eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
1. Solutions
<ol>
	<li>Slicing solution&#8203;
	<ol>
		<li>Refer to Table 1 for the composition of the slicing solution.</li>
		<li>Prepare a 20x stock solution in advance and store it at 4 &#176;C for up to 1 month.</li>
		<li>For the 1x slicing solution, dissolve NaHCO3, glucose, and sucrose in ddH2O and add the 20x stock. Ensure the osmolarity is between 315 and 320 mOsm and store the solution for no more than 1 week at 4 &#176;C.</li>
		<li>Fill two beakers with 100 mL of slicing solution and cover them with parafilm. Chill the solution in a freezer until it becomes partially frozen (approximately 20 min in a -80 &#176;C freezer). Using a gas dispersion tube, bubble both beakers of slicing solution with 95% O2/5% CO2 for 20 min on ice.</li>
	</ol>
	</li>
	<li>aCSF(Artificial cerebrospinal fluid), for slice recovery and maintenance
	<ol>
		<li>Refer to Table 1 for the composition of the aCSF.</li>
		<li>Prepare a 20x stock solution in advance and store it at 4 &#176;C for up to 1 month.</li>
		<li>For 1x aCSF, dissolve NaHCO3 and glucose in ddH2O and add the 20x stock. Ensure the osmolarity is between 298-300 mOsm. Use the solution within 1 day.</li>
	</ol>
	</li>
	<li>aCSF (low Ca2+ for recording)
	<ol>
		<li>Refer to Table 1 for the composition of the low Ca2+ aCSF.</li>
		<li>Prepare a 20x stock solution in advance and store it at 4 &#176;C for up to 1 month.</li>
		<li>For 1x low Ca2+ aCSF, dissolve NaHCO3 and glucose in ddH2O and add 20x (CaCl2 and MgCl2 free) stock, CaCl2 and MgCl2 to specified concentrations. Ensure the osmolarity is between 298-300. Use the solution within 1 day.</li>
	</ol>
	</li>
	<li>aCSF (Sr2+ for recording)
	<ol>
		<li>Refer to Table 1 for the composition of the Sr2+ aCSF.</li>
		<li>Prepare a 20x stock solution in advance and store it at 4 &#176;C for up to 1 month.</li>
		<li>For 1x Sr2+ aCSF, dissolve NaHCO3 and glucose in ddH2O and add 20x (CaCl2 and MgCl2 free) stock, SrCl2 and MgCl2 to specified concentrations. Ensure the osmolarity is between 298-300. Use the solution within 1 day.</li>
	</ol>
	</li>
	<li>Internal solution
	<ol>
		<li>Refer to Table 1 for the composition of the K-gluconate-based internal solution.</li>
		<li>To make 20 mL of internal solution, add 15 mL of molecular biology-grade water to a 50 mL tube. Then, perform the subsequent steps on ice.</li>
		<li>Prepare the following solutions ahead of time to 1 M stock concentrations in molecular biology grade water. Add (in mL): 2.32 K-gluconate, 0.24 Na-gluconate, 0.20 HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.16 KCl, 0.05 K2-EGTA (Potassium ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid), 0.04 MgCl2 to the 50 mL tube.</li>
		<li>Add 100 &#181;L of 0.3 M Na3GTP (Guanosine 5&#8242;-triphosphate sodium salt)</li>
		<li>Weigh 44.08 mg of K2 ATP (Adenosine 5&#8242;-triphosphate dipotassium salt) in a 2 mL microcentrifuge tube and add 1 mL of molecular biology grade water, then add to a 50 mL tube.</li>
		<li>Adjust the pH to 7.2-7.4 with 1 M KOH. Ensure the osmolarity is between 283-289 mOsm.</li>
	</ol>
	</li>
</ol>
2. Slice Preparation
<ol>
	<li>Prepare tools
	<ol>
		<li>Add 200 mL of aCSF to the recovery chamber (constructed from a 250 mL beaker with 4 wells and netting) and place the recovery chamber in a water bath (35 &#176;C).</li>
		<li>Cover the chamber with a paraffin film and constantly bubble the aCSF with 95% O2/5% CO2 using a glass dispersion tube for at least 20 min.</li>
		<li>Set up the dissection tools (scalpel, angled fine scissors, forceps, fine paintbrush, plastic spoon).</li>
		<li>Fill a 60 mL syringe with approximately 15 mL of the ice-cold slicing solution from step 1.1.4.</li>
		<li>Prepare the dissection platform by placing filter paper on the lid of a well plate.</li>
		<li>Prepare the slicing chamber by placing it in the ice tray and filling the tray with ice.</li>
		<li>Set up the vibratome by securing a disposable blade in the blade holder.</li>
		<li>To make a transfer pipette, break the tip of a Pasteur pipette and place a rubber bulb over the broken end.</li>
	</ol>
	</li>
	<li>Dissect mouse brain
	<ol>
		<li>Anesthetize the animal in a chamber saturated with 4% isoflurane until spinal reflexes are absent.</li>
		<li>Decapitate the animal using a guillotine and quickly remove the brain.
		<ol>
			<li>Make a midline incision with a No. 22 scalpel blade from rostral to caudal.</li>
			<li>Laterally, peel the scalp on each side of the head.</li>
