Functional synapse formation among rat cortical neurons grown on three-dimensional collagen gels

To investigate synaptic formation of neurons grown on three-dimensional (3D) collagen gels, neurons dissociated from embryonic rat cerebral cortices were seeded onto type-I collagen gels and cultured in serum-free medium for up to 2 weeks. Double-immunostaining for mitogen-activated protein-2 (MAP-2), a neuronal cell body and dendritic marker, and synapsin I, a synaptic vesicle antigen, was carried out to identify pre- and postsynaptic structural specializations, respectively. MAP-2(+) neuronal soma and dendrites were found to be surrounded by numerous puncta of synapsin I in a 1 week-old culture. Whole-cell patch clamp experiments demonstrated that the neurons grown on 3D gels exhibit sodium and potassium currents similar to those seen in 2D culture. Spontaneous action potentials were found in neurons that had been in culture for 8-12 days. In addition, spontaneous, bicuculline-sensitive gamma-aminobutyric acidergic postsynaptic currents were also present. This is the first demonstration of functional synapse formation among neurons grown on 3D collagen gels, suggesting that type-I collagen can be a promising material for neuronal regeneration.