Whole-Cell Patch Clamp Electrophysiology: A Method to Study Electrical Properties of Neurons

Protocolo

1. Patch Clamp Recording (Whole Cell Mode)

1. All recordings are performed in the whole cell patch clamp mode. Recording chambers are placed onto a microscope stage in an electrophysiology setup. Our stages are fixed and the upright patch clamp microscope is bolted to a movable microscope platform or a microscope translator.
2. Patch clamp pipettes are pulled on a horizontal puller (P-97; Sutter Instruments Co.) from thin-walled, borosilicate glass obtained from WPI. Pipette tip diameters are on the order of 0.2-0.4 μm after fire polishing to a smooth edge, and the shank taper is ~4 mm in length.
3. Attach the pipette to the amplifier head-stage in the electrophysiology setup. It is important to keep the head-stage at roughly 45° angle to the horizontal axis as this ensures an entry angle for the pipette that is suitable for the formation of high resistance seals onto the Mauthner cell.
4. Just before entering the bath solution, apply a small amount of positive pressure to the pipette to reduce the chance of dirtying the tip. When recording mEPSCs, bath solutions include 1 μM TTX to block action potentials, 5 μM strychnine to block glycine receptors and 10 μM bicuculline or 100 μM picrotoxin to block GABAA receptors. When recording mIPSCs, bath solutions include 1 μM TTX, kynurenic acid and either bicuculline or strychnine depending on the receptor activity of interest.
5. 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. 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. Releasing the positive pressure in the pipette allows the seal to be initiated and a small amount of negative pressure coupled with negative pipette potentials results in GigaΩ seals forming within a few seconds.
6. Change the holding potential on the amplifier to -60 mV. Rupture the cell membrane with a series of short pulses of suction. Immediately record membrane potentials and minimize capacitance artifacts. Compensate cell capacitance (Cm) and access (series) resistance (Ra) by 70-85%. Ra should be routinely monitored, every 30 sec to a minute, and if there is a change of 20% or more, abort the experiment.
7. Once the experiment has ended and enough data has been acquired, the preparation is sacrificed by removing the hindbrain with a pair of forceps. At this point, data analysis can begin.

Materiais

Name	Company	Catalog Number	Comments
Pipette Puller	Sutter Instruments Co	P-97
Upright Patch clamp microscope	Leica Microsystems	DMLFSA
Amplifier	Molecular Devices	200B
Micromanipulator	Siskiyou Inc.	MX7500