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Kolkata, India

News Article
Site: http://phys.org/technology-news/

The software giant said net profit for the three months to December 31 rose 12.2 percent to 61.1 billion rupees ($913.78 million) from 54.4 billion rupees for the same period the year before. A Bloomberg survey had expected the Mumbai-based firm to increase its profits to 60.1 billion rupees, but strong orders from its major markets of Europe and the United States propelled earnings higher. "All our industry segments have exhibited growth in a traditionally weak quarter additionally accentuated by the impact of the Chennai floods," TCS chief executive N. Chandrasekaran said in a statement. The floods at the end of last year killed more than 250 people and swamped the offices of many top IT firms, including TCS competitor Infosys. Chandrasekaran remained optimistic about future earnings and said the firm had signed nine major deals in the just-concluded quarter, adding 9,071 new employees. Revenues for the last quarter increased 11.7 percent year-on-year to 273.64 billion rupees. India has become a back office to the world as companies, especially in developed nations, have subcontracted work to firms such as TCS, taking advantage of the country's skilled English-speaking workforce. Explore further: India's TCS Q2 rises 13% on US, European deals


News Article | June 7, 2012
Site: gigaom.com

Solar startup Konarka’s bankruptcy, announced last week, wasn’t just the latest case of the solar industry being hit by the dropping cost of silicon, and cheap modules and panels from China. The company’s technology was just weak and “could not compete on cost, efficiency, or lifetime,” says Lux Research. Lux Research says for at least three years it’s given Konarka a “strong caution” rating for its technology that was “ten times higher cost, and ten times lower efficiency and lifetime compared to alternative solar technologies.” More than technology development, Konarka’s skill was in fund raising, says Lux Research, and its “underlying technology was never market ready.” Lux writes: Driven by the promise of cheap, printed solar modules that can be made colorful and transparent, technically unsavvy investors rushed to invest in Massachusetts organic photovoltaic developer Konarka to the tune of $170 million, with an additional $30 million coming from grant funding. Konarka took that investment and built what it claimed was a 1 GW manufacturing line, although the line would certainly never come close to that capacity. We, too, have long been skeptical of Konarka. It’s amazing that Lux points out that Konarka finally went bankrupt “in the middle of yet another funding round.” Here’s a list of investors that Konarka managed to raise money from: Konica Minolta, Draper Fisher Jurvetson, Good Energies, 3i, Mackenzie Financial Corp., Pegasus Capital, Asenqua Ventures, New Enterprise Associates, Vanguard Ventures, Chevron Ventures, Massachusetts Green Energy Fund, NGEN Partners, Angeleno Group, Total, Good Energies, and 3i.


News Article
Site: http://www.nature.com/nature/current_issue/

The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. FS5, FS4, FS13, FS14, M93-047, UACC-903, and UACC-1273 cells were maintained in RPMI (Invitrogen), supplemented with 10% FBS, 100 units per millilitre penicillin and streptomycin, and 4 mM L-glutamine. WM35, WM793, WM164, WM1799, and 1205 LU cells were maintained in MCDB153 (Sigma)/L-15 (Cellgro) (4:1 ratio) supplemented with 2% FBS and 1.6 mM CaCl (tumour growth media). WM983b and WM3918 cells were maintained in DMEM (Invitrogen), supplemented with 5% FBS, 100 units per millilitre penicillin and streptomycin, and 4 mM L-glutamine. YUMM1.7 cells were maintained in DMEM F-12 (HEPES/glutamine) supplemented with 10% FBS, 1% NEAA, and 100 units per millilitre penicillin and streptomycin. Fibroblasts were maintained in DMEM, supplemented with 10% FBS, 100 units per millilitre penicillin and streptomycin, and 4 mM L-glutamine. Keratinocytes were maintained in keratinocyte SFM supplemented with human recombinant Epidermal Growth Factor 1-53 (EGF 1-53) and Bovine Pituitary Extract (BPE) (Invitrogen). Cell lines were cultured at 37 °C in 5% CO and the medium was replaced as required. Cell stocks were fingerprinted using an AmpFLSTR Identifiler PCR Amplification Kit from Life Technologies at The Wistar Institute Genomics Facility. Although it is desirable to compare the profile with the tissue or patient of origin, our cell lines were established over the course of 40 years, long before acquisition of normal control DNA was routinely performed. However, each short tandem repeat profile is compared with our internal database of over 200 melanoma cell lines, as well as control lines, such as HeLa and 293 T. Short tandem repeat profiles are available upon request. Cell culture supernatants were tested for mycoplasma using a Lonza MycoAlert assay at the University of Pennsylvania Cell Center Services. Organotypic three-dimensional skin reconstructs were generated as previously described30. In each insert, 6.4 × 104 fibroblasts were plated on top of the acellular layer (BD 355467 and Falcon 353092) and incubated for 45 min at 37 °C in a 5% CO tissue culture incubator. DMEM containing 10% FBS was added to each well of the tissue culture trays and incubated for 4 days. Reconstructs were then incubated for 1 h at 37 °C in HBSS containing 1% dialysed FBS (wash media). Washing media were removed and replaced with reconstruct media I. Keratinocytes (4.17 × 105) and melanoma cells (8.3 × 104) were added to the inside of each insert. Media were changed every other day until day 18 when reconstructs were harvested, fixed in 10% formalin, paraffin embedded, sectioned, and stained. Quantification of the invasion was performed using ImageJ software (available at http://imagej.nih.gov/ij/; developed by W. Rasband). Tissue-culture-treated 96-well plates were coated with 50 μl 1.5% Difco Agar Noble (Becton Dickinson). Melanoma cells were seeded at 5 × 103 cells per well and allowed to form spheroids over 72 h. Spheroids were harvested and embedded as previously described using collagen type I (GIBCO, A1048301). For spheroids incubated with fibroblast conditioned media, fibroblasts were seeded onto 75 cm2 flasks at 7 × 105 to 9 × 105 per flask depending on growth rate. Sixteen hours later, media were replaced and incubated for 72 h. Media from young fibroblasts were combined and media from aged fibroblasts were combined. These conditioned media were added to the top of the collagen plug containing the spheroids. Quantitation of invasive surface area was performed using NIS Elements Advanced Research software. Spheroids were generated and embedded as described above. Spheroids were stained using a LIVE/DEAD Viability/Cytotoxicity Kit (L3224, Invitrogen). Briefly, spheroids were washed with PBS and stained with calcein AM/Ethidium homodimer-1. The dyes were diluted in PBS and 300 μl of the solution was added on the spheroid wells for 1 h at 37 °C. The spheroids were washed in PBS and imaged using a Nikon TE2000 Inverted Microscope. Quantitation of fluorescence intensity was performed using NIS Elements Advanced Research software. Matrigel (BD Biosciences, 354234) was diluted in PBS (1:3,000 dilution). One hundred and fifty microlitres of this mixture was pipetted into each insert of the invasion assay plate (Corning, 3422). The plate was incubated at 37 °C for 2 h and then dried at room temperature (25 °C) overnight under sterile conditions. Melanoma cells were pre-treated for 48 h in six-well plates. After 48 h, the cells were harvested and 1.5 × 105 cells were added to each transwell. High concentration serum media (RPMI with 20% FCS, tumour growth media with 10% FCS) were added to the outside (bottom) of the well. The plates were incubated at 37 °C until cells had migrated to the bottom of the well. The migrated cells were fixed in 95% ice-cold methanol and stained with crystal violet (0.5%) for 10 min. The stain was washed and the wells were left to dry. Cells were imaged and quantified using ImageJ software. In a 24-well plate, 5,000 cells in triplicate were plated per day of measurement. Every 2–3 days, cells were counted using a haemocytometer and the total cell number in the well was recorded and plotted on GraphPad Prism. Cells were seeded onto glass cover slips at 1 × 104 to 4 × 104 cells per well, and incubated overnight. After treatment, cells were fixed using 95% methanol. Primary antibodies were diluted as stated above in blocking buffer and incubated overnight at 4 °C. Cells were washed in PBS and incubated with the appropriate secondary antibody (1:2,000, Invitrogen) for 1 h at room temperature. Cells were then washed in PBS and mounted in Prolong Gold anti-fade reagent containing DAPI (Invitrogen). Images were captured on a Leica TCS SP5 II scanning laser confocal system. All antibodies are described in the Supplementary Information. Patient samples were collected under IRB exemption approval for protocol EX21205258-1. Paraffin embedded sections were rehydrated through a xylene and alcohol series, rinsed in H O and washed in PBS. Antigen retrieval was performed using target retrieval buffer (Vector Labs) and steamed for 20 min. Samples were then blocked in a peroxidase blocking buffer (Thermo Scientific) for 15 min, followed by Protein block (Thermo Scientific) for 5 min, and incubated in appropriate primary antibody diluted in antibody diluent (S0809, Dako) at 4 °C overnight in a humidified chamber. For mouse samples to be incubated with anti-mouse antibody, samples were blocked for 1 h in Mouse on Mouse (M.O.M.) Blocking Reagent (MKB-2213, Vector Labs). After washing in PBS, samples were incubated in biotinylated anti-rabbit or polyvalent secondary antibody (Thermo Scientific) followed by streptavidin-HRP solution at room temperature for 20 min. Samples were then washed in PBS and incubated in 3-amino-9-ethyl-l-carboazole (AEC) chromogen and counterstained with Mayer’s haematoxylin for 1 min, rinsed in cold H O, and mounted in Aquamount. Total protein lysate (50–65 μg) was run on a 4–12% NuPAGE Bis Tris gel (Invitrogen). Proteins were then transferred onto PVDF membrane using an iBlot system, and blocked in 5% milk/TBST for 1 h. All primary antibodies were diluted in 5% milk/TBST and incubated over night at 4 °C. The membranes were washed in TBST and probed with the corresponding HRP-conjugated secondary antibody (0.2–0.02 μg ml−1 of anti-mouse, streptavidin, or anti-rabbit). Proteins were visualized using ECL prime (Amersham), or Luminata Crescendo (Millipore). All clones used are described in the Supplementary Information. All short hairpin RNA (shRNA) was obtained from the TRC shRNA library available through the Molecular Screening Facility at The Wistar Institute. Lentiviral production was performed as described in the protocol developed by the TRC library (Broad Institute). Briefly, 293 T cells were co-transfected with shRNA vector and lentiviral packaging plasmids (pCMV-dR8.74psPAX2, pMD2.G). The supernatant containing virus was harvested at 36 and 60 h, combined and filtered through a 0.45 μm filter. For transduction, the cells were layered overnight with lentivirus containing 8 μg ml−1 polybrene. The cells were allowed to recover for 24 h and then selected using 1 μg ml−1 puromycin. Cells (1.5 × 103 per well) were plated in a 96-well plate and treated with PLX4720. After 48 h, cells were incubated with MTS dye (20 μl per well) for 2 h. Absorbance was determined at 490 nm using an EL800 microplate reader (BioTek). The percentage cell proliferation was calculated by converting the experimental absorbance to percentage of control