Plasmid construction

Expression plasmid for R-GECO1 (Addgene plasmid #32444) was obtained from Addgene. For construction of expression plasmids for HA-monSTIM1 and FLAG-monSTIM1, the monSTIM1 sequence from GFP-monSTIM1 [22] was amplified by polymerase chain reaction (PCR) using HA-F, FLAG-F, and HA-R primers. The amplified sequence was then ligated into GFP-monSTIM1 at NheI and SalI sites after excising EGFP. The LOV2(404–546) sequence in OptoCRAC [19] was PCR-amplified using LOV2-F and LOV2-R primers and ligated into the EGFP-C1 vector at BsrGI and HindIII sites to generate the EGFP-LOV2 vector. The STIM1(336–486) sequence in GFP-monSTIM1 was PCR-amplified using STIM1(336–486)-F and STIM1(336–486)-R primers and ligated into the EGFP-LOV2 vector at HindIII and BamHI sites to generate the OptoCRAC expression plasmid. For construction of GFP-CRY2-STIM1(238–448), GFP-CRY2-STIM1(248–448), GFP-CRY2-STIM1(336–448) and GFP-CRY2-STIM1(342–448) expression plasmids, sequences encoding STIM1(238–448), STIM1(248–448), STIM1(336–448) and STIM1(342–448) fragments were PCR-amplified using STIM1(238–448)-F, STIM1(248–448)-F, STIM1(336–448)-F, STIM1(342–448)-F, and STIM1(238–448)-R primers and ligated into monSTIM1 at BspEI and BamHI sites. For construction of GFP-CRY2-STIM1(318–463) and GFP-CRY2-STIM1(318–450) expression plasmids, sequences encoding STIM1(318–463) and STIM1(318–450) fragments were PCR-amplified using STIM1(318–463)-F, STIM1(318–463)-R and STIM1(318–450)-R primers and ligated into monSTIM1 at BspEI and BamHI sites. For generation of the IRES2-CRY2-STIM1(238–448) expression plasmid, the IRES2 sequence was PCR-amplified using IRES2-F and IRES2-R primers and ligated into GFP-CRY2-STIM1(238–448) at NheI and BsrGI sites after excising EGFP. The sequence encoding EGFP was then inserted at NheI and NotI sites to generate the EGFP-IRES2-CRY2-STIM1(238–448) expression plasmid. The STIM1(238–448) fragment was replaced with other STIM1 variants at BspEI and BamHI sites. The CRY2(E281A, A9) sequence in monSTIM1 was PCR-amplified using CRY2-F and CRY2-R primers and ligated into EGFP-N1 at AgeI and BsrGI sites after excising EGFP to generate the CRY2-N1 vector. The vhhGFP sequence in mCherry-CRY2-vhhGFP [42] was PCR-amplified using vhhGFP-F1 and vhhGFP-R1 primers and ligated into CRY2-N1 at NheI and AgeI sites to generate the vhhGFP-CRY2 expression plasmid. The CRY2(E281A, A9) sequence from CRY2-N1 was digested with AgeI and BsrGI and ligated into EGFP-C1 (Clontech) after excision of EGFP to generate the CRY2-C1 vector. The vhhGFP sequence in mCherry-CRY2-vhhGFP was PCR-amplified using vhhGFP-F2 and vhhGFP-R2 primers and ligated into CRY2-C1 at BsrGI and XhoI sites to generate the CRY2-vhhGFP vector. For construction of virus vector for pAAV-CamKIIa-GFP-monSTIM1, an exchange enzyme site was designed by using NheI-BamHI oligomer and inserted into pAAV-CamKIIa-EGFP (addgene #50469). GFP-monSTIM1 was ligated into pAAV-CamKIIa-EGFP at the NheI and BamHI sites after excising EGFP. The W3SL sequence in pAAV-CW3SL-EGFP (Addgene plasmid #61463) was PCR-amplified using W3SL-F and W3SL-R primers and then ligated into pAAV-CamKIIa-GFP-monSTIM1 vector at the BamHI and RsrII sites after the excision of WPRE and hGH poly(A) signal to generate the pAAV-CamKIIa-GFP-monSTIM1-W3SL vector. For construction of virus vectors of pAAV-CamKIIa-FLAG-monSTIM1-W3SL and pAAV-CamKIIa-GFP-STIM1(318–450)-W3SL, FLAG-monSTIM1 or GFP-STIM1(318–450) was inserted into the pAAV-CamKIIa-GFP-monSTIM1-W3SL vector using NheI and BamHI sites after excising GFP-monSTIM1. The GfaABC1D promoter sequence was PCR-amplified using GfaABC1D-F and GfaABC1D-R primers and ligated into pAAV-CaMKIIα-FLAG-monSTIM1 at MluI and XbaI sites after excising CaMKIIα promoter sequence to generate pAAV-GfaABC1D-FLAG-monSTIM1. Oligonucleotides used in this research are listed in Additional file 1: Table S1.

