Sonic hedgehog enhances calcium oscillations in hippocampal astrocytes

Abstract

Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute time scale, indicating a noncanonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a G_i coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP-degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of G_q/11-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, G_i mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.

Keywords: ATP; Sonic hedgehog (SHH); astrocyte; calcium imaging; calcium intracellular release.

Figure 1.

SHH and SAG increased Ca oscillation frequency in mouse hippocampal cultured cells. A, hippocampal cells loaded with a Ca indicator, Fura-2. Scale bar, 100 μm. B and C, addition of SHH (B, a; 500 pm as final concentration) or SAG (C, a; 5 μm) evoked Ca oscillations. Frequency of Ca events in each cell was calculated before (base) and after the agonist applications. B and C, b panels, cumulative histograms during the baseline period and during agonist application periods are shown. Agonist-induced Ca frequency increase was evaluated by subtracting the baseline Ca frequency from that after the drug application in each cell (ΔFrequency). Cumulative histograms of ΔFrequency of cells applied with concentrations of SHH (D) or SAG (E) together with that of DMSO-applied control cells (0.1% as final concentration, n = 820 cells from 7 cultures). Number of cells are: 5 nm SHH, n = 1688 cells from 16 cultures; 500 pm SHH, 510 cells from 5 cultures; 50 pm SHH, 603 cells from 5 cultures; 5 μm SAG. 1131 cells from 8 cultures; 1 μm SAG, 416 cells from 4 cultures; 500 nm SAG, 469 cells from 5 cultures; 100 nm SAG, 560 cells from 6 cultures; and 50 nm SAG, 428 cells from 4 cultures.

Figure 2.

Effects of cyclopamine and apyrase against the SAG-induced enhancement of Ca oscillation frequency in hippocampal primary culture. A, a, CPN (5 μm) was added to the bath, and SAG application followed. b, cumulative histograms of the Ca oscillation frequency during the baseline period (base), CPN and CPN with SAG (n = 768 cells from 6 cultures). c, ΔFrequency of the drug effects are displayed as cumulative histograms, together with that of a control cell group to which 0.1% DMSO was added in place of CPN, which is the same one shown in Fig. 1, D and E. B, a, apyrase (5 units/ml) suppressed the SAG-induced enhancement of Ca oscillation frequency. b, cumulative histograms of Ca oscillation frequency during the baseline period, after SAG application and after apyrase addition (n = 444 cells from 3 cultures). c, cumulative histograms of ΔFrequency. C, as a control, vehicle solution (HBS-TTX) was added in place of apyrase in B. Cumulative histograms of Ca event frequency (C, a) and ΔFrequency (C, b) are shown (n = 729 cells from 7 cultures).

Figure 3.

The SAG-induced Ca oscillation enhancement takes place in astrocytes. A, cell types showing Ca oscillations were characterized by immunohistochemistry. Time lapse Ca imaging of a 10-min baseline (HBS-TTX) and 10 min in SAG was performed in hippocampal cultures (A, a), which were then fixed and stained with anti-MAP2 (red) and anti-S100β (green) antibodies (A, b). Filled arrowheads indicate S100β-positive cells, and open arrowheads indicate MAP2-positive cells. Scale bar, 100 μm. B, a, Fura-2–loaded astrocyte culture. The enhancing effects of SHH (B, 500 pm, n = 465 cells from 5 cultures) or SAG (C, 5 μm, n = 1731 cells from 12 cultures) on Ca oscillation frequency were observed in the astrocyte culture. D, cumulative histograms of ΔFrequency of SHH and SAG together with a DMSO (0.1%) applied control cell group (n = 642 cells from 5 cultures).

Figure 4.

Ca release through IP3R is involved in the SAG-enhanced Ca oscillations. A, a, the SAG-induced Ca oscillation frequency enhancement was tested in Ca-free media, in which Ca-free HBS supplemented with EGTA (1 μm) was used as an extracellular medium throughout the recording period. b, cumulative histograms of Ca oscillation frequency with Ca-free medium during the baseline period and after the application of SAG (n = 431 cells from 5 cultures). c, a cumulative histogram of ΔFrequency(SAG − base) in Ca-free medium and ΔFrequency(DMSO − base) obtained from a control group with DMSO (0.1% in Ca-free medium) in place of SAG (n = 377 cells from 4 cultures). B–D, 2-APB (B, 50 μm, n = 417 cells from 5 cultures), an IP3R inhibitor, Tg (C, 100 nm, n = 416 cells from 5 cultures), an inhibitor to the endoplasmic reticulum Ca-ATPase, or dantrolene (D, 10 μm, n = 505 cells from 5 cultures), a ryanodine receptor antagonist, was applied and addition of SAG followed. B–D, b panels, cumulative histograms of Ca oscillation frequency during the baseline period, after antagonist application and after SAG addition. B–D, c panels, cumulative histograms of ΔFrequency together with those obtained from a control cell group with DMSO in place of the antagonists (n = 384 cells from 4 cultures).

Figure 5.

The SAG-induced enhancement of Ca oscillations requires Gi activation. A, astrocyte cultures were treated with 100 ng/ml of PTX for 24 h prior to Ca imaging. a, SAG increased the Ca oscillation frequency compared with baseline. b and c panels, cumulative histograms of Ca oscillation frequency during the baseline period and after SAG (A, b, n = 714 cells from 7 cultures) or control DMSO (A, c, n = 537 cells from 4 cultures) application. d, cumulative histograms of ΔFrequency together with those from the control cell group shown in Fig. 3D. B, cAMP imaging revealed an increase in [cAMP]_i). a, a cAMP indicator, Flamindo-2 (green), was expressed in astrocytes and Fura-2 (gray) was loaded for simultaneous measurement. Scale bar, 100 μm. b, time course of [cAMP]_i and [Ca]_i were monitored before and after SAG application. c, averaged time course of Flamindo-2 signal from astrocytes to which SAG (n = 97 cells from 8 experiments) or vehicle (0.1% DMSO; n = 58 cells from 4 experiments) was applied.

Figure 6.

Blockers of ATP release channels altered the SAG-induced Ca oscillation frequency enhancement. A, a, CBX (A, 100 μm, n = 231 cells from 4 cultures), a connexin hemichannel inhibitor, 1-octanol (B, 2 mm, n = 274 cells from 5 cultures), an inhibitor to the connexin hemichannel, Gd³⁺ (C, 50 μm, n = 229 cells from 4 cultures), an inhibitor to the maxianion channel, or Brilliant Blue G (D, BBG, 1 μm, n = 292 cells from 5 cultures), a P2X7 receptor antagonist, was applied and addition of SAG followed. b, cumulative histograms of Ca oscillation frequency before application of the blockers, under the blockers, and after SAG addition. c, cumulative histograms of ΔFrequency together with those from a control cell group in which 0.1% DMSO was applied in place of the blockers (n = 368 cells from 5 cultures).

Figure 7.

Enhancement of Ca oscillation in astrocytes in brain slices. A, astrocytes in an acute hippocampal slice were stained with an astrocyte marker, SR101 (red), and a Ca indicator, Fluo-4 (green). Arrowheads point SR101 and Fluo-4 double-positive cells. Scale bar, 100 μm. Ca oscillations by the application of 0.1% DMSO (B, n = 167 cells from 8 slices) and 50 nm SAG (C, n = 175 cells from 9 slices). D, cumulative histograms of ΔFrequency.