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The Journal of Neuroscience, April 15, 2002, 22(8):3251-3261
Brain-Derived Neurotrophic Factor Produces Antidepressant Effects
in Behavioral Models of Depression
Yukihiko
Shirayama,
Andrew C.-H.
Chen,
Shin
Nakagawa,
David S.
Russell, and
Ronald S.
Duman
Division of Molecular Psychiatry, Abraham Ribicoff Research
Facilities, Connecticut Mental Health Center, Yale University School of
Medicine, New Haven, Connecticut 06508
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ABSTRACT |
Previous studies demonstrated that antidepressant treatment
increases the expression of brain-derived neurotrophic factor (BDNF) in
rat hippocampus. The present study was conducted to test the hypothesis
that BDNF in the hippocampus produces an antidepressant effect in
behavioral models of depression, the learned helplessness (LH) and
forced swim test (FST) paradigms. A single bilateral infusion of BDNF
into the dentate gyrus of hippocampus produced an antidepressant effect
in both the LH and FST that was comparable in magnitude with repeated
systemic administration of a chemical antidepressant. These effects
were observed as early as 3 d after a single infusion of BDNF and
lasted for at least 10 d. Similar effects were observed with
neurotrophin-3 (NT-3) but not nerve growth factor. Infusions of BDNF
and NT-3 did not influence locomotor activity or passive avoidance. The
results provide further support for the hypothesis that BDNF
contributes to the therapeutic action of antidepressant treatment.
Key words:
neurotrophin-3; nerve growth factor; dentate gyrus; learned helplessness; forced swim test; hippocampus; MAP kinase
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INTRODUCTION |
Depression is a devastating illness
that affects ~17% of the population at some point in life, resulting
in major social and economic consequences (Kessler et al., 1994 ).
Significant progress has been made in our ability to treat depression,
but not all depressed patients respond to available antidepressants,
and the therapeutic response requires several weeks or months of
treatment (Duman et al., 2000; Wong and Licinio, 2001). In addition,
there is still very little known about the neurobiological alterations that underlie the pathophysiology or treatment of depression.
In recent years, research has been directed at sites beyond the level
of monoamines and receptors to examine potential postreceptor mechanisms in the action of antidepressant treatment. These studies have identified adaptations of intracellular signaling proteins and
target genes that could contribute to the action of antidepressant treatment (Altar, 1999; Duman et al., 2000; Manji et al., 2000; Wong
and Licinio, 2001). One target gene of antidepressant treatment is
brain-derived neurotrophic factor (BDNF). Antidepressant treatment increases the expression of BDNF in limbic structures, most notably the
hippocampus (Nibuya et al., 1995, 1996; Rosello-Neustadt and Cotman,
1999). Upregulation of BDNF occurs in response to chronic but not acute
antidepressant treatment, consistent with the time course for the
therapeutic action of antidepressants. The possibility that BDNF is
also involved in the pathophysiology of stress-related mood disorders
is supported by reports that BDNF expression is decreased by exposure
to stress (Smith et al., 1995; Nibuya et al., 1999). Clinical
brain-imaging studies demonstrate that the volume of the hippocampus is
decreased in depressed patients, consistent with the possibility of
reduced neurotrophic factor support or synaptic remodeling in
depression (Sheline et al., 1996, 1999; Bremner et al., 2000).
BDNF and other members of the neurotrophic factor family, including
nerve growth factor (NGF) and neurotrophin-3 (NT-3), influence cellular
function via activation of their respective tyrosine kinase receptors
and one of at least three effector systems (Chang and Karin, 2001;
Sweatt, 2001). One of the best-characterized neurotrophin-activated
signal transduction pathways is the mitogen-activated protein (MAP)
kinase cascade, which includes extracellular signal-regulated protein
kinase (ERK) as one of the key steps in the pathway (Chang and Karin,
2001; Sweatt, 2001). A recent postmortem study reports that levels of
ERK activity and expression are decreased in hippocampus and cerebral
cortex of depressed suicide patients, providing additional support that
neurotrophic factor function is downregulated in depression (Dwivedi et
al., 2001).
The results of these basic and clinical studies indicate that
regulation of BDNF expression and function in hippocampus could contribute to the pathophysiology and treatment of depression. To test
this hypothesis, we have examined the influence of infusions of BDNF
into hippocampus on two behavioral models of depression, the learned
helplessness (LH) paradigm and the forced swim test (FST). Both are
well established paradigms that are responsive to antidepressant
treatment (Seligman and Beagley, 1975; Porsolt et al., 1977). In
addition, animals exposed to inescapable stress in the LH paradigm
exhibit effects that are seen in depression, including decreased motor
activity, loss of appetite, weight loss, anhedonia, and
immunosuppression (Thiébot et al., 1992; Weiss and Kilts, 1995).
A previous study has reported that infusion of BDNF into the midbrain
results in an antidepressant-like effect in the LH and FST models
(Siuciak et al., 1997). The current study examines the influence of
BDNF infusions into the hippocampus where the expression of this
neurotrophic factor is regulated by stress and antidepressant treatment.
