Conditional Deletion Of Brain-Derived Neurotrophic Factor in the Postnatal Brain Leads to Obesity and Hyperactivity
Maribel Rios,
Guoping Fan,
Csaba Fekete,
Joseph Kelly,
Brian Bates1,
Ralf Kuehn,
Ronald M. Lechan and
Rudolf Jaenisch
Whitehead Institute for Biomedical Research (M.R., G.F., B.B.,
R.J.), Cambridge, Massachusetts 02142; Tupper Research Institute and
Department of Medicine (C.F., J.K., R.M.L.), Division of Endocrinology,
Diabetes, Metabolism and Molecular Medicine, New England Medical
Center, Boston, Massachusetts 02111; Artemis Pharmaceuticals GmbH
(R.K.), Cologne, Germany D-51063; Department of Neuroscience (R.M.L.),
Tufts University School of Medicine, Boston, Massachusetts 02111; and
Department of Biology (R.J.), Massachusetts Institute of Technology,
Cambridge Massachusetts 02139
Address all correspondence and requests for reprints to: Dr. Rudolf Jaenisch, Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142. E-mail:
jaenisch{at}wi.mit.edu
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ABSTRACT
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Brain-derived neurotrophic factor has been associated previously
with the regulation of food intake. To help elucidate the role of this
neurotrophin in weight regulation, we have generated conditional
mutants in which brain-derived neurotrophic factor has been eliminated
from the brain after birth through the use of the cre-loxP
recombination system. Brain-derived neurotrophic factor conditional
mutants were hyperactive after exposure to stressors and had higher
levels of anxiety when evaluated in the light/dark exploration test.
They also had mature onset obesity characterized by a dramatic
80150% increase in body weight, increased linear growth, and
elevated serum levels of leptin, insulin, glucose, and cholesterol. In
addition, the mutants had an abnormal starvation response and elevated
basal levels of POMC, an anorexigenic factor and the precursor for
-MSH. Our results demonstrate that brain derived neurotrophic factor
has an essential maintenance function in the regulation of
anxiety-related behavior and in food intake through central mediators
in both the basal and fasted state.
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INTRODUCTION
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BRAIN-DERIVED NEUROTROPHIC factor (BDNF), a
member of the family of neurotrophins, is essential for the survival
and maintenance of peripheral sensory neurons (1, 2).
Because most Bdnf-/- mutants die
during the second postnatal week, less is known about the in
vivo role of this neurotrophin in the postnatal brain. Previous
findings implicated BDNF in weight regulation as exogenous
BDNF treatment was demonstrated to cause a reduction in weight and
Bdnf+/- animals show an age-related increase in
body weight (3, 4). However, the mode of action of BDNF in
weight regulation remains elusive.
Through the generation and characterization of several mouse genetic
obesity models, a number of central and peripheral factors and their
mechanism of action in food intake and metabolic function have been
identified. Central regulation of food intake is generally associated
with the hypothalamus, where orexigenic factors such as NPY, agouti
related protein (AGRP), orexin, and melanin concentrating hormone (MCH)
and anorexigenic factors such as cocaine and amphetamine-related
transcript, serotonin, TRH, and
-MSH are present
(5, 6, 7, 8, 9, 10, 11, 12). BDNF and its receptor, Trk B, are expressed in
various hypothalamic nuclei associated with eating behavior and obesity
(3). They are present in neurons in the lateral
hypothalamus (feeding center), the ventromedial nucleus (satiety
center), and the paraventricular and arcuate nuclei, both of which are
required for maintenance of normal body weight.
Conclusive evidence that BDNF acts through a central mechanism to
regulate weight is still lacking. In addition, it is necessary to
establish whether this neurotrophin has a developmental or a
maintenance role in weight regulation. To answer some of these
questions and to circumvent the problem of early mortality associated
with global ablation of the BDNF gene, conditional mutant mice were
generated in which BDNF was eliminated in a tissue- and
temporal-specific manner using the cre-loxP recombination system
(13). Analysis of these mice revealed that BDNF affects
locomotor behavior and regulates food intake through a central
maintenance mechanism that is independent from its developmental
function.
