Supplementary Materials
Materials and Methods
Forced Swim Test (FST). The FST was performed according to previously
published methods from our laboratory (Duman et al, 2007) and others (Caldarone et al,
2003). Briefly, C57Bl/6 mice were placed in a glass cylinder (12 cm diameter) filled to a
depth of 10 cm with water (23-25oC). A 10 minute swim test session was videotaped,
and the time spent immobile during the swim session was recorded by an observer
blinded to treatment groups. Using this modified FST paradigm, Caldarone et al. (2003)
have demonstrated that mice treated chronically with an antidepressant have decreased
total immobility compared to vehicle-treated control animals. Immobility was operational
defined as a lack of movement but included the presence of limb movements necessary
to keep the animal’s head above water.
Novelty-Induced Hypophagia (NIH). The NIH pardigm was performed as
described in previous studies (Dulawa et al, 2004). Briefly, C57Bl/6 mice were singlehoused and trained daily to drink a diluted solution of sweetened condensed milk (3:1,
water to milk). Training consisted of presenting the sweetened milk solution in a water
bottle to each mouse for 30 min/day. On the fourth consecutive day, the mice were
tested in their home cage for latency to drink the milk solution. On the fifth day, latency
to drink the milk solution was tested in a novel environment. For testing in the novel
environment, mice were removed from the home cage and placed in an empty standard
cage with no bedding in a novel room with bright light illumination (650 lux).
Chronic Unpredictable Stress (CUS). CUS experiments were performed
according to previously published procedures with minor modifications (Banasr et al,
2007; Mineur et al, 2006). BALB/c mice were used in this experiment because
antidepressant administration has been shown to reverse behavioral deficits in this strain
following CUS exposure (Yalcin et al, 2008). Mice were group-housed (4 mice/cage)
throughout the CUS experiment expect during exposure to restraint and forced
swim stressors and tests of sucrose and water consumption during which
animals were single-housed. Control subjects that received vehicle or peripheral
BDNF but were not exposed to CUS were group-housed (4 mice/cage) in the
vivarium. Briefly, animals receiving peripheral BDNF or vehicle administration were
exposed to a variable, semi-random sequence of mild, unpredictable stressors. Mice
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were transferred to new cages and exposed to stressors in a behavioral space outside of
the colony. Following stress exposure, animals were returned to their home cages
within the animal colony. Following exposure to stressors that required animals be
separated (i.e. forced swim test and restraint), these subjects were returned to
their original cages with the same cagemates that they were group housed with
throughout the duration of the CUS paradigm. The exact stressors, duration of
stress exposure, and sequence of exposure is shown in Supplementary Table 1. Three
stressors were applied daily: once in the morning (beginning at 9:00AM), once in the
afternoon (beginning at 2:00 PM), and overnight. Stress exposure began one day after
implantation of the mini-osmotic pump and lasted for the duration of the experiment.
This sequence of stressors has been shown previously to reduce sucrose consumption
compared to non-stressed controls (Banasr et al, 2007) and was adapted from earlier
studies (Willner et al, 1987). Sucrose consumption was evaluated following 12 days of
CUS exposure in order to determine the long-term consequences of chronic, peripheral
BDNF administration on stress-induced anhedonia (adapted from Gourley et al, 2008).
Briefly, mice were habituated to a 1% w/v sucrose solution for 2 days (days 8 & 9 of
chronic BDNF or saline treatment). Animals were then returned to water drinking and
given one hour of sucrose access following modest (4, 14, then 19 hrs) water restriction
to habituate animals to water restriction (adapted from Gourley et al, 2008; Willner et al,
1996) and prevent neophobia during testing. On the test day, each mouse was
allowed access to the sucrose solution in the home cage for 1 hr in the absence of
cagemates. The 3 cagemates who were not being tested were group housed in a
clean cage within the colony room. Each mouse had 1 hr access to the sucrose
solution such that the average deprivation period across each cage of 4 mice was
13.5 hrs. For example, in a cage of 4 mice, the deprivation period would range
from 12, for the first mouse tested, to 15 hours for the last mouse tested.
Stressed and non-stressed subjects that received vehicle or peripheral BDNF
experienced the same protocol for testing sucrose consumption. Because testing
order could influence sucrose consumption, an ANOVA using testing order as the
independent measure was performed to confirm no effects of this variable on
consumption (F<1). Testing was repeated the following day, except that mice were
given access to water instead of sucrose solution in order to verify that peripheral BDNF
did not influence thirst levels. Body weights did not differ at the time of the test. Total
sucrose consumption on test day was normalized to body weight. Control animals that
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were not exposed to CUS and that received chronic, peripheral BDNF or saline were
group housed in their home cages within the animal colony. These controls received
the same temporal and spatial sequence of habituation and test days that the
stressed animals experienced (i.e. habituation on days 8 & 9 of BDNF or saline
treatment followed by a modest water restriction and then a sucrose test on day
13 and water test on day 14 of treatment). There were no rank order effects of testing
order (data not shown) or significant differences in water consumption between
treatments (see Supplementary Figure 1). Furthermore, there were no significant
differences in bodyweights on test days.