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MATERIALS AND METHODS |
Intrahippocampus surgery. Animal use procedures were
in accordance with the National Institutes of Health Guide for
the Care and Use of Laboratory Animals and were approved by the
Yale University Animal Care and Use Committee. Male Sprague Dawley rats
(225-300 gm; Charles River, Wilmington, MA) were used. The animals
were housed under a 12 hr light/dark cycle with ad libitum
access to food and water. Surgery was performed in a stereotaxic
apparatus (Kopf, Tujunga, CA) under anesthesia with pentobarbital
sodium solution (50 mg/kg, i.p.; Abbott Laboratories, North Chicago, IL). Rats received bilateral microinjection of different amounts of
BDNF (0.05, 0.25, and 1.0 µg/side), NT-3 (0.25 µg/side), NGF (0.25 µg/side), or saline (0.9%) into the dentate gyrus and CA3 and CA1
regions of hippocampus. A total volume of 1.0 µl was infused into
each side over 15 min, and the injection syringe was left in place for
an additional 5 min to allow for diffusion. In some experiments, a
nonspecific tyrosine kinase inhibitor, K252a (0.5 ng/side), was
co-infused bilaterally with BDNF, or an MAP kinase kinase (MEK)
inhibitor, U0126 (0.1 µg in 1 µl of 1% DMSO), was infused
bilaterally 20 min before infusion of BDNF. The coordinates for the
dentate gyrus, CA3, and CA1 (relative to bregma according to the
atlas of Paxinos and Watson, 1997) were as follows:  3.8 anteroposterior (AP), ±2.0 lateral, and 3.1 dorsoventral (DV) from dura (dentate gyrus); 3.8 AP, ±3.8 lateral, and 3.2 DV from
dura (CA3); and 3.8 AP, ±2.0 lateral, and 2.5 DV from dura (CA1).
In one experiment, three injections were made into different anteroposterior sites in the dentate gyrus (bregma, 3.3, 3.8, and
4.3 AP, ±2.0 lateral, and 3.0 DV from dura). For the learned helplessness experiments, animals received surgery 1 d after the postshock screening test (see below). For the forced swim and open
field tests, surgery was performed 3 d before the first day of a
15 min swim-training session and open field test (see below). After the
behavioral tests, the rats were killed by decapitation. The brains were
removed and stored at 80°C until use. Frozen sagittal sections were
cut by cryostat, and cresyl violet staining was conducted to determine
the placement of the injection syringe. Any behavioral test data
generated from animals in which the syringe track was not in the
correct area were excluded.
Learned helplessness paradigm. In this paradigm, an animal
is initially exposed to uncontrollable stress. When the animal is later
placed in a situation in which shock is controllable (escapable), the
animal not only fails to acquire the escape responses but also often
makes no efforts to escape the shock at all. This escape deficit is
reversed by chronic antidepressant treatment (Chen et al., 2001).
Learned helplessness behavioral tests were performed with the Gemini
avoidance system (San Diego Instruments, San Diego, CA). This apparatus
was divided into two compartments by a retractable door. On day 1, rats
were subjected to 60 inescapable electric foot shocks (0.8 mA; 15 sec
duration; average interval, 45 sec). On day 2, a two-way conditioned
avoidance test was performed as a postshock test to determine whether
the rats would show the predicted escape deficits. This screening
session consisted of 30 trials in which an electric foot shock (0.8 mA;
3 sec duration, at random intervals; mean, 30 sec; average, 22-38 sec)
was preceded by a 3 sec conditioned stimulus tone that remained on
until the shock was terminated. Rats with >20 escape failures in the
30 trails were regarded as having reached the criterion and were used
for further experiments. Approximately 75% of the rats reached this
criterion. For antidepressant treatments, imipramine (10 mg/kg, i.p.,
twice per day), fluoxetine (10 mg/kg, i.p., once per day), or saline
(once per day) was administrated 1 d after the postshock screening
test for 7 d until 1 d before the active avoidance behavioral
tests were performed. For neurotrophic factor infusions, 1 d after
the postshock test, rats received bilateral microinjections of BDNF,
NT-3, NGF, or vehicle as described above. At day 6 (3 d after the
surgery) or day 13 (10 d after the surgery), a two-way conditioned
avoidance test was performed. This test session consisted of 30 trials
in which an electric foot shock (0.8 mA; 30 sec duration, at random
intervals; mean, 30 sec; average, 22-38 sec) was preceded by a 3 sec
conditioned stimulus tone that remained on until the shock was
terminated. The numbers of escape failures and escape latency in each
30 trials was recorded by the Gemini avoidance system.
In a later experiment to examine the effects of U0126, the parameters
for the LH paradigm were adjusted to obtain a higher percentage of
animals that reached the criterion of 20 or more failures in 30 trials.
On day 1, animals received sixty 0.65 mA shocks of 30 sec duration. The
postshock test on day 2 was conducted as described above, except there
were 20 trials at 0.65 mA. An additional inescapable shock was
administered on day 2 to reinforce the LH condition. This consisted of
twenty 0.65 mA shocks of 30 sec duration. This paradigm increased the
number of animals reaching criterion to 80-90%. Finally, on day 6, the two-way active avoidance test was conducted as described above,
except the current was set at 0.65 mA instead of 0.80 mA. Infusions of
BDNF produced an antidepressant effect under both paradigms (see Results).
Forced swim test. This paradigm was performed as described
previously (Porsolt et al., 1977; Siuciak et al., 1997). This is a
standard test used to screen compounds for an antidepressant-like effect. On day 1 (3 d after the surgery for infusions of neurotrophins) the animals were placed in a container with water at a depth of 40 cm
(23-25°C) for 15 min. At this depth, the rats cannot touch the
bottom with their hind limbs or tail. On day 2, the rats were placed
back into the water for 10 min, and the sessions were videotaped. The
10 min session on day 2 was subdivided into two 5 min intervals. The
three behaviors scored are defined as follows (Lucki, 1997); (1)
climbing, with the rat making a active attempt to escape from the tank,
including visual searching for the escape routes and diving; climbing
may be more related to attempts to escape than is swimming (Lucki,
1997); (2) swimming, with the rat staying afloat, pedaling, and making
circular movements around the tank; and (3) immobility, with the rat
not making any active movements.
Open field test. Three days after the surgery, spontaneous
locomotor activity was measured in the open field test in a square arena (76.5 × 76.5 × 49 cm) using a standard procedure
(Lacroix et al., 1998). The open field was divided into two areas, a
peripheral area and a square center (40 × 40 cm). Rats were
allowed to explore for 30 min. The test room was dimly illuminated (two
60 W lights, indirect). The computer software (EthoVision;
Noldus) calculated the velocity of movement, the distance of traveling,
and the time spent in the center of the open field. These parameters
are thought to reflect locomotor activity and fear or anxiety, respectively.
