Alcohol drinking alters stress response to predator odor via BNST kappa opioid receptor signaling in male mice

Maladaptive responses to stress are a hallmark of alcohol use disorder, but the mechanisms that underlie this are not well characterized. Here, we show that kappa opioid receptor signaling in the bed nucleus of the stria terminalis (BNST) is a critical molecular substrate underlying abnormal stress responses to predator odor following heavy alcohol drinking. Exposure to predator odor during protracted withdrawal from intermittent alcohol drinking resulted in enhanced prefrontal cortex (PFC)-driven excitation of prodynorphin-containing neurons in the BNST. Furthermore, deletion of prodynorphin in the BNST and chemogenetic inhibition of the PFC-BNST pathway restored abnormal responses to predator odor in alcohol-exposed mice. These findings suggest that increased corticolimbic drive may promote abnormal stress behavioral responses to predator odor during protracted withdrawal. Various nodes of this PFC-BNST dynorphin-related circuit may serve as potential targets for potential therapeutic mediation as well as biomarkers of negative responses to stress following heavy alcohol drinking.


Sample-size estimation
• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Sample sizes were determined using power analysis based on preliminary data or through literature searches. Assuming a 30% change in behavior or physiology is meaningful, with a power level of β=0.80 and a significance level of α=0.05, the sample size used for experiments is at least n=8/group. For these reasons, most of our behavioral data have n=10/group and physiology recordings have at least n=8 cells/group.
Biological replicates are repeated measurements of biologically distinct samples that capture random biological variation. Technical replicates are repeated measurements of the same sample that represent independent measures of the random noise associated with protocols. For behavioral tests, biological replicates (two cohorts of mice) were used to account for availability of transgenic animals, EtOH drinking variability, and adequately powered sample size. For immunohistochemistry, while multiple cohorts of tissue were collected, all biological replicates were run in the same immunostain with the same imaging and analysis parameters. Further, we took four slices per animal and measured bilateral c-Fos and Pdyn expression. For electrophysiology, again, multiple cohorts of transgenetic mice were used as biological replicates. Outliers were tested with Grubb's test. One cell was excluded as it fell outside the inclusion criteria. This information is outlined in the Statistics section. 2 Statistical reporting • Statistical analysis methods should be described and justified • Raw data should be presented in figures whenever informative to do so (typically when N per group is less than 10) • For each experiment, you should identify the statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures (e.g., mean, median, SD, SEM, confidence intervals; and, for the major substantive results, a measure of effect size (e.g., Pearson's r, Cohen's d) • Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.
Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Please indicate the figures or tables for which source data files have been provided: The statistical tests performed for each experiment are described in the Statistics section; however, each statistical comparison is stated in the Figure Legend C57BL/6J mice from Jackson laboratory were randomly assigned to treatment groups. Transgenic mice were bred in-house and litter mates were divided up into different treatment groups. Behavioral data were recorded and quantified by Ethovision XT13, and burying behavior was hand-scored using BORIS (Behavioral Observation Research Interactive Software) by a blind observer. This information is found in the Methods section.