For this study, we used 26 transgenic “VGLUT2-cre” adult male mice (25–30 g; aged 10–11 weeks) obtained by mating male and female homozygote Vglut2-ires-Cre mice from Jackson Laboratories (strain name Slc17a6tm2(cre)Lowl/J, stock #016963). All mice were bred in-house and maintained in standard cages, with food and water ad libitum, in a temperature- and humidity-controlled room under a 12 h light/12 h dark cycle. All the surgical and experimental procedures were performed according to the National Institutes of Health guidelines and the European communities Council Directive of 86/609/EEC and were approved by the University of Aix-Marseille and Lyon University Chancellor's Animal Research Committees.
Four VGLUT2-cre mice underwent stereotaxic injection of rabies virus (RV) within the inner molecular layer of the dDG in order to obtain golgi-like retrograde-labeling of SuM neurons projecting to this region. The strain of RV used was the Challenge Virus Standard (CVS, 4.107 plaque-forming units/mL) (Bras et al. 2008; Ugolini 2010; Coulon et al. 2011). Only vaccinated personnels conducted these experiments at the appropriate biosafety containment level until the sacrifice of the animals as described below.
These animals were used to determine the neurotransmiter phenotype of SuM neurons projecting to dDG at the mRNA level, combining immunohistofluorescent detection of RV with fluorescent in situ hybridization detection of VGAT and VGLUT2 mRNAs.
Adeno-associated virus (AAV) double floxed inverted ORF (DIO) vectors
Thirteen VGLUT2-cre mice underwent stereotaxic injection of the cre-dependent viral vectors: AAV5-EF1a-DIO-EYFP (3.5 × 1012 virus molecules/mL; UNC Gene Therapy Center Vector Core; Dr Deisseroth) into the SuML. These VGLUT2-EYFP mice expressing the Yellow Fluorescent Protein (YFP) in VGLUT2 SuM neurons and their axon terminals were used for the following: (1) to determine the neurotransmitter phenotype of SuM neurons innervating the dDG at the protein level, by simultaneous immunohistofluorescent detection of EYFP labeled axon fibers and terminals, VGAT and VGLUT2 in dDG (n = 3); (2) to determine the synaptic profile of these EYFP labeled axon terminals at the electron microscopy level (n = 3); (3) as control animals (n = 3) for in vitro optogenetic stimulation and patch clamp electrophysiological recordings; (4) as control animals (n = 4) for in vivo optogenetic stimulation and electrophysiological recordings followed by cFos immunolabeling.
Nine VGLUT2-cre mice underwent stereotaxic injection of the cre-dependent viral vectors: AAV5-EF1a-DIO-hChR2(H134R)-EYFP (3.2 × 1012 virus molecules/mL; UNC Gene Therapy Center Vector Core; Dr. Deisseroth) into the SuML. These VGLUT2-ChR2 mice expressing the excitatory opsin, channelrhodopsin 2 (ChR2) and the reporter protein EYFP in VGLUT2 SuM neurons and their axon terminals were used for (1) in vitro optogenetic stimulation and patch clamp electrophysiological recordings experiments (n = 5), (2) in vivo optogenetic stimulation and electrophysiological recordings followed by cFos immunolabeling (n = 4).
Mice were anesthetized by an intraperitoneal injection (i.p.) of Ketamin (50 mg/kg)/Xylazine (5 mg/kg) solution and additional injections were delivered as needed to maintain deep anesthesia during the surgery. Animals were then secured in a stereotaxic frame (David Kopf instruments). The body temperature of mice was monitored and maintained at about 37 °C during the entire procedure by means of an anal probe and heating blanket, respectively. The head was shaved and sanitized with Betadine and 0.9% NaCl. Local anesthesia was performed by infiltration of the scalp with xylocaine (lidocaine hydrochloride 0.5%), and an ophthalmic gel was placed on the eyes to avoid drying. After scalp incision, holes were drilled in the skull, with antero-posterior (AP), medio-lateral (ML) and dorso-ventral (DV) coordinates based on Paxinos and Franklin’s atlas (2005).
