Snail Brain: Ionic Basis of Neuronal Excitability

Introduction

In Lab 8, Snail Brain—Excitability, you recorded from and characterized a variety of neurons from the subesophageal or buccal ganglia of a snail. Although you may not have recorded from every type of cell, you should have seen sodium and calcium action potentials (APs) and several different types of spontaneous activity patterns in your recordings and those of your classmates. The neurons you observed in the last exercise have different mixtures of ionic currents that account for their differences in activity. Some action potentials are based on an influx of sodium, others on an influx of calcium. Some have stable resting potentials, while others have unstable or even oscillating membrane potentials.

In this lab exercise, you will again record from cells in snail ganglia, but instead of trying to characterize a number of cells, you will concentrate on experimental manipulation of only a few. To determine the ionic bases of the rise and fall of action potentials in these cells, you will use the standard techniques of neurophysiology, including ion concentration manipulation, ion substitution, and pharmacological manipulation.

Dissection

The dissection was covered in Lab 8, Snail Brain—Excitability. You can go back to that exercise or watch Video 8.1, Entire Snail Dissection, for a review of the dissection of the land snail Helix aspersa. Consult your instructor if you are using a pond snail for this exercise.

Recording

The recording procedure is the same as in the previous lab. This time, however, you will also need to change salines while recording from a cell. This procedure can be tricky. If your lab has a pump or drip perfusion system, use it to change salines, but be sure to set it up and test it before recording from a cell. If your lab does not have such apparatus, you can change salines with two pipettes. Add the new saline with one pipette while withdrawing saline from the other side of the dish with the other pipette. This may be easiest to do with one person operating each pipette. It will take three or four such changes before the saline is sufficiently replaced.

Another way to reduce the concentration of an ion in the bath is to start with a known relatively small volume of normal saline covering the preparation and then add to it to dilute the ion that is to be reduced. Thus to cut [Na⁺] in half, add an equal volume of Na⁺-free saline to the normal saline already in the dish. This approach will not work, of course, when you need to completely replace the control saline, as with the substitution of Ba²⁺ for Ca²⁺ below. A combination of dilution and pipette exchange techniques may be appropriate in most cases.

It is easiest to see the snail brain cells if you have only a small volume of saline covering the ganglion. In addition, place the light source so that some light shines up through the ganglion to highlight the cells.

Experiments

As in Lab 8, Snail Brain—Excitability, the exact set of experiments you do will depend on the types of cells from which you record. Use the following as a guide, keeping in mind that not every experiment will apply to every cell type.

Ionic Basis of AP Depolarization

Penetrate a cell and record its resting potential (RP), AP amplitude, and AP duration at half-height. It is important to monitor the RP during these experiments, and it is best if it does not change very much (it may change, of course, if the RP depends on Na⁺ or Ca²⁺).

If the cell appears to have a Na⁺-based AP (duration under 10 ms), change the external [Na⁺] by perfusing the ganglion with low Na⁺ saline (start with 25% of the normal Na⁺ concentration). Prepare this by mixing normal saline (120 mM Na⁺) with sodium-free saline (NaCl replaced by choline chloride). After perfusion with low Na⁺ saline, again measure the RP, AP amplitude, and AP duration.

If the cell appears to have a Ca²⁺-based AP (duration over 20 ms), change the external [Ca²⁺] by perfusing the ganglion with low Ca²⁺ saline and do the experiment and analyses described above for sodium (start with 25% of the normal Ca²⁺ concentration). Prepare concentrations by mixing normal saline (6 mM Ca²⁺) with calcium-free saline (CaCl₂ replaced with MgCl₂).

If the AP is of intermediate duration, test both low Na⁺ and low Ca²⁺ salines to determine the contribution of these cations to the AP.

Ionic Basis of AP Recovery

Repolarization of a neuron after an action potential is due to an outward flow of K⁺. This current can be carried by different types of K⁺ channels, including a voltage-activated K⁺ channel (which carries the current I_Kv) and a Ca²⁺-activated K⁺ channel (which carries the current I_KCa). The next two experiments illustrate the contribution of these currents to AP repolarization.

In cells with Ca²⁺-based action potentials, repolarization often involves a Ca²⁺-activated K⁺ channel. Although Ba²⁺ can flow through the Ca²⁺ channels, depolarizing the cell, it does not activate Ca²⁺-activated K⁺ channels. When recording from a cell with a Ca²⁺-based action potential, stimulate a train of APs and measure their amplitudes and durations. Now replace the normal saline with one in which the Ca²⁺ is replaced with Ba²⁺. Repeat the stimulus and measure the AP shape, noting any changes from the control. Because Ba²⁺ is mildly toxic, use gloves while handling Ba²⁺ saline.

