These experiments were modified from a laboratory manual that has been used in a course, orchestrated by Dr. H.L. Atwood, at the Department of Zoology, University of Toronto. The exercises were also used and modified from a manual that was produced for "6th INTENSIVE IBRO WORKSHOP ON BASIC NEUROSCIENCE" and was held at Korea University, Seoul, South Korea in 1993 (Cooper et al., 1993). The current modifications were required to use equipment common to present day student directed laboratories at various universities. Supported by University of Kentucky, Department of Biology, Office of Undergraduate Studies and College of Arts &amp; Sciences.

<ol>
	<li>Bennett, M. V. L., Barrio, L. C., Bargiello, T. A., Spray, D. C., Hertzberg, E., Sdez, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions:+new+tools,+new+answers,+new+questions.">Gap junctions: new tools, new answers, new questions.</a> Neuron. 6, 305-320 (1991).</li><li>Bernardini, G., Peracchia, C., Peracchia, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+effects+of+heptanol+on+gap+junction+structure+and+cell-to-cell+electrical+coupling.">Reversible effects of heptanol on gap junction structure and cell-to-cell electrical coupling.</a> European Journal of Cell Biology. 34 (2), 307-312 (1984).</li><li>Cooper, R. L., Chang, J. J., Ito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+report+on+the,+"SIXTH+INTENSIVE+IBRO+WORKSHOP+ON+BASIC+NEUROSCIENCE".">A report on the, "SIXTH INTENSIVE IBRO WORKSHOP ON BASIC NEUROSCIENCE".</a> , 116-116 (1985).</li><li>Cragg, B. G., Thomas, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+conduction+velocity+and+the+diameter+and+internodal+length+of+peripheral+nerve+fibers..">The relationship between conduction velocity and the diameter and internodal length of peripheral nerve fibers..</a> Journal of Physiology. 136, 606-614 (1957).</li><li>Erlanger, J. G. a. s. s. e. r., S, H., Bishop, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+compound+nature+of+the+action+current+of+nerves+as+disclosed+by+the+cathode+ray+oscillograph.">The compound nature of the action current of nerves as disclosed by the cathode ray oscillograph.</a> American Journal of Physiology. 70, 624-666 (1924).</li><li>Furshpan, E. J., Potter, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+at+the+giant+motor+synapses+of+the+crayfish.">Transmission at the giant motor synapses of the crayfish.</a> Journal of Physiology. 145 (2), 289-325 (1959).</li><li>Johnston, M. F., Simon, S. A., Ramrn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+anesthetics+with+electrical+synapses.">Interaction of anesthetics with electrical synapses.</a> Nature (Lond). 286, 498-500 (1980).</li><li>Loewenstein, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+membrane+junctions.">Permeability of membrane junctions.</a> Annual NY Academy of Sciences. 137, 441-472 (1966).</li><li>Meda, P., Bruzzone, R., Knodel, S., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockage+of+cell-to-cell+communication+within+pancreatic+acini+is+associated+with+increased+basal+release+of+amylase.">Blockage of cell-to-cell communication within pancreatic acini is associated with increased basal release of amylase.</a> Journal of Cell Biology. 103 (2), 475-483 (1986).</li><li>Peracchia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+gap+junction+resistance+with+acidification+in+crayfish+septate+axons+is+closely+related+to+changes+in+intracellular+calcium+but+not+hydrogen+ion+concentration.">Increase in gap junction resistance with acidification in crayfish septate axons is closely related to changes in intracellular calcium but not hydrogen ion concentration.</a> Journal of Membrane Biology. 113 (1), 75-92 (1990).</li><li>Peracchia, C., Dulhunty, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+resistance+junctions+in+crayfish:+structural+changes+with+functional+uncoupling.">Low resistance junctions in crayfish: structural changes with functional uncoupling.</a> Journal of Cell Biology. 70, 419-439 (1976).</li><li>Peracchia, C., Bernardini, G., Peracchia, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+calmodulin+involved+in+the+regulation+of+gap+junction+permeability.">Is calmodulin involved in the regulation of gap junction permeability.</a> Pf&#252;gers Arch. 399, 152-154 (1983).</li><li>Peracchia, C., Lazrak, A., Peracchia, L. L., Peracchia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+models+of+channel+interaction+and+gating+in+gap+junctions.">Molecular models of channel interaction and gating in gap junctions.</a> Handbook of Membrane Channels. Molecular and Cellular Physiology. , 361-377 (1994).</li><li>Spray, D. C., Harris, A. L., Bennett, M. V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctional+conductance+is+a+simple+and+sensitive+function+of+intracellular+pH.">Gap junctional conductance is a simple and sensitive function of intracellular pH.</a> Sciences NY. 211, 712-715 (1981).</li><li>Spray, D. C., Harris, L. L., Bennett, M. V. L., Nuccitelli, R., Deamer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+pH+and+Ca+dependence+of+gap+junctional+conductance.">Comparison of pH and Ca dependence of gap junctional conductance.</a> Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions. , 445-461 (1982).</li><li>Spray, D. C., White, R., De Carvalho, C., Harris, A. L., Bennett, M. L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+of+gap+junction+channels.">Gating of gap junction channels.</a> Journal of Biophysics. 45, 219-230 (1984).</li><li>Watanabe, A., Grundfest, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impulse+propagation+at+the+septal+and+commissural+junctions+of+crayfish+lateral+giant+axons.">Impulse propagation at the septal and commissural junctions of crayfish lateral giant axons.</a> Journal of General Physiology. 45, 267-308 (1961).</li><li>Wiersma, C. A. G., Hughes, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+functional+anatomy+of+neuronal+units+in+the+abdominal+cord+of+the+crayfish,+Procambarus+clarkii.">On the functional anatomy of neuronal units in the abdominal cord of the crayfish, Procambarus clarkii.</a> Journal of Comparative Neurology. 116, 209-228 (1961).</li></ol>

