Microembossing Hydrogel Meso-Circuits for Patterning Dissociated Neurons Promotes Ensemble Formation

Functional networks of wired neurons comprise the basis for neuronal computation and processing. Within neuronal networks, activation of unique ensembles is an important identity of neuronal processing. However, dissociated neuronal networks form homogeneous functional structures with minimal variety in ensemble dynamics. To reintroduce such dynamics, we propose structuring the networks to follow multi-connectivity (micro- and meso-network) paradigms. Here, we use agarose microembossing to physically pattern dissociated neuronal networks across these scales. To perform agarose microembossing, we impress features with poly-dimethylsiloxane (PDMS) stamps into liquid agarose to emboss features which hold under cold gelation. We validate the viability of primary neurons within the hydrogel patterns and interrogate circuit dynamics through calcium imaging. Patterned features presented with robust ensemble dynamics that are dependent on connectivity paradigms. Altogether, this work establishes a platform for investigating how engaging multi-scale features in the physical network informs neuronal ensemble dynamics.