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蚯蚓巨轴索的动作电位 |
Equipment:
1. Background.Giant axons Many invertebrates have evolved giant axons for rapid conduction of action potentials, typically as part of circuits mediating rapid escape reflexes. The squid's giant axon is the best-known example, but the earthworm's giant axons have also been studied extensively, and earthworms have the advantage of being relatively easy to keep in the lab. We will make extracellular recordings from the giant fibers, which will enable us to observe many of the important aspects of action potentials. The experiment is adapted from Welsh, Smith & Kammer, Laboratory Exercises in Invertebrate Physiology, pp. 139-141. An annelid worm's nerve cord is near the ventral surface of the worm, at the bottom in the cross-section on the left. (The large structure that fills most of the body cavity is the intestine.) The giant axons appear as three large profiles (gf) near the dorsal surface of the cord, seen better in the close-up figure. The median giant axon in the center is larger (and conducts more rapidly) than the two lateral giants next to it. The remainder of the nerve cord is largely neuropile (np), where synaptic connections are made. Profiles of ordinary-sized axons can also be seen, and a few neuronal cell bodies appear around the periphery. Each giant axon is formed from many individual neurons whose axons fuse into a single functional unit, but whose cell bodies remain separate. In addition, the two lateral giants are interconnected at many points along their lengths and normally fire together. (They jointly contribute only one spike to an extracellular record.) In mediating the startle response, the median giant receives sensory input from the anterior end of the worm, and the laterals from the posterior, so that normally the median and laterals conduct in opposite directions. However, when action potentials are electrically stimulated (as in our experiment), the spikes propagate away from the stimulating electrodes in both directions. Extracellular recording of action potentials An electrode inside a neuron detects an action potential as a positive-going change from the usually negative resting potential of the cell. The action potential is produced because a few positive (sodium) ions enter the cell from the fluid around the cell. But what would be detected by an electrode outside the neuron? Since the fluid outside a neuron is connected to the other body fluids, at rest its electrical potential is zero (ground). When an action potential passes by, some positive ions leave the extracellular fluid and enter the neuron. For the brief moment of the action potential, the fluid outside the neuron has a deficit of positive ions. A deficit of positive ions is the same as an excess of negative ions. Thus, an electrode outside a neuron sees a negative-going change in potential when an action potential sweeps by. In addition, if two nearby extracellular electrodes (such as the two hooks in our experiment) are connected to the two inputs of a differential amplifier, each electrode will see the same action potential, but with a slight delay as the action potential travels by. The amplifier inverts one of the signals, turning the negative-going spike into a positive potential. The result at the output of the amplifer is a biphasic spike: a negative peak from the non-inverting input, and an upside-down (positive) peak from the inverting input. These two components usually overlap, adding up so that one peak appears bigger than the other. We need substantial amplification because an extracellularly recorded action potential is very small, typically in the range from a few microvolts up to a few millivolts. An electrode in the tissue next to an axon detects only a fraction of the flow of ions around the axon, and axons far away from the electrode will appear to have smaller spikes. In addition, nerves consist of bundles of axons. There may be just a few axons or as many as several thousand, depending on the particular nerve. Axons with large diameters (and large membrane surface areas) will have larger ionic currents flowing around them, and their action potentials will appear bigger to an extracellular electrode than those from small axons. The result of variations in axon distances and diameters is that extracellular records show spikes of many different amplitudes. The extracellular spikes from giant axons are very big. If you set the gain on your amplifier and oscilloscope to see spontaneous activity in ordinary neurons, spikes from the giants will probably go off-screen and may even be clipped by the preamplifier. (Clipping is the squaring off of the top or bottom of any amplified signals that exceed the amplifier's maximum.) You may need to reduce the amplifier's gain to bring the spikes into an appropriate range. 2. Equipment.Before dissecting an earthworm, you should briefly explore the functions of two new pieces of equipment. Grass SD9 stimulators |
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Our stimulators produce two sets of electrical pulses: an "output" stimulus pulse for shocking a nerve, and a synchronizing pulse for triggering the oscilloscope. Begin by setting up the trigger connection: use a bnc-banana cable to connect the stimulator's PREPULSE sync terminal to the oscilloscope's external trigger connector. (Be careful to connect the grounded side of the cable to the stimulator's [green] ground terminal.) Then temporarily use another bnc/banana cable to connect the stimulator's output to channel 1 of the oscilloscope. The output voltage produced by the stimulator appears between the red and black binding posts in its lower right corner. Neither one of these posts is grounded, so the cable's dual-banana plugs could be attached with either of the posts connected to ground. To be consistent with convention, make the red post the "live" one and the black post the grounded one. (Some of the stimulators have a third, green post that is grounded; this is not part of the pulse-producing circuitry.) Set the stimulator's voltage output to the x1 range, and adjust the oscilloscope's vertical sensitivity appropriately. Turn the stimulator's power on (you can leave it on for the rest of the experiment), and set the series of switches across the bottom of the stimulator as follows:
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Set the oscilloscope's trigger source to external, adjust the trigger level, and observe the stimulus pulses as you try out the functions of the various dials and switches. The relationship between the synchronizing pulse (which goes to the trigger circuit of the oscilloscope) and the stimulus pulses (which normally go to electrodes on the nerve) is shown in the following figure. Make sure you know the effect of the TWIN PULSES setting, which you can observe by using a brief pulse DURATION and a long interpulse DELAY. (The oscilloscope sweep must be slow enough to see both of the twin pulses). Vary the DELAY to see how the time between the pair of pulses changes.
