automated image-guided patch clamp technique for studying neurons in brain slices

Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices

In this step, place one brain slice in the center of the recording chamber. Stabilize the brain slice with a slice holdown or a harp. To detect the fluorescent cell, find the area of interest under the four times magnification. Then, move the microscope stage by turning on &#34;click-to-move&#34; mode, and click the center of the area of interest. Next, switch to the high magnification lens and adjust the focus by moving the microscope in the z-axis using RF on the keypad.
The software directs the microscope in the camera to take a series of images at different depths. Then, these images are subjected to computer vision processing to find fluorescently labeled cells. Click the Detect Cell button on the main column, unit zero. A list of cell coordinates will appear in the memory position&#39;s GUI. Remove undesired cells from the list by clicking the X button next to the coordinates.
Then, click on the cell of interest. A yellow dot with a number will appear on the cell, and the coordinates of the cell will appear in the memory position&#39;s GUI. To perform secondary calibration in order to detect pipette offset coordinates, fill one-third of a glass pipette with internal solution. Then, load the pipette onto the pipette holder attached to the head stage.
At low magnification, use one and two on the keypad to switch between unit one and unit two. Then, bring the pipette into the visual field and adjust the focus using the keypad.
Next, load the primary calibration by clicking on Load Calibration. Switch the microscope lens to high magnification, and click 40 times on the main GUI. Bring the pipette tip to the center. Then, click the secondary calibration button on the main GUI under the unit that is in use. To patch a target cell, click on the Patch Control button to open the patch control GUI. Click the Go To button next to the cell of interest on the coordinate list in the memory position GUI.
Subsequently, click on the CTM button of the unit in the main GUI to enable movement. Click on the cell of interest to move the pipette tip to the cell.
Next, use the Unit 1 Selected button to switch the input signal between the two units. Click on the Patch button on the patch control GUI. Automatic patching will begin, and the pressure and resistance can be monitored on the patch control GUI.
The automatic patching algorithm monitors resistance and controls pressure through a series of logic loops to form a gigaseal. A pop-up window notifies the formation of a gigaseal.
The system utilizes resistance change to recognize cell surface contact. In a situation that the cell surface contact is not detected in time, use the Next button to advance patching stage while remaining in the same automatic patching trial.
Manipulate the automatic process at any point by clicking on the respective buttons on the patch control GUI. Then, select Yes to break in with combined zap and suction. Then, save the experiment patch log.

