Research ArticleCalcium signaling

CRAC channels regulate astrocyte Ca2+ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission

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Science Signaling  21 May 2019:
Vol. 12, Issue 582, eaaw5450
DOI: 10.1126/scisignal.aaw5450
  • Fig. 1 Hippocampal astrocytes exhibit SOCE mediated by Orai1 and STIM1.

    (A) Depletion of ER Ca2+ stores with TG (1 μM) in a Ca2+-free Ringer’s solution evoked store release and subsequent SOCE when extracellular Ca2+ (2 mM) was added back. SOCE was blocked by 2 μM LaCl3 and after preincubation with BTP2 (1 μM for 2 hours). The right graph shows summary of the rate of Ca2+ influx in control and BTP2-treated cells. Summary data are means ± SEM of n = 35 to 36 cells for each group from three independent experiments. Ca2+ influx rates were calculated by measuring the initial slope of Ca2+ entry over 18 s after readdition of 2 mM Ca2+ solution (as shown by the dotted line for the control condition). (B) SOCE is abolished in cultured hippocampal astrocytes from mice with brain-specific Orai1 KO. Ca2+ influx rates were attenuated after readdition of external Ca2+. The right graph shows the summary of the rate of Ca2+ influx in WT (Orai1fl/+ and Orai1fl/fl), Orai1 HET (Orai1fl/+ nestin-Cre and Orai1fl/−), and Orai1 KO (Orai1fl/fl nestin-Cre and Orai1fl/− nestin-Cre) cells. Summary data are means ± SEM of n = 39 to 56 cells for each group from four to six independent experiments. (C) SOCE was abolished in cultured astrocytes from STIM1 KO mice (STIM1fl/fl nestin-Cre). Summary data (right graph) are means ± SEM, n = 25 to 30 cells for each group from three to four independent experiments. (D) SOCE was abolished in cultured astrocytes from astrocyte-specific Orai1 KO mice (Orai1fl/fl GFAP-Cre). Summary data are means ± SEM of n = 34 to 40 cells for each group from three independent experiments. **P < 0.01, ***P < 0.001 by analysis of variance (ANOVA) followed by Tukey test for comparison of multiple groups (B) or by unpaired t test for comparison of two groups (A, C, and D).

  • Fig. 2 Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes.

    (A) Cultured hippocampal astrocytes were treated with ATP (100 μM) in a Ca2+-free Ringer’s solution to deplete internal stores. Readdition of 2 mM extracellular Ca2+ elicited SOCE that was significantly decreased in Orai1 KO (Orai1fl/fl nestin-Cre and Orai1fl/fl GFAP-Cre) cells, as measured by the rate of Ca2+ influx. Summary data are means ± SEM of n = 22 to 26 cells for each group from three to four independent experiments. (B) Stimulation of P2Y receptors with UTP (50 μM) activated store release in Ca2+-free solution and subsequent sustained SOCE in 2 mM Ca2+ solution. SOCE was significantly attenuated in Orai1 KO (Orai1fl/fl GFAP-Cre) and STIM1 KO (STIM1fl/fl nestin-Cre) astrocytes. Summary data are means ± SEM of n = 25 to 57 cells for each group cells from three to six independent experiments. (C) Stimulation of PARs with thrombin (1 U/ml) activated store release in Ca2+-free solution followed by SOCE in 2 mM Ca2+ solution. SOCE was significantly attenuated in Orai1 KO (Orai1fl/fl GFAP-Cre) and STIM1 KO (STIM1fl/fl nestin-Cre) astrocytes. Summary data are means ± SEM of n = 24 to 53 cells for each group from three to six independent experiments. ***P < 0.001 by ANOVA followed by Tukey test for comparison of multiple groups (B and C) or by unpaired t test for comparison of two groups (A).

  • Fig. 3 Orai1 channels stimulate vesicular exocytosis after store depletion.

    (A) Fluorescence changes during a single vesicle fusion event monitored with spH. Images were captured every 200 ms, and the time of appearance of the fusion event was set to 0. Scale bar, 1 μm. (B) Location of spH events (shown in blue dots) are mapped onto the footprint of a TG-stimulated WT (Orai1fl/+) astrocyte. Scale bar, 20 μm. (C) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. Stimulation with TG evoked an increase in the rate of exocytosis. (D) Location of the spH events (blue dots) mapped onto the footprint of a TG-stimulated Orai1 KO (Orai1fl/fl GFAP-Cre) astrocyte. Scale bar, 20 μm. (E) Histogram of the number of spH fusion events measured each second. The right plot shows the integral of these events over the time course of the experiment. (F) Summary of the average exocytosis rate during a 2-min unstimulated baseline for each of the indicated conditions. (G) Summary of the average exocytosis rate for each of the indicated conditions. The average exocytosis rate during TG (1 μM) treatment was calculated from the maximum slope of the cumulative events plot over a 200-s window. TG-evoked spH exocytosis was significantly suppressed in Orai1 KO cells, by preincubation with BAPTA–AM (acetoxy methyl ester) (5 μM) or by coexpressing the light chain of tetanus toxin (TeTx) in astrocytes. WT, n = 21 cells; Orai1 KO, n = 17 cells; BAPTA, n = 7 cells; TeTx, n = 5 cells. Bar graphs show means ± SEM. *P < 0.05, **P < 0.01 by ANOVA followed by Tukey test. ns, not significant.

