Astroglial Ca2+ signaling is generated by the coordination of IP3R and store-operated Ca2+ channels

Accepted Manuscript 2+ Astroglial Ca signaling is generated by the coordination of IP3R and store-operated 2+ Ca channels Shigeo Sakuragi, Fumihiro Niwa, Yoichi Oda, Hiroko Bannai, Katsuhiko Mikoshiba PII:

S0006-291X(17)30562-4

DOI:

10.1016/j.bbrc.2017.03.096

Reference:

YBBRC 37483

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 10 March 2017 Accepted Date: 19 March 2017

2+ Please cite this article as: S. Sakuragi, F. Niwa, Y. Oda, H. Bannai, K. Mikoshiba, Astroglial Ca 2+ signaling is generated by the coordination of IP3R and store-operated Ca channels, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.03.096. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Astroglial Ca2+ signaling is generated by the coordination of IP3R and store-operated Ca2+ channels

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Shigeo Sakuragia,1, Fumihiro Niwab,2, Yoichi Odaa, Hiroko Bannaia, b, c, d *, Katsuhiko Mikoshibab *



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a. Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa,

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Nagoya, Aichi, 464-8602, Japan

b. Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute (BSI), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

c. Nagoya Research Center for Brain & Neural Circuits, Nagoya University, Furo-cho, Chikusa, Nagoya, Aichi, 464-8602, Japan

Japan Current affiliation:

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d. Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012,

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1. Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, 980-8577, Japan

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2. École Normale Supérieure, Institut de Biologie de l’ENS (IBENS), INSERM, CNRS, École Normale Supérieure, PSL Research University, 46 rue d’Ulm, 75005 Paris, France


*corresponding author [email protected] [H.B.] [email protected] [K.M.]

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IP3

Gq

SOCC

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PLC

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GPCR

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Ca2+

Ca2+ GPCR PLC

ER

Ca2+

IP3

IP3R

IP3R

SERCA

Inhibition of ER- or SOC-Ca2+ Ca2+ transients

SOCC

Gq

ER

SERCA

ER-Ca2+

Ca2+ transients

SOC-Ca2+

[Ca2+]ER

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ABSTRACT Astrocytes play key roles in the central nervous system and regulate local blood flow and synaptic transmission via intracellular calcium (Ca2+) signaling. Astrocytic Ca2+ signals are generated by

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multiple pathways: Ca2+ release from the endoplasmic reticulum (ER) via the inositol 1, 4, 5-trisphosphate receptor (IP3R) and Ca2+ influx through various Ca2+ channels on the plasma membrane. However, the Ca2+ channels involved in astrocytic Ca2+ homeostasis or signaling have not been fully characterized. Here, we demonstrate that spontaneous astrocytic Ca2+ transients in cultured

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hippocampal astrocytes were induced by cooperation between the Ca2+ release from the ER and the Ca2+ influx through store-operated calcium channels (SOCCs) on the plasma membrane. Ca2+ imaging

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with plasma membrane targeted GCaMP6f revealed that spontaneous astroglial Ca2+ transients were impaired by pharmacological blockade of not only Ca2+ release through IP3Rs, but also Ca2+ influx through SOCCs. Loss of SOCC activity resulted in the depletion of ER Ca2+, suggesting that SOCCs are activated without store depletion in hippocampal astrocytes. Our findings indicate that sustained

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SOCC activity, together with that of the sarco-endoplasmic reticulum Ca2+-ATPase, contribute to the maintenance of astrocytic Ca2+ store levels, ultimately enabling astrocytic Ca2+ signaling.

