1. Patch Clamp Recording (Whole Cell Mode)
<ol>
	<li>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.</li>
	<li>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 &#956;m after fire polishing to a smooth edge, and the shank taper is ~4 mm in length.</li>
	<li>Attach the pipette to the amplifier head-stage in the electrophysiology setup. It is important to keep the head-stage at roughly 45&#176; 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.</li>
	<li>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 &#956;M TTX to block action potentials, 5 &#956;M strychnine to block glycine receptors and 10 &#956;M bicuculline or 100 &#956;M picrotoxin to block GABAA receptors. When recording mIPSCs, bath solutions include 1 &#956;M TTX, kynurenic acid and either bicuculline or strychnine depending on the receptor activity of interest.</li>
	<li>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 Giga&#8486; seals forming within a few seconds.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<table><tbody><tr><td>Pipette Puller</td><td>Sutter Instruments Co</td><td>P-97</td><td></td></tr><tr><td>Upright Patch clamp microscope</td><td>Leica Microsystems</td><td>DMLFSA</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>200B</td><td></td></tr><tr><td>Micromanipulator</td><td>Siskiyou Inc.</td><td>MX7500</td><td></td></tr></tbody></table>

whole-cell patch clamp electrophysiology: a method to study electrical properties of neurons

Source: Roy, B., et al. <a href="https://www.jove.com/v/50551/" target="_blank">Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells</a>. J. Vis. Exp. (2013).
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.

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

Source: Roy, B., et al. Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells. J. Vis. Exp. (2013).
This protocol describes a technique called ...

- 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&#39;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.

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JoVE Encyclopedia of Experiments

Biology

Danio rerio (zebrafish)

Embryo

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Elektrofizjologia klamer krosowych całych komórek: metoda badania właściwości elektrycznych neuronów

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.

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

W tym Artykule

Overview

Protokół

Materiały

Odniesienia

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