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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 filling 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 R1 Red and Green Banana R2 Leads Red Banana Lead Figure 8. Computer-based set up for recording from the sciatic nerve. Stimulator output is sent to Channel # 2 for simultaneous recording. MCB 403 Fall Page 7 of 16
  • 8. NeuroPhysiology Lab. Lab #10 III. Experimental Procedures. Open up both the Scope Template "NeuroScope40310" and the Excel data template "NeuroTemp40310". Settings (Fig.'s 11 & 12) for the following protocols should already be in the Scope template and the data sheet is identical to the data sheet found at the end of this handout. Check with your TA before changing. The data display area File title Page Comment Channel window pop-up menu Range pop- up menu Scale pop-up menu Rate/Time display Samples Amlitude pop-up axis menu Marker Page Comment button Start/Stop button Marker Data Pad Channel information window Time axis separator Figure 10. Scope Window. Note Input Amplifier button MCB 403 Fall Page 8 of 16
  • 9. NeuroPhysiology Lab. Lab #10 Signal amplitude Range pop- up menu Filtering options Signal Input controls Invert the incoming signal Pause/Scroll button Figure 11. Input amplifier for the Channel 1 (A) recording the CAP. Initial Range is set to 100 mV. Later it will be adjusted to 10-20mV. AC Coupling should be on. Differential recording should be chosen. Use this pop-up menu to The shape of the stimulus select the stimulus range waveform is shown here Values set by the controls are displayed above the sliderbar Use this pop- up menu to change the stimulus voltage range Use the sliderbar or text entry button to set values Figure 12. Stimulator setup. This is found under the Setup Menu. Initially set to Pulse (or single). Later a double stimulus will be used. Set Delay at 5 ms, Duration to 0.1 ms, and Amplitude to 10 mV. MCB 403 Fall Page 9 of 16
  • 10. NeuroPhysiology Lab. Lab #10 IIIA. Threshold and Maximal Amplitude determination of the Compound Action Potential in the Sciatic Nerve. IIIA1. Introduction. The purpose of this section is to determine the Threshold2 (lowest level of stimulus that will elicit a recordable response) (Fig. 14) stimulus needed to elicit a recordable CAP. Following that we will determine the Maximal Compound Action Potential (Fig. 14) and its accompanying Maximal Stimulus Level. With this information we will determine the relationship between stimulus level and CAP amplitude. IIIA2. Set up. Using the setup shown in Fig. 8 you will be recording differentially between the R1 and the R2 sites on Channel #1. The stimulus signal will be patched into Channel #2 with a BNC cable. This way the amplitude, duration and delay of the stimulus will be automatically recorded in the file, were otherwise it is not. Initially set the stimulus as seen in Figure 13. Figure 13. Stimulus parameters for Threshold determination. IIIA3. Example of Data. Figure 14. Compound Action Potentials. A series of CAPs are overlaid to show the gradual increase of CAP amplitude as the stimulus is increased to finally produce the Maximal Action Potential with the Maximal Stimulus. IIIA4. Variables. Threshold Level. Maximal Stimulus Level. Maximal Compound Action Potential. 2 See definition in Matthews, pg. 62. MCB 403 Fall Page 10 of 16
  • 11. NeuroPhysiology Lab. Lab #10 IIIA5. Procedures. 1. After checking to see that the set up is correct, start Data Acquisition by pressing the Start button. 2. If the trace on the screen does not show any sign of deflection increase the strength up in 10 mV increments until you get a response. 3. Upon getting a response record the stimulus settings to that page’s notebook and indicate this is Threshold. This is the Threshold Level. Go to the next page by pressing the arrow to the right of the Page indicator at the lower right corner of the window. 