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KEY WORDS

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Astrocyte, Ca2+ signal, SOC channel, IP3 receptor, endoplasmic reticulum

Highlights

・SOCCs are crucial for astroglial Ca2+ signals. ・SOCCs are activated without ER Ca2+ store depletion in hippocampal astrocytes. ・Astroglial Ca2+ signals are generated through the coordination of IP3Rs and SOCCs. Abbreviations Ca2+: Calcium CNS: Central nervous system

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ER: Endoplasmic reticulum [Ca2+]ER: IntraER Ca2+ concentration [Ca2+]i: Intracellular Ca2+ concentration

IP3R: Inositol 1, 4, 5-triphosphate receptor GECI: Genetically encoded Ca2+ indicator SERCA: Sarco-endoplasmic reticulum Ca2+-ATPase

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SOCC: Store operated Ca2+ channel

TRP: Transient receptor potential

INTRODUCTION

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SOCE: Store operated Ca2+ entry STIM: Stromal interaction molecule

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IICR: IP3-induced calcium release

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Astrocytes are the most abundant type of glial cells in the central nervous system (CNS). Astrocytes are well known to play supporting roles in the CNS [1, 2] and are considered to be one of the essential contributors to the maintenance of a healthy CNS. As calcium (Ca2+) imaging techniques have

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advanced, they have clarified that Ca2+ signals, i.e. the dynamics of intracellular Ca2+ concentration ([Ca2+]i) that modulate astrocytic activities. This has led to the discovery of novel astrocytic functions,

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in addition to the support of neurons. [Ca2+]i is elevated by the presence of neurotransmitters [3, 4] and increases in [Ca2+]i trigger the release of neuroactive molecules [5, 6]. Moreover, clamping of astrocytic [Ca2+]i results in the loss of long-term potentiation [7], suggesting that astrocytic Ca2+ signaling has a crucial role in the function of these cells. Therefore, understanding astroglial Ca2+ signaling systems will contribute to the elucidation of the physiological functions of astrocytes. Several lines of evidence indicate that astrocytic Ca2+ signaling is induced by Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ influx from the extracellular region [8–12]. Among them, the most well-characterized astrocytic Ca2+ signaling pathway is Ca2+ release from the ER via

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inositol 1, 4, 5-trisphosphate receptor (IP3R). However, our recent finding that Ca2+ release from the ER in astrocytes was observed less frequently than Ca2+ influx [13] raised the possibility that Ca2+ influx also plays a pivotal role. Indeed, several Ca2+ channels on the plasma membrane, including

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transient receptor potential (TRP) A1, TRPC, and TRPV4, are reported to be involved in astrocytic Ca2+ signaling and physiological functions [11, 14–16]. The molecular mechanisms underlying astrocytic Ca2+ signals have not been characterized yet.

Here, we focused on store-operated calcium channels (SOCCs), plasma membrane Ca2+

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channels responsible for the store-operated calcium entry (SOCE) pathway that supports Ca2+ homeostasis in multiple cell types. SOCCs are activated by the depletion of ER-Ca2+ store and

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contribute to the maintenance of [Ca2+]ER by refilling empty ER-Ca2+ stores after IP3-induced calcium release (IICR) [17, 18]. Astrocytes have been shown to express SOCCs of the Orai families [19-22] as well as stromal interaction molecule (STIM) 1 and 2 [23], which sense ER-Ca2+ depletion. However, how SOCCs contribute to astrocytic Ca2+ signals in the process remains unknown. We report here that

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the SOCE pathway is involved in the mechanism of spontaneous astrocytic Ca2+ signaling together with IICR. Using rat hippocampal primary mixed cultures, we show that astrocytic Ca2+ transients are generated by the co-operation of Ca2+ release from the ER through IP3R and Ca2+ influx through

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SOCCs, resulting from SOCC activity without ER Ca2+ store depletion.

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MATERIAL AND METHODS Ethics statement

All animal procedures were performed in accordance with the guidelines issued by the Japanese Ministry of Education, Culture, Sports, Science and Technology, and were approved by the Animal Experiment Committee of RIKEN and Nagoya University.

Primary culture of rat hippocampal astrocyte Primary cultures of rat hippocampal astrocytes were prepared from Wistar rat embryos (Japan SLC) as previously described [24]. Dissociated cells were diluted to 1.4 × 105 cells/well in a plating medium

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[Minimum Essential Medium (MEM, Gibco) supplemented with 2% NeuroBrew-21 (MACS), 2 mM glutamine (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque), 4.8 U/mL penicillin, and 4.8 µg/mL streptomycin (Nacalai Tesque)] and placed onto 18-mm coverslips at 37 ºC in a CO2 incubator.

