electrophysiological recordings for assessing neuronal regenerations in co-cultured spinal cord slices

Electrophysiological Recordings For Assessing Neuronal Regenerations in Co-cultured Spinal Cord Slices

Put a Petri dish with a coated microelectrode array inside under a stereomicroscope. Bring the array into focus, and center a six-microliter droplet of chicken plasma on the electrode array. Using a small spatula, carefully slide two spinal cord sections with their ventral sides facing each other into the plasma droplet.
Next, add eight microliters of thrombin around the chicken plasma droplet. Then, use the pipette tip to carefully mix and spread the chicken plasma and thrombin mixture. Just before the chicken plasma and thrombin mixture becomes too stringy and starts to coagulate, aspirate the excess liquid. Then, cap the Petri dish and place it in a humidified chamber.
Place the chamber inside an incubator at 37 degrees Celsius for about an hour. After incubation, carefully add 10 microliters of nutrient medium to the sample. Cap the Petri dish and place it back into the incubator for an additional 45 minutes. Next, place each of the multi-electrode array culture assemblies into a roller tube and add three milliliters of nutrient medium. Close the lid tightly and place the roller tube into the roller drum. Rotate the drum at 1 to 2 RPM in the incubator at 37 degrees Celsius.
Using sterile rubber-tipped forceps, remove the multi-electrode array culture assemblies from the roller tube and place them into a Petri dish under a stereomicroscope. Bring the tissue into focus and verify that the two slices are fused. Next, hold the assembly steady and place a scalpel blade in the groove of the multielectrode array close to the tissue slices. Hold the scalpel rather horizontally and then lift the scalpel handle up, but let the scalpel blade stay in the groove of the array in such a way that the blade rolls from base to tip, cutting through the tissue, covering the groove.
Sever any residual tissue connections with a 25-gauge needle tip if necessary. Work only in the area within the groove, and do not touch the tender edges. Put the multi-electrode array culture assemblies back into the roller tube and add three milliliters of fresh nutrient medium to the cultures. Then, place the roller tube back onto the roller drum in the incubator at 37 degrees Celsius.
Mount a multi-electrode array culture assembly into a recording chamber and apply about 500 microliters of extracellular solution to the array. Then, mount the assembly on the microscope. Wait 10 minutes for the system to stabilize, and then, record basic spontaneous activity from each of the activity-detecting electrodes for 10 minutes. Repeat the recordings a total of two times.
To ensure stable extracellular conditions, exchange the extracellular solution after every recording session. If desired, disinhibit the network by applying extracellular solution containing one micromolar of strychnine and 10 micromolar of gabazine and wait for at least two minutes before recording the electrical activity.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Mounting Spinal Cord Tissue Slices on MEAs
<ol>
	<li>Warm up sterile nutrient medium in the incubator (37 &#176;C, lightly unscrewed lid for oxygenation).</li>
	<li>Position the multi-electrode array or MEA with sterile tweezers with rubber-covered tips at room temperature (RT) in the petri dish under a stereo microscope with the electrode array in focus. Center a 6 &#181;l droplet of chicken plasma on the clean, dust-free, and sterile electrode array. Using a small spatula, carefully slide two spinal cord sections with ventral sides facing each other into the plasma droplet. Do not touch the electrodes with the spatula.</li>
	<li>Add 8 &#181;l of thrombin around the chicken plasma droplet. Using the thrombin pipette tip, carefully mix and spread the chicken plasma/thrombin mixture around the two slices. Again, do not touch the brittle electrode array directly. Just before coagulation, aspirate excess chicken plasma/thrombin.</li>
	<li>Cap the petri dish to retain high humidity while the MEA/culture assembly sits for about 1 hr in a humidified chamber inside the incubator at 37 &#176;C.</li>
	<li>Carefully add 10 &#181;l of nutrient medium to the culture chamber, cap the petri dish, and put back into the incubator for about 30 min.</li>
	<li>Place each MEA/culture assembly with sterile, rubber-covered tips into a roller tube, add 3 ml of nutrient medium, and tightly close the lid. Place the roller tube in the roller drum, rotating at 1 - 2 rpm in the incubator in a 5% CO2-containing atmosphere at 37 &#176;C.</li>
	<li>Change half of the nutrient medium after 7 days in vitro (DIV) and afterward 1 - 2 times per week.</li>
</ol>
2. Mechanical Lesions
<ol>
	<li>Take the MEA/culture assembly with sterile, rubber-covered tips out of the roller tube and place in a petri dish without nutrient medium under a stereo microscope with the tissue in focus. Visually verify that the two slices are fused.</li>
	<li>Hold the MEA/culture assembly steady by placing tweezers with rubber-covered tips on the MEA.</li>
	<li>Place a scalpel blade in the groove of the MEA close to the tissue slices. Hold the scalpel rather horizontally.</li>
	<li>Lift the scalpel handle up but let the scalpel blade stay in the groove of the MEA in such a way that the blade &#34;rolls&#34; from base to tip and thereby cuts through the tissue covering the groove.</li>
	<li>Severe any residual tissue connections with a 25 G needle tip if necessary. Work only in the area within the groove and do not touch the tender edges.</li>
	<li>Put the MEA/culture assembly back into the roller tube. Provide 3 ml of fresh nutrient medium to the cultures and place them back into the roller drum in the incubator.</li>
</ol>
3. Electrophysiological Recordings of Spontaneous Activity
<ol>
	<li>To investigate functional regeneration among the two spinal cord slices after the chosen number of DIV, mount the MEA/culture assembly in a recording chamber on a microscope and apply about 500 &#181;l extracellular solution (see materials list).</li>
	<li>Wait 10 min before the first recording to allow the system to stabilize.</li>
	<li>Record basic spontaneous activity 2x for about 10 min from each activity-detecting electrode of the MEA at RT.</li>
	<li>To ensure stable, extracellular conditions, exchange extracellular solution after every recording session.</li>
	<li>To disinhibit the network, apply an extracellular solution containing strychnine (1 &#956;M) and gabazine (10 &#956;M).&#160; Wait for at least 2 min before starting the recording.</li>
</ol>
4. Data Analysis
NOTE: For the detection of the extracellularly recorded action potentials, use a detector based on standard deviations and a subsequent discriminator for each electrode.
<ol>
	<li>Display the detected neuronal activity of each electrode in a raster plot according to standard procedures (Figure 1F).</li>
	<li>Determine and display the total network activity for each slice by summarizing the number of events in the appropriate channels within a sliding window of 10 msec, shifted by 1 msec steps (Figure 1G).</li>
	<li>Detect in each slice individual bursts (clusters of activity that appear on several electrodes). Therefore, set a minimal peak activity threshold in the corresponding total network activity and define each burst start and burst end. For example, an appropriate threshold is 25% of the average burst amplitude, and a burst start can be defined as being the first event in a time window of at least 5 msec, and a burst end by being the last event in a time window of at least 25 msec.</li>
	<li>To quantify functional regeneration, calculate the percentage of synchronized bursts between the two slices. To do this, calculate the latency between a burst in one slice and the following burst in the other slice. 
	NOTE: A burst pair is termed &#34;synchronized&#34; when the latency is smaller than the average burst length. Especially in co-cultures with a lot of activity, the possibility exists that both sides coincidentally initiate a burst in the chosen latency window. This fact has to be taken into account in the quantification of the percentage of synchronized bursts.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Planar multielectrode array</td>
			<td>Qwane Biosciences</td>
			<td></td>
			<td>custom-made according to the design of our lab</td>
		</tr>
		<tr>
			<td>Nutrient medium</td>
			<td></td>
			<td></td>
			<td>For 100 ml</td>
		</tr>
		<tr>
			<td>Dulbeccos modified Eagle&#39;s medium</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td>80 ml</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-070</td>
			<td>10 ml</td>
		</tr>
		<tr>
			<td>distilled, sterile water</td>
			<td></td>
			<td></td>
			<td>10 ml</td>
		</tr>
		<tr>
			<td>Nerve growth factor-7S [5ng/mL]</td>
			<td>Sigma-Aldrich</td>
			<td>N0513</td>
			<td>200 &#181;l</td>
		</tr>
		<tr>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>reconstituted in wash solution with 1% BSA</td>
		</tr>
		<tr>
			<td>Extracellular matrix gel</td>
			<td>BD Biosciences</td>
			<td>356230</td>
			<td>Must stay cold at all times; dilute 1:50 with medium optimized for prenatal and embryonic neurons</td>
		</tr>
		<tr>
			<td>Extracellular solution (pH 7.4)</td>
			<td></td>
			<td></td>
			<td>[mM]</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>145</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>4</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td>1</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td>2</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td>5</td>
		</tr>
		<tr>
			<td>Na-pyruvate</td>
			<td></td>
			<td></td>
			<td>2</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>5</td>
		</tr>
		<tr>
			<td>Gabazine</td>
			<td>Sigma-Aldrich</td>
			<td>SR-95531</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>World Precision Instruments, Inc.</td>
			<td>MF28G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Strychnine</td>
			<td>Sigma-Aldrich</td>
			<td>S0532</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Tissue culture flat tube 10 (= roller tube)</td>
			<td>Techno Plastic Products AG</td>
			<td>91243</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Heidemann, M., et al. <a href="https://app.jove.com/v/53121">Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays.</a> J. Vis. Exp. (2015).
This video presents electrophysiological recordings from organotypic co-cultures of spinal cord slices, captured using a multi-electrode array or MEA device. These recordings assess neuronal regeneration between the slices. The synchronized electrical activity observed confirms successful neural regeneration.

