utilizing multielectrode arrays to measure synaptic physiology in 3d coculture spheres

Utilizing Multielectrode Arrays to Measure Synaptic Physiology in 3D Coculture Spheres

Begin by coating the surface of each multielectrode array or MEA with 1 millimeter of poly-ornithine to render the surface hydrophilic and incubate at 37 degrees Celsius for a minimum of four hours. Following the incubation, remove the poly-ornithine and wash the surface of each MEA with deionized water. Then, add 1 milliliter of extracellular matrix and return to the incubator for a minimum of four hours.
When ready, remove the extracellular matrix, then apply the spheres in 1.5 milliliters of medium onto the MEA surface, ensuring that the spheres are positioned on top of the electrode array. Allow to adhere for two days. To measure the electrical activity of neural spheres, place the MEA on a temperature-controlled head stage.

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Measurement of Live Synaptic Physiology with Multielectrode Arrays (MEAs)
1. Prepare MEAs for cell culture.
<ol>
	<li>Clean the surface of each MEA with 1 mL of detergent for 1 h. Rinse with sterile deionized (DI) water, rinse with 70% ethanol to sterilize, and then air dry in a biosafety cabinet under UV light. 
	NOTE: MEAs can be stored in DI water at 4 &#176;C under sterile conditions until use.</li>
	<li>Prepare a 0.5 mg/mL solution of poly-ornithine (PLO) by dissolving 50 mg of PLO into 100 mL of boric acid buffer. Coat the surface of each MEA with 1 mL of PLO to render it hydrophilic and incubate at 37 &#176;C for a minimum of 4 h. Remove the PLO and wash the surface with DI water.</li>
	<li>Dilute extracellular matrix (ECM) coating solution with basal (DMEM/F12) media to prepare a 1 mg/mL stock solution. For a working solution, resuspend 3 mL of ECM stock into 33 mL of pre-chilled DMEM/F12 media, bringing the total volume to 36 mL for a concentration of 80 &#181;g/mL.</li>
	<li>Add 1 mL of ECM working solution to each MEA and incubate at 37 &#176;C for a minimum of 4 h.</li>
	<li>Remove the ECM and place the spheres in 1.5 mL of media on a MEA surface, ensuring that the spheres are positioned on top of the electrode array (Figures 1A, 1A&#39;). Allow the media to adhere for 2 days. Change half of the media every 2&#8211;3 days (or more often if needed), ensuring that the spheres are not disturbed. 
	NOTE: The addition of growth factors may improve culture survival and maturation.</li>
</ol>
2. Measure the electrical activity of neural spheres.
<ol>
	<li>Set up a multielectrode array (MEA) system with a temperature-controlled headstage. Create a software program with a 5 Hz high-pass filter, a 200 Hz low-pass filter, and a spike threshold of 5x standard deviation (SD).</li>
	<li>Place the MEA on headstage and record spontaneous electrical activity (Figure 1B, B&#39;). Save raw data, including spike frequency and amplitude, for statistical analysis. 
	NOTE: A perfusion system may be utilized with the MEA to add pharmacological agents or to increase the flow of fresh media for long-term recording. As desired, additional post-processing of spikes may be performed.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>10565-042</td>
			<td>With GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>Anti/anti</td>
			<td>Thermofisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149-50KU</td>
			<td>Working concentration: 2 mg/mL</td>
		</tr>
		<tr>
			<td>Matrigel membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.</td>
		</tr>
		<tr>
			<td>MEA 2100 System</td>
			<td>Multichannel Systems</td>
			<td>MEA2100</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>Poly-l-ornithine (PLO)</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>Working concentration: 0.5 mg/mL</td>
		</tr>
	</tbody>
</table>

Source: Cvetkovic, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure of using the multielectrode array (MEA) to measure synaptic physiology in 3D coculture spheres. This method allows to understand the neural network dynamics and synaptic network burst activity within the 3D spheres.

<img alt="Figure 1" src="/files/ftp_upload/22717/22717_Figure1.jpg" /> 
Figure 1: Representative live synaptic physiology of neural spheres on multielectrode arrays (MEAs). (A, A&#39;) MEAs are utilized to measure the live synaptic physiology of neural spheres. Spheres of iNeurons (A) or cocultures of iNeurons and hAstros (A&#39;) can be cultured on MEA surfaces with ECM-mimicking substrates (see step 4.1). (B, B&#39;) Raster plots of representative spontaneous electrical activity measured across all electrodes (vertical axis) during a recording window (horizontal axis). After 4 weeks of culture in neurophysiological basal medium41, iNeuron spheres (B) displayed spontaneous spikes, while coculture spheres (B&#39;) revealed increased network bursts of synchronous firing patterns (orange arrows). (C, C&#39;) Histogram of spikes measured during MEA recording windows from representative electrodes. (D, D&#39;) Raster plots of spontaneous electrical activity were measured across all electrodes after the application of 50 &#181;M CNQX, an AMPA receptor antagonist (same time scale as B, B&#39;). CNQX eliminated the presence of synchronous bursts of spikes observed in cocultures (D&#39;). (E, E&#39;) Histogram of spikes measured during MEA recording windows from representative electrodes after 50 &#181;M CNQX application (same time scale as C, C&#39;). (F) Map of spike tracings of iNeuron spheres from all electrodes over time (left). Demonstrative single electrode displaying multiple clustered traces over time (middle); individual spike traces can be sorted and analyzed individually (right).

Measurement of Live Synaptic Physiology with Multielectrode Arrays (MEAs)
1. Prepare MEAs for cell culture.

	Clean the surface of each MEA with 1 mL of ...

Take a sterile multielectrode array or MEA, a device that records the electrical activity of target cells.
Add a polymer solution to the MEA and incubate.
The polymer coats the surface of the MEA, thereby increasing the water-attracting property of the surface.
Remove the excess polymer and wash with deionized water.
Add extracellular matrix, or ECM, and incubate to allow it to coat the MEA. Remove the excess ECM.
Next, add the 3D coculture spheres comprising neurons and astrocytes in a suitable media.
Incubate to allow the spheres to adhere to the surface of the MEA.
Place the MEA on a temperature-controlled head stage of an MEA system and record the spontaneous electrical activity across the electrodes.
The measured electrical activity corresponds to the communication at the neural junctions within the sphere.

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Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Stimolazione diretta correnti e multi-elettrodo Array registrazione di sequestro simile attività nei topi cervello fetta di preparazione

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

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Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Fabbricazione di array di microelettrodi Ti3C2 MXene per la registrazione neurale In Vivo

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Bioingegneria

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

Utilizing Multielectrode Arrays to Measure Synaptic Physiology in 3D Coculture Spheres

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Utilizing Multielectrode Arrays to Measure Synaptic Physiology in 3D Coculture Spheres