Spaced learning and high unconditioned stimulus saliency are necessary to create a context fear memory independent of...

Spaced contextual fear conditioning can establish a memory that no longer requires contribution from the hippocampus (HPC). It is unclear, however, whether spaced conditioning episodes alone are sufficient or whether a combination of spaced episodes and a high saliency unconditioned stimulus are required to make the context fear memory become HPC independent. To address this, rats underwent spaced contextual fear conditioning with shocks of low (0.4 mA), intermediate (0.7 mA), or high (1.0 mA) saliency, followed by sham or neurotoxic HPC lesions. Two weeks later, retention was assessed using freezing as the memory index, and c-Fos expression was quantified to identify supporting brain structures. In the low-saliency condition, control rats exhibited minimal freezing, limiting interpretation of lesion effects. With intermediate saliency, control rats froze robustly, whereas HPC-lesion rats did not, suggesting maintained HPC dependence despite spaced learning. In contrast, high-saliency conditioning produced comparable freezing in both groups, suggesting that combining spaced episodes with a strong stimulus creates an HPC-independent memory. Consistent with behavior, c-Fos analysis revealed increased expression in perirhinal and anterior cingulate cortices across conditions, regardless of saliency or lesion effects, implicating these regions in context representation. Elevated c-Fos was observed in the basolateral amygdala in rats from the spaced condition that retained fear memory, including HPC-lesioned rats, supporting its role in fear representation. Thus, HPC-independent contextual fear memory emerges when spaced conditioning is paired with a highly salient stimulus and is supported by a perirhinal and anterior cingulate cortices context representation and an amygdalar fear representation.