Circuitoids were established with either 100,000 motor neurons or 100,000 V3 interneurons combined with 0–40,000 V1 interneurons, plated on astrocytes. Networks were imaged 14 days after sorting with a calcium indicator dye and the static frames show pseudocolored calcium intensity (dF/F) images (scale from black to white). (A) Neurons within plated circuitoids (10,000 V1 interneurons and 100,000 motor neurons) fire asynchronously. Static frames during Burst A and B reveal different areas of activity in the network. Traces from two regions of interest (ROI 1 and 2) on opposite corners of the field of view are displayed. (B) Inhibitory antagonists (strychnine + picrotoxin) applied to the network in (A) lead to synchronous bursts across the entire network. (C) Quantification of network complexity (Materials and methods) calculated from network activity. V1 interneurons increase network complexity of motor neuron networks. Median ± bootstrap standard error, n networks in the (spontaneous) and [inhibitory antagonist] condition for V3 networks (10) and MN networks (8)[≥4] at all V1 interneuron concentrations. MN spontaneous vs MN inhibitory antagonist (*) and MN spontaneous vs V3 spontaneous (Δ). Δ/*p<0.05; ΔΔ/**p<0.01; ΔΔΔp<0.001; ΔΔΔΔp<0.0001 Kolmogorov–Smirnov test. (D) 100,000 purified GFP+ motor neurons were cocultured with 10,000 tomato +V1 inhibitory neurons. Calcium imaging was used to detect bursts from five individual GFP+ motor neurons. Individual cells displayed coupled activity (brown), but the entire cohort of motor neurons only became synchronously active when V1 inhibition was blocked with strychnine + picrotoxin (red). (E) 100,000 purified V3 interneurons were cocultured with 100,000 V1 cells, and five individual cells were calcium imaged. Individual interneurons consistently fired together whether V1 cells were active (green) or silenced with inhibitory antagonists (purple). (F) Quantification of neuronal synchrony using pair-wise cross-correlation analysis of motor neurons in motor neuron-V1 networks and interneurons in V3-V1 networks. The activity within motor neuron networks became increasingly less synchronized as V1 cell number increased. Mean±SEM, sample size n=(network number)[neuron number]. V3 network, spontaneous burst condition: 0–40,000 V1 cells (10), 100,000 V1 cells (4). V3 network, inhibitory antagonist condition: 0–40,000 V1 cells (4), 100,000 V1 cells (2). MN network, spontaneous burst condition, each V1 concentration tested (≥7)[≥140]. MN network, inhibitory antagonist condition, each V1 concentration tested (6). MN spontaneous vs MN inhibitory antagonist (*) and MN spontaneous vs V3 spontaneous (Δ) all compared at same V1 concentration, except for spontaneous 100,000 V1 in V3 network vs 40,000 V1 in MN network. Δ/*p<0.05; ΔΔ/**p<0.01; ΔΔΔp<0.001; ΔΔΔΔp<0.0001, unpaired t-test.