Neuronal networks derived from human induced pluripotent stem cells have been exploited widely for modeling neuronal circuits, neurological diseases, and drug screening. As these networks require extended culturing periods to functionally mature in vitro, most studies are based on immature networks. To obtain insights on long-term functional features, we improved a glia-neuron co-culture protocol within multi-electrode arrays, facilitating continuous assessment of electrical features in weekly intervals. By full-field optogenetic stimulation, we detected an earlier onset of neuronal firing and burst activity compared with spontaneous activity. Full-field stimulation enhanced the number of active neurons and their firing rates. Compared with full-field stimulation, which evoked synchronized activity across all neurons, holographic stimulation of individual neurons resulted in local activity. Single-cell holographic stimulation facilitated to trace propagating evoked activities of 400 individually stimulated neurons per multi-electrode array. Thereby, we revealed precise functional neuronal connectivity motifs. Holographic stimulation data over time showed increasing connection numbers and strength with culture age. This holographic stimulation setup has the potential to establish a profound functional testbed for in-depth analysis of humaninduced pluripotent stem cell-derived neuronal networks.