  • Fig. 4 Exocytosis evoked by UTP and thrombin is abrogated in Orai1 KO astrocytes.

    (A) Location of spH events (blue dots) mapped onto the footprint of a WT (Orai1fl/+) astrocyte stimulated with 50 μM UTP (left image). Histogram of the number of spH fusion events measured each second (right plot). UTP was administered after a 2-min baseline. (B) Left: Location of spH events (blue dots) mapped onto the footprint of a WT (Orai1fl/+) astrocyte stimulated with thrombin (1 U/ml). Right: Histogram of the number of spH fusion events measured each second. Thrombin was administered after a 2-min baseline. (C and D) Location of spH events mapped onto the footprint of an Orai1 KO (Orai1fl/fl GFAP-Cre) astrocyte stimulated with 50 μM UTP (C) or thrombin (1 U/ml) (D). Histogram of the number of spH fusion events measured each second (right plot). (E) Cumulative event plots for WT, Orai1 KO, Ca2+-free, and TeTx-expressing astrocytes stimulated by UTP. (F) The average rate of UTP-evoked exocytosis per 1000 μm2 was significantly suppressed in Orai1 KO cells, in Ca2+-free solution, or by TeTx. WT, n = 19 cells; KO, n = 12 cells; Ca2+-free, n = 10 cells; TeTx, n = 7 cells. (G) Cumulative event plots for WT, Orai1 KO, Ca2+-free, and TeTx-expressing astrocytes stimulated by thrombin. (H) Average rate of thrombin-evoked exocytosis per 1000 μm2 in the indicated conditions. WT, n = 18 cells; KO, n = 17 cells; Ca2+-free, n = 7 cells; TeTx, n = 9 cells. Scale bars, 20 μm. Bar graphs show means ± SEM. *P < 0.05, **P < 0.01 by ANOVA followed by Tukey test.

  • Fig. 5 Agonist-evoked ATP secretion is abrogated in Orai1 KO astrocytes.

    (A) SOCE stimulates ATP secretion from cultured astrocytes. ATP levels were measured using a luciferin-luciferase luminescence assay from the supernatant of multiwell plates after 10 min of stimulation. TG-mediated ATP secretion depended on external Ca2+ and was suppressed in Orai1 KO astrocytes and WT astrocytes after preincubation with CRAC channel inhibitor BTP2 (1 μM for 2 hours). n = 9 to 23 wells for each group from three to five independent cultures. (B) Thrombin stimulated ATP secretion from cultured WT astrocytes but not from Orai1 KO astrocytes. n = 10 to 16 wells for each group from three to four independent cultures. Bar graphs show means ± SEM. *P < 0.05 by ANOVA followed by Tukey test.

  • Fig. 6 Orai1 channels generate GPCR-mediated Ca2+fluctuations in astrocyte processes in situ.

    (A) Illustration of the experimental approach. GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaABC1D promoter. After 2 to 3 weeks, to allow for expression, Ca2+ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM. (B and C) Images of GCaMP6f-expressing WT (Orai1fl/fl) (B) or Orai1 KO (Orai1fl/fl GFAP-Cre) (C) astrocytes. Each image is the maximum intensity projection of the time series (540 s). Scale bar, 20 μm. Traces on the right show representative Ca2+ fluctuations measured in individual ROIs from the soma, proximal processes, and distal processes. Thrombin (10 U/ml) was used to activate Gq protein–coupled PARs on astrocytes and evoke Ca2+ signaling. Movies of the Ca2+ signals in these examples are shown in movies S3 and S4. (D and E) Summary of the Ca2+ oscillation frequency (D) and amplitude (E) at baseline and after administration of thrombin in WT (Orai1fl/fl, black bars) and Orai1 KO (Orai1fl/fl GFAP-Cre, orange bars) astrocytes. (WT, n = 11 cells from five mice; Orai1 KO, n = 8 cells from four mice). Statistical analysis was done using paired t test. Prox, proximal processes; Dist, distal processes. (F and G) Cumulative probability plots of the amplitudes of each Ca2+ oscillation in each region of interest (ROI) measured in the proximal (F) and distal (G) processes. (H and I) Comparison of WT and Orai1 KO Ca2+ oscillations, at baseline and after thrombin application. Loss of Orai1 significantly reduced the frequency (H) and amplitude (I) of the Ca2+ fluctuations in the proximal and distal processes. Statistical analysis was done using unpaired t test. Bar graphs show means ± SEM. *P < 0.05, **P < 0.01.

