Figure 5, supplementary figure 7

Figure 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 (BNSTCRF) expressing ERα, ERβ, and both. c) Slc17a6-expressing BNST cells (BNSTVGLUT2) 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 BNSTCRF neurons during slice electrophysiology recordings in high ovarian E2 female CRF-CrexAi9 reporters. 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. 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. 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). *PSupplementary Figure 7: BNST cell population analysis, MPP pharmacology (ex vivo slice electrophysiology and in vivo intra-BNST infusion; related to Fig. 5). a-d) Analysis of single nucleus RNA sequencing of female BNST nuclei (total cells: 38,806; GEO: GSE126836)78. a) Crh (CRF) expression (7.7%) in female BNST cells. b) Slc117a6 (VGLUT2) expression (7.6%) in female BNST cells. c) Gfap (GFAP) expression (0.7%) in female BNST cells. d) ERα (27%) and ERβ (9%) expression (5% both) in female BNSTGFAP cells. e-f) Effects of acute bath application of the ERα antagonist methyl-piperidino-pyrazole (MPP) on excitatory synaptic transmission in BNSTCRF neurons during whole-cell slice electrophysiology recordings in high ovarian E2 female CRF-CrexAi9 reporter mice, as depicted in e. f) Time course of high E2 status BNSTCRF neurons that displayed an increase, decrease, or variable change in spontaneous excitatory postsynaptic current (sEPSC) frequency (left) and amplitude (right) % change from baseline during the 5-minute MPP wash on application period and 5 minute washout. g-i) Effects of acute bath application of MPP on excitatory synaptic transmission in BNSTCRF neurons during whole-cell slice electrophysiology recordings in low ovarian E2 female CRF-CrexAi9 reporter mice, as depicted in g. f) Time course of low E2 status BNSTCRF neurons that displayed an increase, decrease, or variable change in spontaneous excitatory postsynaptic current (sEPSC) frequency (left) and amplitude (right) % change from baseline during the 5-minute MPP wash on application period and 5 minute washout. i) Bath application of MPP (3 μM) had no effect on spontaneous excitatory postsynaptic current (sEPSC) frequency (left) and amplitude (right) in a majority of cells during the 5 min wash on period (Ns = 3 mice, 7 cells). j) Depiction of strategy to site-deliver MPP (10 μM/200 nl/side or saline vehicle (VEH) to the BNST in low E2 females via bilateral indwelling cannulae 10 minutes prior to behavioral testing. k) ERα antagonism via intra-BNST MPP did not alter binge EtOH drinking in low ovarian E2 status females (Ns = 8 VEH, 8 MPP). l) BNST cannula hit map (for Fig. 4d-e, 5j-l); each dot is an individual hit. Data are presented as mean values +/- SEM. Detailed statistics are provided in Supplemental Table 1. Source data are provided as a Source Data file.