ABSTRACT/PROJECT SUMMARY The habenulo-interpeduncular pathway, a forebrain to midbrain conduction system in the vertebrate brain, has been implicated in many essential processes, from sleep to fear/anxiety, pain, learning, motivation, feeding, reproduction, and reward, and in pathological states such as mood disorders and addiction. Despite its importance and diverse roles, little is known about the complete repertoire of neuronal subpopulations and precise connectivity between the bilaterally paired dorsal habenular nuclei (dHb) of the dorsal diencephalon and their major target, the unpaired interpeduncular nucleus (IPN) of the ventral midbrain. A renewed interest in this pathway came from its association with nicotine dependence and withdrawal and from the discovery that, in zebrafish and other vertebrates, the dHb develop with prominent left-right differences in their size, organization, molecular properties and connections to the IPN. In previous work, we described an asymmetric olfactory projection to a subset of neurons in the right dHb that helps mediate the response to aversive cues and determined that altering directional asymmetry of the dHb induces behavioral and physiological changes indicative of enhanced fear/anxiety. Using state-of-the-art genetic, transgenic, imaging, and behavioral approaches, we now aim to identify all of the neuronal populations of the Hb-IPN pathway and further define which groups of neurons are involved in the response to negative cues. We have also extended our studies to characterize the pre-synaptic input and post-synaptic targets of this conduction system to gain a more complete understanding of the underlying neural circuits and their connectivity. The proposed research will shed light on a poorly understood yet highly conserved neural pathway and also address how differential processing of information by neurons on the left and right sides of the brain leads to appropriate behavioral responses.