This work was funded by the National Science Foundation grant CMMI-1300007. We would like to acknowledge previous lab members, who have helped with the design of these protocols and with the maintenance of the cultures for over five years at George Mason University: Dr. Joseph J. Pancrazio, Dr. Hamid Charkhkar, Dr. Gretchen Knaack, Dr. Franz Hamilton, Michael Maquera, and Robert Graham.

<ol>
	<li>Charkhkar, H., Frewin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+cortical+neuronal+networks+for+in+vitro+material+biocompatibility+testing.">Use of cortical neuronal networks for in vitro material biocompatibility testing.</a> Biosens Bioelectron. 53, 316-323 (2014).</li><li>Bologna, L. L., Nieus, T., Tedesco, M., Chiappalone, M., Benfenati, F., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-frequency+stimulation+enhances+burst+activity+in+cortical+cultures+during+development.">Low-frequency stimulation enhances burst activity in cortical cultures during development.</a> Neuroscience. 165 (3), 692-704 (2010).</li><li>Fr&#246;hlich, F., McCormick, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+electric+fields+may+guide+neocortical+network+activity.">Endogenous electric fields may guide neocortical network activity.</a> Neuron. 67 (1), 129-143 (2010).</li><li>Anastassiou, C. A., Perin, R., Markram, H., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephaptic+coupling+of+cortical+neurons.">Ephaptic coupling of cortical neurons.</a> Nature Neurosci. 14 (2), 217-223 (2011).</li><li>Ozen, S., Sirota, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+electric+stimulation+entrains+cortical+neuronal+populations+in+rats.">Transcranial electric stimulation entrains cortical neuronal populations in rats.</a> J. Neurosci. 30 (34), 11476-11485 (2010).</li><li>Wagenaar, D. A., Madhavan, R., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+bursting+in+cortical+cultures+with+closed-loop+multi-electrode+stimulation.">Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation.</a> J. Neurosci. 25 (3), 680-688 (2005).</li><li>Shahaf, G., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+in+networks+of+cortical+neurons.">Learning in networks of cortical neurons.</a> J. Neurosci. 21 (22), 8782-8788 (2001).</li><li>Marom, S., Shahaf, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+learning+and+memory+in+large+random+networks+of+cortical+neurons:+lessons+beyond+anatomy.">Development, learning and memory in large random networks of cortical neurons: lessons beyond anatomy.</a> Q Rev Biophys. 35 (1), 63-87 (2002).</li><li>Marom, S., Eytan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+in+ex-vivo+developing+networks+of+cortical+neurons.">Learning in ex-vivo developing networks of cortical neurons.</a> Prog Brain Res. 147, 189-199 (2005).</li><li>Li, Y., Zhou, W., Li, X., Zeng, S., Luo, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+learning+in+cultured+neuronal+networks+with+antagonists+of+glutamate+receptors.">Dynamics of learning in cultured neuronal networks with antagonists of glutamate receptors.</a> Biophys. J. 93 (12), 4151-4158 (2007).</li><li>Li, Y., Zhou, W., Li, X., Zeng, S., Liu, M., Luo, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+synchronized+bursts+in+cultured+hippocampal+neuronal+networks+with+learning+training+on+microelectrode+arrays.">Characterization of synchronized bursts in cultured hippocampal neuronal networks with learning training on microelectrode arrays.</a> Biosens Bioelectron. 22 (12), 2976-2982 (2007).</li><li>Chiappalone, M., Massobrio, P., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+plasticity+in+cortical+assemblies.">Network plasticity in cortical assemblies.</a> Eur. J. Neurosci. 28 (1), 221-237 (2008).</li><li>le Feber, J., Stegenga, J., Rutten, W. L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+slow+electrical+stimuli+to+achieve+learning+in+cultured+networks+of+rat+cortical+neurons.">The effect of slow electrical stimuli to achieve learning in cultured networks of rat cortical neurons.</a> PloS one. 5 (1), 8871 (2010).</li><li>Ruaro, M. E., Bonifazi, P., Torre, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+neurocomputer:+image+processing+and+pattern+recognition+with+neuronal+cultures.">Toward the neurocomputer: image processing and pattern recognition with neuronal cultures.</a> IEEE Trans Biomed Eng. 52 (3), 371-383 (2005).</li><li>Hamilton, F., Graham, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-Dependent+Increase+in+Network+Response+to+Stimulation.">Time-Dependent Increase in Network Response to Stimulation.</a> PloS one. 10 (11), 0142399 (2015).</li><li>. Guidelines for Euthanasia of Rodents Using Carbon Dioxide Available from: <a target="_blank" href="https://oacu.oir.nih.gov/sites/default/files/uploads/arac-guidelines/rodent_euthanasia_adult.pdf">https://oacu.oir.nih.gov/sites/default/files/uploads/arac-guidelines/rodent_euthanasia_adult.pdf</a> (2016)</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells: Business Source. , (1998).</li><li>Gullo, F., Maffezzoli, A., Dossi, E., Wanke, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-latency+cross-and+autocorrelation+identify+clusters+of+interacting+cortical+neurons+recorded+from+multi-electrode+array.">Short-latency cross-and autocorrelation identify clusters of interacting cortical neurons recorded from multi-electrode array.</a> J. Neurosci. Methods. 181, 186-198 (2009).</li><li>Eytan, D., Brenner, N., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Adaptation+in+Networks+of+Cortical+Neurons.">Selective Adaptation in Networks of Cortical Neurons.</a> J. Neurosci. 23 (28), (2003).</li><li>Bonifazi, P., Ruaro, M. E., Torre, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+properties+of+information+processing+in+neuronal+networks.">Statistical properties of information processing in neuronal networks.</a> Eur. J. Neurosci. 22 (11), 2953-2964 (2005).</li></ol>

