Astrocytic exocytosis vesicles and glutamate: A high-resolution immunofluorescence study
Article first published online: 6 AUG 2004
Copyright © 2004 Wiley-Liss, Inc.
Volume 49, Issue 1, pages 96–106, 1 January 2005
How to Cite
Anlauf, E. and Derouiche, A. (2005), Astrocytic exocytosis vesicles and glutamate: A high-resolution immunofluorescence study. Glia, 49: 96–106. doi: 10.1002/glia.20094
- Issue published online: 10 NOV 2004
- Article first published online: 6 AUG 2004
- Manuscript Accepted: 29 APR 2004
- Manuscript Received: 29 JAN 2004
- Deutsche Forschungsgemeinschaft (DFG). Grant Number: DFG DE 676/2-1
- deconvolution microscopy
Physiological evidence has demonstrated that cultured astrocytes can release glutamate via Ca2+-dependent mechanisms. Also, glutamate released from astrocytes in the hippocampal slice interferes with synaptic neurotransmission. Since these observations suggest vesicular glutamate release from astrocytes, the presence of glutamate-containing exocytosis vesicles was investigated. We applied immunofluorescence techniques combined with high-performance deconvolution microscopy, which yields a resolution of <200 nm and permits evaluation of double labeling in individual vesicles. Using a well-characterized anti-glutamate antiserum and parameters minimizing fixative-induced autofluorescence, glutamate-immunoreactive (ir) puncta were found all over the astrocyte but were conspicuously dense at the cell boundary and in filopodia. Images were very similar with antibodies against vesicular glutamate transporters (vGluT1 and vGluT2). Labeling for the exocytosis markers rab3, synaptophysin, or synaptobrevin was also punctate, particularly dense at the cell boundary, but disappearing toward the perinuclear region. Sections of the cell boundary were delineated by rab3 immunoreactivity. In double-labeled cells, vesicular colocalization of glutamate and any of the exocytosis markers was frequent in filopodia and at the cell boundary. Within the cell, single-labeled glutamate-ir vesicles prevailed; double-labeled vesicles were infrequently present. By resolving single vesicles, in cultured astrocytes we visualize glutamate-containing vesicles, vesicles displaying vGluT1 or vGluT2, and exocytosis vesicles displaying glutamate-ir. This may provide the morphological correlate of Ca2+-dependent glutamate release from astrocytes, possibly occurring at defined sections of the cell membrane and at filopodia. However, since vGluTs and exocytosis markers are classically restricted to nerve terminals in the CNS, glutamate release from astrocytes in the CNS remains to be studied. © 2004 Wiley-Liss, Inc.