This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia/ Minist&#233;rio da Ci&#234;ncia, Tecnologia e Ensino Superior in the framework of the project PTDC/CTM-NAN/3146/2014 (POCI-01-0145-FEDER-016623). Jos&#233; C Mateus was supported by FCT (PD/BD/135491/2018). Paulo Aguiar was supported by Programa Ci&#234;ncia - Programa Operacional Potencial Humano (POPH) - Promotion of Scientific Employment, ESF and MCTES and program Investigador FCT, POPH and Fundo Social Europeu. The microfluidic devices were fabricated at INESC - Microsystems and Nanotechnologies, Portugal, under the supervision of Jo&#227;o Pedro Conde and Virginia Chu.

All procedures involving animals were performed according to the European Union (EU) Directive 2010/63/EU (transposed to Portuguese legislation by Decreto-Lei 113/2013). The experimental protocol (0421/000/000/2017) was approved by the ethics committee of both the Portuguese Official Authority on animal welfare and experimentation (Dire&#231;&#227;o-Geral de Alimenta&#231;&#227;o e Veterin&#225;ria - DGAV) and of the host Institution.
1. Preparation of Culture Media and Other Solutions<...

<ol>
	<li>Scanziani, M., Hausser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+in+the+age+of+light.">Electrophysiology in the age of light.</a> Nature. 461 (7266), 930-939 (2009).</li><li>Nam, Y., Wheeler, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+microelectrode+array+technology+and+neural+recordings.">In vitro microelectrode array technology and neural recordings.</a> Critical Reviews in Biomedical Engineering. 39 (1), 45-61 (2011).</li><li>Obien, M. E., Deligkaris, K., Bullmann, T., Bakkum, D. J., Frey, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+neuronal+function+through+microelectrode+array+recordings.">Revealing neuronal function through microelectrode array recordings.</a> Frontiers in Neuroscience. 8, 423 (2014).</li><li>Blau, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+promotion+strategies+for+signal+transduction+enhancement+in+microelectrode+array+in+vitro+electrophysiology:+An+introductory+overview+and+critical+discussion.">Cell adhesion promotion strategies for signal transduction enhancement in microelectrode array in vitro electrophysiology: An introductory overview and critical discussion.</a> Current Opinion in Colloid &amp; Interface Science. 18 (5), 481-492 (2013).</li><li>Jones, I. L., 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=The+potential+of+microelectrode+arrays+and+microelectronics+for+biomedical+research+and+diagnostics.">The potential of microelectrode arrays and microelectronics for biomedical research and diagnostics.</a> Analytical and Bioanalytical Chemistry. 399 (7), 2313-2329 (2011).</li><li>Bakkum, D. J., 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=Tracking+axonal+action+potential+propagation+on+a+high-density+microelectrode+array+across+hundreds+of+sites.">Tracking axonal action potential propagation on a high-density microelectrode array across hundreds of sites.</a> Nature Communications. 4, 2181 (2013).</li><li>Claverol-Tinture, E., Cabestany, J., Rosell, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisite+recording+of+extracellular+potentials+produced+by+microchannel-confined+neurons+in+vitro.">Multisite recording of extracellular potentials produced by microchannel-confined neurons in vitro.</a> IEEE Transactions on Biomedical Engineering. 54 (2), 331-335 (2007).</li><li>Fitzgerald, J. J., Lacour, S. P., McMahon, S. B., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microchannels+as+axonal+amplifiers.">Microchannels as axonal amplifiers.</a> IEEE Transactions on Biomedical Engineering. 55 (3), 1136-1146 (2008).</li><li>Dworak, B. J., Wheeler, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+MEA+platform+with+PDMS+microtunnels+enables+the+detection+of+action+potential+propagation+from+isolated+axons+in+culture.">Novel MEA platform with PDMS microtunnels enables the detection of action potential propagation from isolated axons in culture.</a> Lab on a Chip. 9 (3), 404-410 (2009).