Figure 2, supplementary figure 2

<b>Figure 2: BNSTCRF neurons are sensitive to ovarian estrogen status.</b> Spontaneous synaptic transmission in BNSTCRF neurons as measured in slice electrophysiology recordings from intact CRF-CrexAi9 reporter females in low vs. high ovarian E2 states, with schematic in a. b) Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs). c) The frequency of sEPSCs in BNSTCRF neurons from high and low ovarian E2 status females (N’s = 6 low E2, 13 cells; 7 high E2, 13 cells; d and e are the same cells). d) sEPSC amplitude in high and low ovarian E2 status female BNSTCRF neurons. e) Excitatory synaptic drive onto BNSTCRF neurons (frequency x amplitude) high and low ovarian E2 status females. f) Representative traces of spontaneous inhibitory postsynaptic currents (sIPSCs). g-i) sIPSC frequency in BNSTCRF neurons high and low ovarian E2 status females (g), amplitude (h), and synaptic drive (i; N’s = 6 low E2, 12 cells; 7 high E2, 11 cells). *P&lt;0.05, **P&lt;0.01 for unpaired t-tests between low E2 and. high E2 groups. Data are presented as mean values +/- SEM. Detailed statistics provided in Supplemental Table 1. Source data are provided as a Source Data file.<b>Supplementary Figure 2: BNSTCRF neurons are necessary for binge alcohol consumption.</b> a-g) Chemogenetic manipulation of BNSTCRF neurons during EtOH Drinking in the Dark (DID) and avoidance behaviors. a-b) Schematic (a) and representative image (b) for viral strategy to bidirectionally manipulate BNSTCRF neurons using a multiplexed Gi+Gq DREADD approach. c-d) Activation of the Gi-coupled KOR DREADD via systemic injection of Salvinorin B (SalB; 10 mg/kg) suppressed binge EtOH consumption compared to vehicle (VEH) injected controls (c; N’s = 7 CON, 5 DREADD) but did not affect the % time in open arms of the elevated plus maze (EPM; left) or distance traveled (right; d; N’s = 6 CON, 5 DREADD). e) DREADD virus hit map in the BNST (for Fig. 2a–f). Each dot is an individual hit and the grey Xs are a miss. f-g) Gq DREADD-mediated BNSTCRF neuron activation via clozapine-N-oxide (CNO; 5 mg/kg i.p.) did not change EtOH consumption (f; N’s = 7 CON, 6 DREADD) or alter avoidance behavior on the open field (OF) measured via % time in center (left) but did reduce distance traveled (right; g; N’s = 14 CON, 9 DREADD).*P &lt; 0.05, unpaired t-tests between CON and DREADD; 2xANOVA main effects and interactions between groups and treatment; 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.

**图2：BNSTCRF神经元对卵巢雌激素状态具有敏感性**
本研究通过脑片电生理记录，检测了完整CRF-CrexAi9报告基因雌性小鼠中BNSTCRF神经元的自发突触传递，对比了卵巢雌二醇（estradiol, E2）低水平与高水平两种状态，示意图详见图a。
b) 自发兴奋性突触后电流（spontaneous excitatory postsynaptic currents, sEPSCs）的代表性轨迹。
c) 卵巢E2水平高低不同的雌性小鼠BNSTCRF神经元的sEPSC频率（低E2组：n=6只小鼠，13个细胞；高E2组：n=7只小鼠，13个细胞；d、e图采用相同细胞）。
d) 卵巢E2水平高低不同的雌性小鼠BNSTCRF神经元的sEPSC幅值。
e) 卵巢E2水平高低不同的雌性小鼠BNSTCRF神经元的兴奋性突触驱动强度（频率×幅值）。
f) 自发抑制性突触后电流（spontaneous inhibitory postsynaptic currents, sIPSCs）的代表性轨迹。
g-i) 分别为卵巢E2水平高低不同的雌性小鼠BNSTCRF神经元的sIPSC频率（g）、幅值（h）以及突触驱动强度（i；低E2组：n=6只小鼠，12个细胞；高E2组：n=7只小鼠，11个细胞）。
*P<0.05，**P<0.01，两组间采用非配对t检验。数据以平均值±标准误（standard error of the mean, SEM）表示，详细统计结果见补充表1，源数据已作为源数据文件提供。

**补充图2：BNSTCRF神经元对于暴饮酒精摄入是必需的**
a-g) 酒精昼夜饮用（Drinking in the Dark, DID）及回避行为实验中对BNSTCRF神经元的化学遗传学操控。
a-b) 采用多重Gi+Gq DREADD（设计性药物激活受体，Designer Receptors Exclusively Activated by Designer Drugs）技术双向操控BNSTCRF神经元的病毒策略示意图（a）及代表性成像图（b）。
c-d) 通过全身注射萨尔维诺林B（Salvinorin B, SalB；10 mg/kg）激活Gi偶联的KOR DREADD，与溶剂（VEH）注射对照组相比，可抑制暴饮酒精摄入（c；对照组CON：n=7，DREADD组：n=5），但不影响高架十字迷宫（elevated plus maze, EPM）中开放臂停留时间百分比（左图）及移动总距离（右图，d；CON组：n=6，DREADD组：n=5）。
e) BNST脑区的DREADD病毒感染定位图（对应图2a-f）：每个圆点代表1个成功感染位点，灰色叉号代表感染失败位点。
f-g) 通过腹腔注射氯氮平-N-氧化物（clozapine-N-oxide, CNO；5 mg/kg）激活Gq DREADD介导的BNSTCRF神经元，未改变酒精摄入水平（f；CON组：n=7，DREADD组：n=6），也未影响旷场实验（open field, OF）中的回避行为（以中央区域停留时间百分比衡量，左图），但可降低移动总距离（右图，g；CON组：n=14，DREADD组：n=9）。
*P<0.05，对照组与DREADD组间采用非配对t检验；组间与处理间的主效应及交互效应采用双因素方差分析（2×ANOVA），并按要求采用H-S校正进行事后t检验。数据以平均值±标准误（SEM）表示，详细统计结果见补充表1，源数据已作为源数据文件提供。