Name: Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication (Video) | JoVE Journal
Uploaded: 2011-02-20T23:59:00.000+00:00
Duration: 8 min 47 s
Description: Learn to fabricate extracellular suction electrodes for spinal cord electrophysiology using PE 90 tubing and an alcohol lamp. This protocol enables precise in vitro recordings from neonatal rodent neural tissue.

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5 minutes

Samuel L. Pfaff is a Professor in the Gene Expression Laboratories at The Salk Institute for biological Studies and an Investigator in the Howard Hughes Medical Institute. This work was supported by the Christopher and Dana Reeve Foundation. Joe Belcovson, Kent Schnoeker and Mike Sullivan in Multimedia Resources at the Salk Institute provided assistance with photography and editing.

Electrophysiological recordings of isolated spinal cords can reveal genetic and developmental changes to neural circuitry 1. We previously demonstrated a method to dissect neonatal mouse spinal cords 2. Here we present a method to prepare suction electrodes useful in recording fictive locomotion in isolated spinal cords 3.
The tip of a plastic tubing electrode can be drawn to a very fine tip by hand using a low-temperature alcohol lamp 3-5. The plastic tubing (PE90, Clay Adams IntramedicTM) is held over the flame to soften the tubing into a malleable form. As the tubing begins to melt and becomes more translucent it is removed from the heat source and the ends are gently pulled apart. The timing of removing the tube from the flame is important to insure the plastic does not collapse or split while being drawn. The thin portion of the tubing is cut with a razor blade according to the desired internal diameter, as dictated by the age of the specimen or the segmental level of the cord. For ease of placing an o-ring on the tubing, a 45° cut is made at the thick end of the tubing with a razor blade. A ferrule and o-ring are installed on the thick end to attach the electrode tip to the barrel of the electrode.
To allow for specifically sized electrodes and avoid the difficulties of hand-pulling electrode tips, electrodes can be constructed using small tubing inserted into a thicker tubing (PE90). The lengths of the tubing are specific to the requirements of the recording setup and are variable. We present the measurements used in our experiments 1-3,5. A 1cm length of PTFE fine tubing (Zeus, Small Parts) and a 10cm length of PE90 tubing are cut with a razor blade. The ends of the small tubing should be examined to be sure they are not crushed or closed. They can be opened using an insect pin (Fine Science Tools). A drop of adhesive (JB Weld) is placed onto the midpoint of the PTFE tubing. The PTFE tubing and adhesive are drawn into the PE90 tubing using mouth suction on the PE90 tubing. A 45° cut is made at the thick end of the tubing and a ferrule and o-ring are attached, as previous.
The ferrule and BNC connector are unscrewed from a commercial suction electrode. The BNC connector is soaked in xylene for one hour to remove the wax seal. The connector is then rinsed in water and dried.
A lower angled side port must be made on the side of the barrel. A metal rod is heated using a Bunsen burner and pressed into the existing hole on the side of the barrel at a low angle. A 15cm length of 0.010 inch silver wire (A-M Systems) is cut and soaked in bleach for fifteen minutes. This is done to create a silver chloride coating that aids in signal conduction 6. The wire is rinsed with water, dried, and then soldered onto the BNC connector. The BNC connector and silver wire are inserted into the suction electrode barrel with enough room for the BNC connector to remain to the side of the barrel. Suction tubing is inserted into the side port 2-3cm. Silicone sealant is injected into the barrel approximately 2-3cm. The BNC connector is threaded into the barrel of the electrode pushing the sealant forward.
The tip of the electrode is now threaded into the front end of the electrode. Shrink tubing can also be added to reinforce the suction line from the side port.
Electrodes are mounted on micromanipulators that are attached to magnetic stands, and placed near the recording dish. BNC coaxial cables are attached to a headstage that is wired to an amplifier. The amplifier is connected to an adaptor (Polyview) that is connected to a analog to digital card (National Instruments) in a PC computer with data acquisition software (Polyview).
When recording electrophysiological data, it is necessary to eliminate all outside interference. The recording dish, amplifier, and head stage are placed on an air table within a Faraday cage to prevent electrical and mechanical interference, respectively.
Representative Results:
<img src="/files/ftp_upload/2580/2580fig1.jpg" alt="Electrode assembly diagram: BNC connector, silicone sealant, silver wire, ferrule, suction tubing." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. A commercially available suction electrode can be modified by adding a heavier gauge silver wire and a plastic tip that is hand drawn on an alcohol lamp, or constructed with commercially available plastic tubing of specific sizes. In addition, the paraffin sealant is replaced with more durable silicone caulk sealant.
<img src="/files/ftp_upload/2580/2580fig2.jpg" alt="Electrophysiology setup diagram with amplifier, dissection dish for neural signals; data processing." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. A schematic of the suction electrode in relation to the air table (for mechanical isolation), Faraday cage (electrical isolation), amplifier and computer. It should be noted that there is a ground referenced to the recording dish and a second ground that is referenced to the air table and Faraday cage.

