Dopamine (DA) neurotransmission is central to multiple behaviors including movement, executive function, reward, and detecting salient stimuli. Multiple drugs of abuse, including cocaine, drive enhanced DA neuron (DAN) firing and increased extracellular DA levels in DAN terminal regions, and the enhanced DA in these regions is required for multiple aspects of cocaine addiction. However, the cellular mechanisms that underlie cocaine’s behavioral response and impact on DAergic signaling are not well defined. In the nucleus accumbens (NAc), where DA is required for cocaine’s rewarding properties, DA released from DAergic terminals modulates the responsiveness of postsynaptic medium spiny neurons (MSNs), which receive glutamatergic afferents from multiple brain regions. Cocaine binds to and inhibits the presynaptic DA transporter (DAT), which blocks DA clearance and increases both the magnitude and duration of the extracellular DA signal, and thereby impacts the downstream MSN excitability and responsiveness to their glutamatergic afferent signals. However, although DA clearly impacts glutamatergic signaling, it is relatively unknown whether glutamate signaling in the NAc conversely impacts presynaptic DA terminals, the DA signal, and DA-dependent behaviors such as cocaine reward. Our preliminary results demonstrate that the Gq-coupled, metabotropic glutamate receptor, mGluR5, is expressed on presynaptic DAergic terminals, where it drives biphasic DAT trafficking and is required for conditioned place preference to cocaine. However, the DAergic circuits and cellular mechanisms through which DAergic mGluR5 (mGluR5DA) mediates cocaine reward, and other cocaine-associated behaviors, are completely unknown. The major goals of the proposed studies are to determine 1) which DAergic circuits require mGluR5DA for cocaine addictive behaviors, 2) how mGluR5DA impacts DA signals and DAN activity, and 3) whether glutamate co-release by DA neurons impacts the DA signal via autocrine mGluR5DA signaling in DAergic terminals. These hypotheses stem from strong preliminary data that demonstrate that conditional mGluR5DA silencing throughout ventral midbrain DANs impacts presynaptic DAergic function and cocaine reward. To pursue this investigative line, we will leverage a variety of state-of-the-art mouse genetic tools, including AAV-mediated conditional gene silencing in DA neurons, coupled with in vivo fiber photometry, and optogenetics. Behavioral studies in mice will specifically test whether mGluR5DA is required for cocaine reward, self-administration, and reward motivation in mesolimbic and mesocortical DAergic circuits. In vivo fiber photometry studies using genetically encoded DA sensors and calcium sensors will test whether mGluR5DA is required for DA signal magnitude and duration, and DAN activity, respectively, during cocaine-associated behaviors. Finally, intersectional viral approaches will be leveraged to selectively test whether glutamate co-release by mesolimbic DANs regulates cocaine-associated behaviors and DA signals via autocrine or paracrine mGluR5DA signaling. The information gleaned from these studies will provide a clearer understanding of how glutamatergic signaling in DA terminal regions impacts the DA signal and cocaine addictive behaviors. Moreover, given that multiple mGluR5-targeted drugs are currently undergoing clinical trial for substance abuse and other DA-related disorders, we anticipate that our findings will greatly impact future strategies aimed at treating DA-related disorders, in which selectively manipulating mGluR5DA may provide therapeutic benefit.