ENGR 093: Biomedical Directed Reading Spring 2004  
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  Materials and Methods

This page is devoted to the basic goals and assumptions that Hodgkin and Huxley made. They had hoped to determine how ions flow in and out of a neuron during an action potential. They assumed that the action potential was associated with an inflow of sodiam and potassium ions. They also assumed that the membrane current could be broken up into a capacitance current and an ionic current.

This page is also devoted to an explanation of the equipment and methods they used to determine how ions flow in and out of the neuron during an action potential. Specifically, we explored the construction and use of the voltage clamp and the construction of the electrodes they placed in the axon.

Goals and Assumptions

Voltage Clamp

Electrodes

Results


The Goals and Assumptions of Hodgkin and Huxley

The goal of Hodgkin and Huxley’s experiments was to determine the laws that govern how ions flow in and out of a neuron during an action potential. At the beginning of their experimentation, Hodgkin and Huxley knew that the action potential is associated with an inflow of sodium and potassium ions. They also knew that the rate and amplitude of the action potential are determined by concentrations of sodium on the outside of the neuron.The main assumption that Hodgkin and Huxley made for their experiments was that the membrane current can be divided into a capacitance current (which is caused by a change in ion density on the outside and inside of the surface of the membrane) as well as an ionic current which depends on the movement of sodium and potassium through the membrane.

The Voltage Clamp

To measure how ions flow through the neuron membrane during an action potential, Hodgkin and Huxley measured the flow of current through a determined area of neural membrane from the axon of a giant squid. They were able to keep the voltage through the membrane constant and uniform via a feedback amplifier

Now, when performing their experiment, an operational amplifier can be used instead of the feedback amplifier used in the experiment.

At the time that Hodgkin and Huxley first performed their experiment, op-amps in their current state were not invented. The negative terminal of the op-amp was connected to the command voltage source that was controlled by the experimenters. The positive terminal was connected to an electrode that was set up to measure the voltage across the membrane. The operational amplifier works using negative feedback to make the voltage across the membrane equal to the command voltage. The output of the op-amp is connected to a second electrode that is meant to measure the current passing through the membrane. Current is injected into the system through the second electrode so that the error signal is as close to zero as possible. The membrane current and voltage as measured from the two electrodes is also passed through an oscilloscope (at the time of the experiment, Hodgkin and Huxley refer to the oscilloscope as a D.C. amplifier and cathode ray oscillograph.

The Electrodes

Hodgkin and Huxley had to develop an interesting way of creating the two electrodes they used in their experiment. The electrodes are made of two silver wires (about 20 microns in diameter). They were places 20-30 mm deep inside the neuron. The outside of the wires was insulated except along the tips of the wires. The axon was surrounded by a guard ring system which contained the external electrodes used in the system (for current, external electrode was connected to ground). The current wire extended along the whole guard system. The voltage wire only extends as far as the center partition. This is to ensure that current is flowing in a straight line where the voltage was being measured. At the ends, current could begin to curve, making the voltage measurements less accurate. For more information on exactly how the electrodes were made, the axons removed, and the guard system applied, please read their paper.


Results:
To test their apparatus, Hodgkin and Huxley ran a few simple experiments. Here are the results of the experiments.

The purpose of this set of experiments was to determine if the action potential could really be measured using their setup. The voltage clamp was turned off. It was found that in general, different amplitude depolarization inputs yielded the same general shaped output. The current pulse consists of a brief surge that is 95% complete within 8 microseconds. The peak amplitude was about 50 mA/cm3. However, depolarizations above a few milivolts yield more non-linear results. It was also found that the membrane capacity was 0.9 microfarads per centimeter squared. It was found that the threshold voltage for an action potential was 15 mV. During the voltage clamp experiment, it was noted that there was a “slight gap” in the output from the membrane. This gap is caused by the capacity current. Hodgkin and Huxley built a table of the graphs of all the current responses for different voltage values. It was found that the amplitude and shape of an impulse did not change with different temperatures, but the rate did change. Higher temperatures yielded faster responses.

 

(Figure 1: Diagram of how the voltage clamp apparatus was set up)

(Figure 2: Schematic of the feedback amplifier originally used in 1952. Now an operational amplifier is used instead)

(Figure 3: Diagram of the internal electrode.The exposed portions of wire areshown with heavier line)

(Figure 4: Diagram to illustrate the arrangement of the internal and external electrodes to create the guard system.A1, A2, A3 and A4 are all partitions. a, b, c, d, and e are electrodes)

(Figure 5: Side view of the guard system through one of the partititions)

(Figure 6: Side view of the guard system through the central channel C. c and d are silver wires. e is a silver sheet)

(Figure 7: Diagram of the time course of the membrane potential following a brief shock (magnitude of shock on the x-axis) at 23 degrees Celcius)

(Figure 7: Diagram of the time course of the membrane potential following a brief shock at 6 degrees Celcius)

(Figure 8: Diagram of the membrane current under voltage clamp conditions. Inward current is represented by upward deflection)

(Figure 9: Memebrane Current at different temperatures. Voltage clamp conditions were not used)

 


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