Animals and genotyping
The c-fos::tetracycline transactivator (tTA) mice were purchased from the Mutant Mouse Regional Resource Center (stock no. 031756-MU). The KA1::Cre mice were purchased from Jackson Laboratory (Jackson Laboratory stock no: 006474, G32–4 Cre). Floxed-NR1 mice (Jackson Laboratory stock no: 005246) were donated by Drs. S. Tonegawa (RIKEN-Massachusetts Institute of Technology) and S. Itohara (RIKEN Brain Science Institute).
The c-fos::tTA/KA1::Cre double transgenic mice for behavioral analyses were generated via in vitro fertilization with eggs from C57BL/6 J mice and embryo transfer techniques . The CA3 pyramidal cell-restricted N-methyl-D-aspartate (NMDA) receptor knock-out (CA3-NR1 KO) mice were generated from KA1::Cre mice and homozygous floxed-NR1 mice via in vitro fertilization.
The c-fos::tTA mice were backcrossed with C57BL/6 J at least 10 times after we purchased from the Mutant Mouse Regional Resource Center. The KA1::Cre mice was mixed C57BL/6 J and C57BL/6 N genetic background when we purchased from Jackson Laboratory. The results of A 32 SNP (single nucleotide polymorphism) panel analysis, which conducted by Jackson Laboratory showed that, at least 3 of 5 markers that distinguish C57BL/6 J from C57BL/6 N were found to be C57BL/6 J type . After we obtained the KA1::Cre mice, the mice were backcrossed with C57BL/6 J at least 5 times to be C57BL/6 J background. The fNR1 mice were backcrossed with C57BL/6 J more than 25 times. Therefore, all experimental mice were C57BL/6 J background. Male mice were used for all the experiment.
The mice were maintained on a 12-h light-dark cycle (lights on 8:00 am) at 24 ± 3 °C and 55 ± 5% humidity with food and water ad libitum and were housed with littermates until the surgeries. All procedures involving the use of animals complied with the guidelines of the National Institutes of Health and were approved by the Animal Care and Use Committee of the University of Toyama.
c-fos::tTA mice were genotyped via polymerase chain reaction (PCR) with genomic DNA isolated from the tails of the pups as described previously . To detect the Cre recombinase transgene in KA1::Cre mice, 5′-ACCTGATGGACATGTTCAGGGATCG-3′ and 5′-TCCGGTTATTCAACTTGCACCATGC-3′ primers were used with PCR conditions of 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 55 °C for 15 s, and 72 °C for 20 s, followed by 72 °C for 7 min. For floxed-NR1 mice, the primers 5′-GCTTGGGTGGAGAGGCTATTC-3′ and 5′-CAAGGTGAGATGACAGGAGATC-3′ to detect the neomycin resistance cassette and 5′-TGTGCTGGGTGTGAGGGTTG-3′ and 5′-GTGAGCTGCACTTCCAGAAG-3′ to detect the NR1 locus were used with PCR conditions of 94 °C for 2 min, 30 cycles of 94 °C for 30 s, 60 °C for 60 s, and 72 °C for 30 s, followed by 72 °C for 7 min.
The pAAV-EF1α::DIO-ChR2(T159C)-mCherry plasmid was donated by Dr. K. Deisseroth, Stanford University. The pAAV-TRE2G::DIO-ChR2(T159C)-mCherry plasmid was constructed by replacing the human elongation factor 1 α (EF1α) promotor sequence with the second generation tetracycline-responsive element (TRE2G) promotor sequence derived from the pLenti6PW-TGB plasmid [12, 34]. The EF1α promoter sequence was removed from the pAAV-EF1α::DIO-ChR2(T159C)-mCherry plasmid using MluI and KpnI sites. The TRE2G promotor sequence was prepared from the pLenti6PW-TGB plasmid using EcoRI and BstXI sites. Obtained TRE2G sequence and adeno-associated virus (AAV) backbone with DIO-ChR2(T159C)-mCherry sequences were blunted with the Klenow fragment of Escherichia coli DNA polymerase I. The TRE2G sequence was then subcloned into AAV backbone with DIO-ChR2(T159C)-mCherry sequences, generating the pAAV-TRE2G::DIO-ChR2(T159C)-mCherry plasmid.