RV and AAV viral vector injections
The RV (200 nL) was pressure-injected unilaterally (n = 2) or bilaterally (n = 2) within the dDG inner molecular layer of VGLUT2-cre mice according to the following coordinates: AP = − 2; ML = ± 1.5; DV = − 1.7. Injections were performed by using a 33-gauge Hamilton syringe connected to a Micro4 injection pump system (World Precision Instruments). After completion of the injection procedures, the syringe was removed and the skin was sutured. Animals were treated with local anesthetic, returned to their cages kept at the appropriate biosafety containment level for a survival period of 38 h to observe an optimal RV retrograde labeling for the dendritic arbor of SuM neurons. This post-injection survival time was adjusted to obtain a Golgi-like labeling of the first-order infected neurons and to limit trans-neuronal spread to second order projection neurons, starting to be detected after 42 h (Fig. 1a–c). AAV5-EF1a-DIO viral vectors (500 nL) were injected bilaterally within the SuML of VGLUT2-cre mice at the following coordinates: AP = − 2.7; LM = ± 1.25; DV = − 4.8 using an 11° angle to avoid the high vascularization at the midline.
Optrode and electrode implantation
After AAV injections, VGLUT2-mice were implanted with an optrode in the left dDG (AP = − 2; ML = − 1.1; DV = − 1.7) for optic stimulation of axon terminals originating from transfected SuML neurons and local field potential (LFP) recordings from the DG. The optrode consisted of an optic fiber (250 μm ∅, 0.39 NA; Thorlabs SAS) and the LFP electrode. The LFP electrodes consisted of two 45 μm-diameter tungsten wires (California Fire Wire Company), twisted and glued together to form a rigid and solid structure, with the two ends of the wires separated by 100 μm from each other (impedance 100 KOhms). Two small screws (1 mm in diameter, Plastics One) each soldered to a wire were fixed on the skull. The first screw was fixed in the parietal part of the skull for EEG recording and the second screw, at the level of cerebellum as reference electrode. Two gold-coated spherical electrodes were inserted in between neck muscles for differential EMG recording. All leads were connected to a miniature plug (Plastics One) that was cemented on the skull.
After completion of the surgery the skin was sutured. Mice were treated with local anesthetic, and an intramuscular injection of antibiotic (Baytril, 5 mg/kg) to prevent any risk of infection. Mice were monitored until waking and returned to their home cages for a survival period of 3 weeks.
Tissue preparation for light microscopy
Animals were deeply anesthetized with ketamine and xylazine and transcardially perfused with 4% paraformaldehyde (PFA) in 0.12 M sodium phosphate buffer, pH 7.4 (PB). After perfusion, the brains were removed from the skull, post-fixed in the same fixative for 1 h at room temperature (RT), rinsed in PB, cryoprotected in 20% sucrose overnight, frozen on dry ice and sectioned coronally at 40 μm with a cryostat (Microm). The sections were rinsed in PB, collected sequentially in tubes containing an ethylene glycol-based cryoprotective solution and stored at − 20 °C until histological processing. One of every ten sections was stained with cresyl violet to determine the general histological characteristics of the tissue throughout the rostro-caudal extent of the brain. Selected sections were processed for the following: (1) simultaneous detection of VGLUT2 mRNA, VGAT mRNA and RV; (2) simultaneous immunohistofluorescent detection of EYFP, VGLUT2 and GAD65 or VGAT; (3) immunohistochemical detection of cFos.
