CNS via neurons. Other neurons carry messages from the CNS to the body’s effectors that respond to the change in the internal or external environment. The nervous system is a highly organized communication system serving the body and the neuron is the basic functional unit of the nervous system. This experiment examines some of the properties of neurons and nerves. Electrical Forces and Transport Some atoms or molecules carry either a positive or negative electrical charge. Any charged atom or molecule is an ion. Two positively charged ions (cations) repel each other, as do two negatively charged ions (anions). However, a positive ion and a negative ion attract each other. The attraction or repulsion of ions in a solution sets up forces, besides the concentration gradient, that drives passive membrane transport mechanisms. Salt solutions, in particular, show electrical forces. Sodium (Na+) and potassium (K+) ions, for example, repel each other in solution because both are positively charged. Yet, each attracts negatively charged chloride (Cl-) ions. In a solution, ions are under the influence of pressure, concentration, and electrical forces. Resting Cell Membrane Potentials In the living organism, the solutions that make up the extracellular fluid are electrically neutral, as the number of negative and positive electrical charges found in the intracellular fluid are equal. Still a voltage, or resting membrane potential, exists across the membrane of all cells. The resting membrane potential of a cell is defined as the electrical potential difference across the plasma membrane when the cell is in a non-excited state and is due to an imbalance of charged particles (ions) between the extracellular and the intracellular surfaces of the membrane. At rest, the membrane has a net positive charge on the outer surface and a net negative charge on the inner surface. For an uncharged, non-ionized molecule, the only passive force that can cause movement of the molecule is the concentration gradient. However, if the particles in the solution are electrically charged, their movement may be influenced by an electrical gradient as well. Electrical gradients as well as concentration gradients exert forces on ions in solution. Therefore, equilibrium across a selectively permeable membrane of electrically charged molecules, with differing concentration gradients on either side of the membrane (Table 2-1), results from two (2) mutually exclusive tendencies. These tendencies include: 1. the attempt to achieve equal concentration of all molecules involved on both sides on the membrane, and 2. the attempt to achieve electroneutrality throughout the system. The movement of individual ions across the cell membrane, either into or out of the cell, is determined by the net electrochemical driving force (i.e., the sum of the movement due to the ion’s concentration and electrical gradients). 2-2 Table 2-1. Concentration of Various Chemicals in Extracellular and Intracellular Fluids. Na+ K+ Ca2+ Mg2+ Cl- Amino Acids Glucose mEq/L mEq/L mEq/L mEq/L mEq/L mg% mg% Extracellular 142 4 5 3 103 30 90 Intracellular 10 140 1 58 4 200 0 OR mM mM mM mM Extracellular 145 5 100 5 Intracellular 15 150 7 0–1 Ion Channels Embedded in the plasma membrane of cells are two (2) major types of ion channels, (1) leakage channels, and (2) gated channels. Ion channels are selective for the type of ion that can pass through. Leakage channels are always open allowing their specific ion (such as K+) to freely cross the membrane as directed by its electro-chemical gradient. There are two (2) types of gated channels. Gated channels usually have a molecular structure that keeps the channel closed until appropriately “stimulated.” Voltage-gated channels open and close in response to changes in the membrane potential. Chemically-gated (also known as ligand-gated) channels are stimulated to open when the appropriate neurotransmitter binds to a receptor associated with the channel. Development of the Resting Membrane Potential In the resting state, K+ ions diffuse down their concentration gradient through leakage channels from the inside of a cell to the outside (Fig. 2-1). Chloride ions also diffuse freely across the membrane from the outside inward. Because there are very few Na+ leakage channels in the membrane (as compared to K+), extracellular Na+ attempts to diffuse down its concentration gradient, to the inside of the cell, but cannot because of the low membrane permeability for sodium ions. In addition, the large negatively charged intracellular proteins and molecules (designated by A-), cannot diffuse across the membrane down their concentration gradient toward the outside of the cell. If the system starts with no potential difference across the membrane, one is soon generated by the diffusion of K and Cl- along their concentration gradients. Potassium ions diffuse out of the cell. Although positively charged, their + outward movement cannot be accompanied by a corresponding movement of A- ions, nor can an equal number of Na+ ions enter the cell in exchange for K+. Potassium ions reach the outside of the membrane alone and are not replaced by Na+. As a result, the outside of the membrane (not the extracellular fluid) acquires a net positive charge, and the inside develops a net negative charge. Since charges of opposite sign attract one another, the excess anions on the inside of the cell attract the excess K+ ions outside the cell. Thus, the excess charges stay in the vicinity of the membrane. The electrical field created retards further outward diffusion of K + or any other cation. The positive charges on the outside of the membrane exert an inward repulsive force on a cation trying to pass through the membrane. The negative charge on the inner surface of the membrane exerts an added inner attractive force.  