Extracellular Nerve Recording device settings modified 10-11-18

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Student Protocol Extracellular Nerve Recording In this experiment, you will explore the basic principles of extracellular nerve recording. Written by staff of ADInstruments. Background Crayfish are excellent organisms for studying neurobiology. For these experiments, you will be using a suction electrode to record from a motor nerve (the 3 rd nerve) that innervates the superficial extensor muscles in the crayfish tail, which contains sensory neurons arising from the muscle receptor organs in the crayfish tail. Required Equipment  LabChart 7 software  PowerLab Data Acquisition Unit  AC/DC Differential Amplifier with headstage  Micromanipulator and appropriate stand for recording electrode  Suction electrode  Electrode puller (if required for your Suction electrode)  Capillary glass for electrodes  ACSF solution  50 mm Petri dish with 5 mm clear silicone layer on the bottom  Ag + /AgCl pellet ground electrode  Tape  One 2-3” (50-76 mm) crayfish ( Procambarus clarkii recommended)  Dissecting microscope and light source  Faraday cage (optional, but recommended) Procedure Extracellular Recording setup 1. Set the AC/DC Differential Amplifier (Figure 1). Enter the settings below:  High Pass: 100 Hz  Notch: Off  Low Pass: 3-10 kHz  Capacity Compensation: As needed  DC Offset: As needed  Gain: 500-1000  Headstage (Figure 2): Attached to Probe Input  Input Select Switch: Diff  Mode Select Switch: Record Figure 1. AC/DC Differential Amplifier Front Panel 2. Make sure the PowerLab is turned off and the USB cable is connected to the computer. 3. Connect the output of the AC/DC Differential Amplifier to Input 1 on the front panel of the PowerLab. Page 1 of 7 ©2010

Extracellular Nerve Recording Student Protocol 4. Attach the capillary glass to the suction electrode, and attach it to the micropositioner. 5. Obtain a Petri dish. Place a small amount of crayfish saline in the dish. 6. Using the micropositioner, lower the suction electrode into the dish. Draw a small amount of saline into the suction electrode so that it contacts the silver wire inside. 7. Attach the cable from the suction electrode to the positive (+) input of the headstage. Figure 2. Schematic of a Monopolar Suction electrode 8. If your suction electrode has an indifferent or external electrode, attach it to the negative (-) input of the headstage (Figure 3). If not, follow the configuration shown in Figure 2. 9. Attach the reference (ground) electrode to the ground (Gnd) input on the headstage. Note: Alternatively, the ground electrode can be attached to the ground (Gnd) on the amplifier chassis. Then you should connect the indifferent lead to the ground input on the headstage. 10. Place the ground electrode pellet in the Petri dish so that the pellet is immersed in saline. A small piece of tape may be needed to secure the wire. 11. Turn on the PowerLab and AC/DC Differential Amplifier. Allow the amplifier to warm up for at least five minutes. 12. Launch LabChart and open the settings file “Crayfish Extracellular Settings” from the Experiments Tab in the Welcome Center . It will be located in the folder for this experiment. 13. Once the equipment has warmed up, Start recording. 14. On the AC/DC Differential Amplifier, turn the “Ω Test” (Ohm Test) to the “on” position. Observe the signal. In the Chart View, select Autoscale and use the View Buttons to adjust the horizontal compression, as necessary, until a square wave is visible. 15. If necessary, adjust the Capacity Compensation on the amplifier to “square up” the shoulders of the wave. 16. Stop recording. Save your data, but do not close the file. Page 2 of 7 ©2010

Extracellular Nerve Recording Student Protocol Figure 3. Schematic of a Bipolar Suction electrode Software Setup (optional) Creating Preset Comments is a useful feature to add comments for each manipulation. They allow you to press one button to add a specific comment instead of typing in the Comments bar each time. 1. Go to the Setup menu and select Preset Comments . A dialog will appear like the one in Figure 4. Figure 4. Preset Comments Dialog with Suggested Configuration 2. Select New from the bottom left of the dialog. 3. In the Event Configuration dialog that appears (Figure 5), select a Shortcut and select “All” under the Channel drop-down list. Write appropriate text in the text box. Click OK . Page 3 of 7 ©2010

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Extracellular Nerve Recording Student Protocol Figure 5. Event Configuration Dialog with Suggested Configuration 4. Repeat steps 2-3 for each comment you want to add. Note: The following steps are optional. Ask your instructor whether or not to complete the rest of the Software Setup. 5. It is possible to set up the Data Pad to record the spike amplitude. Open the Data Pad from the LabChart Toolbar. 6. Click on one of the column headings. The Data Pad Column Setup dialog will appear (Figure 6). Select Selection & Active Point and then Value . Recording mode is “When any channel is selected,” and the Channel is “Channel 1.” Figure 6. Data Pad Column Setup Dialog 7. Repeat step 6 on a different column to set up the Data Pad to record Interspike interval. Follow the instructions for step 6, but instead of selecting Value, select Selection Duration . 8. In Chart View , you can either make a selection or click with your Waveform cursor to make an “Active point”. Then use the keyboard short-cut Control + D to add these values to the Data Pad. Page 4 of 7 ©2010

Extracellular Nerve Recording Student Protocol Note: It is possible to copy-and-paste the data values from the Data Pad to a spreadsheet program, like Microsoft Excel ® , for further analysis. Lab 2: Crayfish Motor Nerve Recording Follow the instructions in the Lab Manual for this experiment. Figure 7 shows a typical setup for extracellular recording from a brain slice Once you have finished the experiment, this Student Protocol provides information to analyze the data using LabChart. Figure 7. Extracellular Recording from a Crayfish Tail Analysis After you have completed Lab 2 and/or Lab 10, you can analyze your data using the LabChart software. If you have completed both experiments, complete each analysis separately. Determining the Amplitude of Individual Spikes Determine the amplitude for each spike observed in your recording. 1. Select the data showing at least three spikes, and select Zoom View . 2. Place the Marker on the first spike peak. Move the Waveform Cursor to the lowest portion of the peak (Figure 8). 3. Record the voltage difference shown at the top of the Zoom View in your lab notebook. Consult your instructor if you are unsure where to record your data. Note: When you use the Marker, the difference (delta) from the Marker is recorded. Page 5 of 7 ©2010

Extracellular Nerve Recording Student Protocol Figure 8. Position of Marker and Waveform Cursor Determining the Frequency of Individual Spikes Determine the average firing rate of the spikes of interest for each experimental condition. 1. Select data showing at least three spikes, and select Zoom View . 2. Place the Marker on the first spike peak. Click the Waveform Cursor on the furthest peak of a similar size. 3. Record the time difference, shown at the top of the Zoom View window, in your lab notebook. Consult your instructor if you are unsure where to record your data. 4. Calculate the firing rate (spikes per minute, SPM) using the following equation: where n = the total number of spikes used in the calculation 5. Calculate the firing frequency for each spike type. Frequency is calculated in the following manner: Literature Cited Crawdad: A CD-ROM Lab Manual for Neurophysiology by Robert A. Wyttenbach, Bruce R. Johnson, and Ronald R. Hoy. The CD is available from Sinauer and Associates, Publishers at http://www.sinauer.com . Copyright © 2010 ADInstruments Pty Ltd. All rights reserved. PowerLab® and LabChart® are registered trademarks of ADInstruments Pty Ltd. The names of specific recording units, such as PowerLab 8/30, are trademarks of ADInstruments Pty Ltd. Chart and Scope (application programs) are trademarks of ADInstruments Pty Ltd. Page 6 of 7 ©2010

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