A naturalistic assay for measuring behavioral responses to aversive stimuli at millisecond timescale

We have designed a Virtual Burrow Assay (VBA) to detect the behavioral responses of head-fixed mice to aversive stimuli. We demonstrate its suitability for measuring novelty detection as well as aversion to both conditioned and innately aversive cues. The VBA simulates a scenario in which a mouse, poised at the threshold of its burrow, evaluates whether to remain exposed to potential threats outside or to retreat inside an enclosure. When presented with aversive stimuli, mice exhibit a stereotyped retreat whose onset is determined by measuring the position of a moveable burrow. This withdrawal, which requires no training, is characterized by an abrupt transition that unfolds within milliseconds--a timescale similar to that of neuronal dynamics, permitting direct comparison between the two. The assay is compatible with standard electrophysiological and optical methods for measuring and perturbing neuronal activity.