Male C57BL/6J mice bred in our animal facility from mice originally obtained from The Jackson Laboratory (Bar Harbor, ME) were used in most experiments. For studies using CREBαΔ mutant mice, experimental mice were male and female wild-type and homozygous products of F1 crosses of CREBαΔ+/−
C57BL/6J with CREBαΔ+/−
129/SvEvTac mice (as by Graves et al., 2002
). The CREBαΔ mutation (Hummler et al., 1994
) had been backcrossed in a heterozygous state in each parental strain for 9–11 generations. Genotyping was performed as described previously (Graves et al., 2002
knock-in mice were generated as described previously (Kasper et al., 2002
; Wood et al., 2006
) and were generously provided by Dr. Paul Brindle (St. Jude Children’s Research Hospital, Memphis, TN). Briefly, the targeting vector for CBP contained the following point mutations: Tyr650Ala, Ala654Gln, and Tyr658Ala. This triple mutation was introduced into the Cbp
locus of embryonic day 14 stem cells derived from 129P2/OlaHsd mice via homologous recombination. Mice carrying the triple-mutation allele of CBP were bred and backcrossed in a heterozygous state onto the C57BL/6J background for at least 10 generations. Experimental animals were generated from heterozygous matings, and wild-type littermates were used as control for homozygous mutant mice (designated cbpKIX/KIX
). The generation of calcium/calmodulin-dependent kinase IIα (CaMKIIα)-CBPΔ1 transgenic mice has been described previously (Wood et al., 2005
). These mice have been bred and backcrossed in a hemizygous state onto the C57BL/6J background for eight generations. Mice were 8- to 16-weeks-old and had access to food and water in their home cages ad libitum
. Lights were maintained on a 12 h light/dark cycle, with all behavioral testing performed during the light portion of the cycle. All experiments were conducted according to National Institutes of Health guidelines for animal care and use and were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. The investigator was blind to the genotype and/or drug treatment of the mice during behavioral or electrophysiological testing.
Bilateral 22 gauge guide cannulas were used to guide an injection cannula into the dorsal hippocampus. The guide cannula placement was as follows: anteroposterior, −1.7 mm; mediolateral, ±1.5 mm; dorsoventral, 1.5 mm below pedestal. Injection cannula extended an additional 0.7 mm below the guide cannula (total depth, 2.2 mm).
Immediately after conditioning, mice received bilateral intrahippocampal injections of 1.0 µl of 16.5 mm trichostatin A (TSA) (A. G. Scientific, San Diego, CA) dissolved in 50% ethanol or 50% ethanol per side [vehicle (VEH)] from a 5.0 µl Hamilton syringe operated by a Harvard Apparatus (Holliston, MA) Pump II Dual Syringe micropump. Injections occurred over 1 min, and injection cannulas were left in place an additional 30 s to allow the fluid to diffuse. Each side was injected individually, one immediately after the other. The entire injection process took ~4 min.
Four hours after intrahippocampal injection with TSA or vehicle, mice were anesthetized with isoflurane and transcardially perfused with PBS, followed by 4.0% paraformaldehyde in PBS using a peristaltic perfusion pump (Rainin Instruments, Oakland, CA). Fixed brains were dissected, postfixed overnight, and then cryoprotected in 30% sucrose at 4°C. Brains were flash frozen in 2-methylbutane on dry ice and mounted on cryostat chucks using OCT. Coronal sections were cut at a thickness of 30 µm and collected in PBS. Floating sections were blocked for 1 h at room temperature in 8% normal goat serum (NGS) (Vector Laboratories, Burlingame, CA) with 0.3% Triton X-100 (Fisher Scientific, Houston, TX) in PBS and then incubated overnight at 4°C in 2% NGS, 0.3% Triton X-100 in PBS with anti-acetyl histone H3 antibody (1:1000; Upstate Biotechnology, Lake Placid, NY) and for 1 h at room temperature with FITC-conjugated anti-rabbit IgG secondary antibody (1:1000; Chemicon, Temecula, CA). Sections were washed three times for 5 min each in PBS before and after each incubation step. 4′,6′-Diamidino-2-phenylindole (DAPI) was added during the last wash for 20 min. Sections were mounted on slides and imaged using a Zeiss (Oberkochen, Germany) Axiovert 200M epifluorescence microscope with CARVII spinning disc confocal unit (BD Biosciences, San Jose, CA) and digitized by using an ORCA-ER CCD camera (Hamamatsu, Bridgewater, NJ). Wide-field (2.5×) and confocal (40×) images of DAPI (cell nuclei) and FITC (acetylated histone) were acquired and combined by using IPLab 4.0 software (BD Biosciences).
