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 × 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 d) 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.