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

Abstract

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.

Outgrowth patterns and directed growth of identified neurons induced by native substrates in culture

“…Indeed, outgrowth patterns on ganglion capsules could be used to distinguish one cell type from another, acting as fingerprints for their identification. Moreover, for a particular cell type, the patterns were consistently very similar but were quite different from the characteristic patterns seen previously in the same cell types plated on Con A or laminin-enriched extract (Chiquet and Acklin, 1986;Grumbacher-Reinert, 1989). Figure 3C shows that Retzius neurons plated on Con A extended broad lamellar processes.…”

Properties of an identified dopamine‐containing neurone in culture from the snail Helisoma

“…Cells maintained on poly-L-lysine exhibit a different pattern of processes from those maintained on Con A (cf. Grumbacher-Reinert, 1989), and even variations in the molecular weight of poly-L-lysine can have pronounced effects. Figure 5 shows the effect of varying the substrate on the growth of the GDN.…”

Axonal sprouting and laminin appearance after destruction of glial sheaths.

“…The same types of leech sensory and serotonin-containing neurons that sprout when desheathed in the connective also extend neurites vigorously in tissue culture on substrates containing leech laminin (15,27,28). Furthermore, extension of neurites on leech laminin is inhibited by antibody against leech laminin (15).…”