ANTERIOR HYPOTHALAMUS–PROJECTING VENTRAL SUBICULAR NEURONS DISPLAY WIDESPREAD BRANCHING IN CIRCUITS ORCHESTRATING FEAR AND ANXIETY BEHAVIOR

The ventral hippocampus is a key regulator of emotional behavior through its projections to hypothalamic defensive circuits. In particular, inputs to the anterior hypothalamic nucleus (AHN) contribute to anxiety, innate fear, and contextual fear responses. Here, we investigated whether hippocampal neurons projecting to the AHN distribute collateral outputs to further brain regions, supporting an extended integrative role. By injecting into the AHN an AAV retrograde vector carrying the enhanced yellow fluorescent protein (EYFP) reporter gene, we mapped the hippocampal neurons projecting to this region and found that they are primarily located along the rostrocaudal extent of the ventral subiculum. To determine their full projection pattern, we combined AHN-targeted retrograde delivery of Cre recombinase along with the Cre-dependent expression of membrane-bound green fluorescent protein (GFP) and synaptophysin-mRuby in the subiculum, enabling visualization of axonal projections and presynaptic terminals. This approach revealed that AHN-projecting ventral subicular neurons exhibit widespread branching projections to multiple brain regions implicated in memory, motivation, arousal, circadian regulation, neuroendocrine control, and goal-directed behavior. These findings demonstrate that hippocampal outputs to the AHN are embedded within a distributed network rather than a single pathway. Such organization positions ventral subicular neurons to coordinate complex behavioral and physiological responses, highlighting their role as integrative nodes in circuits underlying fear and anxiety behavior.