Article Figures & Data
Figures
- Figure 1.
Male and female C57BL/6 mice were exposed to intermittent access to alcohol (IA2BC; Monday, Wednesday, Friday, 24 h/d, 20% v/v ethanol vs water) for 6 consecutive weeks or 18 drinking sessions. A, C, D, Female mice drank more alcohol than males (A), drank more fluids (C), and showed a higher preference for alcohol (D). B, Water intake did not significantly differ between sexes. Data represent the mean ± SEM (n = 10/group, total 20 mice). a, p < 0.05; b, p < 0.01; c, p < 0.001; versus males.
- Figure 2.
A, PACAP immunoreactivity levels are elevated in the BSTld of male and female C57BL/6J mice exposed to intermittent access to alcohol (IA2BC; EtOH), compared with control, water-only mice (Ctrl). B, Data are reported with the two sexes pooled. C, D, Representative PACAP staining (DAB) in mouse BNST of a Ctrl and an EtOH subject, respectively. Magnification, 10×. Scale bar, 200 μm. Mice were killed after 7 weeks of IA2BC, 24 h after the end of the last drinking session. BSTlp, BNST lateral-posterior; BSTlj, BNST lateral-justacapsular. Data represent the mean ± SEM (A, n = 10–13/group; B, n = 5–8/group; total, 23 mice). ***p < 0.001 versus Ctrl. Extended Data Figure 2-1 shows the effect of IA2BC on PACAP immunoreactivity in the CeA.
- Figure 3.
A, The number of PAC1R-immunoreactive cells is not altered in the BNST of IA2BC mice. B, C, Representative PAC1R staining (fluorescence) in mouse BNST of a Ctrl and an EtOH subject, respectively. Magnification, 10×. Scale bar, 200 μm. D, CGRP levels are elevated in the BSTld of IA2BC mice (EtOH), compared with control mice (Ctrl). E, F, Representative CGRP staining (fluorescence) in mouse BNST of a Ctrl and an EtOH subject, respectively. Magnification, 10×. Scale bar, 200 μm. Mice were killed 24 h after the end of the last IA2BC session. BSTlp, BNST lateral-posterior; BSTlj, BNST lateral-justacapsular. Data represent the mean ± SEM (A, n = 11–15/group; D, n = 10–15/group; total, 26 mice). *p < 0.05 versus Ctrl.
- Figure 4.
A, Scheme and timeline of the chemogenetic experiment. B, Representative picture of mCherry fluorescence in the LPB. Scale bar, 100 μm. C–H, Verification (by in situ hybridization) of the selectivity of the retrograde AAV [AAVrg-hSyn-DIO-hM4D(Gi)-mCherry] for Adcyap1, and not Tac1, neurons in the LPB. Filled arrows indicate representative mCherry-expressing cells (where coexpression with Adcyap1 is visible); empty arrows indicate representative Tac1 (where absence of mCherry is visible). Extended Data Figure 4-1 shows the location of the AAVrg-hSyn-DIO-hM4D-mCherry infusions in the BNST.
- Figure 5.
A–F, The effect of inhibition of PACAP neurons afferent to the BNST on ethanol intake (A, B), water intake (C, D), and ethanol preference (E, F). Administration of CNO (0–3 mg/kg, i.p.) dose-dependently reduced 2, 6, and 24 h ethanol intake in AAVrg-hSyn-DIO-hM4D(Gi)-mCherry BNST-infused mice, while it did not affect water intake. CNO also reduced 6 h ethanol preference. Data represent the mean ± SEM (n = 13 mice). *p < 0.05, **p < 0.01, ***p < 0.001; versus Vehicle.
Extended Data
Figure 2-1
A, PACAP levels are unaltered in the CeA of male and female C57BL/6J mice exposed to intermittent access to alcohol (IA2BC; EtOH), compared with control, water-only mice (Ctrl). B, Data are reported with the two sexes pooled. C, D, Representative PACAP staining (DAB) in mouse BNST of a Ctrl and an EtOH subject, respectively. Magnification, 10×. Scale bar, 200 μm. Mice were killed after 7 weeks of IA2BC, 24 h after the end of the last drinking session. CeM, CeA medial. Data represent the mean ± SEM (A, n = 14–15/group; B, n = 6–8/group). Download Figure 2-1, TIF file.
Figure 4-1
A, Illustrations of coronal rat brain slices; numbers represent the distance from bregma (mm). Symbols represent the injection sites of the AAVrg-hSyn-DIO-hM4D-mCherry in the BNST (each dot is the location of one injection, each star the location of 2 or more injections). Download Figure 4-1, TIF file.
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