Patch clamp technique

The gold standard to investigate the electrical response of cells has been the patch clamp technique. It can be used to both record a cell’s response to external conditions and to depolarize or hyperpolarize the cell from within. In this technique, a glass capillary is drawn to a fine taper (1-3 μm in diameter). The pipette is then filled with electrolyte of similar composition to either the intracellular or extracellular fluid. An electrode is inserted in the back of this pipette and used to measure voltages or currents at the tip with reference to an Ag/AgCl bath electrode. The tip of the pipette is brought in close contact with the cell membrane until the lipid bilayer seals onto the glass tip with greater than 1 GΩ resistance. It is then inferred that any current detected at the tip during a constant holding voltage, with reference to the extracellular fluid, is due to opening of ion channels within the patch of membrane attached to the end of the pipette. The graded response of the cell comes as the integration of the conductance of all the channels in the membrane. To investigate this with the patch clamp technique, under pressure is applied to the pipette once it is sealed to the membrane. This ruptures the membrane within the inner diameter of the pipette tip and forms a continuous electrolyte between the cytosol and the patch pipette fluid. The edges of the ruptured membrane remain sealed to the glass with GΩ resistance such that the pipette can be considered an extension of the cell interior connected by the tip resistance (determined by the diameter) and the capacitance of the glass. In this way, the summed action of all the cell’s channels can be measured. Patch clamp has allowed investigation of the affects a single channel type can have on a cell.

The investigation of individual neuron’s behavior is the most widely used application of patch clamp. Excised neurons have been investigated while the holding voltage is varied or current pulses are applied with the pipette. This elucidates the change in response of a single neuron to different environmental conditions or over short periods of time (∼1 hour). To investigate neural processing, small portions of neural networks have been mapped using rabies based reporters loaded into a single neuron via the patch pipette. This allows the investigator to see which neurons provided input to the cell being patched. It is far more complicated to predict exact cell-cell connections for double patch experiments. These double patch investigations allow correlation between the electrical behaviors of the two neurons, but rely on established maps of physiologic connectivity, or carefully planned small in vitro networks where much of the connectivity can be evaluated visually. The patch clamp data has formed the basis of our understanding of neuronal processing at the single cell level.

Contact:

Bogdana Cepkenovic
Tel.:  +49-2461-61-3675
e-mail: b.cepkenovic@fz-juelich.de

Dr. Vanessa Maybeck
Tel.:  +49-2461-61-3675
e-mail: v.maybeck@fz-juelich.de