</li><li>Morin, F., 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=Constraining+the+connectivity+of+neuronal+networks+cultured+on+microelectrode+arrays+with+microfluidic+techniques:+a+step+towards+neuron-based+functional+chips.">Constraining the connectivity of neuronal networks cultured on microelectrode arrays with microfluidic techniques: a step towards neuron-based functional chips.</a> Biosensors and Bioelectronics. 21 (7), 1093-1100 (2006).</li><li>Pan, L., 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=Large+extracellular+spikes+recordable+from+axons+in+microtunnels.">Large extracellular spikes recordable from axons in microtunnels.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 22 (3), 453-459 (2014).</li><li>Lewandowska, M. K., Bakkum, D. J., Rompani, S. B., Hierlemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+large+extracellular+spikes+in+microchannels+along+many+axonal+sites+from+individual+neurons.">Recording large extracellular spikes in microchannels along many axonal sites from individual neurons.</a> PLoS One. 10 (3), e0118514 (2015).</li><li>Narula, U., 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=Narrow+microtunnel+technology+for+the+isolation+and+precise+identification+of+axonal+communication+among+distinct+hippocampal+subregion+networks.">Narrow microtunnel technology for the isolation and precise identification of axonal communication among distinct hippocampal subregion networks.</a> PLoS One. 12 (5), e0176868 (2017).</li><li>Forro, 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=Modular+microstructure+design+to+build+neuronal+networks+of+defined+functional+connectivity.">Modular microstructure design to build neuronal networks of defined functional connectivity.</a> Biosensors and Bioelectronics. 122, 75-87 (2018).</li><li>Frega, M., 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=Rapid+Neuronal+Differentiation+of+Induced+Pluripotent+Stem+Cells+for+Measuring+Network+Activity+on+Micro-electrode+Arrays.">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> Journal of Visualized Experiments. (119), (2017).</li><li>Tukker, A. M., Wijnolts, F. M. J., de Groot, A., Westerink, R. H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC-derived+neuronal+models+for+in+vitro+neurotoxicity+assessment.">Human iPSC-derived neuronal models for in vitro neurotoxicity assessment.</a> Neurotoxicology. 67, 215-225 (2018).</li><li>Hales, C. M., Rolston, J. D., 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=How+to+culture,+record+and+stimulate+neuronal+networks+on+micro-electrode+arrays+(MEAs).">How to culture, record and stimulate neuronal networks on micro-electrode arrays (MEAs).</a> Journal of Visualized Experiments. (39), (2010).</li><li>van Pelt, J., Wolters, P. S., Corner, M. A., Rutten, W. L., Ramakers, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+characterization+of+firing+dynamics+of+spontaneous+bursts+in+cultured+neural+networks.">Long-term characterization of firing dynamics of spontaneous bursts in cultured neural networks.</a> IEEE Transactions on Biomedical Engineering. 51 (11), 2051-2062 (2004).</li><li>Wagenaar, D. A., 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=An+extremely+rich+repertoire+of+bursting+patterns+during+the+development+of+cortical+cultures.">An extremely rich repertoire of bursting patterns during the development of cortical cultures.</a> BMC Neuroscience. 7, 11 (2006).</li><li>Bhattacharya, S., Datta, A., Berg, J. M., Gangopadhyay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+surface+wettability+of+poly(dimethyl)+siloxane+(PDMS)+and+glass+under+oxygen-plasma+treatment+and+correlation+with+bond+strength.">Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength.</a> Journal of Microelectromechanical Systems. 14 (3), 590-597 (2005).</li><li>Heiney, K., 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=.">.</a> &#956;SpikeHunter: An advanced computational tool for the analysis of neuronal communication and action potential propagation in microfluidic platforms. , (2018).</li></ol>

