Animals
The following transgenic (TG) lines were imported from the Jackson (JAX) laboratory: Calcrl-Cre TG (JAX stock, #023014) and Eef1a-LSL-EGFPL10 TG (JAX stock, #030305). Another TG line, Drd2-Cre TG (GENSAT, Clone #ER44), was imported from GENSAT. All TG mice were bred with C57BL/J mice (JAX) to obtain hemizygotes. Drd2-Cre TG (hemizygote) mice were cross-bred with Eef1a-LSLL-EGFPL10 TG (hemizygotes) mice to produce double TG animals. Animals were group-housed 2-5 per cage under a 12 hours light/dark cycle (7 am to 7 pm) with a standard diet (Labdiet™, USA) and water ad libitum. We began all of the animal experiments using age (8-15 weeks)- and gender (male)-matched littermates. All experimental procedures were approved by the Animal Care and Use Committee of the Daegu Gyeongbuk Institute of Science & Technology (DGIST, IACUC #20011503-03).
Dual fluorescence labeling of dMCs and vMCs
The following AAV stocks to express a fluorescence protein in Cre-dependent manner were used for our experiments: AAV1.CAG.FLEX.EGFP (PENN vector core), and AAV2.CAG.FLEX.tdTmato (UNC vector core). Calcrl-Cre TG mice were deeply anesthetized with an intraperitoneal (i.p.) injection of Avertin (250 mg/kg) and placed into a stereotaxic apparatus (Angle Two™, Leica Biosystems, USA). Original AAV stocks were diluted in 1×1012 GC/ml with AAV dilution solution (5% sorbitol in 1X PBS) and 500 nl were injected into either the dorsal hilus (dHil) (-2.1 mm AP, +/-1.4 mm ML, -1.95 mm DV) or ventral hilus (vHil) (-3.3 mm AP, +/-2.7 mm ML, -3.6 mm DV). Flow rate (200 nl/min) was controlled using a Nanopump controller (World Precision Instruments, Florida, USA). The needle was left in the target region after injection for another 5 min. Calcrl-Cre TG mice were allowed at least 3 weeks of rest before the next experimental stage. Any mice with abnormal recovery after stereotaxic surgery were euthanized and thus excluded from the analysis. All injections were verified histologically at the end of the experiments.
Immunohistochemistry and cell counting
All mice were anesthetized by i.p. injection of Avertin (250 mg/kg) and perfused transcardially with PBS, followed by 4% paraformaldehyde (PFA). The brains were extracted and incubated in 4% PFA overnight at 4 °C, before being transferred to 15% sucrose until they sank and then transferred to 30% sucrose overnight at 4 °C. Brains were coronally or horizontally cut into 40 μm sections using a Cryostat (CM3050S, Leica Biosystems, USA). For immunostaining, each slice was incubated with the blocking buffer (1 X PBS, 0.2% BSA, 4% normal goat serum, 0.3% Triton X-100) at room temperature for 1 hour. After blocking, sections were incubated overnight at 4 °C with primary antibodies diluted in the blocking buffer. Primary antibodies for staining were as follows: anti-GluR2/3 (rabbit polyclonal, 1:100; catalog no. ab1506, Millipore, Germany), anti-calretinin (rabbit polyclonal, 1:500; catalog no. 7699/3h, SWANT, Switzerland), anti-parvalbumin (rabbit polyclonal, 1:1000; catalog no. pv27, SWANT, Switzerland), anti-neuropeptide Y (rabbit polyclonal, 1:1000; catalog no. h-049-03, Phoenix Pharmaceutical, USA), and anti-c-Fos (rabbit polyclonal, 1:1000; catalog no. 2250, Cell Signaling, USA). After incubation for 24 hours, sections were washed three times with washing buffer (1 X PBS, 0.3% Triton X-100) for 10 min, before they were incubated with tyramide signal amplification reagent (catalog no. B40955, ThermoFisher, USA) for GluR2/3 and c-Fos and Alexa-flour-conjugated secondary antibodies (1:400; Life Technologies, USA) for calretinin, PV, and NPY at room temperature for 3 hours. The sections were washed three times, counterstained with DAPI, and then mounted using Prolong™ Gold (catalog no. P36930, ThermoFisher) anti-fading mounting medium. To determine cell-type specificity in Calcrl-Cre mice, AAV2.CAG.FLEX.tdTmato was injected into either the dHil or vHil, as described above. Sections were coronally prepared and each representative dorsal and ventral region of the sections stained with cell-type markers (GluR2/3, CRT, PV, and NPY) under standard protocol. Images were taken using a confocal microscope (LSM 780, Zeiss, Oberkochen, Germany). Fluorescence labeled neurons with marker in the hilus were manually quantified using ImageJ (NIH, Maryland, USA).
