Infralimbic projections to the substantia innominata-ventral pallidum constrain defensive behavior during extinction learning.

Fear extinction is critical for decreasing fear responses to a stimulus that no longer poses a threat. While it is known that the infralimbic region (IL) of the medial prefrontal cortex mediates retrieval of an extinction memory through projections to the basolateral amygdala (BLA), IL pathways contributing to extinction learning are not well-understood. Given the dense projection from the IL to the substantia innominata-ventral pallidum (SI/VP), an area that processes aversive and appetitive cues, we compared how the IL-SI/VP functions in extinction compared to the IL-BLA pathway in male mice. Using retrograde tracing, we demonstrate that IL projections to the SI/VP originate from superficial (L2/3) and deep cortical layers (L5), and that they are denser than IL projections to the BLA. Next, combining retrograde tracing with labeling for the immediate early gene cFos, we show increased activity of L5 IL-SI/VP output during extinction learning and increased activity of L2/3 IL-BLA output during extinction retrieval. Then, using in vitro recordings, we demonstrate that neurons in the IL-SI/VP pathway are more excitable during extinction learning than retrieval. Finally, using optogenetics we inactivate the IL-SI/VP pathway, and show that this increases defensive freezing during extinction learning and re-extinction, without affecting memory. Taken together, we demonstrate that the IL-SI/VP pathway is active during extinction learning, when it constrains the defensive freezing response. We propose that the IL acts as a switchboard operator, increasing IL L5 communication with the SI/VP during extinction learning, and IL L2/3 communication with the BLA during extinction retrieval.Significance Statement Fear extinction is a widely used behavioral approach to decrease conditioned fear, and projections from the infralimbic cortex to the amygdala are known to mediate extinction memory retrieval. However, less is known about the role of infralimbic pathways in extinction learning. We use neuroanatomical tracing, behavior, slice recordings, and circuit manipulation to show that infralimbic output to the substantia innominata-ventral pallidum, a region that processes aversive and appetitive stimuli, is denser than to the amygdala, and is more active during extinction learning than retrieval, when it acts to constrain the defensive freezing response. Thus, we posit that during extinction the infralimbic uses several lines of communication, one with the substantia innominata-ventral pallidum during learning one with the amygdala during retrieval.