An important problem in the treatment of cocaine addiction is relapse to cocaine use even years after abstinence. Exposure to stress plays an important role in precipitating relapse. Thus, studying the neuronal circuitry that underlies stress-induced relapse is necessary for the development of effective treatments for addiction. While many brain regions contribute to the expression of stress-induced relapse, very little is known about the precise role of the lateral habenula. Using the self-administration-reinstatement procedure, we propose to directly examine the role of this brain region in stress-induced relapse to cocaine seeking using state-of-the-art genetic and chemical targeting approaches. In this proposal, we will utilize the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology, by which we will manipulate lateral habenula neurons in a manner that is selective, rapid and reversible and study the effect of these manipulations on stress-induced relapse. With the methods currently available in the field of neuroscience, it has been a challenge to study the contribution of specific synaptic connections involved in a specific behavior. We propose to utilize a unique dual-virus strategy in which we will use a combination of floxed, inverted DREADD's introduced into lateral habenula neurons and a retrogradely transported canine adenovirus 2 engineered to express Cre recombinase, to study neuronal circuits involved in cocaine relapse. In specific aim 1, we will investigate whether transient modulation of lateral habenula neuronal activity influences stress-induced reinstatement of cocaine-seeking. In specific aim 2, we will validate the dual-virus approach to dissect synaptic connections and study whether projection neurons from the lateral habenula to monoaminergic nuclei are involved in stress-induced reinstatement of cocaine seeking. In addition to providing novel information on the role of the lateral habenula in cocaine relapse, we will validate the use of the dual-virus strategy which has the potential to be widely used as a valuable tool in studying neuronal circuits in awake, behaving animals. Results from our studies will offer new insights into the neuronal circuitry that underlies stress-induced relapse and possibly open up new avenues for therapeutic options for the treatment of cocaine addiction.