Fig. 3: Ovarian E2 state modulates the activity of BNST^CRF neurons during motivated alcohol drinking.

Fiber photometry recordings of GCamP6s in BNST^CRF neurons, with schematic of unilateral viral injection and optical fiber cannula placement (a; Biorender license: QP27GVY1EL) and representative image of GCaMP expression/fiber placement (b; ac: anterior commissure). c Modified EtOH DID timeline with water access given before (W1) and after (W2) the 2-h EtOH access period, and drinking bouts time course on high and low E2 days. d Total drinking bouts across the session (EtOH + W2; N’s=5 for all sub figures, 13 low E2, 12 high E2). Total time spent displaying motivated EtOH drinking (time in bout; e; 14 low E2, 14 high E2) and average EtOH bout duration (f; 12 low E2, 14 high E2). g Representative traces of GCaMP signal from one mouse on a low E2 and high E2 day. GCaMP signal during high and low E2 days 10 seconds following bout onset for EtOH (h) and water (i; 13 low E2, 15 high E2 for both). j Correlation between GCaMP signal and time spent motivated drinking during the first 30 min of EtOH (25 days). k The number of drinking bouts (13 low E2, 13 high E2) and average GCaMP signal (14 low E2, 14 high E2) during the first 30 min of EtOH (EtOH 1) compared to W1. l Bout number (11 low E2, 9 high E2) and GCaMP signal (11 low E2, 9 high E2) in the first 30 min of W2 compared to the last 30 min of EtOH (EtOH 2) on high and low E2 days. m Representative heat map of the frequency of GCaMP transient events across amplitude bins for a single mouse across W1 and EtOH 1 on low vs. high E2 days. n Frequency distribution of event amplitudes normalized to the amplitude distribution for W1 within each day during W1 and EtOH 1 epochs. o Representative heat map of the frequency of GCaMP events across amplitude bins across EtOH 2 and W2 for the same mouse as in l. p Frequency distribution of event amplitudes normalized to the amplitude distribution for W1 during EtOH 2 and W2. q Area under the curve (AUC) of GCaMP event distributions shown in n and p (W1 and EtOH 1: 15 low E2, 15 high E2; EtOH 2 and W2: 11 low E2, 10 high E2). r The peak of the event amplitude distributions shown in n and p (same N’s depicted in q). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ^&P < 0.10 for unpaired t-tests, 2xANOVA main effects and interactions and their post hoc paired t-tests with H-S corrections, and Pearson’s correlation. Data are presented as mean values +/−SEM. Detailed statistics are provided in Supplemental Table 1. Source data are provided as a Source Data file.