EAAT3 endocytosis activated by psychostimulants in diverse populations of neurons

Abstract

The primary mechanism of action of psychostimulants, including amphetamine (AMPH), has largely been attributed generally to an increase in extracellular dopamine. Dopamine potentiation has been ascribed to several mechanisms including endocytosis of the dopamine transporter, DAT. We have found that this cascade involves entry of AMPH into the cell through DAT, stimulation of an intracellular GPCR for trace amines, TAAR1, and activation of the small GTPase, RhoA. Changes in extracellular glutamate concentrations have also been reported in response to AMPH and we have found that the neuronal glutamate transporter, EAAT3, also undergoes endocytosis via this cascade in dopamine neuron cultures, in acute brain slices and in vivo. EAAT3 is broadly expressed in neuronal populations throughout the brain, suggesting additional actions of AMPH on glutamatergic signaling in cells other than dopamine neurons. AMPH is a transported inhibitor of DAT as well as the norepinephrine transporter, NET, so we explored AMPH‐induced EAAT3 trafficking in NET(+) neurons. By total internal reflection fluorescence (TIRF) microscopy we found that AMPH can cause endocytosis of EAAT3 as well as NET. We developed NET(+) primary cultures derived from E15 murine locus coeruleus. Catecholamine neurotransmitters enter these cells through a desipramine‐sensitive pathway, indicating that this transport is mediated by NET. These cultures also exhibit sodium‐dependent and dihydrokainate‐insensitive tritiated‐glutamate uptake that is mediated by EAAT3. Pretreatment with AMPH decreases both NET and EAAT3 transport capacity in these cultures. Furthermore, through the use of a RhoA inhibitor, we determined that this loss depends on activation of RhoA, as seen previously for AMPH‐mediated internalization of DAT and EAAT3 in midbrain dopamine neuronal cultures. These data indicate that TAAR1 agonists, such as AMPH and dopamine, can affect glutamate signaling through internalization of EAAT3 in noradrenergic as well as dopaminergic neurons.Support or Funding InformationThis work was supported by Intramural Research at the NIH, NIMH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.