In vitro reconstitution of hippocampal cell assemblies
Three-dimensional neuronal cultures, such as brain organoids, have proven effective for in vitro modeling of brain development and disease. However, it remains unclear whether these systems can intrinsically generate the spatiotemporal network motifs that support cognitive function, particularly given their lack of sensory input. To address this, we developed a 3D long-range modular neuronal network approach to test whether dissociated embryonic hippocampal cells can self-organize into neuronal manifolds capable of generating network dynamics analogous to the in vivo hippocampal cell assemblies implicated in cognitive function. Using all-optical interrogation, we demonstrate in vitro recapitulation of cell assemblies and their hallmark features, including recurring sequential activation, hierarchical chaining, multi-day stability, resilience to perturbation, and attractor-like pattern completion. We further investigated the effects of acute ketamine exposure on these assemblies, revealing network-wide reconfiguration reminiscent of in vivo responses. Finally, we show that human iPSC-derived neurons can similarly self-organize into modular network architectures. Together, these findings are consistent with an intrinsically "inside-out" view of the brain, rather than a tabula rasa model in which sensory input is required for the emergence of cognitive network motifs, and open new avenues for in vitro modeling of network-level mechanisms relevant to neuropsychiatric disorders.