iworx Physiology Lab Experiment Experiment AN-7 Cockroach Cercal Sense Organs Note: The lab presented here is intended for evaluation purposes only. iworx users should refer to the User Area on www.iworx.com for the most current versions of labs and LabScribe2 Software. iworx Systems, Inc. www.iworx.com iworx Systems, Inc. 62 Littleworth Road, Dover, New Hampshire 03820 (T) 800-234-1757 / 603-742-2492 (F) 603-742-2455 LabScribe2 is a trademark of iworx Systems, Inc. 2013 iworx Systems, Inc.
Experiment AN-7: Cockroach Cercal Sense Organs Background Cockroaches have a pair of sensory structures at the posterior tip of their abdomen called cercal sense organs, or cerci. The cerci are used to sense predators and effect an escape response. The ventral sides of the cerci are covered with oviform hairs, each of which is connected to a mechanoreceptor. These trigger an action potential in the associated cercal neuron when the hair is moved by wind or sound. The cercal neurons travel through the cercal nerve and synapse with the giant ventral nerve cord interneurons in the terminal abdominal ganglion. The sensory signals travel through the giant interneurons of the ventral nerve cord to the thorax. Here the interneurons synapse with motor neurons that initiate the escape response. The hairs on the cerci are remarkably sensitive to wind. The movement of a toad s tongue is a sufficient stimulus to move the hairs enough to trigger action potentials in the cercal neurons. The hairs are arranged in different orientations so wind from a specific direction only stimulates some of the hairs. Individual interneurons receive the sensory input from a population of hairs that are deflected by wind from one direction. This is how the cockroach senses the direction from which the wind comes. The ventral nerve cord is comprised of two symmetrical connectives. Most sensory input travels up the ipsilateral side of the ventral nerve cord, but there is some crossing over in the terminal abdominal ganglion, as well as at other ganglia along the ventral nerve cord. The action potentials from a number of the sensory neurons in the cercal nerve summate at one interneuron, and it takes the movement of several hairs to trigger an action potential in an interneuron. The sensory hairs are also sensitive to sound. They have a maximal response to a sound frequency of 300-500 Hz. This seems to be related to the length of the hairs, and not a functional response to sound. The sensory signals can be recorded from the cercal nerves, or, more easily, from the ventral nerve cord. The recordings from these two locations are quite different. The sensory neurons of the cercal nerve are all of a similar diameter and the response to a puff of air creates a number of similarly sized extracellular action potentials. The interneurons in the ventral nerve cord are larger, and of varying diameters. They sort into different sized extracellular action potentials. The extracellular action potentials recorded from the ventral nerve cord are fewer in number and greater in amplitude than those in the cercal nerve. The cercal sense organ response adapts to a continuing breeze. Despite the continuation of the wind stimulus, the response in the interneurons of the ventral nerve cord is diminished in a short period of time. This adaptation does not occur in the neurons of the cercal nerve, but instead at the synapses in the terminal abdominal ganglion of the ventral nerve cord. When a quick puff of air of a greater magnitude is applied after adaptation, the response returns despite the adaptation. A cockroach can still respond to a predator threat even after it has habituated to a steady breeze. In this experiment, students will collect data that can be used to discover how animals are able to interpret mechanosensory input. The specific neuronal principles that students will explore include: Coding of stimulus intensity by frequency of action potentials Neuronal adaptation Spatial summation AN-7-1
Experiment AN-7: Cockroach Cercal Sense Organs Equipment Required PC or Mac Computer IXTA data acquisition unit USB cable IXTA power supply NA-100 extracellular amplifier Monopolar extracellular hook electrode C-BNC-N2 Needle electrodes (2) Glass probes (pulled over a Bunsen burner) Dissecting scissors Dissecting microscope Micromanipulator Plastic transfer pipet Aquarium bubbler American cockroaches (Periplaneta americanus) IXTA Setup 1. Place the IXTA on the bench, close to the computer. 2. Check Figure T-1-1 in the Tutorial chapter for the location of the USB port and the power socket on the IXTA. 3. Check Figure T-1-2 in the Tutorial chapter for a picture of the IXTA power supply. 