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Three days after plating, the medium was changed to a maintenance medium [Neurobasal-A medium (Gibco) supplemented with 2% NeuroBrew-21, 2 mM glutamine, 4.8 U/mL penicillin and 4.8 µg/mL streptomycin].

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Gene transfection of genetically encoded calcium indicators (GECIs)

We used GCaMP6f fused with the plasma membrane target sequence (Lck-GCaMP6f) [25, 26],

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G-CEPIA1er (gift from Dr. Masamitsu Iino, Tokyo University) [27] to monitor [Ca2+]ER, and GCaMP6f targeted to the cytosolic side of the ER (OER-GCaMP6f) to monitor Ca2+ release from the ER [13]. All GECIs were expressed under the control of the cytomegalovirus promoter. GECIs were transfected at 0.5 µg/coverslip using Lipofectamine 3000 (Invitrogen) following manufacturer’s

after transfection.

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Ca2+ imaging

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instructions. Transfections occurred after 3–7 days of culture, with experiments performed 2–4 days

We performed Ca2+ imaging using an inverted fluorescent microscope (IX73, Olympus) equipped with

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an oil-immersion objective lens (60×, NA 1.42, Olympus), LED illumination (Cool LED), an EM-CCD camera (ImagEM, Hamamatsu Photonics), and a temperature control system. Recordings were carried out at 2 Hz (100 msec exposure) in 500 µl imaging medium (MEM supplemented with 20 mM HEPES (pH 7.4), 2 mM glutamine and 1 mM sodium pyruvate) at 37 ºC. Pharmacological drugs were applied 2 min after the onset of recording and recorded for 6–8 min. The following drugs were used: dimethyl sulfoxide (DMSO, 0.1%), U73122 [2 µM, a phospholipase C (PLC) blocker, Focus Biomolecules], DPB162-AE (5 µM, a STIM-Orai interaction blocker) [28], GSK-7975A (10 µM, a blocker against Orai1 and Orai3, Aobious) [29], (S)-3,5-dihydroxyphenlglycine (DHPG, 10 µM,

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a Group I mGluR agonist, Tocris) and Thapsigargin [TG, 2 µM, a sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) blocker]. The Ca2+-free imaging medium was prepared by incubating MEM

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with 0.05 g/mL Chelex 100 (Bio-Rad) as previously described [30].

Analysis of Ca2+ signals

We used Metamorph (Molecular device) imaging analytic software or custom made software “TI workbench” [31] to analyze Ca2+ signals. Three to six circular regions of interest (ROIs) 5-µm in

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diameter were set on astrocytic shafts or first to second lateral branches found generating Ca2+ signals

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more than once per minute during the pretreatment period. Events in these ROIs were defined as “Ca2+ transients” when we observed an elevation of Lck-GCaMP6f fluorescent intensity lasting 0.5–5.0 sec and exceeding the summation of the average fluorescence intensity during the entire recording time and its standard deviation (Fig. 1A right). The fluorescent change from the baseline (dF = F – F0) was normalized to the average fluorescent intensity, excluding Ca2+ transients, during the pretreatment

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period (F-2–0). For defined Ca2+ transients, we measured the number and mean dF/F-2–0 of the Ca2+ over 2-min periods. The average value from all ROIs in one cell was calculated and compared before (-2–0 min) and after (2–4 min) the application of drugs or Ca2+. To obtain the average dF/F-2–0 of the

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baseline, which reflects the changes in the baseline Ca2+ level, the average fluorescence intensity,

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excluding Ca2+ transients, during minutes 2–4 was divided by F-2–0.

Statistics

All data are presented as mean ± standard error of the mean. Appropriate statistical methods were selected based on the distribution of each dataset and are noted in the figure legends. Levels of significance are indicated by asterisks: * for P<0.05, ** for P<0.01 and *** for P<0.001 in all figures.