and plotted versus drug concentration. The values were then analysed using a nonlinear dose–response analysis in GraphPad Prism. Transcriptional profiling was determined using Illumina Sentrix BeadChips. Total RNA was used to generate biotin-labelled cRNA using the Illumina TotalPrep RNA Amplification Kit. In short, 0.5 μg of total RNA was first converted into single-stranded complementary DNA (cDNA) with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double-stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in vitro transcription reaction where single-stranded RNA (cRNA) was generated incorporating biotin-16-UTP. A total of 0.75 μg of biotin-labelled cRNA was hybridized at 58 °C for 16 h to Illumina’s Sentrix Human HT-12 v3 Expression BeadChips (Illumina). Each BeadChip has around 48,000 transcripts with approximately 15-fold redundancy. The arrays were washed, blocked, and the labelled cRNA was detected by staining with streptavidin-Cy3. Hybridized arrays were scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner and the image data extracted using the Illumina GenomeStudio software, version 1.1.1). Data are available in the GEO database (accession number GSE57445). Microarray expression data were quantile normalized and probes that showed low expression levels (detection P value > 0.05) across all samples were removed from the analysis. Expression values for each cell line were tested separately in multiple linear regression model with fibroblast age and experiment batches as predictor variables. Matlab version 8.0 ‘regress’ function was used to calculated P values for each probe for association with fibroblast age. False discovery rate was estimated using Benjamini-Hochberg procedure; only probes that showed a false discovery rate < 5% in all three cell lines were considered significant. Heat map was plotted using average expression values for three groups of age (young, middle, and aged) normalized to aged group (100%). All animal experiments were approved by the Institutional Animal Care and Use Committee (112503Y_0) and were performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. From preliminary studies, we observed significant differences (more than 1.8 standard deviations, a very large effect size) in some of the outcomes between young and aged groups. As few as five samples in each group in this study afforded 80% power at a two-sided α of 0.05 to detect a difference of about 1.8 standard deviations in a continuous outcome between young and aged groups, but we increased the sample size slightly to account for potential loss of mice due to health issues associated with ageing. Male C57BL6 mice at 6–8 weeks (young) and 52 weeks (aged) were purchased from Taconic. YUMM 1.7 (1 × 106 cells per 100 μl PBS) or B16F10 (2.5 × 105 per 100 μl PBS) were injected into the tail vein of C57BL6 mice. Alternatively, Yumm1.7 cells were overexpressed with mCherry plasmid (pLU-EF1-MCS-mCherry) using lentivirus. The cells were sorted for mCherry and 1 × 106 cells per 100 μl PBS were injected into tail vein of young C57BL6 mice. After 4 weeks, the mice were euthanized, lungs were harvested, and metastases counted. Lungs were fixed in paraffin and stained with haematoxylin and eosin. Alternatively, lungs were harvested and imaged for presence of metastatic melanoma cells using a Perkin-Elmer IVIS 200 whole body imager. For experiments requiring rsFRP2, mouse rsFRP2 (1169-FR-025/CF, R&D) was diluted in 50 μl PBS and injected at a concentration of 200 ng per mouse twice a week. The levels of sFRP2 were monitored by submandibular blood withdrawal every 2 weeks. All animal experiments were approved by the Institutional Animal Care and Use Committee (112503X_0) and were performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. From preliminary studies, we observed significant differences (more than 1.8 standard deviations, a very large effect size) in some of the outcomes between young and aged groups. As few as five samples in each group in this study afforded 80% power at a two-sided α of 0.05 to detect a difference of about 1.8 standard deviations in a continuous outcome between young and aged groups, but we increased the sample size slightly to account for potential loss of mice due to health issues associated with ageing. YUMM1.7 (2.5 × 105 cells) were suspended in Matrigel (500 μg ml−1) and injected subcutaneously into young (6 week) and aged (52 week) C57/BL6 mice (Taconic). When resulting tumours reached 200 mm3, mice were fed either AIN-76A chow or AIN-76A chow containing 417 mg kg−1 PLX4720. Tumour sizes were measured every 3–4 days using digital callipers, and tumour volumes were calculated using the following formula: volume = 0.5 × (length × width2). Time-to-event (survival) was determined by a fivefold increase in baseline volume (~1,000 mm3) and was limited by the development of skin necrosis. Upon the occurrence of necrosis, mice were euthanized. For subsequent experiments involving sFRP2 manipulation, Yumm1.7 cells overexpressing mCherry were used. One million cells were suspended in PBS and subcutaneously injected into either 6-week-old or 52-week-old male C57/BL6 mice (Taconic). For treatment with rsFRP2, the mice were injected with recombinant protein (200 ng ml−1) through the tail vein every 2 days as described above. For the experiments performed with sFRP2 blocking antibody (clone 80.8.6, MABC539, EMD Millipore), the mice were treated with 1 mg kg−1 antibody (either sFRP2 or isotype control, Biolegend, 400264) once a week through tail vein injections. WM35 and FS5 melanoma cells were seeded in 12-well plates and treated with conditioned media for 48 h. The cells were harvested in ice-cold PBS. The comet assay was performed using CometSlides (Trevigen). Briefly, 75 μl of a 2 × 105 cell suspension was mixed with 500 μl 1% low melting point agarose. Fifty microlitres of cell/agarose mixture was dropped into the wells and allowed to solidify. Slides were incubated in lysis buffer (1.2 M NaCl, 100 nM EDTA, 0.1% Sarkosyl, pH 10.0) for 1 h at 4 °C. Slides were then electrophoresed at 25 V for 12 min in alkaline buffer (0.03 M NaOH, 2 mM EDTA, pH 8.0). After fixation in 70% ethanol, comets were visualized by staining with SYBR Green (Fisher). The extent of DNA damage was measured as the artificial Olive Moment using Cometscore software downloaded from http://www.tritekcorp.com. Five thousand melanoma cells were seeded in a 96-well plate in triplicate. The cells were then treated with conditioned media from young and aged fibroblasts as well as DMEM as control. Hydrogen peroxide was added at 1 mM as control. After 72 h, ROS were measured using a Cell Meter Fluorimetric Intracellular Total ROS Activity Assay Kit (22901, AAT Bioquest) according to the manufacturer’s protocol. The plates were measured using a PerkinElmer EnVision Xcite Multilabel plate reader using the filters for excitation/emission (Ex/Em) = 520/605 nm. Alternatively, samples were imaged using PerkinElmer Operetta and the fluorescent signal was quantified with Harmony 3.0 software. The cell number was determined by Hoescht staining (Hoechst 33342, Invitrogen) and used to normalize the total fluorescence obtained from ROS staining. Topflash vectors were obtained from Addgene (M51 Super 8x FOPFlash/TOPFlash mutant, 12457; M50 Super 8x TOPFlash, 12456). WM35 cells were plated to achieve 70% confluency in six-well plates. Cells were co-transfected with pTK-RLuc (Green Renilla Luciferase) along with either Topflash or Fopflash vectors. After 5 h of transfection, cells were treated as required. After 48 h, cells were harvested and luciferase activity was measured using a Dual-Luciferase Reporter (DLR) Assay System (Promega, E1910). The firefly luciferase signal from each well was normalized to its Renilla luciferase signal. Topflash/fopflash signal was determined from each treatment and graphed using Graphpad/Prism. NAC was obtained from Sigma (A9165) and dissolved in sterile distilled H O (stock 1 M). Cells were treated for 48 h and analysed. After optimization, 10 mM final concentration was used for subsequent experiments. Melanoma cells were seeded into T25 flasks and incubated for 72 h with 6.5 ml conditioned fibroblast media prepared as described above. Cells were then washed in PBS, harvested with TrypLE Express and fractionated using the cellular fractionation kit (NE-PER, Fisher) as per the manufacturer’s protocol. Cell lysates were then separated on an SDS–polyacrylamide gel electrophoresis gel and visualized using standard western blotting procedures. Nunc MaxiSorp ELISA plates (ebiosciences) were coated with 50 μl of 3 μg ml−1 sFRP2 (ab137560, Abcam) overnight at 4 °C. Plates were washed in PBS containing 0.1% Tween and blocked in ELISA diluent (00-4202-56, eBioscience) for 2 h. Serum was diluted 1:100 before addition to the plates and incubated overnight at 4 °C. The next day, the plates were washed in PBS containing 0.1% Tween20 and incubated with detection antibody (MAB6838, R&D Systems) for 1 h at room temperature. Plates were washed and incubated with secondary antibody for 1 h. After washing, 100 μl TMB (00-4201-56, eBioscience) was added to the plates and incubated for 15 min The reaction was stopped using 50 μl of 2 N H SO and absorbance was measured at 450 nm. Fibroblasts were plated into 12-well dishes, incubated for 48 h, washed with PBS, and fixed in 2% formaldehyde/0.2% glutaraldehyde. Cells were then incubated in staining solution (150 mM NaCl, Sigma), 2 mM MgCl (Sigma), 5 mM K Fe(CN) (Millipore), 5 mM K Fe(CN) (Millipore), 40 mM Na PO (Sigma) pH 5.5, 20 mg ml−1 X-gal (Applichem, Darmstadt, Germany) at 37 °C overnight. Stain was removed and cells were stored in 70% glycerol before being imaged. All primers are listed in the Supplementary Information. Mouse tissue was snap frozen in liquid nitrogen immediately after harvesting. Ten milligrams of the lung tissue was homogenized and RNA was extracted using Trizol (Invitrogen) and RNeasy Mini kit (Qiagen) as described previously. One microgram of RNA was used to prepare cDNA using iscript DNA synthesis kit (1708891, Bio-Rad). cDNA was diluted 1:5 before use in further reactions. Each 20 μl well reaction comprised 10 μl Power SYBR Green Master mix (4367659, Invitrogen), 1 μl primer mix (Final concentration 0.5 μM), and 1 μl cDNA. Standard curves were generated for all primers and each set of primers was normalized to an 18 s primer pair. For in vitro studies, a Student’s t-test or Wilcoxon rank-sum (Mann–Whitney) test was performed for two-group comparisons. Estimate of variance was performed and parameters for the t-test were adjusted accordingly using Welch’s correction. An ANOVA or Kruskal–Wallis test with post-hoc Bonferroni’s or Holm–Šídák’s adjusted P values was used for multiple comparisons. For dose–response analysis, Spearman’s correlation was calculated. For in vivo studies, the indicated sample size for each experiment was designed to have 80% power at a two-sided α of 0.05 to detect a difference of large effect size of about 1.25 between two groups on a continuous measurement. The fold change in tumour volume at each time point after treatment relative to baseline was calculated and then the fold change in treatment group relative to the age-matched control group was used with a mixed-effect model to evaluate the treatment effect between age groups. Stata 12.0 (StataCorp) was used for data analysis for in vivo studies and human samples. For other experiments, Graphpad/Prism6 was used for plotting graphs and statistical analysis. Significance was designated as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Extended statistical analyses for patient data are provided in the Supplementary Information.


