The Lateral Habenula to Ventral Tegmental Area Pathway Is Required for Aversive Learning and Defensive Behaviors

Threatening situations require animals to rapidly select appropriate defensive strategies, either disengaging behavior to avoid harm or engaging actions that allow escape or avoidance. The lateral habenula (LHb) is a key hub in aversive processing, and its projection to the rostromedial tegmental nucleus (RMTg) suppresses dopaminergic activity and promotes behavioral disengagement. However, although LHb neurons also project directly to the ventral tegmental area (VTA) and encode aversive signals, how this pathway contributes to learning and behavior remains poorly understood. In this study, we tested the hypothesis that VTA-projecting LHb neurons encode aversive signals that facilitate associative learning and promote escape behavior. Using a retrograde viral strategy, we targeted VTA-projecting LHb neurons and monitored calcium activity during active avoidance training in male and female mice. These neurons were activated by aversive stimuli and predictive cues as mice acquired avoidance responses and showed increased activity at movement onset during the tail suspension test (TST). Silencing LHb→VTA transmission impaired avoidance learning, prolonged escape latency, and reduced persistence and vigor of active responses in the TST, without affecting baseline locomotion. Anatomical and ex vivo electrophysiology revealed that LHb terminals innervate both dopaminergic (TH⁺) and nondopaminergic (TH^-) VTA neurons, exhibiting session-specific synaptic adaptations during avoidance learning. Together, these findings identify the LHb→VTA pathway as a source of aversive predicting signals required for the acquisition of avoidance behavior and the persistence of active responses in aversive contexts, supporting the idea that distinct LHb outputs may differentially regulate behavioral disengagement and active defensive responses.