Methods to induce polarity in constructed neuronal networks

This work focused on the control of neuronal polarity in geometrical patterned neuronal networks. Two different approaches were taken in order to direct polarity within a neuronal network. The first strategy was to chemically pattern two proteins with distinct effects on neurite growth. Aligned two-step microcontact printing (2-step µCP) was developed for accurate aligned printing of two complementary protein patterns under visual control. Netrin-1, laminin-1 and ECM-gel were chosen as axon-guiding proteins, while poly-D-lysine (PDL) was printed as neutral protein. The protein transfer during 2-step µCP and the effect of the second printed pattern on the primary ones was characterized by fluorescence microscopy, ellipsometry and atomic force microscopy (AFM) using manual 2-step µCP substrates. Each method showed, that the first printed pattern consisting of polylysine was not blurred or removed by printing of the second pattern. Moreover, the transfer of ECM-gel, laminin-1 and netrin-1 onto the substrates was inhomogeneous, characterized by a lower protein transfer at areas with preprinted polylysine. A highly irregular protein surface was observed for all proteins by ellipsometry and AFM measurements. Additionally, AFM measurements showed a mesh-like structure in some areas of the transferred laminin pattern. Cell culture experiments on the manual 2-step µCP substrates showed that E18 rat striatal neurons cultured on ECM/PDL substrates and on laminin/PDL had a similar good morphology and viability as neurons cultured on PECM-grids, while on netrin/PDL substrates the amount of dead cells was increased. Electrophysiological measurements showed that E18 rat cortical neurons cultured on the aligned ECM/PDL substrates possessed similar electrophysiological properties as neurons cultured on PECM grid patterns except for the AP-frequency, which was significantly lower in comparison to the neurons on the PECM substrates. Further patch-clamp experiments are required in order to test if neuronal growth on aligned 2-step µCP substrates is guided by the complementary patterns of axon-guiding and neutral proteins. As second strategy for the induction of a defined polarity in neuronal networks the reconstruction of the natural polarity between cortex and striatum in cell culture was chosen. For coculture cell culture of E18 rat striatal neurons was established at the Institute for Bio-and Nanosystems based on the already existing cortical cell culture. About 70% of the cells could be identified as striatal neurons by expressing striatum-specific G-protein coupled receptor-RNA-transcripts. The striatal neurons in monoculture had similar electrophysiological properties as described in literature, except for a significantly higher membrane time constant. For coculture a microstructured coculture chamber was reversibly sealed on glass substrates using activation by UV/ozone plasma and subsequent chemical hydrophilization by sodium hydroxide. Cell culture experiments in the coculture chambers showed, that 25% of the chambers contained viable cortical and striatal neurons at DIV 3. By removing the coculture chamber at DIV 3 and increasing the cell density, neurons in 14% of these chambers could survive up to DIV 11. Electrophysiological analysis at DIV 10-11 showed no significant differences between the electrophysiological properties of the cocultured striatal neurons in comparison to striatal neurons in monocultures. The cocultured cortical neurons showed a significant lower FLHM compared to cortical monocultures on PECM-grids. By double patch-clamp measurements a synapse between two cocultured striatal neurons could be measured, confirming the ability of the cocultured neurons to form synaptic contacts. Further patch-clamp experiments are required in order to test if the natural polarity between cortex and striatum could be reconstructed in cell culture.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2008

Author dc:creator

• Decker, Tanja

Contributors dc:contributor

• Offenhäusser, Andreas

• info:eu-repo/classification/ddc/570
• Nervenzelle
• Zellkultur
• Nervennetz
• Mikrokontaktdruck
• Polarität
• Biowissenschaften, Biologie
• Patch-Clamp
• Kokultur
• Striatum
• Kortex
• Ratte
• coculture
• cortex
• rat

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112649%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A50091