4. Use the USB cable to connect the computer to the USB port on the rear panel of the IXTA. Plug the power supply for the IXTA into the electrical outlet. Insert the plug on the end of the power supply cable into the labeled socket on the rear of the IXTA. Use the power switch to turn on the unit. Confirm that the red power light is on. Start the Software 1. Click on the LabScribe shortcut on the computer s desktop to open the program. If a shortcut is not available, click on the Windows Start menu, move the cursor to All Programs and then to the listing for iworx. Select LabScribe from the iworx submenu. The LabScribe Main window will appear as the program opens. If the shortcut does not appear on the Macintosh desktop, find LabScribe in the Applications and double-click on the icon. 2. On the Main window, pull down the Settings menu and select Load Group. Locate the folder that contains the settings group, IPLMv4Complete.iwxgrp. Select this group and click Open. AN-7-2
3. Pull down the Settings menu again. Select the CockroachCerci-LS2 settings file from Animal Nerve. 4. After a short time, LabScribe will appear on the computer screen as configured by the CockroachCerci-LS2 settings. 5. For your information, the settings used to configure the recording channels in the LabScribe software and IXTA for this experiment are programmed on the Channel windows of the Preferences Dialog, which can be viewed by selecting Preferences from the Edit menu on the LabScribe Main window. 6. Once the settings file has been loaded, click the Experiment button on the toolbar to open any of the following documents: Appendix Background Labs Setup (opens automatically) Cockroach Cerci Equipment Setup 1. Locate the following items in the iworx kit: the HE-100 monopolar extracellular hook electrode (Figure AN-7-S1), a needle electrode to use as a reference electrode (Figure AN-7-S2), and a NA-100 extracellular amplifier and its input cable (Figure AN-7-S3). 2. Plug the input cable of the NA-100 into the input socket on the amplifier. 3. Plug the DIN8 connector of the NA-100 into the Channel 3 input of the IWXIXTA data recorder. 4. Mount the HE-100 hook electrode on a micromanipulator. 5. Insert the plug on the end of the HE-100 hook electrode into the jack on the NA-100 input cable that is color-coded red. 6. Insert the plug on the end of the reference needle electrode into the jack on the NA-100 input cable of the NA-100. Figure AN-7-S1: Monopolar extracellular hook electrode. AN-7-3
Figure AN-7-S2: A set of three color-coded needle electrodes, one can be used as a reference electrode and one as a ground electrode, if needed. Figure AN-7-S3: The NA-100 neuroamp extracellular amplifier with its color-coded input cable. Electrical Noise Electrical noise is the most common problem associated with the recording of bioelectric signals. It radiates through the air and comes from electrical devices in the lab room or building: lights, power outlets, computers, monitors, and the power supplies. Since the source of power for these devices is 60Hz alternating current (AC), this electrical noise appears as a distorted sine wave with a repeating period of 16.7 milliseconds (msec). There are two major sources of electrical noise: pickup and ground loops. Pickup Pickup is caused by electrical radiation that produces currents in the electrodes and wires leading to the amplifiers in the recording system. Because the resistance in the electrodes is high, small currents produce large voltages that may be greater than the biopotential being recorded. The major ways to reduce pickup are: AN-7-4
Faraday Cage: Put a grounded, screened enclosure, known as a Faraday cage, around the preparation and the electrodes. the enclosure separates the source of the radiation from the electrodes. The person operating the equipment might also be a source of noise, and he or she may need to be grounded. Shielded Cables: Use shielded cables to carry the signals from the electrodes to the amplifier and the recorder; this puts a protective ground around the wires carrying the bioelectric signal. Differential Recording: Record using both a positive and a negative recording electrode placed on a nerve or neuron. The noise signals that are equal in magnitude, but opposite in polarity, will cancel each other out and leave a flat baseline. Short Cables: Use the shortest cables available to reduce the length of wiring exposed to electrical noise. Direct Current Equipment: Use equipment, like preamplifiers and illuminators, that are powered by batteries or direct current (DC) transformers. Equipment Removal: Unplug or remove unused alternating current (AC) equipment from the area. Ground Loops Ground loops are a troublesome source of electrical noise caused by the ground cable itself serving as an antenna for the noise radiating in the room. Using a Faraday cage to shield the preparation and the recording electrodes does not remove the electrical noise caused by ground loops. To avoid ground loops, use the following techniques: Ground Hub: Ground all