回路基板と生体膜のモデル化と軸索の電気信号の測定：学生研究室の演習を

The overall goal of this procedure is to demonstrate how biological membranes can be understood using electrical models and to demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish. This demonstration first uses components of a circuit on a breadboard to represent the electrical properties of membranes. The second step of the procedure is to vary the resistance and capacitance on a model circuit to see the effects on the voltage traces.

The third step of the procedure is to apply concepts learned from a model circuit to a live nerve preparation. The final step of the procedure is to record compound action potentials and understand the properties of threshold refractory periods and synaptic transmission. Ultimately, results show how examining models of electrical circuits of biological membranes and properties of live nerves can be useful teaching tools as demonstrated by altering the components of a model circuit and by varying the stimulation of the live nerve.

This method can provide insight into how biological membranes can be understood in terms of electrical models. It can also be applied to other tissues such as muscle Visual. Demonstration of this method is critical as a dissection of the ventral nerve cord and recording setup can be difficult at first and care must be taken to avoid damaging the nerves so as to obtain a good setup for stimulating and recording from the nerve cord.

In the first part of the experiment, resistors and capacitors are used to simulate the electrical activity of the nerve cell membrane. The initial circuit has only resistors. Capacitors are added at a later step.

The passive resistive properties of the nerve membrane are represented by the circuit shown here. The breadboard is set up with resistors in parallel of 33 kilo ohms, representing the transverse membrane resistance and resistors in series of 10 kilo ohms representing the intracellular resistance. This circuit assumes that the resistance of the external fluid is negligible in comparison to the intracellular and transverse membrane resistances.

When your model cell is complete, connect the output leads from the power lab to the breadboard model. The power lab generates the stimulus and has a measuring probe for recording voltage. Next, connect the power lab to the computer.

The computer is used to display the electrical signals recorded from the breadboard. The next step is to set up the computer software that will control the stimulus sent by the power lab to the model nerve cell. Open the lab chart program from the desktop.

Select new file, select setup, and then channel settings. Select one channel to be shown, then select stimulator panel. Long pulses of 1.5 volts are required.

For this portion of the experiment, adjust the amplitude to one volt. The power lab will emit a voltage fluctuating between positive and negative 0.5 volts, which results in a peak tope stimulus of one volt. Set the frequency to 0.75 hertz and the pulse duration to one second.

For channel settings, select channel one to monitor, click on input amplifier and in the box, click on the differential button and set the range to five bolts and select.Okay. The next step is to apply an electrical signal to the nerve cell model and examine the loss of the signal along the length of the model. Apply a voltage at one end of the circuit between A and B by clicking start on the computer.

Measure the voltage for the first unit length along the model axon and monitor it on the computer. Leave the red lead of the voltage measuring probe stationary at point A on the breadboard while moving the other lead to different locations between the resistors in series. Notice the voltage drop at the more remote locations.

Now stop recording by pressing stop on the computer. Measure the saved voltage differences on the traces stored on the computer by using the cursor to move the trace to each pulse recorded. Use the M marker cursor found in the lower left corner to mark the bottom of the trace and place the free cursor at the peak of the trace to measure voltage difference.

Read the voltage difference measurement from the screen and write it in your notebook for each measurement location along the circuitry. The next step is to calculate the length constant for the model cell, which is used to describe the exponential decay of the signal over distance. Plot each value of voltage difference on a graph of potential difference against distance from the source where the membrane resistors are considered to be one unit.

Apart from your graph, determine the length constant of this circuit. The length constant of a nerve fiber is defined as the distance over which a potential difference across the membrane declines to 37%of its original value at the point of origin. In order to better simulate the passive cable properties of nerve and muscle fibers, the model nerve cell will be expanded to contain both resistive and capacitative components.

Not only is there a transverse membrane resistance present in the nerve cell membrane, there is also a surface membrane capacitance, which distorts the nerve cell signal. To simulate this, attach 10 micro fert capacitors in parallel to the transverse membrane. Resistors on the breadboard on the computer.

Set the stimulator to use one second, one volt, which is a 0.5 volt amplitude setting and 0.75 hertz pulses. Also in the upper right hand side of the panel, set the acquisition rate to 20 K per second. Next click on channel one at the right hand side of the screen.

Click on input amplifier in the box. Click on the differential button and set the range to five bolts. Click okay.

To save these changes, click start To begin collecting data. Measure the data at the same points along the breadboard. After data collection, use the zoom window to expand the rise time on one of the square pulses.

Repeat the zoom-in as needed to spread out the trace. Measure the time it takes for the signal to rise from the baseline to the top of the voltage trace. Just as it levels off.

Observe the time course of the potential change and the distortion of the square pulses. Also note if the voltage measured is above 0.2 volts using the M cursor and the free cursor as before, read the change in voltage and record the data in your notebook, assuming the threshold voltage to be 0.2 volts. Measure the time taken for the potential to reach threshold at each membrane resistor.

Plot your results as time against distance and increase in the thickness of the myelin sheath produces a decrease in membrane capacitance. Simulate this by replacing the 10 micro ferd capacitors on the breadboard with smaller 0.1 micro ferd capacitors. Repeat the previous experiment.

Note the change in time to reach threshold at a selected resistor. Superimposed the data with the larger capacitors on the previous graph of time versus distance. Increasing the thickness of the myelin sheath allows electrical signals to rise and fall more quickly in a nerve cell and so increases conduction speed.

