Gene targeting construct for HDAC1 overexpression (OE) mice
The ~1200 nt-long mouse HDAC1 cDNA was amplified from a brain cDNA library and confirmed by sequencing. The cDNA was then cloned upstream of the polyadenylation (pA) signal of pC8N2 with a Spe
I blunt ligation, subsequently HDAC1-pA was cloned into pBSK (Stratagene). A pGKneoLoxP sequence was directionally inserted into the Xho
1 site downstream of the HDAC1-pA in pBSK. The HDAC1-pA-neo was released with Xma
65 and cloned in frame into exon 1 of the Tau
gene. The Tau
targeting arms were taken from pTauKR21
and modified by insertion of a Xma
I and Bsi
WI linker in the unique Nco
I site. The resulting targeting vector (pTH1) containing the in frame fusion of HDAC1 coding sequence with exon 1 of Tau
was confirmed by sequencing. 3–6-month-old mice were used for the behavior test and further analysis.
Gene targeting construct for HDAC2 overexpression (OE) mice
The mouse HDAC2 cDNA was obtained using RT PCR from mouse brain tissue. It was sequenced and subcloned into the XhoI-EcoR1 site of the Topo-TA vector (Invitrogen). The pTH1 targeting vector (described above) was cut open with SmaI-SalI to release HDAC1. The HDAC2 cDNA was cut out from Topo-TA with an EcoRI-XhoI and cloned into the SmaI-SalI site of pTH1, to create the pTH2 targeting vector. The in frame fusion of HDAC2 to exon1 of Tau was verified by sequencing of pTH2. The targeting vectors pTH1 and pTH2 were linearized with SacI and electroporated into V6.5 (129XC57BL/6) F1 embryonic stem (ES) cell line. We picked 96 neomycin resistant clones, of which 46 were analyzed by southern blots. We only used a 3′ external probe, after digestion with BamHI (Left) and EcoRI (Right). Wild-type clones display a 8.8-kb band. The correct targeting event results in a band-shift to 13 kb for the targeted allele. 5 clones were correctly targeted. Two clones were used to generate chimeras by injections into (DBA/2XC57BL/6) F1 blastocysts. Chimeras were mated to C57BL/6 females and offspring was analyzed for germline transmission. The heterozygous knock-in strains were maintained in a mixed background and were mated to obtain homozygous animals. 3–6-month-old mice were used for the behavior test and further analysis.
Generation of HDAC2KO mice
floxed allele was generated by flanking exon 5 and exon 6 with lox
P recombination sites, assuring the deletion of the HDAC-catalytic core of the protein after Cre-recombinase mediated deletion (Supplemental Fig. 7a
). Upon successful targeting of ES-cells and subsequent derivation of chimeric mice, we established a mouse strain carrying a floxed allele of Hdac2
)(FVB). Infection of mouse embryonic fibroblasts with retroviruses expressing Cre-recombinase resulted in complete ablation of HDAC2 only in MEFs carrying two Hdac2
floxed alleles (Supplemental Fig. 7b
). This indicates that the floxed Hdac2
allele is functional and results in an Hdac2
null-genotype upon Cre-recombinase expression. Deletion of Hdac2
in the germline using EIIa-Cre
or Nestin-Cre transgenic mice resulted in viable and fertile Hdac2+/−
mice with no obvious histological abnormalities up to a year of age. Crossing Hdac2+/−
mice gave rise to viable Hdac2
-deficient mice, but these mice were born with a 2-fold lower frequency than expected from a normal Mendelian ratio (9 Hdac2−/−
mice out of 79 littermates, versus 20 out of 79 expected; supplemental Fig. 7c
). Although Hdac2−/−
mice are viable and are capable of producing offspring their fertility is compromised (data not shown). Hdac2−/−
mice (males and females) were approximately 25% smaller compared to wild-type and heterozygote littermates (data not shown). The animals used for behavior tests are in FVBxC57/BL6 background and mated to each other to obtain homozygous animals. 3–6-month-old mice were used for the behavior test and further analysis. There were no difference in behavior tests between males and females.
Fear conditioning tests
Context-dependent fear conditioning. Training consists of a 3 min exposure of mice to the conditioning box (context) followed by a foot shock (2 sec, 0.5/0.8/1.0 mA, constant current). The memory test was performed 24 hr later by re-exposing the mice for 3 min into the conditioning context. Freezing, defined as a lack of movement except for heart beat and respiration associated with a crouching posture, was recorded every 10 sec by two trained observers (one was unaware of the experimental conditions) during 3 min (a total of 18 sampling intervals). The number of observations indicating freezing obtained as a mean from both observers was expressed as a percentage of the total number of observations. For short-term memory test, the memory test was performed 3 hrs after the foot shock training.