the equipment around the preparation to a common grounding point (hub). This includes all the items that are electrically powered or are made of metal, like illuminators or microscopes. Use simple cables, like banana cords equipped with alligator clips, to connect each device directly to the common grounding point. The common ground point is connected to the ground of the recording device with a single cable. The recording device is connected to the building ground. Simple Chain: Ground the devices to the common grounding point using the simplest route that links the first device to the second device, the second device to the third device, and so on. Start the chain at the device that is the farthest from the common grounding point. End the chain by connecting the last device to the common grounding point, which is connected to the ground of the recording device. Free-Floating: In addition to using one of the grounding techniques described earlier, plug all devices powered by alternating current (AC), like illuminators, amplifiers, and recording units to power outlets using three-two prong adapters. High Frequency Noise High frequency noise can also be a problem when recording bioelectric potentials. This type of noise is seen as the thickening of the recorded line. This noise contains many frequencies, and the amplitude of the noise is proportional to the resistance of the electrode. Therefore, intracellular electrodes, with high resistances, pick up a greater amplitude of high frequency noise than extracellular electrodes, with low resistances. AN-7-5
Mechanical Noise Mechanical noise, like vibrations from the ventilation system in the room, can cause the electrodes to vibrate and produce voltage changes with each vibration cycle. The puffs of air in this experiment can cause a good amount of mechanical noise because the air causes the cerci and the ventral nerve cord to move. Because these voltage changes are so slow compared to the action potentials, they can be filtered out through the use of a calculated channel. Grounding The iworx unit has two different connections for grounding: One method uses the ground (C) cable of the NA-100. The other method uses either of the green (Ground or GND) banana jacks on the IXTA. One jack is located on the front panel in the stimulator section; the other jack is located on the back panel of the IXTA. 1. Use a C-ISO-N3 needle electrode as the ground and try recording when the ground wire is inserted into the cockroach and connected to the ground (green) cable of the NA-100. 2. If the recording has a lot of 60Hz noise while grounding the preparation through the NA-100, disconnect the ground wire coming from the prep from the NA-100. Attach an optional green alligator-male banana grounding cable. Attach the alligator clip on one end of this grounding cable to the ground wire coming from the prep. Attach the male banana plug on the other end of this grounding cable to the green banana jack on either the front or back panel of the iworx unit. 3. If neither method mentioned above reduces noise in the recording, try recording without a ground electrode. Sometimes, it is more successful than using a ground. Signal Improvement 1. To improve recordings of the action potential, move the preparation away from sources of 60Hz noise. These sources include outlets, computers, monitors, lights, refrigerators, water baths, and other AC powered devices. 2. If the recording still contains a great deal of electrical noise, apply the digital filtering function to the data. Click on the add function button in the upper margin of the Action Potential channel. Select Filter from the menu of computed functions. 3. In the Filter Setup Dialog window, the Filter Mode is set to the Hamming Window and the Filter Order is set to 51, these are the default settings that should be used. 4. Set the Low Cutoff filter value to 65 and the High Cutoff filter value to 8000. The values for the filter cutoffs can be set by: Typing the values for the filter cutoffs in the boxes to the right of the names of the filter cutoffs. Clicking on the up or down arrows to the right of the boxes displaying the values of the filter cutoffs. AN-7-6