RESULTS Inhibition of PLC or SOCCs attenuates astrocytic Ca2+ transients

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To visualize astrocytic Ca2+ signals, we expressed Lck-GCaMP6f in rat hippocampal primary astrocytes co-cultured with neurons (Fig. 1A). Spontaneous Ca2+ transients were observed mostly in astrocytic processes (Figs. 1A–C), not in the soma, similar to previous reports using cytosolic

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GCaMP2 and 3 or Fluo-4 [12, 32]. When we inhibited the IICR pathway by blocking PLC with U73122, spontaneous Ca2+ transients decreased while the vehicle control (DMSO) had no effect (Figs. 1D–F). These results indicate that blocking Ca2+ release from the ER influences the induction of spontaneous Ca2+ transients, which is consistent with findings of previous studies [8, 9]. Strikingly, the

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inhibition of SOCC by DPB162-AE or GSK-7975A led to a similar decrease, although GSK-7975A inhibition was partial (Figs. 1D–F). These data indicate that SOCC-mediated Ca2+ influx is also

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required for spontaneous astrocytic Ca2+ transients.

Both Ca2+ influx and Ca2+ release are required for the induction of astrocytic Ca2+ transients To further examine the importance of Ca2+ influx on the production of astrocytic Ca2+ transients, we

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monitored astrocytic Ca2+ signals with Lck-GCaMP6f after removing extracellular Ca2+. Ca2+ transients were completely abolished at least 5 min after infusion of the Ca2+-free imaging medium (Fig. 2A, C), but they recovered to the normal condition level (Fig. 1D, E -2 to 0 min) after application

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of 2 mM CaCl2 (Fig. 2A, C). The recovery of Ca2+ transients induced by external Ca2+ was blocked in the presence of DPB162-AE (Fig. 2A, C). Subsequent washout of DPB162-AE resulted in the

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recovery of Ca2+ transients (Fig. 2A), indicating that astrocytic Ca2+ transients are induced by the influx of extracellular Ca2+ via SOCCs. Astrocytes did not generate Ca2+ transients in the presence of U73122 after the addition of external Ca2+ but showed prolonged [Ca2+]i elevation in every part of the process and even in the soma (Fig. 2), suggesting that IICR is also crucial for determination of the spatiotemporal pattern of spontaneous Ca2+ transients. Taken together, these results indicate that cooperation of SOCC-mediated Ca2+ influx and IICR through IP3Rs enables spontaneous astrocytic Ca2+ transients.

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Continuous SOCE is required for the maintenance of [Ca2+]ER and IICR In many cell types, depletion of the ER-Ca2+ store triggers the influx of extracellular Ca2+ via SOCCs [17, 33]. This led us to hypothesize that SOCCs in astrocytes might affect the ER-Ca2+ level when

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astrocytes generate spontaneous Ca2+ transients. To examine this possibility, we analyzed [Ca2+]ER in the absence of extracellular Ca2+. ER-Ca2+ content was monitored by G-CEPIA1er, a GECI targeted to the ER lumen [27]. Although the intensity of G-CEPIA1er remained unchanged in the presence of 2 mM extracellular Ca2+, it decreased remarkably over the course of 5 min after removal of extracellular

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Ca2+ (Fig. S1). These results suggest that astrocytic [Ca2+]ER is highly dependent on the extracellular

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Ca2+ concentration and requires Ca2+ influx to maintain the Ca2+ level in the ER.

Based on the results above, we hypothesized that the constitutive activities of SOCE are required to maintain astrocytic [Ca2+]ER. To test this possibility, we monitored [Ca2+]ER in astrocytes in the presence of extracellular Ca2+ with or without SOCC activity. Although G-CEPIA1er signal did not change after the application of DMSO or U73122, it decreased dramatically in all analyzed cells when

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DPB162-AE was applied (Figs. 3A–D), indicating that blocking SOCCs decreases [Ca2+]ER. To confirm that the DPB162-AE-induced decrease in [Ca2+]ER was not derived from an increase in Ca2+ release, this was monitored using OER-GCaMP6f, a GECI that sensitively detects Ca2+ release from

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the ER [13]. Previously, we have shown that Ca2+ release does not influence [Ca2+]ER when the increase in OER-GCaMP6f fluorescence (dF/F0) is less than 0.75 [13]. When astrocytes were treated

