Appearance of neurofilament subunit epitopes correlates with electrophysiological maturation in cortical embryonic neurons cocultured with mature astrocytes.

E14 rat cortical neurons which have almost no glial progenitors were cocultured with a homogeneous population of mature type 1 astrocytes at a 4/1 ratio in serum free medium. Maturation of neurons was evaluated using a set of well characterized antibodies and two new monoclonal antibodies (MN2E4 and MN3H6) raised against various neurofilament subunits and whole-cell patch clamp experiments. We observed that this coculture method leads to a well-timed and very homogeneous neuronal maturation and that sequential appearance of neurofilament subunits in developing neurons correlates with the electrophysiological maturation. This sequence, early expression of the 68 kDa neurofilament subunit and late appearance of the 200 kDa neurofilament subunit, occurs in normal brain development, which validates this culture model as a useful tool for studying neuronal maturation and differentiation. MN2E4 staining (non-phosphorylated 200 kDa cytoskeletal protein antibody) appeared just before the neurons became excitable. It could thus be used as a functional neuronal marker. MN3H6 staining (phosphorylated 160-200 kDa neurofilament subunit antibody) appeared just after the neurons made synaptic contacts and generated synaptically driven spike bursts. This finding indicated that some phosphorylated epitopes of 160-200 kDa neurofilament followed synaptogenesis. These processes may play a key role in stabilizing the synapses to achieve a functional neuronal network.