DOI: 10.6084/m9.figshare.27316596.v1
title: Figure 5, supplementary figure 7

description: <b>Figure 5: ERα is robustly expressed in the BNST and mediates rapid E2 modulation of binge drinking but not avoidance behavior.</b> 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). *P&lt;0.05, **P&lt;0.01, ***P&lt;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. ##&lt;0.01; one sample t-test. Data are presented as mean values +/- SEM. Detailed statistics are provided in Supplemental Table 1. Source data are provided as a Source Data file.<b>Supplementary Figure 7: BNST cell population analysis, MPP pharmacology (</b><b><i>ex vivo</i></b><b> slice electrophysiology and </b><b><i>in vivo</i></b><b> intra-BNST infusion; related to Fig. 5). </b>a-d) Analysis of single nucleus RNA sequencing of female BNST nuclei (total cells: 38,806; GEO: GSE126836)<sup>78</sup>. a) <i>Crh</i> (CRF) expression (7.7%) in female BNST cells. b) <i>Slc117a6</i> (VGLUT2) expression (7.6%) in female BNST cells. c) <i>Gfap</i> (GFAP) expression (0.7%) in female BNST cells. d) ERα (27%) and ERβ (9%) expression (5% both) in female BNST<sup>GFAP</sup> cells. e-f) Effects of acute bath application of the ERα antagonist methyl-piperidino-pyrazole (MPP) on excitatory synaptic transmission in BNST<sup>CRF</sup> 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 BNST<sup>CRF</sup> 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 BNST<sup>CRF</sup> 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 BNST<sup>CRF</sup> 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.