Fluorescence imaging of dMCs and vMCs
Brains were prepared for immunohistochemistry and fluorescence imaging, as described above. Sections were scanned with a confocal microscope (LSM 780/800, Zeiss, Oberkochen, Germany) under the same imaging conditions. The fluorescent intensity was quantified using ImageJ (NIH, Maryland, USA). The average of fluorescent intensity was measured for the region of interest for each hippocampal subregion, including the hilus, the inner molecular layer (IML), the granule cell layer (GCL), the middle molecular layer (MML), and the outer molecular layer (OML). The linescans were 28 μm (40 pixels) wide.
Three-dimensional imaging of MC projections
Clearing of the intact brain: For imaging of the whole hippocampal region, the posterior two-third of a whole postmortem mouse brain was dissected and polymerized in a 1x PBS solution containing 1% acrylamide (acrylamide:bis-acrylamide=29:1) and 0.1% Azo-initiator (VA-044, Wako) overnight, followed by polymerization for 3 hours at 37 °C (X-Clarity polymerization system, Logos Biosystems, South Korea). Polymerized tissues were cleared in an X-Clarity™ tissue clearing system II (Logos Biosystems, South Korea) for 8 hours at a current of 2 A, temperature of 37.0 °C, and pump speed of 80 rpm. Cleared tissues were stored in a refractive index matching solution (50% sucrose, 20% urea) for imaging with light-sheet fluorescence microscopy.
Three-dimensional (3D) fluorescence imaging: For light-sheet fluorescence microscopy, we used the Ultramicroscope II from LaVision BioTec (Bielefeld, Germany) equipped with Olympus (Tokyo, Japan) MVPLAPO 0.63x lens with dipping cap, NKT Photonics (Birkerød, Denmark) SuperK EXTREME EXW-12 white light laser, Andor Neo sCMOS camera (Thorlabs, New Jersey, USA) and a customized sample holder. Scans were made at 0.63 magnification with a light sheet numerical aperture adjusted at 0.073. For EGFP and tdTomato fluorescence proteins, excitation filters of 470/40, 560/25 and emission filters of 525/50, 620/60 were used. The scan step-size was set at 3 μm and both channels were obtained in two separate scans. For the image post-processing and 3D image rendering, serial tif image files were converted to an Imaris file format and analyzed with Imaris software (Bitplane, Cologne, Germany).
Quantification of dMCs and vMCs subpopulation
For counting the number of MCs, marker (GluR2/3 and CRT) stained cells were counted on every three sections of the dorsal (#1 ~ #3) and twelve sections of the ventral (#4 ~ #8) DG along the longitudinal axis of the hippocampus. The horizontal shape of the DG from the dorsal to the medial part varies greatly, while the ventral part is nearly uniform. To quantitatively analyze a total of dMCs and vMCs in each mouse, MC subpopulations were calculated as: 3 times the number of dMCs (GluR2/3|+CRT-) in each dorsal section (#1 ~ #3) and 12 times the number of vMCs (GluR2/3+|CRT+) in each ventral section (#4 ~#8).
Transcriptional profiling of dMCs and vMCs
Sample preparation and TRAP: TRAP assay was conducted with minor modification of the original procedure 40, as described below. Drd2-Cre female mice (hemizygote) were cross-bred with Eef1a-LSL-EGFPL10 male mice to produce MC-TRAP (Drd2-Cre;Eef1a1LSL.eGFPL10a/+) mice. MC-TRAP male mice around 10 weeks old were used for MC transcriptome analysis. After decapitation, the brains were rapidly soaked in the pre-chilled dissection buffer (1 x HBSS, 2.5 mM HEPES [Ph 7.4], 35 mM glucose, 4 mM NaHCO3, 100 μg/ml cycloheximide, RNase-free Water) for 1 min. The hippocampi of 25 MC-TRAP mice were quickly manually dissected and separated into the dorsal and ventral parts. Five mice per group were pooled for each TRAP to achieve the necessary minimum yield for RNA sequencing. Pooled hippocampal tissues were kept under dissection buffer for 30 min to minimize blood trace and then homogenized in ice-cold polysome extraction buffer (20 mM HEPES [pH 7.4], 150 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 100 µg/ml cycloheximide, protease inhibitors [EDTA-free], 400 unit/ml recombinant RNase inhibitors, and 200 unit/ml Superasin) with a motor-driven Teflon glass homogenizer. Homogenates were cleared by centrifugation at 2000 x g for 10 min at 4 °C. NP-40 (EMD Biosciences, California, USA) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC; Avanti Polar Lipids, Alabama, USA) were added to the supernatant at a final concentration of 1% and 30 mM, respectively, followed by incubation on ice for 5 min. The clarified lysates were centrifuged for 10 min at 20,000 g to pellet insolubilized materials. Monoclonal anti-EGFP antibodies (50 µg each of the clones 19C8 and 19F7) were immobilized onto Dyna magnetic beads (Invitrogen, California, USA) via protein L. These EGFP-affinity beads were added to the supernatant, followed by incubation using an end-over-end rotator for 16 hours at 4 °C. Polysome-bound beads were washed 3 times in the high-salt washing buffer (20 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5 mM dithiothreitol, 100 µg/ml cycloheximide, and RNase-free water) with gentle agitation to resuspend the beads between washing steps. The beads were washed one more time with detergent-free washing buffer and immediately added to RLT buffer, followed by RNA purification with RNeasy Micro Kit (QIAGEN, California, USA) with in-column DNase digestion. The quantity and integrity of purified RNA was determined by using a Quant-iT RiboGreen RNA Assay Kit (ThermoFisher, Massachusetts, USA) and Fragment analyzer (AATI, USA), and only samples with RNA Quality Number (RQN) greater than eight (out of ten) were used for qPCR and RNA-seq.
