Local role of Ca2+ in formation of veils in growth cones

Abstract

A previous study that used high-resolution video (VEC-DIC) microscopy to examine axonal growth cones of Aplysia giant neurons growing in culture had demonstrated that growth occurs by the extension of veils of membrane between filopodia and the subsequent morphological transformation of these veils, in place, into the swollen, organelle-filled central region of the growth cone and then into the cylindrical axon. The possible involvement of Ca2+ in this sequence of events was now examined using VEC-DIC microscopy. Reduction of [Ca2+]o from the normal level of 11 to 1.3 mM or below or the addition of 20 mM Co2+, which blocks Ca2+ channels, caused a large decrease in the area of immature veil (flat and with few organelles) in the growth cone within minutes. Ba2+, 20 mM, which flows well through Ca2+ channels, and 5 microM A23187, a Ca2+ ionophore, caused new immature veil to form in the presence of reduced [Ca2+]o. Maturation of veil into central region was not inhibited by reduced [Ca2+]o. In fact, the disappearance of immature veil was often the result partly, or entirely, of continued veil maturation in the absence of formation of new veil. The next step in maturation, conversion of the central region to cylindrical axon, was also probably not inhibited by reduced [Ca2+]o. Ca2+ was microapplied to large growth cones that had lost their veils by exposure to reduced [Ca2+]o. There was a strong tendency for the first, or only, incidence of veil formation to occur near the micropipette, the rest of the perimeter of the growth cone remaining quiescent. It is concluded that intracellular Ca2+ plays a role in veil formation and that the site of the Ca2+-dependent step is close to the site of veil formation. If this step is exocytosis, veil forms where there is net addition of membrane. Whether a change in [Ca2+]i, rather than some other factor, normally directly triggers veil formation remains uncertain, but, if it does, then the site of formation, which will strongly influence the direction of axon growth, is probably determined by focal changes in [Ca2+]i within the growth cone.