Biology 325 Human anatomy and Physiology Fall 2004 Campos COMPOUND ACTION POTENTIALS FROG SCIATIC NERVE Adapted from Oakley B and R Schafer 1978 Experimental neurobiology Univ Michigan Ann Arbor Introduction In 1850 Hermann von Helmholtz first estimated the velocity of nerve impulses transmitted in a frog nerve muscle preparation with a mechanical kymograph and writing levers During the latter half of the 19th century concepts underlying the modern theory of nerve conduction were developed by Sherrington and others but modern electrophysiological research awaited the development of the cathode ray oscilloscope Using this apparatus Erlanger and Gasser in 1921 first measured the ionic currents of compound action potentials Their studies provided an important foundation for our present understanding of nerve function The frog sciatic nerve was the classical preparation for study of the action potential until experimental researchers developed intracellular recording methods for studying squid giant fibers Action potentials can be elicited simultaneously by electrical stimulation in thousands of axons of a peripheral nerve like the sciatic The collective response is termed a compound action potential CAP In this experiment you will measure compound action potentials CAPs from an isolated frog sciatic nerve to illustrate the basic physiological properties of nerve impulses Indiscriminate as this gross recording technique may seem some basic aspects of neuronal conduction maximal firing rates threshold conduction velocity and the role of axon size and myelination can be demonstrated using the whole nerve approach Procedure a Preliminary setup It is essential to familiarize yourself with the instruments and recording system before beginning the dissection The instrumentation may seem very complex but generally viability issues of the biological preparation often end the experiment Therefore clear up any practical or conceptual problems regarding the equipment before you begin working with the experimental animal Biology 325 Human anatomy and Physiology Fall 2004 Campos b Surgical procedure 1 Remove the skin from the legs and abdomen of the double pithed frog To do this cut around the abdomen and peel the skin downward and off the animal 2 Place the frog in a dissection pan and keep the animal moist at all times with frog Ringer s solution 3 Grasp the urostyle with forceps and cut it free make sure you cut superficially you should be able to observe the nerve plexus below it Figure 1 Note the regions of white fibers that compose the sciatic nerve plexus Each sciatic nerve originates as three spinal nerve roots Be careful not to damage the nerve plexus 4 Carefully tie the roots together with the end of a 10 cm length of Ringer s soaked cotton thread Cut the nerve roots as close to the spinal cord as possible Nerve branches forming sciatic nerve Sacral vertebra Gluteus 5 Using a glass hook gradually lift the sciatic nerve free from the associated fascia and the sciatic artery You may need to use blunt dissection techniques CAUTION Do not stretch the nerve Urostyle Sciatic nerve 6 Using forceps and the glass hook continue to expose the nerve from the animal until you reach the knee joint Semi membranosus Gastrocnemius 7 Holding the nerve with the moist thread reflect the nerve back onto the animal s leg Keep the preparation moist with Ringer s solution 8 Sever the nerve from the gastrocnemius muscle Figure 1 Location of the sciatic nerve in a frog 9 Place the nerve in a Petri dish containing frog Ringer s solution and keep refrigerated 10 While some group members start preparing the nerve for recording other students should dissect the second sciatic nerve as previously described Also immerse the nerve Ringer s solution when done Biology 325 Human anatomy and Physiology Fall 2004 Campos c Setup of equipment for schematic refer to Appendix I 1 Connect the red and black alligator clips from the stimulator electrodes to two of the metal rungs on opposite sides of the MLT012 B Nerve Bath Figure 2 The distance between the electrodes should be 0 5 cm It is not necessary to connect the green ground alligator clip 2 Connect the red positive BNC connector from the stimulator electrode to the positive analog output connector on the PowerLab Connect the black negative BNC connector from the stimulator electrode to the negative analog output connector 3 Connect the red and black leads from the first recording electrode to two of the metal rungs of the MLT012 B Nerve Bath Figure 3 Connect the 8 pin DIN connector to the Pod port on Input 1 of the PowerLab Figure 2 Placement of the stimulus electrodes on the MLT012 B Nerve Bath Figure 3 Placement of first recording electrodes on Nerve Bath Figure 4 Placement of second recording electrodes on Nerve Bath 4 Repeat step 3 for the second recording electrode but place the alligator clips further away from the stimulus electrode Figure 4 Attach the pod connector to the Pod port on Input 2 on the PowerLab 5 Using an eyedropper or Pasteur pipette fill the lower reservoir of the Nerve Bath with frog Ringer s solution Fluid in the lower reservoir must not come in contact with the metal electrode rungs Note OVERFILLING the Nerve Bath in this manner will cause a short circuit in your experiment 6 Cut a strip of filter paper and lay it over the wires in the nerve bath Figure 5 so that it touches both stimulating electrodes and both sets of recording electrodes Moisten the paper strip with frog Ringer s and place the cover on the nerve bath This arrangement will be used to test your connections 7 Turn on the PowerLab and make sure it is connected to your computer Figure 5 MLT012 B Nerve Bath set up for connection test with filter paper and frog Ringer s Biology 325 Human anatomy and Physiology Fall 2004 Campos 8 Open the dialog box for Scope input amplifier Figure 6 and select the following settings Figure 6 Scope input amplifier settings Input A Range 10 20 mV Low Pass Off Positive checkbox checked Negative checkbox checked Input B Range 10 20 mV Low Pass Off Positive checkbox checked Negative checkbox checked Time Base 10 kHz 9 From the Scope Setup menu select the Stimulator dialog box and Figure 7 and apply the following settings Figure 7 Suggested stimulator settings This dialog box is displayed when Stimulator is chosen from the setup menu Before making these settings insure that the Isolated Stim checkbox is off Biology 325 Human anatomy and Physiology Fall 2004