2-3 Figure 2-1. Development of a Resting Membrane Potential. If diffusion continued indefinitely, sodium and potassium concentrations would eventually reach an equilibrium between the inside and outside of the cell. This does not happen because of an active transport mechanism, the sodium- potassium pump. For every cycle of the pump, three sodium ions move out of the cell while two potassium ions are pumped back in. This pump helps to maintain the concentration gradients and therefore the steady state of the polarized cell membrane. To summarize then, the resting cell membrane potential exists because: 1. The cell membrane is more permeable to K+ ions than to Na+ ions. Therefore, the K+, which are relatively more concentrated inside the cell, diffuse out of the cell. Na+, which are relatively more concentrated outside the cell, attempt to diffuse inward. This movement is considerably less than the outward movement of K+ because of the selective permeability of the cell membrane. 2. The cell membrane is essentially impermeable to the large anions (negatively charged protein molecules), which are present inside the cell. Therefore, fewer negatively charged particles move out than positively charged. 3. A sodium-potassium pump transports Na+ to the outside and K+ to the inside, with three (3) Na+ ions moved out for every two (2) K+ ions moved in. Thus, more positive ions are moved out of the cell than are moved into the cell with each cycle of the pump. The potential gradually becomes more negative on the inside with respect to the outside of the cell membrane. Excitable Cells Electrical potentials exist across the membranes of all cells of the body. All membranes have the ability to separate ions, but nerve and muscle cells have, in addition, two distinctive properties. These are excitability (the ability to form action potentials), and conductivity (the ability to propagate action potentials). These properties involve changes in the resting membrane potential. Changes in membrane potential can be produced by changes in membrane permeability to ions or changes in the concentration of ions on either side of the membrane. 2-4 Neurons Nerve cells known as neurons make up nerves (Fig. 2-2). Neurons have three parts: (1) soma (cell body), which contains many cell organelles; (2) a system of dendrites, short cytoplasmic processes that conduct electrical activity toward the soma; and (3) an axon, a long cytoplasmic process that conducts electrical activity away from the soma. We will discuss properties of muscle cells in Experiments #3, #4, and #9. Figure 2-2. The Neuron. Excitability The property of excitability refers to the ability of these nerve and muscle cells to generate and respond to electrical signals. Transient changes in the membrane potential from its resting level can alter cellular activities. These changes allow nerve and muscle cells to process and transmit information. Electrical signals occur in two (2) forms: Graded potentials, which signal over short distances, and Action potentials, which signal over long distances. Graded Potentials Graded potentials are changes in the membrane potential either above or below the resting membrane potential. They are called graded because the amplitude (strength) of the graded potential (Fig. 2-3) is directly proportional to the amplitude of the stimulus that initiates it. Figure 2-3. Changes in Membrane Potential with Differences in Stimuli Strength. Graded potentials are initiated by some change (stimulus) in the neuron’s environment that causes gated channels to open. Gated channels, as discussed earlier, are closed until the appropriate stimulus causes them to open. They remain open only as long as the appropriate conditions exist. The membrane in its resting state (resting membrane potential) is said to be polarized as a result of the separation of charges on either side of the membrane. If the membrane potential as the result of a graded potential becomes more negative, it is said to be hyperpolarized. If on the other hand it becomes less negative on the inside with respect to the outside, it is depolarized. When a membrane potential has been either hyperpolarized or depolarized and then returns to the resting membrane potential, it is said to be repolarized.  2-5 Figure 2-4. Changes in Membrane Potential. The Action Potential The action potential is the basic unit of electrical activity in the nervous system. Action potentials (Fig. 2-5) can be described as the progression of an abrupt change in the resting membrane potential along an excitable cell membrane (nerve or muscle). Figure 2-5. Schematic of the Propagation of an Action Potential. Note that the wave of depolarization moves in both directions from the point of stimulation. The sequence of events (Fig. 2-6) leading to action potentials is: 1. An adequate (threshold) stimulus (chemical, electrical, or mechanical) of the membrane occurs at some point, altering the membrane potential via a graded potential. 2. Voltage gated Na+ channels open, creating an increased membrane permeability for sodium at the point of stimulation. 3. Sodium ions diffuse rapidly inward down their concentration gradients. 4. As sodium ions move inward, the transmembrane potential reaches zero (the membrane becomes locally depolarized). 5. Sodium ions continue to diffuse inward, and the inside of the membrane becomes positively charged relative to the outside (depolarization). 