News Article
Site: http://www.nature.com/nature/current_issue/

All conditional Foxo1- and Myc-mutant mice were on a C57BL/6 genetic background and generated as described16, 18, 24, 25. For constitutive Cre-mediated recombination in ECs, Foxo1fl/fl or Rosa26-Foxo1CA mice were bred with Tie2-cre transgenic mice31. To avoid recombination in the female germline, only Tie2-cre-positive male mice were used for intercrossing. Embryos were collected from cre-negative females at the indicated time points and genotyping was performed from isolated yolk sacs. For inducible Cre-mediated recombination in ECs, floxed mice were bred with transgenic mice expressing the tamoxifen-inducible, Pdgfb promoter-driven creERT2 recombinase32. The degree of Cre-mediated recombination was assessed with the double-fluorescent Cre-reporter Rosa26-mT/mG33 allele, which was crossed into the respective mutant mice. For the analysis of angiogenesis in the postnatal mouse retina, Cre-mediated recombination was induced in newborn mice by intraperitoneal (i.p.) injections of 25 μl 4-hydroxy-tamoxifen (4-OHT; 2 mg ml−1; Sigma-Adrich) from postnatal day (P)1 to P4. Eyes were harvested at P5 or P21 for further analysis. In mosaic recombination experiments, 4-OHT (20 μl g−1 body weight of 0.02 mg ml−1) was injected i.p. at P3 and eyes were collected at P5. To induce Cre-mediated recombination in mouse embryos, 100 μl of 4-OHT (10 mg ml−1) was injected i.p. into pregnant females from embryonic day (E)8.5 to E10.5. Embryos were harvested at E11.5 for the analysis of angiogenesis in the embryonic hindbrain. The Rosa26-Foxo1CA, Rosa26-Myc and Rosa26-mT/mG alleles were kept heterozygous for the respective transgene in all experimental studies. Apart from the mosaic studies, control animals were littermate animals without cre expression. Male and female mice were used for the analysis, which were maintained under specific pathogen-free conditions. Experiments involving animals were conducted in accordance with institutional guidelines and laws, following protocols approved by local animal ethics committees and authorities (Regierungspraesidium Darmstadt). To analyse blood vessel growth in the postnatal retina, whole mouse eyes were fixed in 4% paraformaldehyde (PFA) on ice for 1 h. Eyes were washed in PBS before the retinas were dissected and partially cut into four leaflets. After blocking/permeabilization in 2% goat serum (Vector Laboratories), 1% BSA and 0.5% Triton X-100 (in PBS) for 1 h at room temperature, the retinas were incubated at 4 °C overnight in incubation buffer containing 1% goat serum, 0.5% BSA and 0.25% Triton X-100 (in PBS) and the primary antibody. Primary antibodies against the following proteins were used: cleaved caspase 3 (Cell Signaling Technology, #9664, 1:100), collagen IV (AbD Serotec, #2150-1470, 1:400), ERG 1/2/3 (Abcam, #ab92513, 1:200), FOXO1 (Cell Signaling Technology, #2880, 1:100), GFP (Invitrogen, #A21311, 1:100), ICAM2 (BD Biosciences, #553326, 1:200), MYC (Millipore, 06-340, 1:100), PECAM-1 (R&D Systems, AF3628, 1:400), phospho-histone H3 (Chemicon, #06-570, 1:100), TER119 (BD Biosciences, #553670, 1:100), and VE-cadherin (BD Biosciences, #555289, 1:25). After four washes with 0.1% Triton X-100 in PBS (PBST), retinas were incubated with Alexa-Fluor 488-, Alexa-Fluor 555- or Alexa-Fluor 647-conjugated secondary antibodies (Invitrogen, 1:400) for 2 h at room temperature. For staining ECs with isolectin B4 (IB4), retinas were washed with PBLEC buffer (1 mM CaCl , 1 mM MgCl , 1 mM MnCl and 1% Triton X-100 in PBS) and incubated with biotinylated IB4 (Griffonia simplicifolia, #B1205, Vector Laboratories, 1:100) diluted in PBLEC buffer. After washing, retinas were incubated in Alexa-Fluor-coupled streptavidin (Invitrogen, #S21374, 1:200) for 2 h at room temperature. For nuclear counterstain, retinas were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, #D9542, 1:1,000) for 15 min following washes with PBST and PBS. The labelling of proliferating cells with BrdU was performed in P5 pups. In brief, 50 mg kg−1 of BrdU (Invitrogen, #B23151) per pup was injected i.p. 3 h before they were killed. Retinas were fixed for 2 h in 4% PFA and then incubated for 1 h in 65 °C warm formamide, followed by an incubation of 30 min in 2 N HCl. Afterwards retinas were washed twice with 0.1 M Tris-HCl (pH 8) and then blocked in 1% BSA, 0.5% Tween 20 in PBS and incubated overnight at 4 °C with a mouse anti-BrdU antibody (BD Biosciences, #347580, 1:50). The detection was performed with Alexa-Fluor-488 anti-mouse secondary antibody (Invitrogen, A21202, 1:400). After the BrdU staining, retinas were processed for the IB4 staining as described earlier. The dissection of the embryonic hindbrain was performed as described34. After overnight fixation in 4% PFA, dissected hindbrains were incubated in a blocking solution containing 10% serum, 1% BSA and 0.5% Triton X-100 in PBS at 4 °C. After washes with PBS, hindbrains were incubated for 1 h in PBLEC buffer before the overnight incubation with Alexa-Fluor-conjugated IB4 (Invitrogen, #I21411, 1:100 in PBLEC) at 4 °C. Hindbrains were washed with PBS and stained with DAPI. Retinas and embryonic hindbrains were flat-mounted with Vectashield (Vector Laboratories) and examined by confocal laser microscopy (Leica TCS SP5 or SP8). Immunostainings were carried out in tissues from littermates and processed under the same conditions. HUVECs were seeded on glass-bottom culture dishes (Mattek) and cultured at 37 °C and 5% CO . To detect autophagy, cells were washed and fixed with 4% PFA for 20 min at room temperature. Permeabilization was performed in 1% BSA, 10% donkey serum and 0.5% Tween-20 in PBS. Cells were stained for anti-LC3A/B (Cell Signaling Technology, #12741, 1:400), Phalloidin-TRITC (Sigma Aldrich, #P1951, 1:500) and DAPI in incubation buffer (0.5% BSA, 5% donkey serum and 0.25% Tween-20 in PBS). After washes with PBST, samples were incubated with Alexa-Fluor-conjugated secondary antibodies (Invitrogen, 1:200). Cells were washed and mounted in VectaShield. As a positive control, HUVECs were treated with 50 μM chloroquine overnight before fixation. Stained tissue/cells were analysed at high resolution with a TCS SP8 confocal microscope (Leica). Volocity (Perkin Elmer), Fiji/ImageJ, Photoshop (Adobe) and Adobe Illustrator (Adobe) software were used for image acquisition and processing. For all of the images in which the levels of immunostaining were compared, settings for laser excitation and confocal scanner detection were kept constant between groups. All quantifications were done on high-resolution confocal images of thin z-sections of the sample using the Volocity (Perkin Elmer) software. In the retina, endothelial coverage, the number of endothelial branchpoints, and the average vessel branch diameter were quantified behind the angiogenic front in a region between an artery and a vein. In the embryonic hindbrain, randomly chosen fields were used to quantify the vascularization in the ventricular zone. All parameters were quantified in a minimum of four vascularized fields per sample. Endothelial coverage was determined by assessing the ratio of the IB4-positive area to the total area of the vascularized field (sized 200 μm × 200 μm), and expressed as a percentage of the area covered by IB4-positive ECs. Average vessel diameter was analysed by assessing the diameter of individual vessel branches in a vascularized field (sized 200 μm × 200 μm), which was used to calculate the mean diameter in each field. The diameter of individual vessel branches was averaged from three measurements taken at the proximal, middle and distal part of the vessel segment. The number of filopodial extensions was quantified at the angiogenic front. The total number of filopodia was normalized to a vessel length of 100 μm at the angiogenic front, which was defined and measured according to published protocols35. For quantifying vascular outgrowth in the mouse retina, the distance of vessel growth from the centre of the optic nerve to the periphery was measured in each leaflet of a dissected retina, which was used to calculate the mean value for each sample. The number of ERG/IB4- and BrdU/IB4-labelled cells was counted in at least four fields sized 200 μm × 200 μm per sample. Because of the lower incidence of pHH3-positive ECs, the number of pHH3/IB4- double-positive cells was quantified in larger fields (sized 580 μm × 580 μm). For the quantification of the mosaic control (Pdgfb-creERT2;Rosa26-Foxo1+/+;Rosa26-mTmGfl/+) and Foxo1iEC-CA (Pdgfb-creERT2;Rosa26-Foxo1CA/+;Rosa26-mTmGfl/+) retinas, the GFP/IB4 double-positive area per field was determined and divided by the total IB4-positive area. The percentage of the GFP/IB4 double-positive area per total IB4 area was measured in four fields (400 μm × 400 μm) per sample and used to calculate the mean value. For the quantification of nuclear FOXO1 expression in control and Foxo1iEC-CA mice, high-resolution confocal images were taken with a ×40 objective. The resulting images were analysed with the Bitplane Imaris software. Vessels were first segmented using the Surface module in Imaris. FOXO1 immunofluorescence was then used to set a threshold in the new vascular surface area, in which only CD31-positive nuclei were selected (Surface module). The sum intensity of the nuclear FOXO1 fluorescence was divided by the total vascular area to adjust for differences in vascular density on each image. An average of six images per sample was quantified in three animals per group. All of the images shown are representative of the vascular