Passive avoidance test. Passive avoidance was conducted
according to standard procedures with the following modifications (Ferry et al., 1999). The apparatus was divided into two compartments by a retractable door: a lit safe compartment and a darkened shock compartment (Gemini avoidance test). Three days after the surgery, animals received a single inescapable foot shock (0.8 mA; 1.0 sec
duration). Twenty-four hours later, each rat was placed in the safe
compartment, and the latency to reenter the darkened shock compartment
with all four paws was recorded as the measure of retention.
Immunocytochemistry. At different time points after infusion
of BDNF (0.5, 2, 4, 24, and 72 hr) animals were perfused. All rats were
placed under deep chloral hydrate anesthesia and killed via
intracardial perfusion with 4% paraformaldehyde in 1× PBS, pH 7.4. Brains were removed, post-fixed overnight in the same fixative (with
shaking) at 4°C, and stored at 4°C in 30% sucrose. Serial coronal
sections of the brains were cut (35 µm sections) through hippocampus
on a freezing microtome, and sections were stored at 4°C in 1× PBS
containing 0.1% sodium azide.
BDNF immunocytochemistry was performed as described previously
(Mamounas et al., 2000; Dawson et al., 2001). Free-floating sections
were washed three times for 5 min in 1× PBS and then incubated for 10 min in 1× PBS containing 0.6% hydrogen peroxide to eliminate
endogenous peroxidases. After washing three times for 5 min in 1× PBS,
sections were then incubated for 1 hr in 1× PBS containing 2% bovine
serum albumin (BSA), 5% normal goat serum, and 0.2% Triton X-100 for
blocking. Sections were incubated at 4°C for 72 hr with primary BDNF
rabbit polyclonal antibody (1:1000; Chemicon, Temecula, CA). After
washing six times for 5 min in 1× PBS, sections were incubated for 2 hr with secondary antibody (biotinylated goat anti-rabbit; Vector
Laboratories, Burlingame, CA) followed by amplification with an
avidin-biotin complex (Vectastain Elite ABC kit; Vector Laboratories)
and were visualized with DAB (Vector Laboratories).
Phospho-ERK (pERK) and Fos immunocytochemistry was performed as
described previously with minor modifications (Sgambato et al., 1998).
For pERK, free-floating sections were treated as described for BDNF
immunohistochemistry and then were incubated at 4°C for 72 hr with
primary pERK rabbit polyclonal antibody (1:500; Promega, Madison, WI).
For Fos, sections were prepared as described above and then incubated
for 1 hr in 1× PBS containing 2% BSA, 5% normal horse serum, and
0.2% Triton X-100 for blocking. Sections were incubated at 4°C for
72 hr with primary Fos goat polyclonal antibody (1:300; Santa Cruz
Biotechnology, Santa Cruz, CA). After washing six times for 5 min in
1× PBS, sections for both pERK and Fos were incubated for 2 hr with
secondary antibody (biotinylated goat anti-rabbit or goat anti-goat,
resectively; Vector Laboratories) followed by amplification with an
avidin-biotin complex (Vectastain Elite ABC kit). Immunolabeled
sections were visualized with DAB.
Statistical analysis. Statistical differences among more
than three groups were estimated by a one-way ANOVA, followed by Scheffe's test. For comparison of the mean values between the two
groups, statistical evaluation was done using the two-tailed Student's
t test. For the forced swimming test, two-way repeated measures ANOVA was performed to assess the overall differences for each
behavior between the groups on day 1 of pretest and on day 2. When a
significant interaction in the between-subjects variables (treatment
and time) was determined, a subsequent one-way ANOVA was performed.
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RESULTS |
Infusion of BDNF into the hippocampus decreases escape failure in
the learned helplessness paradigm
In the learned helplessness paradigm, rats that have been exposed
to inescapable foot shock (IES) exhibit a deficit in escape performance
on subsequent conditioned avoidance behavior. Of 30 trials, animals
exposed to IES failed to escape ~20-25 times, with an escape latency
of ~25-30 sec (Fig. 1). Animals not
exposed to IES readily escaped at a much higher rate (approximately
five failures, with an escape latency of ~5-10 sec; data not shown) (Siuciak et al., 1997; Chen et al., 2001). Subchronic administration of
an antidepressant significantly improves the ability of the animals to
escape in the conditioned avoidance test. This effect was observed in
the current study with administration either of a tricyclic
antidepressant, imipramine, or a 5-HT-selective reuptake inhibitor,
fluoxetine (Fig. 1). These results demonstrate that the learned
helplessness paradigm is responsive to antidepressant treatment as
reported previously (Siuciak et al., 1997; Chen et al., 2001).
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Figure 1.
Infusion of BDNF into the dentate gyrus of
hippocampus decreases escape failure in the LH paradigm. Animals were
exposed to IES as described in Materials and Methods and then were
administered the antidepressants or neurotrophic factors as indicated
before conditioned avoidance testing. Imipramine
(IMI) and fluoxetine (FLX)
were administered for 7 d. BDNF, NT-3, NGF, or saline
(SAL) was administered via bilateral infusion into the
dentate gyrus at the doses indicated, and animals were tested in
conditioned avoidance 3 d later. Escape failure and latency to
escape were determined, and the results are expressed as mean ± SEM. The number of animals is listed under each
column. *p < 0.05;
**p < 0.01; ***p < 0.001 when
compared with saline-injected controls (ANOVA and Scheffe's test).
Left top, F(2,15) = 15.38; p = 0.0002; left bottom,
F(2,15) = 9.282; p = 0.0024; right top,
F(5,77) = 10.050; p < 0.0001; right bottom,
F(5,77) = 10.872; p < 0.0001.