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RESULTS
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Generation of Conditional BDNF Mutants
LoxP sites were inserted around the single coding exon of BDNF by
standard gene targeting techniques (14) (Fig. 1A
). Mice carrying the floxed BDNF allele
(Bdnf2lox) were generated using targeted
ES cell clones and homozygous mice for this allele were normal and
fertile. Excision of the BDNF coding sequence in the brain was
accomplished by crossing the Bdnf 2lox
allele with two different lines of mice expressing cre recombinase
under the direction of the
-calcium/calmodulin-dependent protein
kinase II (CamK) promoter, which drives expression in postmitotic
neurons (15). To assess temporal and spatial
activity of cre recombinase, a LacZ reporter transgene that is
activated by cre-mediated recombination (16) was crossed
with the CamK-cre93 strain. We detected only a few blue cells in
various regions of the newborn brain (Fig. 1B
). However, the cre
transgene became widely activated at P21 in the cortex, hippocampus,
hypothalamus, and brainstem with no recombinase activity detected in
glia (Fig. 1B
and data not shown). In the CamK-cre159 transgenic line,
cre recombinase activity began at P15, and the final level of
recombination was reached by the fourth postnatal week (data not
shown). Both lines of conditional mutants showed a similar spatial
pattern of recombination. BDNF conditional mutants obtained by crossing
the floxed BDNF allele with CamK-cre93 and CamK-cre159 transgenic mice
are referred to as Bdnf 2lox/2lox/93
and Bdnf 2lox/2lox/159, respectively.

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Figure 1. Generation and CamK-cre-Mediated Recombination of the
Floxed Bdnf Allele
A, Three lox P sites (triangles) and a selection
cassette (gray box) were introduced into the
Bdnf wild-type allele (Bdnf+) to generate the Bdnf3lox allele. The Bndf2lox allele was produced by removal of the
selection cassette in vitro by cre recombinase
(vertical arrows). Bdnf 2lox
carriers were crossed to mice expressing the cre recombinase under the
CamK-cre promoter, which resulted in the generation of the Bdnf1lox allele in the brain. The white and
black rectangle represents the single coding exon of
Bdnf and the black portion of the box
represents sequence coding for the mature form of BDNF. B, X-gal
staining of coronal sections of hypothalami and hippocampi obtained
from P0 and P21 CamK-cre93/lac Z reporter mice. Lac Z expression
requires cre-mediated recombination. C, Representative Southern blot
containing DNA samples extracted from hypothalamus (1 ), hippocampus
(2 ), cortex (3 ), kidney (4 ), and heart (5 ) from a Bdnf2lox/+/93 mouse, 12 wk of age. D, Representative
Northern blot containing RNA extracted from adult wild-type and
Bdnf 2lox/2lox/93 hypothalami and hippocampi
probed with BDNF and GAPDH. Abbreviations: B, BglII;
CMV, cytomegalovirus; Hypoth, hypothalamus; Hippoc, hippocampus;
3V, third ventricle; DG, dentate gyrus; C, control; CM, conditional
mutant.
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Elimination of the BDNF coding sequence in both lines of conditional
mutants was restricted to the brain (Fig. 1C
and data not shown).
Northern blot analysis showed that BDNF was substantially reduced in
the hypothalamus, hippocampus, and cortex of adult Bdnf2lox/2lox/93 mice (Fig. 1D
and data not
shown). We conclude that the CamK-cre93 and 159 transgenes lead to the
deletion of BDNF postnatally and thus provide a genetic tool to assess
the role of this neurotrophin in the postnatal brain.
BDNF Conditional Mutant Mice Are Anxiety Prone and Obese
Bdnf 2lox/2lox/93 mice were viable,
hyperactive when stressed (Fig. 2
), and
displayed increased intermale aggression (data not shown). When we
examined baseline activity during the light cycle, BDNF conditional
mutants appeared to be marginally more active than the controls but
this was not statistically significant (Fig. 2A
). However, there was a
clear difference in the locomotor behavior of the mutants compared with
the controls when any attempt was made to handle them or their cages.
They became very agitated and active and appeared stressed, and this
behavior was already apparent by the fourth postnatal week. To
investigate this further, mice were individually placed in fresh cages
and their locomotor activity monitored during and after a 1-h
habituation period. Exposure to a novel cage is a mild stressor, which
initially causes an increase in activity in normal mice that is reduced
substantially after a period of habituation. Whereas control mice
appeared relaxed after the habituation hour, both female and male
mutants continued to appear agitated. In normal mice, activity was
reduced by 72% after the habituation hour compared with a 40%
reduction in activity observed in the mutants. We found that mutant
mice were 66% and 250% more active than the controls during the
habituation period and the posthabituation hour, respectively (Fig. 2A
). These results suggested that the absence of central BDNF caused an
increase in anxiety levels in the mutants.