Tone-dependent fear conditioning. Training consisted of a 3 min exposure of mice to the conditioning box (context), followed by a tone [30 sec, 20 kHz, 75 dB sound pressure level (SPL)] and a foot shock (2 sec, 0.8 mA, constant current) 30
. The memory test was performed 24 hr later by exposing the mice for 3 min to a novel context followed by an additional 3 min exposure to a tone (10 kHz, 75 dB SPL). Freezing was recorded every 10 sec by two nonbiased observers as described above.
Morris water maze test
The water maze paradigm 31
was performed in a circular tank (diameter 1.8 m) filled with opaque water. A platform (11 × 11 cm) was submerged below the water’s surface in the center of the target quadrant. The swimming path of the mice was recorded by a video camera and analyzed by the Videomot 2 software (TSE). For each training session, the mice were placed into the maze consecutively from four random points of the tank. Mice were allowed to search for the platform for 60 s. If the mice did not find the platform within 60 s, they were gently guided to it. Mice were allowed to remain on the platform for 15 s. Two training trials were given every day; the latency for each trial was recorded for analysis. During the memory test (probe test), the platform was removed from the tank, and the mice were allowed to swim in the maze for 60 s.
Spatial working memory on elevated T-maze
Mice were maintained on a restricted feeding schedule at 85% of their free-feeding weight. Spatial working memory was first assessed on an elevated plastic T-maze. This consisted of a start arm (47 × 10 cm) and two identical goal arms (35 × 10 cm), surrounded by a 10 cm high wall. A plastic food well was located 3 cm from the end of each goal arm. The maze was located 1 m above the floor in a well-lit laboratory that contained various prominent distal extramaze cues. The mice were habituated to the maze, and to drinking sweetened, condensed milk, over several days before spatial non-matching-to-place testing.
Each trial consisted of a sample run and a choice run. On the sample run, the mice were forced either left or right by the presence of a plastic block, according to a pseudorandom sequence (with equal numbers of left and right turns per session, and with no more than two consecutive turns in the same direction). A reward consisting of 0.07 mL of sweetened, condensed milk (diluted 50/50 with water) was available in the food well at the end of the arm. The block was then removed, and the mouse was placed, facing the experimenter, at the end of the start arm and allowed a free choice of either arm. The time interval between the sample run and the choice run was approximately 15 s. The animal was rewarded for choosing the previously unvisited arm (that is, for alternating). Mice were run one trial at a time with an intertrial interval (ITI) of approximately 10 min. Each daily session consisted of 4 trials, and mice received 24 trials in total.
Suberoylanilide hydroxamic acid (SAHA) was synthesized as described previously in WO 93/07148 PTC/US92/08454 32
. WT-161 was synthesized by J.E.B (manuscript in preparation). Sodium butyrate was purchased from Sigma (#B5887). SAHA and WT-161 were dissolved in DMSO as stock solutions and diluted in saline just before injection. Sodium butyrate was prepared in saline. Mice received intraperitoneal injection daily with either SAHA or saline for 10 days or 21 days, using a vehicle consisting of 10% DMSO/90% saline (water with 0.9% NaCl). SAHA and WT-161 solutions were first prepared by making a 50 mg/mL compound/DMSO stock solution, then diluted with saline in a 1:9 ratio (5 mg/mL final concentration) so the final volume of DMSO is 10% and administered immediately using a 26-guage needle.
were cut to 200 μm thick cross-sections with vibratome and analyzed using a Zeiss 200 Axiovert microscope and Openlab software. The number of apical and basal spines on hippocampal CA1 pyramidal neurons was counted blind to the genotype. For each experimental group, a minimum of 10 cells per slice (animal number n = 3) were analyzed. CA1 hippocampal neurons within the region −1.4 mm to −1.6 mm (relative to the Bregma position) were included for the analysis.
Virus mediated spine labeling
Tomato expressing HSV (0.5 μL, gift from Rachael Neve) was stereo-injected into both sides of area CA1 or dentate gyrus with 0.05 μL/min rate. Mice were sacrificed 48 hrs after injection. Brains were fixed with 4% PFA and sectioned with vibratome (50 μm, Leica). Hippocampal slices were scanned with a confocal microscope. Obtained image stacks were reconstructed and analyzed using Image J (NIH).
