Name: investigating functional regeneration in organotypic spinal cord co-cultures grown on multi-electrode arrays
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This study was supported by the Swiss National Foundation (n&#176; 3100AO-120327 and 31003A_140754/1)

Animal care was in accordance with guidelines approved by Swiss local authorities (Amt f&#252;r Landwirtschaft und Natur des Kantons Bern, Veterin&#228;rdienst, Sekretariat Tierversuche, approval Nrs. 52/11 and 35/14). These guidelines are in agreement with the European Community Directive 2010/63/EU.
Note: Work in a laminar flow hood with sterile instruments and solutions for all steps 1 - 5 including sub-steps.
1. Preparation of MEAs
Note: MEAs are composed of a glass substrate, micro-fabricated metal electrodes and a SU-8 polymer insulation layer (also see Tscherter et al. 3). For the purpose of this study, commercially available MEAs were ordered with a customized electrode array layout (Figure 1 A&#38;B). The 68 electrodes are arranged in a rectangular grid that is split into two zones by a 300 &#181;m wide groove free of electrodes, electrical leads and insulation. Each electrode is 40 x 40 &#181;m in size and they are spaced apart by 200 &#181;m from center to center. Four large ground electrodes are positioned around the recording site. They differ from other commercially available standard MEAs in their size (21 mm x 21 mm) and they have no fixed recording chamber.
<ol>
	<li>For disinfection rinse MEAs in 100% ethanol (2x), 70% ethanol (1x) and distilled water (2x) for at least 30 sec&#160;each. Let dry. Put 10 - 12 MEAs in a clean glass petri dish (150 mm x 25 mm) and close the lid.</li>
	<li>Autoclave MEAs for 20 min at 120 &#176;C. Store at RT.</li>
	<li>Prepare the coating solution (see materials list) for the MEAs and store aliquots at -20 &#176;C.</li>
	<li>Put each MEA in an individual sterile petri dish (35 mm x 10 mm).</li>
	<li>Use a cooled pipette to put 150 &#181;l chilled coating solution on top of the electrodes and close the lid of the petri dish. After about 10 min, check for air bubbles on top of the electrodes and gently remove them with a rubber-covered spatula if necessary. Let rest for 1 hr&#160;at RT.</li>
	<li>Aspirate coating solution residues, wash 1x with medium optimized for prenatal and embryonic neurons and 2x with distilled, sterile water. If MEAs are not used until the next day, keep them in distilled, sterile water at 37 &#176;C in the incubator O/N. Otherwise, let directly dry at RT.</li>
</ol>
2. Ingredients Required for the Preparation and Growth of Organotypic Cultures
<ol>
	<li>Prepare calcium-free wash solution (see materials list).</li>
	<li>Reconstitute chicken plasma in wash solution (1:1, shake gently, avoid formation of bubbles), centrifuge at 3,000 x g for about 20 min and decant the clear content into a sterile tube. Aliquot 200 &#181;l into cryotubes and store at -20 &#176;C.</li>
	<li>Reconstitute thrombin from bovine plasma accordingly (200 U/ml) and sterile-filter (0.2 &#181;m pore filter). Aliquot 200 &#181;l into crytotubes and store at -20 &#176;C.</li>
	<li>For 30 cultures prepare 100 ml of nutrient medium (see materials list).</li>
</ol>
3. Spinal Cord Tissue Dissection
Note: Procedure yields, depending on the number of embryos, spinal cord slices for about 25 - 35 co-cultures and is prepared inside a laminar flow hood under sterile conditions.
<ol>
	<li>Apply a lethal dose of pentobarbital (0.4 ml) to the mother animal by intramuscular injection. Confirm deep anesthesia by checking the pedal withdrawal reflex. Deliver E14 rat embryos by cesarean section and sacrifice by decapitation.</li>
	<li>Transfer the bodies of the embryos into a petri dish filled with sterile, chilled, wash solution.</li>
	<li>Perform one complete transversal cut with a scalpel above the hindlimbs and another one above the forelimbs and remove the limbs from the body. Next, make a cut in the frontal plane to separate viscera from the back piece containing the spinal cord.</li>