Cell culture and transfection

HeLa cells (ATCC) and BV2 cells (ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Cat# 11965092, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Cat# 16,000–044) at 37 °C in a humidified 5% CO2 atmosphere. Hippocampal neurons were prepared from embryonic day 15–16 mice, and the hippocampi from collected embryos were dissected in Hank’s balanced salt solution (HBSS; Gibco, Cat# 14175-095). The collected hippocampus was dissociated by incubating with 0.05% trypsin for 5 min at 37 °C, filtered through a 0.4-μm filter, and then seeded onto a 24-well polymer-coverslip–bottom plate (ibiTreat; ibidi, Cat# 82426, Gräfelfing, Germany) coated with 50 μg/mL poly-D-lysine (Millipore, Cat# A003-E, MA, USA). Neurons were grown in Neurobasal medium Cat# 21103-049) supplemented with 2% B-27, 2% N-2 supplements, 2 mM GlutaMAX (Gibco, Cat# 35050061), and 1000 units/mL penicillin–streptomycin and maintained at 37 °C in a humidified 5% CO2 atmosphere. Primary cortical astrocytes were dissected by removing the adherent meninges from P0-P1 C57BL/6 mouse pups, followed by dissociation into a single-cell suspension by trituration through a Pasteur pipette. Dissociated cells were plated on a 60-mm dish coated with 50 μg/mL poly-D-lysine. Cells were grown in high-glucose DMEM (Gibco) containing L-glutamine, supplemented with 10% horse serum, 10% FBS and 1000 units/mL penicillin–streptomycin, and maintained at 37 °C in a humidified 5% CO2 atmosphere. Cells were transfected using Lipofectamine LTX (Invitrogen, Cat# 15338-100, CA, USA) according to the manufacturer’s instructions.

Live-cell imaging

For live-cell imaging, a Nikon A1R confocal microscope (Nikon Instruments), mounted onto a Nikon Eclipse Ti body and equipped with a CFI Plan Apochromat VC objective (× 60/1.4-numerical aperture (NA)) and digital zoom Nikon imaging software (NIS Element AR 64-bit version 3.21; Laboratory Imaging), was used. Cells were maintained at 5% CO2 and 37 °C during imaging with the microscope-mounted Chamlide TC System (Live Cell Instruments, Inc., Korea). Ca2+ influx in cells co-expressing R-GECO1 was measured using genetically encoded red Ca2+ indicators with each of the monSTIM1 variants; 488 and 561 nm laser sources were used to excite cells at 30-s intervals. Captured images were analyzed using NIS-Elements AR microscope imaging software (NIS-element AR 64-bit version 3.21; Nikon). Time-lapse images of Ca2+ influx were analyzed in defined regions of interest (ROI) in the cells, and changes in R-GECO1 intensity were quantified.