The authors have nothing to disclose.

The steps outlined in this protocol provide sufficient detail for the beginner to plate his/her own neuronal cultures on MEAs and to record network activity. This protocol will help to ensure that the cultures adhere properly, forming a carpet layer of cells over the electrode arrays, and remain healthy and contaminant-free for months.
Although it is best to adhere to all parts of the protocol, there are steps throughout the process that are critical to the successful outcome. The use of aseptic technique throughout the entire process is imperative to prevent the cultures from becoming contaminated. New MEAs must be made hydrophilic, as described in the protocol, or else poor cell adhesion will result. Avoiding harsh pipetting and the formation of air bubbles during dissociation will reduce the number of damaged cells plated and will lead to a higher and healthier yield. Switching from DMEM 5/5 to DMEM+ after the first feeding is also important. DMEM 5/5 contains horse serum, which will cause glial cells to dominate the culture if used continuously and will result in poor neuronal activity, although the cultures will appear healthy17. Feeding the cultures as scheduled and keeping them in proper incubating conditions is also crucial.
Plating cell cultures on MEAs involves many variables that can lead to less-than-optimal results. Although the goal is a perfect &#34;carpet&#34; of cells, failure to address the critical steps mentioned above will result in poor cell maturation or in contamination. Poor cell adhesion, which is different from poor cell maturation, is also a concern. This can be caused by several factors, including poor MEA preparation prior to plating or the use of old medium. If old medium containing a stabilized form of L-glutamine and serum-free supplement for neural cell culture (see Materials Table) is used, the cells initially adhere but then float away after about two weeks. If bacterial contamination is a persistent problem, an antibiotic, such as ampicillin or pen-strep, can be added to the medium. There are also fungicides available to treat fungal contamination. These are some of the more common variables that can affect the outcome of the cultures. There are many others that will only be encountered after time and experience.
In comparison to the use of glass microelectrodes, this technique is excellent for studying network dynamics and pharmacological responses. It enables the use of many different spatio-temporal stimulation patterns and allows for the recording of neuronal responses from multiple areas at once. Previous groups have demonstrated interesting results using protocols similar to the ones described here18. Since the cultures last for weeks or months and the same cultures can be reused, this technique also allows for multiple experiments over time on the same network.
However, there are limitations to this technique. MEAs are non-invasive. Therefore, they can only record extracellular activity, as opposed to patch-clamping or intracellular recording with pipettes. Moreover, since each electrode in an array is covered by several cells, it is not possible to resolve the activity of a single neuron. Conversely, because these are in vitro cultures, they cannot fully reproduce the structural properties of networks in the brain. Also, activity can only be recorded for less than 30 min at a time without some mechanism providing a CO2 atmosphere for the cells to maintain their pH balance.
Once this technique is mastered,pharmacological manipulations with or without electrical stimulation can be explored. New protocols to probe learning and memory formation in neuronal networks can also be designed and tested, along with protocols for hippocampal or spinal cord networks. Protocols for the stimulation and training of networks have been previously published, and some of these were further developed into in vivo protocols, such as the &#34;selective adaptation&#34; proposed by Eytan et al.19. Several protocols were tested. However, only results from a modification to the tetanus procedure proposed by Ruaro in 2005 are presented here14,20.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures1. In contrast to more traditional methods-such as patch-clamping, which can only record activity from a single cell, or field recording with a glass pipette, which can record extracellular responses from the neurons surrounding the electrode at a single site-MEAs can simultaneously record from multiple sites in a cell culture without requiring the arduous task of placing each electrode individually. This allows for the study of the dynamic interactions between groups of cells that form a network within that culture. Moreover, the effects of electrical stimulation on network firing patterns2,3,4,5 and network control6 in neuronal cultures have been well documented, and numerous configurations of electrical stimulation and controls can be easily applied within the same experimental setup, allowing for a broad range of spatio-temporal dynamics to be explored.
One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning7,8,9,10,11,12,13. The Peixoto Lab previously examined the effects of high-frequency training signals, as described in Ruaro et al.14, on networks of mouse neurons plated on microelectrode arrays15. In these experiments, networks displayed an increase in response following training induced by electrical stimulation. The increased response was considered a form of learning via stimulus recognition, whereby the networks responded in a consistent manner to a change in stimulus after the application of a specific stimulation (i.e., training) protocol.
This protocol demonstrates how to culture neuronal cells on MEAs, successfully record from over 95% of the plated dishes, establish a protocol to train the networks to respond to patterns of stimulation, sort single-unit activity, plot histograms, and interpret the results from such experiments. The use of a proprietary system (see the Table of Materials) for the stimulation and recording of neuronal cultures is demonstrated, as well as the application of software packages (see the Table of Materials) to sort neuronal units. A custom-designed graphical user interface (see the Table of Materials) is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol.

