I. Introduction.

I. Introduction.Many physiological processes have regulatory control that maintains these processes with in

precise limits. The nervous, endocrine, and neuro-endocrine system are the primary regulators. One of the fundamental building blocks necessary to understanding of the systemic regulatory systems is the function of the nervous systems basic unit the neu-ron.

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, an 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 as-cending 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 electri-cal 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. 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). 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.

Stimu li

CAPs

Figure 1. Series of Compound Action Potential overlaid on top of each other. Each larger CAP is a response to a stronger stimulus.

Neurophysiology Lab Lab #1

MCB 404 Spring of 16

Figure 2. Compound Action Potential (CAP) with the sodium and potassium conduc-tance 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 poten-tial shift will slow, stop, and begin to reverse as the slower opening voltage gated po-tassium 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 Exercise.Determine the:

A. Threshold Level.Maximal Stimulus level.Maximal Compound Action Potential.

B. Conduction Velocity.C. Refractory Period.

This week we will explore the basic parameters of neuron function of a vertebrate. This will be in preparation for more in depth investigation of systems level control of a variety organs by the nervous system, including cardiovascular, respiratory and renal sys-tems. Keep this in mind for the next two weeks as we look at basic neurophysiology.

There are a variety of tools available to you to supplement this lab course. These include simulations (such as Axon and Cardiovascular simulation) and tutorials (such as Chemical synapses).



II. Dissection and mounting of sciatic nerve.IIA. Removal and mounting of the nerve (sciatic).

1. Decapitate a frog using a guillotine. Decapitating along a line between the eyes and the ears, 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.

Ear

Eye

Level of Decapitation

Figure 3. Decerebration of a frog.

2. Spinal pith the frog by thrusting a probe down the open spinal column and mov-ing it around to completely destroy the spinal cord (Fig. 4).

Figure 4. Pithing the frog.



3. Remove the skin off of one leg (leave the other for the other team to remove the other sciatic nerve) (Fig. 5). Be careful not to bring the skin in contact with tools used on the sciatic since the skin carries neurotoxins that will kill the sciatic nerve!

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.



6. Cut the muscles on ether side of the urostyle (Fig. 7) (careful not to cut the sci-atic).

Urostyle

Cut Attachments Sciatic Nerve Trunks

Former Position of Urostyle

Exposure of Sciatic Nerve

Lift the Urostyle

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. 8) over the layers of grease in the inter-well areas and the electrodes.

Syringe with stub “needle”

Grease filling with stub

Sciatic Nerve

1

2

3

Inter-well area

Beads of

A.

Figure 8. 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 WITH A T.A. THAT YOU ARE SET UP CORRECTLY!!!!



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. 9) system.

R1

R2

Red Banana Lead

BNC Lead Adaptor

StimulusLeads

BNC Lead

Black Banana Lead

BNC Lead Adaptor

Black Banana Lead

Shielded Cable w/DIN-8 Connector

Red and

Green Banana Leads

BNC T-Adaptor

Figure 9. Computer-based set up for recording from the sciatic nerve. Stimulator output is sent to Channel # 2 for simultaneous recording.



III. Experimental Procedures.Open up both the Scope Template "NeuroScope40410" and the Excel data template "Neuro-

Temp40408". Components of the Scope window (Fig .10) will be used to set and con-duct all subsequent activities duering the experiments.

File TitleThe data display area Waveform Cursor in-formation

Waveform Cursor

Scale pop-up menu

Channel pop-up menu

Amplitude axis

Channel units

Marker

Page Comment button

Display pop-up menu

Time axisPage buttons Channel separator

Pag Corner controls

Range pop-up menu

Rate /Time display

Time/ Freq pop-up menu

Samples pop-up menu

Start/Stop controls

Figure 10. Scope Window. Note Input Amplifier button.

Settings (Figs. 11 & 12) for the experimental protocols should already be in the Scope tem-plate and the data sheet is identical to the data sheet found at the end of this handout. Check with your TA before changing



Signal Amplitude

Pause/Scroll button

Amplitude axis

Invert the incoming signal

Filtering options

Signal input con-trols

Range pop-up menu

Units cali-bration op-

tion

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 chossen.

Several stimuli can be recorded on different pages

Stimulus waveform can be directly shaped

Used to set stimulus amplitude

Controls for stimulus

parameters

Stimulus range

Pop-up menu for stimulus

type

Stimulus waveform window

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 0.05ms, Duration to 0.05 ms, and Amplitude to 10 mV.



IIIA. Threshold and Maximal Amplitude determination of the Com-pound Action Potential in the Sciatic Nerve.

IIIA1. Introduction.

The purpose of this section is to determine the Threshold1 (lowest level of stimulus that will elicit a recordable response) (Fig. 13) stimulus needed to elicit a rec-ordable 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. 9 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.


MCB 404 Spring of 161 See definition in Matthews, pg 62.

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?