4. Slowly increase the voltage until the CAP is at its maximum level (i.e. you no longer get increasing CAPs with increasing stimulus amplitude). This was produced using the Maximal Stimulus. Record the CAP amplitude, stimulus and input amplifier settings to that page’s notebook and indicate this is the Maximal Compound Action Potential. Save the file (but don’t close it) and go to the next page. 5. Reduce the stimulus strength to Threshold. Moving up in increments of 25% up to 125% of Maximal Stimulus. Record the CAP amplitude, stimulus and input amplifier settings to that page’s notebook and go to the next page. Activate the Overlay option as you proceed so that you can watch the gradual increase (Fig. 12) of the CAPs with increasing stimulus levels. Record three replicates at each stimulus level. This data will be used to construct a Stimulus/ Response Curve (Fig. 15 and 16). IIIA6. Calculations. Determine the relationship between strength of stimulus (V) and amplitude of the CAP (mV). Is it linear? Can it be described in a simple linear equation? Hint: try the line fit functions in Cricket Graph and be able to explain how well the line fits the data. IIIA7. Data Presentation. Present the data in format similar to that in Figures 15 & 16 supporting what is shown in each. IIIA8. Topics that should be addressed in the report: Discuss the conditions needed to elicit an AP. Explain the difference between a true AP and a CAP. Describe the relationship between stimulus and response in a nerve. How does your data compare to other groups’ data? MCB 403 Fall Page 11 of 16
  • 12. NeuroPhysiology Lab. Lab #10 Stimulus /Response Curve for a Frog Sciatic Nerve. 20 10 0 0.2 0.3 0.4 0.5 0.6 Stimulus (V) Figure 15. Stimulus/Response Curve for a frog sciatic nerve. Percent Stimulus/Response Curve. 120% Frog Sciatic Nerve Percent Maximal CAP 100% 80% 60% 40% 20% 0% 40 60 80 100 120 Percent Maximal Stimulus Level Figure 16. Stimulus/Response Curve using percent Maximal Stimulus Level. This shows the plateau when the Maximal Stimulus level is reached and exceeded, while the Maximal Compound Action Potential no longer increases. MCB 403 Fall Page 12 of 16
  • 13. NeuroPhysiology Lab. Lab #10 IIIB. Conduction Velocity determination of the Compound Action Potential in the Sciatic Nerve. IIIB1. Introduction. Conduction velocity is an important aspect of cellular physiology and is based on a number of factors that determine the fitness of the organism for its environment. These factors include diameter, mylenation (or the lack thereof), temperature etc. Thus, understanding this basic measurement will aid you in formulating a comprehensive view of the functioning of the nervous system. Basically we will measure the time it takes a CAP to travel the stimulating and recording sites. IIIB2. Set up. Use a 50% Maximal Stimulus. IIIB3. Example of Data. CAP from this Stimulus CAP from this Stimulus Figure 17. A recording of a CAP under two different stimulus setups. In the larger CAP the cathode (-) is closer to the recording electrodes (R1 & R2). While the smaller of the CAPs (also shifted to the right) is the product of a stimulus when the cathode is further from the recording electrodes. MCB 403 Fall Page 13 of 16
  • 14. NeuroPhysiology Lab. Lab #10 R1 D R2 Figure 18. The recording chamber and electrodes placement for determining conduction velocity. Where ∆D is the in distance (mm) between the Stimulating electrodes. IIIB4. Variables. Total Distance (∆D) between stimulating sites. Time difference (∆T) between the peaks of the CAPs. IIIB5. Procedures. 1. Generate a single stimulus by using the Pulse setting of the Stimulus, and record a CAP that is 50% of Maximal Stimulus. 2. Switch the polarity of the Stimulating electrodes by reversing the sign of the stimulus from +80 mV to -80mV. 3. Generate a second CAP. 4. Measure the difference in time (∆T) between the peaks. 5. Measure the distance between the stimulating electrodes (∆D) (Fig. 18). 6. Save this page remembering to record the Stimulus parameters and the Input Amplifier parameters (filters etc.) to that page’s comment. 7. Repeat steps 1-6 three time. 8. When you finish and have six to seven pages of data save this file under a descriptive name (like “CondVel/Grp2/Sect3”), including group and section information, and open a New one for the next series of experiments. IIIB6. Calculations. 1. Calculate the Conduction Velocity for each replicate by using the following equation: Conduction Velocity = ∆D(mm)/∆T(ms) 2. Determine the Mean and Standard Deviation for the series of replicates. IIIB7. Data Presentation. Present the data in the form of a table of raw data (i.e. ∆D and ∆T), conduction velocities, and the means and standard deviation of those velocities. MCB 403 Fall Page 14 of 16