Immunohistochemical detection of RV performed in VGLUT2-cre mice (n = 4) injected with RV
Sections at the level of the DG and SuM were processed for immunohistochemical detection of the RV to identify the injection site and the retrograde-labeled neurons within the rostro-caudal extent of the SuM. Sections were pre-treated for 30 min in 1% H2O2, rinsed in PB and in 0.02 M potassium phosphate-buffered saline (KPBS, pH 7.2–7.4). Then, they were processed using a Mouse On Mouse kit (MOM, Vector Laboratories, Burlingame, CA, USA) to eliminate non specific labeling due to use of mouse monoclonal antibody on mouse tissue. Sections were incubated for 1 h at RT in MOM mouse IgG blocking reagent diluted in KPBS containing 0.3% Triton X-100. Sections were rinsed twice with KPBS for 5 min and pre-incubated in MOM diluent for 15 min. They were then incubated overnight at RT in a solution containing the mouse monoclonal antibody directed against RV (1:3000; Raux et al. 1997; gratiously provided By Dr. Patrice Coulon), diluted in MOM diluent. After several rinses in KPBS, sections were incubated for 1 h in horse anti-mouse IgG (1:200, Vector Laboratories) diluted in MOM, rinsed in KPBS and incubated for 1 h at RT in an avidin–biotin–peroxidase complex solution prepared according to the manufacturer's recommendations (Vectastain ABC kit, Vector Laboratories). Sections were then processed for 15 min in 3.3′-diaminobenzidine tetrahydrochloride (DAB, Sigma fast tablets; Sigma), rinsed in KPBS, mounted onto Superfrost Plus slides, dehydrated and coverslipped with Permount.
Simultaneous detection of VGLUT2 mRNA, VGAT mRNA and RV combining fluorescent in situ hybridization (RNAscope technology) and immunohistofluorescent methods performed in VGLUT2-cre mice (n = 4) injected with RV
Selected sections at the level of the SuM were first treated with 1% H2O2 rinsed in PB mounted on SuperFrost Plus slides (Fisher Scientific) and air dried at RT. They were then processed for fluorescent RNAscope in situ hybridization according to the manufacter’s protocol (Advanced Cell Diagnostics). Briefly, sections were treated with 100% ethanol and protease III for 30 min at 40 °C. They were incubated in a solution containing both RNAscope® Probe-Mm-S1c17a6 for detection of VGLUT2 mRNA and Mm-S1c32a1-C3 for detection of VGAT mRNA. After hybridization, sections were processed for visualization using the RNA-scope Multiplex Fluorescent reagent Kit v2 (Advanced Cell Diagnostics) and the Tyramide Signal Amplification (TSA™) Plus Cyanine 3 and TSA Plus Cyanine 5 systems (Perkin Elmer).
After the RNAscope assay, sections were rinsed in KPBS and processed for immnohistofluorescent detection of the RV using the MOM kit as described above. After the overnight incubation in the mouse monoclonal antibody directed against RV (1:3000), sections were rinsed in KPBS and incubated for 2 h in Alexa488-conjugated donkey anti-mouse (1:200; Invitrogen) diluted in MOM diluent. After several rinses in KPBS, all sections were coverslipped with Fluoromount (Electron Microscopy Sciences). The specimens were analyzed with a Zeiss laser-scanning confocal microscope.
Quantification of co-localizing RV, VGLUT2 and VGAT mRNAs
Quantitative analysis was conducted to evaluate the extent of SuM neurons with direct projections to the dDG that co-express VGLUT2 and VGAT mRNAs. For this purpose, the number of triple-labeled neurons was determined for each animal (n = 4), from 3 sections (120 μm apart from each other) across the antero-posterior extent of the SuM. For each section, an image of the entire SuM region was obtained from a single confocal slice using the Tile Scan function with a 20 × objective and sequential acquisition of the different wavelength channels to avoid fluorescent crosstalk with ZEN software (Zeiss). The analysis was then performed with Neurolucida software (version 7, mbfBioscience) as follows: for each confocal image, all RV-labeled neurons were identified on the green channels and examined for colocalization of VGLUT2 mRNA in the blue channel and/or VGAT mRNA in the red one. Triple-, double- and single-labeled neurons were tagged differently and counted by the software. A total of 380 RV-labeled neurons were analyzed.
