NeuroPhysiology Lab

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  • 1. NeuroPhysiology Lab. Lab #10 I. Introduction1. Action Potentials (AP) are the basis of communications in the body. Since for the most part the nervous system is the primary director of homeostasis, and it is the AP that is the signal the nervous system uses in communications, thus understanding the basis of its function is one of the primary goals of physiology. APs travel down neurons, both to and from the CNS. Bundles of these neurons make up nerves. The Sciatic Nerve is made up of hundreds of descending (signals from the CNS to the periphery) and ascending neurons (signals from the periphery to the CNS). The descending neurons of the sciatic, innervate the muscles and other effectors of the leg, while the ascending neurons innervate the sensory structures of the leg. Neurons communicate via electrical signals in the form of an action potential. If one stimulates an isolated sciatic nerve electrically and records from the nerve extracellularly (i.e. from the outside) a Compound Action Potential (CAP) is observed. A CAP is the sum of APs generated by a number of neurons. Figure 1. Series of Compound Action Potential overlaid on top of each other. The CAPs are a response to increasing stimulus. The greater the stimulus, the greater the number of neurons that fire (generate a AP) and the greater the number of firing neurons the larger the CAP (Fig. 1). Finally it must be noted that many of the manipulations you perform today, do not occur within the physiological ranges that are seen within the body. 1 See Chapter 5 Matthews, 1986. MCB 403 Fall Page 1 of 16
  • 2. NeuroPhysiology Lab. Lab #10 Figure 2. Compound Action Potential (CAP) with the sodium and potassium conductances overlaid (measured at the first recording electrode). In an action potential the membrane potential will rapidly shift toward 0 mV as the Na+ ions flood in through the voltage gated (electrically opened, timer closed) sodium channels down the physico-chemical gradients i.e. the sodium conductance (Fig. 2). The potential shift will slow, stop, and begin to reverse as the slower opening voltage gated potassium channels (electrically opened, timer closed) open, allowing K+ ions to flow out of the cell down the physico-chemical gradients returning the membrane potential to the resting state after a slight overshoot. Both of these channels remain inactive for a short time following closure. Objectives of this Laboratory Experiments: Determine the: A. Threshold Level. Maximal Stimulus level. Maximal Compound Action Potential. B. Conduction Velocity. C. Refractory Periods. MCB 403 Fall Page 2 of 16
  • 3. NeuroPhysiology Lab. Lab #10 II. Setup. IIA. Removal and mounting of the nerve (sciatic). 1. Decapitate a frog using a guillotine. Decapitating along a line behind the eyes and cutting off the front of the head in one swift cutting motion (Fig. 3). This is the least stressful way of killing the frog. Then destroy the brain by pithing with a probe. Eye Level of Decapitation Ear Figure 3. Level of decapitation of the frog. 2. Spinal pith the frog by thrusting a probe down the open spinal column and moving it around to completely destroy the spinal cord (Fig. 4). Figure 4. Pithing the frog. MCB 403 Fall Page 3 of 16
  • 4. NeuroPhysiology Lab. Lab #10 3. Remove the skin off of one leg (leave the other for the other team to remove the other sciatic nerve) (Fig. 5). Figure 5. Removing skin from frogs leg. 4. Separate the two major muscles of the thigh to expose the white sciatic nerve (Fig. 6). Figure 6. Exposure of Sciatic nerve in thigh musculature. 5. Gently raise the nerve (without pinching or pulling it) and free it from the sheath. MCB 403 Fall Page 4 of 16
  • 5. NeuroPhysiology Lab. Lab #10 6. Cut the muscles on ether side of the urostyle (Fig. 7) (careful not to cut the sciatic). Exposure of Sciatic Nerve Former Position Urostyle of Urostyle Lift the Urostyle Cut Attachments Sciatic Nerve Trunks Figure 7. Exposure of Sciatic nerve under the urostyle. 7. Gently raise the urostyle and tie off the end of the sciatic as close to the spinal cord as possible and cut between the thread and spinal cord. 8. Lift the sciatic from the thigh, tie a string around the most distal end of the nerve and cut between the thread and the knee joint. 9. First place a small amount of grease in the bottom of the inter-well areas, then carefully lay the sciatic nerve into the nerve chamber (Fig. 9) over the layers of grease in the inter-well areas and the electrodes. MCB 403 Fall Page 5 of 16
  • 6. NeuroPhysiology Lab. Lab #10 Inter-well area A. Beads of Syringe with stub needle Grease lling with stub 3 2 Sciatic Nerve 1 Figure 9. A. Initial placement of grease in the Inter Well areas. B. Placement of nerve in the nerve chamber and the grease filling technique. 10. Finish applying grease to the inter-well area so as to seal the sciatic nerve in. This will allow you to electrically isolate the different sections of the nerve. 11. Fill each well with frog ringers only after applying ALL the grease seals. CHECK THAT YOU ARE SET UP CORRECTLY BEFORE CONTINUING !!!! MCB 403 Fall Page 6 of 16
  • 7. NeuroPhysiology Lab. Lab #10 IIC. Setting up the PowerLab for stimulating and recording from the sciatic nerve. You will be electrically stimulating and recording the electrophysiological response of an isolated sciatic nerve from a frog with the PowerLab (Fig. 8) system. Shielded Cable w/DIN-8 Connector BNC Lead BNC Lead Adaptor Adaptor Black Black Banana Lead Banana Stimulus Lead Leads