perforated patch-clamp recordings of inner ear sensory neuron cell bodies

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

Dip the tip of the pipette into the clean solution of the culture dish, to fill it with a clean solution that does not contain amphotericin. Fill the back of the pipette with the internal solution containing amphotericin. Dip the tip of the pipette back into the clean solution of the culture dish while the amphotericin slowly enters the tip from the back of the pipette. Next, finish filling the pipette with the amphotericin solution up to 2/3rds of the pipette.
Lower the pipette into the bath of the recording chamber, and locate the pipette tip in the middle of the field of view. Ensure that the tip is free of air bubbles or other debris. Position the pipette close to the membrane, and land firmly on the center of the cell. Apply negative pressure or suction using a syringe or mouth pipette.
Turn on the holding potential of minus 60 millivolts. The seal resistance should increase until it passes a gigaohm. Once a gigaohm seal forms, release the negative pressure. Amphotericin begins to work. The input resistance slowly decreases, and the current flowing in response to the 5 millivolts voltage step progressively increases as the amphotericin enters the membrane.

perforated-patch-clamp-recordings-inner-ear-sensory-neuron-cell

Nanyang Technological University

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1. Recording
NOTE: In this procedure, patch-clamp recordings from the isolated ganglion are typically performed 12-24 h after the plating. Other labs have reported results from neurons after much longer periods in culture.
<ol>
	<li>Prepare the recording chamber
	<ol>
		<li>Use the quick exchange recording chamber (or equivalent) that allows for patch-clamp recordings directly in the culture dish. Set up the chamber on the inverted microscope.</li>
		<li>Insert the glass-bottom culture dishes into the chamber.</li>
		<li>Feed the L-15 solution through a perfusion tubing system to the recording chamber. Stabilize the perfusion in-flow and out-flow in the chamber at a rate of 0.5-1 mL per minute.</li>
	</ol>
	</li>
	<li>Prepare the internal solution for loading electrodes
	<ol>
		<li>Thaw an aliquot of the perforated-patch internal solution. Allocate 2.5 mL of the solution onto a 35 mm culture dish. Replace the lid on the culture dish and label as &#34;Tip Dip&#34;. Set aside in a dust-free zone, as it is very important to keep this solution as clean as possible.</li>
		<li>Weigh 1 mg of amphotericin-B and add 20 &#181;L of dimethyl sulfoxide (DMSO). Vortex and spin down the solution until all the amphotericin-B is in the solution. Add 10 &#181;L of the DMSO/amphotericin-B solution to the remaining 2 mL of the defrosted perforated-patch internal solution. Withdraw and dispel the solution with a pipette two or three times to ensure that the DMSO/amphotericin-B has uniformly mixed into the internal solution. The solution will have a mild-yellowish tinge.</li>
		<li>Draw the amphotericin-B perforated-patch internal solution into a 3 mL syringe. Add a 34 G tip to the syringe. Wrap the syringe in aluminum foil and keep the syringe on ice. 
		NOTE: This solution must be remade every 2 h to ensure the effectiveness of the amphotericin-B in perforating the cell membrane. Amphotericin-B is light sensitive, and it must be shielded from light using aluminum foil or other methods.</li>
	</ol>
	</li>
	<li>Patch-clamp recordings
	<ol>
		<li>Visualize the neurons using a 10x or 20x objective. Adjust the illumination and optimize the optics to see the boundaries and shadows around the cell.</li>
		<li>Check the quality of the isolated neurons. Only attempt neurons with smooth and even surfaces that are not too strongly contrasted and have only small, dispersed craters. Also, make sure to avoid pairs of neurons, neurons surrounded by debris, or other cells.</li>
		<li>Under 100x magnification, identify a neuron or field of neurons to patch and line them up in the center of the field of view.</li>
		<li>Fill the tip of the pipette with a clean solution that does not contain amphotericin by dipping the tip (for ~20 s) into the clean solution that was placed in a culture dish. Capillary action will draw a small amount of solution into the tip. 
		NOTE: Perform this step under a stereo dissection microscope, which allows for determining how well the tip holds the Tip Dip solution. If the solution does not hold for ~10-20 s, the shape of the electrode is not ideal. In this case, reconfigure the electrode-pulling program to produce a longer tip.</li>
		<li>Next, fill the back of the pipette with the internal solution containing amphotericin. The filament within the pipette will draw down the solution to meet the clean solution in the tip. Allow the filament to smoothly pull the solution down, and do not attempt to tap out bubbles, as this will force the amphotericin to the tip too fast.</li>
		<li>Use a syringe to draw out solution that may be at the very back of the electrode. 
		NOTE: It is very important to work fast from this point to land and form a high-resistance seal on the desired cell.</li>
		<li>Insert the pipette into the pipette holder. Check that the pipette is snug in the holder to prevent pipette drift. If the pipette is not stable, switch out the sealing O-rings in the front and back of the pipette holder to minimize drift and maintain strong suction. Replace the pipette with one that is freshly filled.</li>
		<li>Lower the pipette into the bath of the recording chamber.</li>
		<li>Locate the pipette tip in the middle of the field of view. Ensure that the tip is free of air bubbles or other debris.</li>
		<li>Apply a voltage step (5 mV) in the membrane test to monitor the pipette resistance. Continue monitoring the input resistance through the entire process of forming a seal on the cell.</li>
		<li>Cancel the pipette offset potential, such that the pipette current reads zero in the membrane test mode of pClamp.</li>
		<li>Correct for the pipette capacitance using the fast capacitance compensation function on the patch-clamp amplifier (Cp fast dials in the Multiclamp Commander soft panel).</li>
		<li>Move the pipette down to the cell.</li>
		<li>Once the pipette is close enough to the neuron, switch to the higher magnification objective and send the image through a camera to a monitor (use a 40x objective, total 400x magnification). Adjust the pipette and re-center the neuron.</li>