Simultaneous immunohistofluorescent detection of EYFP, VGLUT2 and GAD65 or VGAT performed in VGLUT2-EYFP mice (n = 3)
Selected sections at the level of the dDG were processed using the MOM kit as described above. Sections were incubated overnight at RT in a solution containing rabbit anti-GFP (1:2000; Invitrogen), guinea pig anti-VGLUT2 (1:5000; Millipore) and mouse anti-GAD65 (1:100; Millipore) or mouse anti-GFP (1:100; Invitrogen), guinea pig anti-VGLUT2 (1:5000; Millipore) and rabbit anti-VGAT (1:1000; Synaptic System) diluted in MOM diluent. After several rinses in KPBS, they were incubated for 2 h in Alexa488-conjugated donkey anti-rabbit IgG (1:200; Invitrogen), Cy5-conjugated donkey anti-guinea pig (1:100; Jackson ImmunoResearch Laboratories, Inc.), and Cy3-conjugated donkey anti-mouse (1:100; Jackson ImmunoResearch Laboratories, Inc.) or Alexa488-conjugated donkey anti-mouse IgG (1:200; Invitrogen), Cy5-conjugated donkey anti-guinea pig (1:100; Jackson ImmunoResearch Laboratories, Inc.), and Cy3-conjugated donkey anti-rabbit (1:100; Jackson ImmunoResearch Laboratories, Inc.) diluted in MOM diluent. After several rinses in KPBS, all sections were then mounted on superfrost-coated slides, dried overnight at RT and coverslipped with Fluoromount. The specimens were analyzed with confocal microscope (Zeiss).
Quantification of co-localizing GFP, VGLUT2 and VGAT
Two quantification protocols were used in order to evaluate the extent of the different neurochemical phenotypes of axon terminals from the SuM innervating the dDG. Each of these two quantifications was obtained for each mouse (n = 3) from 4 sections (400 μm apart from each other) across the dDG. The first section was selected at rostral level when the granule cell and molecular layers start to form a “V” shaped structure enclosing the hilus corresponding to AP coordinate Bregma − 1.46 mm on Paxinos and Franklin’s atlas (2005). The sections used in the first quantification protocol were adjacent to the sections used in the second protocol. In the first protocol previously described (Soussi et al. 2015), the densities of VGLUT2/VGAT and VGLUT2 only labeled terminals were assessed by the quantification of immunolabeling for VGLUT2/VGAT and VGLUT2 only, respectively. Single optical confocal images were acquired from sections (n = 4) of each mouse (n = 3) with Zeiss LSM 510 laser-scanning microscope and analyzed with the software provided by the microscope manufacturer (LSM 510 Zen, Zeiss). All images were acquired from the suprapyramidal and infrapyramidal blades of the dDG, using identical parameters. The percentages of VGLUT2 labeled terminals containing VGAT were estimated by the Manders’ coefficient (proportion of pixels for VGLUT2 also positive for VGAT) obtained with the JACoP co-localization Plugin for Image J, in the region of interest (ROI) which included granule cell layer (GCL) and the narrow zone superficial to the granule cells defined as the supragranular layer (SGL) following recommendations from Bolte and Cordelières (2006). For each channel, an identical bottom threshold was applied throughout the analyses, and only the pixels with a value above this threshold were counted. When a pixel had a value above the threshold in both channels, it was counted as double positive. The size and the shape of the ROI was the same for each confocal image. The average % of co-localization was calculated for each blade of the DG for each mouse.
