Lab 3 – Propagation of Action Potentials in the Neuron

In this lab, you will examine the propagation of action potentials along the ventral nerve cord of the earthworm Lumbricus in the intact animal.  You will also have the opportunity to look at the effects of changing concentrations of external Na⁺ and K⁺ on action potential generation.

Background

Action Potentials

An action potential (AP) is a rapid, transient change in a cell’s membrane potential. It begins with a depolarization (positive deflection of the membrane potential) and ends 1 or 2 milliseconds later with a return to the resting potential. In neurons, the depolarization is caused by the influx of Na⁺ ions through voltage-gated Na⁺ channels, and is terminated by a combination of the inactivation of the Na⁺ channels and the efflux of K⁺ ions through voltage-gated K⁺ channels.  The normal generation of an AP therefore depends on the correct relative concentrations of Na⁺ and K⁺ inside and outside the cell.

Action potentials are the only known means by which cells can transmit information over distances greater than a few millimeters in less than a tenth of a second. AP’s are propagated along an axon because the voltage changes associated with an initial AP depolarize adjacent regions, activating voltage-gated Na⁺ channels and initiating a new AP a short distance from the initial AP.  This second AP then generates a third, etc.  Because each AP takes a small amount of time to initiate, the propagation of AP’s along the surface of the cell (or group of cells that are coupled by gap junctions) occurs at a measurable rate.  Propagation speeds for unmyelinated neurons range up to 25 meters/sec, while propagation speed in myelinated neurons can be up to 100 m/s.

The initiation of an AP typically involves stimulation of the neuron by another neuron, by a chemical messenger (such as a hormone), or by an appropriate environmental stimulus (in the case of sensory neurons).  However, any phenomenon that depolarizes the cell membrane will initiate an AP.  Physiologists studying AP’s typically use either an electrolyte-filled glass microelectrode (inserted within the neuron or applied to the membrane surface) or a metal electrode (touching or near the neuron) to deliver an electric current sufficient to depolarize the membrane.

Similarly, AP’s can be recorded using either type of electrode.  Only an electrode inserted into the cytosol provides a true measure of the voltage across the cell membrane (V_m).  Electrodes adjacent to the neuron will still record a transient voltage change associated with the AP, but the magnitude of the voltage change will depend on a number of factors, most importantly the distance between the neuron and the electrode.

The anatomy of Lumbricus terrestris

Figure 1 – Cross-section of Lumbricus
 

Figure 2 – Close-up of Lumbricus cross-section
showing details of ventral nerve cord.

Figure 3 – View of ventral nerve cord of
Lumbricus after dorsal dissection.

You will be recording from the ventral nerve cord of an annelid, the earthworm Lumbricus terrestris. The ventral nerve cords of annelids and arthropods are analogous, and possibly even homologous (based upon recent discoveries of homologous proteins involved in early development across phyla), to the dorsal spinal cord of chordates such as ourselves. The nervous systems of annelids and arthropods contain a few axons that are much larger than the rest. These giant axons are invariably involved in escape reflexes. Their large size increases the propagation velocity of the action potential, an obvious advantage for rapid escape reflexes. The appearance of myelin (electrical insulation that greatly increases the speed of action potential propagation in small axons) early in chordate evolution made it possible for our ancestors to coordinate rapid movements without giant axons. Therefore, vertebrates and other chordates usually do not have giant axons. For our experiments, however, giant axons offer a decided advantage in that their large surface area makes it very easy to record action potentials extracellularly, simply by placing recording electrodes in the body near the nerve cord.

The ventral nerve cord of Lumbricus (Figure 1) contains three giant axons, one medial giant axon flanked on either side by lateral giant axons (Figure 2). Each giant axon in Lumbricus is actually a chain of individual cells (one in each body segment) that are electrically coupled end-to-end by gap junction ion channels. This anatomical arrangement allows for the direct and rapid propagation of the action potential from cell to cell. Thus, although each giant “axon” is anatomically several cells, it functions like a single cell. In addition to being electrically coupled lengthwise, the lateral giant axons are also connected to each other via lateral anastomoses and therefore function as a single unit.

Equipment

In this lab you will use a new program to collect data.  Scope is designed to mimic an oscilloscope (just as Chart was designed to mimic a chart recorder), and is better suited than Chart for collecting small samples of extremely fast signals.  Scope is similar to Chart, but does have a number of different features associated with its different functions.  The basic setup and use of the program is covered in the experimental procedures below, but ask an instructor for help if necessary.  We will also be using the PowerLab/PowerLab units to provide stimuli to the ventral nerve cord, and the differential amp and PowerLab to amplify the voltage changes resulting from propagating AP’s.

Because oscilloscopes are used to record very fast events, it is crucial that the recording be correctly timed with respect to the stimulus. When the stimulator and ’scope are independent devices, this is usually done by splitting the stimulus and sending it to both the prep and the ’scope.  When the ’scope “sees” the stimulation, it begins its brief recording of incoming data. In the setup we will use, pushing the “Start” button triggers both the stimulus and the start of data collection.  One channel will monitor the stimulus and the other will record the response.  Once collected by Scope, each set of data is displayed on its own page as graphs of voltage vs. time.