<img alt="Figure 1" src="/files/ftp_upload/22714/22714_Figure1.jpg" /> 
Figure 1. Display and Analysis of Spontaneous Activity. (A) Diagram of a MEA. The platinum-covered electrodes are depicted in black, the transparent wires in red, and the groove in the middle of the MEA in yellow. (B) Close-up of the electrode array located in the center of the MEA. (C) Bright-field image of an 8 DIV old culture. The slices have grown and fused along the sides facing each other. ...

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Mounting Spinal Cord Tissue ...

Take a polymer-coated multi-electrode array or MEA, with co-cultured embryonic spinal cord slices positioned in two separate zones, allowing for simultaneous recording from both slices.
The slice shows regenerated neural connections in the previously lesioned area.
Place the MEA in a recording chamber containing an extracellular solution rich in ions and nutrients to maintain neuronal survival.
Allow the system to stabilize, facilitating neuron adaptation and communication.
Neurons communicate via electrical signals called action potentials, which are recorded by the nearby electrodes in the MEA.&#160;
Individual signals from each electrode are combined, producing an electrical signal from each slice.
Regularly replace the extracellular solution to maintain neuron stability.
Add an extracellular solution with inhibitors that block inhibitory neurotransmitter receptors on neuronal membranes, keeping neurons active for a longer duration.
Compare the electrical activity from both slices. A synchronized electrical signal between the two slices confirms the synaptic connections and successful neural regeneration.

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Electrophysiological Recordings For Assessing Neuronal Regenerations in Co-cultured Spinal Cord Slices

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Electrophysiological Recordings For Assessing Neuronal Regenerations in Co-cultured Spinal Cord Slices