Neurite Regeneration by Aplysia Neurons in Dissociated Cell Culture: Modulation by Aplysia Hemolymph and the Presence of the Initial Axonal Segment1

Neurons from the abdominal ganglion of the mollusc Aplysia californica regenerate neurite processes in dissociated cell culture. Both the nature of neurite outgrowth and the morphology of the cells are influenced by the presence of adult Aplysia hemolymph in the growth medium and the presence of a portion of a cell’s original axonal process. Aplysia hemolymph enhances cell survival, the initiation of neurite outgrowth from multiple sites on the cell body surface, the linear growth of the processes, and the amount of branching by those processes. Hemolymph also decreases the diameter of the outgrowing neurite fascicles and the diameter of the individual neurites within the fascicles. The presence of a cell’s original axon reduces the time required for the initiation of neurite outgrowth and restricts the formation of multipolar processes. In addition, the presence of an initial axonal segment is essential for neurite regeneration from large adult neurons. Before neurons can form synaptic connections with distant targets, they must initiate neurite outgrowth and project these neuritic processes toward the correct target zone. Thus, the pattern of synaptic connections and the Dissociated cell culture techniques have been utilized morphological properties of a neuron are influenced by extensively for studying neurite outgrowth and development of vertebrate neurons. Using this simple system factors which affect neurite outgrowth during early deapproach, investigators have succeeded in identifying a variety of chemical factors which can influence neuron velopment. survival, initiation and direction of neurite outgrowth, neurite extension, and aspects of biochemical differeni This work was supported by National Institutes of Health Grant NS19216 and National Science Foundation Grant 7923602 to S. S., Sloan Foundation Postdoctoral Fellowship to E. P., and National Institutes of Health Grant GM32099 which supports the mariculture facility at the Marine Biological Laboratory, Woods Hole, MA. We wish to thank R. Woolley, L. Shanet, and K. Hilten for technical assistance and preparing figures. We also would like to thank T. Capo and S. Perritt for raising the animals at the Marine Biological Laboratory. We also thank E. Kandel, R. Ambron, V. Castellucci, and J. Camardo for their comments on earlier drafts of this manuscript. ’ To whom correspondence should be sent, at Center for Neurobiology and Behavior, Columbia University, and New York State Psychiatric Institute, 722 West 168th Street, New York, NY 10032. tiation or synapse formation (Levi-Montalcini, 1976; Campenot, 1977; Letourneau, 1978; Patterson, 1978; Varon and Bunge, 1978; Jesse1 et al., 1979; Nishi and Berg, 1981). In some cases, it has been shown that these factors Recently, dissociated cell culture techniques have been extended to studying identified central neurons of invercan differ from one another in terms of tissue specificity, tebrates such as the leech, Helisoma, and Aplysia (Kaczmarek et al., 1979; Ready and Nicholls, 1979; Damechanisms of action, or the number of effects they can gan and Levitan, 1981; Wong et al., 1981). Because many of these invertebrate neurons are large and their propexert on a given cell type. erties have been well characterized in uiuo (Kandel, 1976; Kaneko et al., 1978; Muller et al., 1981), a variety of cell biological methods can be applied to examine regeneration and the specificity of synapse formation under experimentally controlled conditions (Fuchs et al., 1981, 1982; Wong et al., 1981, 1983; Camardo et al., 1983). With regard to neurite regeneration, Kater and his colleagues (Barker et al., 1982) have identified two separate factors released by the adult central nervous system of Helisoma that can affect either neurite extension or certain metabolic activities of Helisoma cells. In Aplysia, the neuroendocrine bag cells of the abdominal ganglion (Kaczmarek et al., 1979) and unidentified buccal ganglion neurons (Dagan and Levitan, 1981) have