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 c ocultured 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 a ntibodies (MN2E4 and MN3H6) raised against various neurofilament subun its and whole-cell patch clamp experiments. We observed that this cocu lture method leads to a well-timed and very homogeneous neuronal matur ation and that sequential appearance of neurofilament subunits in deve loping neurons correlates with the electrophysiological maturation. Th is 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 too l for studying neuronal maturation and differentiation. MN2E4 staining (non-phosphorylated 200 kDa cytoskeletal protein antibody) appeared j ust before the neurons became excitable, It could thus be used as a fu nctional neuronal marker. MN3H6 staining (phosphorylated 160-200 kDa n eurofilament subunit antibody) appeared just after the neurons made sy naptic contacts and generated synaptically driven spike: bursts, This finding indicated that some phosphorylated epitopes of 160-200 kDa neu rofilament followed synaptogenesis. These processes may play a key rol e in stabilizing the synapses to achieve a functional neuronal network .