Steps in the formation of a bipolar outgrowth pattern by cultured neurons, and their substrate dependence

We studied the steps in the formation of the bipolar outgrowth pattern of cultured adult Anterior Pagoda (AP) neurons of the leech growing on a central nervous system (CNS) homogenate as substrate. This pattern, which consists of two primary neurites directed in opposite directions plus some bifurcations, resembles their embryonic pattern but is different from the patterns they develop in culture on leech laminin or Concanavalin A as substrates. In eight neurons that were studied, one primary neurite formed and branched several hours before the second one. Time-lapse video analysis showed that between 12 and 36 h of growth, the more proximal branch of the early neurite migrated retrogradely, rotated, and formed the second primary branch. Both neurites elongated until the total neurite length reached 130-160 microm, when the elongation of primary neurites became synchronous with the retraction of secondary processes, suggesting competition. The substrate dependence of these events was tested by plating AP neurons on leech laminin. On this substrate AP neurons produced multiple independent primary neurites with branches. Retraction of some large branches was followed by their regrowth, and did not correlate with the changes in other neurites. We propose that the dynamics in the formation of the bipolar outgrowth pattern of AP neurons arise from inhibitory extracellular matrix molecules, which reduce the synthesis of precursors for neurite formation.