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| Hauptseite > Publikationsdatenbank > Directing Neuronal Growth And Signal Propagation On MEA |
| Hauptseite > Publikationsdatenbank > Directing Neuronal Growth And Signal Propagation On MEA |
| Poster (After Call) | FZJ-2020-03014 |
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2018
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Please use a persistent id in citations: doi:10.3389/conf.fncel.2018.38.00056
Abstract: Event AbstractBack to EventDirecting Neuronal Growth And Signal Propagation On MEATimm Hondrich1*, Irina Tihaa1, Johannes Lewen1 and Andreas Offenhäusser1 1 Institute of Complex Systems, Forschungszentrum Jülich, Bioelectronics (ICS-8), GermanyMotivation: The brain’s functionality is largely dependent on neuronal ensembles of different scales that are interconnected via long-range projection axons. These projections establish either unilateral or bilateral communication pathways between the different neuronal populations. To form these connections, axons are guided by a diverse set of extracellular signaling molecules. Neuronal networks grown in vitro can be utilized to uncover the underlying principles of the brain’s connectivity on different scales. On the population level, we could show in previous work that directional outgrowth and signal propagation between two neuronal populations can be controlled by culturing neurons on protein patterns with triangular geometries [1]. On the single cell level, we could establish directed axonal outgrowth. With this set of tools, we are now able to address relevant physiological questions ranging from single cell signal integration to neuronal oscillations and network dynamics.Material and Methods: Microcontact printing was utilized to pattern our different designs (for an example, see Fig. 1C) on coverslips and/or nanocavity micro-electrode arrays (MEA). For this, polyolefin plastomer (POP) stamps were fabricated to transfer a mixture of poly-L-lysine (PLL) and extracellular matrix proteins (ECM) onto the substrate. Dissociated rat embryonic neurons (E18) were grown on these modified substrates for 7 to 28 days in vitro (DIV) to form structured networks. Neuronal cultures were analyzed with immunofluorescence stainings against MAP2 (somatodendritic marker) and neurofilament heavy chain or β3-tubulin (axonal markers). Calcium imaging with genetically encoded sensors based on calmodulin was performed to investigate neuronal activity in all patterns. Nanocavity MEAs were shown to have a decreased impedance and improved signal-to-noise ratio [2]. The 64 electrodes of the nanocavity MEAs (Fig. 1A,B) were specifically designed to match the linear chains of triangular populations. MEAs were fabricated in the clean room facilities of Forschungszentrum Jülich and signal recordings were performed with a low-noise amplifier system developed in our institute. For functional analysis of single cell patterns, cells were virally transduced with channelrhodposin (ChR2), stimulated with a 473 nm-laser (Rapp Optoelectronics) and patched in whole-cell configuration with a Heka amplifier system. Results: All scales of patterned networks show a preferred directional axonal and dendritic outgrowth on glass substrates and MEAs (Fig. 1C). This preferred growth depends strongly on the soma position on single cell patterns. Using large triangles, the directional signal propagation led to activity waves along closed-loop structures as a basis for modeling circulating action potentials in vitro. Moreover, we showed via calcium imaging that the directionality and synchronicity of spontaneous network events depends on the scale of the patterns based on triangular geometry. The variable directionality in differently scaled networks observed during calcium imaging is consistent with novel nanocavity MEA recordings (Fig. 1D). Neurons growing on single cell patterns also showed spontaneous calcium activity. Their functional signal propagation was shown using simultaneous optogenetic stimulation and patch clamp recordings. Discussion: Our results indicate that highly interconnected small neuronal populations are, despite their size, able to produce network activity down to the single cell level. The varying synchronicity between smaller triangular populations may result from the different complexity of the structures, enabling a straighter or more diverted growth path for the axons. The star-shaped single cell pattern proved to be a viable platform for investigating the parameters influencing the direction of axon outgrowth. For example, the soma position has a significant impact on axo-dendritic orientation. Thus, we can now investigate parameters like the soma position or cell density and their influence on higher level measures such as activity variations of population grown on the cell pattern or the impact on synchronicity and functional directionality. Conclusion: By implementing triangular geometries into other protein patterns, and by scaling them to different sizes, we are now able to control neuronal populations of many sizes from one cell to about 200 cells both structurally and functionally. In the future, a combination of these patterns with MEA technology and stimulation methodologies can lead to advances in such diverse fields as neuromedicine, neural network algorithms or neuromorphic chip design.
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