In the second quantification protocol, we determined, for each mouse (n = 3), the relative percentages of triple- and double-labeled boutons for the GFP-anterograde tracer and VGAT and/or VGLUT2 in the SGL of the dDG suprapyramidal and infrapyramidal blades. Several z-stacks of 10 confocal slices were acquired, from the different sections (n = 4), with a 100 × objective and a numerical zoom of 8, in each region of interest. The analysis was performed as previously described (Persson et al. 2006; Soussi et al. 2010) and following recommendations from Bolte and Cordelières (2006). For each z-stack, the confocal images obtained from separate wavelength channels (green, red and blue) were displayed side by side on the computer screen together with the images corresponding to colocalized pixels within each optical slice of the z-stack obtained with the colocalization highlighter plugin in ImageJ. The GFP-labeled boutons were identified in the green channel within a probe volume defined by the size of the confocal slice (19.38 μm by 19.38 μm) and the height of the z-stack (2 μm). Each bouton was examined for colocalization through the individual optical slices of the z-stack. Single-, double- and triple-labeled boutons were counted using the Cell Counter plugin in ImageJ. The total number of GFP-labeled terminals analyzed in the two regions of interest was 400.
Immunohistochemical detection of cFos performed in VGLUT2-EYFP (n = 4) and VGLUT2-ChR2 (n = 4) mice
Selected sections at the level of the DG were processed for immunohistochemistry according to previously described protocol (Esclapez et al. 1994). Sections were pre-treated for 30 min in 1% H2O2, rinsed in PB and KPBS, preincubated for 1 h in 3% normal goat serum (NGS, Vector Laboratories) diluted in KPBS containing 0.3% Triton X-100 and incubated overnight at RT in cFos rabbit polyclonal antiserum (1:20,000; Calbiochem) diluted in KPBS containing 1% NGS and 0.3% Triton X-100. After several rinses in KPBS, sections were incubated for 1 h at RT in biotinylated goat anti-rabbit immunoglobulin G (IgG; Vector Laboratories) diluted 1:200 in KPBS containing 3% NGS and then for 1 h at RT in an avidin–biotin–peroxidase complex solution prepared in KPBS according to the manufacturer's recommendations (Vectastain ABC kit, Vector Laboratories). Sections from VGLUT2-EYFP and VGLUT2-ChR2 mice were processed in parallel and for the same period of time (15 min) in 3.3′-diaminobenzidine tetrahydrochloride (DAB, Sigma fast tablets; Sigma), rinsed in KPBS, mounted onto Superfrost Plus slides, dehydrated and coverslipped with Permount.
Quantification of cFos immunolabeled neurons
The number of cFos labeled neurons was calculated in the DG GCL of the right and left (ipsilateral to the optic stimulation) hemispheres in VGLUT2-EYFP control (n = 4), and VGLUT2-ChR2 (n = 4) mice. These analyses were performed using a computer-assisted system connected to a Nikon 90i microscope and the Neurolucida software (MicroBrightField). A total of 4 sections (400 μm apart from each other) surrounding the optrode site were analyzed for each animal. In each section the GCL was delineated and all neurons labeled for cFos were plotted. The number of labeled neurons, obtained from the 4 sections, was calculated in each hemisphere for each animal. The average total number of labeled neurons/hemisphere ± SEM was calculated for each group of control VGLUT2-EYFP and VGLUT2-ChR2 mice. Statistical analysis was performed by Statview software using Wilcoxon Rank Sum Test.