In the next part of the experiment, the electrochemical properties of axons will be examined in the ventral nerve cord of the crayfish. The properties of passive current spread presented in the model circuits are also observed in live preparations. However, an action potential is needed whenever a signal needs to be transmitted over a distance of a few millimeters because the passive electrical properties of cells would attenuate the signals over this distance.

When signals travel along the crayfish ventral nerve cord with different conduction velocities, the fastest ones will be observed first and the slower ones will be observed slightly later. Some of the signals will summate in the recording depending on the population of axons with similar conduction velocities. This summated electrical signal from the multiple axons is referred to as a compound action potential.

The next series of experiments focuses on recording compound action potentials from crayfish nerves to obtain the ventral nerve cord of the crayfish. First, prepare the nerve dish dissection instruments and saline solutions. Place the crayfish on ice for about five minutes to anesthetize it.

Once anesthetized, wrap the crayfish in a moist paper towel and hold the crayfish in one hand with its claws between your thumb and forefinger. Using the other hand, make an incision with the scissors through the rostrum between the eye sockets. Cut downward from the incision until the head is completely severed.

Remove the claws and walking legs. Make an incision at the base of the cephalic thax above the abdomen and detach the cephalic thax. Remove the tail and the swim ettes.

Make a cut along both sides of the ventral side of the abdomen. Be careful not to damage deeper tissue. Peel back the freed section of the exoskeleton to the base of the tail fan in order to expose the ventral nerve cord.

Using the edge of the forceps carefully remove the ventral nerve cord by the tip. Be careful not to stretch crush the nerve cord. Any nodules observed on the nerve cord are crayfish ganglia.

Lay the nerve cord across the metal bars in the nerve chamber. It is imperative to keep the ventral nerve cord moist. To accomplish this, add enough crayfish saline solution to the nerve chamber to reach the level even with the metal crossbars.

Next, pipette out enough of the crayfish saline solution so that the level of the liquid just makes contact with the bottom of the bars. Once the nerve cord is in place, attach the stimulator cable with the two mini hook leads to the nerve chamber. Attach the positive red lead to one of the external metal loops near the end of the nerve cord and attach the negative black lead to the adjacent metal loop that is also in contact with the nerve cord.

Use the manipulator to position the suction electrode near the end of the nerve opposite from the negative and positive leads. Carefully suction the tip of the ventral nerve cord into the recording electrode. Attach the electrode to the head stage probe by attaching one wire from the suction electrode to the positive input of the probe and the other wire to the negative input.

It does not matter which wire goes to which input. The probe can be grounded to the Faraday cage or the saline bath with use of a silver chloride wire, or connect it to the ground input on the differential amplifier to decrease potential 60 hertz noise. Attach the probe to the differential amplifier and connect the amplifier to the power lab.

Check that the settings for the amplifier RS shown in the table in the accompanying article. Use the USB cable to connect the power lab to the computer. Open the scope software on the computer desktop on the top right of the screen.

Select channel two and turn off. Adjust the screen so that only channel one is showing by dragging the bar in the middle to the bottom of the screen. Check the input amplifier with settings given in the accompanying paper.

Then under setup at the top of the screen, select stimulator and turn off isolated stem and set delay to five milliseconds, duration to one millisecond, two pulses, an interval of 15 milliseconds, a range of five volts and an amplitude of 0.5 volts. To begin stimulating the ventral nerve cord, click the start button at the lower left of the screen. A clearly defined action potential should appear on the scope data collection box.

Sketch the general shape of the action potential in your notebook. Slowly decrease the voltage using the tab at the top left of the screen. The action potential should become smaller in magnitude until its characteristic wave pattern disappears.

Decreasing the voltage reduces the number of neurons that are recruited to contribute to the compound action potential to reverse the effect incrementally increase the voltage. As more neurons are recruited, the compound action potential wave grows larger. This exercise illustrates the fact that individual neurons have different thresholds at which their action potentials are elicited.

When a further increase in stimulating voltage does not increase the compound action, all the neurons have been recruited and two distinct peaks in the action potential wave should be visible on the computer screen. The next part of the experiment examines the refractory period of axons. Change the delay between pulses using the tab in the top left corner of the screen.

Keep shortening the delay until the second compound action potential cannot be elicited. The absolute refractory period is defined as the time delay between pulses at which a second action potential is no longer elicited. The relative refractory period is defined as the time from the end of the absolute refractory period to the time when giving a second stimulus results in a full amplitude second action potential.

You can now investigate the properties of a gap junction inhibitor like hept ethanol. The differences in the compound action potential can be compared for the same stimulation settings to determine which signals are conducted via gap junctions along the ventral nerve cord. You could also use cold saline or warm saline and measure differences in the conduction velocities of the various entities that comprise the compound action potential.

Shown here are two compound action potentials recorded from the ventral nerve cord of a crayfish. This figure illustrates the relative refractory period that is the time following a full compound action potential when a second stimulus elicits a smaller compound action potential. This figure shows the effects of using hept ethanol on the compound action potential Following this procedure.

The components of the biological membrane circuit may be varied in order to answer additional questions such as the effect of an altered density of leak channels in the membrane or the function of voltage gated channels After their development. These techniques and procedures pave the way for researchers in the field of physiology to explore how cells function as electrical circuits and how gap junctions function in living tissue in animals.