Astrocytic exocytosis vesicles and glutamate: A high‐resolution immunofluorescence study

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.

[1]  P. Camilli,et al.  Molecular Mechanisms in Synaptic Vesicle Endocytosis and Recycling , 1996, Neuron.

[2]  M. Matteoli,et al.  A Regulated Secretory Pathway in Cultured Hippocampal Astrocytes* , 1999, The Journal of Biological Chemistry.

[3]  S. Jeftinija,et al.  Neuroligand‐Evoked Calcium‐Dependent Release of Excitatory Amino Acids from Cultured Astrocytes , 1996, Journal of neurochemistry.

[4]  F. Conti,et al.  Characterization of antisera to glutamate and aspartate. , 1988, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[5]  M. Frotscher,et al.  Peripheral astrocyte processes: Monitoring by selective immunostaining for the actin‐binding ERM proteins , 2001, Glia.

[6]  V. Subramaniam,et al.  A SNARE involved in protein transport through the Golgi apparatus , 1997, Nature.

[7]  Tullio Pozzan,et al.  Prostaglandins stimulate calcium-dependent glutamate release in astrocytes , 1998, Nature.

[8]  T. Basarsky,et al.  Expression of synaptobrevin II, cellubrevin and syntaxin but not SNAP‐25 in cultured astrocytes , 1995, FEBS letters.

[9]  B. Giros,et al.  The Existence of a Second Vesicular Glutamate Transporter Specifies Subpopulations of Glutamatergic Neurons , 2001, The Journal of Neuroscience.

[10]  R. Weinberg,et al.  Techniques to optimize post-embedding single and double staining for amino acid neurotransmitters. , 1992, Journal of Histochemistry and Cytochemistry.

[11]  B. Jena,et al.  Aquaporin 1 regulates GTP-induced rapid gating of water in secretory vesicles , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[12]  X. Xie,et al.  Structure of the 116-kDa polypeptide of the clathrin-coated vesicle/synaptic vesicle proton pump. , 1991, The Journal of biological chemistry.

[13]  A. Araque,et al.  SNARE Protein-Dependent Glutamate Release from Astrocytes , 2000, The Journal of Neuroscience.

[14]  J. Storm-Mathisen,et al.  Chapter 19: Ultrastructural immunocytochemical observations on the localization, metabolism and transport of glutamate in normal and ischemic brain tissue , 1992 .

[15]  M. Matteoli,et al.  Storage and Release of ATP from Astrocytes in Culture* , 2003, The Journal of Biological Chemistry.

[16]  A. Schousboe,et al.  Multiple compartments with different metabolic characteristics are involved in biosynthesis of intracellular and released glutamine and citrate in astrocytes , 2001, Glia.

[17]  T. Südhof,et al.  Rab3C is a synaptic vesicle protein that dissociates from synaptic vesicles after stimulation of exocytosis. , 1994, The Journal of biological chemistry.

[18]  S. Goldman,et al.  Astrocyte-mediated potentiation of inhibitory synaptic transmission , 1998, Nature Neuroscience.

[19]  F. Fujiyama,et al.  Immunocytochemical localization of candidates for vesicular glutamate transporters in the rat cerebral cortex , 2001, The Journal of comparative neurology.

[20]  O. Ottersen,et al.  Ultrastructural immunocytochemical studies as a means of distinguishing between transmitter and non-transmitter glutamate. , 1993, Biochemical Society transactions.

[21]  W. Volknandt,et al.  A plethora of presynaptic proteins associated with ATP‐storing organelles in cultured astrocytes , 1999, Glia.

[22]  Christian Rosenmund,et al.  Identification of Differentiation-Associated Brain-Specific Phosphate Transporter as a Second Vesicular Glutamate Transporter (VGLUT2) , 2001, The Journal of Neuroscience.

[23]  T. Südhof,et al.  Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein , 1993, Nature.

[24]  Christopher J. Lowe,et al.  Radical alterations in the roles of homeobox genes during echinoderm evolution , 1997, Nature.

[25]  K. McCarthy,et al.  Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue , 1980, The Journal of cell biology.

[26]  M. Frotscher,et al.  Astroglial processes around identified glutamatergic synapses contain glutamine synthetase: evidence for transmitter degradation , 1991, Brain Research.

[27]  Michael M. Halassa,et al.  Fusion-related Release of Glutamate from Astrocytes* , 2004, Journal of Biological Chemistry.

[28]  R. Weinberg,et al.  An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. , 1995, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[29]  C. Barnstable.  Monoclonal antibodies which recognize different cell types in the rat retina , 1980, Nature.

[30]  P. De Camilli,et al.  Association of Rab3A with synaptic vesicles at late stages of the secretory pathway , 1991, The Journal of cell biology.

[31]  A. Araque,et al.  Tripartite synapses: glia, the unacknowledged partner , 1999, Trends in Neurosciences.

[32]  Molecular organization of cerebellar glutamate synapses. , 1997, Progress in brain research.

[33]  D. Bruns,et al.  SNAREs are concentrated in cholesterol‐dependent clusters that define docking and fusion sites for exocytosis , 2001, The EMBO journal.

[34]  Thomas C. Südhof,et al.  The synaptic vesicle cycle: a cascade of protein–protein interactions , 1995, Nature.

[35]  Fang Liu,et al.  Glutamate-mediated astrocyte–neuron signalling , 1994, Nature.

[36]  J. Storm-Mathisen,et al.  Chapter 6 Molecular organization of cerebellar glutamate synapses , 1997 .

[37]  W. Huttner,et al.  Synaptic-like Microvesicles of Neuroendocrine Cells Originate from a Novel Compartment That Is Continuous with the Plasma Membrane and Devoid of Transferrin Receptor , 1997, The Journal of cell biology.

[38]  S. Jeftinija,et al.  Cultured astrocytes express proteins involved in vesicular glutamate release , 1997, Brain Research.