(A) Schematic for viral injections. (B) Expressions of eGFP and mCherry in the PrL. Left, half brain section containing the PrL region; middle, enlarged indicated portion from the left image that was rotated counterclockwise 90°; right, intensity distribution of green (PrL^BLA) and red (PrL^l/vlPAG) fluorescence throughout the different layers in the PrL. a.u., arbitrary unit. Scale bars: 600 μm (left) and 100 μm (middle). (C) Ratios of PrL^BLA and PrL^l/vlPAG neurons. n = 20 slices from 5 mice. (D) Schematic for SNI surgery. (E) Decreased mechanical pain threshold in SNI mice. n = 10 mice per group. (F and H) Representative traces of mice travel in OFT (F) and in EPM (H). (G) Reduced time in center in SNI mice. n = 8 mice per group. (I) Reduced time in open arms in SNI mice. n = 8 mice per group. (J) Time scheme for in-vivo 2P Ca²⁺ imaging on awake mice. (K) Representative somatic Ca²⁺ fluorescent images of PrL^BLA neurons before (BL), 1 week (1W) and 2 weeks (2W) in SNI mice. Bottom, Ca²⁺ fluorescent traces from the numbered neurons (C1, C2, C3), which are circled in the upper images. Scale bars: 10 μm (upper), 500% ΔF/F and 20 s (bottom). (L and M) Increased integrated somatic Ca²⁺ activity (L) and peak amplitude (M) of Ca²⁺ transients of PrL^BLA neurons in SNI mice. n = 131 neurons from 6 mice. (N) Representative somatic Ca²⁺ fluorescent images of PrL^l/vlPAG neurons. Bottom, Ca²⁺ fluorescent traces from the numbered neurons (C1, C2, C3), which are circled in the upper images. Scale bars: 10 μm (upper), 500% ΔF/F and 20 s (bottom). (O and P) Decreased integrated somatic Ca²⁺ activity (O) and peak amplitude (P) of Ca²⁺ transients of PrL^l/vlPAG neurons in SNI mice. n = 104 neurons from 5 mice. Data shown as mean ± SEM or aligned dot plots. *P < 0.05, **P < 0.01, ***P < 0.001. 2-way repeated measures ANOVA followed by posthoc Šidák’s test (E, G, and I); Friedman test with posthoc Dunn’s test (L, M, O, and P).