Tissue preparation for electron microscopy
Three VGLUT2-EYFP mice were perfused intracardially with a fixative solution containing 4% PFA and 0.1% glutaraldehyde in 0.12 m PB. After perfusion, the brain was removed from the skull, post-fixed in the same fixative overnight at 4 °C and rinsed in PB for 1.5 h. Blocks of the forebrain were sectioned coronally at 60 μm with a vibratome. Pre-embedding immunolabeling for GFP was performed on sections at the level of dDG. These sections were pre-treated for 15 min in 1% sodium borohydride prepared in PB and rinsed for 30 min in PB and 3 × 30 min in KPBS. They were incubated for 1 h in normal goat serum diluted in 0.02 M KPBS, then overnight in primary antibody rabbit anti-GFP (1:2000) diluted in KPBS containing normal goat serum at RT. On the following day, sections were rinsed for 1.5 h in 0.02 M KPBS then incubated for 1 h in the secondary antibody goat anti-rabbit (1:200) diluted in KPBS containing normal goat serum. After rinsing in KPBS for 1.5 h, sections were incubated for 1 h in an avidin-biotinylated-peroxidase complex (ABC Elite; Vector Laboratories) prepared in KPBS. After 3 × 30 min rinses in KPBS, sections were incubated for 12 min in 3.3′-diaminobenzidine tetrahydrochloride and 0.01% H2O2, rinses in KPBS, post-fixed in 2% PFA and 2.5% Glutaraldahyde diluted in PB for 3 h, then washed for 1.5 h in PB. After all these steps, sections were treated with 1% osmium tetroxide in PB for 45 min, dehydrated in ethanol, flat embedded in Durcupan resin and polymerized at 56 °C for 24 h (Zhang and Houser 1999). Labeled regions of the DG that contained the molecular and granule cell layers were trimmed from the sections, re-embedded on capsules filled with polymerized Durcupan and further polymerized at 56 °C for an additional 24 h. Ultrathin sections from the most superficial face of the blocks were cut on an ultramicrotome. Serial sections were picked up on nickel mesh grids and stained with uranyl acetate and lead citrate. Sections were examined and photographed with a JEOL electron microscope.
In vitro electrophysiology: optic stimulation and patch clamp recordings
Hippocampal slice preparation
VGLUT2-ChR2-EYFP mice (n = 5) and VGLUT2-EYFP mice (n = 3) were decapitated under isofluorane anesthesia. Brains were quickly removed and placed into an ice-cold (4 °C) cutting solution containing (in mM): 140 potassium gluconate, 10 HEPES, 15 sodium gluconate, 0.2 EGTA, 4 NaCl (pH 7.2). Coronal slices were cut (350 µm) using a vibratome (Leica Microsystem). In order to increase cell survival over time in slices from 10- to 11 week-old adults, slices were incubated first in a solution containing (in mM): 110 choline chloride; 2.5 KCl; 1.25 NaH2PO4; 10 MgCl2, 0.5 CaCl2; 25 NaHCO3; 10 glucose, 5 sodium pyruvate, for 15 min at 20–23 °C. Then, they were transferred to a holding chamber containing an artificial cerebrospinal fluid (ACSF) composed of (in mM) 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 1.3 MgCl2, 2 CaCl2, 25 NaHCO3, 10 d-glucose (pH 7.3–7.4) at RT for at least 1 h before recording. The two last solutions were saturated with 95% O2 and 5% CO2.
