Fig. 6: Activation of ^ILPHF6 cells promotes anxiety-like behaviors and ^ILPHF6 cells receive synaptic inputs from the ^PVNCRH neurons.

a Experimental design for ^ILPHF6 cell activation and assessment of anxiety-like behaviors. b Left: Representative images showing hM3D-mChery (or mCherry) expression in the IL of Phf6-CreER mice, co-stained with c-Fos (green). Right: Quantification revealed significantly higher c-Fos expression in hM3D-expressing ^ILPHF6 cells compared to mCherry controls (mCherry, n = 3; hM3D, n = 3, **** p < 0.0001, unpaired Student’s t-test). DAPI is shown in blue. Scale bars, 50 μm. c Representative locomotor trajectories and quantification of time spent in the center area of the OF test. Activation of ^ILPHF6 cells significantly decreased the time spent in the center area of the OF test (mCherry-saline, n = 8; hM3D-saline, n = 8; mCherry-CNO, n = 10; hM3D-CNO, n = 8; * p < 0.05, two-way ANOVA with Bonferroni’s multiple comparisons test). d Representative locomotor trajectories and time spent in the open arms of the EPM test. Activation of ^ILPHF6 cells significantly decreased the time spent in the open arms of the EPM test (mCherry-saline, n = 8; hM3D-saline, n = 8; mCherry-CNO, n = 10; hM3D-CNO, n = 8; * p < 0.05, two-way ANOVA with Bonferroni’s multiple comparisons test). e Quantification of the latency to feed in the NSF test. Activation of ^ILPHF6 cells significantly increased the latency to feed in the NSF test (mCherry-saline, n = 8; hM3D-saline, n = 8; mCherry-CNO, n = 10; hM3D-CNO, n = 8; * p < 0.05, two-way ANOVA with Bonferroni’s multiple comparisons test). f Experimental design for assessing hormone release following ^ILPHF6 cell activation. g-i Plasma quantification showed significantly elevated α-MSH (g), β-endorphin (h), and corticosterone (i) levels in hM3D-expressing mice compared to controls. (mCherry, n = 10; hM3D, n = 8, * p < 0.05, unpaired Student’s t-test). j Schematic diagram of retrograde monosynaptic tracing using rabies virus in Phf6-CreER mice. Starter cells are labeled in yellow, as indicated by the white arrowheads. Scale bars, 100 μm. k Rabies-labeled upstream inputs to ^ILPHF6 cells. Scale bars, 100 μm. l Percentage of RV-traced tdTomato-positive neurons located in the paraventricular nucleus (PVN), the periventricular zone (Pe), and the arcuate nucleus (ARC) (n = 3). m Representative images of RV-traced tdTomato-positive neurons co-expressing CRH (green) in the PVN. Scale bars, 100 μm (low magnification) and 10 μm (high magnification). Data are presented as mean ± SEM.