We would like to thank the members of the Potter group for providing invaluable comments on the protocol. This work was funded by a clinical research training fellowship from the American Academy of Neurology Foundation to CMH, a fellowship from the National Institute of General Medical Sciences (NIGMS; <a href="http://www.nigms.nih.gov/">http://www.nigms.nih.gov/</a>) to JDR (GM08169), a grant from the NIH National Institute of Neurological Disorders and Stroke (NS054809), a Ruth L. Kirschstein National Research Service Award to JDR (NS060392), an NSF EFRI grant (0836017), the Coulter Foundation Translational Research Award, and a translational research fellowship to JDR (NS007480).

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J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+growth+of+hippocampal+neurons+in+defined+medium+at+low+density:+advantages+of+a+sandwich+culture+technique+or+low+oxygen.">Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen.</a> Brain Res. 494, 65-74 (1989).</li><li>Lelong, I. H., Petegnief, V., Rebel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cells+mature+faster+on+polyethyleneimine+coated+plates+than+on+polylysine+coated+plates.">Neuronal cells mature faster on polyethyleneimine coated plates than on polylysine coated plates.</a> J Neurosci Res. 32, 562-568 (1992).</li><li>Potter, S. M., Pine, J., Fraser, S. 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M. <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+activity-dependent+plasticity+of+action+potential+propagation+delay+and+amplitude+in+cortical+networks.">Long-term activity-dependent plasticity of action potential propagation delay and amplitude in cortical networks.</a> PLoS One. 3 (5), e2088-e2088 (2008).</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>Jimbo, Y., Tateno, T., Robinson, H. 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CMH has nothing to disclose. 
JDR and SMP are paid consultants for Axion Biosystems. 
The submission fees and video processing fees for this article and video were graciously sponsored by Multi-Channel Systems.

This protocol shows instructions on how to culture and maintain neuronal networks on micro-electrode arrays as well as an introduction to recording from and stimulating neuronal networks. Some of the more common problems when recording from MEAs were also discussed in the MEA troubleshooting section. There are occasional variations in the protocol as discussed throughout and oftentimes these changes are utilized to optimize specific conditions required by certain experimental designs.
Although the recording and stimulating system presented here is comprised of our custom designed NeuroRighter15 with the MCS preamp, there are multiple other systems that can provide an interface with the neuronal networks. Choosing a particular setup will depend on the specific experimental needs.
Simplistic recording and stimulation examples were provided in this protocol however these cultures on MEAs can be used to provide insight into complex questions about synaptic activity and neuronal network connectivity. As mentioned above, ongoing work is utilizing this technology to study processes like cellular plasticity, development, excitotoxicity and neurodegeneration.6-10 For example, a recent publication from our laboratory showed reproducible goal directed learning in vitro with real-time closed loop stimulation in response to current neuronal network activity3.
Although MEA cultures are often finicky and delicate, thanks to their simplicity and accessibility (compared to intact animals), they provide a powerful model for studying neuronal activity at a network level.

Supplies
<ul>
 <li>Disposable bottle top filter (150ml, 0.2&mu;m pore size; Nalgene #565-0020)</li>
 <li>Embryonic day 18 timed-pregnant female rat</li>
 <li>Microelectrode arrays (see description in protocol, B1)</li>
 <li>Microelectrode array lids (see description in protocol, B3)</li>
 <li>Nylon cell strainer (40&mu;m, BD Falcon, #352340)</li>
 <li>Polypropylene conical vials (sterile, 15ml)</li>
 <li>Polystyrene Petri dishes (sterile, 35mm and 100x15mm)</li>
 <li>Pipet tips (sterile, 20&mu;l, 200&mu;l and 1000&mu;l)</li>
 <li>Serological pipets (5ml)</li>
</ul>
Equipment:
<ul>
 <li>Biological safety cabinet/laminar flow hood</li>
 <li>Cell counter</li>
 <li>Centrifuge</li>
 <li>Container of ice</li>
 <li>Dissecting scissors and forceps (sterilized)</li>
 <li>Dissecting microscope in a laminar flow hood</li>
 <li>Handheld electric pipetman </li>
 <li>Hemacytometer</li>
 <li>Isofluorane and gas chamber</li>
 <li>Light microscope</li>
 <li>Handheld pipetman (P-20 (20&mu;l), P-200 (200&mu;l), P-1000 (1000&mu;l))</li>
 <li>MEA recording system (see description in protocol, D1)</li>
 <li>Rodent Guillotine</li>
 <li>Tri-gas incubator (see description in protocol, B15)</li>
 <li>Vacuum flask with aspirator attached in the hood</li>
 <li>Vortex</li>
 <li>Water bath (35-37&deg;C)</li>
</ul>
Solutions and Reagents:
BSA solution: 
 
 Make 5% BSA (Sigma A-9418) in phosphate buffered saline pH 7.4 and filtered at 0.2&mu;m. This solution can be prepared ahead of time and stored at 4&deg;C for several months.
Cell Medium: 
 
