Fast Subplasma Membrane Ca²⁺Transients Control Exo-Endocytosis of Synaptic-Like Microvesicles in Astrocytes

Abstract

Astrocytes are the most abundant glial cell type in the brain. Although not apposite for long-range rapid electrical communication, astrocytes share with neurons the capacity of chemical signaling via Ca2+-dependent transmitter exocytosis. Despite this recent finding, little is known about the specific properties of regulated secretion and vesicle recycling in astrocytes. Important differences may exist with the neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca2+ from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We here take advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine epifluorescence and total internal reflection fluorescence imaging to investigate with unprecedented temporal and spatial resolution, the stimulus-secretion coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles (SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in astrocytes proceeds with a time course on the millisecond time scale (τexocytosis = 0.24 ± 0.017 s; τendocytosis = 0.26 ± 0.03 s) and (2) exocytosis is controlled by local Ca2+ microdomains. We identified submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli reaching beneath the plasma membrane and containing SLMVs at which fast (time-to-peak, ∼50 ms) Ca2+ events occurred in precise spatial-temporal correlation with exocytic fusion events. Overall, the above characteristics of transmitter exocytosis from astrocytes support a role of this process in fast synaptic modulation.

Calcium Triggers Exocytosis from Two Types of Organelles in a Single Astrocyte

“…In astrocytes, the types of vesicles used in Ca 2ϩ -regulated exocytosis are under debate (Bezzi et al, 2004;Li et al, 2008;Marchaland et al, 2008;Parpura and Zorec, 2010). Combining TIRFM with glutamate sniffer recording, we found that astrocytes efficiently released two types of Ca 2ϩ -dependent vesicles, small vesicles and lysosomes, following a single stimulation.…”

“…Using immunoelectron microscopy for vesicle glutamate transporter 1 (VGLUT1), Bezzi et al (2004) identified a type of small vesicle ϳ30 nm in diameter. These have characteristics similar to synaptic vesicles with expression of VGLUT1 and synaptobrevin 2 (Syb2)/vesicleassociated membrane protein 2 (Bezzi et al, 2004;Bowser and Khakh, 2007), and are exocytosed in a Ca 2ϩ -dependent manner (Bowser and Khakh, 2007;Marchaland et al, 2008). Cleavage of Syb2 and cellubrevin/vesicle-associated membrane protein 3 by Clostridium toxins greatly inhibits glutamate release from astrocytes (Bezzi et al, 1998;Araque et al, 2000;Pasti et al, 2001;Jourdain et al, 2007).…”

Astrocytes Impose Postburst Depression of Release Probability at Hippocampal Glutamate Synapses

“…The induction of these forms of synaptic plasticity appears to require more extensive synaptic activation than what is required for the induction of the PBD. An interesting possibility is that astrocytes can decode different patterns of synaptic activity into different modes of calcium signaling (Verkhratsky et al, 1998;Perea et al, 2009;Santello and Volterra, 2009) and into release of different constellations of gliotransmitters Bowser and Khakh, 2007;Zhang et al, 2007;Li et al, 2008;Marchaland et al, 2008). The type of calcium signaling as well as the critical gliotransmitters involved in the generation of PBD remains, however, to be determined.…”

Suppression of SNARE‐dependent exocytosis in retinal glial cells and its effect on ischemia‐induced neurodegeneration

“…Studies within the last decade have shown that glial cells can release transmitters (which are called gliotransmitters) in a calcium‐ and soluble N ‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE)‐dependent manner (Marchaland et al, 2008; Martineau, 2013; Montana, Malarkey, Verderio, Matteoli, & Parpura, 2006; Slezak et al, 2012). The physiologic relevance of this process is debated: it may contribute to glial volume regulation (Slezak et al, 2012) and modulate neuronal activity (Araque et al, 2014; Chen et al, 2012; Perez‐Alvarez, Navarrete, Covelo, Martin, & Araque, 2014; Takata et al, 2011).…”