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with DPB162-AE, only 2 out of 15 cells (13.3%) showed an increase in OER-GCaMP6f dF/F0 higher than 0.75, while 13 out of 15 cells (86.7%) showed lower or negative change in dF/F0 (Fig. S2). This suggests that a modification of Ca2+ release cannot fully describe the DPB162-AE-induced reduction in [Ca2+]ER (Fig. 3D, Table S1). To further assess the contribution of SOCCs to astrocytic Ca2+ store functions, we estimated [Ca2+]ER by measuring the cytosolic Ca2+ signal after evoking compulsory Ca2+ release using DHPG, a group I mGluR agonist [32]. When we applied DMSO before DHPG treatment, both the number of Ca2+ transients and the baseline level monitored by Lck-GCaMP6f increased dramatically, with the

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number of Ca2+ transients increasing by a factor of 2.56 ± 0.57 (Fig. 3E–H). However, pretreatment with DPB162-AE abrogated DHPG-induced augmentation of Ca2+ transients (Figs. 3E–H). These results indicate that blocking SOCE in astrocytes depletes Ca2+ stores to a level that impairs Ca2+

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release, suggesting that SOCCs are activated to maintain astrocytic ER function even without ER Ca2+ depletion.

Finally, we examined how extracellular SOCC-mediated Ca2+ influx refills the ER. Starting from Ca2+-free extracellular conditions, [Ca2+]ER was monitored using G-CEPIA1er before and after

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the addition of 2 mM extracellular Ca2+. Fluorescence intensity increased within 3 min after Ca2+ application in DMSO-treated control conditions, suggesting that the ER Ca2+ stores could be refilled

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when extracellular Ca2+ recovered (Fig. 4A–C). However, Ca2+ uptake into the ER was blocked by the presence of DPB162-AE (Fig. 4A–C). This suggests that the increase in [Ca2+]ER by exogenously-applied Ca2+ results from SOCE activity. Thapsigargin, an inhibitor of the ER-Ca2+ pump SERCA, completely prevented the extracellular Ca2+-induced increase in [Ca2+]ER (Fig. 4A–C),

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indicating that Ca2+ that passes through SOCCs refills the ER via SERCA. From these results, we conclude that SOCCs in astrocytes are constitutively active in order to ensure a constant Ca2+ influx that coordinates with the ER-Ca2+ pump SERCA to maintain

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appropriate Ca2+ levels within the ER. This process is required for the maintenance of [Ca2+]ER and the

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IICR function and, ultimately, the induction of Ca2+ signaling in astrocytes (Fig. 4D).

DISCUSSION

In this research, we observed that spontaneous Ca2+ transients in the primary culture of

hippocampal astrocytes were induced by cooperation between Ca2+ release from the ER and Ca2+ influx through SOCCs, which were active even when Ca2+ stores were not depleted. We have shown that pharmacological blockade of SOCCs attenuates spontaneous astrocytic Ca2+ transients. Recently, spontaneous Ca2+ transients in the fine processes of adult mouse astrocytes have been found to occur via Ca2+ influx [12]. Our present data suggest that this Ca2+ influx is partially

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dependent on SOCCs. The specific SOCC responsible for enabling spontaneous astrocytic Ca2+ transients is likely to be a non-TRP SOCC, considering that DPB162-AE does not block SOCE in TRPC3- or TRPC6-expressing HEK293 cells but does reduce the SOCE functions of STIM1- and

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Orai1-expressing cells [34]. We have shown that DPB162-AE, which inhibits Orai1 and Orai2, almost completely inhibits spontaneous astrocytic Ca2+ transients, while GSK-7975A, an inhibitor of Orai1 and Orai3, partially attenuates them. This suggests the possibility that Orai1 and Orai2 are involved in spontaneous Ca2+ transients in astrocytes. However, future studies are needed to precisely identify the

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Orai subtypes involved.

