Fig. 1: ALI activates CRH^PVN neurons.

a Schematic diagram of ALI induced by intranasal LPS instillation (20 mg/kg) and brain activity mapping by c-Fos expression in wild-type mice. b Representative images of c-Fos expression in the PVN of control (PBS) and LPS-treated mice. Scale bar: 100 μm. c Comparison of c-Fos⁺ neurons in PVN between PBS- and LPS-treated mice (n = 5 mice per group) by a 2-tailed unpaired t-test. d Immunofluorescence staining of CRH (red), c-Fos (green), and DNA (blue) in the PVN at 3-h after intranasal LPS administration. Scale bars: 50 μm (low magnification), 20 μm (high magnification). e Proportions of CRH⁺c-Fos⁺ neurons in total c-Fos⁺ cells and CRH⁺ neurons (n = 4 mice). f Schematics of fiber photometry detection of neuronal activity in Crh-IRES-Cre mice. g Schematic of the mouse brain at Bregma −0.82 mm, adapted from The Mouse Brain in Stereotaxic Coordinates, Second Edition, with permission. A representative image of GCaMP6m fluorescence detected by the positioned optical fiber. Scale bar: 100 μm. h Kinetics of the average change of GCamp6m fluorescence (ΔF/F) of CRH^PVN neurons in the PBS-or LPS-treated mice. Heatmaps (i) and quantification (j) of CRH^PVN neurons’ responses in PBS- or LPS-treated mice (n = 6 mice per group), with statistical difference determined by a 2-tailed unpaired t-test. k Schematic of the PRV-531 retrograde tracing experiments in Crh-IRES-Cre mice. l Representative of composite images of PRV⁺ neurons (green) and CRH⁺ neurons (mCherry, red) in the PVN. Scale bar: 100 μm (left), 50 μm (right). m Proportions of EGFP⁺mCherry⁺ neurons in EGFP⁺ cells and mCherry⁺ neurons (n = 4 mice). Each dot represents an individual mouse. Data are represented as mean ± SEM. a, f, k were created in BioRender. Liu, T. (2025) https://BioRender.com/p48gmjd. All experiments were repeated three times, yielding similar results. Source data are provided as a Source data file. ALI acute lung injury, CRH corticotropin-releasing hormone, LPS lipopolysaccharide, PVN hypothalamic paraventricular nucleus, PRV pseudorabies virus, EGFP enhanced green fluorescent protein.