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Synchronizing and stimulus pulses for twin-pulse setting
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When you restore connections to use for the rest of the experiment, you should leave the trigger settings as they are, but you will need to change the oscilloscope's vertical input settings to AC, connect channel one to the output of the preamplifier, and most likely increase the vertical sensitivity considerably. You should also reset the stimulator initially for regular (not twin) pulses, a low frequency, brief delay, brief duration, and low voltage. You can then increase the voltage as you watch the nerve's response. Gould "EasyGraf" chart recorders |
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Our chart recorders use thermal array technology: a fixed writing element composed of many tiny dots heats up a special paper to print the trace, the chart grid, and annotations. The chart recorders can write extracellular action potentials directly from the preamplifier, but today we will use them to plot digitized traces from the oscilloscope screen. The "plot" output from channel 1, on the back of the oscilloscope, is connected to the recorder's channel-1 input. The channel's vertical sensitivity is set by a combination of a pushbutton switch (x100, x1) and a dial whose units are "volts full scale" (this is the total voltage spanned by the entire width of the channel). To get a good representation of the oscilloscope screen, use 10 volts full scale.
The chart speed is controlled by a set of pushbuttons at the top right; 10 mm/sec will give you a good representation of the plotted oscilloscope screen. The last important control is for channel position, just above the exit slot for the paper. The knob on the left controls channel 1, and a green LED array shows you the approximate position of the trace even when the chart is not moving. Set channel 1's position to the far right of the span. When you press "Plot" on the oscilloscope, the trace will jump to the left. You may need to adjust the position when you first plot the screen, but once you have the position set, you won't need to adjust it again. You should set the date and the time of day on the EasyGraf recorder if you have not already so. You should also turn on full annotation, so your chart records will automatically show the time they were made, tics for the time, the vertical sensitivity of each channel, and the chart speed. See the appendix on EasyGraf recorders for information on how to turn on the timer and the annotation.
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3. Dissection. WARNING: It is extremely dry in the laboratory, and an exposed nerve cord will very rapidly become a non-living string. MOISTEN THE NERVE CORD FREQUENTLY!!! Do this more often than you think is necessary. Before beginning the dissection, make or acquire a fine glass rod with a delicate point or hook. Use the rod for probing or lifting the nerve cord. Do not pinch the cord with forceps. Anesthesia. Obtain an earthworm and place it in the ethanol solution provided, to anesthetize it. Earthworm anesthesia is a problem: dilute alcohol acts very slowly, and often leaves a squiggling worm that is difficult to dissect, while concentrated alcohol "pickles" the outside of the worm, knocking out the responses of touch receptors and threatening the response of the giant fibers. Use the minimum anesthesia you can tolerate; it is really for you, not for the worm (which is too simple to care). |
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After suitable anesthesia, rinse the worm in tap water and allow the excess to drain off. Pin out the worm dorsal side up on a flat dissecting dish placed on the stage of your microscope (check that the worm is in the field of view). Place pins only in the middle zone of the worm where you intend to open an incision. Insert the pins at very shallow angles so that they do not get in the way of your dissecting tools. |
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Open an incision and extend it an inch or two in each direction. Use the pins to keep the incision open, and flush out the body cavity from time to time with saline. The accompanying figures will help you identify the nerve cord and other internal structures. |
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Free the cord from its lateral and ventral connections until it can be lifted slightly above the pool of saline (a few centimeters of freed nerve will be long enough). Tilt the dish with a small ball of tackiwax so the saline collects away from the worm. |
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Place the recording electrodes. Start by grounding the preparation: clip one end of an alligator clip lead to one of the dissecting pins (pick one that is out of the way). Clip the other alligator clip to the white (ground) binding post of the amplifier's input block. Then clamp hook electrodes in a micromanipulator (firmly!) and lower the manipulator so that the hooks can be placed under the nerve cord. Use your glass probe to help ease the nerve cord onto both electrodes, and then raise the electrodes to bring the nerve above the saline. Blot or blow away the curtain of saline that clings between the wires of the electrode. Turn on your preamp and audio monitor, and you should hear some spontaneous activity in (non-giant) motor units. Remember to moisten the exposed cord frequently. |
