external excitation of one-dimensional patterned neuronal cultures

External Excitation of One-Dimensional Patterned Neuronal Cultures

Prior to an electric stimulation, place the seated coverslip into an imaging chamber. To stimulate the cells with an electric field of a constant direction, use two parallel electrodes separated by 11 millimeters and located at opposite sides of the culture plated on the 13-millimeter coverslips. Then, assemble the culture imaging chamber and matching electrode holder such that the wire electrodes are immersed in the extracellular media and positioned one millimeter above the culture.
Then, plug the single electrode pair to the operational amplifier that amplifies the signal from the generator. Apply a single pulse of bipolar square wave with a 50% duty cycle and amplitude of 22 volts. Use a bipolar wave with no DC component to avoid electrolysis that may occur at the electrodes. Apply the pulse lasting from 10 microseconds to four milliseconds to ensure effective stimulation, but prevent cell injury.

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

Universidad de Cartagena UDEC

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JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Stimulation and Modulation Techniques

EoE Sub Articles

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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>

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...

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.

19 次观看. 一维模式神经元培养物的外部激发

视频: 一维模式神经元培养物的外部激发 - 实验

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.

用于循环作用电位记录的多井微电极阵列上的人类 iPSC 衍生心肌细胞网络

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

科研

JoVE Journal

生物工程

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.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

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.

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

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

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External Excitation of One-Dimensional Patterned Neuronal Cultures