Clicking on the margins of the colored area in the graphic display of the filter and dragging the margins to the values required. Preparation of the Cockroach 1. Anesthetize the cockroach by placing it in a Petri dish and then covering it with ice. 2. Decapitate the cockroach and remove its legs and wings. 3. Use insect pins to pin the cockroach ventral side down to a wax or Sylgard dish. Use one pin on each side of the thorax. 4. Remove several tergites from the dorsal side of the cockroach. Stabilize the posterior part of the cockroach with two more insect pins. 5. Using small dissecting scissors, remove the gut and fat bodies from the opening you created by removing the tergites. Rinse with saline. 6. The ventral nerve cord extends down the center of the ventral surface, with shiny trachea on either side. 7. Use small dissection scissors to carefully cut the trachea and any fat away from the nerve cord. Clear a section of the nerve cord between two ganglia. 8. Place the reference electrode through the thorax of the cockroach, being careful not to puncture the ventral nerve cord. 9. Using the micromanipulator controls, position the wire hook on the electrode close to the ventral nerve cord. 10. Free a section of the nerve cord anterior to the terminal abdominal ganglion and lift it on the hook electrode.the bend in the hook may have to be adjusted with a pair of fine forceps before attempting to place the nerve cord on the electrode. 11. Insulate beneath the nerve cord with petroleum jelly, injected via a syringe with a blunt needle. See Figure AN-7-S4 for a photograph of the ventral nerve cord situated on the hook electrode and supported by a bed of petroleum jelly. 12. Click Record to check for electrical activity. Use a plastic transfer pipet to puff air at the cerci to see if there is a response. 13. Once you have established electrical activity in response to a stimulus, cover the nerve cord and the electrode with petroleum jelly to keep it from drying out. The experimental set-up is illustrated in Figure AN-7-S5. Note: If you do not see any action potentials, check the wiring; make sure that both the hook electrode and the reference electrode are making contact with the cockroach and that they are not touching each other. It is also possible to stretch the cercal nerves in the dissection. In this case, you will normally see some spontaneous neural activity, but there will be no response to the air stimuli. If this happens, it will be necessary to replace the cockroach with a new prep. AN-7-7
Figure AN-7-S4: The ventral nerve cord situated on the hook electrode and supported by a bed of petroleum jelly. Warning: The cockroach preparation used in this experiment is functional for a limited period of time. To conserve time, complete all the exercises in the experiment before analyzing the data. Figure AN-7-S5: The experimental set-up showing the placement of the recording and reference electrodes. AN-7-8
Experiment AN-7: Cockroach Cercal Sense Organs Exercise 1: Trial Responses Aim: To elicit a ventral nerve cord response to air puffs, and become familiar with what the responses look like. Note: Stimuli can be applied in a number of ways. The simplest method is to use a plastic transfer pipet. Directing it at the cerci and squeezing the bulb provides a stimulus strong enough to trigger the cercal response. A long stream of air can come from an aquarium air pump. If the responses to individual puffs of the same intensity are being compared, it is important to keep the stimuli constant by compressing the pipet bulb with consistent pressure and from a consistent distance from the cerci. Procedure: 1. Click the Record button. Click the Autoscale buttons on both channels. 2. Type Trial Response in the Mark box to the right of the Mark button. Press the Enter key on the keyboard to attach this notation to the recording. 3. Using a plastic transfer pipette, stimulate the cerci with a puff of air and note the response. Note: The action potentials will have an amplitude much greater than the baseline (Figure AN-7-L1). Typically, there is at least some spontaneous activity in the absence of stimuli. The nerve cord contains both sensory and motor neurons. Although you will only be eliciting a sensory response, there is both spontaneous descending information, as well as occasional motor responses to your stimulus, as these stimuli can trigger an escape response. 4. Give a gentler puff of air to the cerci and note that the response is diminished. 5. Click Stop to halt the recording. 6. Select Save As in the File menu, type a name for the file. Choose a destination on the computer in which to save the file, like your lab group folder. Designate the file type as*.iwxdata. Click on the Save button to save the data file. Exercise 2: Stimulus Intensity Aim: To explore the effects of stimulus intensity on the number and frequency of action potentials produced. Stimulus intensity really has two components, amount of air and acceleration. Intensity can be altered in a couple of different ways. It can be changed by producing the air from different distances, or by changing the amount of air produced. The amount of air can be changed by compressing the bulb of the transfer pipet only slightly or giving it a complete compression. AN-7-9