Immunohistochemical analysis was performed as described before 34
. Antibodies were used in a 1:1000 concentration. Anti-HDAC1, and anti-HDAC2 antibodies were purchased from Abcam. Anti-Ac-lysine, anti-Ac-H4K5, anit-Ac-H4K12, anti-Ac-H3K16, anti-CREB, anti-AKT and anti-CaMKIIα antibodies were purchased from Cell Signaling. Anti-Ac-α-tubulin (K40), anti-actin and anti-synaptophysin (SVP-38) antibodies were purchased from Sigma. Anti-NR2A and anti-NR2B were purchased from BD Biosciences. Anti-β-catenin, anti-EGR1, anti-c-FOS, anti-Brn1, anti-TLE4, anti-CDP, anti-ER81 and anti-GAPDH antibodies were purchased from Santa Cruz. Anti-NeuN antibody was purchased from Chemicon. Confocal images (1 μm) were scanned and subjected to three-dimensional reconstruction. LSMeta10 software (Zeiss) was used to calculate the mean synaptophysin intensity. Brain sections with the strongest intensity were scanned first. All other images included in the analysis were scanned using the same settings. Staining was quantified using LSMeta10 software (Zeiss).
Protein extraction and immunoblotting
The hippocampus and forebrain were collected and lysed in RIPA buffer. The lysates were incubated for 15 min on ice and centrifuged for 15 min at 15,000 × g at 4°C. The supernatant was collected as cytosolic protein extract. The lysates were subjected to 10% SDS-PAGE followed by immunoblotting.
Extraction of histone proteins
Hippocampus samples were collected and homogenized in 400 μL TX-buffer (50mM Tris-HCl, pH8.5, 5mM sodium butyrate). The pellets were resuspended in 0.2M HCl/TX buffer and incubated on ice for 30 mins. Samples were spun down at 14000 rpm and the histone-containing supernatants were subjected to western analysis.
3–6 months old HDAC2OE, HDAC2KO or their littermates were killed by cervical dislocation, and hippocampi were rapidly dissected in iced oxygenated artificial CSF (ACSF). Transverse hippocampal slices, 400 μm thick were placed in a chamber and continuously perfused with oxygenated ACSF. A bipolar stimulating electrodes (0.002-inch-diameter nichrome wire; A-M Systems) placed in the stratum radiatum was used to elicit action potentials in CA3 axons. An ACSF-filled glass microelectrode with a resistance between 0.5 and 3 MΩ was placed in the stratum radiatum region of CA1 and was used to record the field excitatory post-synaptic potentials (fEPSP). Data were acquired using HEKA EPC10 and analyzed by patchmaster (HEKA). Peak fEPSP amplitudes from stimulators were required to be at least 2 mV, and stimulus intensity was set to produce 40% of the maximal response. Baseline responses were recorded for 20 min. fEPSP were evoked at the CA1 synapses by stimulating Schaffer collaterals at a low frequency (2 per min) to establish a stable baseline. Immediately following LTP induction with high-frequency stimulation (HFS, 100Hz, 1s), slices from HDAC2OE and control mice showed an increase in fEPSP slope and amplitude, suggesting that short-term potentiation (STP) occurs in all groups. For HDAC2KO and its control WT slices, LTP was induced by applying one train of stimuli at 100 Hz for 1s. For HDAC2OE and its control WT slices, LTP was induced by applying two trains of stimuli at 100Hz for 1s, with an interval of 20s.
Image-based EGR1 expression assay for cultured neurons
Embryonic cortices(E17) of EGR1-GFP BAC transgenic mice (Genesat Project) were isolated using standard procedures and triturated with trypsin/DNAse digestion. Cortical neurons were plated at a density of 10,000 cells per well in black/clear bottom plates coated with poly-D-lysine (Costar) in neurobasal medium (1.6% B27, 2% glutamax, 1% pen/strep and 5% heat inactivated fetal calf serum) and in neurobasal medium without serum 24 hrs later. Under these culture conditions, the percentage of glia was estimated to be in the range of 5–25%. On day 6, HDAC inhibitors or DMSO control (triplicates or quadruplicates) were added to the cultures for ~30hr. BDNF, KCl or forskolin were added to the cultures on day 7 for 8hr.
Cell were fixed in 4% PFA/4% sucrose in PBS. Fixative was washed away with PBS (3 wash cycles) and processed for EGR1-GFP imaging. Cells (3,000 – 5,000 per well) were imaged and analyzed with 5X objective using the Cellomics ArrayScan Image system. The built-in TargetActivation algorithm was optimized to measure average EGR1-GFP expression per cell (mean Fluorescence intensity per cell per well), using the Hoechst dye to mark cells. The data was normalized to control (DMSO addition).