One of the most difficult aspects of trying to record voltages in the microvolt range is that noise from other sources becomes a major problem.  To reduce noise as much as possible, we will

• Use shielded cables for the recording electrodes.
• Ground everything but the electrodes. Grounding means that everything is connected electrically, so there won’t be differences in the electrical state of different parts of the rig.
• Use differential amplification, since this only looks at the difference between the two recording electrodes.  Any noise present in both cables is eliminated by this process.

Initial Setup

In this lab, you will record action potentials from axons and examine some of the properties discussed above. Action potentials are best examined by recording intracellularly from single cells. Unfortunately, it would take a great deal of practice and additional equipment to successfully make intracellular recordings. Therefore you will obtain extracellular recordings. Extracellular voltage changes are caused by the flow of ions into and out of cells during action potentials. Because the extracellular fluid has a low electrical resistance compared to the resistance across the cell membrane, the extracellular voltages are quite small compared to the voltage changes across cell membranes (microvolts vs. millivolts). Therefore we will greatly amplify the voltages in order to record them.

Equipment setup

You will be recording AP’s from your worm as it rests in a dissecting pan.  To reduce electrical noise, you will want to be able to place the pan some distance from any electrical equipment (30 cm or more), so consider that fact as you set up the rest of the equipment.

Setup and test your rig before getting your worm!

Hardware:

• Locate the electrodes.  You should have:
  • One stimulating electrode – a single BNC cable with two needles at the end.
  • Two recording electrodes – each is a BNC cable with one needle at the end.
  Keep the needles covered or stuck in a rubber stopper while setting up the equipment so you don’t stick yourself!.
  You should also have:
  • A lead with an alligator clip at both ends (grounding lead #1).
  • A lead with an alligator clip on one end and a BNC connector on the other end (grounding lead #2).
  • A T-shaped BNC connector.
• With the differential amp turned off, connect the recording electrodes to the plus and minus inputs of channel 1 on the amp.  Then connect output 1 to input 1 of the PowerLab using a standard BNC cable. These electrodes will record the AP as it travels down the nerve.  Set the differential amp as follows:
  • Mode: AC – this setting lets the amp adjust for drift in the baseline voltage
  • Gain: ×1000 - you’ll be measuring tiny voltages
  • Low pass filter: 1 KHz - These filter settings may need to be adjusted later
  • High pass filter: 300 Hz
  • Both inputs on
• Connect one end of grounding lead #1 to the metal dissecting pan using the alligator clip.  Then connect the alligator clip of grounding lead #2 to the pan and the other end to the plus or minus input of channel 2 of the amp.  These are the grounding connections to help reduce noise in the recordings.
• Put the T-connector on output 1 of the PowerLab.  Connect one side of the T to input 2 on the PowerLab. (This will allow you to record the stimulus as you deliver it to the worm).  The other side of the T will be connected to the stimulating electrodes, but do not connect the stimulating electrodes yet!  When connected, these electrodes will be used to depolarize the ventral nerve cord and initiate an AP.

Software:

• Find and start Scope.
• Make sure Input A is set to Channel 1 and Input B to Channel 2.  Set as follows.

Input 1:

Range: 500 mV
Under Input Amplifier…
AC mode checked
Filter off
Positive checked
Negative unchecked

Input 2:

Range: 1 or 10 V (depending on stimulus strength, below)
Under Input Amplifier…
AC mode unchecked
Filter off
Positive checked
Negative unchecked

• Set Time Base to 20 ms to start, with 512 samples.
• Under Setup...Sampling, set the Mode to Single and Source to User (this means the stimulus will be given and data recorded when the user pushes the “Start” button.
• Under Setup...Stimulator: You will be using brief square-wave stimuli, initially at a low voltage, to elicit action potentials in the earthworm’s giant axons. Begin with:

Pulse wave
Delay at 2 ms
Duration at 0.2 ms
Amplitude at 0.1 V
Range at 10 V

• Press the Start button.  You should see your stimulus appear in the lower scope window.
• Have an instructor check your setup.

Earthworm Prep

You will be provided with an anaesthetized earthworm by an instructor.  Once you have the worm:

• Place the worm with its ventral (lighter) side up in the dissecting pan. Firmly pin the worm to the pad by pushing two pins through its anterior end into the pad. The anterior portion of the worm is marked by the clitellum (the thickened area). Gently stretch the worm and insert two pins through its posterior end.
• Stick the stimulating electrodes through the worm so they straddle the worm’s midline, about 2-3 cm from the anterior end.  The midline can usually be identified by the faint ventral blood vessel. Be sure the electrodes are secure in the wax below the worm.
• Place a dissecting pin ~1 cm posterior to the stimulating electrodes, then attach the free end of grounding lead #1 to the pin. (This will minimize the electrical interference picked-up by the recording electrodes.)
• Place one recording electrode about 2 cm from the posterior end of the worm. This is the posterior recording electrode. Place the last electrode (the anterior recording electrode) about halfway between the ground electrode and the posterior recording electrode. Be sure to place the recording and ground electrodes slightly lateral to the midline so they do not puncture the medially-located nerve cord.

