Figure 5
proBDNF reduces dendritic spine density in hippocampal neurons. Cultured neurons were maintained for 3–4 weeks and treated with the indicated reagents (50 ng/ml) for 2 (A, B, and D) or 3 days (C). In A and B, neurons were cultured in serum-containing medium. (A) CR-proBDNF reduced the density of DiI-labeled spines. Representative images of DiI-labeled neurons treated with the indicated reagents for 2 days (left). Summary of spine density (middle) and length (right). Data were collected from 52 (Mock), 59 (matBDNF), and 38 (CR-proBDNF) independent cells (non-parametric test, *P < 0.05, compared to Mock). Note that matBDNF increased spine density, while proBDNF reduced spine density (arrows and arrowheads). (B) proBDNF promoted the shrinkage of phalloidin-labeled spines and p75NTR was involved in the proBDNF action. Application of a functionally blocking antibody against p75NTR, AB1554 was performed and fixed neurons were stained with an anti-MAP2 antibody and FITC-labeled phalloidin. Representative images of the double-stained neurons (left) and a quantitative analysis of phalloidin-labeled spiny structures (right) are shown, n = 30–40 independent cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock (100% as control). (C) CR-proBDNF decreases spine density in low-density hippocampal neurons cultured in serum-free medium. The cultures were incubated with CR-proBDNF in serum-free medium for 2 days and double-stained with an anti-MAP2 antibody and FITC-labeled phalloidin. n = 24–32 cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock. (D) CR-proBDNF decreased spine density in hippocampal slices. Representative images of DiI-labeled neurons at low and high magnification are shown (left). A decrease in spine density was observed in CR-proBDNF-treated slices (arrow heads). Quantification of spine density (right). Data were collected from 7 (Mock) and 6 (CR-proBDNF) independent slices. Note that CR-proBDNF-treated slices showed a decrease in spine density when compared with matBDNF. t-test, *P < 0.05. Scale bar, 10 μm (A, B, and D).