Estrogen receptor-β and G protein–coupled estrogen receptor-1 but not estrogen receptor-α contribute to formalin-induced conditioned place avoidance and place escape/avoidance paradigm acquisition. (A) Schematic of the protocol for the formalin-induced conditioned place avoidance task in rats. (B) Example tracks of the rats before and after conditioning. (C, D) Successful establishment of formalin-induced conditioned place avoidance, as indicated by the time spent in the treatment (intraplantar injection of normal saline or formalin)–paired compartment in the preconditioning and postconditioning tests (C) and the relative avoidance scores (D). **P < 0.01 versus the preconditioning test (two-tailed paired t test); ^##P < 0.01 versus normal saline (two-tailed Student’s t test); n = 8 and 8. (E) Schematic of the protocol for experiments G to I. (F) Schematic of bilateral injections of estrogen receptor antagonists into the rostral anterior cingulate cortex and a photomicrograph of a coronal section showing the cannula placement in the bilateral rostral anterior cingulate cortex. (G, H) Microinjections of the estrogen receptor-β antagonist 4-(2-phenyl-5,7-bis [trifluoromethyl] pyrazolo [1,5-a] pyrimidin-3-yl) phenol (PHTPP; 1 ng/μl, 0.5 μl/hemisphere) or the G protein–coupled estrogen receptor-1 antagonist (3aS*,4R*,9bR*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta [c] quinolone (G15; 1.8 μg/μl, 0.5 μl/hemisphere) into the rostral anterior cingulate cortex significantly blocked formalin-induced conditioned place avoidance, whereas the estrogen receptor-α antagonist 1,3-Bis (4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy) phenol]-1H-pyrazole dihydrochloride (MPP, 1 ng/μl, 0.5 μl/hemisphere) had no effect. **P < 0.01 versus the preconditioning test (two-tailed paired t test); ^##P < 0.01 versus vehicle (10% dimethyl sulfoxide; one-way ANOVA followed by post hoc Dunnett’s test); n = 9, 7, 7, and 7. (I) Example tracks of the rats after conditioning in the post-conditioning day. (J) Schematic of the protocol for experiments K and L. (K) Spared nerve injury rats spent significantly more time on the light side of the chamber in the place escape/avoidance paradigm, indicating that these animals were willing to avoid a preferred area (dark area) to escape stimulation of the affected paw. Control animals preferred to remain in the dark and tended to avoid the light side of the chamber. **P < 0.01 versus the sham control (two-way repeated-measures ANOVA followed by post hoc Bonferroni multiple comparison test); n = 7 and 8. (L) Microinjections of the estrogen receptor-β antagonist PHTPP (1 ng/μl, 0.5 μl/hemisphere) or the G protein–coupled estrogen receptor-1 antagonist G15 (1.8 μg/μl, 0.5 μl/hemisphere) into the rostral anterior cingulate cortex significantly blocked place escape/avoidance, whereas the estrogen receptor-α antagonist MPP (1 ng/μl, 0.5 μl/hemisphere) had no effect. **P < 0.01 versus vehicle (two-way repeated-measures ANOVA followed by post hoc Bonferroni multiple comparison test); n = 9, 8, 8, and 8.