Microtechnologies to fuel neurobiological research with nanometer precision.

Three techniques for isolation of axons in neuronal cell culture. (A) The Campenot chamber is a Teflon ring placed on a layer of silicon grease on top of regular cell culture dish or glass coverslip. Dissociated peripheral neurons such as dorsal root ganglion cells are placed in the middle reservoir. NGF placed in the axonal chambers attracts axons to grow through the silicon grease layer while the cell somas are restricted to the middle reservoir. (B) Microfluidic devices are typically microfabricated from PDMS. The two cell reservoirs are connected via microgrooves (typically appr. 3–10 μm width and hight, >450 μm in length). Axons grow through the microgrooves while somas and dendrites are limited in the cell reservoirs. Many types of neurons including central neurons can be grown in microfluidic devices. (C) Individual neurons can be cultured to create directed neuronal networks using the low-melting agar etching technique. Glass coverslip is coated with a 50 nm-thick indium-tin iodide (ITO) layer below the agar. ITO allows highly localized photo-thermal etching of agar with a 1064-nm infrared laser beam. Based on observed initial axon growth, microchannels can be opened to connect individual neurons to form simple networks. The panel C is reproduced from [7].

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.