Blunted Amphetamine-induced Reinforcing Behaviors and Transporter Downregulation in Knock-in Mice Carrying Alanine Mutations at Threonine-258 and Serine-259 of Norepinephrine Transporter.

ABSTRACT

Altered amine transporter function, phosphorylation, and association with interacting proteins are evident in animals with a history of psychostimulant exposure. Our previous studies have shown that the Thr258/Ser259 motif in the norepinephrine transporter (NET) is involved in amphetamine (AMPH)-mediated NET regulation and behavior. However, the neurobiological consequences of in vivo Thr258/Ser259-dependent NET regulation in an intact animal model are unclear. Therefore, we generated a viable construct-valid NET-Thr258Ala/Ser259Ala (NET-T258A/S259A) mouse model using CRISPR/Cas9 technology by replacing Thr258/Ser259 motif with Ala258/Ala259 motif. NET-T258A/S259A mice have a birth rate consistent with Mendelian inheritance ratios. Both male and female homozygous NET-T258A/S259A mice are viable, display normal growth and general health, and exhibit normal body weight (sex-dependent) and total activity in the open field similar to their wild-type (WT) littermates. NET-T258A/S259A mice showed reduced NET function in the prefrontal cortex (PFC) compared to WT mice while NET function in the nucleus accumbens (NAc) remained unchanged. Compared to respective WT counterparts, NET-T258A/S259A males but not females showed significantly reduced locomotor activation in response to acute AMPH administration and significantly reduced AMPH-induced conditioned place preference (CPP). When tested in the males only, acute AMPH administration inhibited NET function and surface expression in the WT NAc but not in the NET-T258A/S259A NAc while AMPH administration inhibited DAT function and surface expression in the NAc of both WT and NET-T258A/S259A mice. Collectively, our findings reveal that the mice carrying the T258A/S259A mutation in NET gene display brain region-specific differences in NET functional expression and blunted response to AMPH.