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The effect of bilateral microinjections into the hippocampus of BDNF,
as well as other members of the neurotrophin family, NGF and NT-3, was
determined. Antidepressant treatment produces a robust upregulation of
BDNF in the dentate gyrus granule cell layer (Nibuya et al., 1995), and
neurotrophic factor infusions in this subfield of hippocampus were
tested first. For these studies and all subsequent experiments, the
neurotrophic factors were infused bilaterally into the hippocampus
1 d after IES, and 3 d later the animals were subjected to
conditioned avoidance testing. Rats that received bilateral
microinjection of BDNF or NT-3, but not NGF, into the dentate gyrus of
hippocampus demonstrated a significant improvement in the conditioned
avoidance test relative to vehicle-treated controls (Fig. 1). This was
measured as a significant decrease in failure number and latency to
escape in conditioned avoidance testing and was similar to the effects
of antidepressant treatment. Different doses of BDNF were also tested,
demonstrating a significant improvement in escape performance at 0.25 and 1.0 µg but not at 0.05 µg per side (Fig. 1). The influence of
neurotrophic factor infusion into other subfields of hippocampus was
also examined. Infusion of BDNF, but not NT-3 or NGF, into the CA3
pyramidal cell layer also significantly improved performance in the
conditioned avoidance test (Fig. 2). In
contrast, none of the neurotrophic factors tested improved escape
behavior when infused into the CA1 pyramidal cell layer (Fig. 2). The
results demonstrate that the reversal of escape failure is neurotrophic
factor-specific (BDNF and NT-3 but not NGF), is dose-dependent (e.g.,
BDNF), and is region-specific.
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Figure 2.
Influence of neurotrophic factor infusions into
the CA3 and CA1 pyramidal cell layers on the LH paradigm. After
exposure to IES, BDNF, NT-3, NGF, or saline (SAL) was
infused into the CA3 or CA1 pyramidal cell layers of hippocampus, and
conditioned avoidance was conducted 3 d later. Escape failure and
latency to escape were determined, and the results are expressed as
mean ± SEM. The number of animals is listed under
each column. ***p < 0.001 when
compared with saline-injected controls (ANOVA and Scheffe's test).
Left top, F(3,33) = 12.667; p < 0.0001; left bottom,
F(3,33) = 12.045; p < 0.0001; right top,
F(2,15) = 0.824; p = 0.4575; right bottom,
F(2,15) = 1.292; p = 0.3037.
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Next we conducted an experiment to determine whether the effect of BDNF
on conditioned avoidance is long-lasting. Instead of 3 d after
infusion of BDNF, conditioned avoidance was conducted 10 d later
(Fig. 3). Surprisingly, escape behavior
was still significantly improved even 10 d after bilateral BDNF
infusion at a single level of dentate gyrus. Longer periods could not
be tested, because we found that there is a tendency for spontaneous
recovery of escape 14 d after IES training (Chen et al., 2001;
Maier, 2001). Studies were also conducted to determine the influence of
multiple infusions of BDNF on conditioned avoidance and the time course of this effect. Because of the relatively large molecular weight of the
neurotrophic factors (~13 kDa), diffusion from a single site of
injection would not be expected to influence a very large area of the
hippocampus (see below). To determine whether more injections of BDNF
could produce a more robust blockade of escape deficits, bilateral
infusions were made at three anteroposterior levels of dentate gyrus,
separated by 0.5 mm (0.25 µg/infusion site). This multiple injection
paradigm significantly improved performance in the conditioned
avoidance test, but the effect was not greater than that observed after
infusions at one level of dentate gyrus (Fig. 3).
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Figure 3.
BDNF regulation of LH behavior: long-term effects
and multiple infusions. The LH paradigm was conducted as described in
Materials and Methods, except conditioned avoidance testing was
conducted 10 d after BDNF infusion into the dentate gyrus
(left) or 3 d after infusions at three different
rostrocaudal levels of dentate gyrus (right). The
results are expressed as mean ± SEM. The number of
animals is listed under each column.
*p < 0.05; **p < 0.01;
***p < 0.001 when compared with saline-injected
controls (Student's t test). Left top,
F(1,12) = 4.172; p = 0.0013; left bottom,
F(1,12) = 4.022; p = 0.0017; right top,
F(1,10) = 4.824; p = 0.0007; right bottom,
F(1,10) = 2.853; p = 0.0172.
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Infusion of BDNF into the hippocampus decreases immobility in the
forced swim test
The influence of neurotrophic factor infusions on behavior in
another model of depression, the FST, was also determined. In this
model, the immobility that occurs after rats or mice are placed in a
container of water is reversed by antidepressant treatment (Porsolt et
al., 1977), and this effect has been replicated in our laboratory (Chen
et al., 2001). Recent studies also demonstrate that in addition to
immobility behavior, the FST can be further characterized on the basis
of the time animals spend swimming or climbing at the edge of the tank
(Lucki, 1997).
In the current study, the effects of neurotrophic factor infusions on
immobility, swimming, and climbing were determined. BDNF, NT-3, or NGF
(0.25 µg/side) was infused bilaterally into the dentate gyrus, and
3 d later animals were tested. Neurotrophic factor infusion did
not influence immobility measured when animals were first exposed to
water during the training session on the first day (data not shown).
However, infusions of BDNF or NT-3 into the dentate gyrus significantly
decreased immobility and increased swimming on the next, test day (Fig.
4). These effects were observed during
both the first and second 5 min time blocks. There was a tendency for
climbing behavior to be increased, although this effect was not
significant. In contrast, infusion of NGF into the dentate gyrus did
not influence any of the behaviors measured. Bilateral infusion of
BDNF, but not NT-3, into the CA3 pyramidal cell layer significantly
decreased immobility and increased swimming time (Fig. 4). In general,
the neurotrophic factor selectivity and regional specificity observed
in the FST were similar to those found in the LH paradigm. Because of
these similarities, the effects of NGF infusions into the CA3 pyramidal
cell layer or of any of the neurotrophic factors into the CA1 pyramidal
cell layer were not determined.
