Nanoscale Patterning of In Vitro Neuronal Circuits

Abstract

Methods for patterning neurons in vitro have gradually improved and are used to investigate questions that are 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, we present an in vitro 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.

Microfluidic cell engineering on high-density microelectrode arrays for assessing structure-function relationships in living neuronal networks

“…Cell engineering based on microfluidics has become an indispensable technology for studying structure-function relationships and modeling neuronal network functions in vitro , and the high-temporal resolution of the MEA, along with its potential to record LFPs, enables the analysis of novel aspects of the engineered networks. In addition to network modularity, precise control of axon orientation ( Peyrin et al, 2011 ) and even dendritic spines ( Mateus et al, 2022 ) has been demonstrated using microfluidic devices. Combination of such neuroengineering technology with state-of-the-art MEA devices will open new application of in vitro systems as a tool in fundamental neuroscience and pharmacology.…”

Engineering circuits of human iPSC-derived neurons and rat primary glia

“…In the present work, RFP-expressing iNeurons were used, allowing to visualize the neurons’ whole morphology. To get more information about the network’s connections, additional genetically encoded fluorescent markers could be added, for example to locate synapses using the presynaptic terminal marker PreSynTagMA and the postsynaptic marker PSD-95 (Mateus et al, 2022). This should make it possible to precisely link the structure and electrical functional activity of the network.…”

Microfluidic Brain‐on‐a‐Chip: From Key Technology to System Integration and Application

“…This chip can more accurately control the connection and topology of the neural network and ultimately form a fully accessible and connected neural microcircuit. [ 45 ] On most multichamber microfluidic chips, the chambers are connected in the same horizontal plane through microchannels, leading to the same horizontal separation of cell bodies and axons. This horizontal structure cannot fully simulate the 3D microenvironment in the brain.…”