Substrate-dependent interactions of leech microglial cells and neurons in culture.

The principal aim of the present experiments has been to analyze the properties of microglial cells and their role in nerve regeneration. In the leech, damage to the CNS has been shown to be followed by accumulation of laminin and microglial cells at the site of injury (Masuda-Nakagawa et al., 1990. Proc. R. Soc. Lond. B. 241:201-206; and 1993. Proc. Natl. Acad. Sci. USA 90:4966-4970). Procedures were devised for isolating these small, wandering cells from the CNS of the leech. In culture, they were reliably identified by their sizes, shapes, and phagocytotic activity. Their morphology, motility, and interactions with neurons were influenced by the substrate molecules on which they were plated. On the plant lectin concanavalin A (Con A) microglia had a rounded shape and remained stationary. By contrast on extracts of leech extracellular matrix (ECM) enriched with laminin the cells were mobile and spindle-shaped with long processes. On Con A, neuronal growth cones avoided microglial cells, whereas on ECM extract the presence of a microglial cell did not influence neurite growth. Microglial cells showed immunoreactivity on both substrates when stained with a monoclonal antibody against leech laminin. Together these results suggest that microglial cells are influenced in their properties by molecules in the environment and that they could contribute to neuronal outgrowth at the site of an injury.

Influence of substrate on retraction of neurites following electrical activity of leech Retzius cells in culture.

1. The aim of these experiments was to determine how electrical stimulation of identified neurones in culture influences their growth on defined substrates. Single Retzius cells isolated from the central nervous system (CNS) of the leech were plated in culture dishes coated with the plant lectin Concanavalin A or with extracellular matrix extract containing leech laminin to promote neurite outgrowth. Stimuli were applied by a fine tungsten microelectrode placed close to the cell surface. The efficacy of electrical stimulation was checked occasionally by recording intracellularly with a microelectrode. 2. After the period of stimulation had ended, there was a short delay before neurones plated on leech laminin retracted their neurites. Of 112 neurones, only 11 failed to respond to stimulation. Neurite retraction in each cell was non-uniform, some processes retracting while others did not. After having retracted, most neurites subsequently showed clear regrowth. The degree of retraction depended on the duration of the stimulus train: whereas a few minutes was sufficient to produce observable effects, prolonged periods of stimulation caused more extensive retraction. Trains of impulses at 4 s-1 were equally effective when they were delivered in intermittent bursts or continuously. 3. The time in relation to growth at which stimuli were applied was of critical importance. Neurones stimulated during the phase of rapid outgrowth on leech laminin did not retract their neurites, which continued to elongate during and after stimulation. Neurones that had not retracted during the early phase were stimulated again later, when extension and outgrowth of neurites had ceased or slowed. At this stage stimulation was followed by retraction and subsequent regrowth. 4. Retzius cells plated on a substrate of Concanavalin A instead of leech laminin failed to show any retraction after stimulation. 5. To investigate the possible role of Ca2+, cells were grown with raised concentrations of Mg2+ in the bathing fluid. Raised [Mg2+] did not influence the rate or the extent of neurite outgrowth. It reduced, but did not block, the effects of electrical stimulation. Earlier experiments have shown that growth on Concanavalin A occurs without obvious Ca2+ entry following stimulation. Together with the present experiments, the results suggest that Ca2+ entry following impulses in cells grown on laminin is responsible for the massive retraction.

Local influence of substrate molecules in determining distinctive growth patterns of identified neurons in culture.

The growth and branching patterns of individual identified leech neurons in culture depend upon the molecular composition of the substrate. These differences in morphology have been analyzed quantitatively for nerve cells growing on the plant lectin Con A, on extracellular matrix extract (ECM) containing laminin, and on patterned substrates. The total length of neurite outgrowth was about four times greater, and the number of branching points per unit length was three times smaller on ECM laminin extract than on Con A. Single cells placed on a sharp well-defined border separating Con A and ECM-laminin extract sprouted neurites rapidly on both sides of the border without showing preference for either substrate. An individual nerve cell produced neurites with markedly different structure on the two substrates--curved and thick, with a higher branching frequency on Con A, but straight and slender with a lower branching frequency on ECM-laminin substrate. Similar changes were seen in individual neurites that crossed from one substrate to the other. These results show that local contact of the neuron with a particular substrate induces a particular pattern of local outgrowth. Hence, molecules anchored in extracellular matrix could provide information for regenerating or developing axons regarding the type of branching pattern to be produced in that region.