Although Orai1 and STIM1 have been shown to be involved in the generation of Ca2+

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signals in astrocytes [20, 35], the physiological relevance of SOCCs in astrocytic Ca2+ signaling has not been elucidated. In many cell types, SOCC-mediated Ca2+ influx refills the ER after SOCC activation following the depletion of ER Ca2+ stores [36]. Here, we have shown that SOCC inhibition in resting astrocytes leads to the depletion of ER Ca2+ stores. This suggests that astrocytic SOCCs are

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active even when Ca2+ stores are not depleted. To the best of our knowledge, observation of constitutively activated SOCCs has not yet been reported. Our findings have highlighted the novel and important physiological responsibilities of SOCCs in hippocampal astrocytes for the generation of

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spontaneous Ca2+ transients and maintenance of ER Ca2+ store levels. Notably, loss of IICR failed to recover spontaneous Ca2+ transients after the addition of

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extracellular Ca2+ while resulting in uniform elevation of baseline [Ca2+]. This suggests that the unique spatiotemporal patterns of spontaneous Ca2+ transients are facilitated by coordination between IICR and SOCE. Although IICR is thought to be required for Ca2+ signals that propagate over a threshold of network activities [37], our study demonstrates the possibility that IICR may also be necessary for the establishment of local Ca2+ signals coordinated with SOCE. Astrocytic Ca2+ signals are shown to have diverse impact on synaptic transmission [37]. Lines of evidence indicate that the source of Ca2+ signals, whether extracellular Ca2+ or Ca2+ released from internal stores, determines the downstream biological consequences [Refs in 13]. Thus further studies will reveal how this combination of Ca2+ influx with

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Ca2+ release generates diversity in the spatiotemporal pattern of Ca2+ signals at the subcellular level in astrocytes, and induces multiple biological phenomena downstream.

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ACKNOWLEDGMENTS We thank Dr. Masamitsu Iino for the plasmids, Dr. Takafumi Inoue for the data analysis program, and Dr. Charles Yokoyama for his valuable comments on the manuscript.

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FUNDING

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This work was supported by research grants from RIKEN; JST PRESTO [grant number JP15655561]; JSPS, KAKENHI [grant numbers JP16K07316, JP26117509, JP25250002]; Kato Memorial Bioscience Foundation; Naito Foundation; Sumitomo Foundation; Moritani Scholarship Foundation; and

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TOYOBO Biotechnology Foundation. The authors declare no conflict of interest.

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and inhibit store-operated Ca2+ entry via STIM proteins., Cell Calcium. 47 (2010) 1–10. [29]. I. Derler, R. Schindl, R. Fritsch, P. Heftberger, M.C. Riedl, M. Begg, D. House, C. Romanin, The action of selective CRAC channel blockers is affected by the Orai pore geometry, Cell Calcium.

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[30]. H. Bannai, F. Niwa, M.W. Sherwood, A.N. Shrivastava, M. Arizono, A. Miyamoto, K. Sugiura, S.

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Lévi, A. Triller, K. Mikoshiba, Bidirectional Control of Synaptic GABAAR Clustering by Glutamate and Calcium., Cell Rep. 13 (2015) 2768–80. [31]. N. Shafeghat, M. Heidarinejad, N. Murata, H. Nakamura, T. Inoue, Optical detection of neuron connectivity by random access two-photon microscopy., J Neurosci Methods 263 (2016) 48-56. [32]. M. Arizono, H. Bannai, K. Nakamura, F. Niwa, M. Enomoto, T. Matsu-ura, A. Miyamoto, M.W. Sherwood, T. Nakamura, K. Mikoshiba, Receptor-Selective Diffusion Barrier Enhances Sensitivity of Astrocytic Processes to Metabotropic Glutamate Receptor Stimulation, Sci. Signal. 5 (2012) ra27.

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[33]. M.D. Cahalan, STIMulating store-operated Ca2+ entry., Nat. Cell Biol. 11 (2009) 669–77. [34]. E. Hendron, X. Wang, Y. Zhou, X. Cai, J.I. Goto, K. Mikoshiba, Y. Baba, T. Kurosaki, Y. Wang, D.L. Gill, Potent functional uncoupling between STIM1 and Orai1 by dimeric 2-aminodiphenyl

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borinate analogs, Cell Calcium. 56 (2014) 482–492. [35]. C.I. Linde, S.G. Baryshnikov, A. Mazzocco-Spezzia, V.A. Golovina, Dysregulation of Ca2+ signaling in astrocytes from mice lacking amyloid precursor protein., Am. J. Physiol. Cell Physiol. 300 (2011) C1502-12.