 Combine 90 ml Dulbecco's modified Eagle's medium (Irvine Scientific 9024), 10 ml horse serum (Gibco 16050), 1ml sodium pyruvate (100mM, Gibco 11360), 250 &mu;l GlutaMAX (Gibco 35050), and insulin (final concentration 2.5 &mu;g/ml, Sigma I-5500) then pass through a 0.2 &mu;m filter.22 Warm and equilibrate the cell medium in the incubator prior to use. 
Dissection solution for preparing papain solution:23
<table border="1">
 <tr>
 <td>Dissection solution (100ml stock)</td>
 <td>Combine in order, filter 0.22 &mu;m and store at 4&deg;C.</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Component</td>
 <td>Volume ml</td>
 <td>Final Concentration (mM)</td>
 </tr>
 <tr>
 <td>Double-distilled water</td>
 <td>91.175</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1 M MgCl2</td>
 <td>0.58</td>
 <td>5.8 mM</td>
 </tr>
 <tr>
 <td>1 M CaCl2</td>
 <td>0.025</td>
 <td>0.25 mM</td>
 </tr>
 <tr>
 <td>0.5 M HEPES</td>
 <td>0.32</td>
 <td>1.6 mM</td>
 </tr>
 <tr>
 <td>0.5% Phenol red</td>
 <td>0.2</td>
 <td>(0.001%)</td>
 </tr>
 <tr>
 <td>0.1 N NaOH</td>
 <td>0.2</td>
 <td>0.2 mM</td>
 </tr>
 <tr>
 <td>2 M Na2SO4</td>
 <td>4.5</td>
 <td>90 mM</td>
 </tr>
 <tr>
 <td>1 M K2SO4</td>
 <td>3.0</td>
 <td>30 mM</td>
 </tr>
 <tr>
 <td>0.1 M Kynurenic acid</td>
 <td>1.0</td>
 <td>1 mM</td>
 </tr>
 <tr>
 <td>5 mM APV</td>
 <td>1.0</td>
 <td>0.05 mM</td>
 </tr>
</table>
This solution can be prepared ahead of time and stored at 4&deg;C for several months.
DNAse I: 
 
 Invitrogen (#18047019). 50&mu;l aliquots are flash frozen and stored at -80&deg;C. An aliquot is thawed in the laminar flow hood prior to starting the cell dissociation process. 
Hank's balanced salt solution: 
 
 Sigma (#4891). A 1X solution is prepared in deionized water, sterile filtered at 0.2&mu;m and stored at 4&deg;C.
Hibernate solution: 
 
 Prepare on day of culturing. Combine 98 ml L-15 medium (Invitrogen 11415) with 2 ml B-27 supplement (50X, Invitrogen #17504) and filter at 0.2 &mu;m.
Laminin solution: 
 
 Prepare on day of culturing. Dilute 20&mu;l of laminin (1mg/ml, Sigma L-2020) in 980 &mu;l cell medium for a final concentration of 0.02mg/ml. 20 &mu;l aliquots of the laminin (1mg/ml) are stored at -80&deg;C and thawed just prior to use. Thawing should occur on the bench top and not in the water bath as rapid thawing can sometimes lead to gelling of the laminin.
Papain solution:23 
 
 Warm 10 ml of dissection solution (above) to 30-32&deg;C. Add 200 units papain (Sigma P-4762) and 1.6 mg L-cysteine (Sigma C-7880). Adjust pH to 7.4 with about 14 &mu;l of 0.1 N NaOH. Incubate for about 30 minutes until the solution clears, filter 0.2 &mu;m, flash freeze with liquid nitrogen in 2 ml aliquots in 15 ml polypropylene sterile conical vials and store at -80&deg;C. Thaw an aliquot at 35-37&deg;C prior to starting protocol.
Polyethyleneimine (PEI) solution:13 
 
 100 &mu;l of PEI (50% w/v, Sigma P-3143) is diluted in 100 ml sodium borate buffer (0.1 M pH 8.4, Sigma B-9876) for a final concentration of 0.05% w/v. Sterile filter at 0.2 &mu;m. This can be prepared in advance and stored at 4&deg;C for months. 
Sterile de-ionized water: 
 
 Autoclave and allow to cool to room temperature prior to use. 
Trypsin (0.25% with EDTA, Gibco 25200): 
 
 1ml aliquots are flash frozen and stored at -80&deg;C. Aliquots are thawed in the 35-37&deg;C water bath prior to use.

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.

Watch this Scientific Journal Video about How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs at JoVE.com

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.

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 ...

how-to-culture-record-stimulate-neuronal-networks-on-micro-electrode

Research

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Biology

54.1K 조회수.  Emory University School of Medicine. 이 프로토콜은 설정을 돌보는에서 녹음 및 전기 MEAs의 문화를 자극하기 위해 필요한 정보를 제공합니다. 체외에서 네트워크 네트워크 및 두뇌 기능과 기능 장애의 더 나은 이해를 선도하는 세포 수준에서 생리학 관련 질문을위한 수단을 제공합니다.