The Branching Projection Pattern of Ventral Subicular Cells Revealing the Complex Integrative Role of Key Hippocampal Outputs in Driving Fear and Anxiety

Abstract

The ventral regions of the hippocampus play a crucial role in emotional responses through projections to the hypothalamus. In this context, the ventral hippocampus sends extensive projections to components of the medial hypothalamic defensive circuit, where projections to the anterior hypothalamic nucleus (AHN) influence both anxiogenic and innate fear responses, as well as contextual fear reactions. This study aims to examine the branching projection pattern of hippocampal cells that directly project to the AHN and identify the complete range of brain regions affected by key hippocampal outputs that drive fear and anxiety. To accomplish this, we first explored the full range of hippocampal cells that project directly to the anterior hypothalamic area using an AAV retroviral vector carrying the enhanced yellow fluorescent protein (EYFP) reporter gene. We found that the hippocampal cells projecting to the anterior hypothalamus were located primarily along the entire rostro-caudal axis of the ventral subiculum. Next, by combining an AAV retro vector injection into the AHN to insert Cre recombinase specifically in the subicular neurons projecting to the anterior hypothalamic region and subicular injections of an AAV vector expressing membrane-bound GFP and the presynaptic protein synaptophysin fused to the red fluorescent protein mRuby in a Cre-dependent manner, we investigated the collateral branching pattern of subicular neurons that project to the AHN. Our results demonstrated that ventral subicular neurons projecting to the anterior hypothalamus exhibit an extensive branching projection pattern that connects to several brain regions involved in various functions, including memory, motivation, arousal, circadian rhythmicity, neuroendocrine regulation, and goal-oriented behavior. This reveals that ventral subicular anterior hypothalamic-projecting cells display a widespread branching projection pattern, uniquely positioning them to influence complex, integrated responses while driving anxiety and fear behaviors.