automated-image-guided-patch-clamp-technique-for-studying-neurons

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

EoE Sub Articles

eoe sub articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. System Setup
<ol>
	<li>Construct the pressure control unit.
	<ol>
		<li>Assemble the pressure control unit according to the circuit map (Figure 1). Solder the necessary parts onto the Printed Circuit Board (PCB) manufactured according to the electrical circuit schematics (Figure 1b). Use standard resistors, LEDs(Light-emitting diodes), Metal-Oxide Semiconductor field-effect transistors (MOSFETs), capacitors, and connectors. Solder solenoid valves onto the PCB (Printed circuit board). Connect the air pump and air pressure sensor to the PCB with an electrical wire. 
		&#8203;NOTE: After all necessary parts are available, the pressure control unit should be constructed in about 2 hours.</li>
	</ol>
	</li>
	<li>Connect the secondary data acquisition (DAQ) board.
	<ol>
		<li>Connect data outputs from the printed circuit board to the DAQ board, following Table 1. 
		NOTE: The DAQ board will run in &#34;Single-ended mode.&#34; The port map is in the user manual (see the Table of Materials).</li>
		<li>Connect &#34;AIn Pr S&#34; to one of the analog input (AI) channels and &#34;R-Gr&#34; to one of the analog grounds on the secondary DAQ board.</li>
		<li>Connect the primary output from the amplifier to one of the AI channels and the ground to the analog ground of the secondary DAQ board. 
		NOTE: A standard BNC cable can be used to connect the primary output from the amplifier.</li>
		<li>Strip the other end and connect the positive signal (i.e. copper core) to the AI channel and the ground (i.e. the thin wire around the core) to the analog ground. Repeat this step for a second channel if more than one patch channel is used. 
		NOTE: The analog input to the DAQ board will be configured in later steps.</li>
		<li>Connect power to the power output of the secondary DAQ board. Use a separate 12 V power source for the pump.</li>
	</ol>
	</li>
	<li>Connect the tubing.
	<ol>
		<li>Connect the air pump and the two valves.</li>
		<li>In the last step, use a 3-way connector to connect the soft tubing from the valve 2 top port to the pressure sensor and the pipette holder.</li>
		<li>If two pipettes are used, add another 3-way connector to the tubing connected to the pipette holder. When patching, manually switch between the valves and the pipettes in use.</li>
	</ol>
	</li>
	<li>Install Autopatcher IG. 
	NOTE: System requirement: Autopatcher IG was only tested on a PC running Windows 7. It has not been validated for other operating systems. The described procedure applies specifically to the hardware listed in the Table of Materials.
	<ol>
		<li>Download Autopatcher-IG from GitHub (https://github.com/chubykin/AutoPatcher_IG).</li>
		<li>Install Python (see the Table of Materials for the version and download address).</li>
		<li>Uninstall the PyQt4 library by typing &#34;pip uninstall PyQt4&#34; in a command line terminal. 
		NOTE: The system uses an older version of the PyQt4 library to achieve compatibility with the Qwt and Opencv libraries.</li>
		<li>Install Python libraries from historic wheel files (http://www.lfd.uci.edu/~gohlke/pythonlibs/). Find the following files: Numpy (pymc-2.3.6-cp27-cp27m-win32.whl), Opencv (opencv_python-2.4.13.2-cp27-cp27m-win32.whl), Pyqt (PyQt4-4.11.4-cp27-none-win32.whl), and Qwt (PyQwt-5.2.1-cp27-none-win32.whl).
		<ol>
			<li>To install the wheel files, go to the directory where the files are saved and type &#34;pip install ***wheelfilename***.whl.&#34; Substitute &#34;***wheelfilename***&#34; with the actual name of the file. 
			NOTE: &#34;cp27&#34; in the wheel file name indicates Python 2.7, and &#34;win32&#34; indicated Windows 32-bit. If &#34;win32&#34; does not work, try &#34;win64.&#34;</li>
		</ol>
		</li>
		<li>To control the CCD (charge-coupled device) camera, download and install the installer for 64-bit (https://www.qimaging.com/support/software/). Then download MicroManager for 64-bit (https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release) to control the camera in Python.</li>
		<li>To control the manipulators and the microscope stage, install control software provided by the manufacturer. 
		NOTE: By doing this, the driver necessary to control the manipulators is also installed. The installation package is commonly provided in a CD-ROM.</li>
		<li>To control the secondary DAQ board, install the Universal Library from CD-ROM, provided with the purchase of the DAQ board.</li>
	</ol>
	</li>
	<li>Configure the hardware for Autopatcher IG.
	<ol>
		<li>Connect the microscope stage and manipulator controllers to the computer via USB ports.</li>
		<li>Assign COM port numbers to unit 0: microscope stage, unit 1: left manipulator, and unit 2: right manipulator, in this order, in the &#34;ports.csv&#34; configuration file in the &#34;configuration&#34; folder. Leave the other parameters in the ports.csv file (i.e. &#34;SCI&#34; and &#34;1&#34;) unchanged. 
		NOTE: The COM port number information can be found by running the manipulator configuration software provided by the manufacturer. Go to the &#34;settings&#34; tab, select &#34;settings&#34; and the &#34;Motion&#34; page, and read the labels for each tab at the top. Alternatively, this information can be found in the PC Device Manager.</li>
		<li>Assign analog input channel numbers on the DAQ board for a pressure sensor and patch channel 1 and 2 (corresponding to unit 1 and 2). Enter the channel number in the &#34;DAQchannels.csv&#34; file in the &#34;configuration&#34; folder. 
		NOTE: It is recommended to open the .csv files with the Notepad application instead of a spreadsheet, as it may alter the information when saving changes.</li>
	</ol>
	</li>
	<li>Run Autopatcher IG.
	<ol>
		<li>Turn on the amplifier, microscope controller, and manipulator controller. Ensure that the amplifier software is running.</li>
		<li>Run Autopatcher IG with Python from a command line terminal as follows: first, change the directory (command &#34;cd&#34; for most common terminals) where Autopatcher IG is installed, type &#34;python Autopatcher_IG.pyw&#34; in the command line terminal, and hit the &#34;Enter&#34; key. 
		NOTE: Do not run the manipulator control software before running Autopatcher IG because it will occupy the microscope stage and manipulator, causing Autopatcher IG to be unable to find the hardware. Manipulator control software can be run after Autopatcher IG is fully initiated if there are additional modules to be controlled (e.g., the inline heater).</li>
	</ol>
	</li>
	<li>Calibrate the primary pipette.
	<ol>
		<li>Pull patch pipettes. Fill a pulled glass pipette with an internal solution and load it onto the head stage. 
		&#8203;NOTE: Empty glass pipettes have different contrasts under the microscope and may lead to inaccurate calibration.</li>
		<li>Move the pipette tip to the microscope visual field and bring it into focus. If the dial pad is used to move the manipulators and/or microscope stage, update the coordinates by pressing &#34;z&#34; on the keyboard. 
		NOTE: This action is not necessary if the keyboard (microscope stage: A/D - x-axis, W/S - y-axis, R/F - z-axis; manipulators: H/K - x-axis, U/J - y-axis, O/L - z-axis, 1/2 - unit number) is used to control movement because the coordinates will be updated in real-time.</li>
		<li>Click the &#34;Start calibration&#34; button on the main Graphic User Interface (GUI) for the corresponding unit on which the pipette is loaded (Figure 2). 
		NOTE: A pop-up window will appear when the calibration is finished. 
		NOTE: Calibration will be carried out automatically, which will take about 3.5 min. Clicking on the same button (switched now to &#34;cancel calibration&#34; after initiating calibration) will abort the calibration attempt.</li>
		<li>Save the calibration by clicking &#34;save calibration&#34; at the bottom of the main GUI (it saves the current calibration for both manipulators and can be loaded in the future). 
		NOTE: The field of view under low (4 or 10X) and high (40X) magnification must be aligned for secondary calibration to function properly. Please refer to the user manual of the optical system in use for the alignment procedures. 
		&#160;</li>
	</ol>
	</li>
</ol>
2. Automatic Patch Clamp Procedure
<ol>
	<li>Prepare acute brain slices</li>
	<li>Prepare glass pipettes for the patch clamp.</li>
	<li>Place one brain slice in the center of the recording chamber. Stabilize the slice with a slice hold-down or &#34;harp.&#34;</li>
	<li>Detect the fluorescent cell.
	<ol>
		<li>Find the area of interest under the 4X lens. Move the microscope stage by turning on click-to-move (&#34;CTM&#34;) mode and clicking the center of the area of interest. Alternatively, use the keypad to move the microscope stage (A/D - x-axis, W/S - y-axis, R/F - z-axis).</li>
		<li>Switch to the high-magnification lens and adjust the focus by moving the microscope in the z-axis, using R/F on the keypad. 
		NOTE: It is recommended to adjust the water bath level so that the focal plane under the low- and high-magnification lenses are the same or similar in the z-axis.</li>
		<li>Click the &#34;Detect Cell&#34; button on the main GUI column, &#34;Unit 0.&#34; If the LED or laser light source of the setup cannot be controlled by the TTL signal, manually turn on the LED/laser; a pop-up window will appear when the cell detection is finished.
		<ol>
			<li>Turn off the LED/laser if necessary; a list of cell coordinates will appear in the &#34;Memory positions&#34; GUI. Remove undesired cells from the list by clicking the &#34;X&#34; button next to the coordinates.</li>
			<li>Alternatively, if target cells are not fluorescently labeled, click &#34;Mouse mode&#34; on the main GUI. Click on the cell of interest; a yellow dot with a number will appear on the cell, and the coordinates of the cell will appear in the &#34;Memory positions&#34; GUI.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Calibrate the secondary pipette.
	<ol>
		<li>Fill 1/3 of a glass pipette with internal solution. Load the pipette onto the pipette holder attached to the head stage.</li>
		<li>Use the low-magnification lens. Bring the pipette into the visual field and adjust the focus using the keypad (H/K - x-axis, U/J - y-axis, O/L - z-axis). Use &#34;1&#34; and &#34;2&#34; to switch between unit 1 and unit 2.</li>
		<li>Load the primary calibration by clicking on &#34;Load calibration.&#34; Click the &#34;Secondary calibration&#34; button on the main GUI under the unit that is in use. For example, if unit 2 is in use, click the &#34;Secondary calibration&#34; button in the &#34;Unit 2&#34; column. Follow the pop-up window instructions to switch to the high-magnification lens.</li>
	</ol>
	</li>
	<li>Patch a target cell.
	<ol>
		<li>Make sure that the &#34;MultiClamp&#34; (i.e. amplifier) software is running. Click on the &#34;Patch control&#34; button to open the &#34;Patch control&#34; GUI; it may take up to a few min to open this GUI.</li>
		<li>Use the 40X magnification lens by checking &#34;40X&#34; on the main GUI &#34;Unit 0&#34; column. Click the &#34;go to&#34; button next to the cell of interest on the coordinate list in the &#34;Memory position&#34; GUI; the microscope will move to the cell.</li>
		<li>Click on the CTM button of the unit in use in the main GUI to enable movement following a mouse click. Click on the cell of interest; the pipette tip will move to the cell.</li>
		<li>Click on the &#34;Patch&#34; button on the &#34;Patch control&#34; GUI. 
		NOTE: Automatic patching will begin, and the pressure and resistance can be monitored on the &#34;Patch control&#34; GUI.
		<ol>
			<li>Use the &#34;Unit 1 selected&#34; button to switch the input signal between the two units. 
			&#8203;NOTE: The system will approach the target cell, apply negative pressure, match the cell membrane potential, and detect gigaseal formation based on a series of pressure and resistance thresholds and logic.</li>
			<li>Manipulate the automatic process at any point by clicking on the respective buttons on the &#34;Patch control&#34; GUI. For example, click on the &#34;Patch&#34; button again to cancel the patch trial and click on &#34;Next stage&#34; to advance the patching process to the next step, regardless of the threshold. 
			NOTE: A pop-up window will notify the user when a gigaseal has formed, and the option to apply zap along with large negative pressure will be presented.</li>
		</ol>
		</li>
		<li>Select &#34;Yes&#34; to break in with combined zap and suction. Alternatively, select &#34;No&#34; to break in with suction only. 
		NOTE: When a successful whole-cell patch is completed, a pop-up window will remind the user to save the experiment patch log.</li>
		<li>Save the experiment patch log. 
		NOTE: If a patching trial is unsuccessful, a pop-up window will notify the user, and the patch process will reset.</li>
		<li>Go back to step 2.4 and repeat the steps with a different cell.</li>
	</ol>
	</li>
</ol>

