Neurospheres from primary rodent brain cells to probe the 3D organization and function of synapses

To improve our understanding of synapse assembly, there is a need for robust, easy-to-use, and physiologically relevant in-vitro models allowing the controllable formation of neuronal contacts in a reasonable time, whose structure and function can be investigated using advanced microscopy. To address this challenge, we engineered 3D cultures from rodent dissociated hippocampal cells, that spontaneously assemble in low attachment U-bottom wells into compact spheroids of reproducible dimensions (100-300 microns), determined by the number of seeded cells. These neurospheres contain a mix of neurons and glial cells and grow over time in culture, through the combination of cell proliferation and neurite extension. Neurospheres were immunostained in fluid phase, and/or sparsely electroporated for the multi-color visualization of synaptic proteins. Neurons extend an elaborate network of axons and dendrites, forming within 2 weeks numerous excitatory and inhibitory synapses identified at the structural level by confocal and electron microscopy, and at the functional level by electrophysiology. Periodic calcium oscillations throughout neurospheres further highlight network activity. Finally, we demonstrate the potential of neurospheres to study synaptogenesis by modulating and visualizing the adhesion protein neuroligin-1. Overall, neurospheres represent a standardized and cost-effective system to study synapse structure and function at high resolution in 3D, that should be quite appealing to the cellular neurobiology community.