Fig. 4: Hepatic LCN2 elicits anxiety-like behaviors via neuromodulation of mPFC.

a Lcn2 gene transcript levels were remarkably increased in liver, spleen, and intestine tissues but not in brain or other organs in CRS mice. b LCN2 protein was up-regulated only in liver tissues by CRS. Multiple t-test was used for between-group comparison in a two-sided manner, with Holm–Šídák method for correction. N = 5 mice in each group in (a, b). c Outlines for liver-specific LCN2 knocking down assay, in which an shRNA targeting Lcn2 gene was introduced under hepatic promoter TBG. d Fluorescent image of liver tissues showing the viral infection site. Triplicated studies were performed. Scale bar, 500 μm. e Immunofluorescent staining of liver tissue slices for LCN2 expression. Scale bar, 50 μm. f shRNA transfection remarkably depressed Lcn2 gene expression in liver tissues of CRS mice. One-way ANOVA, F(3,16) = 25.83, P < 0.0001. g Serum LCN2 level was also suppressed by shRNA transfection in liver. One-way ANOVA, F(3,16) = 42.00, P < 0.0001. h Hepatic LCN2 silencing did not affect brain LCN2 levels in naïve mice but dampened the LCN2 surge under CRS. One-way ANOVA, F(3,16) = 477.1, P < 0.0001. N = 5 mice in each group in (f–h). i Although knocking down Lcn2 gene in liver did not affect baseline anxiety level, it did help to antagonize CRS as to increase central zone duration. One-way ANOVA, F(3,24) = 18.00, P < 0.0001. j Hepatic LCN2 deficiency recovered normal open arm duration. One-way ANOVA, F(3,24) = 25.94, P < 0.0001. k Light box avoidance behaviors in CRS animals were attenuated after hepatic LCN2 knocking down. One-way ANOVA, F(3,24) = 22.51, P < 0.0001. N = 7 mice in each group in (i–k. l Left), imaging window of 2-photon microscopy. Middle and white, infection sites of GCaMP6s. Triplicated studies were performed. Scale bar, 200 μm and 100 μm. m Heatmaps of relative calcium transient strengths among all groups. Data were processed as z-score and were color-coded. n Although the blockade of liver Lcn2 gene expression decreased mPFC calcium level in naïve animals to some extents, it recovered normal neuronal activity in CRS group. Nonparametric Kruskal–Wallis test statistic = 140.7, P < 0.0001. o Liver Lcn2 gene knockdown also re-elevated calcium peak values of mPFC neurons in CRS mice. Nonparametric Kruskal-Wallis test = 101.3, P < 0.0001. p Distribution of calcium transient frequency among all groups. n = 80 neurons (from 4 animals) in each group in (n–p). Exact P-values were indicated using Tukey’s post-hoc comparison in (f–k), and Dunn’s multiple comparison test in (n, o). All data were presented as mean ± sem. Source data are provided as a Source Data file.