Fear conditioning experiments were performed in chambers using the methods described previously (Wood et al., 2005
). Mice were handled for 3 consecutive days for 1 min each day. For contextual fear conditioning, C57BL/6J mice were placed into the conditioning chamber and received a 2 s, 0.75 mA scrambled footshock 2.5 min after placement into the chamber. Mice were removed from the chamber after 3 min. During testing, mice received one 5 min exposure to the same conditioned context in the absence of shock 24 h after conditioning. For cued fear conditioning, C57BL/6J mice were placed into the chamber, and the cue (white noise) was presented from 2 to 2.5 min after placement into the chamber and coterminated with a 2 s, 0.75 mA footshock. Mice were removed from the chamber after a total of 3 min. On testing day, mice in the cued group received one 5 min exposure to a novel context (another conditioning chamber with smooth flat floor, altered dimensions, and a novel odorant) for 0–2 min [pre-conditioning stimulus (CS)], followed by exposure to the cue from 2 to 5 min (CS), 24 h after conditioning. Conditioning was assayed by measuring freezing behavior, the complete absence of movement (Fanselow, 1980
). Freezing was scored during conditioning as well as testing. The behavior of each mouse was sampled at 5 s intervals, and the percentage of those intervals in which the mouse froze was calculated. Different sets of mice were used for contextual and cued conditioning experiments.
For the contextual fear conditioning experiments using CREBαΔ mutant mice, the conditions were similar to those described above except the shock intensity was lowered to 0.40 mA because our preliminary experiments revealed that the wild-type B6/129 F1 hybrid littermates were reaching a freezing ceiling using the 0.75 mA shock intensity (data not shown).
To analyze the effect of the HDAC inhibitor TSA on early-phase LTP (E-LTP), 6- to 10-week old C57BL/6J mice 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 an interface chamber (Fine Science Tools, Foster City, CA) and continuously perfused with oxygenated ACSF containing either TSA (1.65 µm
or vehicle (same volume of 50% EtOH) while they equilibrated for at least 1.5 h at 30°C before starting electrophysiological recordings (Wood et al., 2005
). To perform two-pathway experiments, two bipolar stimulating electrodes (0.002-inch-diameter nichrome wire; A-M Systems, Sequim, WA) placed in the stratum radiatum were used to elicit action potentials in independent sets of CA3 axons. The independence of these inputs was determined by the absence of paired-pulse facilitation across inputs using a 50 ms interpulse interval. An ACSF-filled glass microelectrode (1.5 × 0.85 mm; A-M Systems) with a resistance between 0.5 and 3 MΩ was placed between the two stimulating electrodes in the stratum radiatum region of CA1 and was used to record the resulting field potentials (fEPSPs). Data were acquired using Clampex 8.2 (Molecular Devices, Palo Alto, CA) and analyzed using Clampfit 8.2 (Molecular Devices). Peak fEPSP amplitudes from both stimulators were required to be at least 3 mV, and stimulus intensity was set to produce 40% of the maximal response. A protocol was used in which stimulations occurred every minute, with a 200 ms delay between the two stimulators. Baseline responses were recorded for 20 min, and E-LTP was then induced by applying one train of stimuli at 100 Hz for 1 s through one of the two stimulators. Recordings continued for 60 min after LTP induction. TSA or vehicle remained present throughout the recording session. Initial fEPSP slopes were normalized against the average of the 20 baseline traces and were expressed as percentage.
