Early life adversity decreases pre-adolescent fear expression by accelerating amygdala PV cell development

Early life adversity (ELA) is associated with increased risk for stress-related disorders later in life. The link between ELA and risk for psychopathology is well established but the developmental mechanisms remain unclear. Using a mouse model of resource insecurity, limited bedding (LB), we tested the effects of LB on the development of fear learning and neuronal structures involved in emotional regulation, the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA). LB delayed the ability of peri-weanling (21 days old) mice to express, but not form, an auditory conditioned fear memory. LB accelerated the developmental emergence of parvalbumin (PV)-positive cells in the BLA and increased anatomical connections between PL and BLA. Fear expression in LB mice was rescued through optogenetic inactivation of PV-positive cells in the BLA. The current results provide a model of transiently blunted emotional reactivity in early development, with latent fear-associated memories emerging later in adolescence.


Sample-size estimation
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Replicates
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A description of experimental design can be found in the methods section. The main behavioral results (decreased freezing at PND 22) was replicated across 3 independent experiments. For all experiments multiple cohorts were used, and the total number of mice (n) for each group is provided within the figure legends. No outliers needed to be removed from the datasets. For optogenetic, and cholera toxin B experiments, exclusion/inclusion was based on placement of optic fiber or injection respectively.

Statistical reporting
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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) Statistical tests and their values are provided in the figure legends. In general, we focused on analyzing the effects of rearing condition at each age tested, instead of across time. This decision was in part based on the difficulty of simultaneously running all ages, sexes and groups in assays such as western blot and immunohistochemistry. However, in behavioral experiments, where large number of animals could be run across ages, 3-way ANOVAS which included effects of age, sex, and rearing condition were conducted.
Litters of mice were randomly assigned to limited bedding or control bedding rearing conditions. All mice of a given litter were thus designated at limited bedding (experimental) or control rearing (control) mice. When possible, automated software was used to eliminate experimenter bias. Any exception to automated analysis is detailed in the figure legend or within the methods section. Within the optogenetic experiments all mice in a litter were implanted, with genotyping for Halo+ status being conducted after the experiment had been completed. This assured no bias in the treatment of Halo+ versus Halo-mice.