phenotype observed in samples from at least two distinct litters per group. Pooled HUVECs were purchased from Lonza and authenticated by marker expression (CD31/CD105 double-positive) and morphology. HUVECs were cultured in endothelial basal medium (EBM; Lonza) supplemented with hydrocortisone (1 μg ml−1), bovine brain extract (12 μg ml−1), gentamicin (50 μg ml−1), amphotericin B (50 ng ml−1), epidermal growth factor (10 ng ml−1) and 10% fetal bovine serum (FBS; Life Technologies). HUVECs were tested negative for mycoplasma and cultured until the fourth passage. The isolation of mouse lung ECs was performed as described36. In brief, adult mice were killed, lungs were removed and incubated with dispase. The homogenate was filtered through a cell strainer, collected by centrifugation, and washed with PBS containing 0.1% BSA (PBSB). The resulting cell suspension was incubated with rat anti-mouse VE-cadherin antibody- (BD Pharmingen, #555289) coated magnetic beads (Dynabeads, Invitrogen, #11035). Next, the beads were washed with PBSB and then resuspended in DMEM/F12 (Invitrogen) supplemented with 20% FCS, endothelial growth factor (Promocell, #C-30140), penicillin and streptomycin. The isolated cells were seeded on gelatin-coated culture dishes and re-purified with the VE-cadherin antibody during the first three passages. Sub-confluent HUVECs were infected with adenoviruses to overexpress constitutively active human FOXO1–Flag (FOXO1CA)37, human c-MYC–HA38 (Vector Biolabs) and GFP or LacZ as a control. HUVECs (70–80% confluent) were incubated in EBM containing 0.1% BSA for 4 h. Prior to infection, adenoviruses were incubated with an antennapedia-derived peptide (Eurogentec) to facilitate the infection. The mixture was then applied to the HUVECs cultured in EBM containing 0.1% BSA and incubated for 4 h. Thereafter, the cells were washed five times and cultured in EBM with 10% FCS and supplements. The adenoviral infection of murine ECs was performed with adenoviruses encoding for Cre or GFP (Vector Biolabs) as a control. To silence FOXO1, MYC or MXI1 gene expression, HUVECs were transfected with a pool of siRNA duplexes directed against human FOXO1, human c-MYC or human MXI1 (ON-TARGETplus SMARTpool, Dharmacon). A negative control pool of four siRNAs designed and microarray-tested for minimal targeting of human, mouse or rat genes was used as a control (ON-TARGETplus Non-targeting pool, Dharmacon). HUVECs were transfected with 50 nM of the indicated siRNAs using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s recommendations. Total RNA quality was verified using the Agilent Bioanalyser and the 6000 nano kit. RNA was labelled according to the Affymetrix Whole Transcript Sense Target Labelling protocol. Affymetrix GeneChip Human Gene 1.0 ST arrays were hybridized, processed and scanned using the appropriate Affymetrix protocols. Data were analysed using the Affymetrix expression console using the RMA algorithm, statistical analysis was done using DNAStar Arraystar 11. Heat maps were generated using GENE-E, publicly available from the Broad Institute (http://www.broadinstitute.org/cancer/software/GENE-E/). For gene set enrichment analysis (GSEA), gene set collections from the Molecular Signatures Database (MSigDB) 4.0 (http://www.broadinstitute.org/gsea/msigdb/) were used for the analysis of the endothelial FOXO1 and MYC transcriptomes. RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed on 2 μg of total RNA using the M-MLV reverse transcriptase (Invitrogen). qPCR was performed with TaqMan Gene Expression Master Mix (Applied Biosystems) and TaqMan probes (TaqMan Gene Expression Assays) available from Applied Biosystems. TaqMan Gene Expression Assays used were as follows: human ACTB Hs99999903_m1; CCNB2 Hs00270424_m1; CCND1 Hs00765553_m1; CCND2 Hs00153380_m1; CDK4 Hs00262861_m1; c-MYC Hs00153408_m1; ENO1 Hs00361415_m1; FASN Hs01005622_m1; FBXW7 Hs00217794_m1; FOXO1 Hs01054576_m1; LDHA Hs00855332_g1; LDHB Hs00929956_m1; MXI1 Hs00365651_m1; PKM2 Hs00987254_m1. Mouse probes were: Actb Mm 00607939_s1; Myc Mm00487804_m1. All qPCR reactions were run on a StepOnePlus real-time PCR instrument (Applied Biosystems) and data were calculated using the ∆∆C method. Western blot analyses were performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, HUVECs were lysed in RIPA buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0) supplemented with a protease inhibitor mix (Complete Mini Protease Inhibitor cocktail tablets, Roche) and phenylmethylsulfonyl fluoride. Proteins were separated by SDS–PAGE and blotted onto nitrocellulose membranes (Bio-Rad). Membranes were probed with specific primary antibodies and then with peroxidase-conjugated secondary antibodies. The following antibodies were used: AMPKα (Cell Signaling Technology, #2532, 1:1,000), caspase 3 (Cell Signaling Technology, #9662, 1:1,000), cleaved caspase 3 (Asp175) (Cell Signaling Technology, #9664, 1:1,000), cleaved PARP (Cell Signaling Technology, #5625, 1:1,000), c-MYC (Cell Signaling Technology, #9402, 1:1,000), FBXW7 (Abcam, #12292, 1:500), Flag M2 (Sigma, #F-3165, 1:1,000), FOXO1 (Cell Signaling Technology, #2880, 1:1,000), HA (Covance, clone 16B12, MMS-101P, 1:1,000), LC3A/B (Cell Signaling Technology, #12741, 1:1,000), MXI1 (Santa Cruz, SC-1042, 1:500), P-ACC (Cell Signaling Technology, #3661, 1:1,000), P-AMPKα (Thr 172) (Cell Signaling Technology, #2535, 1:1,000), PARP (Cell Signaling Technology, #9532, 1:1,000), Tubulin (Cell Signaling Technology, #2148, 1:1,000). The bands were visualized by chemiluminescence using an ECL detection kit (Clarity Western ECL Substrate, Bio-Rad) and a ChemiDoc MP Imaging System (Bio-Rad). The gel source data of the western blot analysis is illustrated in Supplementary Fig. 1. Quantification of band intensities by densitometry was carried out using the Image Lab software (Bio-Rad). Extracellular acidification (ECAR) and oxygen consumption (OCR) rates were measured using the Seahorse XFe96 analyser (Seahorse Bioscience) following the manufacturer’s protocols. Briefly, ECAR and OCR were measured 4 h after seeding HUVECs (40,000 cells per well) on fibronectin-coated XFe96 microplates. HUVECs were maintained in non-buffered assay medium in a non-CO incubator for 1 h before the assay. The Glycolysis stress test kit (Seahorse Bioscience) was used to monitor the extracellular acidification rate under various conditions. Three baseline recordings were made, followed by sequential injection of glucose (10 mM), the mitochondrial/ATP synthase inhibitor oligomycin (3 μM), and the glycolysis inhibitor 2-deoxy-d-glucose (2-DG; 100 mM). The Mito stress test kit was used to assay the mitochondrial respiration rate under basal conditions, in the presence of the ATP synthase inhibitor oligomycin (3 μM), the mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenyl-hydrazone (FCCP; 1 μM), and the respiratory chain inhibitors antimycin A (1.5 μM) and rotenone (3 μM). To measure glycolysis in ECs, HUVECs were incubated for 2 h in growth medium containing 80 μCi mmol−1 [5-3H]-d-glucose (Perkin Elmer). Thereafter, supernatant was transferred into glass vials sealed with rubber stoppers. 3H O was captured in hanging wells containing a Whatman paper soaked with H O over a period of 48 h at 37 °C to reach saturation4. Radioactivity was determined by liquid scintillation counting and normalized to protein content. Lactate concentration in the HUVEC culture media was measured by using a Lactate Assay Kit (Biovision) following the instructions of the manufacturer. Glucose uptake was assessed by analysing the uptake of 2-DG with a Colorimetric Assay (BioVision). ATP was measured from lysates from HUVECs (1 × 106 per ml) with an ATP Bioluminescence Assay Kit CLS II (Roche) according to the instructions of the manufacturer. Intracellular ROS levels were determined using CM-H DCFDA dye (Life technologies). Dye was reconstituted in DMSO (10 mM) and diluted 1:1,000 in PBS containing CaCl and MagCl as working solution. Twenty-four hours after transduction, 1 × 106 cells were incubated in 1 ml working solution for 40 min at 37 °C in the dark. Subsequently the fluorescence of 10,000 living endothelial cells per sample was measured at the BD FACS LSR II flow cytometer. The assays were performed with adenoviruses, which did not co-express fluorescent reporter genes. Data were analysed using BD FACSDiva software (version 8.0.1). To detect senescence-associated β-galactosidase activity in HUVECs, a cellular senescence assay kit (#KAA002, Chemicon) was used according to the manufacturer’s instructions. Briefly, cells were fixed in 1 ml fixing solution at room temperature for 15 min. Two millilitres of freshly prepared SA-β-gal detection solution was added and cells were incubated overnight at 37 °C without CO and protected from light. Then the detection solution was removed and cells were washed and mounted in 70% glycerol in PBS. H O -treated HUVECs were used as a positive control. Statistical analysis was performed by unpaired, two-tailed Student’s t-test, or non-parametric one-way ANOVA followed by Bonferroni’s multiple comparison test unless mentioned otherwise. For all bar graphs, data are represented as mean ± s.d. P values < 0.05 were considered significant. All calculations were performed using GraphPad Prism software. No randomization or blinding was used and no animals were excluded from the analysis. Sample sizes were selected on the basis of published protocols34, 35 and previous experiments. Several independent experiments were performed to guarantee reproducibility and robustness of findings.