Elevated Plus Maze (EPM). The method used for evaluating anxiety-like
behavior in the EPM was adapted from Lister (1987). The maze was elevated 50 cm
from the floor and consisted of two open arms (6.0 cm x 35.0 cm) and two enclosed
arms (6.0 cm x 35.0 cm x 15.25 cm) that all extended from a common platform (6.5 cm x
6.5 cm). The EPM was illuminated equally on all four arms. Mice were placed in the
center of the maze facing an open arm and were allowed to explore the maze for 5
minutes. A blinded observer recorded the total time spent in each arm as well as the
number of entries into each arm. Entries were operational defined as occurring when all
four paws crossed into a particular arm (Pellow et al, 1985). The EPM was conducted
under dim lighting conditions (~25 lx).
Open Field Test (OFT). In this paradigm, a mouse was placed into the center of
an open field (76.5 cm × 76.5 cm × 40 cm) constructed of Plexiglas and allowed to
explore for 10 minutes. Mice were videotaped and activity was measured using the
Noldus (Alexandria, VA) EthoVisionPro Video Tracking System in the absence of the
observer. The duration and distance traveled in the center (10 cm × 10 cm square in
middle of the open field) versus the periphery of the open field were measured and used
as indices of anxiety (i.e., more time spent and more distance traveled in the center of
the open field equals less anxiety). The OFT was conducted in a brightly illuminated
(650 lux) room.
Locomotor Activity. Total locomotor activity was measured for one hour by
video tracking software (EthoVisionPro software, Noldus) in standard mouse cages.
Locomotor activity was videotaped in the absence of the observer.
BDNF Enzyme-linked Immunosorbent Assays (ELISAs)
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Separate cohorts of animals were used for the biochemical studies. These
animals received the same chronic BDNF or saline treatment as those subjects used in
the behavioral assays. A separate group of animals was used to collect hippocampus
and striatal tissue for the ELISA and in situ hybridization studies (i.e., one hemisphere
for ELISA and one hemisphere for in situ hybridization). ChemiKine BDNF Sandwich
ELISAs (Chemicon, Temecula, CA) were performed according to the manufacturers’
instructions. In short, BDNF standards and tissue homogenates (hippocampus and
ventral striatum) or blood plasma from mice receiving chronic, peripheral BDNF or saline
were incubated overnight at 4oC in a 96-well dish pre-coated with rabbit anti-human
BDNF polyclonal antibody. Plates were then incubated with 100 µl of biotinylated mouse
anti-BDNF monoclonal antibody (2 hours) followed by 100 µl of streptavidin-HRP
conjugate solution (1 hour). Finally, the plates were incubated in peroxidase substrate
and tetramethylbenzidine solution to produce a colorimetric reaction. The reaction was
stopped with addition of 1 M HCl. Absorbance was measured at 450 nm using a
microplate reader. Sample BDNF concentrations were then determined by non-linear
regression from the standard curves.
In Situ Hybridization (ISH) Analysis
ISH was performed using radiolabeled riboprobes according to conventional
protocols with minor modifications (Nibuya et al, 1995). Briefly, coronal sections of
frozen brain (16 µm) were cut on a cryostat and stored at ~70°C. Tissue sections were
thaw-mounted on RNase-free Probon slides (Fisher), fixed, and dried. ISH was
performed using radiolabeled riboprobes according to conventional protocols with minor
modifications (Nibuya et al, 1995). Riboprobes were generated by PCR amplification
using gene-specific primers. The reverse primer included a T7 template sequence.
Whole rat brain cDNA was used as the template for PCR, which was performed in a realtime PCR instrument (SmartCycler; Cepheid, Sunnyvale, CA) using the Quantitect Sybr
Green PCR kit (Qiagen). PCR product was purified by ethanol precipitation and was resuspended in TE buffer. One microgram of the 300 bp PCR product was used to
produce radiolabeled riboprobe using a T7-based in vitro transcription kit
(Megashortscript; Ambion). All riboprobes were verified by sequencing of the PCR
product. ISH images were quantified using NIH Image software and statistical analysis
was performed using Statview. Linearity of densitometry was verified using 14C step
standards. For each animal, both hemispheres of 3 coronal sections were analyzed for
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a total of 6 determinations, from which the mean was determined.
