Isolation and analysis of supramammillary nucleus projections to the hippocampus, basolateral amygdala, and prefrontal cortex

The supramammillary nucleus (SuM), located in the ventromedial hypothalamus, is notable for a strong projection to the hippocampus and has been implicated in hippocampal-dependent behaviors. While the SuM is known to connect to many areas, little is known about extra- hippocampal projections. To better understand the function of the SuM in hippocampal-dependent behaviors, we examined the role of the SuM in fear conditioning and open field behaviors. We found that the SuM responds to novel context exposure, aversive stimuli, and contexts carrying an emotional valence. Furthermore, general manipulation of the SuM using chemogenetics altered hippocampal-dependent behaviors. Using an intersectional viral approach, we successfully isolated dorsal hippocampal- and ventral hippocampal-projecting SuM neurons. We found that hippocampal-projecting SuM neurons send collateralized projections to the basolateral amygdala (BLA) and prefrontal cortex (PFC). Interestingly, BLA-projecting SuM neurons are largely distinct from PFC-projecting SuM. Examination of BLA-projecting and PFC-projecting SuM neurons showed collateralized projections to several other areas involved in learning and memory. Manipulation of these projections altered behavior in open field and fear learning paradigms. Our results illustrate a complex network of SuM projections involved in learning and memory and provide a foundation for further studies into the roles of the SuM in neural processing.