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Figure 4.
Infusion of BDNF into the hippocampus has an
antidepressant effect in the FST. BDNF, NT-3, NGF, or saline
(SAL) was infused into the dentate gyrus or CA3 as
indicated. Three days later, the durations of immobility, swimming, and
climbing in the FST were determined. The results are divided into two 5 min time blocks as indicated and are the mean ± SEM of the
number of animals indicated under each
column. *p < 0.05;
**p < 0.01; ***p < 0.001 when
compared with the corresponding saline-injected controls (ANOVA and
Scheffe's test). Left top, Immobility time, 0-5 min,
F(3,49) = 16.348; p < 0.0001; immobility time, 6-10 min,
F(3,49) = 15.823; p < 0.0001; left middle, swimming time, 0-5 min,
F(3,49) = 4.997; p = 0.0042; swimming time, 6-10 min,
F(3,49) = 18.025; p < 0.0001; left bottom, climbing time, 0-5 min,
F(3,49) = 1.807; p = 0.1582; climbing time, 6-10 min,
F(3,49) = 0.477; p = 0.7001; right top, immobility time, 0-5 min,
F(2,30) = 15.749; p < 0.0001; immobility time, 6-10 min,
F(2,30) = 8.219; p = 0.0014; right middle, swimming time, 0-5 min,
F(2,30) = 6.811; p = 0.0036; swimming time, 6-10 min,
F(2,30) = 10.129; p = 0.0004; right bottom, climbing time, 0-5 min,
F(2,30) = 1.243; p = 0.3031; climbing time, 6-10 min,
F(2,30) = 0.812; p = 0.4536. There were no significant treatment × time
interactions.
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Influence of BDNF on additional behavioral tests
Studies were conducted to determine the behavioral specificity of
BDNF infusions into the hippocampus. First, the influence of
neurotrophic factor infusions into the hippocampus on activity in an
open field was determined. The time spent in the center as well as the
distance and velocity traveled were determined. Time spent in the
center of a novel open field is also taken as one measure of anxiety
(Lacroix et al., 1998). Infusions of BDNF or NT-3 into the dentate
gyrus or CA3 subregions of hippocampus, treatments that produced
antidepressant effects in the FST and LH paradigms, did not
significantly influence time in the center, distance, or velocity of
locomotor activity (Fig. 5). There was a
tendency for BDNF infusions to decrease distance traveled and velocity.
This would be opposite to the results expected if a general increase in
locomotor activity were to contribute to the effect of BDNF on
immobility and conditioned avoidance in the FST and LH models of
depression. Infusions of NGF into either dentate gyrus or CA3 subfields
did not influence behavior in the open field test.
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Figure 5.
Influence of neurotrophic factor infusion into the
hippocampus on locomotor activity. BDNF, NT-3, NGF, or saline
(SAL) was infused into the dentate gyrus or CA3, and
3 d later the times in center, distance, and velocity in an open
field were determined. The results are the mean ± SEM of the
number of animals indicated under each
column. Left top, Time in the center,
F(3,30) = 1.285; p = 0.2973; left middle, distance,
F(3,30) = 1.143; p = 0.3479; left bottom, velocity,
F(3,30) = 1.129; p = 0.3529; right top, time in the center,
F(2,15) = 0.073; p = 0.9295; right middle, distance,
F(2,15) = 0.650; p = 0.5359; right bottom, velocity,
F(2,15) = 0.635; p = 0.5438.
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We have also examined the influence of neurotrophic factor infusions on
passive avoidance. For this test, a two-compartment box is used in
which one compartment is illuminated and the other is darkened. A
single inescapable foot shock is administered on day 1 in the darkened
compartment. Twenty-four hr later, animals are placed in the
illuminated compartment and allowed to choose between either
compartment. The darkened compartment is preferred, and the time spent
in the illuminated side is a measure of retention of the memory of the
foot shock on the darkened side. Infusions of BDNF, NT-3, or NGF into
the dentate gyrus failed to change the time spent in the darkened
compartment in the 24 hr retention test (Fig.
6). Similarly, infusions of BDNF or NT-3
into the CA3 pyramidal cell layer of hippocampus did not alter the time
in the darkened compartment in the retention test (Fig. 6). These results provide additional support that BDNF and NT-3 infusions do not
cause a general increase in behavior that could result in the
effects observed in the FST and LH paradigms. If this were the case, we
would have expected to see a decrease in the latency to cross from the
illuminated side to the darkened side in the passive avoidance
test.
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Figure 6.
Influence of neurotrophic factor infusions into
hippocampus on passive avoidance. BDNF, NT-3, NGF, or saline
(SAL) was infused into the dentate gyrus or CA3, and
3 d later, passive avoidance testing was conducted as described in
Materials and Methods. The results are mean ± SEM of the
number of animals indicated under each
column. Left, Day 1 (D1),
F(3,35) = 1.069; p = 0.3746; day 2 (D2),
F(3,35) = 0.962; p = 0.4217; right, D1,
F(2,27) = 1.980; p = 0.1577; D2, F(2,27) = 0.520; p = 0.6002.
|
|
Analysis of BDNF and phospho-ERK immunoreactivity
The regulation of behavior in the LH and FST is somewhat
surprising given the limited diffusion that would be expected after local infusion of a neurotrophic factor into hippocampus. As discussed above, this prompted our experiments to examine the influence of
multiple infusion sites on LH behavior (Fig. 3). To directly address
this point, studies were conducted to determine the extent of BDNF
diffusion by immunohistochemical analysis. In addition, the functional
consequences of BDNF were examined by analysis of pERK and Fos
immunolabeling, both of which are increased by activation of
neurotrophic factor receptors.
Immunolabeling of endogenous BDNF was observed in the major subfields
of hippocampus in saline-infused controls, as reported previously (Fig.
