In this exercise you will record action potentials from earthworm giant nerve fibers. The recordings will differ slightly from the measurements described during lecture because they will be extracellular rather than intracellular. It is just too time-consuming to get microelectrodes into a neuron, even a giant one. Amazingly, you can record action potentials just by sticking simple electrodes (polished steel insect pins) into an earthworm near it's ventral nerve cord. This saves a lot of time and effort.
Because we are recording extracellularly, the
data will look a little different. Remember that a voltage is a difference
in the electrical potential between two points. In this experiment,
rather than comparing the electrical potential between an intracellular
and extracellular electrode at one spot on a neuron, you will measure the
difference between two extracellular electrodes a centimeter or so apart
(Fig. 1). Initially, the extracellular space is slightly positive
(because some K+ has diffused out of the neurons). Since it is positive
all along the neuron, there is no potential difference between the two
electrodes (A). When the nerve is stimulated sufficiently, it fires
an action potential; the Na+ channels open at the point of the stimulus
and Na+ rushes in, leaving the extracellular space more negative.
This depolarization travels down the axon. Therefore, as the action
potential passes the first electrode, it will become more negative than
the second electrode, producing a rise in the recorded voltage (B).
By the time the action potential reaches the second electrode, the area
near the first electrode will be repolarized. Thus, the second electrode
will be more negative, creating a dip in the recording (C). It is
easy to confuse this dip with the hyperpolarization seen in an intracellular
recording, but don't.
Technique:
The technique involved in this experiment is simple,
but it may take some fiddling with electrodes to get a clean recording.
First, get a worm. Anaesthetize it in 10% ethanol (until it stops
wriggling - about 5 min). Put it in a dissecting tray, dorsal side
up (the dorsal side is darker, smoother, and slightly rounder). Then
stick five electrodes along its midline (Fig. 2). Make sure they
are as close as possible to the midline. There are two stimulating
electrodes (positive and negative), a ground electrode, and two recording
electrodes. Keep the worm moist, but don't let the water puddle up.
The tray should stay dry; if you have too much water, you can get
a huge stimulus artifact that drowns out your data. (ie. the stimulus
conducts through the puddle to the other electrode)
Suggested amplifier settings: 1000x gain, filter everything below 100 Hz and above 5 kHz, 60 Hz notch filter out
You may get strong 60 Hz (Hz = cycles/sec) interference if your connections are bad. This is because all your wires are like little antennae that pick up noise from the electrical activity in the room. Since the entire building (the entire country for that matter) is wired with 60 Hz current, your wires pick up a lot of that frequency. You can easily recognize it because 60 cycles/second means it repeats itself every 1/60th of a second (16.6 ms). The main cause for noisy recordings seems to be a weak conducting pathway between the recording electrodes. The best recordings come when you have the electrodes as close as possible to the nerve. Clearly, the closer you are to the nerve, the easier it will be to pick up any voltage changes. So you will want to make sure that all your electrodes are as close as possible to the midline. Since the voltage you record will depend on the electrode placement, and you are not recording the potential across the membrane but between two extracellular spots, don't expect to see the neuron change from -70 to +40 mv like you would see in an intracellular recording.
If you can't get rid of noise, you can often see action potentials anyways. If you see a blip that looks like an action potential you can easily verify it by repeating the stimulus. If it always shows up at the same time with the same magnitude, then it is an action potential. Otherwise it's just random noise.
Use the program NEURON in the "physio lab" folder of Superscope (Fig. 3). This program shows the recorded voltage in the top display, and the applied stimulus in the bottom display. There is a start button, which will output the stimulus and begin recording. There is also pair of buttons labelled marker and draw, which control what the mouse does when it is on the display. If you press marker and move the mouse onto the upper display, you can move a marker back and forth by selecting and dragging it. When you push the marker button again, the computer will show the time and voltage values at whatever point the marker crosses the signal. If you press the draw button, then you can draw changes in the signal that appears on the lower display; this way you can output custom stimuli.
Set the stimulus with the controls labelled voltage
and duration. Use a 0.5 ms stimulus, and try a variety of voltages.
Choose a voltage and then hit the start button and the computer will record
for about 20ms. You will probably see a little jump in the voltage
at the start, then nothing. This jump is simultaneous with the stimulus
(you can tell by comparing it to the lower display), but much weaker (it
doesn't look weaker, because it has been amplified 1,000-10,000x.).
This is the stimulus artifact. It is the result of conduction down
the tissues of the worm. That is why it is simultaneous with the
stimulus (fast conduction), but weak (charge leaks away the further it
goes).
Threshold: Find a voltage that gives a good action potential. Then keep increasing the voltage and restimulating. As the stimulus increases, the stimulus artifact increases. Does the action potential change? Does it get stronger? Does it occur at a different time? Why? At higher voltages, you may see another action potential show up to the right of the first. The first action potential is the median giant fiber, the second is from two lateral giant fibers (combined). They were both triggered at the same time by your stimulus; which conducts faster? Why do you think it is faster? Determine the threshold stimulus for both fibers. This is the lowest stimulus that triggers an action potential.
Threshold for median giant - ___________________ volts
Threshold for lateral giants - ___________________ volts
Velocity: You can calculate the velocity of conduction for each fiber. Measure the physical distance between the positive stimulating electrode (DAC0) and the first recording electrode. Then, using the markers on the computer display, measure the time between the stimulus and the arrival of the action potential at the first electrode. Distance over time is velocity.
Velocity - _______________(Median), _______________(Lateral)
m/sec
Refractory period: Another feature of the action potential is the refractory period. You can demonstrate this by stimulating the neuron with two consecutive pulses. Choose a stimulus that gives a clear action potential for the median fiber, but not the lateral fibers. Use the draw button to draw a second stimulus pulse (on the lower display) about 6 ms after the first. It should be roughly the same voltage and duration as the first stimulus. Then hit start to record again. You should see two action potentials, one for each stimulus pulse. Then erase the second stimulus (reset button) and draw a new stimulus pulse sooner after the first. Record again. Keep moving the second pulse closer and closer to the first until you only get one action potential. What happened to the other action potential?
Duration of refractory period - ___________________ ms