Anxiety‐Like Behaviors in Mice Unmasked: Revealing Sex Differences in Anxiety Using a Novel Light‐Heat Conflict Test

Anxiety and chronic pain afflict hundreds of millions worldwide. Anxiety and pain are more prevalent in females compared to males. Unfortunately, robust sex differences in human anxiety are not recapitulated in rodent tests, and results from rodent pain studies frequently fail to translate clinically. Therefore, there is a need to develop tests that reflect the differential salience of anxiety or pain-related stimuli between the sexes. Accordingly, here we introduce the Thermal Increments Dark-Light (TIDAL) conflict test. The TIDAL test places an anxiety-relevant stimulus (dark vs. illuminated chamber) in conflict with a heat-related stimulus (incrementally heated vs. isothermic chamber); mice freely explore both apparatus chambers. Here, we aim to determine whether the TIDAL conflict test reveals in mice underappreciated sex differences in anxiety and/or heat sensitivity. We establish in four distinct experiments that females on the TIDAL conflict test persist substantially longer on the dark-heated plate, suggesting that female mice exhibit elevated anxiety-like behavior. Mice more strongly prefer the heated-dark plate on the TIDAL conflict test compared to control thermal place preference with both chambers illuminated. We also reveal that an anxiety-relieving drug, paroxetine, reduces mouse preference for the heating dark plate, supporting the validity of the TIDAL test. Therefore, our new TIDAL conflict test reliably unmasks the relative salience of anxiety (vs. heat sensitivity): mice that are female exhibit robust anxiety-like behaviors not consistently observed in classical tests. Future studies should incorporate TIDAL and other conflict tests to better understand rodent behavior and to identify mechanisms underlying anxiety and pain.