Procedure 1. Click the Record button. Click the Autoscale buttons on all channels. Note: Action potentials recorded intracellularly will all be of the same amplitude. Action potentials recorded extracellularly, as in this experiment, may vary greatly in amplitude. This is because an extracellular action potential recording is really a recording of the voltage changes resulting from the current that travels past the recording electrodes on the outside of the nerve as an action potential passes through an axon in the nerve. The recorded action potential amplitude will vary depending on the diameter of the neuron and the distance of the electrodes from the neuron. For example, a large diameter axon will produce a larger extracellular action potential than a small diameter axon because the large diameter axon has a greater surface area. More current will leak across the greater surface area and this will result in a recorded action potential of greater amplitude. Similarly, if the recording electrodes are closer to one axon than another, the current recorded by an action potential in the closer neuron will be greater than that in the other axon because current dissipates over distance. 2. Type Low Intensity in the Mark box to the right of the Mark button. Press the Enter key on the keyboard to attach this notation to the recording. 3. Stimulate the cerci at low intensity three successive times by squeezing the pipet bulb slightly. Leave about 10 seconds between the stimuli. 4. Each stimulus should produce a number of action potentials (Figure AN-7-L1). Figure AN-7-L1: The response of the neurons in the ventral nerve cord to three successive stimuli. 5. Wait for at least 30 seconds. 6. Type Mid Intensity in the Mark box. Press the Enter key to add the mark to your recording. 7. Stimulate the cerci at mid-intensity three successive times by squeezing the pipet bulb slightly more strongly than before. Leave at least 10 seconds between stimuli. AN-7-10
8. Wait at least 30 seconds. 9. Type High Intensity in the Mark box. Press the Enter key to add the mark to your recording. 10. Stimulate the cerci at high intensity three successive times by squeezing the pipet bulb fully. Leave at least 10 seconds between stimuli. 11. Click Stop to halt the recording. Note: It is important to perform replicates of the stimuli in this experiment. There will be some variability of response. Replicates allow the calculation of a more accurate average response. Exercise 3: Spatial Summation Aim: To determine the number of hairs needed to trigger an action potential in the ventral nerve cord. Note: Each cercal hair is connected to an individual neuron, and an action potential is produced when the hair is deflected. However, processing occurs at the terminal abdominal ganglion, and it takes input from a number of hairs before an action potential is triggered in the associated interneuron in the ventral nerve cord. This is an example of spatial summation. Procedure 1. Click the Record button. Click the Autoscale buttons on both channels. 2. Type Deflect one hair in the Mark box, and press the Enter key to add your Mark to the recording. 3. Using a glass probe with a finely drawn tip, immediately deflect one hair several times in succession to see if any action potentials are produced in the ventral nerve cord. 4. Type Deflect two hairs in the Mark box and press the Enter key to add your Mark to the recording. 5. Quickly deflect two neighboring hairs several times in succession to see if any action potentials are produced in the ventral nerve cord. 6. Type Deflect three hairs in the Mark box and press the Enter key to add your Mark to the recording. 7. Deflect three neighboring hairs several times in succession to see if any action potentials are produced in the ventral nerve cord. 8. Type Deflect four hairs in the Mark box and press the Enter key to add your Mark to the recording. 9. Deflect four neighboring hairs several times in succession to see if any action potentials are produced in the ventral nerve cord. 10. Click Stop to halt the recording. 11. Select Save in the File menu. AN-7-11
Exercise 4: Adaptation Aim: To determine the effect of a continuous stimulus on the frequency of action potentials, as well as the response to a single stimulus after fairly complete adaptation. Adaptation is defined as a reduction in neuronal response despite continued stimulation. You will use the air from an aquarium pump to simulate a continuous breeze. Procedure 1. Click the Record button. Click the Autoscale buttons on both channels. 2. Type Pre-adaptation stimulus in the Mark box, and press the Enter key to add the mark to your recording. 3. Using the transfer pipet, stimulate the cerci with the air from one complete compression of the bulb. 4. Type Continuous stimulus in the Mark box and press the Enter key to add the mark to your recording. 5. Allow the aquarium pump to run continuously until you see little response. Stop after five minutes if the response has not been totally eliminated. 6. Type Post-adaptation stimulus 1 in the Mark box, and press the Enter key to add the mark to your recording. 