No conflicts of interest declared.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CCD Camera</td>
			<td>QImaging</td>
			<td>Rolera Bolt</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophysiology rig</td>
			<td>Scientifica</td>
			<td>SliceScope Pro 2000</td>
			<td>Include microscope and manipulators. The manufacturer provided manipulator control software demonstrated in this manuscript is &#34;Linlab2&#34;.</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices</td>
			<td>MultiClamp 700B</td>
			<td>computer-controlled microelectrode amplifier</td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Molecular Devices</td>
			<td>Axon Digidata 1550</td>
			<td></td>
		</tr>
		<tr>
			<td>LED light source</td>
			<td>Cool LED</td>
			<td>pE-100</td>
			<td>488nm wavelength</td>
		</tr>
		<tr>
			<td>Data acquisition board</td>
			<td>Measurement Computing</td>
			<td>USB1208-FS</td>
			<td>Secondary DAQ. 
			See manual at : http://www.mccdaq.com/pdfs/manuals/USB-1208FS.pdf</td>
		</tr>
		<tr>
			<td>Solenoid valves</td>
			<td>The Lee Co.</td>
			<td>LHDA0531115H</td>
			<td></td>
		</tr>
		<tr>
			<td>Air pump</td>
			<td>Virtual industry</td>
			<td>VMP1625MX-12-90-CH</td>
			<td></td>
		</tr>
		<tr>
			<td>Air pressure sensor</td>
			<td>Freescale semiconductor</td>
			<td>MPXV7025G</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice hold-down</td>
			<td>Warner instruments</td>
			<td>64-1415 (SHD-40/2)</td>
			<td>Slice Anchor Kit, Flat for RC-40 Chamber, 2.0 mm, 19.7 mm</td>
		</tr>
		<tr>
			<td>Python</td>
			<td>Anaconda</td>
			<td>version 2.7 (32-bit for windows)</td>
			<td>https://www.continuum.io/downloads</td>
		</tr>
		<tr>
			<td>Screw Terminals (2-Pin)</td>
			<td>Sparkfun</td>
			<td>PRT - 08084</td>
			<td>Screw Terminals 3.5mm Pitch (2-Pin)</td>
		</tr>
		<tr>
			<td>N-Channel MOSFET 60V 30A</td>
			<td>Sparkfun</td>
			<td>COM - 10213</td>
			<td></td>
		</tr>
		<tr>
			<td>DIP Sockets Solder Tail - 8-Pin</td>
			<td>Sparkfun</td>
			<td>PRT-07937</td>
			<td></td>
		</tr>
		<tr>
			<td>LED - Basic Red 5mm</td>
			<td>Sparkfun</td>
			<td>COM-09590</td>
			<td></td>
		</tr>
		<tr>
			<td>LED - Basic Green 5mm</td>
			<td>Sparkfun</td>
			<td>COM-09592</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Barrel Power Jack/Connector (SMD)</td>
			<td>Sparkfun</td>
			<td>PRT-12748</td>
			<td></td>
		</tr>
		<tr>
			<td>Wall Adapter Power Supply - 12VDC 600mA</td>
			<td>Sparkfun</td>
			<td>TOL-09442</td>
			<td></td>
		</tr>
		<tr>
			<td>Hook-Up Wire - Assortment (Solid Core, 22 AWG)</td>
			<td>Sparkfun</td>
			<td>PRT-11367</td>
			<td></td>
		</tr>
		<tr>
			<td>Locking Male x Female X Female Stopcock</td>
			<td>ARK-PLAS</td>
			<td>RCX10-GP0</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Tygon S3 E-3603 Flexible Tubings</td>
			<td>Fisher scientific</td>
			<td>14-171-129</td>
			<td>Outer Diameter: 1/8 in. 
			Inner Diameter: 1/16 in.</td>
		</tr>
		<tr>
			<td>BNC male to BNC male coaxial cable</td>
			<td>Belkin Components</td>
			<td>F3K101-06-E</td>
			<td></td>
		</tr>
		<tr>
			<td>560 Ohm Resistor (5% tolerance)</td>
			<td>Radioshack</td>
			<td>2711116</td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer</td>
			<td>General Valve</td>
			<td>Picospritzer II</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Wu, Q., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56010">Application of automated image-guided patch clamp for the study of neurons in brain slices.</a> J. Vis. Exp. (2017).
In this video, brain slices are immobilized in a recording chamber, and cells of interest are located and focused on using a computer-controlled microscope. The automated image processing system then precisely positions a patch pipette to seal the cell membrane and monitor electrical signals.

