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Microelectrode array and multichannel system for recording the electrical activity of neurons in vitro

Andrey Denisov1, 2*, Pavel M. Bulai1, Taras Pitlik1, Pavel Molchanov1, Margarita Dosina2, Svetlana Pashkevich2, Vladimir Kulchitsky2 and Sergei Cherenkevich1
  • 1 Belarusian State University, Biophysics Department, Belarus
  • 2 Institute of Physiology (NASB), Belarus

Motivation. Cultured dissociated neurons forming connections in vitro is a unique system representing living biological neural network developing in fully artificial conditions. This is a promising model for study of basic mechanisms of the brain functioning that requires special tools for interfacing. The most direct method for interaction with cultured neurons is based on usage of microelectrode arrays for recording and stimulation of electrical activity. Advanced solutions utilizing microelectronic sensors with integrated amplifiers can have tens of thousands recording electrodes [1], but classical approach based on passive microelectrodes embedded into planar substrate is still relevant for modern applications. Main advantages of the conventional planar microelectrode arrays are simple construction and (in case of transparent substrate) possibility to visualize cells development with standard inverted microscope. Typical systems for interfacing with cultured neurons include microelectrode sensors, amplifiers and stimulators, interfacing boards, software for data acquisition and processing. Modern advances in specialized integrated circuits for biopotential recording and data acquisition allow creation of relatively simple designs for multichannel recordings such as Open Ephys platform [2]. We have developed a simple extendable multichannel recording system with modular design suitable for work with living cultured neural networks. Materials and Methods 64-channel microelectrode sensor of electrical activity has been designed for interfacing with cultured neurons (Fig 1A). The sensor consists of planar glass base with transparent indium-tin-oxide conducting tracks serving as electrodes (8x8 grid, 250 um interelectrode distance, 30 um microelectrode diameter). The electrodes are insulated by hard-baked S1813 (MicroChem, USA) photoresist layer. Working electrodes of the sensor were coated by electrodeposited poly-ethylenedioxythiophene for impedance reduction. The chamber for neuronal culture solution has been developed on the basis of 3D printing techniques. The mold was printed from plastic by fused deposition modeling and then PDMS chamber was casted. The sensor and chamber were exposed to oxygen RF-plasma and irreversibly bonded one to another. The chamber was covered by fluorinated ethylene propylene film to optimize culture conditions. 64-channel amplifier has been developed for recording the electrical activity of neurons. The base of the amplifier is a 32-channel integrated circuit RHD2132 (Intan Tech., USA) with built-in analog to digital converter and digital serial interface. The digital interface of RHD2132 chips is connected to the computer via XEM6010 interfacing board (Opal Kelly, USA). Basic recording and visualization operations are controlled by customized RHD2000 interface software (Intan Tech., USA). The amplifier is inserted into sockets of interfacing system assembled from a set of printed circuit boards (Fig 1B). The sensor is inserted into the system and fixed by a board with spring-loaded contacts. Dissociated newborn rat neurons where grown in CO2-incubator on poly-D-lysine coated sensors for at least 10 days in vitro before experiments. Customized open-source module «Tridesclous» [3] was used for spike detection and classification. Results and Discussion Development of the neural network after three weeks in culture is shown at Fig 1C. Neurites growing to the electrode are easily observable. Typical example of recorded multichannel neuronal electrical activity is shown at the Fig. 1D. Marks denote spikes divided into distinct clusters on the basis of amplitude and shape analysis. Neuronal activity on the recordings is represented by background single sparse spikes and spike bursts. Activity generated by «pacemaker» group of neurons at the channel 33 propagates via neurites and synaptic connections to other electrodes indicating formation of functional neural network. The system developed has simple modular design and does not require complex manufacturing processes so that components can easily be modified, upgraded and scaled up. Thus, amplifier/stimulator board supporting 64-channel simultaneous recording and stimulation is being developed. Utilization of 3D printing techniques for culture chamber development allows fast prototyping of different designs with different features and optimal gas diffusion condition. Neural culture requires appropriate concentrations of O2 for breathing and CO2 for pH control so that monitoring of these parameters is advantageous. Miniature optical fluorescent sensors of dissolved gases can be embedded into chamber for noninvasive measurements. Moreover, the approach facilitate creation of CO2-incubator independent devices that can reduce both handling procedures and risk of contamination and also give opportunities for developing of autonomous living neural network applications. The most complicated part of the presented approach is the microelectrode array requiring photolithographic techniques to manufacture. Alternative designs may be based on modern approaches in organic conductors and soft lithography such as [4]. Figure Legend Fig. 1. Planar microelectrode array with photoresist insulation in the center (A). Recording system with sensor and culture chamber installed (B). Neurons growing around microelectrode (C). Network bursting activity (C).

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Acknowledgements

This work is supported by "Science around us" Foundation (London)

References

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2. J. H. Siegle, A. C. López, Y. A. Patel, K. Abramov, S. Ohayon, and J. Voigts. (2017) Open Ephys: an open-source, plugin-based platform for multichannel electrophysiology. J. Neural Eng., 14(4), 45003
3. https://github.com/tridesclous/tridesclous
4. A. Blau, A. Murr, S. Wolff, E. Sernagor, P. Medini, G. Iurilli, C. Ziegler, F. Benfenati (2011) Flexible, all-polymer microelectrode arrays for the capture of cardiac and neuronal signals. Biomaterials, 32(7), 1778–86

Keywords: microelectrode arrays, Multichannel recording, Multichannel amplifier, neural network in vitro, neural activity

Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.

Presentation Type: Poster Presentation

Topic: Microelectrode Array Technology

Citation: Denisov A, Bulai PM, Pitlik T, Molchanov P, Dosina M, Pashkevich S, Kulchitsky V and Cherenkevich S (2019). Microelectrode array and multichannel system for recording the electrical activity of neurons in vitro. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00077

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Received: 19 Mar 2018; Published Online: 17 Jan 2019.

* Correspondence: Dr. Andrey Denisov, Belarusian State University, Biophysics Department, Minsk, Belarus, an.denisov@gmail.com