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Plant Action Potential: Independent Evolution of Excitability

Introduction

Our present understanding of membrane excitability is based mainly on data from neuron and muscle cells of animals. However, plant studies played an early role in development of ideas about cellular excitability (Wayne, 1994; Beilby, 2007). In fact, until the discovery of the squid giant axon as a model preparation, the main advances in the understanding of excitability were accomplished with plant tissue. In this exercise, you will stimulate action potentials in a freshwater plant and investigate their characteristics. You will see that these action potentials have very different properties from the animal action potentials with which you are now familiar.

Why do these plant cells generate action potentials? Even simple plants must respond adaptively to environmental disturbances (Wayne, 1993; Fromm and Lautner, 2007). In plants, as in animals, stimulus-response coupling can be achieved through changes in the flow of ions across cell membranes. Responses such as a mimosa’s leaf folding after being touched or the movements of a Venus flytrap as it captures an insect are produced by such ion flows and accompanying electrical events. In this exercise, you will find that there are mechanisms of action potential generation that do not rely on an influx of Na+ or Ca2+ as the main depolarizing component. Basic mechanisms of cellular excitability probably evolved long before nervous systems existed (Hille, 2001; Greenspan, 2007; Meech and Mackie, 2007).

Preparation

Chara corallina is a freshwater plant that inhabits temperate zone ponds and lakes. Its stem consists of alternating nodes and internodes (Figure 10.1). Each internodal segment is a single large cell, up to 10 cm in length. Because the cell is so large, it has its own circulatory system of sorts, cytoplasmic streaming, to distribute intracellular materials (Figure 10.2).

Choose an internode of Chara that appears a healthy bright green and fairly translucent and that is neither too long nor too short for your recording chamber. Cut off the adjacent cells and trim any cells that project from the ends. Be careful not to bend or cut the cell that you will use. Dry the cell gently with tissue paper and place it in the recording chamber (Figure 10.3). Place the cell flat on the bottom of the recording well so that it does not bend or roll when you try to penetrate the cell wall with the electrode. Electrically isolate the three wells of the chamber with Vaseline from a syringe. Finally, fill the three wells with artificial pond water. The entire procedure is shown in Video 10.1, Chara Preparation.

Before recording, adjust the lighting so that you can see cytoplasmic streaming in the cell. Use the highest magnification and lighting from underneath. You should be able to see particles moving slowly along the length of the cell just below the focal plane of the cell wall. Most of these particles are nuclei.

Recording

Figure 10.4 shows the recording setup. Place your ground electrode in the center well of the chamber. Connect the positive and negative poles of the stimulus isolation unit to electrodes (wires or posts) in each of the two outer wells.

If you have done intracellular recordings from crayfish muscle or snail brain, the procedure for Chara is much the same, except that you can use electrodes of somewhat lower resistance (1 to 10 MΩ), they will be filled with 1.5 M KCl, and the procedure for entering the cell is less delicate.

Pull a pair of glass microelectrodes and place them, sharp end up, in a beaker with a small amount of 1.5 M KCl. After a few minutes, the tip of the electrode should have filled with KCl (Figure C.5). Use a fine syringe to fill the rest of the electrode with KCl (Video C.2, Electrode Filling) and then put it in the electrode holder. With the micromanipulator, place the tip of the electrode in the water over the cell and measure the electrode resistance. (The procedure for measuring resistance varies with different amplifiers; ask your instructor for help.) A resistance of 1 to 10 MΩ is good. Even a broken electrode with resistance in the kΩ range will do if the cell is entered gently. Set the oscilloscope to DC coupling and 0.02 to 0.2 V per division (your amplifier increases the signal 10×, so this is 2 to 20 mV/div of real voltage).

At high magnification, focus the microscope on the tip of the electrode. (The actual tip is too small to be seen and is somewhat lower than what you are focused on.) Adjust lighting for best visibility of the electrode. Now bring the electrode closer to the cell, refocusing along the way, until the visible part of the electrode appears to be a short distance from the cell. With the fine adjustment of the manipulator, push the electrode slowly into the cell. It may appear to indent the cell before it goes in. This is not a problem, but be careful not to tear a large hole in the cell. A gentle tap on the manipulator may help the electrode penetrate the cell wall. If you tear a large hole in the cell, you will see particles streaming out. If this happens, get another strand of Chara and start over. When the electrode enters the cell, the oscilloscope trace should drop considerably, indicating a resting potential between −100 and −200 mV. A good electrode penetration will not affect cytoplasmic streaming. If streaming does stop after penetration, allow the cell to resume streaming before you continue.

