A central challenge in neuroscience is to understand the formation and function of three-dimensional (3D) neuronal networks. In vitro studies have been mainly limited to measurements of small numbers of neurons connected in two dimensions. Here we demonstrate the use of colloids as moveable supports for neuronal growth, maturation, transfection and manipulation, where the colloids serve as guides for the assembly of controlled 3D, millimeter-sized neuronal networks. Process growth can be guided into layered connectivity with a density similar to what is found in vivo. The colloidal superstructures are optically transparent, enabling remote stimulation and recording of neuronal activity using layer-specific expression of light-activated channels and indicator dyes. The modular approach toward in vitro circuit construction provides a stepping stone for applications ranging from basic neuroscience to neuron-based screening of targeted drugs.
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