Background Individual action potentials: The fundamental unit of the nervous system is the neuron.

Neurons and other excitable cells produce action potentials when they receive electrical or chemical stimulation. Recall from lectures that there is a relatively high concentration of sodium (Na+) in the ECF, and a relatively high concentration of potassium (K+) in the ICF (Lluka 2010). The difference in concentrations of Na+ and K+, and their relative permeability through the cell membrane, results in the inside of the cell being more negative than the outside. This difference is around -70 mV in a human neuron and is termed the resting membrane potential (Figure 1, number 1). Diffusion of Na+ into the cell is prevented by the activation gate of the voltage-sensitive Na+ channels being closed; diffusion of K+ out of the cell is prevented by the gate of the voltage-sensitive K+ channels being closed. Upon stimulation, some of the activation gates of the voltage-sensitive Na+ channels are opened. This allows some Na+ to enter the cell, reducing the negative charge. This is termed depolarization (Figure 1, number 2). If the stimulus strength is sufficient, enough Na+ will enter the neuron to reach the threshold potential (around -50 mV in human neurons (Kole and Stuart 2008)). Generation of an action potential is an all or nothing process, once this threshold is reached , a full action potential must be generated. At this point, the activation gates of more voltage-sensitive Na+ channels in the membrane are opened, and there is a larger influx of Na+. This is what causes the rising phase of the action potential (Figure 1, number 3).

Figure 1: a transmembrane recording of an action potential (Campbell et al. 2009).

After a period of delay (around the time that the membrane potential reaches +30 mV), two things happen: the inactivation gates of voltage-sensitive Na+ channels are closed, preventing anymore Na+ from entering the cell; and the voltage-sensitive K+ channels are opened, allowing K+ to leave the cell. As K+ leaves the cell the membrane potential becomes more negative. This is the falling phase of the action potential (Figure 1, number 4). Slow closure of the K+ channels after the falling phase result in the membrane potential becoming more negative than when at rest. This period is called

the undershoot (Figure 1, number 5). After all of the K+ channels are closed, the membrane potential is restored to the resting value of -70 mV. During the falling phase, once the membrane potential gets back below 50 mV, the gates in the voltage-sensitive Na+ channels switch positions: the inactivation gate opens, and the activation gate closes. This still prevents Na+ from entering the cell, but the resetting of the activation gate is a very important event. It is the opening of the activation gates which start the rising phase of the action potential (Figure 1, number 3). Therefore, all the time that these gates are open it is impossible to generate a second action potential gates that are already open cannot be opened again. The period when the activation channels are open is called the absolute refractory period, and no new action potentials can be generated.

Figure 2: the refractory periods of an action potential (Eskandari 2007).

The resetting of the activation gate in the voltage-sensitive Na+ channels means that there is the potential to open the gate again, and thereby generate a second action potential. The period of time between the resetting of the activation gates and the membrane potential returning to the resting state is called the relative refractory period. It is possible to generate a second action potential during this time, but a stimulus of greater strength is required to reach threshold. This is due to 2 reasons: 1) K+ is flowing out of the cell making the membrane potential more negative, more Na+ is therefore required to enter the cell in order to make it more positive and reach threshold; 2) after the K+ channels close the membrane potential is more negative during the undershoot than at rest, so more Na+ must enter to increase this to threshold (-50 mV).

Compound action potentials: Measuring action potentials from single axons requires highly specialised equipment. Instead, you will record from a nerve bundle, the toad sciatic nerve, which contains thousands of individual axons. Keep this in mind when interpreting your results.

Figure 3: nerve bundle - idealised cross section.

