The effect of type 1 astrocytes on neuronal complexity: a fractal analysis

Embryonic, ventral spinal cord neurons were grown on poly(d-lysine) (PDL) or on a monolayer of type 1 astrocytes. At various times from 6 h to 2 weeks postplating, cells were fluorescently labeled and fixed with 4% paraformaldehyde. The cell surface immunoreaction allowed visualization of neurons in their entirety, namely, cell bodies and various membranous extensions that included lamellipodia, growth cones, axons, and dendrites. Outlines were drawn for individual neurons and their fractal dimension (D) was calculated. Neurons on poly(d-lysine) reached a peak D at 3 days in vitro, 1 day later than neurons on astrocytes (2 days in vitro). The maximum D was greater for cells on poly(d-lysine) when compared with neurons on astrocytes. In a second experiment the maximum D was similar for neurons on both surfaces but neurons on PDL maintained a higher D for a much longer period than neurons on astrocytes. An examination of fluorescent images revealed that neurons on poly(d-lysine) exhibited lamellipodia and large growth cones for several days and these structures were likely responsible for the high D seen in these cells. These structures were rarely observed in neurons plated on astrocytes. Interestingly, D on both surfaces decreased to a similar value at between 1 and 2 weeks in vitro. The trend for D in these cultures, an initial increase to a peak value followed by a decrease to a stable value, is discussed in light of the chemical nature of the two surfaces and synapse formation and stabilization.