Bromodeoxyuridine (BrdU) Immunohistochemistry and Quantification
Immunostaining was performed on free-floating sections (35 µm) according to
previously published procedures (Madsen et al, 2005). Mice were transcardially
perfused with ice-cold phosphate-buffered saline (PBS) followed by 4%
paraformaldehyde. Brains were equilibrated in 30% sucrose and cut into 35 µm sections
on a freezing microtome. Every sixth section collected from the prefrontal cortex or
hippocampus was processed for immunoperoxidase staining. This spacing ensured that
the same cell is not counted in two consecutive sections. Briefly, free-floating sections
were denatured, incubated with blocking buffer, and then incubated with mouse antiBrdU antibody (1:100; Becton Dickinson, Franklin Lakes, NJ) in blocking buffer overnight
(4°C). Sections were then incubated with biotinylated goat anti-mouse IgG (1:200)
(Vector Laboratories, Burlingame, CA) for 1 hour followed by amplification and
visualization with diaminobenzidine according to manufacturers specifications (Vector
Laboratories). Neurogenesis was measured by counting BrdU-positive (BrdU+) cells in
the subgranular zone (SGZ) of the dentate gyrus or the prelimibic cortex. The rates of
proliferation and survival of neural progenitor cells were determined using Stereo
Investigator software (MicroBrightField, Williston, VT). A modified, unbiased
stereological procedure was used by an observer blinded to treatment group in order to
quantify BrdU+ cells as described previously (Madsen et al, 2005; Malberg et al, 2000).
For proliferation cell counts, a BrdU+ cell was counted as being in the SGZ if it was
touching or within the SGZ. BrdU+ cells that were located more than two cell diameters
away from the SGZ were excluded. For survival cell counts, all BrdU+ cells that were
located within the granule layer of the dentate gyrus were scored. Brdu+ cells that were
located more than two cells away from the granule layer were excluded. BrdU+ cells
were counted under high-power magnification (400x and 1000x) using a light
microscope. For proliferation and survival cell counts in the prelimibic cortex, a 0.5 x 0.5
mm2 contour was placed over the prelimbic cortex and all BrdU+ cells within the contour
were counted under high-power magnification. Every sixth section was counted
throughout the extent of the hippocampus or prelimbic cortex. The sum of BrdU+ cells
across sections was determined and multiplied by 6 in order to obtain the total number of
BrdU+ cells in the entire dentate gyrus or prelimbic cortex.
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Immunoblotting
A separate group of animals that received the same chronic, peripheral BDNF or
saline treatment as those subjects tested in behavioral assays were used for the
Western blot studies. Standard Western blotting techniques were used to quantify
CREB, pCREB (ser133), ERK1/2, and pERK1/2 (for more detail see Supplementary
Materials) (Kodama et al, 2005). The hippocampus and ventral striatum were rapidly
dissected and immediately frozen on dry ice. Tissue homogenates were made from
frozen tissue sonicated in a solution of 1% sodium dodecyl sulfate (SDS) containing a
cocktail of protease and phosphatase inhibitors (Sigma, MO). Total protein (20 mg) was
loaded into each lane of a tris-glycine gel (Invitrogen, Carlsbad, CA) for electrophoretic
separation. The Odyssey imaging system (LI-COR, Lincoln, NE) was used to scan blots
and bands were analyzed using fluorescent densitometry analysis. Data were
normalized to -actin and subsquently converted to a percent of control samples from
the same membrane in order to control for variance between gels. Findings were
confirmed by analyzing the same samples on independent gels. All identified bands
migrated in patterns consistent with the expected molecular weights for the probed
proteins.
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Supplementary Table 1:
Mice were exposed to three stressors per day (AM, PM, and overnight exposure). The
duration of exposure to each stressor is indicated in parentheses next to the specific
stressor. Animals were exposed to 14 hours of overnight stress (6pm-8am). Sucrose
and water consumption were determined in stressed and non-stressed animals following
12 and 13 days, respectively, of peripheral BDNF or saline administration.
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Supplementary Figure 1
Supplementary Figure 2: Stress exposure does not change water consumption in
mice receiving vehicle or peripheral BDNF. Total water consumption normalized to
bodyweight (mean±SEM) is displayed for stressed and non-stressed animals receiving
saline or 8.0 µg/24 hr BDNF. No significant effects of treatment or stress were found
using a two-way ANOVA. n = 10-16 mice/treatment.
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Supplementary Figure 2
Supplementary Figure 2: Peripheral BDNF administration does not affect total
distance traveled in the perimeter or center zones of a novel, open field
environment. Total duration (mean±SEM) spent in the center (P = 0.95) or the
perimeter (P = 0.87) of an open field is not significantly different between mice receiving
peripheral 8.0µg/24 hr BDNF (n = 12) when compared to saline-treated controls (n = 15).
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