  • Fig. 7 Astrocyte Orai1 channels regulate GABAergic input to CA1 pyramidal cells.

    (A) Administration of thrombin evokes a burst of spontaneous IPSCs on Orai1fl/fl (WT) CA1 pyramidal neurons. Patch-clamp slice recordings were performed from CA1 pyramidal neurons held at −70 mV. (B) sIPSC traces from the experiment in (A) shown on an expanded timescale. (C) Summary of sIPSC frequency and amplitude in CA1 neurons from WT slices before and after application of thrombin. Thrombin evokes an increase in sIPSC frequency with no change in overall amplitude in WT slices (*P = 0.02 by paired t test, n = 8 cells). (D) Amplitude distribution of the sIPSC events in WT slices. (E) Thrombin does not alter the frequency or amplitude of mIPSCs in WT slices. mIPSCs were isolated in the presence of 1 μM TTX (n = 8 cells). (F) The thrombin-induced sIPSC response in CA1 neurons is abolished in Orai1fl/fl GFAP-Cre slices. (G) sIPSC traces from the experiment in (F) shown on an expanded timescale. (H) Summary of sIPSC frequency and amplitude in Orai1fl/fl GFAP-Cre slices before and after application of thrombin (n = 6 cells). (I) Amplitude distribution of the sIPSC events in Orai1 KO slices. (J) The broad-spectrum ATP receptor inhibitor PPADS (30 μM) abolishes the thrombin-mediated increase in frequency of sIPSCs in WT slices (n = 4 cells). Bar graphs show means ± SEM. *P < 0.05 by paired t test.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/582/eaaw5450/DC1

    Fig. S1. Astrocytes exhibit SOCE mediated by Orai1 and STIM1.

    Fig. S2. Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes.

    Fig. S3. Kinetics of individual spH fusion events in WT astrocytes.

    Fig. S4. spH-monitored vesicular exocytosis in AWESAM stellate astrocyte cultures.

    Fig. S5. Local Ca2+ signals contribute to CRAC channel–mediated vesicular exocytosis.

    Fig. S6. Exocytosis as monitored by FM1-43 is impaired in Orai1 KO astrocytes.

    Fig. S7. Ca2+ fluctuations in astrocyte processes in situ.

    Movie S1. spH-monitored exocytotic events in a WT astrocyte.

    Movie S2. spH-monitored exocytotic events in an Orai1 KO (Orai1fl/fl GFAP-Cre) astrocyte.

    Movie S3. GCaMP6f signals monitored by 2PLSM in an astrocyte from a WT hippocampal slice.

    Movie S4. GCaMP6f signals in an astrocyte from an Orai1 KO (Orai1fl/fl GFAP-Cre) hippocampal slice.

    References (75, 76)

  • The PDF file includes:

    • Fig. S1. Astrocytes exhibit SOCE mediated by Orai1 and STIM1.
    • Fig. S2. Stimulation of purinergic and PAR GPCRs activates SOCE in hippocampal astrocytes.
    • Fig. S3. Kinetics of individual spH fusion events in WT astrocytes.
    • Fig. S4. spH-monitored vesicular exocytosis in AWESAM stellate astrocyte cultures.
    • Fig. S5. Local Ca2+ signals contribute to CRAC channel–mediated vesicular exocytosis.
    • Fig. S6. Exocytosis as monitored by FM1-43 is impaired in Orai1 KO astrocytes.
    • Fig. S7. Ca2+ fluctuations in astrocyte processes in situ.
    • Legends for movies S1 to S4
    • References (75, 76)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). spH-monitored exocytotic events in a WT astrocyte.
    • Movie S2 (.avi format). spH-monitored exocytotic events in an Orai1 KO (Orai1fl/fl GFAP-Cre) astrocyte.
    • Movie S3 (.avi format). GCaMP6f signals monitored by 2PLSM in an astrocyte from a WT hippocampal slice.
    • Movie S4 (.avi format). GCaMP6f signals in an astrocyte from an Orai1 KO (Orai1fl/fl GFAP-Cre) hippocampal slice.

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