Chronic stress drives behavioral and physiological changes associated with numerous psychiatric disease states. In rodents, the vast majority of chronic stress models involve imposition of external stressors, whereas in humans stress is often driven by internal cues, commonly associated with a sense of loss. We previously exposed groups of rats to environmental enrichment (EE) for a protracted period (1 month), followed by removal of enrichment (ER), to induce an experience of loss in male rats. ER enhanced immobility in the forced swim test (FST), led to hypothalamic pituitary adrenal (HPA) axis hypoactivity, and caused hyperphagia relative to continuously enriched (EE), single-housed (Scon) and pair-housed (Pcon) groups, most of which were reversible by antidepressant treatment (Smith et al., 2017). Here, we have applied the same approach to study enrichment loss in female rats. Similar to the males, enrichment removal in females led to an increase in the time spent immobile in the FST and increased daytime food intake compared to the single and pair-housed controls. Unlike males, ER females showed decreased sucrose preference, and showed estrus cycle-dependent HPA axis hyperactivity to an acute restraint stress. The increase in passive coping (immobility), anhedonia-like behavior in the sucrose preference test and HPA axis dysregulation suggest that enrichment removal produces a loss phenotype in females that differs from that seen in males, which may be more pronounced in nature.

Glucocorticoid receptors (GR) have diverse functions relevant to maintenance of homeostasis and adaptation to environmental challenges. Understanding the importance of tissue-specific GR function in physiology and behavior has been hampered by near-ubiquitous localization in brain and body. Here we use CRISPR/Cas9 gene editing to create a conditional GR knockdown in Sprague Dawley rats. To test the impact of cell- and region-specific GR knockdown on physiology and behavior, we targeted GR knockdown to output neurons of the prelimbic cortex. Prelimbic knockdown of GR in females caused deficits in acquisition and extinction of fear memory during auditory fear conditioning, whereas males exhibited enhanced active-coping behavior during forced swim. Our data support the utility of this conditional knockdown rat to afford high-precision knockdown of GR across a variety of contexts, ranging from neuronal depletion to circuit-wide manipulations, leveraging the behavioral tractability and enhanced brain size of the rat as a model organism.

26Glucocorticoid receptors (GR) have diverse functions relevant to maintenance of homeostasis and 27 adaptation to environmental challenges. Understanding the importance of tissue-specific GR function in 28 physiology and behavior has been hampered by near-ubiquitous localization in brain and body. Here we 29 use CRISPR/Cas9 gene editing to create a conditional GR knockout in Sprague Dawley rats. To test the 30 impact of cell-and region-specific GR deletion on physiology and behavior, we targeted GR knockout to 31 output neurons of the prelimbic cortex. 32Prelimbic deletion of GR in females caused deficits in acquisition and extinction of fear memory during 33 auditory fear conditioning, whereas males exhibit enhanced active-coping behavior during forced swim. 34Our data support the utility of this conditional knockout rat to afford high-precision deletion of GR 35 across a variety of contexts, ranging from neuronal depletion to circuit-wide manipulations, leveraging 36 the behavioral tractability and enhanced brain size of the rat as a model organism. 37

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