Biological Sciences 300, Smith College Lab 4: Action Potentials in Earthworm Giant Axons
	An updated version of this laboratory uses suction electrodes, which record a large signal and keep the nerve cord moist. This older version uses hook electrodes, which allow the nerve cord to dry out if it is not frequently moistened.
Checklist for Lab 4	View the video: Giant Axons in the Earthworm Nerve Cord
	Equipment: Oscilloscope and audio monitor Preamplifier Stimulator with twin-pulse capability, pin electrodes, synch cable Dissecting microscope Micromanipulator and hook electrodes Shallow dissecting dish Earthworm saline in squeeze bottles Glass probe, cm ruler, dissecting tools 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.
Micrographs of Lumbriculus variegatus by Alanna Morris, from a project in Bio 337, Fine Structure. Click figures to see enlarged images.	Cross-section of an annelid worm   Close-up of nerve cord
	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. 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. Also, even similar sized axons will appear to have smaller spikes if they are far away from the electrode. 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 the: Grass SD9 stimulators
	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 the oscilloscope's channel 2 via the patch panel. 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.) You may need to move the stimulator closer to the equipment rack temporarily for the cable to reach between the stimulator's output terminals and the patch panel. When you have finished examining the stimulator's output pulses, move the stimulator back next to the baseplate so the stimulating electrodes will be able reach the preparation. The synchronizing cable that connects the stimulator to the oscilloscope's external trigger should be able to reach the stimulator in either position. Set the stimulator's voltage output to the x1 range, and adjust the vertical scale of the oscilloscope's CH2 initally to 5 volts per division (you can change it after you see how big the pulse will be). 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: Stimulus: REGULAR Mode: REPEAT Polarity: NORMAL Output: MONO.
	Set the oscilloscope's trigger source to External (Trigger menu), move the trigger point to early in the sweep, adjust the trigger level, and observe the stimulus pulses as you try out the functions of the stimulator's 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 will need to understand later when you explore the refractory period. Use a brief pulse DURATION and an interpulse DELAY of about 20 msec. (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. Two points of caution: (1) Do not use the MOD setting of the Stimulus switch, since it requires an external modulating signal that you are not providing. (2) Do not have the values of Delay plus Duration exceed 50% of the time between pulses. The time between pulses is determined by the Frequency control. For example, at a frequency of 20 pulses per second, there are 50 msec between pulses. The pulse duration plus delay should not exceed 50% of this period, or 25 msec. If the duration were 1 msec, the delay should not be more than 24 msec.
	Synchronizing and stimulus pulses for twin-pulse setting
	When you restore connections to use for the rest of the experiment, you should leave the trigger settings as they are. You will need to: disconnect the stimulator's output terminals from the patch panel (you are finished with that cable), move the stimulator next to the baseplate, turn off the oscilloscope's CH2 and turn on CH1 if it is not already on, and check that the CH1 side of the patch panel is connected to the output of the DAM50 preamplifier (long red cable). You should also reset the stimulator initially for regular (not twin) pulses, a low frequency, brief delay, brief duration, and low voltage. You will later increase the voltage and/or duration as you watch the nerve's response.
View the video: Giant Axons in the Earthworm Nerve Cord	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).
	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.
	With forceps and scissors (not a scalpel), 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.
	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.
	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.
Capturing Oscilloscope Screenshots	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? When you first are exploring for spikes, you will find it helpful to change the trigger menu's Source to CH 1 and adjust the trigger Level knob so that the baseline noise itself triggers sweeps (you can also force sweeps by changing the trigger menu's Sweep setting to Auto). If you find spikes, readjust the trigger Level knob so that large spikes trigger the sweeps. Capture and save a screenshot of one or two examples of the giant spikes (or of ordinary spontaneous spikes if you don't elicit responses in the giants). If you are getting no responses from the giants after a few minutes of exploration, go on immediately to the next section. Electrical stimulation
Capturing Oscilloscope Screenshots	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"). If you changed the oscilloscope's trigger source to CH1 to see spikes elicited by stroking, you will need to return it to EXT source and readjust the trigger level (if necessary) until the stimulator is triggering a sweep. 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) Stop the sweep after capturing a good example of an action potential in the giant axon(s). Transfer the screen image to a USB flashdrive, from which you can transfer it to your computer later for inclusion in the summary page you will make at the end of the afternoon. From the screen, measure the action potential's duration and its apparent amplitude. (You can use the manual Cursor settings to make these measurements, or you can count divisions on the screen.) Divide by the amplification factor (gain) of the preamplifier, and obtain the real amplitude of the action potential at the electrodes. For example, if the height of the action potential on the screen is 120 mV and the amplifier's gain is 1000x, then the potential at the electrodes was really 120/1000 mV, or 120 µV. (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. You can measure the travel time from the same image as in part (a). The negative stimulating electrode actually initiates the action potential, since it is that electrode that decreases the potential between the (negative) inside of the axon and the (now also negative) space around the axon. The decrease in potential (depolarization) opens channels in the axon membrane and initiates the action potential. Reversing the stimulus-polarity switch reverses which of the stimulating electrodes is the negative one. For one of the polarities, the action potential may make a longer trip and take slightly longer to reach the recording electrodes. You can see this effect, but you may need to readjust the stimulus voltage when you reverse the stimulus polarity. If there 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' spikes. (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 until the threshold voltage stops changing. Note that if the stimulus duration lasts so long that it overlaps the elicited spike, it does not make any sense to make it longer. The spike is already underway, and continuing to depolarize the axon cannot contribute to initiating a spike that is already underway. Plot stimulus duration (x-axis) against threshold voltage (y-axis). Use the graph-paper image for plotting strength-duration data. Click the link, display the image, and with your cursor on the image, press the control key to get a pop-up menu. Choose "Save Image As" to download the image to the desktop. Then drag the downloaded file's icon onto an AppleWorks drawing page to put the graph paper in place. You can either plot points and make labels by hand after printing the page, or you can place dots and axis labels in the AppleWorks document itself before printing. If you place dots and labels in AppleWorks, you can group them and the graph paper into one object that you can move around together. Drag across all of the components to highlight them, and then select "Group" from the Arrange menu. Remember that you will be placing other images and data on the finished page.
	(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, the stimulus artifact and the spike will move to the right on the screen. The delay is the time between the prepulse sync pulse (which triggers the sweep) and the actual stimulus pulse (which appears as the stimulus artifact). 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 will still be recovering from generating the previous action potential and will be somewhat refractory. The interval at which the second response first drops out marks the end of the refractory period, although the observed interval will depend on the strength of the stimulus. A stronger stimulus can force a second spike before the refractory period has fully ended. You may also see the amplitude of the second action potential become smaller than normal as the delay is reduced, reflecting the elevated potassium-conductance and the large number of inactivated sodium channels that trail behind the first action potential. If you can, capture images of normal and reduced second spikes and save the screen images to your flashdrive. Two examples are shown here: (e) At the end of the lab, post one neat summary page that you make in AppleWorks showing: a screenshot of an evoked action potential from part (a), the conduction velocity you calculated in part (b), the strength-duration graph from part (c), and a screenshot of one or more traces showing the refractory period (part d). Make sure that the full names of all the team members are on the page as authors. Before you leave, rinse your tools and dissecting dish. Turn off power to all equipment, including the stimulator and the DAM-50 preamplifier.
Links	Appendix: Capturing Oscilloscope Screenshots Appendix: DAM 50 Preamplifier Back to laboratory table of contents