7) (Mamounas et al., 2000; Dawson et al.,
2001). Infusions of BDNF (0.25 µg) into the dentate gyrus
significantly increased immunolabeling for this neurotrophic factor in
the area surrounding the injection site (Fig. 7). Increased BDNF
immunolabeling was observed as early as 30 min after infusion, was most
widespread after 2 hr, and was still present 24 hr later. By 72 hr
after infusion, when behavioral testing was conducted, there was little or no evidence of elevated BDNF immunolabeling. At the 2 hr time point,
the diffusion of BDNF was ~0.5 mm in both the lateral (Fig. 7) and
the rostrocaudal (data not shown) planes from the site of injection.
Diffusion did not reach into the CA3 and CA1 subfields even when higher
doses of BDNF (1.0 µg) were infused (Fig. 7).
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Figure 7.
BDNF immunohistochemistry after local infusion
into the hippocampus. BDNF (0.25 or 1.0 µg) was infused into the
dentate gyrus, and BDNF immunolabeling was determined at the time
points indicated. Representative sections are shown for each time point
and dose of BDNF. Arrows indicate the sites of infusion.
Similar effects were observed in three or four separate animals for
each condition.
|
|
One of the intracellular pathways known to mediate the actions of
neurotrophic factors is the MAP kinase cascade, and the phosphorylation
of ERK (pERK) is a key step in this cascade (Chang and Karin, 2001;
Sweatt, 2001). As one measure of the functional state of the MAP kinase
cascade and the response to infusion of BDNF, the level of pERK
immunoreactivity was determined. In saline-infused animals, very low
levels of pERK were observed, consistent with previous reports
(Sgambato et al., 1998). Infusion of BDNF increased levels of pERK
immunolabeling in dentate gyrus (Fig. 8).
In addition, increased pERK immunolabeling was observed in the CA3 as
well as CA1 subfields (Fig. 8). The pERK immunolabeling was found
primarily in the neuronal processes, although staining was also
observed in cell bodies to a lesser extent (Fig. 8). These results
demonstrate that BDNF infused into the hippocampus activates the MAP
kinase pathway and that the effect spreads to CA3 and CA1 subfields. The latter could result from activity of BDNF at CA3 and CA1 dendrites that extend to the dentate gyrus or via activation of the mossy fiber
pathway to CA3 and then Schaffer colaterals to CA1 pyramidal cell
layers. In any case, this could explain, in part, why local infusion of
BDNF into a specific subfield of hippocampus results in a behavioral
effect similar to that produced by systemic administration of chemical
antidepressants.
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Figure 8.
Influence of BDNF infusion on levels of
phospho-ERK immunolabeling. BDNF (0.25 µg) was infused into the
dentate gyrus, and phospho-ERK immunolabeling was determined at the
time points indicated. Representative low-power (left
panels) and high-power (right panels) sections
are shown for each time point. The locations of the dentate gyrus
(DG) and CA3 and CA1 pyramidal cell layers
(PyrCL) are indicated. Arrows indicate
the infusion sites. Similar effects were observed in three or four
separate animals for each condition.
|
|
To further examine the functional response to BDNF infusions at a
cellular level, the expression of Fos immunoreactivity was examined.
Activation of the MAP kinase pathway increases c-Fos gene expression
via phosphorylation of Elk-1, a transcription factor that
activates the c-Fos promotor (Chang and Karin, 2001; Sweatt, 2001).
Infusion of BDNF into the dentate gyrus significantly increased levels
of Fos immunoreactivity in the granule cell layer, as well as in the
CA3 and CA1 pyramidal cell layers (Fig.
9). The upregulation appears to be a
result of an increase in the number of Fos-immunopositive cells as well
as the amount of Fos per cell. The results provide further evidence
that BDNF infusion produces functional activation of the MAP kinase
pathway.
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Figure 9.
Influence of BDNF infusion on levels of Fos
immunolabeling. BDNF (0.25 µg) was infused into the dentate gyrus,
and Fos immunolabeling was determined 4 hr later. Representative
sections are shown, and similar effects were observed in three or four
separate animals for each condition. The locations of the dentate gyrus
(DG) and CA3 and CA1 pyramidal cell layers are
indicated.
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Inhibition of tyrosine kinase or MEK blocks the antidepressant
effect of BDNF
The neurobiological actions of neurotrophic factors are mediated
by activation of the intracellular tyrosine kinase domain of their
receptors, TrkA, TrkB, and TrkC for NGF, BDNF, and NT-3, respectively.
If the influence of the neurotrophic factors on performance in the LH
paradigm occurs via activation of their respective receptors, then
inhibition of tyrosine kinase activity should block this effect. This
possibility was tested by administration of a broad-spectrum tyrosine
kinase inhibitor, K252a (Tapley et al., 1992; MacKintosh and
MacKintosh, 1994). After IES training, the tyrosine kinase inhibitor
was coadministered bilaterally with BDNF into the dentate gyrus, and
3 d later, animals were exposed to the conditioned avoidance test
as described above. A very low dose of K252a (0.5 ng/side) was chosen
on the basis of previous work with this compound (Pizzorusso et al.,
2000). The results demonstrate that K252a coadministration completely
blocks the effect of BDNF on escape behavior (Fig.
10); that is, animals that have been
exposed to IES and receive infusions of K252a demonstrate the same
escape deficit, although they have received infusions of BDNF. In
contrast, escape performance was significantly improved in animals
receiving infusions of vehicle plus BDNF.
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Figure 10.
Infusion of K252a or U0126 blocks the effect of
BDNF on conditioned avoidance. LH was conducted as in previous
experiments; K252a was co-infused with BDNF, or U0126 was preinfused
before BDNF into the dentate gyrus; and conditioned avoidance was
conducted 3 d later. Escape failure and latency to escape were
determined, and the results are expressed as mean ± SEM. The
number of animals is listed under each
column. *p < 0.05;
**p < 0.01; ***p < 0.001 when
compared with saline-injected controls;
+p < 0.05;
++p < 0.01 when compared with the
BDNF-injected group (ANOVA and Scheffe's test). Left
top, F(3,26) = 7.586;
p = 0.0008; left bottom,
F(3,26) = 8.177; p = 0.0005; right top,
F(3,22) = 10.804; p = 0.0001; right bottom,
F(3,22) = 7.432; p = 0.0013.
