AAV9-TRE-Cre

Extracellular dopamine (DA) levels are limited by the presynaptic DA transporter (DAT), a major psychostimulant target. Using chemogenetics and conditional gene silencing, researchers show that activation of presynaptic Gq-coupled receptors (hM3Dq or mGlu5) drives rapid biphasic DAT membrane trafficking in isolated striatal slices, with region-specific differences between ventral and dorsal striata. DAT insertion requires D2 DA autoreceptors and intact retromers, while DAT retrieval requires PKC activation and Rit2. Ex vivo voltammetric studies revealed that DAT trafficking impacts DA clearance. Furthermore, dopaminergic mGlu5 silencing increases DAT surface expression and abolishes motor learning, which is rescued by DAT inhibition using a subthreshold CE-158 dose. Researchers find that presynaptic DAT trafficking is complex, multimodal, and region-specific, and identify for the first time a cell-autonomous mechanism controlling presynaptic DAT tone. Importantly, these findings are consistent with a role for regulated DAT trafficking in DA clearance and motor function.

Here, researchers tested whether DAergic mGlu5 is required for biphasic DAT transport. Bilateral AAV9-TRE-Cre injection into Pitx3IRES-tTA;mGlu5fl/fl mouse VTA significantly reduced midbrain mGlu5 mRNA and protein expression as compared with AAV9-TRE-enhanced GFP (eGFP)-injected controls (Figure 1A and B). DAergic mGlu5 silencing completely abolished DHPG-stimulated DAT insertion in the VS and DS (Figure 1C and D), consistent with the premise that mGlu5 is expressed presynaptically in DA terminals, where it stimulates DAT trafficking in a cellautonomous manner. The researchers further hypothesized that DAT surface tone in the striatum is a balance between DRD2-stimulated DAT insertion and mGlu5-mediated retrieval, and therefore predicted that mGlu5 silencing in DA neurons would result in enhanced basal DAT surface expression. To test this possibility, basal DAT surface levels were measured after conditional mGlu5 silencing. DAergic mGlu5 loss significantly increased baseline DAT surface levels in VS and DS compared with slices from control mice (Figure 1E), indicating that mGlu5-stimulated DAT retrieval significantly affects striatal DAT surface tone.

Figure 1. mGlu5-mediated DAT trafficking is mediated presynaptically and impacts basal DAT surface expression. (Kearney P J, et al., 2023)