Biology Reference
In-Depth Information
pipette after it is lowered in the bath, gentle positive pressure is applied on the
suction line which is then pinched off until contact with the cell. In addition, bath
solutions are designed to be 10% hyperosmotic to the pipette solution (usually by
adding glucose), so water will stream out of the pipette tip. Axial approaches are
more e
V
ective than lowering the angled pipette straight down on to the cell. Some
manipulators program a fourth axis or you can mount the manipulator at an angle
so one axis is parallel to the pipette. We prefer to monitor the approach of the
pipette to the cell surface by looking at the pipette current trace in response to a
small voltage step. As the pipette touches the cell, the amplitude of the current
diminishes. When it is reduced to 33-50%, we release the positive pressure on the
pipette, and then, if necessary, we apply additional suction. To make these manip-
ulations without disturbing the tip, the small tube connected to the pipette holder
must be firmly anchored to the headstage and the microscope stage to prevent
vibrations. Most of us prefer to apply the suction directly by mouth because the
suction is controlled more easily and changes more evenly. Small flat cells are
obviously harder to patch than large round ones. Visualization of the cell surface
is improved by interference contrast optics, but this can be implemented on very
simple microscopes for the price of two small pieces of black tape (
Axelrod, 1981
).
Most people we have trained find the rat pituitary GH cell lines to be the easiest cells
with which to learn patching. They can be obtained from the ATCC.
D. Making Recordings
1. Cell-Attached Patch Recordings
The two most important things to remember about the cell-attached configura-
tion are that the voltage polarities are reversed relative to traditional whole-cell
recording and that there are two membranes in the current path between your
electrodes. The amplifier sets the voltage between the pipette wire and the ground
electrode in the bath, but, by convention, the cell interior is negative to the outside,
so depolarizing the membrane of a cell-attached patch means making the pipette
more negative. However, the cell membrane also contributes a voltage which is not
clamped in the cell-attached configuration, so to accurately determine the voltage
across the patch, the cell's membrane potential must be set to zero by bathing the
cell in an equimolar potassium solution of 140-150 mM. Unfortunately, that
means all the voltage-gated channels, including the calcium channels, in the
membrane outside the patch will be activated. Therefore, it is essential to reduce
extracellular calcium to avoid flooding the cell with calcium. Some investigators
use calcium bu
ers, but the bath is infinite relative to the cell volume and most cells
are rapidly depleted of calcium. A more physiological solution is to reduce extra-
cellular calcium to 0.1 mM, which is still 100
V
more than resting calcium inside the
cell, but produces negligible currents in the presence of 2-5 mM magnesium.
For cell-attached recordings, the primary goal is increasing the signal and
reducing noise. Increasing the signal
is usually achieved by increasing the