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|
To directly examine the role of the MAP kinase cascade in the actions
of BDNF, we next tested the effect of an inhibitor of MEK, which
phosphorylates and activates ERK (Chang and Karin, 2001; Sweatt, 2001).
After IES, a selective MEK inhibitor, U0126, was infused into the
granule cell layer 20 min before infusion of BDNF. A low dose of U0126
(0.1 µg/side) was chosen on the basis of previous in vivo
studies using this inhibitor (Han and Holtzman, 2000; Kuroki et al.,
2001). U0126 pretreatment alone did not influence responding in the
conditioned avoidance test (Fig. 10). However, U0126 pretreatment
completely blocked the effect of BDNF on failure number and latency to
escape, similar to the effect of K252a. In addition to blockade of the
behavioral effects of BDNF, infusion of U0126 completely blocked the
induction of pERK (Fig. 11). Taken together, the results suggest that BDNF regulation of LH behavior is
mediated by activation of MEK and the MAP kinase cascade.
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Figure 11.
Influence of U0126 on the induction of pERK in
hippocampus. Saline or U0126 was infused 20 min before infusion of BDNF
(0.25 µg) into the dentate gyrus. Phospho-ERK immunolabeling was
determined at the time points indicated. Arrows indicate
the sites of infusion. Representative sections are shown for each time
point and condition. The locations of the dentate gyrus
(DG) and CA3 and CA1 pyramidal cell layers
(PyrCL) are indicated. Similar effects were observed in
three or four separate animals for each condition.
|
|
| |
DISCUSSION |
Recent basic and clinical studies provide evidence for a
neurotrophic hypothesis of depression and antidepressant action. According to this hypothesis, decreased expression of BDNF could contribute to the atrophy of hippocampus in response to stress in
depressed patients, and upregulation of BDNF could contribute to the
action of antidepressant treatment (Duman et al., 1997, 2000). The
results of the present study provide further support for this
hypothesis, demonstrating that infusion of BDNF into the hippocampus
produces an antidepressant effect in two standard behavioral models of depression.
The antidepressant actions of BDNF infusions were dose-dependent,
long-lasting, and region- and neurotrophic factor-specific. Doses as
low as 0.25 µg of BDNF produced a maximal effect in both LH and FST.
The effects of BDNF were observed 3 d after infusion and lasted up
to 10 d, indicating that BDNF produces long-term and persistent
adaptations that underlie the antidepressant behavioral responses. A
similar long-lasting effect on the LH and FST was observed after
transient expression of cAMP response element-binding protein in the
dentate gyrus (Chen et al., 2001). Differential expression of the
neurotrophic factor receptors TrkA, TrkB, and TrkC in hippocampal
subfields could account for the neurotrophic factor and regional
specificity. For example, TrkA expression is extremely low in all
subfields of hippocampus, so it is not surprising that infusions of NGF
had no effect in the models tested (Merlio et al., 1993; Ueyama et al.,
1997). The lack of effect of NGF provides an excellent control for
these studies, because it has approximately the same molecular weight
and similar structure as BDNF and NT-3.
TrkB and its ligand BDNF are expressed at relatively high levels in
dentate gyrus and CA3 pyramidal cell layers, and this could account for
the behavioral effects of this neurotrophic factor in both subfields
(Merlio et al., 1993; Ueyama et al., 1997). TrkC and NT-3 are also
expressed in the dentate gyrus, where NT-3 infusion produced a
behavioral response in both LH and FST. However, TrkC but not NT-3 is
also expressed in the CA3 pyramidal cell layer, suggesting that
expression of the appropriate Trk receptor is not the only factor that
determines a behavioral response. The latter conclusion is further
supported by the presence of TrkB expression in CA1, where there was no
effect of BDNF. It is possible that subcellular localization (i.e.,
cell body vs processes) of Trk receptors or differential expression of
the intracellular signaling machinery necessary to respond to receptor activation contributes to the observed selectivity.
Several additional tests were conducted to determine whether BDNF
infusions into hippocampus result in other behavioral effects. Infusion
of BDNF into the dentate gyrus or CA3 pyramidal cell layer did not
influence the distance traveled or the time spent in the center of a
novel open field, indicating that there is not a general effect on
locomotor activity. Open field activity is also a measure of anxiety,
but studies of additional models of anxiety will be required to further
characterize the effect of BDNF on this behavior. There was also no
effect of BDNF infusions on passive avoidance training. However, one
problem with this test is that the animals receiving saline infusions
perform at near-maximal levels, so it is not possible to conclude that
BDNF does not improve learning in this paradigm. However, this test does provide a good control for the LH paradigm, because the animals must stay in the compartment where they are placed, which is opposite to the response produced by BDNF in the LH paradigm (i.e., animals cross over to the other compartment to escape foot shock). It will be
important to conduct additional tests to further characterize the
behavioral effects of BDNF in hippocampus. For example, we are
currently testing the influence of BDNF on context and cued fear
conditioning as a measure of hippocampal-dependent and -independent learning, respectively.
The area of hippocampus influenced by neurotrophic factor infusion was
examined by immunohistochemical analysis of BDNF. Peak levels of BDNF
immunolabeling were observed 2 hr after infusion and lasted for up to
24 hr. By 72 hr, there was little or no evidence of the exogenous BDNF,
providing additional evidence of long-term adaptations that persist
after the neurotrophic factor returns to basal levels. However, it is
possible that trace amounts of BDNF still exist when behavioral testing
occurs. The diffusion of BDNF from the site of infusion was limited
(~0.5 mm), suggesting that BDNF injections into additional sites
might produce a more robust antidepressant effect. However, bilateral
infusions at three different rostrocaudal levels into the dentate gyrus
did not result in a significantly greater effect than infusion at a
single level.
