Fig. 3: LCN2 disrupted mPFC neural activity.
From: Stress increases hepatic release of lipocalin 2 which contributes to anxiety-like behavior in mice
a Schematics of in vivo 2-photon calcium imaging, with co-infection of AAV-LCN2 and GCaMP6s into mouse mPFC. b Left, imaging window under bright-field microscope. Middle and right, co-expression of LCN2 and GCaMP6s. Triplicated studies were performed. Scale bar, 250 μm on left, and 100 μm. c Heatmap showing calcium activities of pyramidal neurons (PNs) during the 300-s imaging period. Color-coded data were normalized and presented as z-score. A total of 30 neurons (from 4 animals) were displayed. d LCN2 over-expression remarkably suppressed integrated calcium activities. Nonparametric Kolmogorov–Smirnov test D = 0.7750, P < 0.0001. e Decreased amplitudes of single calcium spike. Nonparametric Kolmogorov–Smirnov test D = 0.5959, P < 0.0001. f Elevated frequency of cortical pyramidal neurons after LCN2 transfection. Nonparametric Kolmogorov–Smirnov test D = 0.3000, P = 0.0015. n = 80 neurons (from 4 animals) in each group in (d–f). g Quantification of Slc22a17 and Mc4r gene in mPFC. Two-sided unpaired t-test, Slc22a17: t(8) = 8.394, P < 0.0001; Mc4r: t(8) = 1.210, P = 0.2610. N = 5 mice each group. h Expression of SLC22A17 in mPFC neurons (NeuN+). Scale bar, 100 μm. i Left, most of SLC22A17 proteins were expressed on neurons. Right, the number of SLC22A17+ neurons between naïve and CRS group. Two-sided unpaired t-test, t(12) = 15.22, P < 0.0001. N = 7 mice per group. j Schematic illustration of in vivo fiber photometry recording on the elevated plus-maze under Slc22a17 gene knockdown. k Infection of shRNA and GCaMP6s expressing viruses in mPFC. Triplicated studies were performed. Scale bar, 200 μm on left, and 100 μm on right. l Heatmaps showing calcium activities when the mouse entered into the open arm from the closed arm. Calcium transients were normalized and shown as z-score, and each recording window covered 2 s before and 10 s after the transition. Each line represented the averaged values of 10 trials from one mouse. m Attenuated calcium peak in CRS mice was reversed by knocking down Slc22a17 gene expression in mPFC. One-way ANOVA, F(3.24) = 17.74, P < 0.0001. N = 7 mice in each group. Exact P-values were indicated using Tukey’s post-hoc comparison in (m). All data were presented as mean ± sem. Source data are provided as a Source Data file.
