Abstract
Depolarization of leech neurons growing on extracellular matrix extract (ECM) leads to cessation of neurite outgrowth, rounding up of the peripheral regions of the growth cone, loss of filopodia, and neurite retraction. These responses depend on the influx of calcium (Neely, 1993). The aim of the present experiments was to analyze how the cytoskeleton becomes reorganized as growth cones change their morphology. Immunocytochemistry revealed a loss of microfilaments in the tips of neurites growing on ECM after depolarization. Leech neurons cultured on a different substrate, the plant lectin concanavalin A (ConA), continue to grow during and after depolarization (Grumbacher- Reinert and Nicholls, 1992; Neely, 1993). As expected, we did not observe any change in the distribution of microfilaments after depolarization on ConA. Since there is evidence that this lack of response is due to a reduced calcium influx during depolarization of neurons on ConA (Ross et al., 1988), the effect of the calcium ionophore A23187 on the outgrowth of these cells was analyzed. In the absence of depolarization, this ionophore caused cessation of growth cone motility and a loss of microfilaments, while microtubules were not affected. Cytochalasin D, a microfilament-disrupting agent, induced changes in growth cone morphology and neurite retraction similar to those observed after depolarization and calcium influx. Application of phalloidin, a drug that stabilizes microfilaments, inhibited depolarization-induced retraction of neurites on ECM. By contrast, stabilization of microtubules with taxol did not prevent depolarization from inducing changes in growth cone morphology and neurite growth. These experiments show that changes in growth cone morphology and motility of leech neurons induced by depolarization and calcium influx are accompanied by a dramatic change in the organization of microfilaments, but not microtubules.