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In order to precisely grasp the self-organization process of functional neuronal circuits from individual immature neurons, electrical activity of neuronal circuits should be continuously recorded with their well-defined inner structures and boundary conditions. Standing on this viewpoint, we have developed a series of practical experimental methods for the non-invasive recording of electrical activity from cultured small neuronal circuits by combining a simple micropatterning method of cultured neurons and a site-selective extracellular recording method. The micropatterning was demonstrated with a commercially available spray, and thousands of small neuronal circuits were formed in a 35-mm polystyrene dish by spraying PDL (poly-D-lysine) solution onto the BSA (bovine serum albumin)-coated culture surface. These small neuronal circuits consisted of several neurons and kept well-patterned for more than two weeks. The site-selective recording was demonstrated by means of a handmade mobile microelectrode, and spontaneous firings were detected at multiple recording sites in a small neuronal circuit. This series of experimental methods can be directly applicable to the investigation into the developmental process of the morphology and the functions of various electrically excitable multicellular organisms.

Site-Selective Recording of Spontaneous Activity from Cultured Small Neuronal Circuits by Means of Spray-Patterning and a Mobile Microelectrode