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 substratescurved and thick, with a higher br ng frequency on Con The results of two experiments are presented here. In the first experiment, leech neurons were plated either on Con A or on leech ECM-laminin substrate, and the neurite-sprouting patterns were analyzed quantitatively. In the second experiment, individual nerve cells were placed directly on or adjacent to a well-defined border between Con A and the leech ECM-laminin subtrates. This arrangement made it possible to test whether neurites prefer one substrate over another and whether a single neurite exhibits different structures when crossing from one substrate to the other. The results indicate that local interactions between the growing processes and the substrate determine the numbers and shapes of branches. MATERIALS AND METHODSTissue Culture. Techniques for isolating and culturing individual nerve cells have been described (12,13,18). The connective tissue capsules surrounding leech ganglia were opened in Leibovitz L-15 medium (GIBCO) supplemented with 2 mM glutamine (GIBCO), glucose at 6 mg/ml, gentamicin sulfate at 0.1 mg/ml (Garamycin; Schering), and 2% fetal calf serum (Biological Industries, Kibbutz Beth Haemek, Israel). After digestion of the ganglia with collagenase/ dispase (Boehringer Mannheim; 2 mg/ml in L-15 medium for 45 min), single AP cells, identified by their shape and location within ganglia, were picked up by suction, washed, and plated in microwell dishes 7270The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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.

1. Leech Retzius neurones were isolated by a new technique which allowed investigation of macroscopic currents over the surface of the cell body and the axons using loose patch-clamp. The distribution of ion current densities was measured for neurones that had just been removed from the CNS, and for cultured cells in which neurite outgrowth had begun. To standardize the mapping procedure, the same patch electrode was used at various sites along the neurone. 2. Immediately after isolation of the cell, rapidly activating and inactivating Na+ currents were recorded from distal segments of the axons, but not from the soma or the proximal segment. Na+ currents were isolated by using patch electrodes containing tetraethylammonium (TEA+) and 4-aminopyridine (4-AP) to block K+ channels and Cd2+ to block calcium channels. Na+ currents in all regions of the neurone where they could be recorded were similar in their voltage dependence and kinetics. The Na+ current density was highest at the broken tips of the axon stumps. 3. Neurites began to extend from the broken axon tips approximately 30min after isolation. Newly grown processes showed a high density of Na+ currents at their growth cones. After 2 days in culture the current densities became more uniformly distributed and Na+ currents could then be recorded in the soma and proximal axon segments. 4. In agreement with earlier studies made with conventional two-electrode voltage-clamp, three principal K+ currents were detected in Retzius cells: a rapidly activating and inactivatingA-type current blocked by 4-AP (IA); a more slowly activating and inactivating delayed K+ current blocked by TEA+ (IK1); and a Ca2+-activated K+ current (IC). Immediately after isolation of the Retzius cell, both rapid A-type and slow delayed K+ currents were distributed more uniformly than Na+ currents over the soma and axons. In their voltage sensitivities and kinetics, these two K+ currents were markedly different from each other; their characteristics were, however, constant in different regions of the cell. 5. Ca2+ currents were too small to be measured directly during depolarizing pulses. However, tail currents were large enough to demonstrate the presence of Ca2+ channels in the proximal segment of the axon and in the soma; the currents were not sufficiently large to resolve their spatial distribution. 6. It is concluded that ion channels are present in newly grown membranes and that the density of Na+ channels is highest in the tips of distal axon stumps from which outgrowth begins. By contrast, K+ currents are distributed more uniformly along the neurone.

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.