qPCR analysis: The isolated RNA from the TRAP assay was used to synthesize cDNA with iScript cDNA Synthesis Kit (BioRad, California, USA). 10 ng of cDNA was used for each Quantitative real-time PCR (qPCR) reaction and all samples were run in triplicate. qPCR analysis was carried out using SsoAdvance Universal SYBR Green Supermix (BioRad, California, USA) and AriaMx Real-Time PCR system (Agilent, Technologies, California, USA) following standard cycling conditions (95 °C for 30 s, then 40 cycles of 95 °C for 5 s and then 60 °C for 30 s). The relative quantitation of mRNA was calculated by the comparative Ct method after normalization to mouse Map2. The following primers were used: Calb2, 5′- TTTATGGAGGCTTGGCGGAA-3′ (forward) and 5′-TCATCATAGGGCCTGTTGGC-3′ (reverse); Gria3, 5′-CTCCAAGGACAAGACCAGTGC-3′ (forward) and 5′-GTTTGGACTCTGCCCGTGAT-3′ (reverse). Non-specific amplification was excluded by confirming single melting curve patterns and ethidium bromide staining on 2% agarose gels. All statistical analyses were using Student’s t test.
Data analysis: Full-length cDNA was generated from 500 pg of RNA with Ovation RNAseq V2 kit (NuGene, California, USA). cDNA quality and quantity had been checked on a Fragment Analyzer using DNA High sensitivity assay kit (AATI, USA) prior to sequencing library preparation. Sequencing libraries were constructed by a TruSeq RNA library Prep Kit v2 (Illumina, California, USA) and sequenced for 50 bp paired-end on Illumina HiSeq2500 using Rapid V2 sequencing chemistry (Illumina, California, USA). For each library, the number of detected genes is presented in Table S1. Reads were then aligned to the mouse reference genome (GRCm38) using STAR version 2.5.2b. Read counts per gene were calculated using HTseq-count version 0.7.2. Differential expression analysis on raw read counts was performed in R using the edgeR package. The package implements exact statistical methods based on generalized linear models. The particular feature of edgeR functionality is empirical Bayes methods that permit the estimation of gene-specific biological variation, even for experiments with minimal levels of biological replication. The quasi-likelihood method is implemented for differential expression analyses of bulk RNA-seq data. We first identified ‘expressed’ genes as the genes with counts per million fragments mapped (CPM) larger than 1 under at least two of the biological replicates. Finally, we identified DEGs as the ones that have the adjusted p values < 0.05 and absolute log2-fold-changes > 0.58 (1.5-fold). GO and KEGG pathway analysis were performed in DAVID software using Fisher’s exact test.
Gi-DREADD-dependent MC manipulation
The AAV8.hSyn.DIO.hM4Di-mCherry (PENN vector core) virus was injected in either the dHil or vHil as described above. Control animals were injected with AAV8.hSyn.DIO.mCherry. All MC-specific and region-specific expression of all injections were verified histologically at the end of experiments. The back-metabolized clozapine from CNO may lead to activation of endogenous receptors in non-DREADD animals, thus we applied low-dose CNO (less than 1 mg kg−1) to avoid potential side effects in a higher dose (10 mg kg−1) for our behavioral testing. CNO (Sigma-Aldrich, Missouri, USA) in 0.9% saline was injected i.p. as indicated in Fig. 4 and Fig. 5.
c-Fos Immunohistochemistry in GCs and PV+BCs
For counting c-Fos positive neurons in the DG, mice received CNO (1.0mg kg−1) i.p 30 min before contextual fear conditioning. Then, mice explored the conditioning chamber with a foot shock. After this, mice returned to their home cage in either 90 min or 30 min to examine GCs and PV+BCs activity, respectively. Brains were sectioned coronally and horizontally from each animal. The outline of GCL and SGZ for quantification of GCs and PV+BCs were manually drawn by well-defined anatomical landmarks visualized with staining DAPI. The number of c-Fos positive GCs and PV+BCs were counted on three and two representative dorsal and ventral sections, respectively. The density of neurons in the DG was calculated dividing the number of cells by the total area.
