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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 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.
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 measurements
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 (Direção-Geral de Alimentação e Veterinária - DGAV) and of the host Institution.
1. Preparation of Culture Media and Other Solutions<.......
Using the protocol described here, E-18 rat cortical neurons seeded on µEF 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 µEF (Figure 1). After 15 days in culture, cortical neurons cultured on µEF are expected to exhibit steady levels of activity and propagation of action potentials al.......
The protocol presented here shows how to assemble a µEF, 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.......
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 - Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/CTM-NAN/3146/2014 (POCI-01-0145-FEDER-016623). José C Mateus was supported by FCT (PD/BD/135491/2018). Paulo Aguiar was supported by Programa Ciência - Programa Operacional Potencial Humano (POPH) - Promotion of Scientific Employment, ESF and M....
Name | Company | Catalog Number | Comments |
B-27 Suplement (50X) | Thermo Fisher Scientific | LTI17504-044 | |
Branched poly(ethylene imine) (PEI), 25 kDa | Sigma-Aldrich | 408727 | Purify branched PEI by dialysis using a 2.5 kDa cut-off membrane for 3 days at 4°C against a 5 mM HCl solution (renewed daily). Freeze-dry the purified PEI. |
Cell strainer (40 µm) | Falcon | 352340 | |
Conical microtubes (1.5 ml) | VWR | 211-0015 | |
Disposable diaper, 60x40 cm | Bastos Viegas SA | 455-019 | |
Forceps Dumont #5, straight | Fine Science Tools | 91150-20 | |
Forceps Dumont #5/45 | Fine Science Tools | 11251-35 | |
Forceps Dumont #7, curved | Fine Science Tools | 91197-00 | |
Heat Inactivated Fetal Bovine Serum Premium | Biowest | S181BH-500ML | |
Laminin from Engelbreth-Holm-Swarm | Sigma-Aldrich | L2020-1MG | 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 °C in small aliquots (20 µL) to avoid repeated freeze/thaw cycles. |
L-Glutamine 200mM | Thermo Fisher Scientific | LTID25030-024 | |
Neubauer improved counting chamber (hemocytometer) | Marienfeld | 630010 | |
Neurobasal Medium (1X) | Thermo Fisher Scientific | 21103-049 | Basal medium used for neuronal cultures |
PDMS microfluidic devices | not applicable | not applicable | Composed of two cell seeding compartments interconnected by 20 microgrooves with 450 μm length × 10 μm height × 14 μm width dimensions and separated by 86 µm (total interspace of 100 μm). |
Penicillin-streptomycin (P/S) solution (100X) | Biowest | L0022-100 | |
PES syringe filter unit (Ø 30 mm), 0.22 µm | Frilabo | 1730012 | |
Polypropylene conical tubes, 15 ml | Thermo Fisher Scientific | 07-200-886 | |
Polypropylene conical tubes, 50 ml | Thermo Fisher Scientific | 05-539-13 | |
Polystyrene disposabel serological pipets, 10 ml | Thermo Fisher Scientific | 1367811D | |
Polystyrene disposabel serological pipets, 5 ml | Thermo Fisher Scientific | 1367811D | |
Standard Regenerated cellulose membrane (2 kDa) | Spectrum labs | 132107 | |
Standard surgical scissor | Fine Science Tools | 91401-14 | |
Substrate-integrated planar MEAs (256 microelectrodes) | Multi Channel Systems | 256MEA100/30iR-ITO | 252 titanium nitride (TiN) recording electrodes and 4 internal reference electrodes organized in a 16 by 16 square grid. Each recording electrode is 30 µm in diameter and interspaced by 100 µm. |
Syringe luer-lock tip, 10 ml | Terumo Europe | 5100-X00V0 | |
Syringe luer-lock tip, 50 ml | Terumo Europe | 8300006682 | |
Terg-A-Zyme | Sigma-Aldrich | Z273287 | Enzyme-active powdered detergent used for MEAs cleaning |
Tissue culture plates, 35 mm | StemCell Technologies | 27150 | |
Tissue culture plates, 90 mm | Frilabo | 900095 | |
Trypan Blue solution (0.4%) | Sigma-Aldrich | T8154 | |
Trypsin (1:250) | Thermo Fisher Scientific | 27250018 | |
Vinyl tape 471 | 3M | B40071909 |
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