Fig. 5: ERα is robustly expressed in the BNST and mediates rapid E2 modulation of binge drinking but not avoidance behavior.

a–c Analysis of single nucleus RNA sequencing (snRNA-seq) of female mouse BNST nuclei (total cells: 38,806; GEO: GSE126836)⁷⁸. a BNST cells expressing estrogen receptor α (ERα; Esr1) and estrogen receptor β (ERβ; Esr2) and both. b Crh-expressing BNST cells (BNST^CRF) expressing ERα, ERβ, and both. c Slc17a6-expressing BNST cells (BNST^VGLUT2) expressing ERα, ERβ, and both. d, e RNAscope fluorescence in situ hybridization (FISH) probing for ERα (Esr1), ERβ (Esr2), CRF (Crh), and VGLUT2 (Slc17a6) in the BNST in females, with red boxes indicating the location of confocal z-stack images taken; representative images pseudocolored for visibility. d ERα and ERβ expression in CRF+ and CRF- cells (N’s = 4). Dashed arrow: cell expressing CRF/ERα/ERβ; solid arrow: cell expressing CRF/ERα. e ERα and ERβ expression in VGLUT2+ cells (N’s = 4). Dashed arrow: cell expressing VGLUT2/ERα/ERβ; solid arrow: cell expressing VGLUT2/ERα. f Bath application of the ERα antagonist methyl-piperidino-pyrazole (MPP) on BNST^CRF neurons during slice electrophysiology recordings in high ovarian E2 female CRF-CrexAi9 reporters (Biorender license: NF27GVWGQV). g, h Effects of bath application of MPP (3 μM) on spontaneous excitatory postsynaptic current (sEPSC) frequency and amplitude (N’s = 4, 7 cells). i Depiction of strategy to site-deliver MPP (10 μM/200 nl/side), the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; 10 μM/200 nl/side), or saline (VEH) to the BNST in high E2 females (Biorender license: ZS27GW07FP). j Effects of intra-BNST MPP (N’s=8, 8 VEH, 8 MPP) or PHTPP (N’s = 7, 7 VEH, 7 PHTPP) on binge EtOH consumption (Biorender license: VB27JLIIQ2). k Effects of intra-BNST MPP on avoidance behavior in the light:dark box (LDB; N’s = 8 VEH, 7 MPP). l Effects of intra-BNST PHTPP on avoidance behavior in the open field (OF; N’s = 5 VEH, 6 PHTPP). *P < 0.05, **P < 0.01, ***P < 0.001 for 2xANOVA main effects and interactions between receptors; paired t-tests between VEH vs. ER antagonist treatment prior to DID; unpaired t-tests between VEH vs. ER antagonist treatment prior to avoidance assays; post hoc t-tests with H-S corrections as indicated. ##<0.01; one sample t-test. Data are presented as mean values +/−SEM. Detailed statistics are provided in Supplementary Table 1. Source data are provided as a Source Data file.