Neurite outgrowth and synapse formation by identified leech neurones in culture

Abstract

After injury, neurones in the central nervous system (CNS) of the leech regenerate with a high degree of specificity. The aim of our experiments has been to study the sequential steps involved in neurite growth and synapse formation using isolated identified neurones in culture. An important requirement for sprouting of leech neurones is the substrate. Neurites grow only slowly and sparsely on polylysine or vertebrate laminin. The extracellular matrix of leech ganglion capsules contains a protease-sensitive factor which can be extracted with urea. With this material as substrate, growth proceeds rapidly in defined medium. Another neurite-promoting substrate is provided by the plant lectin concanavalin A (Con A). The activity of Con A, but not of the capsule matrix factor, is blocked by the Con A-specific hapten methyl alpha-D-mannoside. The morphology and branching pattern of the neurites in culture depend on the specific substrate and on the type of neurone. During stimulation, less Ca2+ uptake occurs into growth cones than in cell bodies. The mechanism of neurite growth seems not to depend on activity-mediated Ca2+ influx or on interactions between neuronal cell surfaces. However, even without profuse outgrowth, electrical and chemical synapses develop between neighbouring neurones. The type of synapse depends predictably on the types of neurones within the cell pair. Since the development of a synapse can be followed with time in culture, the sequential events can each be studied separately for this multi-step process.