Figure 5From: Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survivalproBDNF 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).Back to article page