The recombinant AAV vectors were then produced as described previously [35, 36]. Mice were injected with AAV9-TRE2G::DIO-ChR2(T159C)-mCherry at a titer of 1.3 × 1013 viral genomes (vg)/ml or AAV9-EF1α::DIO-ChR2(T159C)-mCherry at a titer of 1.8 × 1012 to 5.4 × 1012 vg/ml.
Stereotactic surgery and cannula placement
Surgeries were carried out as described previously [12, 13, 17, 37]. The mice were anesthetized with isoflurane, given intraperitoneal injections of medetomidine hydrochloride (0.75 mg/kg of body weight), midazolam (4 mg/kg of body weight), and butorphanol tartrate (5 mg/kg of body weight) and then placed in a stereotactic apparatus (Narishige, Tokyo, Japan). For the optical stimulation experiments, 0.3 μl of AAV9-TRE2G::DIO-ChR2(T159C)-mCherry was bilaterally injected into the CA3 region (anterior-posterior [AP], − 2.0 mm; medial-lateral [ML], ±2.3 mm from bregma; doral-ventral [DV], − 2.0 mm from dura) using a glass micropipette filled with mineral oil attached to a 10-μl Hamilton microsyringe. A microsyringe pump (Narishige, Tokyo, Japan) and its controller were used to control the speed of the injection (0.1 μl/min). The needle was slowly lowered to the target site and remained there for 3 min after the injection. Then, stainless steel guide cannulas (internal diameter, 0.29 mm; outer diameter, 0.46 mm; Plastics One, Roanoke, VA) were bilaterally implanted in the CA3 areas (AP, − 2.0 mm; ML, ±2.3 mm; DV, − 1.0 mm from bregma). Microscrews were anchored in the skull near bregma and lambda, and the guide cannulas were fixed in place with dental cement. After the surgery, dummy cannulas with caps were inserted into the guide cannulas as protective covers. Mice were 11–18 weeks old at the time of surgery, and were allowed to recover 16–19 weeks before being used in behavioral experiments.
To establish the labeling system (for ChR2-mCherry-positive cell counting), the same surgical procedure was used except that the cannulas were not implanted. For this, the mice were 17–27 weeks old at the time of surgery and allowed to recover 5–10 weeks before being used in behavioral experiments.
For in vivo recording experiments, 0.3 μl of AAV9-EF1α::DIO-ChR2(T159C)-mCherry was injected into the CA3 regions of the right hemispheres (AP, − 2.0 mm; ML, + 2.3 mm from bregma; DV, − 2.0 mm from dura) using a glass micropipette. The mice were 13–18 weeks old at the time of surgery and were allowed to recover 21–41 weeks before being used in in vivo recording experiments.
Behavioral analyses and optical stimulation
The home cage of each mouse was placed on a desk in the animal housing room for approximately 10 min before being transferred to the adjoining experimental room. For behavioral tests, each mouse was gently caught at the base of its tail and transferred to each context described below.
Context A was a cylindrical chamber (diameter, 180 mm; height, 230 mm) with a white acrylic floor and walls covered with black tape (see Additional file 1). Context B was a square-type chamber (175 × 165 mm; height, 300 mm) with a transparent acrylic board front, white sides and back walls, and a floor consisting of 26 stainless steel rods with a diameter of 2 mm placed 5 mm apart with a scented tray containing 0.25% benzaldehyde underneath (Additional file 1). The rods were connected to a shock generator via a cable harness. Constant minimal illumination was provided by a small light in the chamber. Context C was a square-type chamber (290 × 250 mm; height, 290 mm) with a transparent acrylic board front wall partially covered with white tape, grey side and back walls, and a floor consisting of grey acrylic board covered with white Kimtowels (Additional file 1). The room lights were off for context B but on for contexts A and C.
