PROJECT SUMMARY Opioid use disorder (OUD) is a major epidemic in the United States and current pharmacological treatments result in ~50% relapse. Alternative strategies are needed to isolate, target, and repair the specific brain circuits implicated in craving and relapse. Low intensity focused ultrasound (LIFU) is a new method of non-invasive neuromodulation that can be focused anywhere in the brain with high spatial resolution. Ultrasound can be used to up or down regulate activity in specific brain regions and is currently approved for treating other disorders in humans. The application of LIFU to modulate specific brain circuitry as an intervention for addiction is unknown. The goals of this application are to determine whether LIFU has utility as an anti-relapse intervention in a rat model of OUD and to learn about the neurochemical mechanisms that underlie its effects. One specific region implicated in addiction is the dorsomedial prefrontal cortex (dmPFC). dmPFC glutamatergic projections to the nucleus accumbens core (NAcc) underlie craving for many drugs, including opioids, as well as time-dependent increases in craving (“incubation”) over protracted withdrawal. During early withdrawal, this circuit is hypoactive and interventions that excite the dmPFC block the incubation effect. During late withdrawal, this circuit is hyperactive and inhibition down-regulates activity and reduces craving. We propose to use inhibitory or excitatory LIFU during the different withdrawal periods to modulate neuronal activity in the dmPFC – NAcc circuit and reduce craving/vulnerability to relapse. In Aim 1, we will apply different doses of inhibitory and excitatory LIFU to the dmPFC and measure neurochemical markers of dmPFC activity as well as specific markers for dopamine and glutamate transmission in the dmPFC-NAcc pathway. Effects will be examined acutely during early or late withdrawal, and then adapted to a 7-day treatment regimen. Next, using a 7-day treatment regime, we will examine the effect of LIFU to the dmPFC during early (Aim 2) and late withdrawal (Aim 3) on vulnerability to relapse. Neurochemical markers will also be measured to relate brain activity to behavior. Both males and females will be included. Based on previous research and preliminary data, our overall hypothesis is that during early withdrawal, LIFU-induced excitation, but not inhibition, of the dmPFC will offset deficits in neuronal activity and block the incubation of craving; whereas, during late withdrawal, LIFU-induced inhibition, but not excitation, will decrease neuronal activity and block craving. Our long-term goal is to translate LIFU into human clinical trials as an anti-relapse intervention for OUD. This application is a major step toward this goal since the results will provide the necessary preclinical proof-of-concept data, as well as preliminary evidence for its neuromodulatory effects.