a. Sparse labeling of a 1-year ALI-CO with Sendai-emGFP reveals extensive dendritic trees with dendritic spines (arrows). Maximum intensity projection shown. b. ALI-COs from organoids electroporated with fGFP to label neuronal processes harbour mature synapses with juxtaposed pre- (SYT1, Bassoon, SYP) and post- (Homer1, PSD95) synaptic termini (arrowheads). ALI-CO age: 55 + 40 days ALI (95 total) and 89 + 23 (112 total). c. Immunostaining for the markers VGAT and Calretinin reveals numerous interneurons and VGAT positive punctae suggesting GABA-ergic synapses. ALI-CO age: 57 + 49 day ALI. Maximum intensity projection shown. d. Staining for Somatostatin and GAD67 demonstrates the presence of other interneuron types. ALI-CO age: 57 + 49 day ALI. e. ALI-CO electroporated with fGFP reveals a pyramidal neuron with dendritic spines decorated by punctae of the presynaptic structural protein PCLO and the presence of VGAT punctae suggesting GABA-ergic synapses onto the pyramidal neuron (arrowhead). ALI-CO age: 55 + 40 day ALI. Sparse labelling in a-e with emGFP or fGFP was performed on three ALI-COs from three organoids with similar results. Staining for pre- and post-synaptic markers was performed on three samples, and staining for interneuron markers in c and d was performed on three samples. Lower magnification overview is a maximum intensity projection. f. c-Fos staining for active neurons and MAP2 (dendrites) in a 54 + 60 day ALI-CO. Representative image shown of three experiments on three independently prepared ALI-COs with similar results. g. Image of an ALI-CO after 65 days at ALI (125 days total) then placed on the 3D multielectrode array (MEA). h. Five-second trace from an individual electrode recorded from a 40 + 85 day ALI-CO with threshold (red line) for detected spikes (black hash marks above trace); inset, expanded view of a single spike. i. Heatmap of average spike frequency per electrode over 75-second recording before (top) and after (bottom) application of 2 μM tetrodotoxin (TTX) showing spatial arrangement of spontaneous activity. j. Rasterplot of spike frequency (averaged in 5-ms time bins for each electrode) in the ALI-CO 75 seconds before (left) and after (right) TTX illustrating the temporal distribution of spontaneous activity. k. Quantification of number of active electrodes before and after TTX application in one such ALI-CO recording where TTX was applied. l. Quantification of average spike frequency (error bars are s.e.m.) in the eight active electrodes of k. before and after TTX application. Scale bars, 20 μm (a, c, d), 10 μm (b, e), 50 μm (f), 1 mm (g), 1 μm (b insets, e inset) and 5 μm (c inset, d insets).