Human physiology: compound action potential lab, Summaries of Physiology

Download Human physiology: compound action potential lab and more Summaries Physiology in PDF only on Docsity! lOMoARcPSD|2805715 L. Sherwood, Human physiology: compound action potential lab Biology 342L Compound Action Potential of the Sciatic Nerve of Leopard Frog, Rana pipiens Introduction The fundamental unit of the nervous system is the neuron. Neurons and other excitable cells (e.g., muscle) produce action potentials when they receive electrical or chemical stimulation. The action potential (AP) occurs as a large-scale depolarization when positive ions (sodium ions) rapidly enter the neuron via specialized voltage-gated membrane channel proteins. APs are “all-or-none” events, meaning that in any given axon, all action potentials are of the same amplitude and duration, regardless of the strength of stimulus. Once an AP begins, it propagates down the length of the axon (the specific processes involved are described below, under “Background”). When the AP reaches the end of the axon, a neurotransmitter is typically released into the synapse. After an AP occurs, the neuron must repolarize. During this time, called the refractory period, the neuron is incapable of producing another AP. Measuring APs from single neurons requires highly specialized equipment. In this lab, you will record compound action potentials (CAPs) from the isolated frog sciatic nerve. A CAP represents the summed action potentials of the multitude of neurons that are present in this ‘compound’ nerve. Background In 1944, two physiologists (Erlanger and Gasser) won a Nobel Prize for their development of a cathode ray oscilloscope, that they then used to carry out experiments on nerve conduction. They used the sciatic nerve of the bullfrog, Rana catesbeiana, and characterized the conduction velocity of different families of nerve axons --the sciatic nerve contains many individual neurons. Electrodes were placed at two different positions outside of the nerve to record the electrical activity on the oscilloscope. The original data were thus generated from extracellular recordings of complex nerves: the “compound action potential” (CAP). Since then, progressively more sophisticated and sensitive recording techniques, such as the use of intracellular probes and computer- aided analyses, have been used to advance our understanding of neuron cell function. In the present laboratory, we use the original approach of placing electrodes against the outside of a nerve, with the modernization of using computer-aided analysis utilizing the PowerLab interface and Chart 7.3, which simulates a chart recorder. We will study the sciatic nerve of the leopard frog, Rana pipiens. Nerve Conduction: The resting membrane potential (Vrest) of most neurons is typically between –90 to –40 mV (i.e., the cell’s interior is negative with respect to the outside). To cause an AP, the inside of the membrane must be depolarized (made less negative) to the point that the threshold voltage (-55mV) is reached. At threshold, the activation gates of voltage-gated Na+ channels open and Na+ rushes inward, down its electrochemical gradient. The upstroke phase (i.e., Na+ rushing in) of the AP brings the membrane potential (Vm) to a peak between +20 to +40 mV. At the peak, inactivation gates on the Na+ channels close, preventing further influx of Na+, and voltage gated K+ channels open allowing an efflux of K+ from the cell. This results in repolarization (the downstroke phase), in which the membrane potential becomes negative once again. The K+ channels are slow to close, resulting in hyperpolarization, where the membrane potential becomes more negative than resting potential for a short period of time. During this time, the gates on the sodium channel reset (inactivation gates open, close, the Na+/K+ pumps re- establish resting membrane pot tory period, characterized by an inability to generate another AP, e time when the Na+ gates reset. During this time, it is impossible e reversed (Na+ inside, K+ outside) and b) the gates on th losed. A relative refractory period occurs from the time tha tial is re-established. During this time, the K+ channels are still o quired to generate an action potential. 1 5) Cut a strip of kimwipe and lay it over the wires in the nerve bath so that it touches both stimulating electrodes and both sets of recording electrodes. Moisten the paper strip with frog Ringer’s, and place the cover on the nerve bath. This arrangement will be used to test the connections. 6) Turn on the PowerLab. 7) Open the LabChart program, and open the settings file “Frog Nerve CAP Settings”. If you are not sure where to find this file, it should be under the Experiments tab of the Welcome Center. 8) LabChart will open up in Scope View. From the toolbar, select the Macro menu and choose Test Connection. LabChart will now automatically record data for one second. A series of stimulus pulses will be recorded (Figure 3). You may need to adjust the axes or auto scale to see the signal. 9) If no signal is recorded, check to make sure the clips are secure and the filter paper is moist and draped over all the active wires in the Nerve Bath. 10) Once the connections are tested and working, remove the kimwipe and proceed to next step. 11) Place your dissected sciatic nerve into the Nerve Chamber. Lift the nerve by grasping the thread tied to the end of the nerve (again, avoid grasping the nerve with the forceps). As illustrated in Figure 6, lay the nerve across the wire electrodes, make sure it is in contact with each. The fatter end of the nerve should be placed at the end where the stimulator electrodes are located. Adjust the position of the recording electrodes as necessary. Place the cover on the Nerve Chamber to avoid drying of the nerve. 