The sucrose gap technique is used to create a conduction block in nerve or muscle fibers. A high concentration of sucrose is applied to the extracellular space, which prevents the correct opening and closing of sodium and potassium channels, increasing resistance between two groups of cells. It was originally developed by Robert Stämpfli for recording action potentials in nerve fibers, and is particularly useful for measuring irreversible or highly variable pharmacological modifications of channel properties since untreated regions of membrane can be pulled into the node between the sucrose regions.
History
The sucrose gap technique was first introduced by in 1954 who worked with Alan Hodgkin and Andrew Huxley between 1947 and 1949. From his research, Stämpfli determined that currents moving along nerve fibers can be measured more easily when there is a gap of high resistance that reduces the amount of conducting medium outside of the cell. Stämpfli observed many problems with the ways that were being used to measure membrane potential at the time. He experimented with a new method that he called the sucrose gap. The method was used to study action potentials in nerve fibers. Since its introduction, many improvements and alterations have been made to the technique. One modification of the single sucrose gap method was introduced by C.H.V. Hoyle in 1987.
The double sucrose gap technique, which was first used by Rougier, Vassort, and Stämpfli to study cardiac cells in 1968, was improved by C. Leoty and J. Alix who introduced an improved chamber for the double sucrose gap with voltage clamp technique which eliminated external resistance from the node.
Method
A classic sucrose gap technique is typically set up with three chambers that each contain a segment of the neuron or cells that are being studied. The test chamber contains a physiological solution, such as Krebs or Ringer's solution, which mimics the ion concentration and osmotic pressure of the cell's natural environment. Test drugs can also be added to this chamber to study the effect that they have on cellular function. Ag-AgCl or platinum wire electrodes are generally used for stimulating the cells in the test solution. The sucrose chamber (or gap) is the middle chamber that separates the two other chambers, or sections of the nerve fiber or cells. This chamber contains an isotonic sucrose solution of a high specific resistance. Specific resistance describes the ability of a material or solution to oppose electric current, so a sucrose solution of a high specific resistance is effective in electrically isolating the three chambers. The third chamber usually contains a KCl solution that mimics the intracellular solution. The high potassium concentration in this chamber depolarizes the immersed segment of the tissue, allowing potential differences to be measured between the two segments separated by the sucrose gap. Vaseline, silicon grease, or a silicon-vaseline mixture is used to seal the nerve or tissue in position and prevent diffusion of solution between the chambers. A pair of agar-bridged Ag-AgCl electrodes are placed in the test and KCl chambers to record the changes in membrane potential. This allows all of the current originating on one side of the gap to flow to the other side only through the interior of the nerve or tissue. Changes in electrical potential between the two groups relative to each other can be measured and recorded. When used with proper electronics, the double sucrose gap can be used to voltage clamp the membrane potential of the nerve or tissue segment contained in the test chamber.
Limitations
A major limitation of the single sucrose gap is that it cannot determine the real values of the membrane potential and action potential amplitudes. It can only measure the relative changes in the potential between the regions separated by the sucrose solution because of the shunting effect. Double sucrose gap, however, can measure the membrane potential and resistance. Another limitation is that membrane potentials cannot be obtained from tissues where there is no electrical coupling between the cells (i.e. when the spatial constant, λ, is close to zero).
The sucrose-gap technique has been applied to determine the relation between external potassium concentration and the membrane potential of smooth muscle cells using guinea-pig ureters. It has also been used to rectify inaccurate membrane potential measurements resulting from leakage currents through the membrane and extracellular resistance. Correction of an inaccurate membrane current reading is also possible through utilization of the sucrose-gap method.
Developments in the sucrose-gap method have led to double sucrose-gap techniques. A double sucrose-gap is generally advantageous when used to electrically isolate smaller segments of nerve fibers than would be possible with a single sucrose-gap, The double sucrose-gap technique is also utilized over the single sucrose-gap to study cardiac muscle, where it allows for clearer resolution of early currents, those occurring within the first 10-100 milliseconds of depolarization.