Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy.

Abstract

Abstract. The regenerative growth in culture of the axons of two giant identified neurons from the central nervous system of Aplysia californica was observed using video-enhanced contrast-differential interference contrast microscopy. This technique allowed the visualization in living cells of the membranous organelles of the growth cone. Elongation of axonal branches always occurred through the same sequence of events: A flat organelle-free veil protruded from the front of the growth cone, gradually filled with vesicles that entered by fast axonal transport and Brownian motion from the main body of the growth cone, became more voluminous and engorged with organelles (vesicles, mitochondria, and one or two large, irregular, refractile bodies), and, finally, assumed the cylindrical shape of the axon branch with the organelles predominantly moving by bidirectional fast axonal transport. The veil is thus the nascent axon. Because veils appear to be initially free of membranous organelles, addition of membrane to the plasmalemma by exocytosis is likely to occur in the main body of the growth cone rather than at the leading edge.Veils almost always formed with filopodial borders, protruding between either fully extended or growing filopodia. Therefore, one function of the filopodia is to direct elongation by demarcating the pathway along which axolemma flows. Models of axon growth in which the body of the growth cone is pulled forward, or in which advance of the leading edge is achieved by filopodial shortening or contraction against an adhesion to the substrate, are inconsistent with our observations. We suggest that, during the elongation phase of growth, filopodia may act as structural supports.

Cytoplasmic Linker Proteins Regulate Neuronal Polarization through Microtubule and Growth Cone Dynamics

“…For the extension, the axonal growth cone repeatedly undergoes a cycle of specific steps (Goldberg and Burmeister, 1986;Lowery and Van Vactor, 2009) (Fig. 8).…”

Microtubule-based filopodium-like protrusions form after axotomy

“…Neurons from buccal ganglia ofjuvenile Aplysia calijbrnica were cultured for video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy as previously described (Goldberg and Burmeister, 1986). Cell bodies were extracted from ganglia with lengths of their original axons attached.…”

“…VEC-DIC microscopy was performed as previously described (Goldberg and Burmeister, 1986;Burmeister et al, 199 1). In a few experiments, we used a long working distance condenser (NA 0.65) rather than the usual short-working-distance condenser (NA 1.4) to allow greater access to the cells.…”

Tyrosine Phosphorylation of Paxillin and Focal Adhesion Kinase during Insulin-like Growth Factor-I-stimulated Lamellipodial Advance

“…Formation of adhesions on the lamella is necessary not only for lamellar stability but also for continued lamellar advance (10, 11). Actively advancing regions of lamella are known as lamellipodia and mediate cell migration and growth cone translocation (15)(16)(17).In our laboratory, we have been studying the role of FAK and paxillin in the regulation of neuronal morphology. We showed recently that FAK and paxillin are tyrosine-phosphorylated during integrin-mediated cell spreading and neurite formation in human neuroblastoma .…”

“…Formation of adhesions on the lamella is necessary not only for lamellar stability but also for continued lamellar advance (10,11). Actively advancing regions of lamella are known as lamellipodia and mediate cell migration and growth cone translocation (15)(16)(17).…”