Encyclopedia of Experiments: Biology
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Transcript
- Start by placing a recording chamber containing a sample in a bath solution, whose composition matches the physiological extracellular environment or the cytoplasm, onto a microscope stage. Advance the recording micropipette, a pipette containing a silver electrode bathed in an electrolyte solution, which mimics the intracellular conditions, toward the neuron by applying positive pressure, and make contact with the cell membrane.
Gently switch from positive to negative pressure to form a tight seal between the membrane and the pipette. Set a holding potential on the amplifier to maintain the cell at a constant voltage. Then rupture the cell membrane within the micropipette by applying suction.
Once the cell membrane ruptures, the holding potential causes the flow of ions through voltage-gated ion channels, creating a membrane potential, which is recorded by the recording electrode. Immediately record the membrane potential. The recording electrode transmits the signal to the feedback amplifier, which subtracts the membrane potential from the holding potential-- an oscilloscope, which presents a visual display of the membrane potential, and a computer. In the following protocol, we will perform a patch clamp measurement on the Mauthner cell in a zebrafish embryo.
- To prepare for patch clamping, begin by pulling some patch clamp pipettes with thin-walled borosilicate glass in a horizontal puller. Pipette tip diameters are about 0.2 to 0.4 micrometers after fire polishing to a smooth edge, and the shank taper is about 4 millimeters long.
Fill the pipette tip with intracellular solution by dipping the tip into the intracellular fluid. Then insert a syringe needle into the pipette and gently expel intracellular solution from the syringe to finish filling the pipette. Attach the pipette to the amplifier head stage in the electrophysiology setup. It is important to keep the head stage at a roughly 45 degree angle to the horizontal axis, as this ensures an entry angle for the pipette that is suitable for the formation of high resistance seals with the Mauthner cell.
Right before the pipette lowers into the bath solution, apply a small amount of positive pressure to the pipette to reduce the chance of the tip blocking. Continue to approach the M-cell with a small amount of positive pressure in the pipette. The positive pressure gently pushes the cell from side to side, and when positioned immediately over the cell, forms a small dimple on the cell membrane.
Now, leave the pipette in place for a few seconds to gently clean the cell surface so that a strong seal between the pipette and the membrane can be formed. Then release the positive pressure in the pipette to initiate the seal. A small amount of negative pressure, coupled with negative pipette potential, results in giga-ohm seals forming within a few seconds.
Subsequently, change the holding potential in the amplifier to minus 60 millivolts. Rupture the cell membrane with a series of short pulses of suction. Then immediately record membrane potentials and minimize capacitance artifacts. Compensate the cell capacitance and access resistance by 70% to 85%.
Access resistance should be monitored every 30 seconds to a minute, and if there's a change of 20% or more, abort the experiment. Once the experiment has ended and enough data has been acquired, sacrifice the embryo by removing the hindbrain with a pair of forceps.