Table 1. Printed Circuit Board (PCB) to secondary data acquisition (DAQ) board connection configuration. Use this table to connect PCB outputs (first column from left) to ports on the DAQ board (second column from left). The port name and number on the secondary DAQ refer to single-ended mode.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Outlet on the PCB</td>
			<td>Port name on the DAQ board</td>
			<td>Port # on DAQ board</td>
			<td>Remark</td>
		</tr>
		<tr>
			<td>DOUT V1</td>
			<td>Port A channel 1</td>
			<td>22</td>
			<td>Control valve 1</td>
		</tr>
		<tr>
			<td>DOUT V2</td>
			<td>Port A channel 2</td>
			<td>23</td>
			<td>Control valve 2</td>
		</tr>
		<tr>
			<td>DOUT P</td>
			<td>Port A channel 3</td>
			<td>24</td>
			<td>Control air pump</td>
		</tr>
		<tr>
			<td>Gr</td>
			<td>Ground</td>
			<td>29</td>
			<td>Ground</td>
		</tr>
	</tbody>
</table>
<img alt="Figure 1" src="/files/ftp_upload/22838/22838_Figure1.jpg" /> 
Figure 1: Pressure Control Unit. A: Printed Circuit Board (PCB) for connecting the valves, pressure sensor, and air pump. The left shows details on the PCB, labeling locations of outputs that are mentioned in the protocol. The right shows the connection between the PCB and the air pump, USB port, and tubing. B: Circuit map for the PCB.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. System Setup