To analyze the effect of TSA on E-LTP in CREBαΔ mutant mice and cbpKIX/KIX
mice, homozygous mutants and wild-type littermates were used. However, in this protocol, only one stimulating electrode was used, and, after recording baseline CA1 fEPSPs for 20 min in the presence of control ACSF, slices were perfused with ACSF containing either TSA (1.65 µm
) or vehicle (0.05% EtOH) for another 20 min before singletrain stimulation as above. Recordings continued for 120 min after tetanus, and drug remained present throughout the recording period. We also examined the effect of the transcriptional inhibitor actinomycin D (ActD) (Tocris, Ellisville MO), dissolved in 100% DMSO, on the enhancement of E-LTP by TSA in C57BL/6J mice. This experiment was performed as above, except with either ActD (25 µm
) or vehicle (0.05% DMSO) added to the ACSF containing TSA. We also performed experiments using another HDAC inhibitor, sodium butyrate (NaBu) (Upstate Biotechnology) in CREBαΔ mutant and wild-type mice. NaBu was bath applied at 300 µm
for 40 min beginning 20 min before tetanization, as by Levenson et al. (2004)
Preparation and purification of mRNA
Hippocampi were harvested from each animal and placed in 500 µl of RNAlater (Ambion, Austin, TX). Aerosol Barrier ART tips, DEPC-treated sterile water, and autoclaved 1.5 ml microcentrifuge tubes were used for all subsequent steps. Tissue was homogenized in 1 ml of Trizol (Invitrogen, Carlsbad, CA) and transferred to 1.5 ml tubes, in which samples were incubated at room temperature for 5 min. Phenol–chloroform (300 µl) addition was followed by vigorous mixing and room temperature incubation for 3 min. Samples were transferred to phase-lock gel tubes (Eppendorf, Westbury, NY) and were centrifuged at 4°C and full speed for 15 min. The aqueous phase was transferred to new tubes, in which 2 vol of ethanol, 1:10 vol of NaOAc, and 1 µl of glycogen were added. After gentle mixing, the samples were incubated at −20°C for 10 min. Centrifugation at 4°C and full speed was followed by aspiration of the supernatant. Pellets were washed with 300 µl of 70% ethanol and then centrifuged at 4°C and full speed for 5 min. The supernatant was aspirated and samples were air dried for ~ 10 min. After resuspension in 100 µl of water, RNA was purified using the RNeasy system (Qiagen, Valencia, CA) according to the protocol of the manufacturer. Residual DNA was removed by treatment with DNA-free (Ambion). Optical density measurement at 260 and 280 nm by the SmartSpec 3000 (Bio-Rad, Hercules, CA) spectrophotometer was used to assess the concentration and quality of mRNA in each sample.
Preparation of cDNA
Generation of cDNA was performed by the RETROscript (Ambion) kit. Briefly, each reaction was performed with 1 µg; of RNA in a total volume of 20 µl composed as follows: 50 mm Tris-HCl, pH 8.3, 75 mm KCl, 3 mm MgCl2, 5 mm DTT, 500 µm each dNTP, 5 µm random decamers, 10 U of RNase inhibitor, and 100 U of Moloney murine leukemia virus reverse transcriptase. As controls, additional reaction mixtures were generated that lacked either reverse transcriptase or template mRNA. Subsequent reactions were allowed to proceed at 44°C for 1 h, followed by heat inactivation at 100°C for 10 min.
Quantitative real-time reverse transcription-PCR
The cDNA samples were diluted to 2 ng/µl in nuclease-free water. Reactions were prepared in 96-well optical reaction plates (Applied Biosystems, Foster City, CA) with optical adhesive covers (Applied Biosystems). Each well contained 11.5 µl of cDNA, 1 µl of 5 µm
primer solution, and 12.5 µl of Quantitect SYBRgreen PCR mastermix (Qiagen). Four biological and three technical replicates were used in the TSA treatment experiments. Reactions were performed in the Applies Biosystems ABI Prism 7000 with an initial activation by incubation at 50°C for 2 min followed by incubation at 95°C for 15 min and 40 subsequent cycles of 95°C for 15 s, 56°C for 30 s, and 72°C for 30 s. For a list of primers and their corresponding sequences, see supplemental Table S1
(available at www.jneurosci.org
as supplemental material
). Data was normalized to actin G (Actg
), hypoxanthine phosphoribosyltransferase (Hprt
), and Tubulin
before calculation of differences. PCR products were cloned using the TA cloning kit (Invitrogen) following the protocol of the manufacturer. Cloned fragments were then sequenced to confirm the identity of the amplified gene. The Icer
primers are the same as described previously (Conti et al., 2004
). Relative quantitation of gene expression was performed according to Applied Biosystems User Bulletin #2. Fold change was calculated from the ΔCt cycle threshold values with corrections for standard curve data from each gene and housekeeping gene expression levels for each sample based on the relative standard curve method described in the Applied Biosystems manual. For each sample, the ΔCt was calculated against the mean for the sample set of that gene. Next, each of these ΔCt values was corrected with the slope of the standard curve for the relevant primer to account for any variation in primer amplification efficiency. The efficiency-corrected ΔCt value was normalized to the similarly corrected ΔCt from housekeeping genes for each sample to account for variability in mRNA input. The difference between corrected ΔCt for each sample was then grouped according to condition (TSA or vehicle) and the average ΔCt calculated. Because Ct values are on a logarithmic scale, fold change is equal to two raised to the difference between experimental and control ΔCt values. Because correction has been made for primer efficiency, we presented the data as fold change. The data presented are the calculated mean for the biological replicates, with n
being equal to the number of biological replicates (i.e., the number of mice examined).
