Figure 4. Flx-induced bone loss is brain serotonin-dependent and mediated by the sympathetic tone.

(a) Expression of Nfatc1 in long bones of WT females treated for 6 w. (b) Analysis of NF-κB signaling in RAW264.7 osteoclasts treated with RANKL (ng/ml), and with veh or Flx for 30 min. Top: Representative image of a western blot (n = 2 western blot per conditions). Bottom: NFκB-p65 (green) subcellular localization by immunocytochemistry. AF-548 phalloidine (actin, red), DAPI (nuclei, blue) (n = 10 images per group). Scale bar, 50 μm. Arrows indicate osteoclast nuclei. (c–f) WT female mice treated with Flx or veh for 3 or 6 weeks. Urine E and NE concentration (c), Ucp1 expression in brown adipose tissue (d), Bche expression in hypothalamus (left) and NE content in brainstem (right) (e), western blot analysis of hypothalamus extracts (n = 1 technical replicate of the number of mice indicated in the bar graphs on the right, each lane shown corresponds to a single mouse) (left) and quantification of band intensities normalized with GAPDH and reported to veh (right) (f). (g) Western blot analysis of Neuro2A (N2A) neuroblastoma cultures treated for 20 min with serotonin (μM) (veh n = 4, 25 and 200 5HT n = 3 western blots per condition). (h,i) Urine E (left) and NE (middle) concentrations and the Tnfrsf11b/Tnfsf11 ratio in long bones (right) (h), and vertebrae analysis, with representative images (n = 4 images/mouse; scale bars, 400 μm) and quantification of BV/TV (left) and bone histomorphometric analyses (right) (i) in Tph2^−/− females treated for 6 w. (veh n = 10, Flx n = 8). (j) Representative images of vertebrae (n = 4 images/mouse; scale bars, 400 μm) and quantification of BV/TV in Slc6a4^−/− females treated for 6 w (veh n = 8, Flx n = 4). Values are mean ± SEM compared to veh *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. One-way ANOVA followed by Dunnet’s test (f, middle), followed by Turkey’s test (f, right; g), or Student’s test (all other panels).