Light-Addressed Stimulation and Simultaneous Calcium Imaging for Probing Spatio-Temporal Activity of Cultured Neural Network

In order to probe the spatio-temporal activity of cultured neural network, microelectrode arrays (MEAs) have been widely used. MEAs, however, have limitations of their electrode numbers and densities, resulting in low spatial resolutions of stimulation and recording. Here, to overcome this problem, we propose and develop an experimental setup for light-addressed stimulation and simultaneous fluorescence calcium imaging, using the previously published light-addressable electrode. The electrode has a translucent thin-film-laminated structure and allows optical access from both sides of the substrate. We, thus, provided the fluorescence excitation light from the topside and an addressing illumination from the bottom. By instantly shutting out the fluorescence excitation light during the stimulus application, we prevented the excitation light from interfering with the addressing illumination. With this experimental setup, we successfully measured spatio-temporal patterns of neuronal activities evoked by light-addressed stimuli. Evoked fluorescence transients with hundred-millisecond latencies suggested the possibility that some neurons were activated by recurrent synaptic inputs, which were possibly overlooked by previous MEA studies.