Animals were cannulated and fear conditioned as described for behavioral experiments. Two hours after fear conditioning, hippocampi were dissected onto a dry ice-cooled cutting surface. The tissue was finely chopped and incubated in freshly prepared 4% paraformaldehyde in PBS for 20 min with gentle rocking. Crosslinking was halted by addition of 2.5 m
glycine and an additional 5 min of gentle rocking. Samples were washed three times in PBS plus protease inhibitors (Sigma, St. Louis, MO), frozen on dry ice, and stored at −80°C. Tissue was thawed on ice, resuspended in 1 ml of cell lysis buffer (10 mm
Tris, 10 mm
NaCl, and 0.2% NP-40), and Dounce homogenized to release nuclei. Nuclear lysis buffer (1% SDS, 10 mm
EDTA, and 50 mm
Tris-HCl, pH 8.1) was added to release chromatin. Chromatin was sheared into 400–600 bp fragments using the Sonic Dismembrator 100 (Fisher Scientific) by five 10 s pulses at 10 W output with 1 min on ice between pulses. Samples were diluted fivefold in chromatin immunoprecipitation (ChIP) dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm
EDTA, 16.7 mm
Tris-HCl, pH 8.1, and 167 mm
NaCl) and precleared with Protein-G plus (Santa Cruz Biotechnology, Santa Cruz, CA). Samples were incubated overnight at 4°C with primary antibodies for acetylated H3 (Upstate Biotechnology) and acetylated H4 (Upstate Biotechnology). Mock immunoprecipitation was performed in parallel with normal rabbit IgG (Upstate Biotechnology). Immunoprecipitation was performed with 100 µl of blocked protein-G plus agarose beads at 4°C for 2 h. Beads were washed twice each with low-salt buffer (0.1% SDS, 1% Triton X-100,2 mm
EDTA, 150 mm
NaCl, and 20 mm
Tris-HCl, pH 8.1), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mm
EDTA, 500 mm
NaCl, and 20 mm
Tris-HCl, pH 8.1), LiCl buffer (250 mm
LiCl, 1% NP-40, 1% deoxycholate, 1 mm
EDTA, and 10 mm
Tris-HCl, pH 8.1), and Tris–EDTA. Chromatin fragments were released from beads in elution buffer (1% SDS and 0.1 m
). Formaldehyde crosslinking was reversed by overnight incubation at 95°C in the presence of 200 mm
NaCl, followed by treatment with proteinase K for 1 h at 55°C. DNA was extracted using Qiaquick spin columns (Qiagen) and analyzed by gel electrophoresis and spectrophotometry (Nanodrop, Wilmington, DE) to confirm DNA quality. Quantitative real-time PCR was performed as described above for mRNA quantification with primers specific to the Nr4a1
(nuclear receptor subfamily 4, group A, member 1) and Nr4a2
promoters (supplemental Table S1
, available at www.jneurosci.org
as supplemental material
ANOVAs were performed in all experiments using SigmaStat (version 2.03; Systat Software, Port Richmond, CA), Systat (version 7.0.1), or Statistica 7 (StatSoft, Tulsa, OK). Repeated-measures ANOVA were performed when appropriate. For fear conditioning data analysis, Student–Newman–Keuls post hoc tests were used. For analysis of electrophysiology experiments, Tukey’s post hoc tests were used. Simple planned comparisons were made using Student’s t test. For the quantitative real-time reverse transcription (RT)-PCR data, we used a Kruskal–Wallis statistic test to determine significance. Experimenters were blind to genotype, and genotypes were confirmed after behavioral and electrophysiological tests. ChIP data analysis was performed using a factorial ANOVA and Newman–Keuls post hoc test.