For a more global block of the K⁺ channels that contribute to AP repolarization, inject a mixture of tetraethylammonium (TEA) and cesium (Cs⁺) into a cell. When applied externally at lower concentrations (10 to 20 mM), TEA blocks the voltage-gated K⁺ channel in most animals. In snails, however, TEA must be applied inside the cell. Intracellular Cs⁺ will help block any K⁺ channels missed by the TEA. Fill an electrode with a solution of 2M TEA and 2M CsCl (this mixture is toxic, so wear gloves). This electrode will have a higher resistance than a KCl-filled electrode. Use this electrode to record from a cell with either Na⁺, Ca²⁺, or mixed action potentials. Immediately depolarize the cell to elicit a train of APs. Record the amplitude and duration of these control APs. Now pass constant depolarizing current through the electrode to force TEA and Cs⁺ (which are positively charged) into the cell. This will take about 5 minutes with +1 to +5 nA of current. At one-minute intervals, elicit another train of APs and measure their amplitudes and durations. You should see an effect within 5 min. Note any changes from the control APs. When TEA/Cs is applied in this way, its concentration is high enough to block many types of K⁺ channels in addition to those activated by Ca²⁺.

Further Exploration

• Examine in detail the dependence of snail APs on Na⁺ and Ca²⁺ concentration. Repeat the measurements above with several different concentrations of Na⁺ or Ca²⁺ (depending on the type of AP). Graph AP amplitude vs. log of external Na⁺ or Ca²⁺ concentration. What is the slope of this line?
• Alter currents involved in action potential generation:
1. The voltage-gated Na⁺ channels involved in APs are sensitive to tetrodotoxin (TTX), a toxin from the Japanese puffer fish (Fugu). Test the contribution of voltage-gated Na⁺ channels to the snail AP by adding 10⁻⁷ M TTX to the saline. (TTX is highly toxic, so wear gloves and use it under an instructor’s supervision.)
2. More than one type of K⁺ current may participate in the action potential (Jan and Jan, 2012). You can test the participation of the early K⁺ current (IA) in repolarization of APs, for example, by adding 4-aminopyridine (5 mM), a blocker of this current’s channel. (This blocker is toxic, so wear gloves while handling it.)
• Alter currents involved in postinhibitory rebound:
1. A hyperpolarization-activated current (I_h) is thought to be responsible for postinhibitory rebound. The channel for this current is blocked by cesium (Cs⁺). Test the role of I_h by adding 5 mM Cs⁺ to the saline while recording from a cell that shows postinhibitory rebound. (Cesium is toxic, so wear gloves while handling it.)
2. Test the role of IA in post-inhibitory rebound using 4-aminopyridine as above. Why might this current have a role in post-inhibitory rebound?
• Tonic firing and bursting involve a number of different currents (McCormick, 2009). For a spontaneously firing cell, note changes in the firing frequency and AP characteristics in each of the following tests. For a bursting cell, use these tests to examine what currents shape burst characteristics (burst frequency and the duration, amplitudes, and number of spikes in each burst).
1. Use reduced Na⁺ saline or reduced Ca²⁺ saline.
2. Add Cs⁺ to normal saline to block the hyperpolarization-activated current (I_h).
3. Add the K⁺ channel blocker 4-aminopyridine to normal saline to block the early K⁺ current (I_A).
• You may have examined the effects of modulators on the properties of bursting cells as suggested in Lab 8, Snail Brain—Excitability. Based on those results, develop and test hypotheses about which ion currents are affected by each modulator.

Lab Cleanup

During the lab, be sure to immediately discard used glass electrodes in the appropriate container. After the lab, remove your last electrode from its holder and discard it as well. Clean up any spilled saline and rinse the ground electrode with distilled water. Put the snail brain in the trash and rinse the dissecting dish with fresh water. Ask your instructor how to dispose of TEA/Cs or other chemicals you used.

Questions

In your lab write-up, report and explain the results of the above experiments.

1. Compare AP amplitudes in normal saline and in reduced Ca²⁺ saline. Does the peak of the AP in control saline approach the Nernst potential for Ca²⁺ (calculate the Nernst potential using internal ion concentrations from the crayfish, Table 4.1)? Is the difference between the AP amplitude in normal and reduced Ca²⁺ saline what you would expect from the Nernst potential? Explain your observations and conclusions.
2. Do the same for Na⁺.
3. If you were able to compare action potential amplitudes with a complete series of different external Na⁺ or Ca²⁺ concentrations, what would you expect the slopes of the relationships between log of [Na⁺] or [Ca²⁺] and action potential amplitude to be?
4. What effect did substitution of barium for calcium have? What would explain this?
5. What effect did intracellular TEA/Cs have on the cell from which you recorded? How do you explain it?
6. Why is it important that the RP remain fairly constant when you compare AP amplitudes at different ion concentrations? How would a depolarized or hyperpolarized RP affect the rising and falling phases of the AP?
7. What is the most important property of ion channels involved in action potentials?

References

• Adams DJ, Smith SJ, Thompson SH (1980). Ionic currents in molluscan soma. Annu Rev Neurosci 3:141-167. [doi]
• Jan LY, Jan YN (2012). Voltage-gated potassium channels and the diversity of electrical signalling. J Physiol 590:2591-2599. [doi]
• Johnston D, Wu SM-S (1995). Foundations of Cellular Neurophysiology (MIT Press, Cambridge MA), ch. 7.
• Kerkut GA, Gardner DR (1967). The role of calcium ions in the action potentials of Helix aspersa neurones. Comp Biochem Physiol 20:147-162. [doi]
• McCormick DA (2009). Membrane potential and action potential. In: Byrne JH, Roberts JL (eds.), From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience (Academic Press, San Diego), ch. 5.