After imaging, cells were processed for antibody staining: cells were permeabilized with 0.25% TritonX100 (10–15min). Triton was washed away by 3 PBS wash cycles, cells were blocked in PBS containing 10% goat or horse serum (1hr, 37°C). Cells were exposed to anti-acetyl-Lysine -histone H3 or H4 antibody. Then washed 5 times with PBS followed by secondary antibody conjugated to Alexa594, and Hoechst (1hr, RT). Secondary antibody was washed 5 times with PBS, and assayed on Cellomics ArrayScan Image system.
Chromatin immunoprecipitation (ChIP)
ChIP was performed using mouse forebrains fixed with 4% PFA solution and stored at −80°C prior to use. Brains were chemically cross-linked by the addition of one-tenth volume of fresh 11% formaldehyde solution for 15 min at room temperature, homogenized, resuspended, lysed in lysis buffers, and sonicated to solubilize and shear crosslinked DNA. Sonication conditions vary depending on cells, culture conditions, crosslinking, and equipment. We used a Misonix Sonicator 3000 and sonicated at power 7 for 10 × 30 s pulses (90 s pause between pulses) at 4°C while samples were immersed in an ice bath. The resulting whole-cell extract was incubated overnight at 4°C with 100 μL of Dynal Protein G magnetic beads that had been preincubated with 10 μg of the appropriate antibody. Beads were washed five times with RIPA buffer and one time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C with occasional vortexing and crosslinking was reversed by overnight incubation at 65°C. Whole-cell extract DNA (reserved from the sonication step) was also treated for crosslink reversal. Immunoprecipitated DNA and whole-cell extract DNA were then purified by treatment with RNaseA, proteinase K, and multiple phenol:chloroform:isoamyl alcohol extractions. Purified DNA samples were normalized and subjected to PCR analysis. Antibodies used for pull downs were: anti-HDAC1 (#31263), anti-HDAC2(#12169) from Abcam; anti-AcH4 (#06-866), anti-AcH3(# 06-599) from Upstate. After IP, recovered chromatin fragments were subjected to semiquantitative PCR or Real-time PCR for 32–40 cycles using primer pairs specific for 150–250bp segments corresponding to mouse genes promoter regions (regions upstream of the start codon, near the first exon).
Real-time PCR was carried out with SYBR-Green-based reagents (Invitrogen, express SYBR GreenER) using a CFX96 real-time PCR Detection system (BioRad). The relative quantities of immunoprecipitated DNA fragments were calculated using the comparative CT method. Results were compared to a standard curve generated by serial dilutions of input DNA. Data were derived from three independent amplifications. Error bars represent standard deviations.
Primer sequences used for PCR:
- BDNF PI:
- BDNF PII:
- BDNF PIV:
- 5′-ACGGAAACAGCCGAGCTC -3
- PKM zeta (100bp upstream of the PKMzeta mRNA initiation site 35, which contains a cAMP response element (CRE) consensus sequence):
- Creb binding protein (CBP):
- Neurexin I:
- Neurexin III:
- p21 (WAP/CIP1):
- GLUTAMATE RECEPTOR 1 PRECURSOR (GLUR-1/ AMPA 1)
For preparation of nuclei, fresh mouse forebrains were washed with PBS, homogenized in 0.32 M sucrose, 1mM MgCl2, 0.5mM CaCl2 and1mM NaHCO3, and then were centrifuged at 710g for 10 min to get nuclei pellet. The nuclear envelope was removed by addition of 1% Triton X-100 and extracted with lysate buffer (1mM EDTA, 0.5mM EGTA, 10 mM Tris (pH8), 100mM NaCl, 0.1%Na-Deoxycholate, 0.5%N-lauroyl sarcosine). All buffers contained complete protease inhibitor (Roche Molecular Biochemicals). For immunoprecipitation, 2–5μg antibody were added into the nuclear lysates and incubate for 1hr at 4C, then 60 uL Sepharose beads conjected with protein-G (GE healthcare Bio-Sciences AB) were added and incubated for 4 hrs. Beads were spin down at 2000 rpm and washed 4 times with 1 ml RIPA buffer (50 mM Hepes (pH7.6),1mM EDTA, 0.7% DOC, 1% NP-40, 0.5M LiCl). Protein were eluted by sample buffer (containing 1% SDS, 2% 2-Mercaptoethanol) and analyzed.
Antibodies used here: HDAC1, HDAC2, mSin3A from Abcam; MTA-2, CoREST from Upstate.