DOI: 10.6084/m9.figshare.27316566.v1
title: Figure 3, supplementary figure 3

description: <b>Figure 3: Ovarian E2 state modulates the activity of BNSTCRF neurons during motivated alcohol drinking. </b>Fiber photometry recordings of GCamP6s in BNSTCRF neurons, with schematic of unilateral viral injection and optical fiber cannula placement (a) 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-hour 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). e-f) 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. h-i) 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 minutes 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 minutes 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 minutes of W2 compared to the last 30 minutes 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&lt;0.05, **P&lt;0.01, ***P&lt;0.001, ****P&lt;0.0001, &P&lt;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.<b>Supplementary Figure 3: Fiber photometry additional measures (related to Fig. 3).</b> a) Hit map of fiber photometry cannulae in the BNST. Each line corresponds to the fiber placement and the grey X corresponds to a surgical miss that was not used in experiments due to poor GCaMP signal. b) Fiber photometry signals in the 465 nm excitation (GCaMP), isobestic 405 nm (control) wavelength channels, and normalized z-score during EtOH-DID in low E2 status and high E2 status female drinking days (c). d) There was no difference in total licks (TTLs) across epochs between low E2 and high E2 status drinking days (N = 5, 14 low E2 days, 14 high E2 days). e) Time course of transient event frequency as a % of W1 baseline across the 30 minutes of W1, first 30 minutes of EtOH (EtOH 1), last 30 minutes of EtOH (EtOH 2), and the first 30 minutes of W2 in 5 minutes bins. f) Time course of transient event peak amplitudes as a % of W1 baseline across the 30 minutes of W1, EtOH 1, EtOH 2, and the first 30 minutes of W2 in 5 minutes bins. g) There was a positive correlation between time spent drinking EtOH (s) and g/kg EtOH intake during the EtOH epoch (N = 5, 24 drinking days). h) There was an increase in transient event frequency during low and E2 status drinking days during the transition from the last 5 minutes of W1 to EtOH 1 (left; N = 5, 15 high E2 days, 15 low E2 days) and the last 5 minutes of EtOH 2 and W2 (right; N = 5, 11 low E2 days, 9 high E2 days; same Ns for i). i) There was an increase in transient event amplitude in the first 5 minutes of EtOH 1 compared to the last 5 minutes of W1during high E2 status drinking days but not during low E2 status drinking days (left), with no change during the transition from EtOH 2 to W2 (right). j) There was a trend towards increased % time spent in the open arms of the elevated plus maze (EPM) on high E2 status days compared to low E2 status days (left), with no change in distance traveled (center), and a trend towards increased normalized GCaMP signal in the open arms compared to the closed arms of the EPM on low E2 status days but not high E2 status days (right; N’s = 5 low E2, 6 high E2). k) There was a trend towards increased % time spent in the center of the open field (OF) on high E2 status days (left), with no effect on distance (center), and a trend towards increased normalized GCaMP signal in the center compared to the corners of the OF on low and high E2 status days (right; N’s = 4 low E2, 6 high E2). *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001; unpaired t-tests between low vs high E2; 2xANOVA main effects and interactions between groups and time/compartment; post hoc t-tests with H-S corrections as indicated. $P &lt; 0.10 for post hoc t-tests with H-S corrections between groups. Data are presented as mean values +/- SEM. Detailed statistics are provided in Supplemental Table 1. Source data are provided as a Source Data file.