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@INPROCEEDINGS{Tihaa:829528,
      author       = {Tihaa, Irina and Jonas, Albers and Offenhäusser, Andreas},
      title        = {{N}euronal guiding: {D}esigning {I}n {V}itro {N}etworks
                      {O}n {MEA}},
      volume       = {10},
      issn         = {1662-453X},
      address      = {Lausanne},
      publisher    = {Frontiers Research Foundation},
      reportid     = {FZJ-2017-03213},
      pages        = {1},
      year         = {2016},
      abstract     = {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.},
      month         = {Jun},
      date          = {2016-06-28},
      organization  = {10th International Meeting on
                       Substrate-Integrated Electrode Arrays,
                       Reutlingen (Germany), 28 Jun 2016 - 1
                       Jul 2016},
      cin          = {ICS-8},
      ddc          = {610},
      cid          = {I:(DE-Juel1)ICS-8-20110106},
      pnm          = {552 - Engineering Cell Function (POF3-552)},
      pid          = {G:(DE-HGF)POF3-552},
      typ          = {PUB:(DE-HGF)8},
      doi          = {10.3389/conf.fnins.2016.93.00080},
      url          = {https://juser.fz-juelich.de/record/829528},
}