Cultured astrocytes were prepared from P0-P3 of C57BL/6 mice. The cerebral cortex was dissected free of adherent meninges, minced and dissociated into single cell suspension by trituration through a Pasteur pipette. Dissociated cells were plated onto either 12 mm glass coverslips or six-well plates coated with 0.1 mg/ml poly d-lysine. Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 25 mM glucose, 10% heat-inactivated horse serum, 10% heat-inactivated fetal bovine serum, 2 mM glutamine and 1000 units/ml penicillin-streptomycin. After three days later, cells were vigorously washed with repeated pipetting using medium and the media was replaced to get rid of debris and other floating cell types.

For Ca²⁺ imaging, astrocytes with HEK293T cells transfected with P2X2-V343Q were incubated with 5 µM Fura-2 AM (mixed with 5 µl of 20% Pluronic acid) (Invitrogen, Grand Island, NY, USA) for 30 min and washed at room temperature, and subsequently transferred to a microscope stage for imaging. External solution contained (in mM): 150 NaCl, 10 Hepes, 3 KCl, 2 CaCl₂, 2 MgCl₂, 5.5 glucose, 20 Sucrose, pH adjusted to pH 7.3. Intensity images of 510 nm wavelength were taken at 340 nm and 380 nm excitation wavelengths using either iXon EMCCD (DV887 DCS-BV, ANDOR technology, UK). Two resulting images were used for ratio calculations in Axon Imaging Workbench version 6.2 (Indec System, CA, USA). P2X2-V343Q-mediated currents were recorded from HEK293T cells expressing P2X2-V343Q under voltage clamp (Vh=-70 mV) using Multiclamp 700B amplifier (Molecular Devices), acquired with pClamp 9.2. Recording pipettes were filled with (mM): 110 Cs-Gluconate, 30 CsCl, 0.5 CaCl₂, 10 HEPES, 4 Mg-ATP, 0.3 Na₃-GTP and 10 BAPTA (pH adjusted to 7.3 with CsOH). For simultaneous recording, Imaging Workbench was synchronized with pClamp 9.2.

To make mechanical stimulation glass pipette was placed right above the membrane of cultured astrocyte. With monitoring its resistance, pipette generally pressed cell membrane until the value of membrane resistance reaches to approximately as double. When the pressure by pipette evoked Fura2 signals, the position of pipette was returned to original place.

All data are given as mean±SEM; n denotes the number of individual cells assessed in sniffer patch-clamp studies. Statistical differences were determined by two-tailed unpaired Student's t test and considered significant at p<0.05.

We performed sniffer patch to detect ATP release from astrocyte. On the experimental date, HEK293T cells transfected with P2X2-V343Q, known as a non-selective cation permeable channel [22], and GFP (sensor cells) were mixed with cultured astrocytes, allowing sensor cells to be located nearby astrocytes. For monitoring of the astrocytic Ca²⁺ signal, Fura-2 intensity from the astrocyte was recorded from the beginning of the experiment. To make mechanical stimulation, glass pipette placed above a cultured single astrocyte and monitored pipette resistance to determine when the pipette touched the cell membrane. Pipette was slowly lowered until the value of resistance doubled. Once the Fura-2 signal started to change, the pipette was immediately returned to original height. Whole cell patch recording from HEK 293T cell expressing P2X2-V343Q nearby astrocyte was performed to sense the ATP release from astrocyte (Fig. 1A). Fig. 1B shows a representative Fura-2 intensity images of before and after mechanical stimulation on cultured astrocyte reflecting the increase of Ca²⁺ signals in cytoplasm of astrocyte evoked by mechanical stimulation.

MS-induced ATP release and Ca²⁺ signals were simultaneously monitored while performing whole-cell patch clamp recording from sensor cell nearby a single astrocyte and Fura-2 ratiometric imaging (Fig. 1Ca and 1Cb). At the end of each experiment, we bath-applied 100 µM ATP to measure the full extent of expression of biosensor, P2X2-V343Q. The amount of ATP release was expressed by measuring the peak amplitude of MS-induced ATP release and taking the percent of full activation. We found that mechanical stimulation elevates Ca²⁺ in astrocytes and triggers a release of ATP from astrocyte that was detected in sensor cell as an inward current (Fig. 1Ca). Some astrocytes showed Ca²⁺ oscillation which caused ATP release from astrocytes with a similar time course (Fig. 1Cb).

