Mechanisms of transmitter release from astrocytes

The Journal of Physiology

2011

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Non-technical summary Astrocytes have been shown to release transmitters by vesicle fusion, in a manner similar to that of neuronal exocytosis. The details of this process in astrocytes are not well understood, so we used a fluorescently labelled vesicle protein, synapto-pHluorin (spH), to track how these fusions occurred. When astrocytes were mechanically stimulated we saw a slow burst of fusions, while other stimuli caused a relatively even sustained rate of fusion. We observed two distinct types of events, transient and full fusions, the proportion of which was stimulus dependent. Similarly, stability of the vesicle fusion pore with the plasma membrane varied with the stimulus. We describe the effects on fusion events resulting from expressing variants of exocytotic proteins, synaptotagmin 1 and SNAP25B. Studying the characteristics of astrocytic exocytosis will aid in the general understanding of this process and also events at the tripartite synapse, both in health and disease.Abstract Astrocytes can release various gliotransmitters in response to stimuli that cause increases in intracellular Ca 2+ levels; this secretion occurs via a regulated exocytosis pathway. Indeed, astrocytes express protein components of the vesicular secretory apparatus. However, the detailed temporal characteristics of vesicular fusions in astrocytes are not well understood. In order to start addressing this issue, we used total internal reflection fluorescence microscopy (TIRFM) to visualize vesicular fusion events in astrocytes expressing the fluorescent synaptobrevin 2 derivative, synapto-pHluorin. Although our cultured astrocytes from visual cortex express synaptosome-associated protein of 23 kDa (SNAP23), but not of 25 kDa (SNAP25), these glial cells exhibited a slow burst of exocytosis under mechanical stimulation; the expression of SNAP25B did not affect bursting behaviour. The relative amount of two distinct types of events observed, transient and full fusions, depended on the applied stimulus. Expression of exogenous synaptotagmin 1 (Syt1) in astrocytes endogenously expressing Syt4, led to a greater proportion of transient fusions when astrocytes were stimulated with bradykinin, a stimulus otherwise resulting in more full fusions. Additionally, we studied the stability of the transient fusion pore by measuring its dwell time, relation to vesicular size, flickering and decay slope; all of these characteristics were secretagogue dependent. The expression of SNAP25B or Syt1 had complex effects on transient fusion pore stability in a stimulus-specific manner. SNAP25B obliterated the appearance of flickers and reduced the dwell time when astrocytes were mechanically stimulated, while astrocytes expressing SNAP25B and stimulated with bradykinin had a reduction in decay slope. Syt1 reduced the dwell time when astrocytes were stimulated either mechanically or with bradykinin. Our detailed study of temporal characteristics of astrocytic exocytosis will not only aid the general understanding of this process, but also the interpretat...

Section: Resultssupporting

confidence: 91%

“…Astrocytes can release gliotransmitters using various mechanisms, which can result in signalling to neurons (Ni et al . 2007; Malarkey & Parpura, 2008, 2009). Exocytosis is one of the prominent mechanisms underlying gliotransmitter release from astrocytes (Parpura et al .…”

Section: Introductionmentioning

confidence: 99%

Temporal characteristics of vesicular fusion in astrocytes: examination of synaptobrevin 2‐laden vesicles at single vesicle resolution

Malarkey

The Journal of Physiology

2011

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“…As alluded to above, glutamate release from astrocytes can occur by several different mechanisms [reviewed in (Malarkey and Parpura 2008)]. Future work will be necessary to determine whether the same glutamate release mechanisms that operate under physiological conditions operate during pathophysiological conditions or whether there are specific release mechanisms that operate under particular conditions.…”

Section: Discussionmentioning

confidence: 99%

Dual regulation of Ca²⁺‐dependent glutamate release from astrocytes: Vesicular glutamate transporters and cytosolic glutamate levels

2009

Glia

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Vesicular glutamate transporters (VGLUTs) are responsible for vesicular glutamate storage and exocytotic glutamate release in neurons and astrocytes. Here we selectively and efficiently over-expressed individual VGLUT proteins (VGLUT1, 2 or 3) in solitary astrocytes and studied their effects on mechanical stimulation-induced Ca 2+- dependent glutamate release. Neither VGLUT1 nor VGLUT2 over-expression changed the amount of glutamate release, while over-expression of VGLUT3 significantly enhanced Ca 2+- dependent glutamate release from astrocytes. None of the VGLUT over-expression affected mechanically-induced intracellular Ca2+ increase. Inhibition of glutamine synthetase activity by L-methionine sulfoximine in astrocytes, which leads to increased cytosolic glutamate concentration, greatly increased their mechanically-induced Ca 2+- dependent glutamate release, without affecting intracellular Ca2+ dynamics. Taken together these data indicate that both VGLUT3 and the cytosolic concentration of glutamate are key limiting factors in regulating the Ca 2+- dependent release of glutamate from astrocytes.

“…In particular, ER-originated Ca 2+ signals are critical for inducing exocytotic release of neurotransmitters (such as, for example, ATP, glutamate or D-serine) from astrocytes (see [79,80] for review and references). Inhibition of Ca 2+ accumulation into the ER by specific blockade of SERCA pumps with thapsigargin, that leads to exhaustion of the ER Ca 2+ content due to an unopposed leak through the endomembrane, effectively eliminated Ca 2+ -dependent release of glutamate from cultured astrocytes [71].…”

Section: Ca2+ Signalling In Astrogliamentioning

confidence: 99%

Homeostatic function of astrocytes: Ca2+ and Na+ signalling

Translational Neuroscience

Verkhratsky

2012

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111

The name astroglia unifies many non-excitable neural cells that act as primary homeostatic cells in the nervous system. Neuronal activity triggers multiple homeostatic responses of astroglia that include increase in metabolic activity and synthesis of neuronal preferred energy substrate lactate, clearance of neurotransmitters and buffering of extracellular K+ ions to name but a few. Many (if not all) of astroglial homeostatic responses are controlled by dynamic changes in the cytoplasmic concentration of two cations, Ca2+ and Na+. Intracellular concentration of these ions is tightly controlled by several transporters and can be rapidly affected by the activation of respective fluxes through ionic channels or ion exchangers. Here, we provide a comprehensive review of astroglial Ca2+ and Na+ signalling.