Whole-cell voltage-clamp recordings
Slices were submerged in a low-volume recording chamber and continuously superfused with 32–34 °C ACSF at 5 mL/min perfusion rate. For each mouse, four slices containing the dDG were selected for patch clamp recordings. DG neurons were visualized by infrared video microscopy using an upright microscope (SliceScope, Scentifica Ltd). Patch pipettes were pulled from borosilicate glass tubing (1.5 mm outer diameter, 0.5 mm wall thickness) and filled with an intracellular solution containing (in mM) 20 CsCl, 115 CsGlu, 10 HEPES, 1.1 EGTA, 4 MgATP, 10 Na phosphocreatine and 0.4 Na2GTP as well as 0.2% biocytin for post-hoc morphological identification of the recorded neuron (see below). The pipette resistance was 4–6 MΩ. Recordings were performed in the apex, suprapyramidal and infrapyramidal blades of the dDG. Signals were fed to a Multiclamp 700A (Molecular Devices), digitized (10 kHz) with a DigiData 1550 (Molecular Devices) interface to a personal computer and analyzed with ClampFit software (Molecular Devices). Optical stimulation of ChR2-expressing axon terminals was performed by pulses of 470 nm blue light delivered by a LED (pE-2, CoolLED) through a 40 × objective attached to microscope (SliceScope, Scientifika Ltd). Stimulations consisted of paired 5 ms pulses (500 ms between pulses, every 30 s). For VGLUT2-ChR2 mice, the light intensity corresponded to 20–30% (1.6–2.5 mW) of the LED maximum power (7.5 mW) and for the VGLUT2-EYFP control mice, it varied between 20 and 90% (6.9 mW) of LED maximum power. Postsynaptic current (PSC) responses to optic stimulations were recorded at different holding potentials ranging from − 70 mV (close to reversal potential of GABA-A currents but far below the reversal potential of glutamate receptor-mediated currents) to + 10 mV (close to reversal potential of glutamate receptor-mediated currents but far above the reversal potential of GABA-A-mediated currents). Pharmacological characterization of inhibitory PSCs (IPSCs) and excitatory PSCs (EPSCs) was achieved using antagonists of GABA-A, AMPA and NMDA receptors. We used Gabazine or bicuculline (antagonist of GABA-A receptors, 10 µM), D-AP5 (antagonist of NMDA receptors, 40 µM) and NBQX (antagonist of AMPA and Kainate receptors, 10 µM). The co-release of glutamate and GABA was further demonstrated in several DG granule cells (n = 5), by recording first light stimulated PSC at − 70 mV (Fig. 4k). At this holding potential the recorded currents were essentially generated by glutamate receptors. Then the glutamate component was abolished by NBQX and D-AP5, and the GABA component was revealed at + 10 mV holding potential. This PSC was completely inhibited by bicuculline application that confirmed the GABA-A receptor origin of these remaining currents (see also Fig. 4j).
Double immunohistofluorescent labeling for Biocytin and GFP
After recordings, slices were processed for simultaneous detection of the biocytin-filled neurons and GFP-labeled axon fibers and terminals in order to identify the recorded cells and evaluate the efficiency of the transfection, respectively. Slices were fixed overnight at 4 °C in a solution containing 4% PFA in PB. Then they were rinsed in PB, cryoprotected in 20% sucrose and quickly frozen on dry ice. After several rinses in KPBS, slices were incubated in a solution containing normal donkey serum (NDS, 1:30; Vector Laboratory) diluted in KPBS with 0.3% Triton- X100, for 2 h at RT. They were incubated in a solution containing goat anti-biotin (1:200) and rabbit anti-GFP (1:2000), diluted in KPBS containing 0.3% Triton-X100 and NDS (1:100), overnight at RT. After several rinses in KPBS, slices were incubated for 2 h in Alexa488-conjugated donkey anti-goat IgG (1:200; Invitrogen), and Cy3-donkey anti-rabbit IgG (1:100) diluted in KPBS with 0.3% Triton-X100. After rinses in KPBS, slices were mounted on slides and coverslipped with Fluoromount. The specimens were analyzed with a fluorescence microscope (Nikon 50i) or confocal microscope (Zeiss).
In vivo electrophysiology: optic stimulation, LFP and EEG recordings
All mice were placed for 7 days in a recording box in order to get them used to the recording conditions. The recording box was ventilated, as well as electrically and sound isolated. The temperature was regulated at 21 °C, and a 12 h light/12 h dark cycle imposed. Mice were accustomed to the cable connecting them to the recording device. The recording cable connected the micro-connector implanted on the head of the animal to a collector, which ensured the continuity of the recorded signals without hindering the movements of the mouse. At the end of this habituation, the control recordings begun. EEG and EMG recordings were digitized at 1 kHz, amplified 5000 times with a 16-channel amplifier (A-M System) and collected on a computer via a CED interface using Spike 2 software (Cambridge Electronic Design). The signal was band-pass filtered online between 1 and 300 Hz for EEG, and between 10 and 100 Hz for EMG. The 50 Hz signal was removed with a notch filter. The EEG and LFP signals were acquired by monopolar derivation (differential between the recording electrode and the reference electrode located above the cerebellum). The EMG bipolar signals were calculated by measuring the differential between the two EMG electrodes. Mice were recorded for 24 h of baseline followed by optogenetic manipulation.