<table><tbody><tr><td>Ascorbic Acid</td><td>Sigma Aldrich</td><td>A4403</td><td>Make 4 mg/mL aliquots</td></tr><tr><td>B-27</td><td>Invitrogen</td><td>17504-044</td><td>Serum-free supplement for neural cell culture. Make 1 mL aliquots and freeze</td></tr><tr><td>Beakers - glass - 100 mL</td><td>VWR</td><td>13912-160</td><td></td></tr><tr><td>Beakers - glass - 500 mL</td><td>VWR</td><td>10754-956</td><td></td></tr><tr><td>Blunt-tipped thumb forceps</td><td>World Precision Instruments</td><td>500365-G</td><td></td></tr><tr><td>Cryogenic vial - sterile, 2 mL</td><td>Fisher Scientific</td><td>10-500-26</td><td></td></tr><tr><td>Cryogenic vial - sterile, 5 mL</td><td>Fisher Scientific</td><td>10-500-27</td><td></td></tr><tr><td>DMEM with Glutamax</td><td>Gibco Life Technology</td><td>10569-010</td><td>Cell culture media that contains a stabilized form of L-glutamine</td></tr><tr><td>DNase</td><td>Worthington Biochemical</td><td>LK003172</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>04-355-451</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>ATCC</td><td>30-2030</td><td></td></tr><tr><td>Fine-tipped thumb forceps</td><td>World Precision Instruments</td><td>501324-G</td><td></td></tr><tr><td>Glass Pipets</td><td>VWR</td><td>14673-010</td><td></td></tr><tr><td>GlutaMAX -- I (100X)</td><td>Gibco Life Technology</td><td>35050-061</td><td>A stabilized form of L-glutamine used as a suplement</td></tr><tr><td>Hemocytometer chip</td><td>Fisher Scientific</td><td>22-600-101</td><td></td></tr><tr><td>Hibernate EB complete</td><td>BrainBits</td><td>D00118</td><td>Ambient CO&lt;sup&gt;2&lt;/sup&gt; cell storage media</td></tr><tr><td>Horse Serum</td><td>Atlanta Biologicals</td><td>S12195H</td><td></td></tr><tr><td>Laminin</td><td>Sigma Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>MCS filter amplifier</td><td>MultiChannel System</td><td>FA60SBC</td><td></td></tr><tr><td>MCS headstage/pre-amplifier</td><td>MultiChannel System</td><td>MEA1060-INV</td><td></td></tr><tr><td>MCS microelectrode array</td><td>MultiChannel System</td><td>60MEA200/10iR-ITO</td><td></td></tr><tr><td>MCS power supply</td><td>MultiChannel System</td><td>PS20W</td><td></td></tr><tr><td>MCS signal divider</td><td>MultiChannel System</td><td>SDMEA</td><td></td></tr><tr><td>MCS stimulus generator</td><td>MultiChannel System</td><td>STG4002</td><td></td></tr><tr><td>MCS temperature controller</td><td>MultiChannel System</td><td>TC02</td><td></td></tr><tr><td>Media storage bottle - glass 500 mL</td><td>VWR</td><td>10754-818</td><td>Autoclave</td></tr><tr><td>Microcentrifuge tubes - 0.4 mL</td><td>Thermo Fisher</td><td>3485</td><td>Autoclave</td></tr><tr><td>Microcentrifuge tubes - 2 mL</td><td>Thermo Fisher</td><td>3434ECONO</td><td>Autoclave</td></tr><tr><td>Micropipette tips - 10 &amp;mu;L</td><td>VWR</td><td>37001-166</td><td>Autoclave</td></tr><tr><td>Micropipette tips - 1,000 &amp;mu;L&amp;nbsp;</td><td>VWR</td><td>13503-464</td><td>Autoclave</td></tr><tr><td>Micropipette tips - 200 &amp;mu;L&amp;nbsp;</td><td>VWR</td><td>37001-596</td><td>Autoclave</td></tr><tr><td>Micropipetter - 10 &amp;mu;L&amp;nbsp;</td><td>Thermo Fisher</td><td>4641170N</td><td></td></tr><tr><td>Micropipetter - 1,000 &amp;mu;L&amp;nbsp;</td><td>Thermo Fisher</td><td>4641210N</td><td></td></tr><tr><td>Micropipetter - 200 &amp;mu;L&amp;nbsp;</td><td>Thermo Fisher</td><td>4641230N</td><td></td></tr><tr><td>Papain - 0.22 Filtered</td><td>Worthington Biochemical</td><td>LK003178</td><td></td></tr><tr><td>Penicillin-Streptomycin (Pen Strep)</td><td>Thermo Fisher</td><td>15070063</td><td></td></tr><tr><td>Petri dishes -100 mm disposable</td><td>Fisher Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>Petri dishes -100 mm glass</td><td>VWR</td><td>10754-788</td><td></td></tr><tr><td>Petri dishes -35 mm&amp;nbsp;</td><td>Fisher Scientific</td><td>08-757-100A</td><td></td></tr><tr><td>Phosphate buffer saline (PBS) (1x)</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Plasma Cleaning System</td><td>Plasma Etch, Inc.