Fura-2 imaging

HeLa cells were loaded with Fura-2 AM (Invitrogen, Cat# F6774), dissolved in dimethyl sulfoxide and diluted to 2 μM in DMEM, by incubating at room temperature for 30 min. The cells were then washed three times for 5 min at each step. Fura-2 imaging was performed using a LAMBDA DG-4 lamp (Sutter Instrument Company) and a × 40/0.75 NA CFI Plan Fluor objective, with intermittent excitation using 340 and 380 nm filtered fluorescent light. The emitted light was collected with a Nikon DS-Qi1 monochrome digital camera after passing through a 510-nm emission filter.

Mice

Male C57BL/6 mice, 12–15 weeks old, were group-housed on a 12-h light/dark cycle with ad libitum access to food and water. Experimental protocols were approved by the Institutional Animal Care and Use Committee of IBS (Daejeon, Korea). Mice were randomly assigned to experimental groups.

Stereotaxic surgery and in vivo light-stimulation conditions

Surgical procedures were performed under IBS IACUC guidelines. Anesthesia in mice was induced with 5% isoflurane and maintained with 1–2% isoflurane during the stereotaxic surgery. After securing the skull in a stereotaxic apparatus (RWD), the skin was shaved and the scalp was sterilized with povidone-iodine, and a small craniotomy was performed. The following coordinates were used for microinjections into the CA1: AP, 2.0 mm, ML, ± 1.5 mm, DV, -1.4 mm. All microinjections were carried out using a glass capillary Picospritzer (Parker). Following each injection, the capillary was kept in place for an additional 10 min to allow for diffusion, and then slowly withdrawn. In vivo light-stimulation experiments were performed 3 weeks post injection using 473-nm light, delivered via a solid-state LED excitation system (Live Cell Instrument) [22]. The duration of light illumination was 30 min.

Immunohistochemistry

Mice were anesthetized with mixture of Alfaxan (40 mg/kg) and xylazine (10 mg/kg) 90 min after dark or light-illumination conditions and transcardially perfused first with PBS and then with PBS containing 4% paraformaldehyde (PFA). Brains were extracted, postfixed overnight in 4% PFA at 4 ℃, and then sectioned to a thickness of 30 μm using a vibratome (Leica). For immunostaining, free-floating sections were washed in PBS, incubated for 1 h in blocking solution (PBS containing 5% normal goat serum and 0.3% Triton X-100) and incubated overnight at 4℃ with primary antibodies (see below) in blocking solution. Sections were then washed in PBS, incubated for 2 h at room temperature with secondary antibodies (see below) in PBS. Finally, brain tissue sections were washed in PBS and slide-mounted with Vectashield antifade mounting medium containing DAPI (4’,6-diamidino-2-phenylindole) (Vector Laboratories, Cat# H-1200, CA, USA). For CA1 imaging, fluorescence images were obtained using a Leica Stellaris 8 confocal microscope with 20×, 40×, and 60 × objectives. For all brain regions, fluorescence images were acquired using a Zeiss Axio scan Z1 with 10 × objectives. Images were analyzed using ImageJ (NIH). More than three coronal brain sections were analyzed for each mouse sample for quantification of cFos+ and FLAG+ cells.

AntibodiesPrimary antibodies

The following primary antibodies were used: rabbit anti-cFos (diluted 1:2000; Cell Signaling Technologies, Cat# 2250, MA, USA), rat anti-FLAG (diluted 1:500; BioLegend, Cat# 637303, CA, USA), chicken anti-GFAP (diluted 1:1000; Millipore, Cat# AB5541), chicken anti-GFP (diluted 1:1000; Invitrogen, Cat# A10262).

Secondary antibodies

The following secondary antibodies were used: Alexa Fluor 488-conjugated goat anti-rabbit (diluted 1:1000; Invitrogen, Cat# A11034), Alexa Fluor 594-conjugated goat anti-rat (diluted 1:1000; Invitrogen, Cat# A11007), Alexa Fluor 647-conjugated goat anti-chicken (diluted 1:1000; Invitrogen, Cat# A32933), Alexa Fluor 488-conjugated goat anti-chicken (diluted 1:1000; Invitrogen, Cat# A11039), and Alexa Fluor 594-conjugated goat anti-rabbit (diluted 1:1000; Invitrogen, Cat# A11037).