The protocol presented here shows how to assemble a &#181;EF, comprised of a microfluidic device and a MEA with standard commercially-available designs, and how to analyze the recorded data.
When designing an experiment, researchers must take into account that the in vitro model is limited by the MEA fixed grid, which constrains microgroove arrangements. The use of a particular microfluidic or MEA design will depend on the specific experimental needs but, in general, the same procedur...

Understanding electrical communication in neuronal circuits is a fundamental step to reveal normal function, and devise therapeutic strategies to address dysfunction. Neurons integrate, compute and relay action potentials (APs) which propagate along their thin axons. Traditional electrophysiological techniques (e.g., patch clamp) are powerful techniques to study neuronal activity but are often limited to the larger cellular structures, such as the soma or the dendrites. Imaging techniques offer an alternative to study axonal signals with high spatial resolution, but they are technically difficult to perform and do not allow long-term measurements1. In this context, the combination of microelectrode arrays (MEAs) and microfluidics can make a powerful contribution in disclosing the fundamental properties of neuron&#180;s activity and signal transmission within neuronal networks in vitro2,3.
MEA technology relies on extracellular recordings of neuronal cultures. The main advantages of this electrophysiological methodology are its ability to support long-term, simultaneous stimulation and recording at multiple sites and in a non-invasive way3. MEAs are made of biocompatible, high conductive and corrosion resistant microelectrodes embedded in a glass wafer substrate. They are compatible with conventional cell culture bio-coatings, which by promoting cell adhesion significantly increase the sealing resistance between the substrate and cells3,4. Moreover, they are versatile in design and may vary in microelectrodes size, geometry and density. Overall, MEAs work as conventional cell culture vessels with the advantage of allowing concurrent live-imaging and electrophysiological recordings/stimulation.
The use of MEA technology has contributed to the study of important features of neural networks5. However, there are inherent features that limit the performance of MEAs for studying communication and APs propagation in a neuronal circuit. MEAs enable recordings from single cells and even subcellular structures like axons, but when compared to somal signals, axonal signals have a very low signal-to-noise ratio (SNR)6. Moreover, the characteristic of sensing extracellular field potentials from all sources around every microelectrode hampers the tracking of signal propagation in a neuronal circuit.
Recent studies have demonstrated, however, that better recording conditions can be achieved by having the microelectrodes aligned within narrow microchannels into which axons can grow. This configuration provides a significant increase in the SNR such that propagating axonal signals can be easily detected7,8,9,10,11,12,13. The strategy of allying microfluidic devices with MEA technology creates an electrically isolated microenvironment suitable to amplify axonal signals11. Moreover, the presence of multiple sensing microelectrodes along a microgroove is fundamental for detection and characterization of axonal signal propagation.
Such in vitro platforms with highly controllable neuronal network topographies can be adapted to many research questions14. These platforms are suitable to be used in the context of neuronal cultures but can be expanded to engineer co-culture configurations, where the communication between neurons and other cell types can be monitored and assessed. This setup thus provides very interesting conditions to explore a number of neural-related studies such as neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration. Furthermore, its combination with emerging models of human induced pluripotent stem cells15,16 can open new avenues in the development of potential therapies for human diseases that affect the nervous system.