</td><td>PE-50</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide (PDL)</td><td>Sigma Aldrich</td><td>P6407-5MG</td><td></td></tr><tr><td>Polyethylene conical (centrifuge) tube -15 mL</td><td>Fisher Scientific</td><td>05-527-90</td><td></td></tr><tr><td>Polypropylene conical (centrifuge) tube -50 mL</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Procedure masks</td><td>Imco</td><td>1530-imc</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma Aldrich</td><td>P4562-5G</td><td></td></tr><tr><td>Scalpel blades</td><td>World Precision Instruments</td><td>&amp;nbsp;500237-G</td><td></td></tr><tr><td>Scissors - iris</td><td>World Precision Instruments</td><td>14111-G</td><td></td></tr><tr><td>Scissors - large</td><td>World Precision Instruments</td><td>14214</td><td></td></tr><tr><td>Scissors - small surgical</td><td>World Precision Instruments</td><td>&amp;nbsp;501733-G</td><td></td></tr><tr><td>Serological pipette - sterile, 1 mL&amp;nbsp;</td><td>VWR</td><td>89130-892</td><td></td></tr><tr><td>Serological pipette - sterile, 2 mL&amp;nbsp;</td><td>VWR</td><td>89130-894</td><td></td></tr><tr><td>Serological pipette - sterile, 25 mL&amp;nbsp;</td><td>VWR</td><td>89130-900</td><td></td></tr><tr><td>Serological pipette - sterile, 50 mL&amp;nbsp;</td><td>VWR</td><td>89130-902</td><td></td></tr><tr><td>Software: Matlab GUI</td><td>Peixoto Lab</td><td>npeixoto@gmu.edu</td><td>Used to analyze .mat files.&amp;nbsp; Available for free upon request.&amp;nbsp; Contact npeixoto@gmu.edu to request a copy.</td></tr><tr><td>Software: MCS MC_Rack</td><td>MultiChannel System</td><td>N/A</td><td>Used for data acquisition</td></tr><tr><td>Software: NeuroExplorer</td><td>Plexon&amp;nbsp;</td><td>http://www.plexon.com/products/neuroexplorer</td><td>Used to convert .nex files to .mat</td></tr><tr><td>Software: Offline Sorter</td><td>Plexon&amp;nbsp;</td><td>http://www.plexon.com/products/offline-sorter</td><td>Used to sort neural spikes and convert data files to .plx and then to .nex</td></tr><tr><td>Software Manual: MCS MC_Rack</td><td>MultiChannel System</td><td></td><td>https://www.multichannelsystems.com/sites/multichannelsystems.com/files/documents/manuals/MC_Rack_Manual.pdf</td></tr><tr><td>Software Manual: NeuroExplorer</td><td>Plexon&amp;nbsp;</td><td></td><td>https://www.neuroexplorer.com/downloads/Nex3Manual.pdf</td></tr><tr><td>Software Manual: Offline Sorter</td><td>Plexon&amp;nbsp;</td><td></td><td>www.plexon.com/system/files/downloads/Offline%20Sorter%20v2.8%20Manual.pdf</td></tr><tr><td>Spatula - small double-ended</td><td>World Precision Instruments</td><td>&amp;nbsp;503440</td><td></td></tr><tr><td>Stericup 0.22 &amp;micro;m pore filter - 250 mL</td><td>Millipore</td><td>SCGVU02RE</td><td></td></tr><tr><td>Transfer pipette - large bore&amp;nbsp;</td><td>Thermo Fisher</td><td>335-1S</td><td></td></tr><tr><td>Transfer pipette - small bore&amp;nbsp;</td><td>Thermo Fisher</td><td>242-1S</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma Aldrich</td><td>T8154-20ML</td><td></td></tr><tr><td>Whatman filter paper</td><td>Whatman</td><td>1442 150</td><td>Cut into 8 pie wedges and autoclave in a glass Petri dish</td></tr></tbody></table>

