Overview
This protocol describes a technique called whole-cell patch clamp electrophysiology, which is a method to study electrical properties of Mauthner neurons and other reticulospinal cells in zebrafish embryos.
Protocol
1. Patch Clamp Recording (Whole Cell Mode)
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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Materials
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 |