SUPPLEMENTAL MATERIALS AND METHODS Animals All experiments were conducted in accordance with the 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 California at Irvine. Mice (male, C57Bl/6J, 6 weeks; Jackson Laboratory) were acclimated to the vivarium for one week prior to any experimental procedures, and were individually housed with food and water provided ad libitum. Lights maintained on 12:12 light/dark cycle, and all behavior testing was carried out during the light phase of the cycle. Running Wheels Animals in the exercise group had individual access to a running wheel (11.5 cm diameter, Mini Mitter, OR) for three weeks, and voluntary running distance was monitored by automated counters that were interfaced with computer software (VitalView, Mini Mitter) as described previously (Adlard et al. 2004). Exercised animals were housed singly in polyethylene cages (48 x 27 x 20 cm) designed to accommodate the running wheel apparatus. Mice initially ran 1-2 km per night, and progressively increased this distance to 3-5 km per night within one week, maintaining this distance thereafter. Sedentary mice were housed in individually in standard cages without running wheels. Object Location Memory (OLM) OLM training and testing procedures were performed as described previously (Stefanko et al., 2009). Mice were handled for 2 min/d for 5 days, followed by habituation to the experimental apparatus (white rectangular open field measuring 30 x 23 x 21.5 cm) for 5 min/d for 4 consecutive days prior to training. For training trials, each mouse was placed in the apparatus and allowed to explore two identical objects (100 mL glass beakers, 2.5 cm diameter, 4 cm height) for 3 min. All animals were subsequently placed in clean home cages without access to running wheels. Spatial cues around the experimental apparatus, and those on the interior walls of the apparatus, were held constant throughout the experiment. The training trial was limited to 3 min to provide a sub-threshold duration that does not result in longterm memory in sedentary animals, but that does result in long-term memory in sedentary animals treated with the HDAC inhibitor NaB, as shown previously (Stefanko et al., 2009; McQuown et al., 2011). All locations of objects were counterbalanced to reduce bias due to location preference, and all objects were cleaned with 70% ethanol to eliminate odor cues between trials. After 24 h or 7 d, animals were administered a 5-min retention test consisting of re-exposure to the apparatus with one object moved to a novel location. An illustration of the experiment procedures is provided (Fig. 1). OLM Data Analysis The acquisition trial, 24 h and 7d retention tests were recorded using a video camera mounted above the apparatus. For each set of video data, two investigators blind to the experimental conditions scored the time the animal spent exploring each object as indicated by the head oriented towards the object within 1 cm. Object exploration was defined by animals licking, sniffing, or touching the object with the forepaws while sniffing, but not leaning against, standing, or sitting on the object as described previously (Snigdha et al., 2010). To account for differences in exploratory behavior among individual animals, behavior was expressed as the discrimination index (DI). The DI was calculated from the difference between the time exploring each object over the time exploring both objects (DI = (tnovel − tfamiliar)/((tnovel + tfamiliar) × 100) as reported previously (McQuown et al., 2011). Animals with a total exploration time less than 10 s in acquisition trials or retention tests were excluded from analyses in accord with previous studies (Roozendaal, 2002; Okuda et al., 2004 ). The training trials were also screened to ensure that neither the total time exploring the identical objects, nor the preference for object location differed between groups. In vivo siRNA Experiment 2 featured intrahippocampal infusions with SMART pool siRNAs (Dharmacon; Thermo Fisher Scientific, Lafayette, CO) two days prior to training using methods as described previously (McQuown et al. 2011). Briefly, mice were anesthetized using isoflurane and siRNAs were prepared with JetSI transfection reagent (Polyplus Transfection, New York, NY) to a final concentration of 4 µm. Bilateral infusion needles were positioned in the dorsal CA1 region of the hippocampus (anteroposterior −2.0 mm; mediolateral ±1.5 mm from bregma) and lowered 0.2 mm/15 s to a depth of −1.5 mm (dorsoventral from bregma). Needles were set in position for 2 min, and then 1.0 μl of bdnf siRNA or RNA-induced silencing complex-free siRNA (Control) was injected at a rate of 6 μl/h. The solution was allowed to diffuse for 2 min prior to needle removal at a rate of 0.1 mm/15 s. The bdnf siRNA efficacy was confirmed by knockdown of bdnf mRNA and protein via quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively, and lack of knockdown was used as criteria for exclusion from experimental groups. Tissue Preparation Following the final retention test, all animals were briefly anesthetized with CO2 and brains were collected and rapidly frozen in powdered dry ice. Brains were stored at −80⁰ C until they were cryosectioned (Microm Cryostat HM 500, Heidelberg, Germany). Two 300-µm sections were taken extending from rostral to caudal hippocampus (bregma −1.3 mm to −1.9 mm; Paxinos and Franklin 2001). The hippocampus was dissected from these frozen sections using razor blades and stored in 1.5mL tubes at -80⁰ C until further processing. Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) Hippocampal tissue was homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) using RNAse-free tissue pestles and RNA was isolated using the RNeasy® Lipid Tissue Kit (Qiagen, Valencia, CA) and manufacturer’s protocol. 