time-dependent increase in the network response to the stimulation of neuronal cell cultures on micro-electrode arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Watch this Scientific Journal Video about Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays at JoVE.com

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

time-dependent-increase-network-response-to-stimulation-neuronal-cell

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

9.8K Views.  George Mason University. Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Video: Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

Developmental Biology

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop working in various diseases. Studies have included neuropathological observation, fluorescent microscopy with genetic labeling, and intracellular recording in both dissociated neurons and slice preparations. This protocol discusses another technology, which involves growing dissociated neuronal cultures on micro-electrode arrays (also called multi-electrode arrays, MEAs).
There are multiple advantages to using this system over other technologies. Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with electrical stimulation sequences through the array's multiple electrodes. Because the network is small, the impact of stimulation is limited to observable areas, which is not the case in intact preparations. The cells grow in a monolayer making changes in morphology easy to monitor with various imaging techniques. Finally, cultures on MEAs can survive for over a year in vitro which removes any clear time limitations inherent with other culturing techniques.1
Our lab and others around the globe are utilizing this technology to ask important questions about neuronal networks. The purpose of this protocol is to provide the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

Biology

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

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.

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 ...

Applications of 3D-Printing for Micro-Scale Neuronal Cell Culture Devices

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Author Spotlight: Modular Neuronal Networks for Analyzing Brain Functions

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

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

Source: Cvetkovic, C., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

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 ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Summary

Explore More Videos

Chapters in this video

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

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