SUMMARY Untreated and inadequately treated pain has substantially increased as a problem in the US over at least the last 20 years. Increasing fears around prescribing opioids have also contributed to inadequate analgesic treatment. Importantly, often chronic pain and major depressive disorder (MDD) are comorbid. Therefore, discoveries of previously unknown neural circuits that can be targeted to address not only nociceptive discomfort but also the affective component of chronic pain and its interactions with depression remains an important area of focus for research. We recently showed that the lateral habenula (LHb) is a brain region that contributes to both acute nociception and the aversiveness of chronic pain. The LHb also plays a role in behavioral models of depression in rodents and has been targeted for deep brain stimulation to treat MDD in humans. LHb neurons generally fire more in response to noxious stimuli and aversive behavioral states. We recently found that a glutamatergic input from the lateral preoptic area of the hypothalamus (LPO) to the LHb is activated by noxious stimuli, and inhibiting LPO → LHb neurons relieves the mechanical hypersensitivity induced by a neuropathic pain model (spared nerve injury, SNI) in male rats. Inhibiting LPO → LHb also generated a conditioned place preference in male and female animals with SNI, but not the control animals. This suggests that targeting this circuit should have less abuse liability compared to agents that act on neural circuits that produce reward in control animals. Here we propose to investigate the projections out of the LHb that transmit the pain signal compared to the projections that modulate depression-model-related changes in behavior. While many have studied the direct and indirect projections from the LHb to the dopaminergic systems and dopamine systems contribute to pain and relief signaling, the LHb also sends a direct excitatory projection to the parabrachial nucleus (PBN), an important component of the pain processing circuitry. The PBN receives nociceptive inputs from the dorsal horn of the spinal cord and transmits pain signals to the central nucleus of the amygdala. Therefore an LHb input to the PBN is a direct route for the LHb to have a strong impact on both sensory and affective components of pain. The LHb also projects to the dorsal raphe nucleus (DRN), which is strongly implicated in MDD in humans and depression models in rodents. Our preliminary findings show that the projections to the PBN and DRN are from mostly non-overlapping sets of LHb neurons, making interactions possible but not necessary. Here we propose to systematically investigate the contributions of the LHb → PBN and LHb → DRN to nociception and motivated behaviors at the anatomical, neurophysiological, and behavioral levels. We will make these measurements in control animals, animals with SNI, and animals treated with repeated aversive restraint stress (a model for inducing depression-related be...