Contributions of associative and non-associative learning to the dynamics of defensive ethograms

Defensive behavior changes based on threat intensity, proximity, and context of exposure, and learning about danger-predicting stimuli is critical for survival. However, most Pavlovian fear conditioning paradigms focus only on freezing behavior, obscuring the contributions of associative and non-associative mechanisms to dynamic defensive responses. To thoroughly investigate defensive ethograms, we subjected male and female adult C57BL/6J mice to a Pavlovian conditioning paradigm that paired footshock with a serial compound stimulus (SCS) consisting of distinct tone and white noise (WN) stimulus periods. To investigate how associative and non-associative mechanisms affect defensive responses, we compared this paired SCS-footshock group with four control groups that were conditioned with either pseudorandom unpaired presentations of SCS and footshock, shock only, or reversed SCS presentations with inverted tone--WN order, with paired or unpaired presentations. On day 2 of conditioning, the paired group exhibited robust freezing during the tone period with switching to explosive jumping and darting behaviors during the WN period. Comparatively, the unpaired and both reverse SCS groups expressed less tone-induced freezing and rarely showed jumping or darting during WN. Following the second day of conditioning, we observed how defensive behavior changed over two extinction sessions. During extinction, the tone-induced freezing decreased in the paired group and mice rapidly shifted from escape jumping during WN to a combination of freezing and darting. The unpaired, unpaired reverse, and shock-only groups displayed defensive tail rattling and darting during the SCS, with minimal freezing and jumping. Interestingly, the paired reverse group did not jump to WN, and tone-evoked freezing was resistant to extinction. These findings demonstrate that non-associative factors promote some defensive responsiveness, but associative factors are required for robust cue-induced freezing and high-intensity flight expression.