			<li>Use fine scissors to cut the skull on one side from caudal to rostral (including the side of the frontal bones), using caution not to damage the brain.</li>
			<li>Use forceps to lift the skull piece off the brain and quickly cool the brain with 15 mL of ice-cold slicing solution using the syringe from step 2.1.4.</li>
			<li>Lift the brain out of the skull.</li>
			<li>Place the brain in one of the beakers filled with ice-cold slicing solution (from step 1.1.4) bubbled with 95% O2/5% CO2.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Prepare slices of mouse hypothalamus.
<ol>
	<li>Block the brain for the desired brain area and cut angle (e.g., for coronal hypothalamic slices, trim off the tissue rostral to the optic chiasm and caudal to the pons using a blade and ensure the caudal block has a flat surface perpendicular to the base of the brain).</li>
	<li>Using a cut piece of filter paper, pick up the brain from the anterior side and glue the posterior side to the holding plate using instant glue.</li>
	<li>Quickly place the holding plate into the slicing chamber and fill it with the slicing solution from the second beaker in step 1.1.4.</li>
	<li>Secure the slicing chamber and ice tray on the vibratome.</li>
	<li>Define the slicing area (anterior and posterior to the brain) and begin slicing 250 &#181;m thickness coronal slices. Recommended parameters: speed 0.10 mm/s, amplitude 2 mm.</li>
	<li>Trim the slices to the appropriate size for the desired brain area.</li>
	<li>Recover the slices at 35 &#176;C for 30-45 min. Then, remove the recovery chamber from the warming bath and allow the slices to recover at room temperature for an additional 30 min. Keep slices at room temperature for the rest of the day and continue to bubble the bath constantly with 95% O2/5% CO2.</li>
</ol>
3. Whole-cell Patch ClampRecording
<ol>
	<li>Pull the patch pipettes.&#8203;
	<ol>
		<li>Using the suggested parameters for the whole-cell recording from the pipette puller&#8217;s manual, pull patch pipettes from thick-walled glass to a pipette resistance of 3-5 M&#8486;.</li>
		<li>Using a microsyringe (commercial or homemade), fill a pipette tip with a filtered internal solution. To make a micro syringe, burn the tip of a 1 mL syringe and allow the tip to fall, creating a long, fine tip.</li>
	</ol>
	</li>
	<li>Obtain the whole-cell configuration.
	<ol>
		<li>Place the recording pipette just above the slice and offset the pipette current in the voltage clamp mode. Apply slight positive pressure to the pipette and lock the stopcock.</li>
		<li>Select a healthy cell with an intact membrane and approach it with the pipette. The positive pressure should cause a slight disturbance in the tissue (i.e., a slow wave in the tissue when entering).</li>
		<li>Slowly continue to bring the pipette closer to the cell using a diagonal motion until the pipette forms a small dimple on the cell surface.</li>
		<li>Release the positive pressure lock. The cell will begin to form a seal, and the resistance will increase above 1 G&#8486;. In the voltage clamp, hold the cell at -68 mV.</li>
		<li>Slightly pull away from the cell diagonally to remove excess pressure from the cell.</li>
		<li>Compensate for the fast and slow pipette capacitance.</li>
		<li>Apply a brief suction through the tube connected to the pipette holder to break through the cell and obtain a whole-cell configuration.</li>
		<li>Switch to Cell mode on the membrane test window in an electrophysiology Data acquisition and analysis software (e.g., Clampex).</li>
		<li>Before each voltage clamp recording, perform a membrane test using the same software and record the relevant parameters in a lab book (membrane resistance, access resistance, and capacitance).</li>
		<li>Maintain the temperature of the recording bath at 27&#8211;30 &#176;C and the flow rate at 1.5&#8211;2.0 mL/min for subsequent experiments.</li>
	</ol>
	</li>
</ol>
Table 1: The composition of various solutions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="5">Solution Concentrations (mM)</td>
		</tr>
		<tr>
			<td></td>
			<td>Slicing</td>
			<td>Normal aCSF</td>
			<td>Low Ca2+ aCSF</td>
			<td>Sr+ aCSF</td>
			<td>Pipette/Internal</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>87</td>
			<td>126</td>
			<td>126</td>
			<td>126</td>
			<td>-</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.5</td>
			<td>2.5</td>
			<td>2.5</td>
			<td>2.5</td>
			<td>8</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>0.5</td>