You will need to keep the worm moist, but not too wet. Moisten the preparation by dripping 10% EtOH in worm Ringer’s on it. Use the eyedropper and beaker provided. Wick away excess fluid with a paper towel or Kimwipe. Repeat wetting occasionally. Double check your wiring, then attach the stimulating electrodes’ BNC connector to the T-connector on the PowerLab’s output 1.

Experiments

Experiment 1 – Basic observations of AP propagation

Keep an eye on the worm during the experiments. If the worm begins to rouse slightly, you can apply 10% EtOH.  If the worm rouses completely, remove the pins and exchange the worm for a freshly anaesthetized one.

You may find that the giant axons “fatigue” with continued stimulation. If this happens, stop stimulating for a few minutes to give the neurons time to recover, then try again.

Procedure:

• Begin stimulation at 0.1 V. You should see the stimulus on channel 2.  The voltage trace on channel 1 should be somewhat noisy but smooth enough to recognize an AP’s signal if one occurs.
• Increase the voltage with each stimulation until you think you see an AP.  Stimulate a few more times at that voltage to confirm you are generating an AP. You should see one hump as the AP passes the anterior electrode and another as it passes the posterior electrode.
  
  Note:
  • A true AP will always appear at the same time; voltage changes caused by noise will jump around.
  • You may have to increase voltage close to 10 V to see an AP, depending on where your electrodes are located. If you see no signal at 10 V, try repositioning your electrodes or ask for help from an instructor.
  • At higher stimulating voltages, you may begin to see a stimulus artifact at the recording electrodes.  This is caused by the cable properties of the neurons carrying the stimulus voltage without generating an AP.  You will need to differentiate between this effect and a true AP.
• With luck, you may be able to find a voltage that triggers just the medial giant axon, as opposed to the slightly higher voltage that triggers the medial giant axon and the two lateral giant axons.  To tell when both axons are triggered, look for two humps in the AP (at each electrode), or a difference in the overall size of the AP.
• Answer the questions on the worksheet, carrying out further experiments with the stimulus variables as needed.

Choose either experiment 2 or 3 below to do if time allows

Experiment 2 – Testing the ionic basis of the AP

Dissection:

• Remove all pins and electrodes from your worm and flip it over so that its dorsal surface is up (if your worm is clearly dead, you will want to get another one).
• Using dissection scissors and forceps, open up the dorsal surface of the worm to expose the intestine and, underneath that, the ventral nerve cord (Be careful to keep the tips of your scissors superficial so that you do not damage the internal organs). Pin open the flaps of skin, and drip Normal worm saline onto the prep with a the provided dropper.
• Tease away the intestine from the nerve cord over much of the length of the worm. Do not damage the nerve cord.

Electrode setup:

• Setup the electrodes in the position that you did for the preceding experiments. Be sure that both stimulating and recording electrodes are physically contacting but not puncturing the nerve cord.

Experimental procedure:

• Obtain beakers of both low-Na+ and low-K+ worm saline, in addition to the normal worm saline.
• Test the effects of each solution on the generation and appearance of the action potential. Take care to rinse thoroughly between each solution with Normal worm saline, and blot off any extra solution with Kimwipes (only add enough solution, dropwise, to thoroughly soak the nerve cord. You do not want it to be submerged).
• Briefly document your results on the worksheet.

Experiment 3 – Mechanical stimulation and giant fiber responses

Setup:

• Place a worm, ventral side up, on the foam pad and pin it out using a minimal number of pins. Again, make sure that you avoid the middle of the worm and, also, avoid pinning the anterior end of the worm (the goal is to stimulate this end, so obviously you don’t want to skewer it!).
• Set-up only the recording electrodes in the positions you had them previously.
• In Scope, turn the Stimulator off, set Sampling to Multiple, with 50 or 100 sweeps.  This setup causes Scope to continuously record using sequential sweeps or “pages” until the requested number is reached.  Click Start to begin recording.

Experimental procedure:

• Record a set of sweeps and note the presence of any spontaneous activity in the nerve cord (that is, any AP’s generated without physical stimulation of the worm).
• Start another set of sweeps and, using a blunt probe, mechanically stimulate (gently!) the anterior end of the worm. What do you observe? You may see multiple discharges, but can you determine whether or not one or more types of giant nerve spikes are seen as a result of mechanical stimulation?
• If you do not see anything dramatic, you may want to consider opening up a small section of the worm (being careful to not damage the nerve) and place the recording electrodes in direct contact with the nerve cord. Once you are able to get a consistent response as a result of anterior stimulation, see if stimulating other parts of the worm causes a different response (one or other of the giant fiber discharges). Are some parts more sensitive than others?