Once you have a recording, allow the cell to rest for a few minutes. The resting potential may continue to become more negative during this time. Keep the oscilloscope on DC coupling and set its time scale to 0.5 to 1 s/div. Start with the stimulator set to produce a fairly long pulse (100 to 200 ms). Set the voltage output of the stimulus isolation unit to 5 V and stimulate the cell. You should at least see a stimulus artifact and possibly also a large slow action potential. If there is no action potential, increase the output a few volts at a time and repeat the stimulus until you see an action potential. Once you find a voltage at which you can elicit an action potential, decrease the duration of the stimulus pulse to 10 ms and increase the voltage. The shorter pulse reduces disruption of the action potential waveform by the stimulus artifact. Wait a few minutes after each stimulus. Since the action potential depends on ion distribution across the cell membrane, change the external solution occasionally to refresh the ionic gradients.

The recording procedure is shown in Video 10.2, Chara Recording. Note that you can see the cytoplasmic streaming before the cell is stimulated and that the streaming stops after the stimulus.

Experiments

Action Potential Waveform

Characterize the action potential. Note its shape; measure its rise time, amplitude, and duration at half-amplitude. Increase the stimulus voltage and determine whether this changes the amplitude or duration. Is the action potential an all-or-none event (Shimmen et al., 1994)? Elicit 5 to 10 action potentials at least 5 min apart and measure the variability in their amplitudes and half-amplitude durations. Does the threshold for stimulation vary between action potentials?

Refractory Periods

Stimulate two action potentials 3 min apart. If they are similar in amplitude, repeat the pair of stimuli with a shorter interval. Do this several times with shorter intervals and note the longest interval at which the second action potential is smaller than the first. This interval is the relative refractory period. Plot the ratio of action potential amplitudes (second divided by first) against stimulus interval. At short intervals, you may find that the second stimulus does not elicit an action potential at all. The longest interval at which this happens is the absolute refractory period.

Further Exploration

You can follow up on the above experiments to examine the ionic basis of the action potential in Chara cells:

Lab Clean Up

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 water and rinse the ground electrode with distilled water. Clean out the recording chamber and wipe off any Vaseline that remains on it.

Questions

  1. How does the resting potential of Chara compare with that of animal neurons or muscle? Calculate the Nernst potentials of the ions that might contribute to the resting potential See Table 10.1, below, for the composition of internal and external fluids (Lunevsky et al., 1983). Note each ion has two Nernst potentials, cytoplasm-external and cytoplasm-vacuole. Use the Nernst potentials for the cytoplasm-external interface to decide which ion is likely to be most important in setting the resting potential. Is this the same ion that is important for the resting potentials of neurons and muscle fibers?
    Table 10.1 Chara corallina Ion Concentrations (mM) [open in a new window]
     [K+] [Na+][Ca2+][Cl−]
    Pond water0.10.10.10.4
    Cytoplasm110.05.00.001 22.0
    Vacuole103.034.012.0162.0
    Data from Wayne (1994).
  2. Describe the waveform of the Chara action potential and compare it with action potentials of animal neurons. Given the cytoplasm-external Nernst potentials you calculated above, determine which ions could contribute to the Chara action potential. Suggest a mechanism for the fall of the Chara action potential based on your knowledge of animal action potentials.
  3. Do the Chara action potential waveform and the stimulation threshold for its activation show more variability than you might see with animal action potentials? Suggest a mechanism that might account for this variability. (Hint: See Figure 10.2 and consider how deeply the electrode might penetrate the cell.)
  4. Show the results of your tests of refractory periods. Compare the refractory periods of neurons and Chara. What differences in excitability mechanisms might account for these differences in refractory period?
  5. You saw that when the electrode penetrated the Chara cell or when an action potential occurred, the cytoplasmic streaming stopped (Video 10.2, Chara Recording). Suggest one function of the Chara action potential.
  6. Consider the different types of ion channels found in animal neurons (like our brain cells; see McCormick, 2009) and how some of these channel types might also contribute to excitable properties of Chara. What does the presence of similar channel types in both plant and animal cells suggest about the evolution of excitability?
  7. Imagine that a manned expedition to Mars has discovered a unicellular amoeboid creature in a sheltered pocket of water. An astroneurophysiologist finds that action potentialilike events can be recorded across the creature’s cell membrane, despite a lack of Na+ and Ca2+ in the water. Imagine that you have analyzed the ionic composition of the creature’s cytoplasm and the water and found differences in concentrations of specific ions across the membranes (be creative about the ions and concentrations you make up). Present the results of your analyses and from them propose a mechanism for action potentials in this creature.

References