			<td>2.5</td>
			<td>0.5</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>SrCl2</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>2.5</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>7</td>
			<td>1.5</td>
			<td>2.5</td>
			<td>1.5</td>
			<td>2</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>1.25</td>
			<td>1.25</td>
			<td>1.25</td>
			<td>1.25</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>25</td>
			<td>26</td>
			<td>26</td>
			<td>26</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>25</td>
			<td>10</td>
			<td>10</td>
			<td>10</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>75</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>116</td>
		</tr>
		<tr>
			<td>Na-gluconate</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>12</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>10</td>
		</tr>
		<tr>
			<td>K2-EGTA</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>1</td>
		</tr>
		<tr>
			<td>K2ATP</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Na3GTP</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>0.3</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>10 blade</td>
			<td>Fisher Scientific/others</td>
			<td>35698</td>
			<td></td>
		</tr>
		<tr>
			<td>22 blade</td>
			<td>VWR/others</td>
			<td>21909-626</td>
			<td></td>
		</tr>
		<tr>
			<td>22 uM syringe filters</td>
			<td>Milipore</td>
			<td>09-719-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson foreceps</td>
			<td>Harvard Instruments</td>
			<td>72-8547</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled sharp scissors</td>
			<td>Harvard Instruments</td>
			<td>72-8437</td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex</td>
			<td>Molecular Devices</td>
			<td>pClamp 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge blade</td>
			<td>VWR</td>
			<td>74-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Sigma/others</td>
			<td>1001090</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine paintbrush</td>
			<td>Fisher/various</td>
			<td>15-183-35/various</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Dispersion Tube</td>
			<td>VWR</td>
			<td>LG-8680-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fresenius Kabi/others</td>
			<td>M60303</td>
			<td></td>
		</tr>
		<tr>
			<td>Krazy glue</td>
			<td>various</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini analysis</td>
			<td>Synaptosoft</td>
			<td>MiniAnalysis 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmomoter</td>
			<td>Wescor Inc</td>
			<td>Model 5600</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>VWR</td>
			<td>14672-200</td>
			<td></td>
		</tr>
		<tr>
			<td>ph meter</td>
			<td>Mettler Toledo</td>
			<td>FE20-ATC</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bulb</td>
			<td>VWR</td>
			<td>82024-550</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle No. 3</td>
			<td>Harvard Instruments</td>
			<td>72-8350</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle No. 4</td>
			<td>Harvard Instruments</td>
			<td>72-8356</td>
			<td></td>
		</tr>
		<tr>
			<td>Single edge blade</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome slicer</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Purification System</td>
			<td>Millipore</td>
			<td>Milli-Q Academic A10</td>
			<td></td>
		</tr>
		<tr>
			<td>Well plate lid</td>
			<td>Fisher/various</td>
			<td>07-201-590/various</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>CdCl2</td>
			<td>sigma</td>
			<td>202908</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Sigma</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>K2-ATP</td>
			<td>Sigma</td>
			<td>A8937</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma</td>
			<td>G4500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology grade water</td>
			<td>Sigma</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3GTP</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>SOD001.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-gluconate</td>
			<td>Sigma</td>
			<td>S2054</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>71504</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>sigma</td>
			<td>P1675</td>
			<td></td>
		</tr>
		<tr>
			<td>SrCl</td>
			<td>Sigma</td>
			<td>255521</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Bioshop</td>
			<td>SUC507.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing a whole-cell voltage-clamp configuration for electrophysiological recordings in brain slices

