Figure 2

Topographic control of axon/dendrite connections by stepwise formation of neuronal network pattern using photothermal etching. By applying stepwise photo-thermal etching to agar microchambers during cultivation, the direction of synaptic connectivity in a living neuronal network can be controlled. This allows the tunnels in which axons were elongated to be flexibly extended by melting the narrow micrometer-order grooves (microchannels) in steps through photo-thermal etching where a portion of the agar layer is melted with infrared laser beam. (A) Phase-contrast images of single hippocampal neurons during the stepwise etching procedure. When cultivation started, single cells were placed into the agar microchambers and cultivated for 2 days in vitro (DIV). The first single neurites that elongate from the cells into the microchannels are typically axons, whereas the second and latter neurites are typically dendrites. When the elongations of neurites are sufficiently stable, additional photo-thermal etching steps can be applied to connect two adjacent agar microchambers. (B) Fluorescent staining of neurites with axon- and dendrite-specific markers at DIV 5 (red, synapsin I for axons; green, MAP2 for dendrites). (C) A combination of microelectrode and agar microchambers for an individual-cell-based neural network patterning and electrophysiological recording. 8-μm microelectrode-size agar microchambers and 2-μm microtunnels were arranged on the MEA chip to control the positions of neurons and their connections.