Figure 4, supplementary figure 6

<b>Figure 4: Rapid E2 signaling in the BNST recapitulates the pro-drinking but not anxiolytic effects of ovarian E2 and modulates BNSTCRF neurons. </b>a) Systemic E2 synthesis inhibition in high ovarian E2 mice using the aromatase inhibitor letrozole (LET, 10 mg/kg). b) Effects of LET administration on EtOH consumption (N’s = 13 saline VEH, 19 LET). c) Effects of LET administration on elevated plus maze (EPM) % time spent in the open arms (left) or distance traveled (right; N’s = 15 VEH, 14 LET). d) Intra-BNST infusion of LET (1 μg in 200 nl/side) in high E2 status females. e) Effects of intra-BNST LET on EtOH consumption (N’s = 7 VEH, 6 LET). f) Intra-BNST infusion of E2 (20 pg in 200 nl/side) or membrane-impermeable E2 (mE2, 55 pg in 200 nl/side) in low E2 status females. g) Effects of intra-BNST E2 on EtOH consumption (N’s = 8 VEH, 7 E2). h) Effects of intra-BNST mE2 on EtOH consumption (N’s = 12 VEH, 11 mE2). i) Effects of intra-BNST E2 on EPM % time spent in the open arms (left) or distance traveled (right; N’s = 7 VEH, 8 E2). j) Effects of bath application of E2/mE2 on excitatory synaptic transmission in BNSTCRF neurons during slice electrophysiology recordings in low ovarian E2 status female CRF-CrexAi9 reporters. k) Spontaneous excitatory postsynaptic currents (sEPSCs) maximum delta from % baseline during E2/mE2 wash on (E2 nM: 0.01: N = 2, 2 cells; 1: N = 4, 4 cells; 10: N = 5, 7 cells; 100: N = 4, 4 cells; 1000: N = 3, 5 cells; 100 nM mE2: N = 3, 6 cells; l and m are the same cells). l) Time course of BNSTCRF neurons that displayed increase/decrease/no change in sEPSC frequency and amplitude (m) % change from baseline during E2 application and proportion of responding categories (pie charts). *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001 for unpaired t-tests between VEH vs. LET treatment and VEH vs E2/mE2 treatment; Fisher’s exact test frequency vs amplitude; post hoc t-tests with H-S corrections as indicated. 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 6: E2 sEPSC frequency responsive BNST</b>^{<strong>CRF</strong>}<b> cells have a specific cellular phenotype (related to Fig. 3). </b>a) Proportion of responder categories during 10-minute E2 wash on across E2 doses (0.01, 1, 10, 100, 1000 nM) for frequency (top) and amplitude (bottom). b) Raw average frequency (top) and amplitude (bottom) during baseline, E2 wash on, and washout for each cell. c) Maximum % change from baseline for cells categorized as increased and decreased during E2 wash on for frequency (top) and amplitude (bottom). Amplitude did not differ between BNST^CRF neurons that had increased vs. decreased frequency (top) and vice versa (bottom, N = 17 mice, 22 cells). d) % change from baseline in frequency (top) and amplitude (bottom) during E2 did not correlate with baseline frequency and amplitude. Data are presented as mean values +/- SEM. Detailed statistics are provided in Supplemental Table 1. Source data are provided as a Source Data file.