6. Depolarization at the original site of stimulation causes a local current that acts as a stimulus to the adjacent region of the membrane and the action potential is propagated along the axonal membrane. 7. Meanwhile, at the point originally stimulated, there is now a decrease in the membrane’s permeability to sodium, as Na+ gated channels are inactivated. 2-6 8. An increased membrane permeability for potassium follows as voltage gated K+ channels open. 9. Potassium ions rapidly diffuse outward, again making the inside of the membrane again negative with respect to the outside (repolarization). 10. Following an action potential, the membrane potential becomes more negative than the resting potential for several milliseconds. This hyperpolarized state is the result of more K+ moving out of the cell than necessary to restore the resting potential. 11. Now the cycle repeats from Step 1, relative to the advanced site along the axonal membrane. 12. After the action potential, the on-going action of the sodium-potassium pump transports Na+ back out of and K+ back into the cell. Figure 2-6. Changes in Membrane Potential (Action Potential). If we place one recording electrode inside a nerve or muscle cell and a second recording electrode on the surface of the cell, then measure and graph the changes in potential across the membrane against time, we get the tracing seen in Figure 2-6. Threshold Stimulus The resting membrane potential of a nerve cell is -70 mV1, i.e., the inner surface of the membrane is normally 70 millivolts less than the outer surface. A threshold stimulus sufficiently increases the permeability of the membrane to Na +. This raises the membrane potential to a negative 55 mV. Once this threshold potential is reached, complete depolarization and repolarization occurs and generates an action potential (Fig. 2-7). 1 The resting membrane potential (RMP) varies with the type of cell. For example, skeletal muscle has a RMP of ~ -85 mV.  2-7 Figure 2-7. Changes in Membrane Potential in Response to Increasing Stimulus Strength. All or None Law The action potential is an all or none phenomenon. This means that if a stimulus is adequate to cause the membrane potential to reach the threshold potential, a maximal response is evoked. Sub-threshold stimuli do not cause any response and stimuli greater than threshold do not cause a greater response than that evoked by the threshold stimulus. Refractory Period In the interval from the onset of an action potential until repolarization is complete, no new stimulus can elicit another response. The non-responsive period is the absolute refractory period. During the absolute refractory period, the Na+ channels are inactivated and cannot be opened. Following the absolute refractory period is an interval during which the cell membrane will not respond to a normal threshold stimulus. It will, however, respond to a supra-threshold stimulus. This period of less than normal responsiveness is the relative refractory period and results partially because the membrane is hyperpolarized. Factors Influencing Conduction Velocities Several factors influence the speed at which axons of nerve cells conduct impulses. 1. Diameter of the conducting fiber: Conduction velocity is directly proportional to the fiber diameter. Large fibers conduct action potentials faster. 2. Temperature of the cell: Warmer nerve fibers conduct action potentials at higher speeds. 3. Presence of myelin sheath: Myelinated fibers conduct impulses more rapidly than unmyelinated. The action potentials are generated only at the nodes of Ranvier, instead of progressing from point to point along the axon. This gives the appearance of a leaping or jumping of the impulse along the axon and is called saltatory conduction. Saltatory conduction is not only faster, but also consumes less energy, because the pumping of sodium and potassium ions need occur only at the nodes. 4. Pharmacologic agents: Some drugs affect the stability of the nerve cell membrane. Procaine or Novocain stabilizes the neuronal membrane and prevents the initiation and propagation of action potentials by blocking the entry of Na+ into the cell. It is used clinically as a local anesthetic. 2-8 The Synapse A synapse (Fig. 2-8) is the specialized junction through which impulses pass from one neuron to another. It is the synapse that makes nerve transmission unidirectional. If an action potential is experimentally initiated anywhere along the axon, it can travel in both directions from the point of stimulation, but in only one direction through the synapse to another neuron or an effector. In the body most action potentials originate at the axon hillock. The steps in the process of synaptic transmission are: 1. An action potential spreads over the axon terminal causing a change in membrane potential. 2. Ca2+ voltage gate channels open allowing an influx of Ca2+ from the extracellular fluid into the cell. 3. Calcium, serving as an intracellular messenger causes synaptic vesicles to fuse with the membrane in the axon terminal. Ca2+ ions are removed from the intracellular fluid via active transport mechanisms. 4. Vesicles release neurotransmitter via exocytosis into the synaptic cleft. 5. Neurotransmitter diffuses across synaptic cleft to the post-synaptic membrane. 6. Neurotransmitter combines with specific receptors on chemically gated channels on the post-synaptic membrane. 