ChAT::Cre transgenic rats show sex‐dependent altered fear behaviors, ultrasonic vocalizations and cholinergic marker expression

The cholinergic system is a critical regulator of Pavlovian fear learning and extinction. As such, we have begun investigating the cholinergic system's involvement in individual differences in cued fear extinction using a transgenic ChAT::Cre rat model. The current study extends behavioral phenotyping of a transgenic ChAT::Cre rat line by examining both freezing behavior and ultrasonic vocalizations (USVs) during a Pavlovian cued fear learning and extinction paradigm. Freezing, 22 kHz USVs, and 50 kHz USVs were compared between male and female transgenic ChAT::Cre+ rats and their wildtype (Cre‐) littermates during fear learning, contextual and cue‐conditioned fear recall, cued fear extinction, and generalization to a novel tone. During contextual and cued fear recall ChAT::Cre+ rats froze slightly more than their Cre‐ littermates, and displayed significant sex differences in contextual and cue‐conditioned freezing, 22 kHz USVs, and 50 kHz USVs. Females showed more freezing than males in fear recall trials, but fewer 22 kHz distress calls during fear learning and recall. Females also produced more 50 kHz USVs during exposure to the testing chambers prior to tone (or shock) presentation compared with males, but this effect was blunted in ChAT::Cre+ females. Corroborating previous studies, ChAT::Cre+ transgenic rats overexpressed vesicular acetylcholine transporter immunolabeling in basal forebrain, striatum, basolateral amygdala, and hippocampus, but had similar levels of acetylcholinesterase and numbers of ChAT+ neurons as Cre‐ rats. This study suggests that variance in behavior between ChAT::Cre+ and wildtype rats is sex dependent and advances theories that distinct neural circuits and processes regulate sexually divergent fear responses. This study examined freezing and ultrasonic vocalizations (USVs) during a fear conditioning and fear extinction paradigm, plus expression of vesicular acetylcholine transporter (VAChT), in both male and female transgenic ChAT::Cre rats compared with their wildtype littermates. Results showed that fear responses in ChAT::Cre+ and wildtype rats related to the VAChT overexpression are sex‐dependent. During fear recall ChAT::Cre+ rats froze slightly more than their Cre‐ wildtype littermates, and displayed significant sex differences in contextual and cue‐conditioned freezing, 22 kHz USVs, and 50 kHz USVs. Females showed fewer 22 kHz distress calls than males during fear learning and recall, but produced more 50 kHz USVs durin