Currently, there are no proven medications to treat stimulant abuse, addiction or relapse. Therefore, new knowledge on the neuronal substrates that are modified by psychostimulant drugs will greatly aid in identifying novel targets for developing therapeutic strategies to intervene in these processes. Altered noradrenergic transmission and presynaptic norepinephrine transporter (NET) expression have long been associated with depression and drug-addiction. Indeed, amphetamine (AMPH) targets NET and downregulates its function. We have shown that AMPH downregulates NET function and surface expression via transporter T258/S259 trafficking motif. We have also shown that activation of the neurokinin-1 receptor (NK1R) negatively regulates NET via PKC phosphorylation of the same motif. These findings are of physiological significance because (i) Substance P, the endogenous NK1R agonist, is released following AMPH treatment. SP enhances AMPH stimulant effects, whereas NK1R antagonists block this effect; (ii) NK1R knockout mice exhibit ADHD-like phenotype; and (iii) AMPH is a therapeutic agent for treating ADHD in the human. Thus, there is, apparently a physiologically relevant relationship between NET, NK1R and AMPH. In this regard, our discovery that both NK1R and AMPH mediate NET downregulation and that both are linked to the T258/S259 motif suggests that this pathway may play a significant role in regulating AMPH-elicited behaviors. In support, our preliminary in vivo studies suggest that AMPH-induced NET downregulation and locomotor activation are sensitive to NK1R antagonists and to manipulations targeting the T258/S259 motif. We propose two specific aims to test our hypothesis that the NK1R-mediated T258/S259-specific NET downregulation contributes to AMPH behavioral effects. Aim 1 will test the hypothesis that NK1R mediates AMPH-induced NET regulation and reward-like behavior in mice. We will identify the role of ventral striatal NK1R in modulating AMPH-induced synaptic NET regulation and conditioned place preference by brain nuclei-specific microinjections of agonists and antagonists alone or in combination. Aim 2 will test the hypothesis that NET T258/S259 motif is required for AMPH-mediated reward-like behavior and NET downregulation in mice. We will study the role of the T258/S269 motif by ventral striatal microinjections of cell permeable TAT-NET peptides that interfere with the AMPH-dependent trafficking of NET. Since dopamine transporter (DAT) is also downregulated by both NK1R and AMPH, and both NET and DAT share highly conserved sequence homology at the T258/S259 region, DAT-KO mice will also be utilized in both aims to dissociate the role of NET regulation from that of DAT. Thus, understanding how NK1R regulates amine transporters may help us identify the specific mechanisms (which could be therapeutic targets) whereby AMPH disrupts catecholamine transporter function and hence animal behavior.