An Infralimbic Cortex Engram Encoded during Learning Attenuates Fear Generalization

Generalization allows previous experience to adaptively guide behavior when conditions change. The infralimbic (IL) subregion of the ventromedial prefrontal cortex plays a known role in generalization processes, although mechanisms remain unclear. A basic physical unit of memory storage and expression in the brain is a sparse, distributed group of neurons known as an engram. Here, we set out to determine whether an engram established during learning contributes to generalized responses in IL. Generalization was tested in male and female mice by presenting a novel, ambiguous, tone generalization stimulus following Pavlovian defensive (fear) conditioning. The first experiment was designed to test a global role for IL in generalization using chemogenetic manipulations. Results show IL regulates defensive behavior in response to ambiguous stimuli. IL silencing led to a switch in defensive state, from vigilant scanning to generalized freezing, while IL stimulation reduced freezing in favor of scanning. Leveraging activity-dependent "tagging" technology (ArcCreER^T2 x eYFP system), an engram, preferentially located in IL Layer 2/3, was associated with the generalization stimulus. Remarkably, in the identical discrete location, fewer reactivated neurons were associated with the generalization stimulus at the remote timepoint (30 days) following learning. When an IL engram established during learning was selectively chemogenetically silenced, freezing increased. Conversely, IL engram stimulation reduced freezing, suggesting attenuated fear generalization. Overall, these data identify a crucial role for IL in suppressing generalized conditioned responses. Further, an IL engram formed during learning functions to later attenuate a conditioned response in the presence of ambiguous threat stimuli.<b>Significance statement</b> Generalization refers to the ability for organisms to use previous experience to guide behavior when environmental conditions change. Despite the immense importance of generalization in adaptive behavior, the precise brain mechanisms remain unknown. Here we identified a small population of neurons, known as an engram, in a discrete region of the frontal cortex that was associated with the expression of generalization related to a threatening situation. When these cells were turned off, generalization increased. When they were turned on, generalization decreased. Considering that over-generalization of threatening stimuli is a known fundamental dimension of both anxiety and post-traumatic stress disorders, these findings have implications not only for our understanding of intrinsic generalization processes but also highly prevalent clinical disorders.