Properties of Ca²⁺‐dependent exocytosis in cultured astrocytes

Abstract

Astrocytes, a subtype of glial cells, have numerous characteristics that were previously considered exclusive for neurons. One of these characteristics is a cytosolic [Ca2+] oscillation that controls the release of the chemical transmitter glutamate and atrial natriuretic peptide. These chemical messengers appear to be released from astrocytes via Ca(2+)-dependent exocytosis. In the present study, patch-clamp membrane capacitance measurements were used to monitor changes in the membrane area of a single astrocyte, while the photolysis of caged calcium compounds by a UV flash was used to elicit steps in [Ca2+]i to determine the exocytotic properties of astrocytes. Experiments show that astrocytes exhibit Ca(2+)-dependent increases in membrane capacitance, with an apparent Kd value of approximately 20 microM [Ca2+]i. The delay between the flash delivery and the peak rate in membrane capacitance increase is in the range of tens to hundreds of milliseconds. The pretreatment of astrocytes by the tetanus neurotoxin, which specifically cleaves the neuronal/neuroendocrine type of SNARE protein synaptobrevin, abolished flash-induced membrane capacitance increases, suggesting that Ca(2+)-dependent membrane capacitance changes involve tetanus neurotoxin-sensitive SNARE-mediated vesicular exocytosis. Immunocytochemical experiments show distinct populations of vesicles containing glutamate and atrial natriuretic peptide in astrocytes. We conclude that the recorded Ca(2+)-dependent changes in membrane capacitance represent regulated exocytosis from multiple types of vesicles, about 100 times slower than the exocytotic response in neurons.

Synaptotagmin IV regulates glial glutamate release

“…If these receptors are localized adjacent to release sites they could provide locally elevated calcium to activate a low-affinity Ca 2ϩ sensor. Although the K d for glutamate release is not known for astrocytes, the K d for exocytosis from these cells is Ϸ20 M (19). Thus, it is entirely feasible that the slow integrated release of transmitter detected with our assay is due to the summation of temporally asynchronous release events that individually are due to Ca 2ϩ released through an inositol trisphosphate receptor locally associated with the release machinery to provide the Ca 2ϩ elevation necessary to activate a low-affinity Ca 2ϩ sensor.…”

“…Although more than one mechanism recruited under different physiological or pathological conditions likely mediates gliotransmitter release, recent evidence supports an exocytosismediated pathway for glutamate release from astrocytes (18). This evidence includes the findings that vesicular glutamate transporters are expressed within astrocytes in situ, that perturbation of N-ethylmaleimide-sensitive factor attachment protein receptor complex formation prevents astrocyte glutamate release, and that whole-cell capacitance measurements demonstrate that receptor-induced Ca 2ϩ elevations lead to a clostridial toxin-sensitive fusion of vesicles with the plasma membrane, which is coincident with the release of glutamate (18,19). Although this recent evidence supports a vesicle-mediated mechanism of Ca 2ϩ -evoked glutamate release from astrocytes, the protein target of action for elevated internal Ca 2ϩ is undefined.…”

Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy

“…9 F and G). We also estimated the [Ca 2ϩ ] i changes evoked by ADP␤S and thrombin with ratiometeric fura-2 fluorescence imaging, and estimate that the peak [Ca 2ϩ ] i increases to Ϸ0.3-0.5 M for both ADP␤S and thrombin, i.e., to levels expected to affect transmitter release (11,12,30). In subsequent experiments we used ADP␤S (100 M) and thrombin (1-10 units/ml) to evoke astrocyte [Ca 2ϩ ] i transients in astrocytes.…”

“…Astrocyte [Ca 2ϩ ] i transients are known to be triggered by neuronal activity (8) and are known to occur independently of neuronal activity because of intracellular Ca 2ϩ release (9,10). [Ca 2ϩ ] i increases, between a few nanomolar (11) and tens of micromolar (12) triggered by flash photolysis of caged Ca 2ϩ or pharmacological means (13), trigger release of transmitters from astrocytes into the extracellular space, which may then activate receptors on other nearby cells, including neurons. For instance, recent experiments suggest that [Ca 2ϩ ] i increases within single astrocytes can release glutamate, which activates extrasynaptic NMDA receptors on neurons (14,15), leading to synchrony (14).…”

Two Forms of Astrocyte Calcium Excitability Have Distinct Effects on NMDA Receptor-Mediated Slow Inward Currents in Pyramidal Neurons

“…Moreover, it is now well established that astrocyte [Ca 2ϩ ] i increases trigger release of transmitters into the extracellular space. The evidence in favor of this is derived from a variety of approaches, including biochemical, amperometric, and capacitance methods, as well as single-vesicle imaging (Araque et al, 2000;Krzan et al, 2003;Bezzi et al, 2004;Evanko et al, 2004;Kreft et al, 2004;Zhang et al, 2004a,b;Bowser and Khakh, 2007a;Jaiswal et al, 2007). One of the transmitters known to be released from astrocytes is glutamate.…”