	<li>For slicing, transfer the back pieces containing the spinal cord one at a time on a mounting disk and cut at a thickness of 225 - 250 &#181;m with a tissue chopper. Put a drop of wash solution on the chopped tissue and transfer slices with a spatula in 35 mm x 10 mm petri dishes filled with sterile, chilled, wash solution.</li>
	<li>For each slice separately, dissect spinal cord away from remaining tissue. Leave dorsal root ganglia attached.</li>
	<li>Let the slices rest for 1 hr at 4 &#176;C.</li>
</ol>
4. 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 MEA with sterile tweezers with rubber-covered tips at 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 afterwards 1 - 2 times&#160;per week.</li>
</ol>
5. 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 &#8220;rolls&#8221; 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>
6. 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 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>
7. Data Analysis
Note: For the detection of the extracellularly recorded action potentials use for each electrode a detector based on standard deviations and a subsequent discriminator. This procedure is described in detail in Tscherter et al. 3.
<ol>
	<li>Display the detected neuronal activity of each electrode in a raster plot according to standard procedures 3 (Figure 1F).</li>
	<li>Determine and display the total network activity for each slice by summing up the number of events in the according channels within a sliding window of 10 msec, shifted by 1 msec steps (Figure 1G and see Tscherter et al. 3).</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 by being the first event in a time window of at least 5 msec, a burst end by being the last event in a time window of at least 25 msec (see Heidemann et al. 10).</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 &#8220;synchronized&#8221; 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. For a detailed description of the calculations see Heidemann et al.10.</li>
</ol>
8. Fixation of the Cultures and Immunohistochemistry
<ol>
	<li>For all steps that include washing or incubation make sure to put the cultures on a tilting table to ensure proper diffusion of the solution through the tissue.</li>
	<li>Directly after the recording session is finished take the MEA/culture assembly out of the setup, put it in a petri dish (35 mm x 10 mm) and add about 2 ml of extracellular solution.</li>
	<li>To separate the slices from the surrounding cell layers that have formed during the time in vitro, take a 10 &#181;l pipette tip and circle the slices. Pay attention not to damage the cultures. It is possible to omit this step but embedding later on is easier when it is performed.</li>
	<li>Use a thin nonmetallic syringe needle (see materials list) in combination with a 1 ml syringe filled with extracellular solution to gently blow the culture off the MEA.</li>
	<li>Remove the tip of a Pasteur pipette and fit the rubber bulb on the slivered end. Use the back end to transfer the culture into fixative (4% paraformaldehyde with phosphate buffered saline (PBS, 0.1 M)). Do not use the standard tip of the Pasteur pipette for the transfer because it is too small and the cultures would be folded and probably damaged. Fix for 1 - 2 hr at 4 &#176;C. Rinse the MEA with distilled water and remove tissue residues with fingertips but do not scratch with the nail. Store MEAs in distilled water at 4&#176; C.</li>
	<li>Wash the cultures 3x in PBS for at least 10 min each. Store in PBS at 4 &#176;C or directly start with the staining procedure.</li>
	<li>Incubate the cultures for at least 90 min in blocking solution (5% goat serum, 1% bovine serum albumin, 0.3% detergent in PBS).</li>
	<li>Add the first antibody diluted in blocking solution and incubate for 3 nights at 4 &#176;C.</li>
	<li>Wash 3x in PBS for at least 30 min each.</li>
	<li>Add the second antibody diluted in PBS for 2 hr at RT.</li>
	<li>Wash 3x in PBS for at least 30 min each.</li>
	<li>Transfer the slices with a Pasteur pipette with removed tip on an object slide. Remove excess PBS and embed with mounting medium.</li>
</ol>

