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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We thank E. Callaway (Salk Institute) for the lentiviral DNA constructs (HIV-CS-CG-SynapsinPr-GFP), R. Tsien (University of California, San Diego) for the pRSETB_tdTomato plasmid, K. Kolstad and J. Flannery (University of California, Berkeley) for providing us with the AAV-SynapsinPr-LiGluR virus and for assisting in the preparation of the lentivirus, the Molecular Imaging Center and H. Aaron for help with the confocal microscopy, and M.M. Poo, M. Shelly, M.B. Forstner and S. Kohout for helpful discussions and comments. This work was supported by the US National Institutes of Health Nanomedicine Development Center in Optical Control of Biological Function (PN2 EY1018241). C.W. was supported by a Marie Curie Outgoing International Fellowship funded through Laboratoire de Neurosciences et Systèmes Sensoriels, Centre National de la Recherche Scientifique, Uniteé Mixte de Recherche 5020.
Supplementary Figures 1–2 (PDF 795 kb)
Animation of 3D reconstruction of macroscopic 3D neuronal network. Animation showing confocal z-series imaging presented in Figure 2. (MOV 749 kb)
Animation of 3D reconstruction of neuronal processes on the guiding layer. Reconstruction of axons and dendrites on the 45 μm cAMP beads after 7 days using the filament tracer of the Imaris software (Bitplane Inc.). (MOV 2438 kb)
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Pautot, S., Wyart, C. & Isacoff, E. Colloid-guided assembly of oriented 3D neuronal networks. Nat Methods 5, 735–740 (2008) doi:10.1038/nmeth.1236
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