  • 15. NeuroPhysiology Lab. Lab #10 IIIC. Refractory Period determination of the Compound Action Potential in the Sciatic Nerve. IIIC1. Introduction. Information in the nervous system is often encoded in the frequency of the signal since the amplitude of APs tend to be constant for a give population of neurons with similar physical characteristics. The maximum frequency at which neurons can produce APs is dependent on the characteristics of certain ion channels, most notably the sodium (Na+) and potassium (K+) channels. The minimum time between APs is defined as the refractory period. There are two types of this period. The Absolute Refractory Period (Fig. 20) is the minimum amount of time between APs regardless of the strength of the stimulus. The Relative Refractory Period (Fig. 20) is the minimum amount of time between APs generated by a physiologically relevant stimulus strength. See Fig. 2 for the actions of the ion channels and their relation to these two refractory periods. IIIC2. Set up. Use a 50% Maximal Stimulus (Fig. 19). 5.0 ms 0.1 ms ms 84 mV 84 mV Figure 19. Stimulus parameters for Refractory Period determination. Note the change to Double Stimulus. IIIC3. Example of Data. Figure 20. Series of CAPs from a frog sciatic nerve illustrating both the Relative and Absolute Refractory Periods. As the interval between the stimuli drops, the point when the first reduction in the second CAP’s amplitude occurs is the Relative Refractory Period. When the interval between the stimuli causes a complete abolition of the second CAP, this is the Absolute Refractory Period. MCB 403 Fall Page 15 of 16
  • 16. NeuroPhysiology Lab. Lab #10 IIIC4. Variables. Absolute Refractory Period. Relative Refractory Period. IIIC5. Procedures. 1. Use the Double Mode to generate two stimuli. This pair of CAPs should be of identical amplitude (mV) using identical size (mV) stimuli of 50% Maximal 2. Slowly reduce the stimulus Interval until the second CAP’s amplitude just starts to be reduced when compared to the first CAP (Fig. 21). Save this page, recording the stimulus settings in the notebook before going to the next page. This Interval is the Relative Refractory Period in ms. 4. Continue to slowly reduce the Interval until the second CAP’s completely disappears. Save this page, recording the stimulus settings in the notebook before going to the next page. This Interval is the Absolute Refractory Period in ms. 5. Repeat steps 1-4 at least three times. 6. When you finish and have six to ten pages of data save this file under a descriptive name (like “RefracPrd/Grp2/Sect3”), including group and section information. IIIC6. Calculations. Determine the Mean Absolute and Relative Refractory Periods along with SDs. IIIC7. Data Presentation. Present the data in the form of a table of both raw and calculated (mean and SEM) data. IV. References. Aidley, D.J. 1989. The Physiology of Excitable Cells. Cambridge University Press., Cambridge, New York, Port Chester, Melbourne, Sidney. Baker, P.F. 1966. The Nerve Axon. Scientific American. March. pp 74. Mathews, G.G. 1986. Cellular Physiology of Nerve & Muscle. Blackwell Scientific Publications. Boston, Oxford, London, Edinburgh, Victoria. Stevens, C.F. 1979. The Neuron. Scientific American. September. Vol. 48. pp 54. MCB 403 Fall Page 16 of 16
  • 17. NeuroTemp40310 Page 1 NeuroPhysiology MCB 403 - Fall Names: Date: Group Section: IIIA. Threshold & Maximal CAP & Maximal Stimulus. Replica te Thres hold 25% Max 50% Max 75% Max 100% Max 125% Max CAP Stim CAP Stim CAP Stim CAP Stim CAP Stim CAP Stim 1 2 3 Mean #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! SD #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! Stim Duration (ms): Stim. Delay (ms): Notes IIIB. Conduction Velocity.. Latency Stim. Polarity Stim. Polarity Replica te + - - + Add a Latency CAP (ms) CAP Latency (ms) ∆T ∆D 50 % Max Stimulus 1 Stim. Amplitude (mV): 2 Stim Duration (ms): 3 Stim. Delay (ms): Mean #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! SD #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! Notes IIIC. Refractory Periods. Inter-Stimulus Intervals (ms) & CAP Amplitudes (mV). Absolute Relative Replica te Interval CAP #! CAP #2 Relative CAP #! CAP #2 1 50 % Max Stimulus 2 Stim. Amplitude (mV): 3 Stim Duration (ms): Mean #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! Stim. Delay (ms): SD #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! #DIV/0! Notes