It has been recently shown that acutely dissociated astrocytes release ATP by Ca²⁺ elevation via metabotropic and ionotropic receptors or Ca²⁺-uncaging [18]. To increase astrocytic Ca²⁺, we applied various agonists of GqPCRs by pressure-mediated agonist application (puffing) onto cultured astrocyte. TFLLR, an agonist for Protease Activated Receptor 1 (PAR1), increased Ca²⁺ in cultured astrocyte (Fig. 2A). Contrary to previous report [18], TFLLR did not induce ATP release in our experimental system (Fig. 2B). We also found that bradykinin, an agonist for B2 receptor, and 2MeSADP, an agonist for P2Y receptor, did not induce ATP release from cultured astrocytes (Figs. 2C and 2D). Bath application of 100 µM ATP was used for the full activation of P2X2-V343Q channels expressed in the sensor cell again (Fig. 2B, 2C, and 2E). Fig. E shows summary bar graph of Ca²⁺ increase induced by GPCR agonists (% of full activation, TFLLR: 1.02±0.43, n=10 cells; Bradykinin: 0.38±0.33, n=8 cells; 2-(Methylthio) adenosine 5'-diphosphate trisodium salt hydrate (2MeSADP): 1.62±1.58, n=4 cells). This result indicates that, unlike MS-induced Ca²⁺ increase, GPCR-induced Ca²⁺ increase might not be sufficient to cause ATP release from cultured astrocyte.

Since Ca²⁺ increase by mechanical stimulation, but not by activation of GPCRs, causes ATP release, we hypothesized that the concentration of intracellular Ca²⁺ is different between mechanical stimulation and activation of GPCRs. To test our hypothesis, we measured the ratio of the Ca²⁺ transient by application of TFLLR and mechanical stimulation from Fura-2 loaded astrocytes (Fig. 3). The ratio of Ca²⁺ increase by mechanical stimulation was significantly greater than by application of TFLLR (ratio of Ca²⁺ response, peak: TFLLR, 0.14±0.03, M.S., 0.18±0.02, n=5 cells; area: TFLLR, 8.40±2.15, M.S., 16.09±1.83, n=6 cells; Fig. 3B), suggesting the ATP release from astrocyte might be regulated by concentration of intracellular Ca²⁺.

To determine the source of Ca²⁺ increase required for MS-induced ATP release from astrocyte, we tested whether MS-induced ATP release can be detected in the absence of extracellular Ca²⁺. Under normal condition, MS-induced Ca²⁺ increase and ATP release were evoked by two sequential mechanical stimulations (ratio of 2^nd/1^st, Ca²⁺, 0.72±0.06, n=3 cells; ATP, 0.70±0.05, n=3 cells) (Fig. 4A and B). However, when the extracellular Ca²⁺ was omitted, ATP release was dramatically reduced, but a small portion of Ca²⁺ increase was still detected in astrocyte (Fig. 4C). Because this residual Ca²⁺ increase might come from endoplasmic reticulum (ER) store, we used 1µM thapsigargin (TG) to deplete intracellular Ca²⁺ from ER in Ca²⁺ free extracellular bath solution and performed sniffer patch. Under this condition, mechanical stimulation did not induce Ca²⁺ elevation or ATP release (Fig. 4D). Fig. 4E shows summary of these experiments with bar graph (% of full activation, Ca²⁺, 55.97±9.83 and Ca²⁺ free, 9.83±4.92, n=6 cells; Ca²⁺, 63.92±5.94 and Ca²⁺ free + TG, 0.001±0.01, n=7 cells). These results strongly indicate that accumulated cytoplasmic Ca²⁺ is required for ATP release and external Ca²⁺ is critical source for initiating ATP release from astrocyte.