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[36]. J. Soboloff, B.S. Rothberg, M. Madesh, D.L. Gill, STIM proteins: dynamic calcium signal

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FIGURE LEGENDS

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Figure 1. Influence of PLC or SOCC inhibition on astroglial Ca2+ transients in rat hippocampal primary cultures.

A: Representative images of spontaneous astroglial Ca2+ transients reported by Lck-GCaMP6f. Red

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circles (5-µm diameter) represent examples of ROIs located on astrocytic processes. Ca2+ transients were generated locally (arrowheads). Scale bars: 30 µm. B, C: Representative time-course of dF/F0

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(B) and number of Ca2+ transients (C) at the soma and processes. D: Representative time-course of dF/F0. Drugs were applied at 0 min as indicated by horizontal bars. Numbers at the left of DMSO traces correspond to the ROIs shown in (A). E: Quantification of Ca2+ transients before and after drug application. F: Average dF/F-2–0 of Ca2+ transients at 2–4 min. Scale bar (B, D): 1 dF/F0 (vertical). Statistics: Mann-Whitney U test (C), Wilcoxon signed-rank test (E), one-way ANOVA followed by Tukey-Kramer test (F).

Figure 2. Induction of Ca2+ transients by extracellular Ca2+ application in Ca2+-free conditions.

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A: Representative time-course of dF/F-2–0 before and after addition of 2 mM CaCl2 at 0 min. Numbers at the left of U73122 traces correspond to ROIs in (B). Scale bar: 1 dF/F0 (vertical). B: Representative Ca2+ elevation monitored by Lck-GCaMP6f after addition of extracellular Ca2+ in U73122-treated

before and after CaCl2 application. Statistics: Steel-Dwass test.

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astrocytes. Scale bar: 30 µm. C, D: Number of Ca2+ transients (C) and average dF/F-2–0 of baseline (D)

Figure 3. Influence of SOCC inhibition on ER-Ca2+ stores and mGluR activation.

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A: Representative images of ER-Ca2+ imaging with G-CEPIA1er. All reagents were applied at 0 min.

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Scale bar: 30 µm. B: Examples of dF/F-2–0 time-courses using G-CEPIA1er. Scale bar: 0.2 dF/F0 (vertical). C: Time-course of average dF/F0 after addition of SOCC or PLC inhibitors. D: Box plot of dF/F0 at 4 min. Values are shown in Table. S1. E: Representative time-course of Lck-GCaMP6f dF/F0 after 10 µM DHPG treatment in presence of DMSO or DPB162-AE. DMSO or DPB162-AE was applied at 0 min and DHPG at 4 min. Scale bar: 1 dF/F0 (vertical). F: Time-course quantification of

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Ca2+ transients. G: Quantification of DHPG-induced Ca2+ transients normalized to DHPG pre-treatment. H: Average dF/F-2–0 of baseline. Statistics: One-way ANOVA followed by

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Tukey-Kramer test (C), Mann-Whitney U test (G, H).

Figure 4. Contribution of SOCC and SERCA to the maintenance of astrocytic [Ca2+]ER.

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A: Representative images of ER-Ca2+ imaging. Drugs and 2 mM CaCl2 were simultaneously applied at 0 min. Scale bar: 30 µm. B: Representative time-course of G-CEPIA1er dF/F0. Scale bar: 0.2 dF/F0 (vertical). C: Time-course of average dF/F0. D: Proposed model of astrocytic [Ca2+]ER maintenance. Statistics: Steel-Dwass test.