		<li>Move the recording electrode close to the soma. Locate the center of the neuron on the monitor and position the recording electrode above the neuron.</li>
		<li>Approach the neuron from above, such that the electrode will land in the center of the spherical cell. Adjust the pipette offset to zero the constant DC potentials in the system.</li>
		<li>Position the pipette close to the membrane. Land firmly on the center of the cell. 
		NOTE: When landing, a small dimpling on the surface of the neuron and a doubling/tripling of the input resistance will occur.</li>
		<li>Apply negative pressure (suction) using a syringe or mouth pipette. Turn on the holding potential of -60 mV. The seal resistance should increase until it passes a giga-ohm. 
		NOTE: Work fast to reduce the time between filling an electrode and landing on a cell. There is a limited time before the amphotericin added to the perforated-patch internal solution reaches the electrode tip. Once the tip is contaminated with amphotericin, it is difficult to form high-resistance seals, and the resistance often plateaus to ~200 megaohms.</li>
		<li>Once a giga-ohm seal forms, release the negative pressure. As soon as the seal forms, apply fast-capacitance compensation to reduce the amplitude of the pipette capacitance transients in membrane test mode as much as possible.</li>
		<li>As amphotericin begins to work, watch as the input resistance slowly decreases and the current flowing in response to the 5 mV voltage step progressively increases as the amphotericin enters the membrane. A sudden decrease in series resistance instead of gradual stabilization suggests that spontaneous rupture has occurred.</li>
		<li>Estimate the whole-cell capacitance and the series resistance using the amplifier&#39;s capacitive transient nulling function. Document and monitor the series resistance at the beginning of, and regularly during, the experiment. 
		NOTE: The series resistance can take anywhere from 5-20 min to stabilize, depending on the size of the electrode and how much clean solution is drawn into the pipette. Voltage-clamp and current-clamp modes can be used once the series resistance has stabilized below 30 megaohms. Swelling or shrinking of the neurons during recording is sometimes observed, but rarely when the osmolarity and pH of the solutions are correctly adjusted.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin</td>
			<td>Sigma-Aldrich</td>
			<td>A4888-100MG</td>
			<td>For perforated patch recordings.</td>
		</tr>
		<tr>
			<td>ATP di-sodium</td>
			<td>Sigma-Aldrich</td>
			<td>A7699</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>J.T. Baker</td>
			<td>1311-01</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Biotium</td>
			<td>90082</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E0396</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>Electrode Puller</td>
			<td>Narashige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td>for cleaning head and around dissection bench</td>
		</tr>
		<tr>
			<td>Filamented Borosilicate Capillaries for electrodes</td>
			<td>Sutter Instruments</td>
			<td>BF140-117-10</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-100G</td>
			<td>for pH buffering all solutions in protocol</td>
		</tr>
		<tr>
			<td>Hot plate / magnetic stirrers</td>
			<td>VWR</td>
			<td>76549-914</td>
			<td></td>
		</tr>
		<tr>
			<td>K2SO4, Potassium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>P9458-250G</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P93333</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>KOH (1 M)</td>
			<td>Honeywell</td>
			<td>319376-500ML</td>
			<td>To bring internal solution to desired pH.</td>
		</tr>
		<tr>
			<td>Low-profile-bath recording chamber for culture dishes</td>
			<td>Warner Instruments</td>
			<td>64-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2-Hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>microFil needle for filling micropipettes - 34 gauge</td>
			<td>World Precision Instruments</td>
			<td>MF34G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narashige</td>
			<td>MF-90</td>
			<td>For electrode polishing.</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Additive to internal solution</td>
		</tr>
		<tr>
			<td>NaOH (1 M)</td>
			<td>Thomas Scientific</td>
			<td>319511-500ML</td>
			<td>for titration pH</td>
		</tr>
		<tr>
			<td>Oxygen, Medical grade, with adequate regulator and tubing</td>
			<td>USC Material Management</td>
			<td>MEDOX200 (Identifier: 00015)</td>
			<td>for dissolving into dissection and bath solutions</td>
		</tr>
		<tr>
			<td>Poly-d-lysine coated glass bottomed 
			culture dish</td>
			<td>Mattek</td>
			<td>P35GC-0-10-C</td>
			<td>to plate neurons for culture</td>
		</tr>
		<tr>
			<td>PES membrane filters , 0.2 micrometer</td>
			<td>Nalgene</td>
			<td>566-0020</td>
			<td>for filtering solutions</td>
		</tr>
		<tr>
			<td>PES membrane sterile syringe filters, 0.22 um, 30 mm</td>
			<td>CELLTREAT</td>
			<td>229747</td>
			<td>for filtering solutions drawn into syringes</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo</td>
			<td>Model S20</td>
			<td></td>
		</tr>
		<tr>
			<td>Reference Cell</td>
			<td>World Precision Instruments</td>
			<td>RC1T</td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific Scale</td>
			<td>Mettler Toledo</td>
			<td>XS64</td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric flask, 1000 milliliter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water, sterile u ltrapure, R&#62;18.18 megaOhms cm (e.g., filtered by a Millipore-Sigma water purification system)</td>
			<td>Millipore-Sigma</td>
			<td>CDUFBI001</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Iyer, M. R., et al. <a href="https://app.jove.com/v/64908">Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.</a> J. Vis. Exp. (2023).
This video demonstrates the patch-clamp technique for measuring ionic currents in inner ear sensory neuron cell bodies. It details the preparation and use of a polished patch-clamp pipette filled with an internal solution containing amphotericin B, which perforates the membrane to allow ion movement while preserving intracellular conditions. Voltage pulses are applied to trigger and record ion channel activity, providing insights into their properties and behavior.

