DISRUPTION OF MICROFILAMENTS IN GROWTH CONES FOLLOWING DEPOLARIZATIONAND CALCIUM INFLUX

Depolarization of leech neurons growing on extracellular matrix extrac t (ECM) leads to cessation of neurite outgrowth, rounding up of the pe ripheral regions of the growth cone, loss of filopodia, and neurite re traction. These responses depend on the influx of calcium (Neely, 1993 ). The aim of the present experiments was to analyze how the cytoskele ton becomes reorganized as growth cones change their morphology. Immun ocytochemistry revealed a loss of microfilaments in the tips of neurit es growing on ECM after depolarization. Leech neurons cultured on a di fferent 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 th e distribution of microfilaments after depolarization on ConA. Since t here is evidence that this lack of response is due to a reduced calciu m 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 c aused 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 neurit e retraction similar to those observed after depolarization and calciu m influx. Application of phalloidin, a drug that stabilizes microfilam ents, 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 neu rite growth. These experiments show that changes in growth cone morpho logy and motility of leech neurons induced by depolarization and calci um influx are accompanied by a dramatic change in the organization of microfilaments, but not microtubules.