All experimental protocols were approved by the RIKEN Institutional Animal Care and Use Committee.

Generation of transgenic mice

G-CaMP7-WPRE-polyA DNA15 was subcloned into a pCR-FRT-Amp-FRT plasmid39. A bacterial artificial chromosome (BAC) clone, RPCI-23-361H22 (BAC PAC Resources), containing the GLT1 gene was modified by the Red/ET recombination system (Gene Bridges) to insert the PCR fragment of G-CaMP7-WPRE-polyA-FRT-Amp-FRT immediately downstream of the initiation codon. After selection of recombined colonies by ampicillin resistance, the Amp cassette was removed from the recombined BAC clones by introducing the Flp recombinase expression plasmid 706-FLP (Gene Bridges). The resulting BAC construct was amplified, purified with the Large-Construction kit (Qiagen) and digested with AscI. Correct modification of the BAC was verified by pulsed-field gel analysis of restriction digests and direct sequencing of the insert. The linearized BAC DNA was purified, adjusted to be ∼1 ng μl−1 in a microinjection buffer (10 mM Tris-Cl, 0.1 mM EDTA, 100 mM NaCl, pH 7.4), and individually injected into the pronuclei of 590 C57BL/6 J-fertilized embryos. As a result, 88 founders were born, of which 14 founders were positive for the transgene. Resulting founder mice were identified by PCR using the following primer pair: 5′- CGAGGCGCTAAAGGGCTTACC-3′ and 5′-GTACCGCCCTTGTACAGCTC-3′. Positive founder mice were crossed with C57BL/6 J mice to obtain germline transmission and the mouse lines were maintained on this genetic background. Despite the well-known-specific promoter activity of GLT-1 in astrocytes in the cerebral cortex22,33,40, we could not obtain any founders that had astrocyte-specific visible fluorescence expression levels with this construct. Rather, we obtained several lines in which visible amounts of fluorescence were seen in neurons of the cerebral cortex, hippocampus, striatum and other regions of the brain. Of these lines, line 817, which we term G7NG817, exhibited high fluorescence levels in the cerebral cortex. Homozygous transgenic mice were bred and used in the current study except for the experiments described in Supplementary Fig. 11, where heterozygous mice were used.

Surgical procedures for virus innoculation

pAAV-hGFAP-G-CaMP7 was synthesized by modifying the pAAV-hSyn-EGFP vector (Addgene plasmid #50465, a gift from Bryan Roth) with a human GFAP promoter41 and G-CaMP7 complementary DNA. AAV9-hGFAP-G-CaMP7 was purified at a titre of 1.2 × 1014 vg ml−1. AAV2.1-hSyn1-G-CaMP7 (ref. 42) (7.1 × 1013 vg ml−1) is a gift from Masanori Matsuzaki (National Institute for Basic Biology, Japan). The viruses were diluted to 5–7 × 1012 vg ml−1 with phosphate-buffered saline (PBS) for microinjection. Mice were anaesthetized with isoflurane (1.5%) or ketamine+xylazine (intraperitoneal (i.p.), 56 and 8 mg kg−1, respectively), and fixed in a stereotaxic frame. A small craniotomy was made at the site of prospective imaging and a glass micropipette containing AAV was inserted to a depth of 250 μm below the surface of the cortex. Microinjection of 300 nl was made over 5 min using a Femtojet injector (Eppendorf). Imaging experiments were performed at least 2 weeks later.

Surgical procedures for acute experiments

Male and female C57BL/6 wild type, G7NG817 and IP 3 R2 KO17 mice of postnatal 8–12-week old were used. The background strain of these mice is C57BL/6. Mice were housed under a 12 h/12 h light/dark cycle and raised in a group of up to five. Mice were anaesthetized with urethane (1.6 gkg−1) and the body temperature was maintained at 37 °C with a heating pad (BWT-100 A, Bio Research Center or TR-200, Fine Science Tools) during surgery and recording. After skull exposure, a metal frame was attached to the skull using a dental acrylic (Fuji LUTE BC, GC Corporation, Super Bond C&B, Sunmedical). For transcranial imaging, the skull was treated by a mixture of paraffin oil and glycerol (mixture composition) to increase transparency.

For two-photon imaging, a craniotomy (2 mm in diameter) was made above the visual cortex (anterior-posterior (AP) —2.0 mm, and mediolateral (ML) 3.0 mm). The dura mater was surgically removed. Sulforhodamine 101 (100 μM in PBS) was topically applied to label astrocytes and washed with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered artificial cerebrospinal fluid (ACSF) after 1 min. After the dye loading, the craniotomy was covered with agarose (1.5% w/v in ACSF) and gently sealed with a thin glass coverslip (3 × 3 mm, thickness: 0.12 mm, Matsunami Glass). The cranial window was secured by dental cement. For experiments involving LFP recording, a screw electrode (diameter, 0.7 mm; SUS-XM7, no. 00PH+14046, Matsumoto Industry) was implanted in the interparietal bone to serve as reference.

