An electron microscopic analysis of hippocampal neurons developing in culture: early stages in the emergence of polarity.

In culture, hippocampal neurons initially establish several short, minor processes. The initial step in the emergence of polarity is marked by the rapid and selective growth of one of these processes, which becomes the axon. Subsequently the remaining processes become dendrites. We examined the ultrastructure of hippocampal neurons before and after the emergence of the axon. The minor processes in cells that had not yet formed axons were somewhat variable in appearance, but we found no ultrastructural feature that indicated which minor process might become the axon. The emergence of the axon was marked by several changes in its ultrastructure. The axon contained a sevenfold lower density of polyribosomes than the minor processes. In addition, axonal growth cones contained a pronounced concentration of membranous elements that resembled endoplasmic reticulum, elements that were rare in the growth cones of minor processes. Axons and minor processes did not differ in microtubule density. In order to gauge how rapidly these ultrastructural changes occur, we examined cells with short axons that, from their length, were estimated to have emerged only hours earlier. The preferential exclusion of polyribosomes from the axon and the concentration of reticular membrane in the axonal growth cone were already evident in such cells. These observations demonstrate that exclusion of ribosomes from the axon occurs early in development, about as soon as the axon can be identified. In contrast, previous work has shown that the differences in microtubule polarity orientation that distinguish mature axons and dendrites, and that have been proposed to account for the selective segregation of some constituents in neurons, first appear at a later stage of development (Baas et al., 1989). These observations also demonstrate that the accumulation of reticular membrane elements in growth cones, which has been noted previously, occurs preferentially in axonal growth cones and is closely correlated in time with the initial specification of the axon. The selective concentration of these elements in axonal growth cones could be associated with the uniquely rapid rate of axonal growth.