No conflicts of interest declared.

Our goal in the on-line video presentation and this paper is to demonstrate that the biophysical properties of cells can, in part, be modeled as electrical circuits. In addition, with live neural tissue that is relatively easily obtained, fundamental principles of conduction velocity, refractory periods and electrophysiological recording techniques are possible for undergraduate student laboratories with modest investment of equipment. The themes and fundamental paradigms presented can be readily modified for the requirements of different courses.
Maintenance of crayfish and their abundance makes them attractive models for student-driven experimentation. Crustacean ventral nerve cords are generally robust and retain physiological integrity in a minimal saline for hours, which is adequate for a 3 hour student laboratory.
Given that some of the large axons in the VNC of the crayfish are connected via gap junctions, additional experimentation on their contribution can be conducted, and different properties than found in the standard frog sciatic nerve preparation can be demonstrated. The sciatic nerve is a classic model for addressing compound action potentials and conduction properties. It might even be an interesting comparative experiment for students to compare conduction properties, axon recruitment, and refractory periods between these two preparations.

Circuit board
<ol>
 <li>Electronics listed below for the breadboard experiments can be obtained at an local electronics store such as Radio Shack.</li>
 <li> Bread board, resistors, capacitors, batteries and wires that can serve as junctions for the breadboard as outlined in the experiments.</li>
 <li>A common voltmeter (Wavetek Meterman voltmeter)</li>
 <li>A/D board for on line recording to a computer. Electrical signals are recorded on line to a <a href="http://www.adinstruments.com/products/pl3504#overview" target="_blank">PowerLab/4s interface</a> (ADInstruments, Colorado Springs, CO, USA). We use standard software from ADInstruments named Chart or Scope.</li>
</ol>
Physiology experiments
<ol>
 <li>Crayfish (Procambarus clarkii). Atchafalaya Biological Supply Co., Raceland, LA., USA.</li>
 <li>Standard crayfish saline: Modified from Van Harreveld's solution (1936). (in mM) 205 NaCl; 5.3 KCl; 13.5 CaCl22H2O; 2.45 MgCl26H2O; 5 HEPES and adjusted to pH 7.4. All saline chemicals were obtained from Sigma chemical company (St. Louis, MO).</li>
 <li>Dissection tools: Fine #5 tweezers, fine scissors, knife blade holder, #26002-20 insect pins (all obtained from Fine Science Tools (USA), Inc., 373-G Vintage Park Drive, Foster City, CA 94404-1139)</li>
 <li>A nerve chamber dish (ADInstruments, Colorado Springs, CO, USA) to hold the nerve. The wires that come with the nerve chamber dish can be used to stimulate the nerve.</li>
 <li> A suction electrode is used to record the signals. </li>
 <li>The manipulator or a clamp on a ring stand will also serve as a holder for the suction electrode.</li>
 <li>Faraday Cage</li>
 <li>Dissecting Microscope</li>
 <li>High Intensity Illuminator (light source)</li>
 <li>Microscope Platform</li>
 <li>AC/DC Differential Amplifier (A-M Systems Inc. Model 3000)</li>
 <li><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML856/" target="_blank">PowerLab 26T</a> (AD Instruments)</li>
 <li><a href="http://www.adinstruments.com/products/hardware/education/product/FE185/" target="_blank">Extracellular amplifier</a> (AD Instruments)</li>
 <li><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">LabChart 7</a> (ADI Instruments)</li>
 <li> Dissecting tools</li>
 <li>A ring stand and clamp to serve as a holder for the recording suction electrode</li>
</ol>

modeling biological membranes with circuit boards and measuring electrical signals in axons: student laboratory exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

Watch this Scientific Journal Video about Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises at JoVE.com

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

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22.3K Görüntüleme.  University of Kentucky. Bu biyolojik membranlar elektrik modelleri kullanarak nasıl anlaşılabilir bir gösteri. Ayrıca öğrenci yönelik laboratuarlar için kerevit ventral sinir kordonundan aksiyon potansiyelleri kayıt prosedürleri göstermektedir.