The c-fos::tTA/KA1::Cre double transgenic mice were 11–18 weeks old and 17–27 weeks old at the time of surgery for the optical stimulation experiment and for the establishment of the labeling system (ChR2-mCherry-positive cells counting), respectively. Cannula-implanted and AAV-injected c-fos::tTA/KA1::Cre double transgenic mice were maintained on 40 mg/kg Dox food pellets in a microisolation rack system (FRP BIO2000, CLEA Japan) consisting of 16 individually ventilated boxes (1–4 cages/box) with glass fiber filters. Mice were used for behavioral analysis 16–19 weeks and 5–10 weeks after surgery for the optical stimulation experiment and for the establishment of the labeling system (ChR2-mCherry positive cells counting), respectively. Before starting behavioral experiment, the mice were kept under the condition of Dox withdrawal (OFF Dox) for 2 days and then exposed to context A for 6 min. One day later, the mice were subjected to contextual fear conditioning (CFC) in context B, consisting of 3 unsignaled foot shocks (2-s duration, 0.4 mA, 1 min apart) beginning 2 min after acclimation. After the last shock, the mice remained in the context for 1 min and were then returned to their home cages. One day later, the mice were anesthetized with approximately 2.0% isoflurane, and the dummy cannulas were replaced with two-branch-type optical fiber units comprising a plastic cannula body and a tightly connected 0.25-mm-diameter optic fiber (COME2-DF2–250; Lucir, Ibaraki, Japan). The tip of the optical fiber was targeted slightly above CA3 (DV, − 1.5 mm from bregma). The mice were then returned to their home cages for 1–1.5 h. For the optical stimulation session, the mice were moved to the experimental room, and the fiber unit was connected to an optical swivel (COME2-UFC; Lucir), which was connected to a laser (200 mW, 473 nm, COME-LB473/200; Lucir) via a main optical fiber. The delivery of laser pulses was controlled by a schedule stimulator (COME2-SPG-2; Lucir) operating in time-lapse mode. The mice in their home cages were subjected to ten trains of laser pulses, each consisting of 300, 500-μs pulses at 20-Hz of 473 nm light (approximately 10 mW output from the fiber tip) with 45-s intertrain intervals. Approximately 1–1.5 h after the laser stimulation, the mice were anesthetized with approximately 2.0% isoflurane, the optic fiber unit was detached, and the mice were again returned to their home cages. The mice were then given food containing 1000 mg/kg Dox for 2 days and then maintained on food containing 40 mg/kg Dox.
To test fear memory, the mice were placed in contexts A, B, and C for 3 min each at 1, 2, and 3 days after the optical stimulating session, respectively. At the end of each session, the mice were returned to their home cages and the contexts were cleaned with water and 80% ethanol. A video tracking system (Muromachi Kikai, Tokyo, Japan) was used to measure the freezing behavior of the animals, as described in previous studies [12, 13, 17, 38, 39]. Freezing was defined as no movement detected for > 1.5 s. All training and testing were conducted during the light phase of the light-dark cycle. The mean values of the freezing responses during each session were analyzed except for the context A pre-exposure session, for which freezing responses during first 3 min were analyzed.
For the animals used for ChR2-mCherry-positive cell counting, the behavioral experiments were conducted as described above until CFC in context B. One day after CFC, the mice were perfused and histologically analyzed.
