Fig. 3.
Increased c-Fos expression in central amygdala γ-aminobutyric acid–mediated neurons and ventrolateral periaqueductal gray glutamatergic neurons of mice show depression-related behavior and pain sensitization. (A) Experimental design: Mice underwent a 3-week restraint stress protocol, 6 h per day; the sucrose preference test was conducted every 7 days, the mechanical and thermal withdrawal thresholds were tested pre- and postchronic restraint stress, and the forced swim test was carried out 1 day after the last Hargreave test. (B) Chronic restraint stress decreased preference for sucrose (n = 7 per group; two-way repeated-measures ANOVA: time, F[3, 36] = 0.713, P = 0.551; chronic restraint stress, F[1, 12] = 11.17, P = 0.006; time × chronic restraint stress, F[3, 36] = 1.209, P = 0.32; Bonferroni post hoc test). (C) Chronic restraint stress decreased time struggling in the forced swim test (n = 7 control, n = 10 chronic restraint stress, unpaired two-tailed Student’s t test, P = 0.029). (D) The Von Frey test showed that chronic restraint stress induced profound mechanical hypersensitivity (n = 7 control, n = 9 chronic restraint stress; two-way repeated-measures ANOVA: time, F[2, 28] = 9.201, P = 0.001; chronic restraint stress, F[1, 14] = 8.83, P = 0.01; time × chronic restraint stress, F[2, 28] = 4.482, P = 0.021; Bonferroni post hoc test). (E) Chronic restraint stress elicited robust c-Fos expression in the central amygdala. Scale bar, 100 µm. (F) Chronic restraint stress elicited robust c-Fos expression in the ventrolateral periaqueductal gray. Scale bar, 100 µm. (G) Representative images of c-Fos (green, top left and bottom left), γ-aminobutyric acid (GABA; purple, top middle), and glutamate (purple, bottom middle) staining in the central amygdala from chronic restraint stress c-fos-tTA mice. Scale bar, 25 µm. (H) Quantification of (G) shows most c-Fos+ central amygdala cells expressing GABA. n = 6–8 slices (0.016 mm2 imaging area per slice) from three mice per group. (I) Representative images of c-Fos (green, top left and bottom left), GABA (purple, top middle), and glutamate (purple, bottom middle) staining in the ventrolateral periaqueductal gray from chronic restraint stress c-fos-tTA mice. Scale bar, 25 µm. (J) Quantification of (I) suggests that most c-Fos+ ventrolateral periaqueductal gray cells displayed glutamate immunoreactivity. n = 7 or 8 slices (0.016 mm2 imaging area per slice) from three mice per group. Aq, aqueduct; BL, paw withdrawal threshold baseline; BLA, basolateral amygdala; CeA, central amygdala; CRS, chronic restraint stress; DMPAG, dorsomedial periaqueductal gray; FST, forced swim test; SPT, sucrose preference test. *P < 0.05. All data are expressed as mean ± SD.