Fig. 4. Netrin 1 induces Dcc-dependent process extension by immature oligodendrocytes, and Dcc-dependent process branching and myelin-like membrane sheet extension by mature oligodendrocytes, in vitro. (A) Addition of netrin 1 (100 ng/ml, 24 hours) to cultured rat oligodendrocytes results in a 4% decrease in the ratio of immature cells expressing Cnp only (arrowheads) as compared with more mature cells expressing Cnp and Mbp. Netrin 1 did not change the ratio of cells expressing Mbp only versus Mbp and Mag. (B) Cnp-positive immature oligodendrocytes are multipolar cells with one major process. Dashed red lines indicate examples of the processes measured. (C) The length of the major process increased following the addition of netrin 1 (100 ng/ml) for 24 hours. Application of Dcc function-blocking antibody (Dccfb) to immature oligodendrocytes blocks the netrin-1-induced increase in process extension, whereas Dccfb alone does not have a significant effect on process extension. (D) A mature Mbp- and RIP-positive oligodendrocyte extending both myelin-like membrane sheets and branched processes. Sheet area was quantified by tracing the Mbp-positive myelin-like membrane sheets (left, red outlines). Branching was quantified by measuring the number of intersections that processes made with concentric circles (right, red), which are numbered 1-5 to reflect increasing distance from the cell body (and as labeled on the x-axis of bar charts displaying branching complexity). (E) Netrin 1 (100 ng/ml, 24 hours) increased the area of Mbp-positive sheets compared with the control. This effect was blocked by Dccfb. (F) Processes of mature oligodendrocytes exposed to netrin 1 for 24 hours exhibited an increase in branching. Dose-response analysis indicated maximal branching at 100 ng/ml netrin 1. (G) Addition of Dcc function-blocking antibody together with netrin 1 prevented the netrin-1-dependent increase in branching, and decreased branching compared with controls. A, 20x0.5 n.a. objective; B,D,F, 40x0.75 n.a. objective. Scale bars: 40 µm in A; 20 µm in B,D,F. A.U., arbitrary units. ^*P<0.05, ^**P<0.005, versus control. Error bars indicate s.e.m.