CLUSTERING AND DYNAMIC REMODELING OF A GLUTAMATE TRANSPORTER, GLT-1A, IN HIPPOCAMPAL ASTROCYTES.

A.M. Benediktsson¹, G.S. Marrs¹, J.C. Tu³, P.F. Worley³, J.D. Rothstein³, D.E. Bergles³, and M.E. Dailey^1,2.

¹Program in Neuroscience,  ²Dept. of Biological Sciences, Univ. of Iowa, Iowa City, IA, and  ³Dept. of Neuroscience, Johns Hopkins Univ. Med. Sch., Baltimore, MD, USA.

Astrocytic glutamate transporters are essential for normal CNS function.  Transporters are in high concentration in astrocytic membranes, but little is known about their spatial organization in situ.  Here we examined the subcellular distribution of one of the major glial transporters, GLT-1a (EAAT2), in GFAP+ astrocytes of P7 rat organotypic hippocampal slices cultured for 3-8d.  Astrocytes within these cultures retain their complex morphology and close relationships to neuronal synapses.  Immunostaining with anti-GLT-1 antibodies showed punctate staining patterns in hippocampal tissue slices.  To visualize the subcellular distribution of GLT-1a in individual astrocytes, we used particle-mediated gene transfer to express a functional GLT1a-GFP fusion protein.  Confocal fluorescent imaging and 3-D reconstruction revealed that GLT1a-GFP is found in distinct clusters or "hotspots" that line the edges of astrocyte processes and cell body.  Co-transfection with cytoplasmic DsRed2 confirms that GLT1a-GFP is targeted to the plasma membrane and that hotspots are true sites of GLT1a-GFP protein concentration.  Clusters are distributed non-uniformly along astrocyte processes, and are often found in fine lamellae or spine-like processes that have GLT-1a clusters at their tips.  Time-lapse confocal imaging in live tissue slices reveal that clusters can be remodeled dynamically:  individual clusters can move along the surface of growing processes, and spine-like structures containing GLT1a-GFP clusters can extend and retract, carrying along the cluster.  These studies suggest that the astrocyte glutamate transporter, GLT-1a, is organized into a pattern of clusters that can be dynamically remodeled on a time scale of minutes.

Supported by NS43468 (MED).