Source: Sunstrum, J. K., et al. <a href="https://app.jove.com/v/59461">Evaluation of synaptic multiplicity using whole-cell patch-clamp electrophysiology.</a> J. Vis. Exp. (2019)
This video demonstrates positioning a fine-pointed pipette near brain slices, forming a seal with a neuronal cell. Suction breaks the cell membrane, thereby creating a whole-cell patch clamp that enables current measurements for electrophysiology experiments.

Establishing a Whole-Cell Voltage-Clamp Configuration for Electrophysiological Recordings in Brain Slices

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review ...

Take a recording pipette with a wire electrode in an electrolyte solution.
Position the pipette above an immobilized brain slice.
Adjust the pipette current to reduce background signals.
Use a microscope to locate the target neuron and move the pipette toward the neuron.
Apply a slight positive pressure and lock it.
The positive pressure disturbs the tissue surface, indicating proximity to the cell.&#160;&#160;&#160;&#160;
Bring the pipette closer until a dimple forms on the membrane, signifying correct positioning.
Release the positive pressure.
Observe an increase in resistance as the cell forms a seal with the pipette.
Hold the membrane potential at a constant negative voltage for stability.
Pull the micropipette away to remove excess pressure.
Adjust the pipette&#39;s fast and slow electrical responses for accuracy.
Apply brief suction to break through the cell, establishing a direct electrical connection.
The whole-cell voltage clamp configuration is now ready for electrophysiology recording.

establishing-whole-cell-voltage-clamp-configuration-for

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

88 ビュー. 出典:Sunstrum, J. K., et al.全細胞パッチクランプ電気生理学を用いたシナプス多重度の評価。J. Vis. Exp.(2019年) このビデオでは、細い先の尖ったピペットを脳スライスの近くに配置して、ニューロン細胞とシールを形成する方法を示します。吸引により細胞膜が破壊されるため、電気生理学実験の電流測定を可能にする全細胞パッチクランプが形成されます。 

ビデオ: 脳スライスにおける電気生理学的記録のための全細胞電圧クランプ構成の確立 - 概念

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

精巣上体から分離した一次上皮細胞の全細胞パッチクランプ記録

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

JoVE Journal

発生生物学

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

スライス パッチ ・ クランプの技術学習によって誘発される可塑性を分析するため

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

神経科学

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers easy identification and characterization of these cell types, prerequisites to study their specific functions. This article describes the multiplex single cell reverse transcription polymerase chain reaction (RT-PCR) protocol, which allows, after patch-clamp recording in slices, to detect simultaneously the expression of tens of genes in a single cell. This simple method can be implemented with morphological characterization and is widely applicable to determine the phenotypic traits of various cell types and their particular cellular environment, such as in the vicinity of blood vessels. The principle of this protocol is to record a cell with the patch-clamp technique, to harvest and reverse transcribe its cytoplasmic content, and to detect qualitatively the expression of a predefined set of genes by multiplex PCR. It requires a careful design of PCR primers and intracellular patch-clamp solution compatible with RT-PCR. To ensure a selective and reliable transcript detection, this technique also requires appropriate controls from cytoplasm harvesting to amplification steps. Although precautions discussed here must be strictly followed, virtually any electrophysiological laboratory can use the multiplex single cell RT-PCR technique.

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

パッチ ・ クランプ後単一のセル多重逆のトランスクリプション ポリメラーゼ連鎖反応

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...