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.
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