The reason that a single infusion produces an effect similar to that of
multiple infusions may be explained by the finding that the functional
response to BDNF is more widespread than BDNF immunolabeling. Induction
of pERK and Fos immunoreactivity is taken as a measure of activation of
the MAP kinase cascade, one of the primary intracellular pathways
stimulated by BDNF and TrkB (Chang and Karin, 2001; Sweatt, 2001). ERK
is a key mediator of the MAP kinase pathway, and the activity of this
kinase is regulated by phosphorylation. There are many cellular
proteins that are regulated by ERK, including the gene transcription
factor Elk-1, and one of the gene targets of Elk-1 and the MAP kinase
cascade is Fos. Infusion of BDNF into dentate gyrus increased pERK
immunolabeling in the surrounding area of the dentate gyrus, as well as
in CA3 and CA1 pyramidal cell layers. Similarly, we found that Fos
immunoreactivity was upregulated in the dentate gyrus and CA3 and CA1
pyramidal cell layers by infusion of BDNF into the dentate gyrus.
The induction of pERK and Fos by BDNF distal to the infusion site could
occur via one of several mechanisms. One possibility is that the
dendritic fields of CA3 and CA1 pyramidal cells extend far enough to be
influenced by the spread of BDNF from the dentate gyrus. These
pyramidal cells have extensive dendritic processes with an estimated
total dendritic length of 13-16 mm. More than 20% of the processes
are in the stratum lacunosum-moleculare, the terminal region of the
perforant pathway where the dendritic tree of the granule cells also
extends (Amaral and Witter, 1995). Another possibility is that the
induction of pERK and Fos occurs in response to BDNF modulation of
neurotransmission. There are numerous reports of neurotrophic
factor-mediated enhancement of synaptic transmission, primarily via
modulation of presynaptic transmitter release but also by modulation of
postsynaptic sites (Poo, 2001). In addition, a recent study
demonstrated that neurotrophic factors can cause membrane
depolarization via TrkB activity-dependent regulation of a novel
sodium channel (Kafitz et al., 1999). The induction of both pERK and
Fos is known to be activity-dependent, and the response to BDNF could
result from enhancement of synaptic transmission or depolarization of
granule cells. Enhanced synaptic activity in the granule cell layer
could then spread to CA3 via the mossy fiber pathway and to CA1 via
Schaffer collaterals. BDNF could also increase synaptic activity of CA3
and CA1 pyramidal cells via regulation of synaptic transmission at
dendrites that extend to the dentate gyrus as discussed above.
Regardless of the mechanism, induction of pERK and Fos demonstrates
that BDNF infusion produces a functional activation of the MAP kinase
pathway that could contribute to the effects of BDNF in the LH and FST paradigms.
The induction of pERK and Fos suggests that the behavioral effects of
BDNF could occur via activation of Trk receptors and the MAP kinase
cascade. To test this possibility the influence of a broad-spectrum
protein kinase inhibitor, K252a, was determined. Co-infusion of K252a
with BDNF into the dentate gyrus completely abolished the
antidepressant effect of BDNF in the LH paradigm. K252a is an inhibitor
of receptor-tyrosine kinases, and these results are consistent with the
possibility that activation of Trk receptors is required for the
actions of BDNF. However, K252a also inhibits other classes of tyrosine
kinase receptors, as well as Ser/Thr kinases, including those in the
MAP kinase cascade (MacKintosh and MacKintosh, 1994). To further
examine the role of the MAP kinase cascade, we also tested the effect
of a selective inhibitor of MEK, the kinase responsible for activation
of ERK. Infusion of U0126 completely blocked the effect of BDNF on
escape behavior in the LH paradigm as well as the induction of pERK. These results provide additional evidence that activation of MEK and
ERK are necessary for the antidepressant-like effects of BDNF in the LH
behavioral model.
There are several possible neurobiological mechanisms that could
contribute to the antidepressant effects of BDNF, including regulation
of synaptic plasticity and learning and memory. The LH paradigm is
thought to result from the learning and memory of a stressful
experience, and modulation of these processes could influence
responding in the conditioned avoidance test. Studies of long-term
potentiation (LTP), a cellular model of learning and memory, suggest
that BDNF is necessary, but not sufficient, for both the early and late
phases of LTP (Poo, 2001). Therefore, infusion of BDNF several days
after exposure to stress should not influence the memories that have
already been formed. However, it is possible that enhancement of the
learning and memory of new experiences or modulation of hippocampal
synaptic transmission by BDNF could interfere with the memory of the
stressful experience and thereby result in an antidepressant effect.
Another possibility is that BDNF makes hippocampal neurons more
resilient and able to oppose the effects of stress (e.g., oppose the
downregulation of neurotrophic factor support, inhibit potential
excitoxic damage, and block neuronal atrophy; Duman et al., 2000;
McEwen, 2000; Sapolsky, 2000). The ability of BDNF to increase neuronal
survival, function, and synaptic remodeling is consistent with this
possibility (McAllister et al., 1999; Poo, 2001). Future studies will
be needed to further identify the molecular and cellular adaptations
that underlie the actions of BDNF and how these adaptations translate into antidepressant effects at a systems level.
| |
FOOTNOTES |
Received Sept. 20, 2001; revised Jan. 25, 2002; accepted Feb. 1, 2002.
This work is supported by United States Public Health Service Grants
MH45481 and 2 PO1 MH25642, a Veterans Administration National Center
grant for post-traumatic stress disorder, and the Connecticut
Mental Health Center.
Correspondence should be addressed to Ronald S. Duman, Division of
Molecular Psychiatry, Abraham Ribicoff Research Facilities, Connecticut
Mental Health Center, Yale University School of Medicine, 34 Park
Street, New Haven, CT 06508. E-mail: ronald.duman{at}yale.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
