Synaptic Transmission on Speed

Science Signaling  29 Jul 2014:
Vol. 7, Issue 336, pp. ec200
DOI: 10.1126/scisignal.2005738

The addictive drug amphetamine is a psychostimulant and is associated with increased extracellular concentrations of the neurotransmitters dopamine and glutamate. Glutamate is cleared by excitatory amino acid transporters (EAATs) on the surface of neurons and astrocytes to limit excitatory neurotransmission. Underhill et al. found that amphetamine uptake into dopamine neurons triggered the internalization of EAAT3 from the neuron surface, reducing glutamate clearance. In cultures of primary mouse midbrain neurons, or in human embryonic kidney 293 (HEK 293) cells expressing the dopamine transporter (DAT) and EAAT1, EAAT2, or EAAT3, amphetamine decreased the uptake of tritium-labeled glutamate selectively through EAAT3. Amphetamine decreased the surface abundance of enhanced green fluorescent protein (eGFP)–tagged EAAT3, but not EAAT2, in HEK 293 cells coexpressing DAT. Using a nonpermeable biotin labeling method, lysates from adult mouse midbrain slices bathed in artificial cerebrospinal fluid containing amphetamine showed decreased surface abundance of EAAT3 compared with that of lysates from slices bathed in fluid without amphetamine. Cocaine, a DAT antagonist, prevented the amphetamine-induced decrease in surface EAAT3. A pH-sensitive, fluorescently tagged streptavidin showed that biotin-tagged eGFP-EAAT3 was internalized into endocytic vesicles in HEK 293 cells cultured with amphetamine. The guanosine triphosphatases dynamin and RhoA mediate a mechanism of endocytosis. Dynasore (a dynamin inhibitor), C3 exotoxin (a RhoA inhibitor), or expression of a dominant-negative RhoA mutant prevented the amphetamine-induced reduction in glutamate uptake in HEK 293 cells coexpressing EAAT3 and DAT. Expressing chimeras of EAAT2 that contained C-terminal fragments of EAAT3 in HEK 293 cells revealed that the amphetamine-dependent internalization of EAAT3 was mediated by a short motif in its cytoplasmic tail. In HEK 293 cells coexpressing EAAT3 and DAT, or in mouse brain slices, addition of a cell-permeable peptide containing this motif competed with endogenous EAAT3 and prevented the suppression of glutamate uptake and the increased glutamatergic synaptic currents in dopamine neurons that was otherwise induced by amphetamine. Thus, amphetamine increases excitatory neurotransmission by reducing the clearance of glutamate through EAAT3 in dopamine neurons.

S. M. Underhill, D. S. Wheeler, M. Li, S. D. Watts, S. L. Ingram, S. G. Amara, Amphetamine modulates excitatory neurotransmission through endocytosis of the glutamate transporter EAAT3 in dopamine neurons. Neuron 83, 404–416 (2014). [PubMed]