Physiological requirement for the glutamate transporter dEAAT1 at the adult Drosophila neuromuscular junction

Abstract

L-glutamate is the major excitatory neurotransmitter in the mammalian brain. Specific proteins, the Na+/K+-dependent high affinity excitatory amino acid transporters (EAATs), are involved in the extracellular clearance and recycling of this amino acid. Type I synapses of the Drosophila neuromuscular junction (NMJ) similarly use L-glutamate as an excitatory transmitter. However, the localization and function of the only high-affinity glutamate reuptake transporter in Drosophila, dEAAT1, at the NMJ was unknown. Using a specific antibody and transgenic strains, we observed that dEAAT1 is present at the adult, but surprisingly not at embryonic and larval NMJ, suggesting a physiological maturation of the junction during metamorphosis. We found that dEAAT1 is not localized in motor neurons but in glial extensions that closely follow motor axons to the adult NMJ. Inactivation of the dEAAT1 gene by RNA interference generated viable adult flies that were able to walk but were flight-defective. Electrophysiological recordings of the thoracic dorso-lateral NMJ were performed in adult dEAAT1-deficient flies. The lack of dEAAT1 prolonged the duration of the individual responses to motor nerve stimulation and this effect was progressively increased during physiological trains of stimulations. Therefore, glutamate reuptake by glial cells is required to ensure normal activity of the Drosophila NMJ, but only in adult flies.

DrosophilaGlial Glutamate Transporter Eaat1 Is Regulated by Fringe-Mediated Notch Signaling and Is Essential for Larval Locomotion

“…This mimics the effects we observed with acute pharmacological manipulations of glutamate transport, and resembles a published report on mutants of the AMPA-type glutamate receptor KaiRIA (also known as GluR-IID or brec) (Featherstone et al, 2005). Second, Eaat1 has been reported to be absent from neuromuscular junctions in larvae (Rival et al, 2006). Third, crawling was rescued with a Gal4 driver expressed exclusively in CNS glia.…”

“…Previous approaches using RNAi had uncovered no function for Eaat1 in larvae (Rival et al, 2004;Rival et al, 2006); this is likely due to insufficient knockdown at larval stages since here we found Eaat1 mutants have a severe crawling deficit. We narrowed the requirement for this glutamate transporter to a subpopulation of CNS glia and found that the crawling defect could be induced by conditional inactivation of Eaat1 after embryogenesis.…”

“…Previous research in adult flies has shown that reduction of Eaat1 in glia using RNA interference (RNAi) increases sensitivity to oxidative stress, and results in fewer dopaminergic neurons, degeneration of the brain neuropil, and decreased life span (Rival et al, 2004). However, our understanding of the importance of this glutamate transporter for CNS development and function remains incomplete since RNAi approaches to study Eaat1 function did not reveal phenotypes at embryonic and larval stages (Rival et al, 2004(Rival et al, , 2006.…”

Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment

“…The known lethality of null mutations in Eaat1 (Stacey et al, 2010) and pronounced phenotypes in flies with pan-glial-cell RNAi knockdown of Eaat1 (repo-GAL4) (Rival et al, 2004;Rival et al, 2006) led us to test alrm-GAL4> Eaat1 RNAi larvae for gross locomotor defects. We did not detect any changes in contraction rate (mean +/− SD contractions per minute = 40.2 +/− 20.0 for UAS Eaat1 RNAi controls, 46.6 +/− 10.5 for alrm-GAL4 controls, and 41.2 +/− 13.1 for alrm-GAL4> Eaat1 RNAi ; n = 7 in each group, n.s.).…”

“…Given this cellular phenotype, it is somewhat surprising that the larvae exhibit grossly normal locomotion and development. In contrast, flies carrying null mutations in Eaat1 do not survive beyond the first-instar larval stage (Stacey et al, 2010), and those with pan-glial-cell RNAi knockdown of Eaat1 (repo-GAL4) are viable but exhibit a shortened lifespan, neuropil degeneration, and locomotor phenotypes (namely, they can walk but cannot fly; Rival et al, 2004;Rival et al, 2006). This is unexpected given that Eaat1 immunolabeling is localized almost entirely to astrocytes; and alrm-driven RNAi knockdown eliminates any detectable labeling.…”