Figure 5.

Reversal of Directed Arbor Growth in Neighboring Sub-arbors

(A) Analysis of spatially biased growth of sub-arbors. Sub-arbors were defined as rostral or caudal according to the gravity center of the branch positions. Spatially biased growth was quantified as the difference in the change in branch length between the rostral and the caudal sub-arbors over the imaging interval. All branching points were analyzed, and the data were pooled (Figures S3 and S4).

(B) An image of an axon expressing tdTomato and synaptophysin-EGFP to identify synaptic puncta.

(C) Reconstruction of the axon in (B) with branches color-coded according to the intensity of the synaptophysin-EGFP signal. Red dots identify the puncta locations. Synaptophysin-EGFP intensity per branch length was measured and normalized to mean value across the axon. Right: overlay of puncta.

(D) Plot of synaptophysin-EGFP intensity per unit branch length between neighboring rostral and caudal branches. Data are from animals treated with or without DL-APV and before and after 2 days of visual stimulation with the dt = ±15-ms protocol. No significant difference was detected. See Table S2 for data and p values.

(E) Differences in branch lengths between the sibling sub-arbors across DL-APV doses for axons from earlier- or later-stimulated conditions. In controls, sub-arbors in earlier-stimulated axons showed a rostral spatial bias in sub-arbor growth, but in 5 μM APV, sub-arbors in later-stimulated axons showed a rostral growth bias. ∗∗∗p < 0.001. Bootstrap test, N = 10,000. (0 μM: earlier group: N = 100 branch points, 7 animals; later group: N = 132 branch points, 7 animals. 5 μM: earlier group: N = 213 branch points, 7 animals; later group: N = 120 branch points, 7 animals). See Table S2 for data and p values.