7. Immediately stimulate the cerci with the air from one complete compression of the bulb. 8. Type Post-adaptation stimulus 2 in the Mark box. 9. Wait 10 seconds, press the Enter key to add the mark to your recording, and give another single stimulus. 10. Type Post-adaptation stimulus 3 in the Mark box. 11. Wait 10 seconds, press the Enter key to add the mark to your recording, and give a third single stimulus. 12. Click Stop to halt the recording. 13. Select Save in the File menu. Data Analysis Exercise 2: Stimulus Intensity 1. Click the Marks button in the LabScribe toolbar (Figure AN-7-L2) to go to the Marks window. Select the mark, Low intensity stimulus, by clicking on its number in the column on the left side of the Marks window. Click on the Go To Mark button at the bottom of the window. 2. Scroll to the first response that resulted from a low intensity stimulus, and click on the Double Cursors icon in the LabScribe toolbar. AN-7-12
Figure AN-7-L2: The LabScribe toolbar. 3. Place one cursor as closely as possible to the beginning of the response and the other cursor as closely as possible to the end of the response. You can place the cursors more accurately after expanding the response to the full width of the Main window by clicking the Zoom between cursors icon on the LabScribe toolbar. Note: It may be difficult to determine exactly where the sensory response begins and ends if there is a good deal of spontaneous activity. The response can usually be distinguished from spontaneous activity because the response has action potentials that are larger and of similar amplitude. 4. Click on the Analysis Window icon in the toolbar or select Analysis from the Windows menu to transfer the data displayed in the Main window to the Analysis window (Figure AN-7-L3). 5. Look at the Function Table that is above the uppermost channel displayed in the Analysis window. The mathematical functions, Mean and T2-T1, should appear in this table. The values for Mean and T2-T1 are seen in the table across the top margin of each channel. 6. In the Analysis window, place one cursor at the beginning and one cursor at the end of the response. The parameters that should be measured with the cursors in these positions are: Mean action potential frequency (Mean on the Action Potential Frequency channel). Duration of the response (T2-T1 on the Action Potential Frequency channel). 7. Once the cursors are placed in the correct positions, the values of each of the parameters can be recorded in the on-line notebook of LabScribe by typing its name and value directly in the Journal, or on a separate data table (Table AN-7-L1). 8. The functions in the channel pull-down menus of the Analysis window can be used to enter the values of the parameters from the recording to the Journal. To use this function: Place the cursors at the locations used to measure the values of the parameters. Transfer the names of the mathematical functions used to determine the values of the parameters to the Journal using the Add Title to Journal function in the Action Potential Frequency Channel pull-down menu. AN-7-13
Transfer the values for Mean and T2-T1 to the Journal using the Add All Data to Journal function in the Action Potential Frequency Channel pull-down menu. Figure AN-7-L3: A single sensory response displayed in the Analysis window, with the cursors positioned at the beginning and end of the response. Table AN-7-L1: Ventral Nerve Cord Response to Stimuli of Different Intensities. Action Averaged Action Potential Frequency (Hz) Averaged Response Duration (s) Low Intensity o Mid Intensity o High Intensity o 9. Perform Steps 1 through 8 for the responses from a low intensity stimulus. These values can be used to find an average as well as the degree of variance for low intensity stimulations. Enter the values in the Journal by using one of the techniques in Step 8 and in Table AN-7-L1. 10. Repeat steps 1-9 for the Mid and High Intensity stimuli. 11. Select Save in the File menu. AN-7-14
Questions 1. Graph the response changes as a function of intensity. 2. How does the response change as a function of intensity? 3. Explain how the response differences would be important to the cockroach in its environment. Exercise 3: Spatial Summation 1. Click the Marks button in the LabScribe toolbar to go to the Marks window. Select the mark, Deflect one hair, by clicking on its number in the column on the left side of the Marks window. Click on the Go To Mark button at the bottom of the window. 2. Determine if there were any large action potentials immediately following the Deflect one hair mark. You deflected the hair several times. Was there a response to any of the deflections? How many action potentials are in each response? You should be able to determine this manually by counting the number of large action potentials in each response. 3. Record whether there was any response and if there was, the average number of action potentials in the on-line notebook of LabScribe by typing its name and value directly in the Journal, or on a separate data table (Table AN-7-L2). 4. Repeat the procedure for the deflection of two, three, and four hairs. Table AN-7-L2: Response to Individual Hair Deflections Number of Deflected Hairs Was there a response? Number of Action Potentials 1 2 3 4 Questions 1. What was the minimum number of hairs required to elicit a response? 2. Why might you see no response in the ventral nerve cord if only one or two hairs are deflected? 3. What function might this serve in the natural history of the cockroach? AN-7-15