1. Recording
NOTE: In this procedure, patch-clamp recordings from the isolated ganglion are typically performed 12-24 h after the plating. Other labs have ...

Place a culture of inner ear sensory neuron cell bodies in a recording chamber.
Front-fill a patch-clamp pipette with an internal solution.
Backfill with the same solution containing a membrane-perforating antibiotic, allowing it to gradually reach the tip.
Fill two-thirds of the pipette with the antibiotic solution, insert it into a holder containing a recording electrode, and lower it into the chamber.
Visualize under a microscope to approach a neuron.
As the pipette tip contacts the neuron, the neuronal surface forms a dimple, and the resistance increases.
Apply negative pressure to form a tight seal between the pipette and the membrane.
Maintain the holding potential close to the resting membrane potential.
The antibiotic perforates the membrane, allowing ion movement between the cell and electrode to establish a stable ionic environment.
Apply voltage steps to open voltage-gated ion channels, allowing ion influx and generating currents proportional to the ion channel function, which are recorded by the electrode.

16 조회수. 내이 감각 뉴런 세포체의 천공된 패치 클램프 기록

비디오: 내이 감각 뉴런 세포체의 천공된 패치 클램프 기록 - 실험

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

성인 쥐에서 본래 등쪽 뿌리 신경절에 패치 클램프 녹음

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

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생화학

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

본래 Neuroepithelium에서 마우스 강한 감각 뉴런의 천공 패치 클램프 녹음 : 확인 된 부 취제 수용체를 발현 뉴런의 기능 분석

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

신경과학

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ion channels, voltage-gated potassium (Kv) channels are closely associated with some serious heart diseases, such as drug-induced myocardial damage and myocardial infarction. In the present study, the whole-cell patch-clamp technique was employed to determine the effects of 1.5 mM 4-aminopyridine (4-AP, a broad-spectrum potassium channel inhibitor) and aconitine (AC, 25 &#181;M, 50 &#181;M, 100 &#181;M, and 200 &#181;M) on the Kv channel current (IKv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the IKv by about 54%, while the inhibitory effect of AC on the IKv showed a dose-dependent trend (no effect for 25 &#181;M, 30% inhibitory rate for 50 &#181;M, 46% inhibitory rate for 100 &#181;M and 54% inhibitory rate for 200 &#181;M). Due to the characteristics of higher sensitivity and precision, this technique will promote the exploration of cardiotoxicity and the pharmacological effects of ethnomedicine targeting ion channels.

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

The present protocol describes an efficient method for the real-time and dynamic acquisition of voltage-gated potassium (Kv) channel currents in H9c2 cardiomyocytes using the whole-cell patch-clamp technique.

전체 세포 패치 클램프 기술을 통한 H9C2 심근 세포에 전압 의존적 칼륨 전류 기록

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ...

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

내레이션 대본

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies