Name: Video: Excitación externa de cultivos neuronales con patrones unidimensionales - Concepto
Uploaded: 2024-10-31T13:46:39.000+00:00
Duration: 1 min 26 s
Description: 25 vistas. Fuente:Stern, S., et al. Excitación externa de neuronas utilizando campos eléctricos y magnéticos en cultivos unidimensionales y bidimensionales. J. Vis. Exp. (2017) Este video muestra una técnica para estimular cultivos neuronales con patrones de líneas con un campo eléctrico uniforme y unidireccional.

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1. Imaging of spontaneous or evoked activity in neuronal cultures with fluorescent dyes.
<ol>
	<li>Prepare a solution of 50 &#181;g calcium sensitive fluorescent dye (see Table of Materials/Reagents) in 50 &#181;L DMSO (dimethyl sulfoxide).</li>
	<li>Prepare extracellular recording solution (EM) containing (in mM) 10 HEPES, 4 KCl, 2 CaCl2, 1 MgCl2, 139 NaCl, 10 D-glucose, 45 sucrose (pH 7.4).</li>
	<li>Incubate neuronal culture in 2 mL EM with 8 &#181;L of the calcium-sensitive fluorescent dye solution for 1 h. Protect from light and gently rotate to ensure a homogenous spread of the dye to the cells.</li>
	<li>Replace the solution with fresh EM prior to imaging. The fluorescent imaging is demonstrated in Figure 1.</li>
	<li>Image in a fluorescence microscope with optical filters for calcium fluorescence imaging (excitation peak at 488 nm, emission peak at 520 nm), using a camera and software capable of quantifying the intensity of any region of interest (ROI) within the field of view of the microscope.</li>
</ol>
2. Electric Stimulation of Cultures
NOTE: The basic setup for electric stimulation is shown in Figure 2. A cover slip on which the neuronal culture has been grown for about 14 days is placed in a Petri dish under a fluorescence microscope. Electrical activity of the neurons is imaged using calcium sensitive dyes while a voltage is applied via two pairs of bath electrodes that are positioned outside the culture. The electrodes are driven by a signal generator whose output is amplified by a dual channel amplifier. Voltage control for stimulation is preferred over the more standard current control, because the electric field vectors are determined directly, thus enabling straightforward vector addition and combination. This does require a careful check of the uniformity of the electric field, which can be performed over the whole sample for the case of voltage control. When using voltage control care should be taken to avoid any ground loops and the homogeneity of the electric field should be verified.
<ol>
	<li>For electric stimulation with a homogeneous electric field use a pair of parallel electrode wires.
	<ol>
		<li>Use electrodes made of platinum with a thickness on the order of 0.005&#39;&#39; (127 &#181;m). When used with the 13 mm coverslips, ensure that the distance between the two electrodes is around 11 mm, and position the electrodes 1 mm above the culture. 
		&#8203;NOTE: To make the electrode holder (Figures 3A), use polytetrafluoroethylene (PTFE). Drill narrow holes through the PTFE to insert the electrodes. The device should be higher than the extracellular solution so that the top end, where the electrodes are exposed, will never come in contact with the solution. For insulation, use epoxy glue on any part of the electrode leads that might be exposed.</li>
		<li>Use a square pulse shape with a 50% duty cycle, with no DC component to avoid electrolysis. Vary pulse duration between 10 &#181;s and 4 ms to cause effective stimulation without burning the culture. Ensure that the amplitude is in the order of &#177; 22 V (see Figure 3). The square pulse can be observed on an oscilloscope connected in parallel to the electrodes. 
		NOTE: For easy programming of any desired waveform, use a commercial waveform editing software (see Materials list). Enter graphically the desired waveform and send it to the waveform generator.</li>
	</ol>
	</li>
	<li>To test for field homogeneity use a probe electrode. Use a grid of at least 1 mm x 1 mm to allow the probe to be moved in the area between electrodes and measure electric potential.
	<ol>
		<li>Measure electric potential. Use <img alt="Equation 1" src="/files/ftp_upload/22715/22715eq01.jpg" style="margin: auto;" /> to calculate the electric field. Use one of the electrodes as a reference electrode. Measure the electric field with varying pulse durations between 100 &#181;s to 4 ms (see Figure 3B for an example of 100 &#181;s pulse duration) to verify that the field is homogeneous within the range of stimulating durations. 
		NOTE: See Figure 3D for an example of a measured electric field homogeneity when the pulse duration was 1 ms.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcium chloride , 1M</td>
			<td>Fluka</td>
			<td>21098</td>
			<td>Extracellular recording solution.</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>65146</td>
			<td>Plating medium, Extracellular recording solution.&#160;</td>
		</tr>
		<tr>
			<td>Hepes, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>Extracellular recording solution.</td>
		</tr>
		<tr>
			<td>KCl, 3M</td>
			<td>Merck</td>
			<td>1049361000</td>
			<td>Extracellular recording solution.&#160;</td>
		</tr>
		<tr>
			<td>Magnesium chloride , 1M</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Extracellular recording solution.&#160;</td>
		</tr>
		<tr>
			<td>NaCl, 4M</td>
			<td>Bio-Lab</td>
			<td>19030591</td>
			<td>Extracellular recording solution .&#160;</td>
		</tr>
		<tr>
			<td>Sucrose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>S1888</td>
			<td>Extracellular recording solution.</td>
		</tr>
		<tr>
			<td>Electrodes wires</td>
			<td>A-M Systems, Carlsborg WA</td>
			<td>767000</td>
			<td>Electric stimulation of neuronal cultures.&#160;</td>
		</tr>
		<tr>
			<td>Signal generator</td>
			<td>BKPrecision</td>
			<td>4079</td>
			<td>Shaping of the electric signal.&#160;</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Homemade</td>
			<td></td>
			<td>Voltage amplification of the signal from the signal generator to the electrodes.&#160;</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Matrix</td>
			<td>MPS-3005 LK-3</td>
			<td>Power supply to the sputtering machine.&#160;</td>
		</tr>
		<tr>
			<td>Platinum wires 0.005&#39;&#39; thick; A-M Systems,</td>
			<td>Carlsborg WA</td>
			<td>767000</td>
			<td>Electric stimulation of neuronal cultures.&#160;</td>
		</tr>
	</tbody>
</table>

external excitation of one-dimensional patterned neuronal cultures

Source:Stern, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a technique for stimulating line-patterned neuronal cultures with a uniform, unidirectional electric field.

<img alt="Figure 1" src="/files/ftp_upload/22715/22715_Figure1.jpg" /> 
Figure 1: Example Traces of Calcium Transients Imaged During Synchronous Network Bursts. A. An image of neurons that were dyed previous to the experiment with a calcium dye. B. Traces of intensity vs. time of the ROIs in A with the color of the trace representing the color of the border of the ROI in A. A large increase in intensity synchronized within the three ROIs represents a...

External Excitation of One-Dimensional Patterned Neuronal Cultures

1. Imaging of spontaneous or evoked activity in neuronal cultures with fluorescent dyes.

	Prepare a solution of 50 &#181;g calcium sensitive fluorescent ...

Take a coverslip containing a 1D patterned neuronal culture.
The neurons are loaded with a calcium indicator dye, which fluoresces upon calcium binding.
Transfer the coverslip to a fluorescence microscope imaging chamber containing an extracellular recording solution.
Assemble a holder with two parallel wire electrodes separated by a distance.
Invert the holder and position it above the neuronal culture with the electrodes immersed in the recording solution.
Connect the electrodes to an amplifier. Apply a non-DC pulsed bipolar square wave to generate a uniform uni-directional electric field across the neurons without causing injury.
The generated electric field excites the neurons, triggering an action potential.
The action potential travels along the axon, activating the voltage-gated calcium channels and causing calcium influx into the neuron terminal. Further, neurotransmitters are released into the synapse, facilitating synaptic transmission.
Following calcium ion influx, these ions bind to the intracellular dye and fluoresce, indicating neuronal activity in response to electrical stimulation.

external-excitation-of-one-dimensional-patterned-neuronal-cultures

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Stimulation and Modulation Techniques

25 vistas. Fuente:Stern, S., et al. Excitación externa de neuronas utilizando campos eléctricos y magnéticos en cultivos unidimensionales y bidimensionales. J. Vis. Exp. (2017) Este video muestra una técnica para estimular cultivos neuronales con patrones de líneas con un campo eléctrico uniforme y unidireccional. 

Video: Excitación externa de cultivos neuronales con patrones unidimensionales - Concepto

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

Redes de cardiomiocitos derivados de iPSC humanos en matrices de microelectrodos multipolos para grabaciones potenciales de acción recurrente

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Investigación

JoVE Journal

Bioingeniería

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Medición del potencial de acción óptica unicelular en cardiomiocitos derivados de células madre pluripotentes inducidas por humanos

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

En Vivo Medición electrofisiológica de potencial de acción compuesto del músculo de las extremidades anteriores en modelos de ratón de la degeneración de la motoneurona

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

External Excitation of One-Dimensional Patterned Neuronal Cultures

Transcripción

External Excitation of One-Dimensional Patterned Neuronal Cultures