Figure 1. Rapid increase in PVN CRF neuronal activity by aversive stimuli.

(a) Viral construct and implantation scheme for fiber-photometry on PVN-CRF neurons. Middle: a representative image validates GCaMP6s expression in CRF neurons and optical fiber tract above the PVN. Scale bar: 100μm; Further right – images depict the overlap between GCaMP6s expressing cells (green) and anti-CRF positive cells (red). Scale bar: 20μm. Right: Bar graph showing the percentage of GCaMP6s+ neurons co-expressing CRF. (N=4 mice) (b) Schematic for forced-swimming test (FST) and a representative trace illustrating an increase of PVN-CRF^GCaMP6 signal during FST (red bar, above) and decreasing activity while in back to home cage (white bar). Behavioral epochs, swimming (light blue) and climbing (blue), annotated in color-coded shaded bars. (c) Plot (left) and heat map (right) across animals aligned to the start and end of FST, and the following rest in home cage. Black bold line and grey shadow in this and following figures indicate mean and s.e.m., respectively. (N=6 mice) (d) Schematic for tail-restraint test (TRT) and a representative trace showing increases of PVN-CRF^GCaMP6 signal during restraint (red bars, above). Color-coded shaded bars depict the periods during which mice were chased by a hand (grey) and struggled (beige). (e) PETH plot (left) and heat map (right) across animals aligned to the start of TRT. (N=7 mice) (f) Schematic for presenting an overhead object and a representative trace showing increases of PVN-CRF^GCaMP6 signal during the presentations (red bars, above). Shaded bars depict the epochs during which mice exhibited flight (red) and freezing (beige). (g) PETH plot (left) and heat map (right) across animals aligned to the onset of overhead presentation. (N=7 mice) (f’) Schematic for a looming shadow disk and a representative trace showing increases of PVN-CRF^GCaMP6 signal during the presentations (red bars, above). Shaded bars depict the epochs during which mice exhibited flight (red), freezing (beige), and hiding in a nest (yellow). (g’) PETH plot (left) and heat map (right) across animals aligned to the onset of looming presentation. (N=8 mice) (h) Schematic of odor presentation and a representative trace showing increases of PVN-CRF^GCaMP6 signal during TMT exposure (red bar, above). Shaded bar depicts the epoch during which mice exhibited freezing (beige). (i) Plot across animals aligned to the start and end of TMT exposure (N=6 mice); 6 out of 9 mice that displayed a defensive response showed an increase in GCaMP signal. (j) Comparison of average ΔF/F of PVN-CRF^GCaMP6 in response to different aversive stimuli. The measurements shown in blue dots are illustrated in the representative traces in (b, d, f, h). (k) Schematic of intraperitoneal (i.p.) administration under anesthesia and a representative trace showing increases of PVN-CRF^GCaMP6 signal after injection of LiCl. Shaded bars depict the period during which a mouse was placed in an anesthesia chamber (grey) and the period during which the mouse received LiCl injection (red). (l) Plot across animals before and 30mins after LiCl injection (black line) and saline (grey line) (N=8 mice). Arrow head depicts the increase GCaMP signals. (m) Bar graphs showing average ΔF/F of PVN-CRF^GCaMP6 for 30mins after injection of LiCl or saline. The measurement shown in a blue dot is illustrated in the representative traces in (k). Paired two-tailed student’s t-test for GCaMP signals before vs. during exposure. *p<0.05, **p<0.01. ***p<0.001. Data are presented as mean ± s.e.m. See Supplementary Table 1 for detailed description of statistics for this figure and subsequent figures.