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Wild-type C57Bl6/J male mice were group housed three to five to a cage and kept on a reverse 12 h light/dark cycle with ad libitum food and water (except in virtual reality behaviour experiments, for which water was restricted; details later). Experimental protocols were approved by Stanford University Institutional Animal Care and Use Committee (IACUC) and meet the guidelines of the National Institutes of Health guide for the Care and Use of Laboratory Animals. The target number of subjects used in each experiment was determined based on numbers reported in published studies. No statistical methods were used to predetermine sample size. Viral injections were carried out under protocols approved by Stanford University IACUC and were performed in mice anaesthetized with 1–2% isoflurane using a stereotaxic apparatus (Kopf Instruments). For retrograde tracing, 4–5-week-old wild-type male mice were injected slowly (50 nl min−1) with small amounts (200 nl) of highly concentrated glycoprotein-deleted rabies virus tagged with tdTomato (RV-tdTomato)22 in the dorsal hippocampus (A/P: −1.5 mm; M/L: +1.75 mm; D/V: −1.8 mm) with a 1 μl Hamilton syringe and a 35-gauge bevelled needle (World Precision Instruments) under the control of a UMP3 syringe pump (WPI). Following injections, the incisions were closed using Vetbond tissue adhesive (Fischer), and mice were allowed to recover and were housed for 5 days to allow for expression before their brains were collected for histological analysis. In the case of anterograde tracing, 4–5-week-old wild-type male mice were injected (150 nl min−1) with 500 nl of AAV5-CaMKIIα::eYFP (titre: 2 × 1012 vg ml−1) in dorsal anterior cingulate (A/P: +1; M/L: −0.35; D/V: +1.2) and were housed for 30 days to allow for expression in terminals before collection of brains for histological analysis. For histological analysis, injected mice were transcardially perfused with ice-cold 1× PBS, immediately followed by perfusion of 4% paraformaldehyde (PFA). Brains were fixed overnight in PFA, then transferred to a 30% sucrose/PBS solution. Coronal sections of either 40 µm (for retrograde tracing with RV) prepared using a freezing microtome (Leica) or 300 µm (for anterograde tracing with AAV5) prepared using a vibratome (Leica) were collected and stored in a cryoprotectant solution (25% glycerol, 30% ethylene glycol, in PBS) until further processing. For DAPI staining, slices were washed in PBS, incubated for 20 min with DAPI at 1:50,000, washed again in PBS, then mounted with PVA-DABCO (Sigma). A scanning confocal microscope (TCS SP5, Leica) and LAS AF software (Leica) was used to obtain and analyse images. Acute brain slices were prepared from mice 6–8 weeks following viral injection with AAV5-CaMKIIα::ChR2(H134R)-eYFP, to allow sufficient time for channelrhodopsin to express in axon terminals. After lethal anaesthesia, mice were transcardially perfused with cold sucrose slicing solution (see later) before decapitation, following which the brain was rapidly extracted and submerged in ice-cold sucrose-based slicing solution (234 mM sucrose, 26 mM NaHCO , 11 mM glucose, 10 mM MgSO ·7H O, 2.5 KCl, 1.25 mM NaH PO ·H O, 0.5 mM CaCl ·2H O). Coronal hippocampal slices (300 μm thick) were cut on a Leica vibratome (Leica VT1000S) in sucrose solution and then submerged in a hypertonic recovery solution (artificial cerebrospinal fluid (ACSF) at an 8% increased osmolarity) at 33 °C for 15 min before being transferred to standard ACSF (123 mM NaCl, 26 mM NaHCO , 11 mM glucose, 3 mM KCl, 2 mM CaCl ·2H O, 1.25 mM NaH PO ·H O, 1 mM MgCl ·6H O) for a further 45 min at 33 °C, at which point they were transferred to room temperature. Whole-cell patch-clamp recordings from CA3/CA1 hippocampal neurons were performed on an upright Leica DM-LFSA microscope. Borosilicate glass (Sutter Instruments) pipette resistances were pulled to 3–6 MΩ and filled with potassium gluconate intracellular solution (130 mM KGluconate, 10 mM KCl, 10 mM HEPES, 10 mM EGTA, 2 mM MgCl , pH adjusted with KOH to 7.3). Voltage and current-clamp recordings were performed using pClamp (Axon Instruments). Cells with leak current greater than −200 pA or series resistance greater than 35 MΩ were excluded. Light stimulation was performed using a 300 W DG-4 lamp (Sutter Instruments) with an external filter for blue light (wavelength in nm/bandwidth in nm: 470/20). Light pulses (2–5 ms pulse width) were delivered through a ×40, 0.8 NA water-immersion objective at 4–10 mW mm−2 light power density. Latencies were measured as light pulse start to EPSC initiation. After injection with the indicated virus (for example, CAV or RV, expressing ChR2, eNpHR3.0, or eYFP) at the appropriate location (for example, cingulate, hippocampus, or medial septum), as described in Fig. 2 and Extended Data Figs 2 and 4, 5-week-old wild-type male mice were implanted with implantable fibre-optic lightguides (IFLs) consisting of a 2.5-mm-diameter metal ferrule with 0.22 NA and a 200-µm-thick protruding cleaved bare optic fibre cut to the desired length (Thorlabs) as previously described36, either at the injection site (typically ~0.2 mm dorsal to the injection site) or at the terminals for stimulation experiments as indicated in the figure legends. For inhibition experiments, dual fibre-optic cannulas of 200 μm thickness and 0.22 NA spaced 0.7 mm apart were used to target anterior cingulate bilaterally, and two-ferrule cannulas spaced 3 mm apart were used to target hippocampus bilaterally. Mice were typically allowed to recover and housed for 1 month to allow for adequate expression before behavioural testing. All animals undergoing behavioural experiments were acclimated to a 12 h reverse light/dark cycle, handled for several days, and before behavioural testing, were acclimated to the room in which experiments were to be conducted for at least 30 min. The fear conditioning apparatus consisted of a square conditioning cage (18 × 18 × 30 cm) with a grid floor wired to a shock generator and a scrambler, surrounded by an acoustic chamber (Coulburn Instruments). The apparatus was modified to enable light delivery during retrieval testing. Contextual fear conditioning was performed by placing mice in the conditioning cage (visual cues: bare walls; tactile cues: grid floor; odour cues: 70% ethanol) for 6 min, while receiving four 2 s shock pulses of 7 mA each at 1 min intervals, with the first shock presented 2 min after placing the mouse in the conditioning context. A fraction of animals of the same cohort were not fear conditioned, and instead served as a control group that were just exposed to the conditioning context for the same amount of time (6 min) but did not receive any associated shocks. The following day, all mice were tested in a different ‘neutral’ cage (visual cues: coloured shapes; tactile cues: smooth paper towel covered plexiglass floor; odour cues: 1% acetic acid) for light-mediated fear retrieval. For stimulation experiments, optical stimulation through the fibre-optic connector was administered by delivering light through a patch-cord connected to a 473 nm laser in 30 s light-on/1 min light-off sessions. During light-on sessions, stimulation was delivered at 20 Hz, 15 ms pulses, with 8–10 mW power at the fibre tip. On the third day, all mice were then returned to the original conditioning context for 2.5 min to assess intact natural fear memory retrieval. In some cases, subsequent extinction of fear memory was performed by placing mice in the original conditioning chamber for three consecutive days, for 5 min each, without shock. Light-induced fear retrieval was then tested in the neutral context 24 h following the last extinction training session. Subsequent reinstatement was performed by again placing the animals back in the conditioning context for one 6 min interval and providing four 2 s shock pulses of 7 mA each at 1 min intervals. A final light-induced fear retrieval testing was performed 24 h later as described earlier. For loss of function experiments, optical inhibition through a fibre-optic connector was administered by delivering light through a dual patch-cord connected to a 589 nm laser. Constant light at 8–10 mW was used at the fibre tip to deliver inhibition either at cell bodies or terminals. On the first day, both eNpHR3.0 and eYFP control groups were trained to contextual fear conditioning as described earlier, and on the second day, mice were allowed to perform retrieval as usual during light off for the first 2 min, to assess baseline freezing in each animal. Then light was turned on for the next 30 s (not longer, as the potential for extinction related unfreezing could confound light-related unfreezing at time points succeeding the typical 2–3-min retrieval protocol). Freezing scores during the 30 s light sessions were compared with the per cent freezing during 30 s of the immediately preceding light-off sessions. On the third day, all mice underwent retrieval in the conditioning context for 2 min with light off to test for reversal of light-induced behaviour. After context conditioning and retrieval, all mice subsequently underwent auditory-cued conditioning (cued conditioning was done separately from context conditioning to ensure robust conditioning to both context and cue, since when performed together, mice often develop robust conditioning to tone (the more salient cue) and only weak conditioning to context). To perform auditory-cued fear conditioning, mice were placed in a different context (with coloured shapes as visual cues and a smooth floor), for 6 min, where after the first 2 min, four 20 s auditory cues consisting of 2.9 kHz tone was played at 1 min intervals, each followed by a 2 s 7 mA shock. Retrieval on the subsequent day was performed by presenting the tone four times (two during light off and two during light on) at 1 min intervals and per cent freezing was assessed during the 20 s post-tone compared with the immediately preceding 20 s during tone, for both light-off and light-on conditions. Latency measures were performed as separate experiments, using the same cohorts; after finishing contextual and cued conditioning, these mice were retrained (contextually fear conditioned) to the first conditioning context. On the following day, 2 min retrieval was performed in the conditioning context with light on the entire time to test for latency to freezing, where latency was defined as the first instance in time that the animal was immobile for 5 consecutive seconds. Freezing in all experiments was scored by an experimenter blinded to the treatment group. Randomization of animals to experimental and control groups was performed by an experimenter with no explicit randomization algorithm used. All of the results were analysed by Student’s t-test or two-way ANOVA, followed by post-hoc tests, as applicable. C57BL/6J male mice were injected with 500 nl of AAVdj-CaMKIIα::GCaMP6m in CA3 (A/P: −1.7, M/L: +1.9, D/V: −1.9) and allowed to recover for at least 1 week before surgical implantation of a cranial window above CA2/CA3 for optical access similar to previously described hippocampal preps24. Briefly, mice were injected with 80 mg kg−1/6 mg kg−1 of ketamine/xylazine intraperitoneally, and maintained under 1.0–2.0% isoflurane throughout. For optimal window placement to access CA2/CA3, the mouse’s head was angled during surgery such that the skull location at the CA2/CA3 injection site was level and exactly perpendicular to dorsal views of the head. A circular titanium headplate (7 mm in diameter) was centred over CA2/CA3 and adhered to the skull with adhesive cement (Metabond; Parkell) and a ~3 mm craniotomy was made in the centre using a trephine (Fisher). Parts of cortical region S1 and of parietal association cortex were vacuum-aspirated, with care taken to avoid the ventricle, until white matter was visible above the hippocampus. Vacuum aspiration was done with a 27-gauge blunt needle while irrigating with chilled 1× PBS. The top layer of white matter above the hippocampus was further removed by vacuum aspiration with a 31-gauge blunt needle, but care was taken to preserve deep layers of external capsule and the alveus (to preserve afferents and efferents to hippocampus). A forceps was used to manually insert a