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The authors have nothing to disclose.

Several elements in the presented procedure are fundamental for the accomplishment of accurate and reproducible experiments. All steps during the preparation of the MEAs, solutions and the production of the slice cultures are performed under sterile conditions in a laminar flow hood. Antibiotics are not applied to the nutrient medium because they can affect the spontaneous activity 14. During the MEA coating, the most important part is to make sure that the extracellular matrix gel solution stays cold at all times. Thaw it in the fridge and keep it on ice for the whole procedure to ensure a maximal temperature close to 0 &#176;C. During the preparation of the slices quick isolation and sectioning, as well as paying attention to a low temperature by using ice cold dissection buffers increases the percentage of healthy cultures. Moreover, the plasma/thrombin coagulation is one of the most critical steps. First, make sure that the plasma is properly mixed with the thrombin. Second, pay special attention to removing as much residue as possible to ensure proper attachment of the slices to the MEAs. This step is extremely time-sensitive; if the time frame is too short, too much of the plasma/thrombin mixture is removed. If the time frame is too long, coagulation already happened, increasing the risk of removing the slices along with the residual plasma/thrombin.
If mechanical lesions are planned, it is important to use MEAs with an accurate design. The area where the lesion is intended needs to be free of electrodes, wires and insulation. Otherwise, the electrics of the MEA are going to be damaged and therefore, no recordings can be performed anymore.
Another crucial step concerns the size of the lesion. It has been shown previously that reconnecting two young slices after lesion takes about two days 10. For all experiments, the rule of thumb was used that the space between the slices after lesion should not be bigger than the width of the MEA groove (300 &#181;m). A bigger lesion size might result in a longer time frame for reconnection or, if the lesion is too big, no reconnection at all. To annotate, former experiments revealed that an initial placing of the two slices further away than 1 mm results in a very low connection rate.
Before the start of the recordings, an adjustment time of at least 10 min to RT, instead of 37 &#176;C of the incubator, and to the extracellular solution, instead of the nutrition medium is recommended. Close observation of the activity pattern during these initial minutes ensures that the measured data represent the bursting pattern of the culture appropriately after the recording was started.
For the detection of the activity, data acquisition and further processing, homemade hardware (recording chamber, connections to the amplifiers and amplifiers), programs in LabVIEW, C++ and IgorPro are used. Commercial MEA setups and other software packages are suited as well for these operations. Here, the signals seen by the electrodes are amplified 1001 times, and then digitized at a rate of 6 kHz.
The presented model can be a helpful tool to examine propriospinal connections because in vivo, they are difficult to study without the interference of ascending and descending fibre tracts. The co-culturing of two spinal cord slices can elegantly circumvent this issue. The cultures provide a microenvironment that can be tightly controlled and easily manipulated. On the other hand, supraspinal and sensory input is of course missing. Additionally, the process of the culture preparation represents a situation of CNS damage. Glial cells like astrocytes and microglia are known to respond to lesions with increased proliferation and therefore, the tissue is to a certain extent reorganized. Relating to the presented model the formation of a glial scar after performing mechanical lesions was not observed. One explanation could be that adaptive mechanisms of astrocytes were already triggered during the preparation process and that lesions of the cultures did not result in a further response. Also, there is no evidence for the involvement of myelin associated inhibitors, e.g., Nogo-A, in the low regeneration potential of cultures lesioned at an old age. However, it was shown that treatment with the phosphodiesterase 4 inhibitor Rolipram, starting directly after performing lesions at 23 DIV increases functional regeneration between the slices 10. These results demonstrate that functional recovery can be increased in old cultures. Noticeably, when counting the number of axons crossing the lesion site, no difference between cultures lesioned at a young age and cultures lesioned at an old age was discovered. These findings suggest that lack of axonal regeneration is not the major reason for the loss of recovery after late lesions in culture and therefore emphasize the need for a model that allows functional analysis of regeneration. Synaptic formation and synaptic plasticity could play a major role in the recovery process 10. These mechanisms gained a lot of attention during recent years and it is nowadays widely accepted that they have a major impact on the outcome after spinal cord injury. Therefore, a model that provides stable conditions to investigate these processes in isolation and in great detail can certainly help to obtain insight about functional regeneration in the spinal cord.

