Neuroanatomical and behavioral characterization of corticotropin-releasing factor-expressing lateral Habenula neurons in mice

Abstract The lateral habenula (LHb) is a critical hub for stress-related behaviors, yet the sources of its corticotropin-releasing factor (CRF) inputs remain poorly defined. Using high-resolution imaging, RNAscope, and viral tracing, we identified a novel, intrinsic population of CRF-expressing LHb neurons (LHb CRF ). These neurons are primarily VGLUT2+, though a rostral subpopulation co-expresses GAD2. While chemogenetic activation of LHb CRF neurons did not impact place preference or anxiety-like behaviors, it selectively biased defensive strategies toward passive action-locking during the Visual Looming Shadow Test (VLST). Notably, this activation prolonged escape latencies in males and post-escape shelter stays in females. Electrophysiological and optogenetic characterization revealed significant sexual dimorphism: male LHb CRF neurons are more numerous and intrinsically excitable, whereas female LHb CRF neurons exhibit stronger local excitatory connectivity. These findings establish LHb CRF neurons as a sexually dimorphic circuit component that could modulate sex-specific defensive strategies under threat via divergent cellular and synaptic mechanisms between the sexes.