cylindrical borosilicate glass implant until the floor of the implant rested against the hippocampus. The implant was constructed from a 3.0-mm-diameter glass capillary tube (Friedrich & Dimmock) custom cut to 1.5 mm length, adhered on one end to a 3.0 mm diameter coverslip of #0 thickness (Warner Instruments) using UV-curing optical glue (Norland Products). The top of the implant extruding from the craniotomy was then secured to the skull using Metabond adhesive cement. After surgery, mice were given 5 mg kg−1 carprofen subcutaneously and allowed to recover for at least 1 week before behaviour training. To ensure that the above manipulations (including GCaMP6m virus injection into CA3, GCaMP6m expression, and surgical excavation of certain regions of cortex) did not affect normal physiological properties of the hippocampus, we performed control experiments to assess Ca2+-dependent physiology in weakly versus strongly expressing CA3 neurons in vitro, spontaneous activity in weakly versus strongly expressing CA3 neurons in vivo, and behavioural measurements before and after placement of the cannula (Extended Data Fig. 3). We used a custom built virtual reality environment, modified from previously reported versions24, 51. A 200-mm-diameter styrofoam ball (Graham Sweet Studios) was axially fixed with a 6-mm-diameter assembly rod (Thorlabs) passing through the centre of the ball and resting on 90° post holders (Thorlabs) at each end, allowing free forward and backward rotation of the ball. Mice were head-fixed in place above the centre of the ball using a headplate mount52. Virtual environments were designed in game development software Unity3d (http://www.unity3d.com). The virtual environment was displayed by back-projection onto projector screen fabric stretched over a clear acrylic hemisphere with a 14-inch diameter placed ~20 cm in front of the centre of the mouse. The screen encompasses ~220° of the mouse’s field of view. The virtual environment was back-projected onto this screen using two laser-scanning projectors (Microvision), each projector covering one half of the screen. To create a flat image on the three-dimensional screen, we warped the two-dimensional image of the virtual environment using video manipulation software (Madmapper). The game engine allowed scripts written in JavaScript or C# to trigger external events based on the mouse’s interactions with the virtual environment by communicating over a TCP socket to custom Python control software. A LabJackU6 (http://labjack.com) was used to time-lock virtual environment events and imaging frame times, to record mouse licking behaviour with incoming TTL pulses from the lickometer (Island Motion), and to send TTL pulses to deliver solenoid-gated water rewards (delivered from a gravity-assisted syringe attached to tubing connected to the lickometer) and aversive air puffs (from a compressed air tank to a tube ending in a pipette tip facing the mouse’s snout). Tactile and odour cues were fixed directly to each of two Styrofoam balls representing the two separate contexts. Auditory stimuli were presented through speakers situated behind the animal. The mouse’s movements on the ball were recorded using an optical computer mouse (Logitech) that interfaced with the virtual environment software. For fear conditioning in the virtual environment, mice were water restricted (>80% pre-deprivation weight) and habituated to handling, head-fixation, and the virtual environment for at least 2 weeks, with free access to small water rewards (~0.5 μl per 10 licks) while on the ball. By the end of 2 weeks (one 5-min session per day), mice appeared comfortable and alert on the ball. After habituation, mice underwent a 4-day fear conditioning training and testing protocol. On day 1, mice were exposed to two contexts that differed in visual (blue triangles versus pink vertical stripes), tactile (smooth side of Velcro versus sharp side of Velcro fixed onto running ball), odorant (acetic acid versus ethanol), and auditory cues (8 kHz phasic tone versus 3 kHz pure tone) for 5 min each. On day 2, mice were provided with 8 aversive air puffs to the snout (500 ms, 10 psi) at randomly timed intervals throughout the 5 min while in the fear context, but not while in the neutral context for 5 min. On days 3 and 30, mice were placed back in each of the two contexts for 5 min for retrieval. Five mice were imaged on all days, in 5-min sessions, during exposure, training, and retrieval. We used a resonant galvanometer two-photon microscope (Prairie Technologies). We used the genetically encoded calcium indicator GCaMP6m in all experiments (GCaMP6m was amplified from Addgene plasmid #40754 by PCR and subcloned into an AAV backbone under the control of the CaMKIIa promoter.) All experiments were performed using a Coherent Ultra II Ti-Sapphire pulsed laser tuned to 920 nm to excite GCaMP6m through a ×20 0.5 LUMPlanFL/N (Olympus) water-immersion objective interfacing with the implanted cannula through a few drops of distilled water. Fluorescence was detected through gallium arsenide phosphide (GaAsP) photomultiplier tubes (PMTs) using the PrairieView acquisition software. High speed z stacks were collected in the green channel (using a 520/44 bandpass filter, Semrock) at 512 × 512 pixels covering each x–y plane of 500 µm × 500 µm over a depth of ~100 µm (3–7 z slices ~10–20 µm apart) by coupling the 30 Hz rapid resonant scanning (x–y) to a Z-piezo to achieve ~6 Hz per volume. Later, we describe the methods to extract cells (pre-processing), obtain cellular-level activity (ΔF/F) measures (processing), and evaluate population-level activity measures (post-processing). In statistical analysis of the post-processed data, both parametric and non-parametric tests were employed as appropriate. In cases where normality could not be assessed (low sample sizes), we ensured that there were no significant outliers (by Grubbs’ test) and that the variance between groups was not significantly different (by Levene’s Test). Time series data sets were x–y motion corrected with ImageJ plug-in Stack Reg using rigid body transformations. Cell extraction was then performed sequentially, by first computing cell segments automatically followed by manual quality control for missed cells, non-cells, or conjoined cells. For initial automatic extraction, we used a metric based on image threshold intensity, variance and skewness. Images with high contrast-to-noise ratio, wherein clear thresholds in maximum intensity separated cells and background, were fully segmented with the former. In the remainder of cases, cells were distinguished from background based on standard deviation across time (high for active cells), or skewness (asymmetry) in intensity across time53. This resulted in a general mathematical criterion to define cell-masks at each voxel location (i, j, k): where is the indicator function (=1 if the condition is satisfied); is the standard deviation of intensity over time defined as and skewness is defined as E is the expectation operator; F , σ and s represent cut-offs for image intensity, standard deviation and skewness respectively. Coefficients α , β and γ are chosen on an image-specific basis; if thresholding is sufficient β and γ are chosen to be zero, otherwise coefficients are iterated to obtain a cell mask containing the largest population of active cells (evaluated by inspection). Automatic cell extraction was then followed by manual cell-by-cell curation to identify cells that were not extracted using the automated algorithm. This occurs when cell boundaries may not be captured due to non-translational motion artefact in the original imaging, and/or lack of clear cut-offs F , σ and s differentiating cell and background. For these cases, cell detection is performed with a manual editing step involving comparison of the automated cell-mask to the raw image data, and by using a Gaussian filter was applied on the edited image to smooth edges, and edge-detection54 was used to define cell boundaries. The interior of the resulting cells were filled, and the final cell masks were eroded to minimize contamination from neuropil signal. Each cell was labelled with a unique cell identifier for the next stage; custom-written MATLAB scripts were used for all steps, and are available on request. Calculation of ΔF/F. For each cell identified in step 1, the intensity value F was obtained by averaging over all pixels inside the ROI to compute a space-averaged value for each frame (corresponding to a single time point). These are used to define ΔF/F in each cell as where is the baseline fluorescence, calculated as the mean of the fluorescence values for a given cell, continuously acquired over a 20 s moving time window to account for slow time-scale changes in fluorescence. Given the sparse firing of neurons in our data set, the mean served as an accurate estimate of baseline activity (fluorescence). Furthermore, the main results of the study were not influenced by using the median or 8th percentile as the baseline (and correlations were independent of baseline definition). We used an approach similar to that outlined previously24 to identify significant transients in each neuron, as well as to estimate and remove effects that may be related to motion artefacts. Briefly, to estimate the occurrence rate of potential motion-related fluorescence changes in the signal, all negative deflections in the ΔF/F trace were assumed to be due to motion. Because motion-related fluorescence changes should be equally likely to generate positive- or negative-going changes, positive and negative deflections in the ΔF/F curve that are attributable to motion should occur at the same frequency and can be subtracted out of the signal by using the rate of occurrence of the negative-going transients as an estimate of the rate of motion-related positive-going transients. To determine statistically significant transients, we first calculated an estimate of the noise for each cell using an iterative approach: (1) initialize a cut-off value that separates signal and noise, (2) calculate the standard deviation (σ) of all ΔF/F values that fall below the cut-off, and (3) compare 3σ to the cut-off. In this analysis, the goal is to find an estimate of standard deviation (σ) of the noise, defined for time periods that are unlikely to contain neural events (that is, using the iterative approach to estimate the σ of the noise, rather than calculate standard deviation for the entire time epoch, which would contain real events). For each iteration of the analysis, if |cut-off − 3σ| < tolerance, the program terminates (where tolerance = 0.02). If cut-off >3σ, the program increases the cut-off by 10% and goes back to step 1. If cut-off <3σ, it reduces the cut-off by 10% and goes back to step 1. This approach helped ensure that neuronal activity-generated events in ΔF/F are not included in the estimation of noise and avoided the need for manually selecting epoch intervals on a cell-by-cell basis that did not contain an event in order to estimate noise. Subsequently, we analysed positive- and negative-going transients to further determine the false positive rate. Transient onsets are defined as the times when the ΔF/F exceeds 2σ and offset is defined as the time at when a given transient falls below 0.5 σ3. A histogram of the number of transients that exist for each σ threshold value (that is, >2σ, >3σ, >4σ), for various durations, is extracted, where negative-going transients are to the left of the ordinate and plotted in red (Extended Data Fig. 4c–f). The ratio of the number of negative to positive going transients is calculated for different transient durations across three amplitude levels (2σ, 3σ, 4σ), and serves as our estimate of false positive rate. Following from the reasoning described earlier, this ratio will be 50% when the motion-based noise significantly exceeds the signal. We plot the false positive ratio for the different scenarios described earlier, and choose the amplitude (in σ) and duration cut off (Extended Data Fig. 4c–f) needed to reduce the false positive rate to below 5%. As mentioned previously3, it is important to note that this estimate of noise represents an upper bound, and could be influenced by other sources of noise apart from motion (that is, photon shot noise). The