<ol><li>Gallarda, B. W., Sharpee, T. O., Pfaff, S. L., Alaynick, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+rhythmic+locomotor+burst+patterns+using+a+continuous+wavelet+transform%5BTitle%5D)+AND+%22Ann+N+Y+Acad+Sci%22%5BJournal%5D)">Defining rhythmic locomotor burst patterns using a continuous wavelet transform.</a> Ann N Y Acad Sci. 1198, 133-139 (2010).</li>
<li>Meyer, A., Gallarda, B. W., Pfaff, S., Alaynick, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+cord+electrophysiology%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Spinal cord electrophysiology.</a> J Vis Exp. , (2010).</li>
<li>Gallarda, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Segregation+of+axial+motor+and+sensory+pathways+via+heterotypic+trans-axonal+signaling%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Segregation of axial motor and sensory pathways via heterotypic trans-axonal signaling.</a> Science. 320, 233-236 (2008).</li>
<li>Landmesser, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+motor+projection+patterns+in+the+chick+hind+limb%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">The development of motor projection patterns in the chick hind limb.</a> J Physiol. 284, 391-414 (1978).</li>
<li>Myers, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cholinergic+input+is+required+during+embryonic+development+to+mediate+proper+assembly+of+spinal+locomotor+circuits%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Cholinergic input is required during embryonic development to mediate proper assembly of spinal locomotor circuits.</a> Neuron. 46, 37-49 (2005).</li>
<li>Chanin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+determination+of+chloride+by+use+of+the+silver-silver+chloride+electrode%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The determination of chloride by use of the silver-silver chloride electrode.</a> Science. 119, 323-324 (1954).</li>
<li>Goulding, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circuits+controlling+vertebrate+locomotion%3A+moving+in+a+new+direction%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">Circuits controlling vertebrate locomotion: moving in a new direction.</a> Nat Rev Neurosci. 10, 507-518 (2009).</li>
</ol>

No conflicts of interest declared.

Nervous system development can be studied using isolated rodent spinal cords. In the presence of neurotransmitters, fictive locomotion can be generated from the spinal cord in the form of patterned electrical activity 1,3. These rhythmic bursts are produced at 0.2 to 0.5 Hz and are patterned in left-right and flexor-extensor alternations. At different developmental stages, the robustness and patterns of this activity varies 1. Genetic mutations can also disturb the patterning of this activity 3...

<table><tbody><tr><td>PTFE Sub Lite Wall Tubing (Small tubing)</td><td>Zeus</td><td>36AWG</td><td>0.005&amp;#8221;ID x 0.003&amp;#8221; Wall (Small Parts) Also available in 0.003&amp;#8221; to 0.006&amp;#8221;</td></tr><tr><td>Large tubing (0.86mm (0.34&amp;#8221;))</td><td>BD Biosciences</td><td>427420</td><td>0.86mm (0.34&amp;#8221;) O.D. 1.27mm (.050&amp;#8221;)</td></tr><tr><td>Electrode Barrel</td><td>A-M Systems</td><td>573000</td><td></td></tr><tr><td>Adhesive</td><td>JB Weld</td><td></td><td></td></tr><tr><td>Adhesive: Silicone caulk</td><td></td><td></td><td></td></tr><tr><td>Solder and soldering iron</td><td></td><td></td><td></td></tr><tr><td>Bleach</td><td></td><td></td><td></td></tr><tr><td>Xylene</td><td></td><td></td><td></td></tr><tr><td>Silver wire: 0.010&amp;#8221;</td><td>A-M Systems</td><td></td><td></td></tr><tr><td>Insect pins: Austerlitz 0.1mm</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Magnetic Stand</td><td>Narishige International</td><td>GJ-8</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige International</td><td>MN 151</td><td></td></tr><tr><td>Miniboard (Headstage)</td><td>Grass Technologies</td><td>F-15EB/B1</td><td></td></tr><tr><td>Polyview Adaptor Unit</td><td>Grass Technologies</td><td>PVA 8</td><td></td></tr><tr><td>Bipolar Portable Physiodata Amplifier System</td><td>Grass Technologies</td><td>15LT</td><td></td></tr><tr><td>ANALOG TO DIGITAL CARD</td><td>National Instruments</td><td>6035E</td><td></td></tr><tr><td>Air Table; Vibraplane</td><td>Kinetic Systems</td><td></td><td></td></tr></tbody></table>

