Journal: Advanced Science

Article Title: Astrocytic PCBP1 Suppresses Ferroptosis to Restore Glutamatergic Homeostasis and Mitigate Stress‐Induced Depression in Male Mice

doi: 10.1002/advs.202513438

Figure Lengend Snippet: Astrocytic PCBP1 enhances glutamatergic neuronal activity and neurophysiology. a) c‐Fos expression in vHip of mice following CUMS. Above: Representative images and quantification of c‐Fos‐positive glutamatergic neurons. Below: Representative images and quantification of c‐Fos‐positive GABAergic neurons. Scale bars = 50 µm. n = 9 slices from 3 animals per group. b) Timeline of fiber photometry recording Ca 2+ and glutamate signals from vHip c) Left: Representative images validate GCaMP6s expression in vHip. Scale bar = 500 µm. Right: Representative images showing the overlap between GCaMP6s‐expressing cells (green) and glutamatergic neurons (violet). Scale bar = 50 µm. d) Representative images of c‐Fos‐positive glutamatergic neurons. Scale bars = 100 or 20 µm. e) Statistical analysis of c‐Fos co‐labeled glutamatergic neuron in vHip of mice. f) Schematic of fiber photometry recording in TST and representative Ca 2+ signals photometric traces in response to the TST during struggling and immobility phases. Scale bars = 10 s. g) Ca 2 ⁺ signal recording in vHip during TST. Left: Representative heatmaps showing Ca 2 ⁺ signal in response to TST during struggling. Right: Z‐score traces and quantification of average Z‐scores during TST. n = 5 mice per group. h) Representative images of the AAV vectors engineered to express the glutamate sensor iGluSnFR under the hSyn promoter in the vHip. Scale bars = 500 µm / 50 µm. i) Representative trace of glutamate signal event detection during TST. Red dots indicate detected events. j) Event‐based quantification. Event frequency (left), AUC (middle) and decay tau (right). n = 5 mice per group. k) Timeline of electrophysiological recordings from vHip glutamatergic neurons. l) Quantification of mEPSC frequency (left) and amplitude (right) recorded from vHip glutamatergic neurons. n = 10–12 cells from 3 mice per group. m) Left: Evoked firing rates of action potentials recorded from eGFP⁺ neurons in the vHip. Right: Quantifications of rheobase and resting membrane potential recorded from eGFP + neurons in vHip. n = 10 cells from 3 mice per group. Statistical analyses included unpaired t‐test, one‐way/two‐way ANOVA followed by Tukey's post hoc test and Kruskal‐Wallis test. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For immunofluorescence analysis, the 30‐μm‐thick sections were incubated overnight at 4 °C with primary antibodies against NEUN (94403S, Cell Signaling Technology), GFAP (GB11096, Servicebio), IBA‐1 (GB12105, Servicebio), CaMKII (11533‐1‐AP, Proteintech), GAD67 (PA5‐21397, Invitrogen), c‐Fos (OB‐PGP080, Oasis biogarm), GPX4 (ab125066, Abcam), and PCBP1 (14523‐1‐AP, Proteintech).

Techniques: Activity Assay, Expressing, Labeling, Membrane