Pearson correlation coefficient was calculated between each pair of cells, c and c , as This metric measures linear dependence between signals in the two cells, and is invariant with respect to scaling or amplitude translation of the cell signals. We define a matrix of correlation coefficients of size N × N wherein each entry corresponds to correlation between the cells identified by the corresponding row and column. To avoid accumulation in correlated signal due to slow drifts (for example, the long decay curve of GCaMP6m), we set all ΔF/F values lying outside the window of a significant transient (as defined earlier) to 0. The property of high correlation (HC) was tested for in each neuron by finding the number of correlated neurons with which the Pearson’s correlation coefficient was above 0.3 (a Pearson correlation cut-off of 0.3 was used as a conservative estimate of connectivity since previous studies using in vivo two-photon calcium imaging followed by paired whole-cell recordings reported a greater than 50% chance of connectivity when correlations of Ca2+ signals exceeded 0.3 in vivo)55, 56. Histograms were obtained by binning this number across neurons in steps of 5 and calculating the number of neurons that fell into each bin, with the resulting histogram representing the degree distribution of all neurons in the network. HC neurons were defined as those neurons that had more correlated partners than that of the average neuron in the same volume by >1 standard deviation. To identify network population activity measures that best distinguished fear and neutral contexts, we used a space of graph theoretic parameters (described later), which together can be used to define an optimally separating hyperplane between the two contexts. Mathematically, this is posed as a constrained optimization problem, with the objective function seeking to maximize the sum of distances of the hyperplane to the nearest data points in each context, and the constraint being that the hyperplane separates the two contexts. This constrained optimization problem was solved using Lagrange multipliers. To analyse the spontaneous activity of the entire network, we computed the onset and duration of each activity transient (where event onsets and offsets are calculated as described earlier) for each neuron, and then combined transients from all cells into raster plots and collapsed these raster plots into activity histograms, which indicated the percentage of active cells as a function of time. To identify epochs of synchronous activity that included more active cells than would be expected by chance at each frame, we used interval reshuffling (randomly reordering of intervals between events for each cell), performed 1,000 times for each mouse in each context, such that a surrogate histogram was constructed for each reshuffling. The threshold percentage of active neurons corresponding to a significance level of P < 0.05 (appearing only in 5% of histograms) was taken to be the per cent of coactive cells required in a single frame to be considered a synchronous event, and this threshold ranged between 2.5% and 5% active neurons per frame across all mice and fields of view. At least three consecutive frames with activity above the significance threshold were required to be considered a synchronous event, and all subsequent contiguous frames above this threshold were grouped together into the same synchronous event. To plot the cumulative distribution function of event onsets for HC and non-HC neurons during synchronous events, all synchronous events across all mice were identified, and the onset times of HC versus non-HC neurons were binned per frame and plotted cumulatively as a function of the percentage of time elapsed during the synchrony window. To quantify whether the activity of HC neurons was leading or lagging their correlated pairs, the event onset of the HC neurons (defined as the first instance when the signal exceeded 3σ for time consecutive frames) was first fixed at t = 0. The event onsets of all correlated pairs were then binned into 0.167 s time windows immediately preceding or succeeding the onset of the hub neuron at t = 0. PCA was used to describe and visualize population activity of all neurons over time in each context. This was done by transforming the of each cell (typically ~500 cells per mouse per context), over all time points in a given context (typically 1,800 frames) to a different coordinate system characterized by linearly independent eigenvectors, where each eigenvector represents a weighted combination of the different cells. Eigenvalues were sorted in decreasing order to reveal the most energetic (contributory) eigenvectors as well as the magnitude of their overall contribution. PCA was performed using eigenvalue decomposition of the correlation matrix. The corresponding eigenvalues and eigenvectors were calculated using custom MATLAB scripts. An undirected graph is defined based on the cell correlations in the population. An edge, E, is defined between neurons if they are correlated beyond the threshold described earlier. The undirected neuronal graph G (V, E) is defined using all the cells, which are denoted by V (vertices), and E (edges). Mean and maximum cell correlations are calculated using aggregate average and the maxima over all of the measured correlations We also fit an exponential distribution between n (the number of correlations) and n (the degree distribution described earlier) to quantify how closely the graph mimics small world networks, which are characterized by a power law degree distribution for which the power law parameters, a and b, are calculated by transforming the above equation into a logarithmic scale and performing a minimum least-squares fit. A neighbourhood is defined for each cell as a sphere of radius 30 μm. The clustering coefficient for a vertex is defined as the ratio of number of edges within its neighbourhood to the maximum number of connections possible. If there are k nodes in the neighbourhood, k(k − 1)/2 is the maximum number of possible connections9. The clustering coefficient of the entire network is defined as the mean clustering coefficient across all vertices. The mean path length is defined as the average path between any two randomly selected vertices of the graph. The mean path length (mpl) is calculated by first constructing an adjacency matrix, which is an n × n matrix, and all correlated vertex pairs are given a value of one in the corresponding row and column, and zero otherwise. The minimum path from i to j can be recursively calculated using Small-world networks are characterized by high clustering coefficient and low mean path length, quantified using the ratio of clustering coefficient to mean path length, where each term is normalized to a purely random graph with the same number of vertices. Betweenness centrality is a measure of the centrality of nodes in the network, and indicates how central a node is to communication between all pairs of node. Betweenness centrality is computed by calculating all possible paths between two nodes and calculating the number of those that pass through a given node. Strength of a graph quantifies how strongly different subcomponents of a graph are connected and is a measure of resistance of the graph to attack on its edges. Let P = (V , V  … V ) denote all possible partitions of the graph into a mutually exclusive set of vertices V , V  … V , such that the union of all the vertices is V. Let E denote the number of edges that needs to be removed from G to create the partition P. Then the strength is defined as , where the minima are calculated over all possible partitions P. In other words, the strength quantifies how to remove minimal edges to create maximal separation among vertices of the graph. The strength is calculated using MATLAB code based on algorithms described previously57, 58. Deconvolution algorithms enable the estimation of spike rate trains from fluorescence data. Here, we use deconvolution to estimate activity-event onset, not to detect single spikes, since GCaMP6m is assumed to neither have the linear response kinetics nor the sensitivity needed to detect single spikes from bursting neurons in the hippocampus. We used this analysis to help confirm our main results regarding synchronous events and timing of highly correlated neurons, since these analyses offer an alternative method to identify event onsets, while helping to remove noise (for example, long Ca2+ signal decays) from the analyses. Many deconvolution algorithms exist. Early methods to deconvolve fluorescence data used either thresholding to infer event onset59 or optimizations to match a chosen spike profile60. More robust algorithms such as the Wiener linear filter are promising61 but with practical value diminished since negative-going spikes are allowed. In 2010, Vogelstein and colleagues provided a fast non-negative deconvolution method that is, in addition to imposing a non-negative constraint on the spike trains, scalable on a large population of neurons32. Since our imaging involves hundreds of neurons over multiple contexts and days, we use the algorithm from Vogelstein et al. to deconvolve fluorescence signals. Three classes of parameters were optimized in this algorithm to fit data: (1) GCaMP-related parameters, namely sensitivity of fluorescence to elevations in intracellular Ca2+ concentration (α) and baseline concentration (β); (2) acquisition parameters, namely the size of the time bin (δ) and the noise (σ) in the ΔF/F signals; and (3) system (hippocampus/CA3)-related parameters, namely expected spike rate per second (ϕ) and the time constant (τ), or the length it takes for Ca2+ concentrations to decay. For the GCaMP-related parameters, β is set to the baseline of the ΔF/F traces as described earlier, and α is set to 1 as a default value (since varying α broadly around this value did not affect the deconvolution results). δ was set to 1/3 s because image acquisition was at least 3 Hz per optical slice, and σ was estimated to be 0.16 as explained earlier. The main challenge resides in choosing parameters for ϕ and τ since (1) the expected spike rate (close to 0.1 Hz on average, but >10 Hz when bursting) is bimodal and insufficiently captured by the Poisson distribution of spikes as assumed by this model, and (2) the time constant expected for Ca2+ signals in hippocampus is not fully understood. Therefore, we optimized these two parameters by iterating over multiple combinations of time constants and expected spike rates to yield spike events consistent with good fits to our data (Extended Data Fig. 8). The final parameters chosen were: α = 1; β = baseline; δ = 0.33 s; σ = 0.16; ϕ = 5 Hz; τ = 2 s. These values were not exactly the same as, but were comparable to, values reported by others in cortical regions49, 55, 62. Importantly, varying ϕ and τ within a fairly broad range (ϕ ~ 5–10 and τ ~ 0.67–2) did not significantly alter the main conclusions of the subsequent analyses. The ΔF/F signals for all the mice, contexts and days were deconvolved. Correlation coefficients were calculated on the deconvolved signals, and metrics that rely on accurate estimate of event onsets were recomputed, such as synchrony, lead-lag, and identification of HC neurons. The only difference from the methods described earlier was that there were no additional noise filters since the noise is filtered in the process of finding the optimal spike rate (here, event rate), and the onset time was characterized by the first instance that the signal became non-zero. Further analysis of the various specific deconvolution parameters would be of interest but would probably require combined in vivo imaging and single-cell patching experiments, beyond the scope of the current study, and unlikely to significantly affect the specific analyses applied here given the robustness of results to broad ranges of parameters. Furthermore, we performed the analyses described earlier only to help ensure robustness in results obtained from using the raw ΔF/F for measurements relying on precise timing (correlations, leading versus lagging, and synchrony). Lick rates and movements on the ball were captured in XML log files storing timestamps of behavioural data. These were then parsed with custom Python