a. Rasterplot of spike frequency (averaged in 5-ms time bins for each electrode) during a 6-minute recording. Network burst shown in Fig. 6b is marked by the red arrowhead. b. High resolution 50-ms traces from 12 electrodes at the time of a network burst. c. Correlation matrix shows clusters of synchronous activity. Correlated activity in the 59 electrodes of the ALI-CO shown in Figure a-b was determined using the spike time tiling coefficient as detailed in methods. d. Human ALI-CO (lower tissue)-mouse spinal (upper right) co-culture at 36 days at the ALI, before (blue box) and after (red box) axotomy (red dash line). Representative image of three such co-cultures with axotomy performed. Spontaneous contractions (blue trace, Supplementary Video 9) show a decrease in amplitude after axotomy (red trace, Supplementary Video 10). Total organoid age is 100 days. e. ALI-CO and mouse co-culture at 40 days at the ALI (total organoid age: 104 days) in which innervation has not occurred (blue box) and image after axotomy-style cut was made (red box, red dashed line) near mouse tissue. This axotomy-style cut on non-innervated co-culture was performed once. Spontaneous small amplitude muscle contractions before (blue trace) and after (red trace) cut show similar amplitude and frequency, suggesting that disturbing the mouse tissue through nearby manipulation is not sufficient to eliminate these low-amplitude contractions. Yellow box indicates ROI used for quantification of displacement due to muscle contraction. f. Image of 31-day ALI-CO-mouse co-culture (total organoid age: 79 days) with a stimulation electrode (yellow dashed line) placed on axon tracts projecting from the organoid. Large amplitude muscle contractions, recorded from ROI (yellow), were evoked by brief current pulses (upper trace, black arrow and dotted line, 3.2 mA, 120 µs-long) applied at 30, 60, 75, 90, 105, 120, 135 s. Repeated stimulation at 1 Hz (lower trace, black hash marks, 3.2 mA, 120 µs-long) could also drive muscle contractions. g. Higher magnification image of the mouse spinal section shown in white-dashed box in Fig. 6h, stained with Map2 for mouse neuronal cell bodies and dendrites (green). STEM121 stains human axon bundles (magenta) revealing innervation of the mouse spinal cord (white arrows). Maximum intensity projection shown. h. Staining of the same co-culture as in f. and i. after axotomy and fixation showing human axon tracts (STEM121+) innervating (arrows) the Map2+ mouse spinal cord. Maximum intensity projection shown. i. Precise electrode placement (yellow dashed line outlining the electrode) determines the ability to evoke muscle contractions in a 31-day mouse spinal cord-ALI-CO co-culture. Top-left image shows correct electrode placement in the innervating tract with evoked contractions traced at bottom-right (blue trace), while electrode displacement from the axon tracts (top-right panel, light blue outline), or axotomy (bottom-left, red outline) abolishes the ability to elicit a response. Red dashed line indicates the site of axotomy. Stimulation (hash marks above traces) before and after electrode displacement and axotomy was done with 1 Hz TTL-stimulated current pulses (120 µs, increasing current amplitude every 10 s for 0.2, 0.8, 1.6, 3.2 mA) with the final 1Hz TTL stimulation at 3.2 mA lasting 14 s for the control trace (dark blue hash marks). f-i are representative results of six such co-culture experiments with stimulation, two of which included axotomy. j. Confirmation of stimulation electrode placement before (dark blue outline), after TTX application (orange outline), and after wash-out (green outline) for recording shown in Fig. 6n. Shown are representative results for two independent experiments with TTX application. Yellow boxes denote the ROI used for quantification of muscle contractions. Purple-dashed box with corresponding inset shows the axonal tracts (arrowhead) joining the organoid and the mouse spinal co-culture. Scale bars, 500 μm (d, e, f, I, j), 100 μm (g, j inset) and 200 μm (h).