Nanoscale Patterning of <i>In Vitro</i> Neuronal Circuits.

Methods for patterning neurons <i>in vitro</i> have gradually improved and are used to investigate questions that are difficult to address <i>in</i> or <i>ex vivo</i>. Though these techniques guide axons between groups of neurons, multiscale control of neuronal connectivity, from circuits to synapses, is yet to be achieved <i>in vitro.</i> As studying neuronal circuits with synaptic resolution <i>in vivo</i> poses significant challenges, we present an <i>in vitro</i> alternative to validate biophysical and computational models. In this work we use a combination of electron beam lithography and photolithography to create polydimethylsiloxane (PDMS) structures with features ranging from 150 nm 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 postsynaptic protein, PSD-95, we demonstrate functional synapses can form in this region.