  In vitro neuronal culture is an important research platform in cellular and
network neuroscience. However, neurons cultured on a homogeneous scaffold form
dense, randomly connected networks and display excessively synchronized
activity; this phenomenon has limited their applications in network-level
studies, such as studies of neuronal ensembles, or coordinated activity by a
group of neurons. Herein, we develop polydimethylsiloxane-based microfluidic
devices to create small neuronal networks exhibiting a hierarchically modular
structure resembling the connectivity observed in the mammalian cortex. The
strength of intermodular coupling was manipulated by varying the width and
height of the microchannels that connect the modules. Using fluorescent calcium
imaging, we observe that the spontaneous activity in networks with smaller
microchannels (2.2$-$5.5 $\mu$m$^2$) had lower synchrony and exhibit a
threefold variety of neuronal ensembles. Optogenetic stimulation demonstrates
that a reduction in intermodular coupling enriches evoked neuronal activity
patterns and that repeated stimulation induces plasticity in neuronal ensembles
in these networks. These findings suggest that cell engineering technologies
based on microfluidic devices enable in vitro reconstruction of the intricate
dynamics of neuronal ensembles, thus providing a robust platform for studying
neuronal ensembles in a well-defined physicochemical environment.
