Significant advances have been made in developing analytical methods to monitor neuronal activity on a single cell level (fluorescence imaging, voltammetry, ion-selective electrodes, microelectrode arrays, combination of separation techniques with mass spectrometry) [18-20]. To meet the spatial resolution of those single cell level-monitoring technologies, micropatterning techniques for controlling of adequate spatial arrangements of neurons and neurites have been developed and applied [21,22]. While most of micropatterning techniques such as microcontact printing and microetching-based fabrication techniques are suitable for controlling the populations of dissociated neurons with randomly arranged network patterns, those conventional micropatterning techniques can just control the orientation of spatial arrangements of neurites in pre-fabricated (ready-made) micropatterns, and, in principle, cannot control the directions of neurites’ elongation and connections. To overcome those problems, agar-microetching technique has been developed to fully control of spatial arrangements of single neurons and the direction of their connectivity by flexible stepwise-fabrication of additional microstructures (Figure 1C) [6,7,23]. This technique provides a constructive approach for spatial direction control and neuronal network formation during cultivation.