spinal cord electrophysiology ii: extracellular suction electrode fabrication

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Hi.My name is Resh Gary Dory, and I'm a research assistant in the faf Lab of the Salk Institute for Biological Studies. Today I'll be showing you how to fabricate extracellular suction electrodes using two methods. These electrodes can be used to take electrophysiological recordings of my spinal cords in vitro.Use an alcohol lamp as a heat source to melt the PE 90 plastic tubing into the desired width. Hold the tubing over the lit flame and remove as it begins to melt, gently pull the ends apart, being careful not to break the middle. This process is shown again.Remove the tubing as it softens and melts and gently pull the ends apart. Cut the tubing with a sharp razor blade at a point corresponding to the approximate width of the sample to be measured at the thick end of the tubing. Make a cut at a 45 degree angle.A feral and O-ring must now be installed onto the thick end. Insert this end of the tubing through the hexagonal opening of the feral. Then insert the end of the tubing into the rubber O-ring.This setup will be screwed into the front of the electrode. Cut a one centimeter length of Zeus small tubing with a sharp razor blade and inspect the ends for a clean cut and an open boar Cut a 10 centimeter piece of the PE 90 tubing using a sharp razor blade. Place a drop of JB weld onto the midpoint of the small tubing using mouth suction.Draw the small tubing and the weld into the bore of the large tubing and allow this setup to cure overnight. Although it is not shown, a 45 degree cut should be made at the thick end of this tubing, and a feral and o-ring should be installed onto this end. In the same way shown previously.Disassemble a standard suction electrode unscrew the feral from the front end of the electrode and the BNC connector from the rear. A lower angled side port must now be made on the side of the electrode barrel. Heat up a piece of metal using a bun and burner.Press the heated metal into the existing hole on the side of the barrel at a low angle. Remove the silver wire from the BNC connector by pulling on the wire where it meets with the connector. Soak the BNC connector in xylene for one hour to remove the wax.Once the wax is removed, rinse the connector in water and dry with paper towels. Cut a 15 centimeter length of silver wire. Soak the wire in bleach for 15 minutes.Then rinse and dry with paper towels. Solder the silver wire to the BNC connector. Insert the silver wire into the back end of the suction electrode barrel, and leave enough room for the BNC connector to remain to the side.Insert the suction tubing into the side port approximately three centimeters. Inject silicone sealant into the back opening of the suction electrode while preventing it from spilling out of the side port by covering it with your finger. Inject sealant approximately two to three centimeters into the electrode barrel.Once failed, tightly screwed the BNC connector into place, which will push the sealant forward. Let the sealant cure overnight. Screw the feral of the tubing created by either the pulled tip or cut tip method onto the front of the electrode straightening.The tubing will help ease the silver wire through the electrode is now complete. The recording setup is placed inside a faraday cage to reduce outside interference. Our setup has also been placed on an air table to eliminate mechanical interference.The electrodes are mounted on magnetic stand micro manipulators and placed near the recording dish. The BNC coaxial cables are connected to a head stage that is connected to an amplifier. The amplifier is wired to a poly view adapter, which is wired to the computer.This setup is shown closeup. The BNC coaxial cables are connected to a head stage. The head stage is connected to the amplifier.The amplifier output exits the Faraday cage and is connected to a poly view adapter. The poly view adapter is connected to the computer which has the appropriate recording software installed. The entire setup must be grounded properly.An external ground is established by connecting the Faraday cage and air table to a ground. An internal ground is established by wiring the dissection dish to the head stage. It is also necessary to ground the doors of the cage to the main body.With the proper setup data can now be collected. Thank you for watching this video and to help It assist you with your future experiments.

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Learn to fabricate extracellular suction electrodes for spinal cord electrophysiology using PE 90 tubing and an alcohol lamp. This protocol enables precise in vitro recordings from neonatal rodent neural tissue.

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

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