In vivo optogenetic stimulation
Optical stimulations were delivered via a patch cable connected to a 100 mW 473-diode (Laserglow). Stimulations were performed during 4 days: in the first 3 days, mice were stimulated during one specific vigilance state par day: waking (WK), slow wave sleep (SWS) or PS. Each day, stimulations were delivered during the same circadian period (10 AM–2 PM). Stimulations were applied 10 s after the occurrence of a stable WK, SWS or PS event as detected by real-time observation by the experimenter. For WK and SWS, stimulations were spaced apart by at least 1 min and for PS, by at least 15 s.
Blue exciting stimulations consisted of 10-s trains of 10-ms pulses at 20 Hz. Light power at the fiber tip was 10 mW.
The 4th day of experiments, 4 VGLUT2-EYFP and 4 VGLUT2-ChR2 animals were stimulated for 15 min at 20 Hz (10-ms pulses). All mice were killed 90 min after the beginning of the stimulation by transcardiac perfusion of 4% PFA. Brain tissues were processed for immunohistochemical detection of cFos expression (see above).
Analysis of the sleep wake states
Polysomnographic recordings were visually scored by 5-s epochs for WK, SWS and PS as previously described (Sapin et al. 2009). Hypnograms were obtained by using a custom Matlab script. For each animal, the number of awakenings during SWS and PS optogenetic stimulations was counted and expressed as percentage of the total number of stimulations.
LFP and EEG analysis
LFP and EEG signals were analyzed using a custom Matlab script using the Chronux toolbox. The time–frequency spectrograms were computed with the same toolbox and expressed in arbitrary units. The mean power spectral density in the 10 s before the stimulation was compared to that in the 10 s during the stimulation (sliding window: 1 s), in order to obtain a mean spectral power ratio (PR) ± SEM for frequencies equal or above 1 Hz. The frequency spectra were grouped into frequency bands commonly used Delta: 1–4 Hz, Theta: 6–12 Hz, Sigma: 12–14 Hz, Beta: 15–30 Hz, Gamma: 30–100 Hz. Power spectral values at 20 Hz and its harmonics were excluded from the analysis.
To analyze the evolution of LFP and EEG theta and gamma bands during optogenetic stimulation, the mean PR of these spectral bands and the respective 95% confidence intervals were calculated from 10 s before to 10 s after the photostimulation. In order to compute these intervals, we used a bootstrap procedure, which allows creating artificial groups from the original data, with replacement. The mean of each artificial group derived from the original data was then computed. This operation was repeated 10,000 times and the 95% confidence interval was the 5th and the 95th percentile of the means of the randomly constructed samples. Finally, during WK and PS the peak of theta frequency (6–12 Hz) in the 10 s before and during the optogenetic stimulation was identified in all animals.
Analysis of variance (Mann–Whitney test) was performed on the percentage of awakenings, the mean spectral power ratios and the theta frequency peaks. These statistics were performed using Statview software (StatView Inc, Nestbit, NS).
EMG signals during WK were analyzed using a custom Matlab script. The mean EMG value in the 10 s before the stimulation was compared to that in the 10 s during the stimulation, in order to obtain a mean EMG ratio ± SEM. In the two groups of animals we performed a sequential analysis per 0.5 s on the mean of the absolute EMG values from 20 s before to 10 s after the photostimulation. The respective bootstrap 95% interval (computed as described above) was calculated for each sequential value.
Further, analysis of variance (Mann–Whitney test) was performed on the EMG mean by using Statview software.