The habenulo-interpeduncular pathway, a highly conserved conduction system in the vertebrate brain, has been implicated in many processes, from sleep to fear/anxiety, pain, learning, motivation, feeding, reproduction and reward, and in pathological conditions in humans such as mood disorders and addiction. Despite their importance and diverse roles, little is known about the repertoire of neuronal subtypes in the bilaterally paired medial habenular nuclei (mHb) of the dorsal diencephalon or their precise connections with their major target, the unpaired interpeduncular nucleus (IPN) in the ventral midbrain. A renewed interest in this pathway has come from its association with nicotine dependence and withdrawal and from the discovery that, in zebrafish and other vertebrates, the dorsal Hb (equivalent to the mHb of mammals) show prominent left-right differences in their size, organization, molecular properties and connections to the IPN. Indeed, we have identified novel, asymmetrically distributed neuronal subtypes in the dHb that project to restricted regions of the IPN. Preliminary data support the hypothesis that dHb efferents form a precise connectivity map with specific IPN neurons, which will now be validated by experiments proposed in Specific Aims 1 and 2. In previous work to explore the functional significance of differences between the left and right dHb, we found that altering directional asymmetry induces behavioral and physiological changes in larval and adult zebrafish that are indicative of enhanced fear/anxiety. We recently discovered that neuronal activity predominantly in the left dHb attenuates freezing, a larval fear response, thereby promoting recovery of swimming activity. Using state-of-the-art techniques, experiments proposed in Aim 3 will combine optogenetic, transgenic and behavioral approaches to identify the responsible neurons. The proposed research will not only increase our knowledge of an essential but understudied neural pathway, they will also elucidate how differential processing of information by neurons on the left and right sides of the brain leads to appropriate behavioral responses.