Electrophysiology Of The Sciatic Nerve – II.

Strength-Duration Relationship and Refractory Period

 

Experimental Techniques

Once you have activated the simulation, make sure that the Pulse Control is set to the “Single” mode.  Select Recording Electrode #1 by clicking on the “Recording Electrode #1” button at the bottom of the display. This will cause the blue 'wire' to connect the Recorder to the first (blue) recording electrode in the nerve chamber. 

Click the “Go” button to stimulate the sciatic nerve and cause it to generate an action potential. When you click the “Go” button, the product of the stimulus’ Strength and Duration is displayed in the white area on the Stimulator, and an orange stimulus indicator ‘light’ flashes briefly to inform you that a stimulus has been sent to the stimulating electrode (can you describe in electrical terms what is really happening?).  If the stimulus delivers enough power to the sciatic nerve, it will generate a compound action potential which progresses along the nerve to the recording electrodes.  When the action potential reaches the location of the recording electrodes, a graph of the action potential at that location will be generated on the display’s axes. 

Each time you click “Go”, a new color is used to depict the resulting action potential, and a corresponding Legend entry is displayed at the top of the display.  This will facilitate your comparing the results of various experimental treatments.

Note:  just as would happen if you were really running this experiment (i.e., using living experimental animals and an oscilloscope to record the action potentials), the numerical data you need to complete the exercise will not be displayed for you on the computer screen.  As in real life, you’ll have to measure and/or estimate times and amplitudes.

 

Important Note:  the timer for the x-axis starts when the “Go” button is clicked and the compound action potential is initiated at the point of stimulation.  Thus, the x-axis represents the time since the compound action potential was initiated on the sciatic nerve.  Consequently, you will not see anything displayed on the graph until the compound action potential reaches the recording electrodes.

    Determination Of The Strength-Duration Relationship

Once you are comfortable with the workings of the simulation vary the Strength and Duration of the applied stimulus and determine the combinations that (i) supply just enough stimulus to the membrane to trigger an action potential and (ii) are sufficient to give a maximum-amplitude action potential.  Use the attached data sheet to record data on the maximum amplitude of the action potential (mV), the time of maximum amplitude (ms after stimulation), and peak width at half-height (W½).


     Determination Of The Sciatic Nerve's Refractory Periods

Click the “Dual” checkbox.  Note that when you do this, the 1st recording electrode is automatically selected.  You cannot record from other electrodes during this exercise.

Set the Strength and Duration parameters at values that give you a maximum-amplitude action potential.  Select the “Dual” pulse mode.  Vary the Delay setting and conduct enough runs to allow you to determine the length of the absolute and relative refractory periods, and to get a clear picture of the amplitude of the action potential at different stages in the relative refractory period.  Use the attached data sheet to record your data. 

Tips & hints

1.  The recording electrodes are 10mm apart, and the first recording electrode is 20mm from the stimulating electrode.  Use this information when calculating conduction velocity of the compound action potential.

2.  When determining the refractory period of the nerve, you may – depending on the resolution of computer’s monitor – be able to see a very small action potential at a pulse delay of 2 ms. 

3.  For the strength-duration exercise, start with a stimulus strength of 5 V, and work ‘up and down’ from that value.

4.  You should note how the numerical value displayed in the Stimulator’s white panel changes as you change the value of the stimulus’ Strength and/or Duration

5.  In this simulation, the Stimulus Strength is measured in units of volts, but in many cases it’s measured in units of amperes.  We’ll use volts because from Ohm’s Law:

V = I×R

V and I are linearly proportional to each other, so we may use voltage as a surrogate for current in this case (we are, of course, assuming that R is constant).

Questions

    Basic Compound Action Potential Recording

1.  What is the general appearance of the action potential?  Is it a symmetrical curve?  Does it show a hyperpolarized afterpotential phase that you are used to seeing in the case of an action potential recorded from a single neuron? 

2.  How do your values for W½ compare with the 0.1 – 0.5 ms value for W½ that typifies the action potentials recorded from most individual axons?  What do you think might explain the difference?

3.  Does the appearance of the action potential change when you reposition the recording electrode further from the stimulating electrode?  If so, how?  Does the change, if any, make sense, given what you learned during the Compound Action Potential simulation?

4.  Does the appearance of the action potential alter as you change the duration and/or strength of the applied stimulus? 

5.  Select Recording Electrode #5 and set the Strength and Duration to their maximum values.  Click the Go button and observe the resulting action potential.  Describe its appearance and suggest an explanation.

