Abstract The long-term goal of this study is to determine how vagus nerve stimulation (VNS) regulates basal dopamine transmission and methamphetamine regulation of dopamine neurons. Methamphetamine abuse is a major public health issue around the world, yet there are no effective pharmacotherapies for the treatment of methamphetamine addiction. Methamphetamine is a potent psychostimulant that increases extracellular dopamine levels by targeting the dopamine transporter (DAT) in the midbrain and striatum. In the previous cycle of this grant, we shown that methamphetamine competes with the DAT-mediated dopamine uptake, increases dopamine efflux via the DAT, increases the DAT mediated inward depolarizing current leading to increased firing activity of dopamine neurons. Methamphetamine increases Ca2+ levels in the dopamine neurons that enhances both action potential dependent and independent dopamine release (i.e., dopamine efflux). We also found that neuronal depolarization induces DAT internalization leading to decreased dopamine and methamphetamine uptake. Multiple studies have shown that VNS increases dopamine levels in the midbrain region and reduces cocaine seeking behavior, albeit with an unknown mechanism via a multi-synaptic connection between vagus nerve and midbrain region. We found that optogenetic stimulation of vagal sensory neurons innervating the upper gastrointestinal tract depolarizes dopamine neurons and increases basal firing activity of midbrain dopamine neurons lasting for at least 30 minutes. Histological analyses revealed a reduction in somatodendritic DAT in the c-fos positive neurons. These data are consistent with our previous report showing neuronal depolarization induces DAT internalization, that can reduce the efficacy of methamphetamine regulation of dopamine transmission. Our preliminary data also revealed that optogenetic VNS increases extracellular dopamine levels that does not reach ceiling levels, but it reduces the methamphetamine stimulation of dopamine neurons, by depolarizing dopamine neurons, reducing DAT levels. These preliminary data and the literature support the overarching hypothesis that VNS increases basal dopamine levels by depolarizing dopamine neurons, reducing DAT levels, and decreasing methamphetamine-stimulation of dopamine transmission thereby attenuating methamphetamine’s behavioral and cellular responses. Our proposed studies will determine the underlying cellular mechanism for VNS-regulation of dopamine transmission and methamphetamine-induced responses.