Our lab is using this platform combining microelectrodes with microfluidics (&#181;EF) to understand neuronal processes at the cellular and network level and their implication in the physio- and pathologic nervous system. Given the value of such platform in the field of neuroscience, the purpose of this protocol is to demonstrate how to create a compartmentalized neuronal culture over substrate-integrated MEAs, how to culture neurons in this platform and how to successfully record, analyze and interpret the results from such experiments. This protocol will certainly enrich the experimental toolbox for neuronal cultures in the study of neural communication.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B-27 Suplement (50X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>LTI17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Branched poly(ethylene imine) (PEI), 25 kDa</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td>Purify branched PEI by dialysis using a 2.5 kDa cut-off membrane for 3 days at 4&#176;C against a 5 mM HCl solution (renewed daily). Freeze-dry the purified PEI.</td>
		</tr>
		<tr>
			<td>Cell strainer (40 &#181;m)&#160;</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical microtubes (1.5 ml)</td>
			<td>VWR</td>
			<td>211-0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable diaper, 60x40 cm</td>
			<td>Bastos Viegas SA</td>
			<td>455-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5, straight</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5/45</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #7, curved</td>
			<td>Fine Science Tools</td>
			<td>91197-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Fetal Bovine Serum Premium</td>
			<td>Biowest</td>
			<td>S181BH-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin from Engelbreth-Holm-Swarm&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td>Prepare laminin stock solution at 1 mg/mL by dissolving the powder in the respective volume of non-supplemented medium. Store laminin solution at -20 &#176;C in small aliquots (20 &#181;L) to avoid repeated freeze/thaw cycles.</td>
		</tr>
		<tr>
			<td>L-Glutamine 200mM&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>LTID25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer improved counting chamber (hemocytometer)</td>
			<td>Marienfeld</td>
			<td>630010</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103-049</td>
			<td>Basal medium used for neuronal cultures</td>
		</tr>
		<tr>
			<td>PDMS microfluidic devices&#160;</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>Composed of two cell seeding compartments interconnected by 20 microgrooves with 450 &#956;m length &#215; 10 &#956;m height &#215; 14 &#956;m width dimensions and separated by 86 &#181;m (total interspace of 100 &#956;m).</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (P/S) solution (100X)</td>
			<td>Biowest</td>
			<td>L0022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PES syringe filter unit (&#216; 30 mm), 0.22 &#181;m&#160;</td>
			<td>Frilabo</td>
			<td>1730012</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene conical tubes, 15 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>07-200-886</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene conical tubes, 50 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-539-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene disposabel serological pipets, 10 ml&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene disposabel serological pipets, 5 ml&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Regenerated cellulose membrane (2 kDa)&#160;&#160;</td>
			<td>Spectrum labs</td>
			<td>132107</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard surgical scissor</td>
			<td>Fine Science Tools</td>
			<td>91401-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate-integrated planar MEAs (256 microelectrodes)</td>
			<td>Multi Channel Systems</td>
			<td>256MEA100/30iR-ITO</td>
			<td>252 titanium nitride (TiN) recording electrodes and 4 internal reference electrodes organized in a 16 by 16 square grid. Each recording electrode is 30 &#181;m in diameter and interspaced by 100 &#181;m.</td>
		</tr>
		<tr>
			<td>Syringe luer-lock tip, 10 ml&#160;</td>
			<td>Terumo Europe</td>
			<td>5100-X00V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe luer-lock tip, 50 ml&#160;</td>
			<td>Terumo Europe</td>
			<td>8300006682</td>
			<td></td>
		</tr>
		<tr>
			<td>Terg-A-Zyme</td>
			<td>Sigma-Aldrich</td>
			<td>Z273287&#160;</td>
			<td>Enzyme-active powdered detergent used for MEAs cleaning</td>
		</tr>
		<tr>
			<td>Tissue culture plates, 35 mm&#160;</td>
			<td>StemCell Technologies</td>
			<td>27150</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plates, 90 mm</td>
			<td>Frilabo</td>
			<td>900095</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution (0.4%)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (1:250)</td>
			<td>Thermo Fisher Scientific</td>
			<td>27250018</td>
			<td></td>
		</tr>
		<tr>
			<td>Vinyl tape 471</td>
			<td>3M</td>
			<td>B40071909</td>
			<td></td>
		</tr>
	</tbody>
</table>