Procedures for awake mouse recording

One to two weeks before the imaging session, mice were anaesthetized with a ketamine–xylazine cocktail (70 mg kg−1 ketamine and 10 mg kg−1 xylazine). After skull exposure, a stainless metal frame was attached to the skull using a dental acrylic. Following recovery, the mice were on a 24-h water-deprivation schedule and trained to be restrained under the microscope using a mechanical fixture that rigidly fixes the head frame once a day for 5–7 days. During training, mice have access to water once the head frame is fixed to the apparatus, thereby making an association between the head fixture and satiation of thirst.

In vivo transcranial fluorescence imaging

Mice were fixed to a stereotaxic stage by clamping the head frame and placed under a fluorescence stereo microscope (MZ10F, Leica). The GFP3 filter set (excitation 470±20 nm, emission 525±25 nm, Leica) was used with the EL6000 light source (Leica). Images were acquired using the ORCA-Flash 2.0 CMOS camera (Hamamatsu Photonics) using HC Image software (Hamamatsu Photonics). The HC Image software also controlled a shutter unit to illuminate the skull only during imaging. Images were acquired with a size of 512 × 512 pixels and 16 bit resolution. For sensory stimulation experiments (Fig. 4; Supplementary Figs 3, 4 and 11), images were acquired with a 30-Hz frame rate. For tDCS experiments (Fig. 1; Supplementary Figs 5–7B), images were acquired at 10 Hz. For displaying purposes, the ΔF/F signal is thresholded at mean+s.d. for Figs 1d and 4b; Supplementary Fig. 3A, B and 4. Pseudocolouring was done using Origin 9.0 (Origin lab).

In vivo two-photon imaging

Two-photon imaging was performed with urethane-anaesthetized adult mice (as above), using a resonant scanner-based B-Scope (Thorlabs) with a Chameleon Vision 2 laser (Coherent, wavelength 920 nm) and an Olympus objective lens (XLPlan N × 25). The B-Scope is equipped with a reverse dichroic mirror (ZT405/488/561/680-1100rpc, Chroma) and the emission light was separated by using a dichroic mirror (FF562-Di03, Semrock), with band-pass filters FF03-525/50 and FF01-607/70 (both from Semrock) for the green and red channels, respectively. Images were acquired using the ThorImage software with a frame rate of 30 Hz.

Sensory stimulation

Sensory stimulation was presented to mice as follows. Visual flash stimulation: a flash of light from a red light-emitting diode (UR5365S, 20 mA, Stanley Electric) was presented to the right eye and then to the left eye 10 s later in darkness. This sequence was repeated every minute for 20 min. tDCS (see below) was applied 1 min after the visual flash stimulus. No visual flash stimulus was applied during tDCS. The same visual flash stimuli were presented 1 min after tDCS. For VEP plasticity experiments, the flash signal was presented to the left eye at an interval of 30 s in a room light condition. Whisker stimulation: individual whiskers were deflected at a frequency of 10 Hz for 5 s by a custom device made out of an audio speaker (Supplementary Fig. 4A). For whisker evoked response plasticity experiments, single air puffs were presented to the left whisker pad at an interval of 30 s (Fig. 3e,f). Auditory stimulation: 5-kHz pure tone for 500 ms (76 dB) was presented over a speaker using a custom LabView program (Supplementary Fig. 4C). Tail pinch: tail pinch was manually applied in anaesthetized mice via blunt tongs for several seconds.

Transcranial d.c. stimulation

An anode was placed on a conductive (PBS with 1.5% agarose) gel interface spreading over a 2 mm2 area above the primary visual cortex. A cathode was inserted to the neck muscle for experiments with anaesthetized mice. For experiments with unanaesthetized mice, a cathode was placed on the neck skin. D.c. was applied from the anode to the cathode with a stimulus isolator (ISO-Flex, AMPI) or a custom-made isolated constant-current supply, powered by a 9-V battery. Unless otherwise noted, current intensity and duration for tDCS are set to be 0.1 mA and 10 min, respectively. The potential difference between the anode and cathode was typically around 0.5 V.