 

Strength-duration Relationship

1.  Construct a graph of the Strength-Duration combinations that were just sufficient to elicit an action potential.  Now, do the same thing for the Strength-Duration combinations that were required to elicit a maximum-amplitude action potential. 

2.  Using the instructions, estimate rheobase and chronaxie values for the graphs you generated in Question #1. 

a.  The fact that nerves (indeed, as you will see, all excitable cells) exhibit a rheobase and a chronaxie tells us something fundamental about what’s involved in depolarizing a membrane to threshold.  Can you deduce what that fundamental something is?

b.  Speculate about the possible significance of differences in the rheobase and chronaxie values in the two curves.

2.  The chronaxie is felt by most investigators to represent a good measure of membrane capacitance.  Does this seem like a reasonable interpretation to you?  Justify your answer.

3.  Compare the strength-duration curves for minimal and maximal action potential amplitude.  How are they similar?  How do they differ?  

4.  Is the power required for a maximal action potential the same at all electrode positions?

5.  Based on the strength-duration curves for minimal and maximal action potential amplitude, select a value for duration that yields a decent ‘spread’ between the voltages required for minimal and maximal action potential amplitude (hint:  0.5 sec should be about right).  Then, using small increments in stimulus strength (hint:  try 0.1V), gather data that will let you establish the relationship between stimulus strength (the independent variable) and action potential amplitude (the dependent variable).  What is the nature of this relationship?  What does the ‘shape’ of the curve suggest about the distribution of neuron thresholds in the sciatic nerve?

6.  Construct a graph of W½ vs. maximum amplitude.  Is there any relationship between maximum amplitude and W½?  If so, what is the nature of the dependence of W½ on amplitude (linear, exponential, etc.)?

7.  Remember that we’re measuring the stimulus’ strength in volts, as a surrogate for current, thereby ignoring the resistance (Tips & Hints #5, above).  Given that, is the choice to use the product of the actual values of the Duration and the Strength of the stimulus for display by the Stimulator seem justifiable? 

Hint:  this is a tricky question that will test your understanding of electric current, membrane potentials, and what causes membranes to depolarize.

 

    Refractory Period

1.  Enter your data for the amplitude of the second action potential as a function of the Delay into an Excel spreadsheet.  What is the shape of the graph?  Use Excel’s curve-fitting algorithms to find which model (linear, power, exponential, logarithmic, or polynomial) fits the data best.  What do those results suggest about the recovery of the sciatic nerve’s neurons from their refractory period?

2.  Use your spreadsheet’s curve-fitting functions to fit various regression models to your data (click here ***data_handling.html*** for instructions on how to do this with Microsoft Excel).  Which model (linear, logarithmic, exponential, or power) fits the data best?  Do any of them fit the data well?

a.  Now, copy the data from the following table to your spreadsheet and use its graphing function to overlay graphs of Y1 and Y2 on the plot of your data:

 

Stimulus

Delay

( ms )

 

 

Y1

 

 

Y2

0

0

0

1

.2

.2

2

1.4

1.2

3

7.9

6.5

4

34.8

26.9

5

76.9

42.1

6

95.4

18.6

7

99.2

3.8

8

99.8

.7

9

99.9

.1

19

99.95

.05

11

100

.05

12

100

0

13

100

0

14

100

0

15

100

0

 

The values for Y1 were generated by fitting a sigmoid curve to the data. 

The values for Y2 are simply the increase in Y1 from the previous Stimulus Delay entry.  That is, Y2 for a Stimulus Delay of 5 ms is simply Y15 – Y14 = 76.9 – 34.8 = 42.1.   

b.  Does a sigmoid curve fit the data better than  the models your spreadsheet provided?  What does this suggest about the recovery of the sciatic nerve’s neurons from their refractory period? 

c.   What is the general shape of the graph of Y2?  Careful analysis of this graph will significantly enhance your understanding of the recovery of compound nerves from their refractory period.   (Hint:  The Y2 entries basically tell you what proportion of the neurons have recovered from their refractory period during each Delay interval.  I.e., 42.1% of the sciatic nerve’s neurons recovered from their refractory period during the 4 to 5 ms Stimulus Delay interval)

DATA SHEET FOR STRENGTH-DURATION EXPERIMENT

 

 

Recording

Electrode #

Stimulus Duration

( ms )

Stimulus Strength

( V )

 

Peak Time

( ms )

Peak

Amplitude

( mV )

 

W½

(ms)

 

 

Notes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DATA SHEET FOR REFRACTORY PERIOD EXPERIMENT

 

 

Stimulus Delay

( ms )

1st Action Potential

Amplitude

( mV )

2nd Action Potential

Amplitude  ( mV )

 

 

Notes