interfacing microfluidics with microelectrode arrays for studying neuronal communication and axonal signal propagation

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

Using the protocol described here, E-18 rat cortical neurons seeded on &#181;EF are able to develop and remain healthy in these culture conditions for over a month. As soon as 3 to 5 days in culture, cortical neurons grow their axons through microgrooves towards the axonal compartment of &#181;EF (Figure 1). After 15 days in culture, cortical neurons cultured on &#181;EF are expected to exhibit steady levels of activity and propagation of action potentials al...

Watch this Scientific Journal Video about Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation at JoVE.com

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

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

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

The overall goal of this protocol is to demonstrate how to combine the use of micro fluidics with micro electrode arrays to generate the platform suitable for studying neuronal communication and axonal signal propagation. Hello, I'm Paulo Aguiar and I'm the PI of the Neuro engineering and Computation Neuroscience Lab at INEB, used to code for biomedical engineering at University of Porto, Portugal. Understanding information transmission in neuro circuits remains one of the biggest challenges in neuroscience.Today, I'm going to show you how we can combine individual micro actuator arrays with micro fluidics and software tools developed here at our lab, to detect and characterize propagating action potentials. We use this setup in a variety of studies, namely neural coding and decoding, and in communications between sensory neurons and other cell types. We believe that the visual demonstration of this protocol is crucial.Since micro fluidic and micro electrode arrays assembly is that difficulty in managing, and difficult to reproduce just by reproducing protocols. For the market, cultary sales on this platform is not straightforward, as the conventional ABAs, and it requires special attention to the cell sitting stuff, and culture magnets. This setup allows us to study several communication related properties, such as propagation velocity and direction, in a well controlled environment.Although the recording procedure is simple, data analysis requires knowledge and the use of the right computational tools. On the day before cell sitting, start by preparing the micro fluidic devices, by cleaning down with vinyl tape. Gently press the tape against the device to reach all areas of the micro fluidic.Air plasma clean the MEA for three minutes to clean the surface and make it more hydrophilic. Inside a laminar flow hood, submerge the micro fluidics in 70 percent ethanol. Allow them to air dry.Place each MEA in a petri dish, and sterilize by 15 minutes of UV light exposure. Then, go to MEA's central area with 500 microliters of PEI solution. Incubate for at least one hour.Inside a laminar flow hood, aspirate the PEI coating solution from the MEA surface. Then, wash the MEAs four times with one milliliter of sterile water. With the help of a sterile microscope, placed inside the laminar flow hood.Center the MEA chip, and add one milliliter of sterile water to the central area of the MEA. Next, place the micro fluidic device on top of the MEA surface, and carefully align the microgrooves micro active grid of the MEA. Aspirate the excessive water, and incubate overnight at 37 degrees, to allow complete attachment.On the next day, load the wells with laminin, and incubate again. After collecting the embryo susseds, proceed to the prefrontal cortex dissection. Start by carefully exposing the brain, using a pair of forceps.Then, gently remove the brain from it's cavity. Cover it with cold H-HBSS. When present, remove and discard the olfactory bulbs.Finally, dissect the prefrontal cortex. Ensure the complete removal of the meninges. Repeat this procedure for both hemispheres.For the number of brains required for your study. Inside a laminar flow hood, collect the cortex fragments previously dissected in a total of five milliliters of H-HBSS. Then, transfer these fragments to a sterile 15 milliliter chronical tube.Pipette, and have 2.3 milliliters of freshly prepared trypsin solution to the tube containing the cortex fragments. Mix the suspension, and incubate at 37 degrees for 15 minutes. Agitate every five minutes.Then, discard the supernatant containing the trypsin, add H-HBSS containing FBS, to inactivate the remaining trypsin. Gently agitate. Let the cortex fragments settle down, and discard the supernatant.Wash two times with H-HBSS, to remove the FBS, and discard the supernatant again. Add supplemented neural basal medium, and mechanically dissociate tissues by slowly pipetting up and down. Discard the remaining tissue by filtering the salt suspension through a cell strainer.Then, mix 20 microliters of cell suspension with 20 microliters of trypan blue. Transfer 10 microliters to a Neubauer chamber, and count the number of viable cells. Immediately before cell sitting, remove laminin from all wells of the micro EF.Then, add the small volume of supernatant medium to each well of the axonal compartment.Slowly seed five microliters of cell suspension to the somal compartment. Incubate at 37 degrees for one hour to allow cell attachment to the substrate. After one hour, gently fill each well with warm supplemented medium.Add small volumes to avoid cell detachment. Then, transfer the micro EF to an incubator. Here is shown a culture at five days in vitro.After one week, cultures start exhibiting spontaneous activity. Before performing recordings, use a temperature controller to eat the pre amp fire base plate. Then, place the micro EF on the preamplifier, and close and latch the lid.To record, open the multi channel experimental software. Set the sampling rate to 20 kilohertz. Drag the filter and recorded boxes, and connect them sequentially to record both raw and filtered data.Start the data acquisition to visualize the signals, and quick record. The recorded data can be quickly explored using multi channel analyzer. Here, the signal propagation along the microgrooves is already clear.To identify and characterize this propagating events, open your recording in micro spike hunter. Click the file info button to access the recording settings, and select the set of micro electrodes for analysis. Set the threshold for propagating event detection.And quick read data. The software will list all the detected sequences. The user can then assess the propagation velocity as the information, between pairs of micro electrodes, and based on their inter distance and cross correlation.The chemo graph tool allows the user to manually estimate the propagation velocity. Here is shown a propagating event. Detected along four micro electrodes within a microgroove.These events can be represented in a Rasser plot. Here is the propagating activity along 11 microgrooves for the first two minutes of recording. The high and the low activity microgroove are colored in yellow and red, respectfully.Notice synchronization of the activity along the 11 microgrooves. The synchronization is independent of the rate of activity. As can be seen in the variations of the instantaneous firing rate of the two microgrooves.Microgrooves also function as axonal signal amplifiers, as shown in the box and whiskers plot. We have shown you how to combine micro fluidics with MEAs, and how to culture neurons on them. Also, how to record and extract meaningful data from those recordings.We hope this demonstration to be useful for your research, why you may wanted to study the communication in neural circuits.

interfacing-microfluidics-with-microelectrode-arrays-for-studying

Research

JoVE Journal

Bioengineering

8.0K visualizações.  Universidade do Porto. O objetivo geral deste protocolo é demonstrar como combinar o uso de micro fluidez com microeletos para gerar a plataforma adequada para o estudo da comunicação neuronal e propagação de sinais axonacionais. Olá, sou Paulo Aguiar e sou o PI do Laboratório de Neurociências em Neuroengenharia e Computação do INEB, usado para codificação para engenharia biomédica na Universidade do Porto, Portugal. Entender a transmissão de informações em circuitos neurológicos continua sendo um ...