7. Permeability of the post-synaptic membrane is altered, as chemically gated channels open allowing the rapid influx of Na+ into the post-synaptic cell. 8. If enough transmitter binds so that the membrane reaches threshold, an action potential is generated on the post- synaptic neuron. The action potential is the result of voltage gated channels opening allowing a rapid influx of Na+ ions. 9. Neurotransmitter is removed from the synapse (1) by being enzymatically degraded, (2) by being taken back up into the pre-synaptic terminal, or (3) by diffusing out of the synaptic region. Similar events occur between a neuron and the cell membranes of the peripheral nervous system effectors (skeletal, smooth, or cardiac muscle and glands). Figure 2-8. The Synapse.  2-9 Compound Nerves Nerves are made up of several populations of axons rather than a single axon. As a result, action potentials recorded from whole nerves are complicated by two factors. First, the action potential amplitude is graded with stimulus intensity. In each axon of the nerve, the action potential is an all-or-none response, but as more and more axons depolarize to threshold by increases in stimulus intensity, the compound nerve action potential amplitude increases until all of the axons have been stimulated (Fig. 2-9). Second, the axons within a nerve exhibit differences in conduction velocity because of differences in axon diameter and myelination. As a result, the action potential may show humps in its falling phase corresponding to these different groups of axons. Figure 2-9. Recruitment of Neurons in Compound Nerve with Increasing Stimulus Voltages. 2-10 For Your Information: Studying Action Potentials Action potentials recorded from whole nerves are compound nerve action potentials (CNAP) and represent the sum of electrical activity occurring at any given time in the compound nerve. To record compound potentials, the recording electrodes are placed extracellularly as shown in Figure 2-10. In this arrangement, a difference of electrical potential is recorded between the “active” electrode as conducted action potentials pass it and the “reference” electrode. We define electrical potential as the relative voltage at a point in an electric field with respect to some reference point in the field. Figure 2-10 shows diagrammatically an electrode arrangement suitable for recording the compound action potential of a nerve. Two recording electrodes, A and B, are attached to the nerve’s outer surface. In the resting state, the fibers under both recording leads are externally electropositive, and no difference of potential between them is recorded. If the nerve fibers are stimulated, action potentials along the fibers (hatched area) approach electrode A from the left and reach the position indicated in Panel 1, where a difference of potential is recorded between A and B. The active fibers at A are externally electronegative to the yet quiescent portion of the same fibers at B. This difference in potential registers as an upward deflection of the oscilloscope recording beam. In Part 2 of Figure 2-10, the conducted volley progressed so that both electrodes are in contact with equally depolarized fibers. Consequently, the recording beam returns to zero potential. In Part 3, the wave of depolarization progresses beyond A, i.e., the fibers under A repolarize, so that B is now relatively negative to A and the recording beam deflects downward. As the depolarization passes beyond B, the beam returns to zero. The tracing resulting from this process is a diphasic compound action potential. Figure 2-10. Recording of Diphasic Action Potentials by External Electrodes. Left: Gray area represents action potential progressing from left to right in 1, 2, and 3. In 1, electrode A is negative with respect to electrode B. In 2, A and B are equipotent. In 3, B is negative with respect to A. Right: Line trace recorded is diphasic action potential. Numbered arrows below indicate instantaneous potential differences corresponding to three stages of conduction shown at left.  2-11 CLINICAL APPLICATIONS Multiple sclerosis is a disease that results in the progressive loss of myelin sheaths of neurons in the central nervous system. The sheaths deteriorate to scleroses, or hardened scars. It is thought to be an autoimmune disease triggered by a viral infection. The average age of onset is 33 years. As a result of demyelination, normal transmission of nerve impulses is impaired. Neurological defects include muscular weakness, tremors, paralysis, impaired vision and speech, and decreased sensation. Sciatica is an inflammation of the sciatic nerve or its tracks. This results in pain that passes from the back or thigh down its length into the leg, foot, and toes. The most common cause of sciatica is a slipped intervertebral disc. Epilepsy is thought to result from nerve impulses reaching synaptic knobs at rapid rates. These impulses originate from certain brain cells and result in violent contractions of skeletal muscle. In time the synaptic knobs run out of neurotransmitter substances and the seizure subsides. Dilantin, a drug used to treat epilepsy, stabilizes the neuron membranes and increases the effectiveness of the Na+ active transport system. As Na+ moves from inside the neurons, the thresholds of the membranes lower and stabilize against excessive stimulation. EXPERIMENTAL OUTLINE I. THE NERVE IMPULSE VIDEO II. NERVE PHYSIOLOGY SIMULATION A. Introduction B. Methods: Sciatic Nerve Preparation C. Simulation Program 1. Using the Program 2. Experiments [Titles in brackets reference topic in Simulation Program] a. The Compound Nerve Action Potential [Bi Phasic/Mono-Phasic Action Potential] b. Response to Stimulus Strength [Stimulus strength - response relationship] c. Refractory Period d. Conduction Velocity e. The Effect of Temperature f. The Effect of Procaine g. Directionality of Propagation (optional)