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4. Experiments. Mechanical stimulation Touch or stroke the anterior and posterior ends of the worm with the blunt tip of your glass rod. Are there any large spikes in response? If so, can you distinguish the median and lateral units, based on which end of the worm must be stimulated to elicit them? Capture and plot one or two examples of the giant spikes. Make sure to label your plots with the vertical sensitivity and the sweep speed of the oscilloscope (do this right away, before you risk changing one or more settings). If you are getting no responses after a few minutes of exploration, go on immediately to the next section. Electrical stimulation |
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Connect two straight-pin electrodes to the output (red and black terminals) of a stimulator (the adjacent ground terminal should not be used). Insert the electrodes across from each other through the body wall at the head or tail end of the worm. The body surface and the dissecting dish must be dry near the stimulating electrodes, or the saline will "short out" the stimulus and reduce its effectiveness. Check that the stimulus control is set to "Regular," not "Twin Pulse," and that the stimulator's delay is brief (1 or 2 msec). With the pulse rate at a frequency of several per second, a duration of 0.1 msec, and the voltage at its lowest setting, turn on the stimulus ("repeat"), adjust the trigger level (if necessary) to get a sweep, and gradually increase the stimulus voltage. You will see the stimulus artifact increase gradually as you increase the voltage (the artifact represents stimulus current that has travelled to the recording electrode through the saline and the tissue fluids; it is not a biological response). When the stimulus reaches threshold, an action potential from one or both giant axons will suddenly appear. Then do the following: (a) Use digital storage to capture, display and plot a good example of an action potential in the giant axon(s). From the screen, measure the action potential's time course and its apparent amplitude. Divide by the amplification factor of the preamplifier, and obtain the real amplitude of the action potential at the electrodes. Label a chart record of the action potential with calibration bars showing the time/division and volts/division of the plot. Save the record to post with your data from the next sections. (b) Measure the conduction velocity in the giant axons. The conduction velocity is the distance between the stimulating electrode and recording electrodes divided by the time it took the action potential to travel that distance.
Reversing the stimulus-polarity switch reverses which of the stimulating electrodes is the initiating one. For one of the polarities, the action potential will make a longer trip and will take slightly longer to reach the recording electrode. You can see this effect, but you may need to readjust the stimulus voltage when you reverse the stimulus polarity. If their is a noticeable difference in conduction time, set the polarity to make the pin closest to the recording electrodes the active stimulating electrode. Use your ruler to measure the distance between the stimulating pin and the nearer recording (hook) electrode. (You may need to estimate parts of the path if the worm is curved.) Measure the time on the screen between the stimulus artifact and each spike. Divide the distance (in mm) by the time (in msec) to find the conduction velocity (meters/second) of both the medial and the lateral giants. (c) Measure and plot a strength-duration curve for the stimulus. Begin with the briefest stimulus duration that your stimulator can provide, and find and record the stimulus voltage that is just above threshold for one of the giant axons. Then double the stimulus duration, find the new threshold voltage for the same axon, and proceed in that way to measure the relation between duration and voltage. (Keep doubling the stimulus duration till you reach the stimulator's maximum.) Plot stimulus duration (x-axis) against threshold voltage (y-axis), leaving room on the page for the data from parts (b) and (d).
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(d) Measure the refractory period that follows an action potential. Adjust the Delay control of the stimulator to about 20 msec. As you increase the delay, you should see the stimulus artifact and the spike move to the right on the screen.
Now switch the stimulus mode from "Regular" to "Twin Pulses." Two stimuli will now be delivered for each sweep, one pulse at the start of the sweep, and a second one at a time governed by the delay control. If the second pulse occurs 10 or 20 msec after the first pulse, the axon will have fully recovered from its response to the first pulse. Gradually decrease the delay between the two stimulus pulses, and observe the action potentials. When the second pulse is only a few msec later than the first, the axon at the stimulus site will still be recovering from generating the previous action potential and will be somewhat refractory. The interval at which the second response drops out is the refractory period.
(e) At the end of the lab, post one neat summary page (with your team's full names on it) that has:
Post next to it the chart record you made in part (a), with your names on it, and any other chart records (or superimposed tracings of records) that you obtained in part (d). Before you leave, rinse your tools and dissecting dish. Turn off power to all equipment, including the stimulator and the DAM-50 preamplifier. |
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