eoe sub articles

EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Electrophysiological Recordings to Investigate Spontaneous and Electrode Stimulation Dependent Activity
<ol>
	<li>Wash the multielectrode array (MEA) culture with extracellular solution, at room temperature (RT).</li>
	<li>Dry the contacts with a tissue and mount the MEA on the MEA setup. 
	NOTE: To keep the culture humid during the mounting, add a small drop of extracellular solution to the culture.</li>
	<li>Add 300 &#181;l of extracellular solution and wait for 10 min before recording, to allow the system to stabilize.</li>
	<li>Record spontaneous activity for 2 min from all electrodes by pressing on the record/acquire button of the software and identify recording electrodes.</li>
	<li>MEA electrode stimulation: Select the amplitude/duration/shape of the stimulus on the appropriate software and apply it to several electrodes consecutively. Select the electrodes based on the ones showing spontaneous activity (as in step 1.4). Record from all the remaining electrodes.</li>
	<li>To exclude stimulation artifact, stimulate from the same electrode 10 times. If the culture responds at least 8 out of 10 times, it can be assumed as a positive response upon electrode induced stimulation.</li>
	<li>To identify background noise, apply Tetrodotoxin (TTX) to the culture at a concentration of 1 &#181;M, to block voltage-gated sodium channels and record for 2 min. Use this to perform spike detection (2.1 and 2.2).</li>
</ol>
2. Data analysis
<ol>
	<li>Using the appropriate software detect spontaneous activity for each electrode employing a detector based on standard deviations and a subsequent discriminator. Activity appears as fast voltage transients (&#60;5 msec).</li>
	<li>Choose a threshold that results in no activity when analyzing the TTX treated samples. Adapt the threshold value for each experiment to discriminate between false positive and false negative detections.</li>
	<li>Using appropriate software observe the detected neuronal activity of each electrode as a raster plot according to standard procedures (see Figure 1A-C).</li>
	<li>Determine and display the total network activity by summing up all detected events within a sliding window of 10 msec, shifted by 1 msec steps.</li>
	<li>Detect stimulation-induced activity by displaying the raw data using appropriate analysis software. Identify the single spikes offline manually. Single spikes appear as fast voltage transients, occurring after the stimulation artifact (arrowhead in Figure 1E). An example is shown in Figure 1E.</li>
	<li>Depending on the experiment, analyze the number of responding electrodes, the threshold needed to achieve a response, number of action potential per electrodes and other parameters of interest.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Extracellular solution (pH 7.4)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>145 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>4 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Na-pyruvate</td>
			<td></td>
			<td></td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEA electrodes</td>
			<td>Qwane Biosciences</td>
			<td></td>
			<td>(Lausanne, Switzerland)</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td></td>
			<td>Switzerland</td>
		</tr>
		<tr>
			<td>IgorPro</td>
			<td>WaveMetrics</td>
			<td></td>
			<td>Lake Oswega, USA</td>
		</tr>
	</tbody>
</table>

analyzing neural activity in primary murine spiral ganglion neurons using multielectrode arrays

Source: Hahnewald, S., et al. <a href="https://app.jove.com/v/54538">Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays.</a> J. Vis. Exp. (2016).
This video demonstrates the recording of spontaneous and stimulation-dependent neural activity from primary murine spiral ganglion neuron (SGN) cultures on a multielectrode array (MEA). It involves washing and preparing the MEA, recording spontaneous neural activity, and applying controlled electrical stimuli to identify stimulation-dependent neuronal activity.

<img alt="Figure 1" src="/files/ftp_upload/22706/22706_Figure1.jpg" /> 
Figure 1. Data recordings on MEA. (A) Traces of original recordings of six out of 63 electrodes showing spontaneous activity. (B) Raster plot of the six electrodes of Figure 1A after spike detection. Each bar represents one action potential. (C) Raster plot including all electrodes (as in A and B...

Analyzing Neural Activity in Primary Murine Spiral Ganglion Neurons Using Multielectrode Arrays

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

Begin with a spiral ganglion neuron culture on a multielectrode array, or MEA.
The MEA consists of recording electrodes connected to contacts via a circuit.
Wash the MEA to remove media and dry the contacts to avoid moisture-induced noise during recording.
Mount the MEA on its holder and install it in the recording setup.
The MEA contacts align with the holder connectors that transmit electrical signals to the recording setup.
Add an extracellular environment-mimicking solution, allowing normal physiological activity in the neurons.
Begin recording. The electrodes detect spontaneous signals or action potentials generated by the neurons, visualized as spikes on the software interface.
Now, apply an electrical pulse to one electrode exhibiting spontaneous activity.
The pulse generates an electric field, stimulating the neurons on neighboring electrodes.
Record the stimulation-dependent activity.
Finally, add a neurotoxin that blocks voltage-gated ion channels, preventing action potential generation.
Record the signal to identify background noise.

analyzing-neural-activity-primary-murine-spiral-ganglion-neurons

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

91 ビュー. 出典:Hahnewald, S., et al. Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays. J. Vis. Exp. (2016). このビデオは、一次マウススパイラル神経節ニューロン(SGN)培養からの自発的および刺激依存的な神経活動を多電極アレイ(MEA)で記録する方法を示しています。これには、MEAの洗浄と準備、自発的な神経活動の記録、および刺激依存性のニューロン活動を特定するための...

ビデオ: 多電極アレイを用いた初代マウス渦巻神経節ニューロンの神経活動の解析 - 概念

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

マウスにおける心内ペース作りの欠損を同定するための、中房ノードの発火率のマイクロ電極アレイ記録

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

JoVE Journal

医学

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

パッチクランプ記録のための新生児げっ歯類からの前庭およびらせん状神経節体細胞の分離と培養

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

神経科学

Laser-Induced Action Potential-Like Measurements of Cardiomyocytes on Microelectrode Arrays for Increased Predictivity of Safety Pharmacology

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small proarrhythmic membrane potential fluctuations. The shape and time course of the repolarizing fraction of the AP can be pivotal for the presence or absence of arrhythmia.
Microelectrode arrays (MEA) allow easy access to cardiotoxic compound effects via extracellular field potentials (FP). Although a powerful and well-established tool in research and cardiac safety pharmacology, the FP waveform does not allow to infer the original AP shape due to the extracellular recording principle and the resulting intrinsic alternating current (AC) filtering.
A novel device, described here, can repetitively open the membrane of cardiomyocytes cultivated on top of the MEA electrodes at multiple cultivation time points, using a highly focused nanosecond laser beam. The laser poration results in transforming the electrophysiological signal from FP to intracellular-like APs (laser-induced AP, liAP) and enables the recording of transcellular voltage deflections. This intracellular access allows a better description of the AP shape and a better and more sensitive classification of proarrhythmic potentials than regular MEA recordings. This system is a revolutionary extension to the existing electrophysiological methods, permitting accurate evaluation of cardiotoxic effect with all advantages of MEA-based recordings (easy, acute, and chronic experiments, signal propagation analysis, etc.).

The combination of laser poration and microelectrode arrays (MEA) allows action potential-like recordings of cultivated primary and stem cell-derived cardiomyocytes. The waveform shape provides superior insight into test compounds' mode of action than standard recordings. It links patch-clamp and MEA readout to further optimize cardio safety research in the future.