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Figure 2. BDNF Conditional Mutants Have Increased Levels of
Anxiety
A, Locomotor activity of control (white columns) and
BDNF conditional mutant (black columns) mice at baseline
(n = 9) and after exposure to a novel chamber (n = 6) was
monitored. Baseline activity was measured for 1 h during the light
cycle, and activity after exposure to a novel chamber was measured
during the first hour immediately after placement into a fresh cage and
for an additional hour after the animals were allowed to habituate for
1 h to the new chamber. *, P < 0.05; **,
P < 0.03. B, Latency of first entry into the light
zone and total time spent in the light chamber during the dark/light
exploration test. Each control (white columns) and
conditional mutant (black columns) mouse (n = 12)
was tested for a period of 5 min. *, P < 0.004;
**, P < 0.044. C, Number of dark-light compartment
transitions during the dark/light exploration test (n = 12). *,
P < 0.001.
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To further investigate the anxiety-related behavior of the mutants, the
light/dark exploration test was performed. For this test, animals were
placed in a box consisting of a dark chamber and a larger brightly
illuminated second chamber. The latency for the first entry into the
light compartment, total time spent in the light compartment, and the
number of dark-to-light chamber transitions were monitored for each
animal for a period of 5 min. Because mice have a natural aversion to
open and bright spaces, animals that are more anxiety prone will have
longer latency times for the first transition into the light zone, will
spend less total time in the light chamber, and will make less
dark-to-light zone transitions. Conditional mutants exhibited a
reduction in exploratory behavior and an increase in anxiety-like
behavior during the light/dark exploration test (Fig. 2
, B and C). It
took mutants 3.6 times longer to make the first transition from the
dark to the light compartment, and they spent half as much time in the
light zone compared with the controls (Fig. 2B
). In addition, mutants
only made 35% the number of dark-to-light zone transitions compared
with the control animals (Fig. 2C
). These results show that the absence
of normal levels of central BDNF has an anxiolytic effect.
In addition to changes in locomotor activity and anxiety-related
behavior, conditional mutants had increased body weights compared with
littermate controls, and this difference reached statistical
significance at 8 wk of age (Fig. 3
, A and
B). By 30 wk of age, mutant males and
females were 80 and 150% heavier than age-matched controls,
respectively. Weight of mutant females at 30 weeks was 64.6 g ±
3.8 compared with 25.8 g ± 1.5 for the controls
(P < 0.0004) (Fig. 3A
). Conditional mutant males had a
weight mean value of 56.5 g ± 1.3 compared with 31.4 g ±
2.9 for the controls (P < 0.002) (Fig. 3B
).

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Figure 3. Increased Body Weight, Food Intake, and Body Length
of the BDNF Conditional Mutants
A, Growth curve of control (open circles) and
Bdnf 2lox/2lox/93 (solid
circles) females (n = 15). B, Growth curve of control
(open circles) and Bdnf2lox/2lox/93 (filled circles) males
(n = 14). C, Measurement of daily food intake by controls and
conditional mutant mice (n = 15). *, P <
0.0003. D, Measurement of body length of control and Bdnf2lox/2lox/93 mice (n = 11). *,
P < 0.01. Abbreviations: C, controls; CM,
conditional mutants.
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To assess whether the increased body weight in the mutants was
associated with hyperphagia, food consumption was monitored in control
and mutant animals fed a standard chow diet ad libitum. We
found that food intake in Bdnf2lox/2lox/93 mice was 74% higher than that
of the controls (P < 0.0003) (Fig. 3C
). Furthermore,
when Bdnf 2lox/2lox/93 mutants were
pair fed with control littermates, they lost 19.3% of their weight in
a period of 9 d. Alterations in linear growth were also examined
in the mutants. They had a 10% increase in naso-anal length compared
with the controls (P < 0.01) (Fig. 3D
).
BDNF is an important survival and differentiation factor during
development of the nervous system (1, 2). As BDNF was
removed from the brain postnatally in BDNF conditional mutants, it was
unlikely that the phenotypes observed were the result of the disruption
of developmental functions normally carried by this neurotrophin. To
confirm that the obesity and hyperactivity in the mutants arose from
the lack of a maintenance function of BDNF in the brain, the weights of
Bdnf 2lox/2lox/159 mice were measured
and their locomotor activity monitored. Recombination of the floxed
BDNF allele in this line of mice begins at P15 and is completed during
the fourth postnatal week. Similar to Bdnf2lox/2lox/93 mice, Bdnf2lox/2lox/159 mutants were obese and
hyperactive (data not shown), confirming that the phenotype was caused
by the loss of BDNF as a maintenance factor for central neurons
involved in food intake and locomotor behavior.
