Neuronal network formation is an intricate process by which individual neurons connect into a functional circuitry. At the subcellular level, neuronal connectivity is characterized by the number, size and strength of synapses. At the cellular level, in vitro network characterization remains a challenge due to the large number of neurons involved, spreading widely across a culture dish. Here, we demonstrate a pipeline using high-content confocal microscopy and automated image analysis to study spatial organization of individual neurons in an in vitro cellular network. With this approach, we enable analysis of thousands of neurons in one well, and of multiple wells simultaneously. Using this workflow, we compared the spatial organization of primary mouse neuronal networks derived from the hippocampus, cortex and cerebellum. We also demonstrate how to extract morphological details, such as size of the nucleus and axon initial segment number, orientation and length from our data. This workflow can be applied to study underlying molecular mechanisms of circuitry formation, to assess network formation of neurons derived from mouse or human iPSC models for neurological diseases, and serve as a future platform for drug development.

In the present study, the influence of topographic and mechanical cues on neuronal growth cones (NGCs) and network directionality in 3D-engineered cell culture models is explored. Two-photon polymerization (2PP) is employed to fabricate nanopillar arrays featuring tunable effective shear modulus. Large variations in mechanical properties are obtained by altering the aspect ratio of the nanostructures. The nanopillar arrays are seeded with different neuronal cell lines, including neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs), I³Neurons, and primary hippocampal neurons. All cell types exhibit preferential orientations according to the nanopillar topology, as shown by neurites creating a high number of oriented orthogonal networks. Furthermore, the differentiation and maturation of NPCs are affected by the topographic and mechanical properties of the nanopillars, as shown by the expression of the mature neuronal marker Synapsin I. Lastly, NGCs are influenced by effective shear modulus in terms of spreading area, and stochastic optical reconstruction microscopy (STORM) is employed to assess the cytoskeleton organization at nanometric resolution. The developed approach, involving laser-assisted 3D microfabrication, neuro-mechanobiology, and super-resolution microscopy, paves the way for prospective comparative studies on the evolution of neuronal networks and NGCs in healthy and diseased (e.g., neurodegenerative) conditions.