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S

77

73

ROI

P

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3

81

P

SC

4 85

-2

93 (s)

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89

E

D

number of Ca2+ transient / 2 min

1

10

-2 to 0 min 2 to 4 min

7.5

2

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3

0

U73122

2

4 -2 0 time (min)

2

DPB162-AE -2

0

2

0 4 -2 time (min)

2

4

0 2 4 6 time (min)

***

***

5

2.5 0

Process (n=12) Soma (n=12)

***

5

0

DMSO

U73122

(n=29)

F

GSK-7975A

***

2.5

4

AC C

-2

DMSO

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4

2 to 4 min

10 7.5

P

ROI

2

C

B

1

number of Ca2+ transient / 2 min

0.1 dF/F0

Lck-GCaMP6f

0.5

ave. dF/F-2–0

A

0 -0.5 -1.0

(n=17)

DPB162-AE GSK-7975A

2 to 4 min **

** ***

*

DMSO (n=29) DPB162-AE (n=17) U73122 (n=17) GSK-7975A (n=17)

(n=17)

(n=17)

pre-expt. (2 mM Ca2+) 1

4

dF/F0 0.6

Ca2+ 2 mM Ca2+ free

0.1

-2

0

2

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washout 4 -2 0 time (min)

8

2

4

60

120

240 (s)

*** ***

6

AC C

DPB162-AE

10

number of Ca2+ transient / 2 min

C

30

4 2 0

Ca2+ free 2 mM Ca2+ (-2 to 0 min) (2 to 4 min) DMSO (n=15) DPB162-AE (n=14) U73122 (n=15)

D

2 to 4 min * *

0.75 0.50 0.25 0

U7

2

3 (n= 1 2 2 15 )

Ca2+ 2 mM Ca2+ free

0

20

1 (n= 6214 AE )

4 -2 time (min)

10

D

2

0

M (n= SO 15 ) B

0

-120

-420

U73122

DP

-2

2

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DMSO

3

4

3 4

2 mM Ca2+

ave. dF/F-2–0 of baseline

1 2

U73122-treated condition

Ca2+ free

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2 mM Ca2+

SC

Ca2+ free

Ca2+ 2 mM Ca2+ free

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B

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A

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1

2

4 (min)

U73122

-2

0 2 time (min)

4

4 min

0.1

dF/F0

U73122

4

drugs

0

-0.1

2

4

6

DHPG

0 2 4 time (min)

6

H 3.0

**

2.0 1.0 0

DMSO (n=9) DPB162-AE (n=10)

0.2 0.1

***

TE D -2

U73122 DPB162-AE

G

DMSO (n=9) DPB162-AE (n=10)

drugs

DMSO

ave dF/F-2–0 of baseline

6 -2 0 time (min)

-0.3

0.3

0

-0.1 -0.2

DM (n SO DP =9) B (n 162 =1 -A 0) E

4

DHPG

-0.2

0

number of Ca2+ transient (norm. with pre-DHPG)

2

DPB162-AE

AC C

DMSO

DHPG

14 12 10 8 6 4 2 0

number of Ca2+ transient / 2 min

F

EP

E

0

0.2

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D

0 2 time (min)

SC

DPB 162-AE

-2

dF/F0 -0.3

-2

drugs

0.2 0.1 0 -0.1 -0.2 -0.3 -0.4 -0.5

*** ***

G-CEPIA1er

DMSO (n=10) DPB162-AE (n=10) U73122 (n=10)

dF/F0

0

DMSO

–2

RI PT

A

C

DPB DMSO 162-AE

B

drugs -2

DHPG

0 2 4 time (min)

6

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A

G-CEPIA1er

0

1

Ca2+ free

4

0.1

D

drugs -2

Ca2+

GPCR *** ***

dF/F0

AC C

TG

0 2 time (min)

0.5

2 mM Ca2+

EP

0.3 0.2 0.1 0 -0.1 -0.2 -0.3 -0.4

drugs -2

dF/F0

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C

2 mM Ca2+

DPB DMSO 162-AE

Ca2+ free

4 (min)

SC

DPB 162-AE TG

B

2

RI PT

DMSO

-2

0 2 4 time (min) DMSO (n=10) TG (n=12) DPB162-AE (n=11)

PLC

Gq

SOCC

Ca2+

IP3 IP3R

ER

SERCA

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Highlights ・SOCCs are crucial for astroglial Ca2+ signals. ・SOCCs are activated without ER Ca2+ store depletion in hippocampal astrocytes.

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・Astroglial Ca2+ signals are generated through the coordination of IP3Rs and SOCCs.