The ventrolateral periaqueductal gray neurons innervated by central amygdala bidirectionally modulate nociception. (A) Schematic showing the injection of AAV-Cre-eGFP into the central amygdala and of chemogenetic virus into the ventrolateral periaqueductal gray in a wild-type mouse (left). The experimental timeline (right). (B) hM4Di-mCherry expression in the ventrolateral periaqueductal gray. Scale bar, 50 µm; inset, 25 µm. (C) Clozapine-N-oxide caused mechanical hypersensitivity of the hM4Di group mice (n = 7 mCherry, n = 8 hM4Di; two-way repeated-measures ANOVA: time, F[9,117] = 10.85, P < 0.001; clozapine-N-oxide, F[1, 13] = 24.51, P < 0.001; time × clozapine-N-oxide, F[9, 117] = 8.595, P < 0.001, Bonferroni post hoc test). (D) Clozapine-N-oxide had no effect on the mechanical threshold of the hM3Dq group mice (n = 7 mCherry, n = 8 hM3Dq; two-way repeated-measures ANOVA: time, F[9, 117] = 1.192, P = 0.306; clozapine-N-oxide, F[1, 13] = 0.278, P = 0.607; time × clozapine-N-oxide, F[9, 117] = 0.473, P = 0.89, Bonferroni post hoc test). Aq, aqueduct; CeA, central amygdala; CNO, clozapine-N-oxide; hM3Dq, Gq-coupled human M3 muscarinic receptor; hM4Di, Gi-coupled human M4 muscarinic receptor; lPAG, lateral periaqueductal gray; vlPAG, ventrolateral periaqueductal gray. **P < 0.01, ***P < 0.001. All data are expressed as mean ± SD.