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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.

Sulfated glycosaminoglycans (sGAGs) are vital molecules of the extracellular matrix (ECM) of the nervous system known to regulate proliferation, migration, and differentiation of neurons mainly through binding relevant growth factors. Alginate sulfate (AlgSulf) mimics sGAGs and binds growth factors such as basic fibroblast growth factor (FGF-2). Here, thin films of biotinylated AlgSulf (b-AlgSulf n ) are engineered with sulfation degrees (DS = 0.0 and 2.7) and the effect of polysaccharide concentration on FGF-2 and nerve growth factor (β-NGF) binding and subsequent primary neural viability and neurite outgrowth is assessed. An increase in b-AlgSulf n concentration results in higher FGF-2 and β-NGF binding as demonstrated by greater frequency and dissipation shifts measured with quartz crystal microbalance with dissipation monitoring (QCM-D). Primary neurons seeded on the 2D b-AlgSulf n films maintain high viability comparable to positive controls grown on poly-d-lysine. Neurons grown in 3D AlgSulf hydrogels (DS = 0.8) exhibit a significantly higher viability, neurite numbers and mean branch length compared to neurons grown in nonsulfated controls. Finally, a first step is made toward constructing 3D neuronal networks by controllably patterning neurons encapsulated in AlgSulf into an alginate carrier. The substrates and neural networks developed in the current study can be used in basic and applied neural applications., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Theoretical and in vivo neuroscience research suggests that functional information transfer within neuronal networks is influenced by circuit architecture. Due to the dynamic complexities of the brain, it remains a challenge to test the correlation between structure and function of a defined network. Engineering controlled neuronal networks in vitro offers a way to test structural motifs; however, no method has achieved small, multi-node networks with stable, unidirectional connections. Here, we screened ten different microchannel architectures within polydimethylsiloxane (PDMS) devices to test their potential for axonal guidance. The most successful design had a 92% probability of achieving strictly unidirectional connections between nodes. Networks built from this design were cultured on multielectrode arrays and recorded on days in vitro 9, 12, 15 and 18 to investigate spontaneous and evoked bursting activity. Transfer entropy between subsequent nodes showed up to 100 times more directional flow of information compared to the control. Additionally, directed networks produced a greater amount of information flow, reinforcing the importance of directional connections in the brain being critical for reliable communication. By controlling the parameters of network formation, we minimized response variability and achieved functional, directional networks. The technique provides us with a tool to probe the spatio-temporal effects of different network motifs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bottom-up neuroscience, which consists of building and studying controlled networks of neurons in vitro, is a promising method to investigate information processing at the neuronal level. However, in vitro studies tend to use cells of animal origin rather than human neurons, leading to conclusions that might not be generalizable to humans and limiting the possibilities for relevant studies on neurological disorders. Here we present a method to build arrays of topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons. The circuits consist of 4 to 50 neurons with well-defined connections, confined by microfabricated polydimethylsiloxane (PDMS) membranes. Such circuits were characterized using optical imaging and microelectrode arrays (MEAs), suggesting the formation of functional connections between the neurons of a circuit. Electrophysiology recordings were performed on circuits of human iPSC-derived neurons for at least 4.5 months. We believe that the capacity to build small and controlled circuits of human iPSC-derived neurons holds great promise to better understand the fundamental principles of information processing and storing in the brain. [ABSTRACT FROM AUTHOR]

We present a stimulate and record paradigm to examine the behavior of multiple neuronal networks with controlled topology in vitro. Our approach enabled us to electrically induce and record neuronal activity from 60 independent networks in parallel over multiple weeks. We investigated the network performance of neuronal networks of primary hippocampal neurons until 29 days in vitro. We introduced a systematic stimulate and record protocol during which well-defined 4-node neural networks were stimulated electrically 4 times per second (4Hz) and their response was recorded over many days. We found that the network response pattern to a stimulus remained fairly stable for at least 12 h. Moreover, continuous stimulation over multiple weeks did not cause a significant change in the stimulation-induced mean spiking frequency of a circuit. We investigated the effect of stimulation amplitude and stimulation timing on the detailed network response. Finally, we could show that our setup can apply complex stimulation protocols with 125 different stimulation patterns. We used these patterns to perform basic addition tasks with the network, revealing the highly non-linear nature of biological networks' responses to complex stimuli. • Platform to stimulate and record the spiking activity of neurons cultured on MEAs. • Small neuronal networks respond to electrical stimulation in a highly defined manner. • Topologically constrained networks have distinct responses to different stimuli. • The spike responses of the network remain fairly stable for multiple hours. • The non-linear nature of our networks is revealed by performing arithmetic operations. [ABSTRACT FROM AUTHOR]