Local field potential recording

Extracellular recordings were performed with an ELC-03XS amplifier (NPI electronic). A glass micropipette (2-μm tip diameter, 1B150F-4, World Precision Instruments) was filled with HEPES-ACSF (pH 7.4) and placed to an electrode holder with a headstage preamplifier. The headstage is then mounted to a remote-controlled micromanipultor (Sensapix). Under a stereo microscope, the glass micropipette was inserted to the primary visual cortex (250 μm below the pia) at a 30° insertion angle. Recording sessions started 1–2 h after insertion of the electrode for the stabilization of evoked responses43. After amplification (2000 ×, 0.1 Hz to 3 kHz), the signal was digitized at 20 kHz and stored on a hard drive using a LabVIEW-based data acquisition system. The field potential experiments have been performed in a room light condition.

Drug application

For pharmacological experiments with cranial window (that is, two-photon imaging or LFP recording), reagents were dissolved in HEPES-ACSF (in mM:125 NaCl, 5 KCl, 10 glucose, 10 HEPES, 2 CaCl 2 and 2 MgSO 4 ) and applied onto the brain surface from 20 min immediately preceding imaging and the solution was kept in the cranial window until the end of the experiment. The concentrations of the applied drugs were: atropine sulfate monohydrate (2–3 mM, catalogue no. 015-04853, Wako Pure Chemical Industries)7, prazosin hydrochloride (200 μM, catalogue no. P7791-50MG, Sigma–Addrich)19 and AP-5 (50 μM, Tocris).

For i.p. administration of prazosin performed in transcranial imaging experiments, injection was made at a dosage of 10 mg kg−1 (1% w/v, dissolved in 0.9% NaCl). DSP-4 treatment was performed by a single i.p. injection of DSP-4 (50 mg kg−1, 12.5% w/v, dissolved in 0.9% NaCl) 7 days before the imaging experiment.

Behavioural test

Chronic restraint stress and tail suspension test (TST) were performed as described in previous papers44,45. In brief, metal frame-attached mice were restricted to water access and socially isolated for 12 days (Supplementary Fig. 8A). Furthermore, each mouse was subjected to 8–9 h of chronic restraint stress for 5 consecutive days before behavioural test using a plastic cylinder with a radius of 8 cm and a height of 8 cm. After each session of restraint, animals were returned to their home cage with free access to food. For the behavioural test, all mice were exposed to a 9-min TST and the immobility time was counted (Supplementary Fig. 8B). Each mouse was individually suspended by their tail from a metal bar fixed 60 cm above the surface of a table with a soft vinyl tape. The test sessions were recorded by a video camera. The subjects were split into five groups: sham (n=10), tDCS (n=10), prazosin+tDCS (n=10), DSP-4+tDCS (n=9) and IP3R2 KO+tDCS (n=9). The protocols of tDCS and drug application were the same as described above.

The repeated TSTs were performed in six successive trials (Supplementary Fig. 8A). TST1: 5 days before tDCS as control; TST2: after experienced 5-day chronic restraint stress; TST3: 180 min after tDCS; TST4: 1 day after tDCS; TST5: 3 days after tDCS; and TST6: 1 week after tDCS.

Histology

Mouse brains were perfusion-fixed with 4.0% paraformaldehyde (pH 7.4 in 0.1 M phosphate buffer). Following overnight postfixation in the same fixative, coronal slices (60 μm) were prepared using a microslicer (PRO 7, Dosaka). For GFP, NeuN, GFAP and IBA-1 staining, sections were incubated with primary antibodies (1:2,000 for anti-GFP, NeuN, GFAP and IBA-1 antibodies, Tris-buffered saline (TBS) with 0.1% Triton X-1000) overnight. For S100B staining, sections were first treated with 10% normal goat serum in 0.1% Triton X-100 TBS for 1 h, and then incubated with anti-S100B antibody (1:1,000) in 10% normal goat serum (in 0.1% Triton X-100 TBS) overnight. For GAD67 staining, we blocked the sections for 1 h using the Mouse on Mouse Polymer IHC kit (Abcam), and then incubated with anti-GAD67 antibody (1:500) in 0.1% Triton X-100 TBS for overnight. The sections were subsequently washed in phosphate buffer and incubated with Alexa 488- and 594-conjugated secondary antibodies (Invitrogen) for 2 h for fluorescent labelling. Immunolabeled sections were examined using a FV1000 confocal microscope (Olympus). Confocal images were taken with a 60 × oil immersion lens (UPlanSApo, numerical aperture (NA)=1.35). For Supplementary Fig. 9, the images were acquired with a × 20 objective (UPLanSApo, NA=0.75) with a z-step size of 2 μm and a × 60 water immersion lens (UPlanSApo, NA=1.2) with a z-step size of 0.5 μm, respectively. Primary antibodies were obtained from the following sources: GFP46, NeuN (Chemicon, MAB377), S100B (Sigma, S2532), GAD67 (Millipore, MAB5406), GFAP(DAKO, Z0334) and IBA-1 (Wako, 019-19741).

