Skip to main content
Figure 4 | Molecular Brain

Figure 4

From: Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival

Figure 4

Neurite growth of basal forebrain cholinergic neurons is inhibited by CR-proBDNF, but elicited by matBDNF. BFCNs were cultured in serum-containig (A-E) or serum-free (F) medium for 2 weeks and treated with 100 ng/ml CR-proBDNF or matBDNF in the same medium condition for 2 days. Histochemistry and quantitation of AChE-positve neurites were done as described in Methods. (A) BFCNs were double-stained using AChE histochemistry and Hoechst 33258 (arrows). (B) The survival rate of BFCNs (%) = 100 × [living cells]/([living cells] + [dead cells]). Data were normalized to mock cultures (100% as control). n = 159 (Mock), 151 (matBDNF), and 149 (CR-proBDNF) from three independent coverslips. Results were replicated in three independent experiments. (C) Low- and high-magnification images of AChE-stained BFCNs. (D) The number of neurites extending outwards from the cell body shown in C. n = 38 (Mock), 37 (matBDNF), and 40 (CR-proBDNF) cells from three independent coverslips. (E) Neurite complexity as revealed by Sholl analysis. n = 30 (Mock), 30 (matBDNF), and 30 (CR-proBDNF) cells from three independent coverslips. In multi-bar graphs, ANOVA followed by post-hoc analysis was used. **P < 0.01. Results were replicated in at least three independent experiments. (F) The opposing effects of matBDNF and CR-proBDNF on neurite fiber density were confirmed by a distinct quantitative method. The maximal threshold of AChE-positive cholinergic fiber intensity was defined as 70% above the background. The total intensity of the fibers was determined in an optical field and was divided by the number of AChE-positive neurons in the same field. Data were collected from four independent fields in a single chamber. t-test, **P < 0.01, compared to Mock (100% as control); n = 6 independent culture dishes. Scale bar, 5 μm (A and C). (G) Effect of CR-proBDNF on neurite density of BFCNs in serum-free condition and a dose-dependency test of CR-proBDNF. Cell survival (left) and the neurite number of BFCNs (right) were determined. Note that proBDNF negatively regulates the neurite density of BFCNs in serum-free conditions and at subnanomoler concentration. n = 3 independent coverslips. t-test, **P < 0.01, significantly different from Mock.

Back to article page