Organotypic slice cultures of the central nervous system (CNS) have been used extensively to study various physiological and pathological phenomena reaching from neuronal development to neurodegeneration. Compared to dissociated cell cultures, organotypic slices offer the advantage of more complexity with an intact cytoarchitecture and local synaptic circuitry. At the same time, the tissue can be analyzed in an isolated way without the need to implicate the intricacy of the whole in vivo context. Moreover, easy and repeated access to the cells of choice is warranted, the extracellular environment can be precisely controlled and usually, in vitro models are less costly than their in vivo counterparts. In recent years, various studies presented striking similarities between the developmental profiles of long term cultures versus the corresponding living animals 1,2. It has been shown that neuronal circuits of various CNS regions express spontaneous network activity during development and that organotypic slices partially maintain this phenomenon 3&#8211;7. Isolated spinal cord preparations have been particularly used to investigate rhythm generation 6 and the formation of neuronal circuits 1.
A way to record the spontaneous activity of organotypic slices is to culture them on top of multi-electrode arrays (MEAs). These devices contain multiple electrodes that can monitor the extracellular potentials generated by action potentials from numerous cells simultaneously (multi-unit recording). The high temporal resolution of the MEA recordings can be used to reconstruct the precise activity dynamics within a given neuronal circuit. Additionally, in contrast to patch-clamping the technique is non-invasive, which permits long-term measurements and results in the fact that neurons are not affected in their behavior.
For the purpose of this study, the combination of organotypic spinal cord co-cultures and multisite recordings was used to investigate functional regeneration within the spinal cord. Since adult higher vertebrates have limited potential to recover from damage of the spinal cord, different strategies have been studied to promote regeneration after spinal cord injury. So far, the corticospinal tract has usually been the model system of choice for such investigations. These experiments are typically time-consuming, costly and require large animal cohorts due to high variability within single groups. Moreover, evidence suggests that propriospinal connections (neurons that are entirely confined within the spinal cord) can play a pivotal role in the recovery process following spinal cord lesions 8. These fibers are difficult to study in vivo without the interference of ascending and descending fiber tracts. Bonnici and Kapfhammer 9 used longitudinal spinal cord slice cultures of postnatal mice as an alternative approach. After performing mechanical lesions they analyzed semi-quantitatively the morphological regeneration of axons between spinal cord segments. They observed less but not none axons crossing the lesion site in cultures cut at a mature age. In contrast, cultures that were lesioned at a younger stage displayed a high amount of axonal regeneration 5 - 7 days after the damage. However, proof for functional connections was not presented.
Therefore, the development of a representative in vitro model of propriospinal fibers that allows the investigation of functional regeneration can be a valuable tool to extend our knowledge about regenerative processes in the spinal cord.

<table><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&#039;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&amp;nbsp;</td></tr><tr><td>Nerve growth factor-7S [5 ng/ml]</td><td>Sigma-Aldrich</td><td>N0513</td><td>200 &amp;micro;l</td></tr><tr><td colspan="4">reconstituted in wash solution with 1% BSA</td></tr><tr><td>Coating solution</td><td></td><td></td><td></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>medium optimized for prenatal and embryonic neurons</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Wash solution</td><td></td><td></td><td>[mg/L]</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;-6H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>100</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;-7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>100</td></tr><tr><td>KCl</td><td></td><td></td><td>400</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td>60</td></tr><tr><td>NaCl</td><td></td><td></td><td>8,000</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; anhydrous</td><td></td><td></td><td>48</td></tr><tr><td>D-Glucose (Dextrose)</td><td></td><td></td><td>1,000</td></tr><tr><td colspan="4">According to the protocol on http://www.lifetechnologies.com/ch/en/home/technical-resources/media-formulation.152.html</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>1</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>Blocking Solution</td><td></td><td></td><td></td></tr><tr><td>Detergent: Triton X-100</td><td></td><td></td><td>0.3%, harmful if swallowed</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td>1%</td></tr><tr><td>Goat serum</td><td>Gibco</td><td>16210-064</td><td>5%</td></tr><tr><td>Chicken Plasma</td><td>Sigma-Aldrich</td><td>P2366</td><td>Lyophilized, reconstitute with wash solution</td></tr><tr><td>Gabazine</td><td>Sigma-Aldrich</td><td>SR-95531</td><td></td></tr><tr><td>Mecamylamine hydrochloride</td><td>Tocris</td><td>2843</td><td>toxic if swallowed</td></tr><tr><td>Micropipette</td><td>World Precision Instruments, Inc.</td><td>MF28G-5</td><td></td></tr><tr><td>Paraformaldehyde</td><td></td><td></td><td>harmful, 4%</td></tr><tr><td>SMI-31</td><td>Covance</td><td>SMI-31P</td><td></td></tr><tr><td>SMI-32</td><td>Covance</td><td>SMI-32R-500</td><td></td></tr><tr><td>Strychnine</td><td>Sigma-Aldrich</td><td>S0532</td><td>toxic</td></tr><tr><td>Thrombin</td><td>Merck Millipore</td><td>1123740001</td><td>irritant, sensitizing, reconstitute with wash solution</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>

investigating functional regeneration in organotypic spinal cord co-cultures grown on multi-electrode arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

