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Keywords:

  • astrocyte;
  • GLT-1;
  • EAAT2;
  • biolistics;
  • time-lapse;
  • activity

Abstract

Glutamate transporters (GluTs) maintain a low ambient level of glutamate in the central nervous system (CNS) and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here, we examined the subcellular distribution and dynamic remodeling of the predominant GluT GLT-1 (excitatory amino acid transporter 2, EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. Green fluorescent protein (GFP)-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse three-dimensional confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of GluTs in developing astrocyte membranes and their position relative to synapses. © 2011 Wiley Periodicals, Inc.