In vivo recordings
In vivo recordings were carried out as described previously [17, 38,39,40,41,42], with modification for the optogenetic stimulation. Mice previously infected with AAV9-EF1α::DIO-ChR2(T159C)-mCherry were anesthetized with urethane (1.2 g/kg) and placed in a stereotactic apparatus. An optic fiber (COME2-DF1–250; Lucir) was glued to the recording tungsten electrode (WE 40 mm 0030.1B5, 100 kΩ; MicroProbes, Gaithersburg, MD) so that the tip of the fiber was 500 μm above the tip of the electrode. The optrode was slowly inserted into the CA3 region of the right hemisphere, and the optic fiber unit was connected to an optical swivel (COME2-UFC) connected to a laser (COME-LB473/200). The body temperatures of the mice were maintained by keeping them on a heating pad (MK-900; Muromachi Kikai, or ATC-TY; Unique-Medical Inc., Tokyo, Japan) during the recording sessions.
The delivery of laser pulses was controlled by a Master 8 device (A.M.P. Instruments, Jerusalem, Israel). Optically evoked field excitatory postsynaptic potentials (fEPSPs) was recorded by using optical test pulses (stimulation frequency, 0.033-Hz, 500-μs pulses). After establishing a stable baseline at the recording site for 30 min, a 20-Hz optical stimulating protocol was conducted, which was followed by 0.033-Hz test pulses for 3 h. The 20-Hz optical stimulating protocol was identical to that used in the behavioral analysis, except the laser power was the same as that used for the optical test pulses. The stimulation laser power was adjusted to approximately half of the maximum fEPSP amplitude.
Signals were amplified and filtered from 5-Hz to 1-kHz with a Bioelectric amplifier (MEG-1200; Nihon Kohden, Tokyo, Japan), digitized by Digidata (1322A or 1550B; Axon Instruments, Molecular Devices, San Jose, CA), and sampled at 10-kHz using Clampex software (version 9.2 or 10.7). The data were analyzed with Clampfit 10.7 software. All animals were perfused after the recordings, and the positions of the recording sites were verified.
The mice were deeply anesthetized with an overdose of pentobarbital solution and perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). The brains were removed and further post-fixed by immersion in 4% paraformaldehyde in PBS for 24 h at 4 °C. Each brain was equilibrated in 25% sucrose in PBS and then frozen in dry-ice powder. Coronal sections of 30 μm (for ChR2-mCherry-positive cell counting) or 50 μm thickness (after in vivo recording) were cut on a cryostat and transferred to 12-well cell culture plates (Corning, Corning, NY) containing PBS. After washing with PBS, the floating sections were treated with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/ml, 10,236,276,001; Roche Diagnostics) at room temperature for 20 min and then washed with PBS three times (3 min per wash). The sections were mounted on slide glass with ProLong Gold antifade reagent (Invitrogen of Thermo Fisher Scientific, Waltham, MA). Images were acquired on a fluorescence microscope (BZ9000; Keyence, Osaka, Japan) with a Plan-Apochromat 20× objective lens (Nikon, Tokyo, Japan) for ChR2-mCherry-positive cell counting or with a Plan-Apochromat 10× objective lens (Nikon) for verification of the recording site. To quantify the number of ChR2-mCherry-positive cells, images of CA3 were acquired by collecting z-stacks (2.4 μm apart, 5–6 images). Maximum intensity projections of the images were created with the image analysis software (BZ-II; Keyence). Two sections (AP, approximately − 1.9 and − 2.0 mm from bregma) corresponding to each region of interest (ROI) (CA3 pyramidal cell layers from both hemispheres within 540 × 720 μm2) were chosen from each mouse, and the ChR2-mCherry-positive cells in the ROI were counted manually. The average number of ChR2-mCherry-positive cells per section from one hemisphere are presented throughout the text.
Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA). Comparisons of data between two groups were analyzed with Student’s t tests (two tailed) or Welch’s t test (two tailed, without assuming homogeneity of variances), and multiple-group comparisons were assessed using one-way analyses of variance (ANOVAs) followed by Tukey’s post hoc multiple-comparisons tests when significant main effects were detected. In Fig. 3e, data were analyzed with two-way repeated measures (RM) ANOVA. When significant main effects were detected, Bonferroni’s multiple comparisons test were used for post hoc tests. Quantitative data are expressed as the means ± standard errors of the means (SEMs).