Project Summary/Abstract: Amphetamines (AMPHs) are psychostimulants commonly used for the treatment of neuropsychiatric disorders (e.g. attention deficit disorders). They are also abused, with devastating outcomes. The abuse potential of AMPHs is associated with their ability to cause mobilization of cytoplasmic dopamine (DA), which leads to an increase in extracellular DA levels. This increase is mediated by the reversal of the DA transporter (DAT) function, which causes non-vesicular DA release, here defined as DA efflux. Notably, inhibition of DA efflux reduces both the ability of AMPH to increase motor activity and AMPH preference8-10. Imbalances in the gut microbiome (dysbiosis) have been suggested to participate in the pathogenesis of substance use disorders11. In addition, psychostimulant abuse promotes dysbiosis12-15. Therefore, it is possible that changes in the gut microbiome and its metabolites may not only be a consequence of substance use disorders, but may play a role in mediating the behavioral responses to drugs of abuse11. Microbial products, such as short-chain fatty acids (SCFAs), are thought to play a fundamental role in regulating the gut-brain axis16. Among SCFAs, butyrate is known to cross the BBB and directly act on neurons and glial cells17. Fusobacterium nucleatum (F. nucleatum) is an anaerobic, filamentous, gram-negative bacterial species that secretes butyrate18, the growth of which is stimulated by AMPH abuse13-15. Our data demonstrate that in germ-free (gnotobiotic) Drosophila, colonization with F. nucleatum enhances AMPH-induced DA effluxes (recorded in isolated fly brains), as well as AMPH behaviors. This potentiation of AMPH actions by F. nucleatum is paralleled by oral administration of butyrate. To understand, mechanistically, how F. nucleatum promotes AMPH actions, it is important to consider that changes in DAT expression regulate both AMPH-induced DA efflux and psychomotor actions19. Also, butyrate is a potent inhibitor of histone deacetylases (HDACs)20-22; inhibition of HDACs robustly increases expression of both DAT mRNA and proteins21-23. Our hypothesis is that F. nucleatum enhances AMPH actions by elevating DAT expression. This is mediated by secretion of butyrate, HDAC inhibition, and enhanced Drosophila DAT (dDAT) promotor acetylation. We will test this hypothesis through the following specific aims: S.A. #1. To determine how F. nucleatum regulates dDAT expression. S.A. #2. To test, mechanistically, how F. nucleatum increases AMPH-induced DA efflux. The experiments of S.A. #1-2 will be performed in isolated Drosophila brains, a model our laboratory developed to study AMPH actions ex vivo. Furthermore, using adult Drosophila, we will be able to translate the molecular discoveries of S.A. #1-2 to specific AMPH behavioral phenotypes. Thus, S.A. #3 focuses on behavior. S.A. #3. To define how F. nucleatum enhances AMPH behaviors.

Narrative The dopamine transporter and related family members are targets for medications, and secondary targets for psychostimulants such as amphetamine. In this proposal, we will determine how imbalances in the gut microbiome support the pathogenesis of amphetamine abuse and the role played by the dopamine transporter.