Abstract
The central nervous system consists of an unfathomable number of functional networks enabling highly sophisticated information processing. Guided neuronal growth with a well-defined connectivity and accompanying polarity is essential for the formation of these networks. To investigate how two-dimensional protein patterns influence neuronal outgrowth with respect to connectivity and functional polarity between adjacent populations of neurons, a microstructured model system was established. Exclusive cell growth on patterned substrates was achieved by transferring a mixture of poly-l-lysine and laminin to a cell-repellent glass surface by microcontact printing. Triangular structures with different opening angle, height, and width were chosen as a pattern to achieve network formation with defined behavior at the junction of adjacent structures. These patterns were populated with dissociated primary cortical embryonic rat neurons and investigated with respect to their impact on neuronal outgrowth by immunofluorescence analysis, as well as their functional connectivity by calcium imaging. Here, we present a highly reproducible technique to devise neuronal networks in vitro with a predefined connectivity induced by the design of the gateway. Daisy-chained neuronal networks with predefined connectivity and functional polarity were produced using the presented micropatterning method. Controlling the direction of signal propagation among populations of neurons provides insights to network communication and offers the chance to investigate more about learning processes in networks by external manipulation of cells and signal cascades.
Highlights
Large populations of neurons have the ability to carry out multiple complex processes in parallel facilitated by the highly ordered architecture of the network
For all experiments performed in this study, triangular protein structures as depicted in Figures 1A–D were transferred onto silanized glass coverslips by μCP, as previously described (Fricke et al, 2011; Wendeln and Ravoo, 2012; Albers et al, 2015)
Cortical embryonic rat neurons were cultured on the substrates for 10–14 days in vitro (DIV) for immunofluorescence analysis and 14–24 DIV for recordings of spontaneous activity by calcium imaging
Summary
Large populations of neurons have the ability to carry out multiple complex processes in parallel facilitated by the highly ordered architecture of the network. Setting up these intricate networks necessitates the control of precise wiring of neuronal circuits. The concept of neural microcircuits is widely accepted based on experimental and computational studies (Bastos et al, 2012; Kwan and Dan, 2012). These circuits form more complex networks termed macrocircuits that connect various brain regions. Monitoring every neuron’s input and output is crucial for understanding its function in these circuits but is technically impossible and does not allow to unravel the structure–function relationship (Feldt et al, 2012)
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