12) Make sure the nerve is in contact with each of the active connections. If the nerve is too short, adjust the position of the recording electrodes as necessary. Place the cover back on the Nerve Bath. C. Determination of threshold voltage and maximal CAP amplitude 4 In this part of the experiment, you will give the nerve a series of electrical stimuli, each increasing in amplitude from a minimum of 20 mV and progressively increasing to 400 mV. You will then be able to determine the threshold voltage (“threshold potential”) for the nerve, as well as the voltage required to generate a maximum CAP. 1) From the LabChart toolbar select Macro: Threshold. 2) LabChart will automatically stimulate the nerve and record 20 pages of data. 3) Fill in Table 1 below, following the directions from the Analysis section below. Analysis: First, set the screen magnification to 5:1 –this is done at the bottom right of the screen. Secondly, be sure to scroll leftward to the beginning of the data for this experiment (the programming carries out the experiment in terms of stimulations, etc., and the data move rightward as they are recorded, leaving some of the earlier data hidden to left!). From your data trace, use the waveform cursor to measure CAP amplitude at each stimulus voltage. To do this more easily, use your mouse to scroll over each “event” and then use the zoom window to expand the view (also, the values you need will appear at the top of this window). The program runs the experiment automatically, with increasing stimuli from a beginning of 20 mV and going up to 400 mV in 10 mV steps; you will see that the stimulator settings (from program) are already entered in the first column of Table 1. Record your measured CAP peak amplitudes in Table 1. Note the stimulus level where you first see a CAP —this is your threshold potential (voltage). Also, you will see later that the CAP amplitude does not increase, despite increasing stimulator voltages, and this is your maximum CAP amplitude. [Be sure that you understand why you see increasing CAP amplitudes after threshold is reached, which is not expected if you were looking at one axon, and why the amplitudes level off.] Table 1. CAP amplitude versus stimulus intensity. Stimulus amplitude (mV) CAP amplitude (mV) Stimulus amplitude (mV) CAP amplitude (mV) 20 0 220 5.673 40 0 240 6.234 60 0.537 260 6.248 80 2.291 280 6.159 100 5.084 300 6.070 120 5.936 320 6.120 140 6.241 340 6.103 160 6.000 360 5.323 180 6.327 380 6.275 200 5.541 400 6.156 Threshold stimulus voltage: 60 mV Maximum CAP amplitude: 6.327 mV D. Determination of the refractory period Before you perform this experiment, it is important that you complete the analysis for Part C (all of Table 1). In this part of the experiment, the PowerLab will stimulate the nerve with a series of pulses. In each block of 5 data, the interval between each stimulus pulse will decrease. You will be able to use this recording to determine the relative and absolute refractory periods of your nerve. 1) From your results in Table 1, determine the minimum stimulus voltage required to elicit a maximal CAP from your nerve. Indicate this voltage here: 180 mV, which will elicit a strong CAP response in your nerve. 2) From the LabChart toolbar, select Macro: Refractory Period. You will be instructed to enter the above voltage. 3) Chart will now record data in a series of 15 “time-blocks” (separated by dark lines). Each time-block is 10 milliseconds in duration and two pulses are presented to the nerve. The program will progressively (in each new time-block) speed the rate at which stimulations are given (in other words, the interval between stimulations will be decreased progressively). 4) Follow the directions from the Analysis section below. Fill in Table 2 on next page. Analysis: Using the mouse, select (scroll over) the first two CAPs* recorded in each time-block of data recorded, and open the zoom window [*Do not pay attention to the 3rd or later CAPs, as their size can be complicated by many factors!] Using the waveform cursor, find the amplitude for the second CAP and record the value in Table 2. The stimulus intervals (used by program) in this experiment are already recorded in Table 2; record your appropriate value in the adjacent column. Determine the stimulus interval where the amplitude of the second CAP shows a decrease (to ~1/2 of the first peak): this is the relative refractory period. Record this value at bottom of Table 2. Determine the stimulus interval where the second CAP completely (or nearly) disappears: this is the absolute refractory period. Record this value at bottom of Table 2. Table 2. CAP amplitude versus stimulus interval Stimulus interval (ms) Amplitude of second CAP 4.0 5.873 3.5 5.848 3.0 4.211 2.5 3.530 2.0 3.684 1.9 3.775 1.8 2.609 1.7 1.977 1.6 0.888 1.5 0.244 1.4 0 1.3 0 1.2 0 1.0 0 Relative refractory period: 1.8 ms Absolute refractory period: 1.4 ms E. Determination of nerve conduction velocity In this part of the experiment, you will calculate the velocity of the CAP as it travels down the nerve. 1) Using a ruler, measure the distance in mm between the negative (black) lead of the stimulating electrode and the negative lead of the recording electrode. Record this value in Table 3. 6