Neuronal guiding: Designing In Vitro Networks On MEA
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1
Forschungszentrum Jülich GmbH, ICS/PGI-8 Bioelectronics, Germany
Motivation
The central nervous system is subdivided in different parts connected to each other via multiple paths. The directional neuronal outgrowth is fundamental for the functionality of this complex organ and influenced by various environmental conditions like signaling molecules and chemical gradients. The study of the emerging functionality of the brain is impeded by the complex structure and the manifold influencing factors. Formation of neural network in vitro is one way to decipher the performance of hierarchically organized neuronal networks. In this study, we developed a simple and effective platform to pattern neurons in neural circuitries by focussing on the influence of the geometrical structure on these network formations. By reducing the number of interconnections, the impact of the neuronal orientation can be examined in detail. Our idea was, that with a controlled geometry we can control the anatomy of the network and thereby the resulting signal propagation directionality.
Material and Methods
We created interconnected networks of a few hundred neurons utilizing the microcontact printing technique. In this process, cell-adhesive protein patterns consisting of a mixture of poly-L-lysine (PLL) and laminin are transferred onto a hydrophobic, cell-repellent glass surface. We chose a triangular shaped pattern as single building block. Multiples of these single triangles are stringed together to form a loop. Dissociated rat embryonic neurons (E18) are grown on these modified substrates for at least 14 days in vitro and form structured networks. We investigated the influence of the geometrical parameters on the directionality of neuronal populations. Therefore neuronal cultures were analyzed with immunofluorescence stainings against MAP2 (somatodendritic marker) and the axon specific marker Anti-200 kD neurofilament Heavy. Additionally Calcium imaging with the fluorescent dye Fluo-4-AM was performed to investigate the spontaneous neuronal activity. To further analyze the electrical signal transmission among and within these populations we apply proteins patterns onto Multi-electrode Arrays (MEAs). This enables tracing single cell activity as well as network activity with up to 64 electrodes simultaneously. MEAs with electrode diameters of 24 µm were fabricated in the clean room facilities of Forschungszentrum Jülich (fig. A, B). Signal recordings were performed with a low-noise amplifier system developed in our institute.
Results
The results of the immunofluorescence stainings showed a polar anatomy of the shaped networks (fig. D). Spontaneous neuronal activity analyzed with Calcium Imaging revealed that the anatomy of the networks can be used to predict the direction of the signal transmission. The analysis of connected populations on MEAs (fig. C) confirm our observation that the geometry of the given pattern has a major impact on the directionality of signal propagation.
Discussion
Our results demonstrate that the shape of transferred protein patterns has a great influence on the emerging higher network structure and its direction of signal propagation. In conclusion the orientation of connected neuronal networks and their information processing can be controlled by choosing the right geometrical parameters. As an outlook we want to further analyze geometrical parameters affecting the hierarchy of the network such as the size of a single building block.
Conclusion
Here, we show a simple and effective platform to pattern primary neurons in hierarchical populations for long-term study of neural circuitry. By interfacing these circuitries with MEA devices will allow to build neuron based logic devices for computational applications.
References
Albers, J., Toma, K., Offenhäusser, A., Engineering connectivity by multiscale micropatterning of individual populations of neurons. Biotech. J. 2015, 10 (2), 332 - 338
Figure Legend
Designed in vitro Network on MEA. A) Multi-Electrode Array (MEA) fabricated in the cleanroom facilities of Forschungszentrum Jülich. B) Enlarged electrode area of MEA, grey: feedlines, black: electrode openings. C) Phase contrast image of a designed neuronal network. D) Immunofluorescence image of a designed neuronal network, green: PLL-FITC, yellow: MAP2, magenta: NFH.
Acknowledgements
We thank Marco Banzet and Michael Prömpers for chip and stamp fabrication. Many thanks to Bettina Breuer and Rita Fricke for neuron preparations.
Keywords:
Neurons,
MEA,
directed networks,
microcontact printing,
neuronal guiding
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Tihaa
I,
Albers
J and
Offenhäusser
A
(2016). Neuronal guiding: Designing In Vitro Networks On MEA.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00080
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
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Correspondence:
Dr. Irina Tihaa, Forschungszentrum Jülich GmbH, ICS/PGI-8 Bioelectronics, Jülich, Germany, i.tihaa@fz-juelich.de