scripts and imported into MATLAB for synchronizing with microscope imaging frames with kHz precision, and for subsequent analysis. To quantify differences in licking between fear and neutral contexts during retrieval, the number of licks per second (each lick causing a beam-break resulting in TTL pulse output of at least 1V), was integrated over the first 2 min in the context. Total licking amounts were normalized to the highest lick rate, observed from any mouse in any context, and presented as a fraction of this value for each mouse and each context. Lick suppression data are presented as mean values across all mice in each experimental group; significance values of differences between contexts were evaluated by Student’s t-test. Lick rates during optogenetic stimulation experiments were scored by quantification during the 15 s of light delivery, which was then normalized to the corresponding value from the 15 s just before light delivery. Significant differences in licking for fear versus neutral context, and for neutral/stimulated versus neutral context alone, were evaluated using Student’s t-test. Lick rates and velocity on ball during synchronous population activity events were calculated by comparing the amount of licking and distance travelled in the 5 s window beginning at the start of a synchronous event, and then normalizing to the amount of licking and distance travelled in the 5 s window before synchronous event. Similar quantitative results were observed with this time window set to 1–10 s after synchrony compared with before, with no significant difference in lick rate and velocity during versus before synchrony. Simultaneous 1-photon (1P) stimulation (594 nm) and 2-photon (2P) imaging (920 nm) was performed by injecting the new red-shifted opsin with improved trafficking and kinetics (bReaChES) via AAV8 in cingulate and GCAMP6m via AAVdj in CA3, and positioning a cranial window above CA2/CA3 for optical access. The 2P imaging and full-field optogenetic stimulation setup is shown in Fig. 4b. Briefly, a resonant galvanometer 2P microscope using an NIR pulsed laser set to 920 nm is combined with simultaneous, full field stimulation using a 594 nm continuous wave laser that is coupled into the system with an optical fibre, lenses and dichroic beam splitters. A 2P compatible NIR reflecting dichroic designed with an additional 594 nm band-pass filter was used for 1P yellow light stimulation during 2P imaging. GCaMP6m signals (green channel) and stimulation artefact (red channel—used to precisely blank stimulation time points) are recorded using standard 2P resonant scanning imaging. 1P stimulation artefacts were removed offline from the 2P images. Stimulation parameters: 591 nm light, 20 Hz, 15 ms pulses, 15 s, 8–10 mW mm−2 laser power at sample after the objective. In total, 4 mice (separate cohort from those used in the imaging-only experiments) were used for the combined stimulation and imaging experiments. The same cells and the same FOV are captured for before-training stimulation trials as well as after-training stimulation trials (conducted 5–7 days later). For a neuron to be considered responsive to (recruited by) the stimulus, at least one significant transient as defined earlier was required to occur during the stimulation window. For latency measurements provided in Fig. 4, event onsets were defined as the first time frame at which the response surpassed three standard deviations above noise, and increased for at least two consecutive frames; if occurring within the first frame, then only neurons with responses increasing from the previous frame are considered, to exclude responses decaying into the stimulation window. Responding neurons were assigned to latency bins of 333 ms. DSI is dependent on the increase of postsynaptic intracellular Ca2+ to suppress GABA release from presynaptic inhibitory neurons expressing cannabinoid receptors63. We performed patch-clamp recordings from CA3 neurons expressing GCaMP6m and examined spontaneous inhibitory postsynaptic currents (sIPSCs) before and following a depolarizing pulse to induce Ca2+ influx. Electrophysiological recordings were performed 4–6 weeks post-injection of AAVdj-CaMKIIα::GCaMP6m into CA3 (in 4–5-week-old mice). Coronal slices (300 μm) from injected mice were prepared after intracardial perfusion with ice-cold, sucrose-containing artificial cerebrospinal fluid solution (ACSF; in mM): 85 NaCl, 75 sucrose, 2.5 KCl, 25 glucose, 1.25 NaH PO , 4 MgCl , 0.5 CaCl and 24 NaHCO . Slices recovered for 1 h at 32–34 °C, and then were transferred to an oxygenated recording ACSF solution (in mM): 123 NaCl, 3 KCl, 26 NaHCO , 2 CaCl , 1 MgCl , 1.25 NaH PO and 11 glucose, at room temperature. Excitatory synaptic transmission blockers (d-2-amino-5-phosphonovaleric acid (APV; 25 μM) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX; 10 μM)) were added to isolate GABAergic postsynaptic currents, and 5 μM carbachol was used to enhance sIPSC frequency to facilitate detection of DSI. Recordings were performed at 32–34 °C under constant perfusion of the oxygenated recording ACSF solution. Slices were visualized with an upright microscope (BX61WI, Olympus) under infrared differential interference contrast (IR-DIC) optics, and a Spectra X Light engine (Lumencor) was used for viewing GCaMP6m expression. Recordings of CA3 neurons were made after identifying GCaMP6m expression, and functional GCaMP6m activity was verifiable (in cells without BAPTA), by observing the increase in GCaMP6m fluorescence during the depolarizing pulse used to induce DSI. The following intracellular solution was used for the patch-clamp electrodes (in mM): 40 CsCl, 90 K-Gluconate, 1.8 NaCl, 1.7 MgCl , 3.5 KCl, 10 HEPES, 2 MgATP, 0.4 Na GTP, 10 phosphocreatine (pH 7.2, 270–290 mOsm). For the BAPTA experiments, 40 mM BAPTA was added to the intracellular solution. Series resistance was monitored for stability, and recordings were discarded if the series resistance changed significantly (by >20%) or reached 20 MΩ. Resting membrane potential was taken at rest, and the reported values incorporate a liquid junction potential of +11.2 mV. Input resistance was calculated from a 100 pA pulse. MiniAnalysis (Synaptosoft) and pClamp10.3 (Molecular Devices) was used to calculate charge transfer (area under sIPSCs) and analyse data. Baseline charge transfer was measured during a 4 s pre-pulse period, DSI was examined during a 4 s period following the depolarizing pulse, and charge transfer after recovery from DSI was measured during a 4 s window. The pulse used to evoke DSI was a 500 ms step to 0 mV from holding potential of −65 mV. DNA sequences of ReachR and bReaCh were synthesized (GenScript) and cloned into AAV vectors containing the CamKIIα promoter for expression in neurons. All constructs were fused to eYFP DNA to detect protein expression in neurons by fluorescence microscopy. The Glu123Ser mutation was introduced using QuickChange Site-Directed mutagenesis kit (Agilent). Plasmid DNA was purified with QIAprep Spin Miniprep Kits (Qiagen) after transformation and amplification in Escherichia coli. Electrophysiological recordings in neuronal cultures were prepared as described64. Patch pipettes (4–6 MΩ) were pulled from glass capillaries (Sutter Instruments) with a horizontal puller (P-2000, Sutter Instruments) for whole-cell recordings in voltage and current clamp. Recordings were made using a MultiClamp700B amplifier (Molecular Devices). The external recording solution contained (in mM): 127 NaCl, 10 KCl, 10 HEPES, 2 CaCl , 2 MgCl , 30 d-glucose, pH 7.3, including synaptic blockers (25 µM d-APV, 10 µM NBQX). The patch pipette solution contained (in mM): 140 K-gluconate, 10 HEPES, 10 EGTA, 2 MgCl , pH 7.3. All measurements were corrected for a liquid junction potential of +15 mV. Series resistance was monitored throughout recordings for stability. A Spectra X Light engine (Lumencor) was used to excite eYFP and to apply light for opsin activation. Yellow and red stimulation light was filtered by 575/25 or 632/22 band-pass filters (Chroma) and applied through a ×40 objective (Olympus) at 5 mW mm−2 light intensity. Light power density was measured with a power meter (ThorLabs). The functionality of all constructs was determined by comparing stationary photocurrents at −80 mV to 1 s continuous light pulse. Spikes were optically evoked in current-clamp mode with light pulses (5 ms) delivered at 633 nm, 5 mW mm−2 and 1–20 Hz. The activation spectra for C1V1, bReaCH-ES and ChR2 was measured by recording stationary photocurrent in voltage clamp mode at −80 mV and light intensities of 0.65 mW mm−2 at each wavelength. Light was delivered through 20 nm band-pass filters (Thorlabs) at (in nm): 400, 420, 440, 460, 470, 480, 490, 500, 520, 540, 560, 570, 580, 590, 600, 620, 630, 650. Photocurrents were normalized to maximum values respectively: 480 nm for ChR2, 560 nm for C1V1 and 570 nm for bReaCh-ES. Kinetics of channel closure were determined by fitting the decay of photocurrents after light off, with mono-exponential functions. Channel kinetics were quantified by corresponding τ values respectively. pClamp10.3 (Molecular Devices) and OriginLab8 (OriginLab) software was used to record and analyse data. The following adeno-associated viruses (AAVs) with serotype DJ were produced at the Stanford Neuroscience Gene Vector and Virus Core: AAVDJ-CaMKII::bReach-E162S-TS-eYFP; and AAVDJ- CaMKII::C1V1(E122T/E162T)-TS-eYFP. Four-to-six-week-old mice were injected bilaterally with 1 µl of either virus in the medial prefrontal cortex, at the following coordinates (from Bregma): A/P: +1.7 mm; M/L: +0.3 mm; D/V: −2.5 mm. Titre was matched at 1.5 × 1012 vg ml−1 for both viruses. Electrophysiological recordings were performed 12–14 weeks post-injection for opsin expression at the mPFC terminals. Coronal slices (300 μm) from injected mice were prepared after intracardial perfusion with ice-cold, sucrose-containing ACSF (in mM): 85 NaCl, 75 sucrose, 2.5 KCl, 25 glucose, 1.25 NaH PO , 4 MgCl , 0.5 CaCl and 24 NaHCO . Slices recovered for 1 h at 32–34 °C, and then were transferred to an oxygenated recording ACSF solution (in mM): 123 NaCl, 3 KCl, 26 NaHCO , 2 CaCl , 1 MgCl , 1.25 NaH PO and 11 glucose, at room temperature. Electrophysiological recordings were performed at 32–34 °C under constant perfusion of the oxygenated recording ACSF solution. For mPFC recordings, synaptic transmission blockers (APV (25 μM), NBQX (10 μM) and gabazine (10 μM)) were added to the recording ACSF solution. Slices were visualized with an upright microscope (BX61WI, Olympus) under IR-DIC optics. A Spectra X Light engine (Lumencor) was used both for viewing fluorescent protein expression and delivering light pulses for opsin activation. Light power density was obtained with a power meter (Thorlabs). Recordings of mPFC neurons were made after first identifying regions of eYFP+ expression, and recordings of postsynaptic basolateral amygdala (BLA) neurons were obtained after confirming eYFP+ expression in both mPFC and the mPFC axonal fibres at the BLA. Whole-cell voltage-clamp recordings were performed at −65 mV, and current-clamp recordings were performed at rest. Patch-clamp pipettes contained the following internal solution (in mM): 125 K-gluconate, 10 KCl, 10 HEPES, 4 Mg -ATP, 0.3 Na-GTP, 10 phosphocreatine, 1 EGTA. Recordings were conducted using MultiClamp700B amplifier and pClamp10.3 software (Molecular Devices). pClamp10.3, OriginLab8 (OriginLab), and SigmaPlot (SPSS) were used to analyse data. Stationary photocurrent of the opsins was measured at the end of a 1 s light pulse in voltage-clamp mode. Light-evoked spike probability in the mPFC neurons and in the postsynaptic BLA neurons was calculated as the fraction of successful action potentials evoked in the recorded neuron upon various light stimulation frequencies. Light-evoked EPSC amplitude in the postsynaptic BLA neurons was measured at the peak of the evoked response to light stimulation of the opsin-expressing mPFC fibres. Series resistance was monitored for stability, and recordings were discarded if series resistance changed significantly (by >20%) or reached 20 MΩ. Statistical analysis was performed with two-tailed t-test, with significance set at P < 0.05.

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