Cell cycle kinetics and immunohistochemical characterization of dissociated fetal neocortical cultures: evidence that differentiated neurons have mitotic capacity

Abstract Neurons in the neocortex (regardless of their developmental state) are considered to be post-mitotic and incapable of dividing. We used dissociated primary cultures derived from the neocortices of 16-day-old fetuses to test the counter-hypothesis, that is, differentiating neocortical neurons can divide. The cultured cells experienced considerable cell death, yet the number of viable cells remained relatively constant over the first 5 days in vitro. The implication was that the cultures contained proliferating cells. This was confirmed with a [ 3 H]thymidine ([ 3 H]dT) incorporation study and cumulative bromodeoxyuridine labeling. In fact, over 1/4 of the cells were cycling and the length of the cell cycle was 20.0 h; kinetics which mirror those of the developing cortex in vivo. This population of proliferating cells was eliminated by 48 h treatment with fluorodeoxyuridine. Immunohistochemical procedures determined that most cultured cells (≥90%) expressed proteins associated with differentiating or mature neurons, e.g., neurofilament (NF) 200 and isoforms of microtubule-associated protein (MAP) 2. Markers for immature neurons (e.g., nestin) were expressed by 10% of the cells. In contrast, markers for glia and their precursors were expressed by ≤2% of the population. Double-labeling with [ 3 H]dT and a neural-specific antibody showed that cells expressing an antigen for immature neurons constituted most of the proliferating cells, however, a considerable number of [ 3 H]dT-labeled cells expressed markers for differentiating neurons (e.g., NF200 and MAP2). Thus, differentiating neocortical neurons can be mitotically active and it appears that differentiating neurons are derived from both the ventricular and subventricular proliferative zones.