eoe sub articles

EoE Sub Articles

1. Recording Neuronal Network Activity
<ol>
	<li>On the day of acquisition, exchange the culture medium with fresh neurobasal medium and return the MEAs to the CO2 incubator for a minimum of 2 h before recording. Start the recording software and set the temperature of the MEA to 37 &#176;C by clicking on the temperature icon. 
	NOTE: Recording can begin as early as day 3 of plating and can continue as long as the cultures are alive.</li>
	<li>Set up the acquisition parameters by right-clicking on the &#34;Muse&#34; icon (under Streams in the left window panel) and select &#34;Add Processing&#34; and &#34;Spike Detector&#34; and click OK in the pop up window. &#34;Spike Detector (6 x STD)&#34; will appear below the file name.</li>
	<li>Next right click on the Spike Detector, select &#34;Add Processing&#34; and &#34;Burst Detector&#34; and click OK in the pop up window. &#34;Burst Detector&#34; (ISI) will appear below the Muse icon.</li>
	<li>Next right click on Burst Detector, select &#34;Add Processing&#34; and &#34;Neural Statistics Compiler.&#34; In the pop up window, ensure that File Header, Aggregated Well Statistics and Synchrony are selected. Click OK. &#34;Statistics Compiler&#34; will appear below. 
	NOTE: This will set the adaptive threshold crossing (6x STD filter), the inter-spike interval at 100 ms with a minimum number of spikes at 5, the mean firing rate detection at 10 s with a synchrony parameter at 20 ms, and the minimum spike rate at 0.083333 spikes per min.</li>
	<li>Set up the scheduled recording for the recording time of 5 minutes (300,000 ms) by clicking on the clock icon at the bottom. This is achieved by changing the information in the setting section to record every 5.1 minutes and record for 5 minutes. This will be started immediately and will be executed once.</li>
	<li>Ensure that the temperature of the MEA has reached 37 &#176;C before moving the MEA. Remove the MEA from the incubator, place it on the recording unit, and lock down the MEA. Start recording the network activity by clicking on the start record in the scheduled recording section or the record icon in the &#34;File Play&#34; window. Typically, a recording time of 5 minutes (300,000 ms) is sufficient, although longer recordings of up to 10 minutes can be obtained.</li>
	<li>After recording, return the MEA to the incubator. 
	NOTE: Care must be taken to maintain sterility and to minimize the time the MEA is outside the incubator.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Axion Muse MEA</td>
			<td>Axion Biosystems</td>
			<td>M64-GL1-30Pt200</td>
			<td>Will be called MEA system in manuscript</td>
		</tr>
		<tr>
			<td>Axis Software</td>
			<td>Axion Biosystems</td>
			<td></td>
			<td>Will be called recording software in the manuscript</td>
		</tr>
		<tr>
			<td>BPA</td>
			<td>Sigma-Aldrich</td>
			<td>239658-250g</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH</td>
			<td>Sigma-Aldrich</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>BrainBits</td>
			<td>Nb4-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuroexplorer statistical software</td>
			<td>Nex Technologies</td>
			<td>Neuroexplorer version 5</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing the effects of toxins on chick embryo neural network development using multielectrode arrays

Source: Sanchez, K. R., et.al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56300">Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays.</a> J. Vis. Exp. (2018).
This video demonstrates the use of multi-electrode arrays (MEA) to study the effects of toxins on early embryonic chick neuronal cultures. By monitoring the synchronous activity of the neurons in the neuronal network, the MEA captures the functional dynamics of the neuronal network in response to toxin exposure. Reduced synchrony and firing rates upon toxin exposure highlight its detrimental impact on neuronal network maturation and function.

Assessing the Effects of Toxins on Chick Embryo Neural Network Development Using Multielectrode Arrays

1. Recording Neuronal Network Activity

	On the day of acquisition, exchange the culture medium with fresh neurobasal medium and return the MEAs to the ...

Begin with adherent cultures of early embryonic chick neurons on extracellular matrix-coated multielectrode arrays, or MEAs.
The base of the MEAs contains grids of tiny electrodes that record neuronal activity.
In the test culture, neurons grow in the presence of a toxin, while in the control culture, neurons grow without the toxin.
Before recording, replace the media in both MEAs with toxin-free media and incubate briefly.
Next, place one MEA in the recording device and begin recording.
Neurons communicate via electrical impulses called action potentials or spikes.
At the synapse, these action potentials trigger neurotransmitter release, propagating the signal to the next neuron.
The MEA electrodes record the spikes generated by the synaptically connected neurons.&#160;
In the control condition, the healthy mature neuronal network exhibits synchronous spiking activity.
In the test culture, reduced spiking and synchrony suggest impaired neuronal network maturation caused by the toxin.

assessing-effects-toxins-on-chick-embryo-neural-network-development

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

32 Views. Source: Sanchez, K. R., et.al. Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays. J. Vis. Exp. (2018). This video demonstrates the use of multi-electrode arrays (MEA) to study the effects of toxins on early embryonic chick neuronal cultures. By monitoring the synchronous activity of the neurons in the neuronal network, the MEA captures the functional dynamics of the neuronal network in response to to...

Video: Assessing the Effects of Toxins on Chick Embryo Neural Network Development Using Multielectrode Arrays - Concept

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

JoVE Journal

Developmental Biology

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Assessing the Effects of Toxins on Chick Embryo Neural Network Development Using Multielectrode Arrays

Transcript

Assessing the Effects of Toxins on Chick Embryo Neural Network Development Using Multielectrode Arrays

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation