<p>by Yi-Ling Sun, Yi-Ting Peng, Xue Zhang, Cui Yin, Cheng Xiao, Junping Chen, Chunyi Zhou</p> The lateral hypothalamus (LH) is fundamentally involved in motivated behaviors, yet the precise functional segregation of its GABAergic projections in modulating pain and its affective dimensions remains poorly understood. Here, using male and female mice, we revealed that LH GABAergic neurons sent parallel projections to two pivotal pain and affect processing centers: the lateral parabrachial nucleus (LPB) and the paraventricular nucleus of the thalamus (PVT). In vivo fiber photometry revealed that LH GABAergic terminals in both the PVT and LPB are robustly activated by noxious, aversive, and rewarding stimuli, indicating their roles in encoding salient and affective information. Critically, we uncovered a remarkable functional segregation of these LH GABAergic pathways. Optogenetic activation of the LH-PVT projection exerted anxiogenic effects without altering pain sensitivity in both naïve and neuropathic pain mice; whereas optogenetic inhibition of this pathway exerted significant analgesic and anxiolytic effects specifically in neuropathic pain, highlighting a context-dependent modulation of pain and affect. In contrast, optogenetic activation of the LH-LPB projection induced contralateral pronociception without impacting affective behaviors, conversely, inhibition of this pathway significantly elevated contralateral pain thresholds in both physiological and neuropathic pain conditions. We also demonstrated that stimulation of the LH-LPB projection acted via a disinhibitory mechanism involving local LPB GABAergic interneurons to enhance activity of LPB glutamatergic neurons. Collectively, our findings delineate two parallel and functionally segregated LH GABAergic output pathways that differentially control distinct aspects of pain experience, thereby offering novel circuit-specific targets for the treatment of chronic pain and its debilitating emotional comorbidities.