0.60.50.40.30.20

10

20Stimulus /Response Curve for a Frog Sciatic Nerve.

Stimulus (V)

Figure 15. Stimulus/Response Curve for a frog sciatic nerve.

1201008060400%

20%

40%

60%

80%

100%

120%Percent Stimulus/Response Curve. Frog Sciatic Nerve

Percent Maximal Stimulus Level

Percent Maximal CAP

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.



IIIB. Conduction Velocity determination of the Compound Action Po-tential 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 environ-ment. These factors include diameter, mylenation (or the lack thereof), tem-perature etc. Thus, understanding this basic measurement will aid you in for-mulating a comprehensive view of the functioning of the nervous system. Basi-cally we will measure the time it takes a CAP to travel the stimulating and re-cording sites.

IIIB2. Set up.

Using the setup shown in Figure 9 and setting the stimulus to achieve a 50% Maximal CAP as in Figure 17.

Figure 17. Stimulus parameters for Conduction Velocity determination.

IIIB3. Example of Data.

CAP from this s

etup

Stimulus+-

Electrodes

R1 R2

Nerve

Stimulus+ -

Electrodes

R1 R2

Nerve CAP from This setup

ΔΤ

Figure 18. 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 prod-uct of a stimulus when the cathode is further from the recording elec-trodes.



R1

R2

ΔD

Figure 19. The recording chamber and electrodes placement for determining conduc-tion velocity. Where ∆D is the in distance (mm) between the Stimu-lating 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 CAP.

2. Switch the polarity of the Stimulating electrodes by reversing the sign of the stmulus from 80 mV to -80mV. (Fig. 17).

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. 19).6. Save this page remembering to record the Stimulus parameters and the Input

Amplifier parameters (filters etc.) to that page’s notebook.7. Repeat steps 1-6 three time.8. When you finish and have six to seven pages of data save this file under a descrip-

tive name (like “CondVel/Grp2/Sect3”), including group and section informa-tion, 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 equa-tion:

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 veloci-ties, and the means and standard deviation of those velocities.



IIIC. Refractory Period determination of the Compound Action Poten-tial 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 be-tween APs is defined as the refractory period. There are two types of this pe-riod. The Absolute Refractory Period (Fig. 21) is the minimum amount of time between APs regardless of the strength of the stimulus. The Relative Refrac-tory Period (Fig. 21) 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.

Using the setup shown in Figure 9 and setting the stimulus as in Figure 20.

Figure 20. Stimulus parameters for Refractory Period determination. Note the change to Double Stimulus.

IIIC3. Example of Data.

Figure 21. 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 ampli-tude occurs is the Relative Refractory Period. When the interval be-tween the stimuli causes a complete abolition of the second CAP, this is the Absolute Refractory Period.



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, re-cording 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 disap-pears. 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 descrip-

tive name (like “RefracPrd/Grp2/Sect3”), including group and section informa-tion.

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.

V. References.

Principles of Physiology. by Berne and Levy, C.V. Mosby Company, 1990.

The Physiology of Excitable Cells. by D.J. Aidley, Cambridge University Press, 1984.

Neural and Integrative Animal Physiology. edited by C. Ladd Prosser, Wiley-Liss, 1991.



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A B C D E

Neurophysiology AlphaMCB 404 - Spring

Name(s):Section: Date:

A. Threshold & Maximal AmplitudeStim. Durat. (ms):

Sample Stimulus (mV) CAP (mV)SubThreshold

Threshold10% Maximal Stimulus20% Maximal Stimulus30% Maximal Stimulus40% Maximal Stimulus50% Maximal Stimulus60% Maximal Stimulus70% Maximal Stimulus80% Maximal Stimulus90% Maximal Stimulus100% Maximal Stimulus110% Maximal Stimulus120% Maximal Stimulus130% Maximal Stimulus

B. Conduction VelocityTo produce a Compound action Stimulus Strength (mV): NotesPotential of 50% Maximal CAP CAP Amplitude(mV):The following stimulus is used: Stimulus Duration (ms):

Time (ms)Replicate ∆t ∆D (mm) Velocity (mm/ms)

1 #DIV/0!2 #DIV/0!3 #DIV/0!4 #DIV/0!5 #DIV/0!

Mean #DIV/0!SEM #DIV/0!

C. Refractory PeriodTo produce a Compound action Stimulus Strength (V): NotesPotential of 50% Maximal CAP CAP Amplitude(mV):The following stimulus is used: Stimulus Duration (ms):

Refractory Period (ms)Replicate Absolute Relative

123

Mean #DIV/0! #DIV/0!SEM #DIV/0! #DIV/0!

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CAP

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Stimulus (mV)."

Stimulus-Response Curve, Alpha"

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Neurophysiology BetaMCB 404 - Spring











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Stimulus-Response Curve, Beta."

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Neurophysiology GammaMCB 404 - Spring











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Stimulus-Response Curve, Delta."

I. Introduction.

Documents

Transcript of I. Introduction.