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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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

Protocol

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<.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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....

Materials

NameCompanyCatalog NumberComments
B-27 Suplement (50X)Thermo Fisher ScientificLTI17504-044
Branched poly(ethylene imine) (PEI), 25 kDaSigma-Aldrich408727Purify 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) Falcon352340
Conical microtubes (1.5 ml)VWR211-0015
Disposable diaper, 60x40 cmBastos Viegas SA455-019
Forceps Dumont #5, straightFine Science Tools91150-20
Forceps Dumont #5/45Fine Science Tools11251-35
Forceps Dumont #7, curvedFine Science Tools91197-00
Heat Inactivated Fetal Bovine Serum PremiumBiowestS181BH-500ML
Laminin from Engelbreth-Holm-Swarm Sigma-AldrichL2020-1MGPrepare 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 ScientificLTID25030-024
Neubauer improved counting chamber (hemocytometer)Marienfeld630010
Neurobasal Medium (1X)Thermo Fisher Scientific21103-049Basal medium used for neuronal cultures
PDMS microfluidic devices not applicablenot applicableComposed 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)BiowestL0022-100
PES syringe filter unit (Ø 30 mm), 0.22 µm Frilabo1730012
Polypropylene conical tubes, 15 mlThermo Fisher Scientific07-200-886
Polypropylene conical tubes, 50 mlThermo Fisher Scientific05-539-13
Polystyrene disposabel serological pipets, 10 ml Thermo Fisher Scientific1367811D
Polystyrene disposabel serological pipets, 5 ml Thermo Fisher Scientific1367811D
Standard Regenerated cellulose membrane (2 kDa)  Spectrum labs132107
Standard surgical scissorFine Science Tools91401-14
Substrate-integrated planar MEAs (256 microelectrodes)Multi Channel Systems256MEA100/30iR-ITO252 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 Europe5100-X00V0
Syringe luer-lock tip, 50 ml Terumo Europe8300006682
Terg-A-ZymeSigma-AldrichZ273287 Enzyme-active powdered detergent used for MEAs cleaning
Tissue culture plates, 35 mm StemCell Technologies27150
Tissue culture plates, 90 mmFrilabo900095
Trypan Blue solution (0.4%) Sigma-AldrichT8154
Trypsin (1:250)Thermo Fisher Scientific27250018
Vinyl tape 4713MB40071909

References

  1. Scanziani, M., Hausser, M. Electrophysiology in the age of light. Nature. 461 (7266), 930-939 (2009).
  2. Nam, Y., Wheeler, B. C. In vitro microelectrode array technology and neural recordings. Critical Reviews in Biomedi....

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MicrofluidicsMicroelectrode ArraysNeuronal CommunicationAxonal Signal PropagationNeural CircuitsNeural CodingNeural DecodingSensory NeuronsCell CultureData AnalysisComputational ToolsPEI CoatingMEA Preparation

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