200-ng of total RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcriptase Kit (Roche, Indianapolis, IN) and manufacturer protocol. RT-qPCR primer sets were designed using the Roche Universal Probe Library Assay Design Center and obtained from Integrated DNA Technologies (Coralville, IA) as listed in Supplemental table 1. The relative abundance of these targets was normalized to levels of glyceraldehydes-3-phosphate dehydrogenase (Gapdh) and β-actin after determining that levels of neither housekeeping gene differed across treatments, using multiplexed reactions containing probes conjugated to LightCycler®555 Yellow. These reactions were carried out using reagents from FastStart TaqMan Probe Master (Agilent Technologies, Santa Clara, CA). Total bdnf primers were designed to the common 3’ coding exon. Primers for bdnf transcripts I, IV, and VI correspond to each unique exon sequence (Aid et al. 2007) as shown in Fig. 2B. Brilliant III UltraFast RT-qPCR Master Mix (Agilent Technologies) was used for reactions containing primers for bdnf I, bdnf, IV, and bdnf VI). Standard curves for all primer sets ensured efficiency of 96-100%, and primer specificity was validated using melting curve analyses and by resolving products via 2% agarose gel electrophoresis. All RT-qPCR reactions were run in triplicate using a Stratagene MX3005P thermocycler (Agilent Technologies) programmed as follows: 95⁰ C for 10 min followed by 40 cycles of 95⁰ C for 10 s and 58⁰ C for 15 s. Fluorescence measurements were obtained with the aid of MxPro™ RT-qPCR analysis software and the 2−ΔΔCt method was used to analyze the data as described previously (Livak and Schmittgen 2001). ChIP We focused on histone H4 and unique bdnf transcript promoters in the hippocampus based on their involvement in synaptic plasticity (reviewed by (Sweatt, 2009)). In order to measure relative levels of histone acetylation at bdnf promoter regions, chromatin extraction from hippocampal tissue was performed using the EZ-Magna ChIP A Chromatin Immunoprecipitation Kit and manufacturer’s protocol (Millipore, Billerica, MA). Briefly, hippocampi were cross-linked in 1% formaldehyde (Sigma, St. Louis, MO) for 15 min at 25⁰ C, lysed, and sonicated using a Sonic Dismembrator 60 (Fisher Scientific; Pittsburgh, PA). Sheared chromatin was immunoprecipitated overnight using antibodies: anti-acetylated H3 (positive control; Millipore), non-immune rabbit IgG (negative control; Millipore), anti-acetyl histone H4 lysine 8 (H4K8Ac; Millipore), or anti-acetyl histone H4 lysine 12 (H4K12Ac; Abcam, Cambridge, MA). Immunocomplexes were collected using protein A magnetic beads (Millipore) and chromatin was washed, eluted from the beads, and reverse cross-linked using Proteinase K. DNA was column-purified prior to RT-qPCR with SYBR Green detection and Brilliant III Ultra-Fast RT-qPCR Master Mix (Agilent Technologies). Reactions contained primer sequences for the promoter regions of bdnf I, bdnf IV and bdnf VI as indicated in Fig. 2A. RT-qPCR reactions to amplify the Gapdh promoter were also run for quality control, and all primer sets were selected and optimized to achieve 96-100% amplification efficiency. Oligonucleotide primer sequences were obtained from Integrated DNA Technologies and are provided in Table 2. RT-qPCR reactions were run in a Stratagene MX3005P thermocycler programmed as follows: 95⁰ C for 3 min, followed by 45 cycles of 95⁰ C for 10 s, and 58⁰ C for 15 s. Each RT-qPCR run included all samples run in triplicate and a standard curve. Data were analyzed by the 2−ΔΔCt method and expressed as fold change over control after normalizing with input samples as described previously (Sahar et al., 2007). bdnf ELISA Tissue was processed using the bdnf Emax Immunoassay ELISA kit (Promega, WI, USA) and manufacturer’s protocol. Briefly, frozen tissue was thawed and mechanically homogenized in 18 μl/mg of lysis buffer prepared according to manufacturer's specifications. All procedures were performed at room temperature. Homogenized samples were centrifuged (14,000 rpm, 5 min), the supernatant was assayed for total protein concentration by bicinchoninic acid assay (Thermo Fisher Scientific), and samples were diluted to a uniform concentration using Dulbecco’s phosphate-buffered saline. To this buffer, 1N HCl was added to a pH of 2.5 and the solution was incubated for 15 min before neutralization with an equivalent amount of 1N NaOH. Pre-treated lysate was then assayed by pre-treating Nunc 96-well flat bottom MaxiSorp plates with coating antibody in 0.5 M bicarbonate buffer overnight. Samples and standards were loaded into 96-well plate in duplicate for 2 h, followed by incubation with anti-bdnf polyclonal antibody. HRP-conjugated anti-IgY was added for a 2-h incubation followed by color development with the tetramethylbenzidine reaction. The colorimetric reaction was stopped after 30 min using 1N HCl and absorbance was read on a 96-well plate reader at 450 nm. Statistics Treatment effects were detected using one-way analysis of variance (ANOVA) followed by post hoc Bonferroni’s t-tests to delineate between-group differences. References McQuown SC, Barrett RM, Matheos DP, Post RJ, Rogge GA, Alenghat T, Mullican SE, Jones S, Rusche JR, Lazar MA, Wood MA (2011) HDAC3 is a critical negative regulator of long-term memory formation. J Neurosci 31:764-774. Okuda S, Roozendaal B, McGaugh JL (2004) Glucocorticoid effects on object recognition memory require training-associated emotional arousal. Proceedings of the National Academy of Sciences of the United States of America 101:853-858. Roozendaal B (2002) Stress and memory: opposing effects of glucocorticoids on memory consolidation and memory retrieval. Neurobiology of learning and memory 78:578-595. Sahar S, Reddy MA, Wong C, Meng L, Wang M, Natarajan R (2007) Cooperation of SRC-1 and p300 with NF-kappaB and CREB in angiotensin II-induced IL-6 expression in vascular smooth muscle cells. 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