To investigate the potential for functional recovery of the co-cultures derived from the spinal cord of E14 rat embryos, lesions were performed in a time window of 8 to 28 DIV. 2 to 3 weeks later, the spontaneous neuronal activity was recorded with the MEAs (Figure 1 C&#38;D). The extracellularly recorded action potentials were identified offline for each individual electrode (Figure 1E). From these data raster plots were generated (Figure 1F), followed by network activity plots to visualize the total activity separately per side (Figure 1G). In each slice the spontaneous activity is usually organized in bursts. If the slices are functionally connected, these bursts can propagate from one slice to the other. Therefore, the amount of synchronized bursts between the two slices was calculated to quantitatively measure functional regeneration. For a detailed description of how the transformation into the different plots is performed and how the formula for calculating the percentage of synchronized activity was derived, please see Heidemann et al. 10.
In all experiments, the activity was first recorded under standard conditions. In a second step, synaptic inhibition was blocked by adding strychnine (1 &#181;M) and gabazine (10 &#181;M) to the extracellular bath solution. This disinhibition leads usually to a more regular activity pattern with prolonged bursts and burst intervals, which facilitates the definition of bursts and thus the determination of burst synchronization between the slices.
Cultures lesioned at a young age (7 - 9 DIV) displayed a high amount of synchronized activity 2-3 weeks after lesion whereas cultures lesioned at later stages (&#62;19 DIV) showed a distinct reduction in the ability to regenerate (Figure 2 A - F). These findings indicate that the regeneration ability of functional connections in spinal cord slice cultures decreases from a high level, representing the embryonic state in vivo, to a low level, representing the further developed state in vivo, within three weeks in culture.
What kinds of connections are actually involved in burst synchronization between the two slices? Besides propriospinal connections, the fibers could also arise from motoneurons or dorsal root ganglion neurons. It has been shown previously that dorsal root ganglion neurons are not relevant for the functional connectivity between the spinal cord slices 10. However, the involvement of motoneurons remained a possibility. Since motoneurons are known to form cholinergic connections to spinal cord neurons through nicotinic receptors 13, the activity of spinal cord co-cultures was recorded at 8 DIV, a time when the cultures show highly synchronized activity 10, and the nicotinic antagonist Mecamylamine (MEC, 100 &#181;M) was added to the extracellular bath solution. A participation of motoneurons in the connection between the two slices would result in a decreased percentage of synchronized activity. However, this was not the case (MEC: 91.0 &#177; 4.5%, n = 7; control: 93.6 &#177; 4.6%, n = 7, p &#62;0.05, Wilcoxon matched pairs test). These results suggest that cholinergic synapses do not contribute to the functional connection between slices. However, since motoneurons have also been shown to release glutamate at synapses within the CNS 11, 15, these experiments do not entirely exclude their involvement.
To identify motoneurons immunohistochemical stainings against the non-phosphorylated neurofilament H with SMI-32 were performed. They revealed several labelled large cell bodies typical for motoneurons, distributed over the slices (Figure 3A). The SMI-32 antibody has been reported to label the cell bodies of motoneurons 12 and this finding holds also true for the used cultures 13. Also, stainings of neuronal cell bodies in general, e.g., against b-III-tubulin or NeuN as well as glial cell bodies, e.g., astrocytes with GFAP antibody are in line with other reports and match the morphological descriptions of these cell types (Figure 3 B - D). Nevertheless, the labelling of axons with the SMI-32 antibody is concerning (Figure 3E). Against expectations, a huge network of fibers appears when using this antibody and it looks similar to the SMI-31 staining that labels phosphorylated neurofilament H (Figure 3F). One interpretation of this result could be that the developmental regulation of neurofilament phosphorylation/dephosphorylation is not as tight in the used cultures compared to the in vivo situation. A mixed occurrence of phosphorylated and non-phosphorylated neurofilaments in the same axon could explain this finding. Another possibility is that the SMI-32 labelled axons arise from projection neurons. Tsang and colleagues 16 have shown that e.g., Clark&#8217;s column and intermediolateral cell column neurons are stained by SMI-32 besides motoneurons. Pronounced neurite proliferation of such neurons could also explain the abundance of SMI-32 positive fibers.
<img alt="Figure 1" src="/files/ftp_upload/53121/53121fig1.jpg" /> 
Figure 1.&#160;Display and Analysis of Spontaneous Activity.&#160;(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. The diagrams are kindly provided by Dr. M. Heuschkel. (C) Bright-field image of an 8 DIV old culture. The slices have grown and fused along the sides facing each other. The yellow bar represents the electrode- and insulation-free groove of the MEA. Scale bar = 400 &#181;m (D) Timeline of experiments. Two spinal cord slices of E14 rat embryos are placed next to each other on MEAs. Within a few days, the slices grow and fuse along the sides facing each other. In a time frame of 8 - 28 DIV, complete lesions are performed through the fusion site. Two to three weeks later the spontaneous activity is recorded and the cultures are fixed for immunohistochemical stainings. (E) Spontaneous activity traces of each individual electrode of a 23 DIV old culture. For clearer visualization, only every second trace is illustrated. Orange traces depict activity that has been recorded from the slice on the right side, blue traces from the left side. Most of the bursts are synchronized between the two. The arrow points to a burst occurring in the left slice that only partially propagated to the right slice. The activity in the right slice however did not reach the chosen threshold of at least 25% of the averaged maximal peak activity of the according side and therefore, is not detected as a burst. Magnifications on the right depict the last synchronized burst pair. (F) Raster plot of the activity shown in (E). (G) Network activity plot with defined bursts (bars below baseline) of the activity shown in (E). Adapted from Heidemann et al. 10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53121/53121fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53121/53121fig2.jpg" /> 
Figure 2.&#160;Synchronized versus Not Synchronized Activity.&#160;(A, B) Raster plots of cultures lesioned at a young age (at 7 - 9 DIV), showing synchronized activity under standard conditions (A) and under disinhibition (B). (C, D) Raster plots of cultures lesioned at an old age (at&#160;&#62;19 DIV), showing not synchronized activity under standard conditions (C) and under disinhibition (D). (E, F) Average percentage of synchronized activity plotted for each individual lesion day recorded under standard conditions (E) and under disinhibition (F). Recordings were taken 2 - 3 weeks after the day of lesion. Adapted from Heidemann et al. 10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53121/53121fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53121/53121fig3.jpg" /> 
Figure 3.&#160;Immunohistochemical Characterization.&#160;(A) Staining of motoneuronal cell bodies with SMI-32. (B) &#946;-III tubulin labeling of mature neuronal cell bodies and their processes. (C) Identification of astrocytes with GFAP. (D) Labeling of mature neuronal cell body with NeuN. (E) SMI-32 staining of non-phosphorylated neurofilament H. (F) Phosphorylated neurofilament H identified by SMI-31. Adapted from Heidemann et al. 10. Note that with both, SMI-31 and SMI-32, a large network of fibers is visible in the cultures. Bars A-D = 20 &#181;m, H&#38;F = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53121/53121fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays at JoVE.com

