Gut microbiota influence numerous aspects of host biology, including brain structure and function. Growing evidence implicates gut microbiota in aversive conditioning and anxiety‐related behaviors, but research has focused almost exclusively on males. To investigate whether effects of gut dysbiosis on aversive learning and memory differ by sex, adult female and male C57BL/6N mice were orally administered a moderate dose of nonabsorbable antimicrobial medications (ATMs: neomycin, bacitracin, and pimaricin) or a control over 10 days. Changes in gut microbiome composition were analyzed by 16S rRNA sequencing. Open field behavior, cued aversive learning, context recall, and cued recall were assessed. Following behavioral testing, the morphology of basolateral amygdala (BLA) principal neuron dendrites and spines was characterized. Results revealed that ATMs induced gut dysbiosis in both sexes, with stronger effects in females. ATMs also exerted sex‐specific effects on behavior and neuroanatomy. Males were more susceptible than females to microbial modulation of locomotor activity and anxiety‐like behavior. Females were more susceptible than males to ATM‐induced impairments in aversive learning and cued recall. Context recall remained intact, as did dendritic structure of BLA principal neurons. However, ATMs exerted a sex‐specific effect on spine density. A second experiment was conducted to isolate the effects of gut perturbation to cued recall. Extinction was also examined. Results revealed no effect of ATMs on cued recall or extinction, suggesting that gut dysbiosis preferentially impacts aversive learning. These data shed new light on how gut microbiota interact with sex to influence aversive conditioning, open field behavior, and BLA dendritic spine architecture.

Gut microbiota influence numerous aspects of host biology, including brain structure and function. Growing evidence implicates gut microbiota in aversive conditioning and anxiety-related behaviors, but research has focused almost exclusively on males. To investigate sex-specific effects of gut dysbiosis on aversive learning and memory, adult female and male C57BL/6N mice were orally administered a moderate dose of non-absorbable antimicrobial medications (ATMs; neomycin, bacitracin, pimaricin) or a control over 10 days. Changes in gut microbiome composition were analyzed by 16S rRNA sequencing. Open field behavior, cued aversive learning, context recall, and cued recall were assessed. Following behavioral testing, the morphology of basolateral amygdala (BLA) principal neuron dendrites and spines was characterized. Results revealed that ATMs induced distinct but overlapping patterns of gut dysbiosis across sex, with stronger effects in females. There were also sex-specific effects on behavior and neuroanatomy. Treated males but not females exhibited altered locomotor and anxiety-like behavior in the novel open field test. Treated females but not males showed impairments in aversive memory acquisition and cued recall. Context recall remained intact in both sexes, as did dendritic structure of BLA principal neurons. However, ATMs exerted sex-specific effects on spine density. A second experiment was conducted to isolate gut perturbation to cued recall. Results revealed no effect of ATMs on recall of a previously consolidated fear memory, suggesting that gut dysbiosis preferentially impacts aversive learning. These data shed new light on how gut microbiota interact with sex to influence aversive conditioning, anxiety-like behavior, and BLA dendritic spine architecture.SignificanceGut microbiota can influence brain function and behavior, including trauma and anxiety-related disorders. Although these disorders disproportionately affect women, preclinical research has focused almost exclusively on male rodent models. We investigated the impact of antimicrobial administration on gut microbiome structure, aversive conditioning, open field behavior, and basolateral amygdala principal neuron morphology in female and male mice. Results showed that treatment exerted wide-ranging effects, many of which were sex-specific. Our findings underscore the importance of studying sex differences and support a role for microbial modulation of aversive learning, anxiety-like behavior, and amygdala spine patterning.