	Construct ...

Begin with an immobilized brain slice in a recording chamber.
Using the camera-controlled software of a microscope, focus on the target area
Turn on the excitation light and take multiple pictures at different depths.
The software detects the coordinates of fluorescently labeled cells.
Now, position the solution-filled patch pipette containing the recording electrode into the chamber.
Use suitable software to calibrate the position controls.
Under high magnification, recalibrate the controls to precisely target the fluorescently labeled cell.
Select the coordinates of the target cell.
Start the automatic lowering of the patch pipette.
The computer monitors the resistance via the electrode. Apply a negative pressur, an increase in resistance due to the pipette&#39;s contact with the cell membrane indicates gigaseal formation.
Following this, the software further decreases the pressure to puncture the cell membrane and obtain a whole-cell patch configuration.

18 Views. Tecnica automatizzata di patch clamp guidata da immagini per lo studio dei neuroni nelle fette di cervello

Video: Tecnica automatizzata di patch clamp guidata da immagini per lo studio dei neuroni nelle fette di cervello - Experiment

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Analisi elettrofisiologica dei cardiomiociti derivanti da cellule staminali pluripotenti umane (hPSC-CMs) Utilizzando matrici multi-elettrodo (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

Ricerca

JoVE Journal

Biologia dello sviluppo

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Applicazione di morsetto di patch automatico guidato per immagini per lo studio dei neuroni in fettine di cervello

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Neuroscienze

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Applicazioni tecniche di microelectrode array e patch clamp recordings su cardiomiociti derivati da cellule staminali pluripotenti indotte umane

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices

Trascrizione

Automated Image-Guided Patch Clamp Technique for Studying Neurons in Brain Slices