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

investigating-functional-regeneration-organotypic-spinal-cord-co

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.0K Views.  University of Bern.  Hier presenteren we een protocol dat is gebaseerd op gekweekt ruggemerg segmenten op multi-elektrode arrays functionele regeneratie van propriospinal verbindingen in vitro bestuderen. 

Video: Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Bioengineering

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

Studying the Regeneration of Functional Connections between Spinal Cord Slices Using a Multi-Electrode Array

Source: Heidemann, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53121">Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays.</a> J. Vis. Exp. (2015).
The video demonstrates the functional regeneration of propriospinal connections using a multi-electrode array (MEA)-based assay. The spinal cord slices are placed on the MEA and are allowed to fuse. The fused slices are mechanically separated by creating a lesion, inducing propriospinal neurons to grow over the lesion and connect the slices. The electrical activity of the slices is assessed to identify the regeneration of functional connections.

1. Preparation of MEAs
Note: MEAs are composed of a glass substrate, micro-fabricated metal electrodes and a SU-8 polymer insulation layer. For the ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

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

Source: Heidemann, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.

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

Neurophysiology

Electrophysiology Techniques

Culturing Murine Spiral Ganglion Neuron Explants on Multielectrode Arrays

Source: Hahnewald, S., et al <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54538">Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays.</a> J. Vis. Exp. (2016)
This video demonstrates the process for isolating spiral ganglion explants from the inner ear and co-culturing them with the organ of Corti on a multielectrode array. Spiral ganglia are carefully extracted and sectioned into smaller tissue sections or explants. These explants are cultured on the multielectrode array&#39;s active area to promote neuronal process outgrowth.

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

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