Cytoskeletal movements and substrate interactions during initiation of neurite outgrowth by sympathetic neurons in vitro

Abstract

The initial outgrowth of neurites from chick sympathetic neurons grown in vitro was investigated by time-lapse microscopy with laser-scanning and conventional light microscopes. Video-enhanced contrast, differential interference contrast optics (VECDIC) were used to monitor movements of neuronal cytoplasm, as well as the movements of small beads attached to the surface membrane, and interference reflection microscopy (IRM) was used to determine the concomitant pattern of attachment to the growth substrate (polyornithine or laminin). Related changes in the distributions of actin filaments, microtubules, and neurofilaments were determined by fluorescence labeling methods. Neurite formation on both substrates entailed invasion of the actin cores of filopodia by cytoplasm containing microtubules and neurofilaments. Small beads attached to the surface membrane surrounding the cytoplasm moved outward simultaneously with the cytoplasm. Cytoplasm invaded filopodia of neurons plated on laminin soon after attachment to the substrate or, for neurons generated in vitro, within as little as 3 min after cytokinesis. However, cytoplasm invaded filopodia of neurons grown on polyornithine only when they contacted a three-dimensional object such as another cell or a large, polyornithine-coated polystyrene bead. The observation that adhesion of filopodia to polyornithine-coated beads can initiate neurite formation is inconsistent with the commonly held view that neurite formation requires adhesion mediated by specific cell adhesion molecules. Simultaneous IRM and DIC imaging showed that cytoplasm invaded filopodia when only their tips were closely apposed to a substrate but not when they were closely apposed to a substrate along their entire lengths. These findings help to elucidate the mechanisms by which interactions between the cytoskeleton and the growth substrate initiate and produce the neuronal movements that lead to the formation of neurites.

The Steroid Hormone 20-Hydroxyecdysone Enhances Neurite Growth ofDrosophilaMushroom Body Neurons Isolated during Metamorphosis

“…Very short neurites (less than ϳ8 m) could not be measured reliably because of limitations in the precision of cursor placement at the ends of a measured segment. Furthermore, on Manduca motor neurons in vitro such short neurites are transient, actin-based filopodia that rarely contain microtubules (Matheson and Levine, 1999) that stabilize neuritic branches (Ahmad et al, 1993;Smith, 1994). Therefore, those neurites with a measured length Ͻ7.8 m were not included in the length and branch number totals for a cell but were tallied separately.…”

Filopodia: The Fingers That Do the Walking

“…Dissociated primary neurons on glass cover slips undergo a standard progression of morphologies to reach their mature state with one axon and several dendrites (23). Filopodia appear to be essential for neurite initiation, when the precursors for axons and dendrites first form and extend from the neuronal cell body (24,25). This suggests that filopodia function to establish neural circuitry during development.…”

Contact with Isolated Sclerotome Cells Steers Sensory Growth Cones by Altering Distinct Elements of Extension

“…Veils and filopodia may engorge with cytoplasm. Because not all extensions engorge (Goldberg and Burmeister, 1986), cues may also steer by influencing engorgement (Smith, 1994). Third is consolidation.…”