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Three-dimensional (3D) neural microtissues are a powerful in vitro paradigm for studying brain development and disease under controlled conditions, while maintaining many key attributes of the in vivo environment. Here, we used primary cortical microtissues to study the effects of neuroinflammation on neural microcircuits. We demonstrated the use of a genetically encoded calcium indicator combined with a novel live-imaging platform to record spontaneous calcium transients in microtissues from day 14–34 in vitro. We implemented graph theory analysis of calcium activity to characterize underlying functional connectivity and community structure of microcircuits, which are capable of capturing subtle changes in network dynamics during early disease states. We found that microtissues cultured for 34 days displayed functional remodeling of microcircuits and that community structure strengthened over time. Lipopolysaccharide, a neuroinflammatory agent, significantly increased functional connectivity and disrupted community structure 5–9 days after exposure. These microcircuit-level changes have broad implications for the role of neuroinflammation in functional dysregulation of neural networks.

Lipopolysaccharide-induced neuroinflammation disrupts functional connectivity and community structure in primary cortical microtissues