Anxiety-related behavioral testing
All experiments were conducted during the light cycle (7 am to 7 pm). Mice were handled for 1 min on three consecutive days before testing and were randomly assigned to a blinded investigator. Mice were administrated CNO i.p. 120 min before the test was conducted
Elevated plus maze test: the elevated plus maze consisted of two open arms and two enclosed arms (30 × 5 cm) with a center platform (5 × 5 cm). The entire apparatus was placed 50 cm above the floor. Mice were habituated in the testing room in their home cages for 60 min before the test was conducted. Each mouse was placed in the center platform and facing the enclosed arm. The time spent in the open arm was measured using an automated video-tracking system (EthoVision XT, Noldus, USA) for 5 min. Between sessions, the apparatus was cleaned using 70% ethanol.
Open field test: locomotor activity was measured in an open-field area (40 × 40 × 40 cm) using EthoVision XT for 30 min. Mice were transferred to the testing room and acclimated for 1 hour before the test. The center zone index was defined as the center travel distance (20 × 20 × 20 cm) versus total travel distance. After every session, the open field was cleaned with 70% ethanol.
Contextual fear conditioning
The chamber for Context A (paired with shock) was a 15 × 15 × 20 cm chamber with a metal grid, white lighting, and background noise (provided by a fan) that was covered by a transparent acrylic lid. For context B (with no shock), the chamber had a checkered cylindrical wall, grid pattern, no background noise, and the odor of 0.25% benzaldehyde (in 100% ethanol) provided using a paper dipped in the solution and placed beneath the chamber during experiments. All mice were acclimated in the anteroom for 1 hour before the test. Mice were allowed to train the context for 180 sec and then received a foot shock (2 sec, 0.5 mA), followed by a post-shock period of 60 sec. Freezing scores were measured using Freezeframe 4 (Actimetrics Software, Evanston, IL, USA). The threshold was set at 20, and the freezing bout was set at 1 sec. The chamber and grid were cleaned with 70% ethanol between sessions.
Memory encoding test: mice were administrated CNO i.p. 120 min before training context. Mice were placed in context A, followed by a foot shock and a 60 sec post-shock period. After training, mice were returned to their home cage. 24 hours later, mice explored in the shock-associated context again to recall fear memory for 4 min. Freezing scores were quantified for 3 min using Freezeframe 4 (Actimetrics Software, Evanston, IL, USA).
Contextual discrimination: this procedure was based on a protocol described in detail previously 34. For contextual fear acquisition, mice were trained in context A to associate fear memory for consecutive 3 days (days 1-3). Mice were allowed to explore context A for 180 sec, were administered a foot shock (2 sec, 0.5 mA), and were then returned to cage 60 sec later. For contextual generalization, mice were placed in context A or context B without shock for 240 sec and then were placed in the opposite context for 240 sec 1.5–2 hours later (counterbalanced order) on consecutive 2 days (days 4-5). For contextual discrimination training, mice were administrated CNO i.p. 120 min before discrimination of contexts. They were placed in context B (not paired with shock) for 240 sec. following 1.5-2 hours context B, they were placed in context A (again paired with shock) for 180 sec, received a 2 sec 0.5 mA shock, left in the chamber for 60 sec following the shock. Mice were trained to discriminate in both contexts on each day for 12 days. The alternative training orders followed a BAAB -> ABBA pattern (day 6, B → A; day 7 A → B, day 8, A → B; day 9, B → A; day 10, A → B; day 11, B → A; day 12, B → A; day 13, A → B; days 6 through 17). Freezing scores were measured for an initial explored time of 180 sec. For contextual discrimination presentation, the freezing score was combined consecutive 2 days into the block, so that each block consisted of a freezing score of consecutive 2 days in both context A and B. The inhibition of each MC subpopulation and their control group was alternatively performed.
Data analysis and statistics
All data are represented as mean ± standard error of the mean (SEM). Statistical parameters and analysis performed can be found in the figure legends and Supplementary Table 3. Statistical analyses were performed using Prism 7.0a (GraphPad, La Jolla, CA). A comparison of two groups was analyzed by Student’s t-test (paired and unpaired, two-tailed). Two-way ANOVA or repeated measures ANOVA for more than two groups were used to investigate main effects and Bonferroni-corrected post hoc comparisons.