Methods for patterning neurons in vitro have gradually improved and are used to investigate questions difficult to address in or ex vivo. Though these techniques guide axons between groups of neurons, multiscale control of neuronal connectivity, from circuits to synapses, is yet to be achieved in vitro. As studying neuronal circuits with synaptic resolution in vivo poses significant challenges, an in vitro alternative could serve as a testbed for in vivo experiments or as a platform for validating biophysical and computational models. In this work we use a combination of electron beam and photolithography to create polydimethylsiloxane (PDMS) structures with features ranging from 150 nanometers to a few millimeters. Leveraging the difference between average axon and dendritic spine diameters, we restrict axon growth while allowing spines to pass through nanochannels to guide synapse formation between small groups of neurons (i.e. nodes). We show this technique can be used to generate large numbers of isolated feed-forward circuits where connections between nodes are restricted to regions connected by nanochannels. Using a genetically encoded calcium indicator in combination with fluorescently tagged post synaptic protein, PSD-95, we demonstrate functional synapses can form in this region. Although more work needs to be done to control connectivity in vitro, we believe this is a significant step in that direction.

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