Nerve bundles contain both sensory nerves and motor nerves. The individual axons vary in diameter, myelination, excitability, threshold and speed of conduction. It is important to appreciate that the threshold voltage required to generate an action potential in each individual axon will be different. Thus the compound action potentials that you will record represent the summed 'all or nothing' action potentials from only those axons that are excited by the stimulus. As the stimulus voltage is increased, more and more individual axons will be excited until eventually all of the axons in the nerve are excited. Thus the peak of the compound action potential will increase as more individual action potentials are generated. Note that compound action potentials arise from extracellular stimulation of the axons in the bundle, and are recorded by extracellular electrodes.
4 3 2 1 0 -1 -2 -3 0 1 2 3 4 5 6 Time (msec)
Duration Latency Peak Stimulus artifact

Figure 4: representation of (A) the electrodes used and (B) the recording produced for a compound action potential.

The shape of the compound action potential (Figure 4 B) is not related to the classical pictures that you see of single action potentials (Figures 1 and 2). What you are recording here is the difference between the readings of two extracellular recording electrodes. In the absence of a stimulus, there is no difference and we have a baseline recording of 0 mV difference. Following a stimulus, individual action potentials will propagate down each axon. As individual action potentials pass under the first electrode, it becomes negative compared to the second electrode, this is recorded as an upward deflection in the recording trace. As more individual action potentials pass under the first electrode, this difference increases (i.e. the peak gets higher). Then, when individual action potentials pass under the second electrode, it now becomes negative to the first electrode, resulting in a downward deflection in the recording. When analysing compound action potentials it is important to understand what biological activity each aspect of the recording relates to: The stimulus artifact is the electrical activity produced by applying the stimulus to the outside of the nerve; it occurs exactly when the stimulus was applied, and has nothing to do with the biology of action potentials. The latency is the time between applying the stimulus and the fastest traveling individual action potentials passing under the first recording electrode; it relates to how quickly the nerve bundle responds to a stimulus. The peak relates to the number of individual action potentials passing under the first recording electrode; the higher the peak, the more action potentials are under the first electrode. The duration is the time between the fastest individual action potentials passing under the first recording electrode and the slowest traveling action potentials passing under the second recording electrode; it relates to how quickly all the axons in the bundle complete the response to a stimulus.

Experimental design: Now that you have a good understanding of the background material, you can proceed to design your own experiment. In the practical class, your experimental set up will allow you to deliver multiple electric stimuli to the toad sciatic nerve. You will all do a brief preliminary investigation to analyse the effect of altering stimulus strength on the peak of compound action potentials. You will then be asked to complete a further experiment of your own design. You should have a detailed outline of your experiment before you come to class, this will enable you to discuss materials and methods with your group members and tutor at the start of the class. The following equipment will be available for your use during the action potential practical class. You should base your hypothesis and materials and methods on what is achievable in the 3-hour time frame (it is better to have time spare at the end). The equipment provides a range of different methods which can be used. You are not expected to use all of the equipment provided. Whole leg of a cane toad, Bufo marinus. Powerlab and recording electrodes. 8cm strip of filter paper. Stimulating electrode - deliver single or multiple stimuli of varying strengths and intervals. Frog Ringers solution (a substitute for extracellular fluid) of different temperatures.

A frozen block of frog Ringers solution, which the nerve can be exposed to for a short time. Lignocane a local anaesthetic (must be administered by a tutor).

To help you further understand all of the processes involved in this class, Dr Jim Hanan has made an interactive computer model which you can access at any time (http://www.cbit.uq.edu.au/jim/EducationalModels/ActionPotential.htm). It is strongly suggested that you make use of this to apply some of the theory you have learned. References: Campbell, N. A., J. B. Reece and N. Meyers (2009). Biology. Frenchs Forest, NSW, Pearson Education Asutralia. Eskandari, S. (2007). "The action potential." Retrieved January 15th, 2007, from http://www.csupomona.edu/~seskandari/physiology/lecture_6.html. Kole, M. H. P. and G. J. Stuart (2008). "Is action potential threshold lowest in the axon?" Nature Neuroscience 11(11): 1253-1255. Lluka, L. (2010). Lecture 2.2: Neural Excitability. BIOL1040 Module 2 Lectures, The University of Queensland.