Exercise 4: Adaptation 1. Click the Marks button in the LabScribe toolbar to go to the Marks window. Select the mark, Pre-adaptation stimulus, by clicking on its number in the column on the left side of the Marks window. Click on the Go To Mark button at the bottom of the window. 2. Scroll through the data to find the response to this stimulus and use the same techniques described in the analysis section of Exercise 1 to measure the action potential frequency and the duration of this response. 3. Enter the values for this response in the Journal, or on a separate data table (Table AN-7-L3). 4. Click the Marks button in the LabScribe toolbar to go to the Marks window. Select the mark, Continuous Stimulus, by clicking on its number in the column on the left side of the Marks window. Click on the Go To Mark button at the bottom of the window. 5. To determine the time course of adaptation, scroll to the beginning of the adaptation process, and click on the Double Cursors icon in the LabScribe toolbar. 6. Click on the Analysis Window icon in the toolbar or select Analysis from the Windows menu to transfer the data displayed in the Main window to the Analysis window. 7. On the Analysis Window, place one cursor at the beginning of the adaptation process and, using the T2-T1 function, the other cursor 50 msec later. 8. Once the cursors are placed in the correct positions, the Mean on the Action Potential Frequency channel can be recorded in the on-line notebook of LabScribe by typing its name and value directly in the Journal, or on a separate data table (Table AN-7-L3). 9. Using the time bar across the bottom of the Analysis Window, move the cursors to the start of the second minute of the adaptation process. Place one cursor at the beginning of minute 2 of the adaptation process and, using the T2-T1 function, the other cursor 50 msec later. 10. Once the cursors are placed in the correct positions, the Mean on the Action Potential Frequency channel can be recorded in the on-line notebook of LabScribe by typing its name and value directly in the Journal, or on the separate data table. 11. Repeat Steps 7 and 8 for the first 50 msec of minutes 3, 4, and 5. 12. Click the Marks button in the LabScribe toolbar to go to the Marks window. Select the mark, Post-adaptation stimulus 1, by clicking on its number in the column on the left side of the Marks window. Click on the Go To Mark button at the bottom of the window. 13. Scroll through the data to find the response to this stimulus and use the same techniques described in the analysis section of Exercise 1 to measure the action potential frequency and the duration of this response. AN-7-16
Table AN-7-L3: Time Course of Adaptation Time interval Action Potential Frequency in first 50 msec Minute 1 Minute 2 Minute 3 Minute 4 Minute 5 14. Enter the values for this response in the Journal, or on the separate data table (Table AN-7-L4). 15. Repeat Steps 13 and 14 for the responses to Post-adaptation stimulus 2 and Post-adaptation stimulus 3. Table AN-7-L4: Responses to Stimuli Before and After Adaptation Response Pre-adaptation stimulus Action Potential Frequency (Hz) Response Duration (s) Post-adaptation stimulus 1 Post-adaptation stimulus 2 Post-adaptation stimulus 3 Questions 1. How complete was the adaptation to a continuous stream of air? Did the action potential frequency change gradually over the course of adaptation or did most of the adaptation occur within the first minute or two? Was the response completely extinguished within five minutes? 2. Compare the response to a single stimulus before the adaptation to the response to the stimulus immediately after adaptation. AN-7-17
3. Compare the three responses to the stimuli after adaptation. How do they change? 4. If the continuous air was a steady outdoor breeze, and the stimulus after adaptation was the approach of a predator, what conclusions can you draw? Appendix Recipe for Cockroach Ringer s Solution. Concentration (mmolar) Salt Grams/liter DI H 2 0 160 Sodium Chloride 9.35 4.96 Potassium Chloride 0.37 1.97 Calcium Chloride 2H 2 O 0.29 0.54 Sodium Phosphate 7H2O 0.14 Adjust ph to 7.5 with Tris or Maleic acid AN-7-18