Ca²⁺ Entry is Required for Mechanical Stimulation-induced ATP Release from Astrocyte

Abstract

Astrocytes and neurons are inseparable partners in the brain. Neurotransmitters released from neurons activate corresponding G protein-coupled receptors (GPCR) expressed in astrocytes, resulting in release of gliotransmitters such as glutamate, D-serine, and ATP. These gliotransmitters in turn influence neuronal excitability and synaptic activities. Among these gliotransmitters, ATP regulates the level of network excitability and is critically involved in sleep homeostasis and astrocytic Ca2+ oscillations. ATP is known to be released from astrocytes by Ca2+-dependent manner. However, the precise source of Ca2+, whether it is Ca2+ entry from outside of cell or from the intracellular store, is still not clear yet. Here, we performed sniffer patch to detect ATP release from astrocyte by using various stimulation. We found that ATP was not released from astrocyte when Ca2+ was released from intracellular stores by activation of Gαq-coupled GPCR including PAR1, P2YR, and B2R. More importantly, mechanical stimulation (MS)-induced ATP release from astrocyte was eliminated when external Ca2+ was omitted. Our results suggest that Ca2+ entry, but not release from intracellular Ca2+ store, is critical for MS-induced ATP release from astrocyte.

Stretch‐induced Ca2+ independent ATP release in hippocampal astrocytes

“…Although we did not record quantal ATP spikes in cultured astrocytes (but see Lee et al . ) similar to those proposed by Lalo et al . (), it is possible that non‐quantal and small‐vesicle quantal ATP release coexists in astrocytes in brain slices or in vivo .…”

Stage-Specific Transcription Factors Drive Astrogliogenesis by Remodeling Gene Regulatory Landscapes

“…eA and lA were incubated with 10 μM OGB1, diluted in culture medium, for 45 min at 37°C in a humidified atmosphere. After the incubation time, cells were washed with warm culture media and analyzed within 2 hr ( Lee et al., 2015 ). Cells were placed in a recording chamber mounted on a fluorescent microscope (Zeiss Axio Imager A2) and superfused with artificial cerebrospinal fluid (ACSF) (in mM): NaCl, 125; KCl, 2.5; NaHCO 3 , 25; NaH2PO 4 , 1.25; CaCl2, 2; MgCl 2 , 1 and glucose, 25, saturated with 5% CO 2 and 95% O 2 (pH 7.4, 30-32°C).…”

“…Cells were placed in a recording chamber mounted on a fluorescent microscope (Zeiss Axio Imager A2) and superfused with artificial cerebrospinal fluid (ACSF) (in mM): NaCl, 125; KCl, 2.5; NaHCO 3 , 25; NaH2PO 4 , 1.25; CaCl2, 2; MgCl 2 , 1 and glucose, 25, saturated with 5% CO 2 and 95% O 2 (pH 7.4, 30-32°C). Cells were visualized with a 40x (0.7 NA) objective and movies (15 s) were acquired with a frequency of 8.9 Hz with a Hamamatsu Orca-0.3G camera and HCImage software ( Lee et al., 2015 ). During acquisition, we tested the generation of Ca 2+ waves in astrocytes using mechanical stimuli with a glass pipette which was placed right above the membrane of cultured astrocyte (resistance of 5-10 MΩ, which corresponds to a tip opening of 1-2 mm) ( Lee et al., 2015 ).…”

“…Cells were visualized with a 40x (0.7 NA) objective and movies (15 s) were acquired with a frequency of 8.9 Hz with a Hamamatsu Orca-0.3G camera and HCImage software ( Lee et al., 2015 ). During acquisition, we tested the generation of Ca 2+ waves in astrocytes using mechanical stimuli with a glass pipette which was placed right above the membrane of cultured astrocyte (resistance of 5-10 MΩ, which corresponds to a tip opening of 1-2 mm) ( Lee et al., 2015 ). For quantification, we defined regions of interest (ROIs) in cells surrounding the site of mechanical stimulus.…”

Activity-triggered tetrapartite neuron–glial interactions following peripheral injury

“…What chemical signals do microglia pick up as a sign of injury? Among a handful of candidates, purines, as neurotransmitters, appear on the top of the list [15, 24-28*], although ATP could also be derived from other sources such as astrocytes [29]. Microglia-neuronal contact alters with changes in neuronal input.…”