Data analysis

Extracellular field recording. For spectral power analyses (Supplementary Fig. 10), the power spectral density was estimated by the Welch periodogram method using a Hamming window (window size=0.82 s; overlap=0.41 s). Recordings within 10 s from the onset or offset of tDCS were excluded from the analysis. The slope of evoked visual field response was calculated as follows. First, the initial deflection of the LFP response was isolated. Next, the region for slope calculation was defined as the interval within 20–80% of the peak-to-peak amplitude of the negative deflection 7 (Fig. 3a). The slope was computed by linear regression of the selected region.

Transcranial imaging. The original 512 × 512 pixel images were reduced to 64 × 64 pixels by binning. For sensory stimulus response imaging (Fig. 4; Supplementary Figs 2,3A,4 and 11), ΔF/F was calculated by taking the baseline F as the signal 500 ms before the stimulus presentation. For tail pinch or tDCS experiments (Figs 1 and 2; Supplementary Figs 3 and 7), the baseline F is defined as the mean intensity of the 50 s period ending 10 s before tail pinch or tDCS. For the analysis of the spontaneous slow oscillations (Supplementary Figs 2 and 6), the baseline F is defined as the mean intensity for the entire imaging period. Cross correlation was computed with the Z scores of the mean intensities of the region of interests (ROIs), so that the result is the Pearson correlation coefficient. Ca2+ events are defined as the events that had ΔF/F larger than the mean+1 s.d. for longer than 1 s and classified into two classes: those which had ΔF/F larger than mean +1 s.d. for shorter than 10 s (<10 s) and others that lasted longer (≥ 0 s; Fig. 1i,l; and Supplementary Fig. 11C). Time to Ca2+ onset is defined as the time where ΔF/F exceeded the mean+3 s.d. since the start of tDCS (Fig. 1h; Supplementary Fig. 11B). For the analysis of visual evoked response area, the active area is defined as the region that exceeds 90% of the mean peak response during the control period (Fig. 4b,e; and Supplementary Fig. 11F). To compute the decay slope, the mean visual evoked responses for respective periods were normalized to their peak value (Fig. 4f). The slope was computed similarly to VEP (Fig. 3a). All data are expressed as mean±s.e.m.

Two-photon imaging. The peak amplitude and duration statistics were made on cells that had at least one Ca2+ elevation during the course of 600 s imaging (Fig. 2c,d,j; Supplementary Fig. 3D). For each ROI, the baseline mean and s.d. of the signal were calculated for the first 60 s. The relative fluorescence change ΔF/F was computed, where the ΔF is the difference from the baseline mean and F is the baseline mean. Signal fluctuations in an ROI larger than the mean+3 s.d. were considered to be Ca2+ events (Fig. 2e,g,k; and Supplementary Fig. 7D). The duration is computed as the full-width half-maximum of the Ca2+ event (Supplementary Fig. 3E). For tail pinch experiments, the ‘during tail pinch’ period is defined as the 100-s period starting from the onset of tail pinch. For tDCS experiments, the ‘tDCS’ period is defined as the entire period of tDCS (that is, 600 s). All data are expressed as mean±s.e.m. Time to Ca2+ onset is defined as the time where ΔF/F exceeded the mean+3 s.d. since the start of tDCS. Ca2+ events for individual cells were detected with a threshold of the mean+3 s.d. Ca2+ events of astrocytes in G7NG817 mice were detected after applying a low-pass filter (cutoff frequency 0.5) to exclude possible neuronal signals from the contiguous neurogliopil (Fig. 2e,g,k). Ca2+ events of neurons in G7NG817 mice were detected after applying a band-pass filter (bandpass: 0.5–3 Hz) to exclude possible astrocytic signals from the contiguous neurogliopil (Fig. 2e–g,k). For the analysis of astrocyte-Ca2+ on versus off period (Fig. 2f), the ‘on’ period is defined as the period where at least one astrocyte is detected for a Ca2+ event.

Statistics

For comparisons of two sample means (Fig. 2c,e–g; Supplementary Figs 3C,E,F,7D), two-sample t-tests and paired t-tests (Fig. 3d) were performed using ORIGIN (OriginLab). For comparisons of multiple groups, statistical tests based on analysis of variance (ANOVA) are performed followed by Tukey s post-hoc tests. Two-way (Fig. 1g,i, and Fig. 4d-f) or one-way (Supplementary Fig. 10B) repeated measures ANOVA was computed using R (http://www.r-project.org) with a software add-on for ANOVA (ANOVAKUN, http://riseki.php.xdomain.jp/). One-way ANOVA was computed using ORIGIN (Fig. 1l and Fig. 3b,f; and Supplementary Fig. 8B). Unless otherwise noted, mean values are presented with the s.e.m.