Background: Social isolation (SI) is an established rat model of chronic stress. We applied this to the stress‐sensitive Wistar Kyoto (WKY) strain to explore brain‐to‐gut interactions associated with mood. Whether SI stress‐induced behavioral changes are sex‐specific or if they affect the microbiome in WKY is unknown. We hypothesized individually housed (IH) animals would be more anxious than pair‐housed (PH), with sex differences. Male and female rats were either IH or PH from 70 to 112 days old and behavior was assessed in modified open field (OFTmod), elevated plus maze (EPM), and novel object recognition (NOR) tests. Cecal content DNA was analyzed by shotgun metagenome sequencing.Results: IH rats, particularly females, spent more time in the center of the OFTmod where the semi‐novel feed was presented compared to PH group rats. There was a tendency for greater distance traveled, or potential hyperactivity, in IH female rats. Males stayed in the EPM closed arms more than females. No treatment difference occurred for recognition memory. SI altered cecal microbiome composition in females where housing was associated with seven differentially abundant taxa and 49 differentially abundant KEGG Level 3 ortholog/gene categories. Several relationships were noted between behavioral traits and relative abundance of microbiome taxa. There was a greater shift in female microbiome composition.Conclusions: In summary, behavioral responses to the housing treatment were minimal. IH animals, particularly females, spent more time in the center of an OFT that contained food; this may have been an indication of depression, as opposed to anxiety. Housing status had a differential impact on the microbiome for females compared to males. The associations between cecal microbiota and activity in the modified OFT suggest that dietary interventions that influence the relative abundance of Bifidobacteria, Alistipes, and Muribaculaceae should be explored.

Although effects of stress-induced anxiety on the gastrointestinal tract and enteric nervous system (ENS) are well studied, how ENS dysfunction impacts behaviour is not well understood. We investigated whether ENS modulation alters anxiety-related behaviour in rats. We used loperamide, a potent μ-opioid receptor agonist that does not cross the blood–brain barrier, to manipulate ENS function and assess changes in behaviour, gut and brain gene expression, and microbiota profile. Sprague Dawley (male/female) rats were acutely dosed with loperamide (subcutaneous) or control solution, and their behavioural phenotype was examined using open field and elevated plus maze tests. Gene expression in the proximal colon, prefrontal cortex, hippocampus, and amygdala was assessed by RNA-seq and caecal microbiota composition determined by shotgun metagenome sequencing. In female rats, loperamide treatment decreased distance moved and frequency of supported rearing, indicating decreased exploratory behaviour and increased anxiety, which was associated with altered hippocampal gene expression. Loperamide altered proximal colon gene expression and microbiome composition in both male and female rats. Our results demonstrate the importance of the ENS for communication between gut and brain for normo-anxious states in female rats and implicate corticotropin-releasing hormone and gamma-aminobutyric acid gene signalling pathways in the hippocampus. This study also sheds light on sexually dimorphic communication between the gut and the brain. Microbiome and colonic gene expression changes likely reflect localised effects of loperamide related to gut dysmotility. These results suggest possible ENS pharmacological targets to alter gut to brain signalling for modulating mood.