Figure 7.
Effect of ephrin-B ligands on the aggregation and axon bundling of rat hippocampal neurons. A, Hippocampal neurons of E18 embryos (105 per well of 12-well dish) were cocultured with control NIH-3T3 cells. B, C, E18 hippocampal neurons were cocultured with ephrin-B3-G6 (B) or ephrin-B3-G10 (C), two lines of ephrin-B3-expressing cells, respectively. The cocultures were maintained for 2 d and then stained with anti-neuron-specific enolase antibody. Arrows indicate aggregates with similar sizes from different cultures that were chosen for quantification. Scale bar, 20 μm. D, Relative bundle thickness. The diameter of all axonal bundles was measured from aggregates that had similar size in both control and ephrin-B3-expressing cell lines. E, Neuron dissociation index. Dissociation index is the ratio of the number of individual unaggregated neurons in cocultures of ephrin-B3-expressing and control cells. Because the total number of neurons seeded on each well is the same (105 per well of 12-well Corning tissue culture dishes), the number of unaggregated individual cells is a measure of how aggregated the neurons are in the presence of ephrin-B3 compared with the controls. The dissociation index on control cells was regarded as 1. Asterisk indicates statistical significance (p < 0.05; t test).