Finally, to ascertain if the reproductive capability of conditional
mutants was compromised, vaginal smears were performed to determine
whether females were cycling and mutant animals were bred to wild-type
controls. After examining vaginal smears from two lean and three obese
mutants, we found that only the former were cycling normally. Moreover,
when three lean and five obese mutant females were bred to wild-type
males, only the lean mutants became pregnant and produced progeny.
These lean mutants were young animals that subsequently became obese
and sterile. These data show that sterility in the obese female mutants
is a secondary effect of the obesity. Mutant males were also examined
and found to be fertile independently of obesity (data not shown). We
conclude that BDNF has a maintenance function in weight regulation
through a central mechanism.
BDNF Conditional Mutants Are Hyperleptinemic, Hyperinsulinemic, and
Hyperglycemic
Leptin, a satiety signal produced in adipose tissue, and insulin,
an important factor in regulation of glucose homeostasis, lipid
metabolism, and energy balance, are often altered as a response to
feeding, fasting, and obesity (17, 18, 19, 20, 21). Elevated levels of
these hormones were associated with the obesity observed in BDNF
conditional mutants. Serum levels of leptin and insulin were 15-fold
and 6-fold higher, respectively, in obese mutants as compared with
controls (Fig. 4
, A and B). Serum levels
of leptin and insulin in mutants that were not yet obese were
determined to be normal (data not shown), indicating that the elevated
levels detected in the obese mutants were secondary effects of
obesity.

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Figure 4. BDNF Conditional Mutant Mice Are Hyperleptinemic,
Hyperinsulinemic, and Hyperglycemic
A, A 15-fold elevation in the serum levels of leptin was detected in
Bdnf 2lox/2lox/93 mice compared with the
controls (n = 10). *, P < 0.0001. B, Fasting
insulin levels were increased by 600% in the conditional mutants
(n = 6). *, P < 0.05. C, A 70% elevation in
the fasting levels of serum glucose was detected in the conditional
mutants (n = 8). *, P < 0.02. D, Cholesterol
levels were increased by 54% in the mutants, and triglyceride levels
were similar to those of the controls (n = 8). *,
P < 0.005. Abbreviations: C, controls; CM,
conditional mutants.
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Glucose levels in the conditional mutants were 70% higher than in the
controls (Fig. 4C
). As changes in lipid metabolism and synthesis have
been associated with obesity, we investigated whether levels of
cholesterol and triglycerides were altered in the conditional mutants.
Cholesterol levels in control mice were 130.3 ± 3.3 mg/dl and
200.5 ± 11.4 mg/dl in the mutants, representing a 54% increase
in the latter (Fig. 4D
). There was no significant difference in
triglyceride levels in the conditional mutant serum compared with that
from the controls (Fig. 4D
). These findings indicate that BDNF
conditional mutants are leptin and insulin resistant and that
resistance is a secondary effect of the obesity.
Expression of Hypothalamic Weight-Regulating Factors Is Normal in
BDNF Conditional Mutants
The hypothalamus is a known center for the regulation of food
intake and metabolic function (22). To examine the overall
organization of the mutant hypothalamus, brains from BDNF conditional
mutants were obtained for histological and immunohistochemical
examination. Brain sections stained with cresyl violet failed to
uncover any gross morphological abnormalities (data not shown).
Expression of hypothalamic orexigenic and anorexigenic factors known to
regulate eating behavior and metabolic function were also examined in
control and mutant mice 14 to 16 wk of age. Expression levels and the
pattern of distribution of NPY, MCH, orexin, AGRP,
-MSH, serotonin,
and TRH appeared normal in all of the mutants examined (Fig. 5
and data not shown), suggesting that
BDNF does not affect expression of these factors.

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Figure 5. Immunohistochemical Analysis of the BDNF Conditional
Mutant Hypothalamus
Immunohistochemical delineation of NPY (A and F), MCH (B and G), orexin
(C and H), AGRP (D and I) and -MSH (E and J) in the hypothalamus of
conditional mutant (AE) and control (FJ) mice. No differences could
be distinguished between the animal groups (n = 3). PVN,
Paraventricular nucleus; 3V, third ventricle; Arc, arcuate nucleus; ME,
median eminence.
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Enhancing Levels of Serotonin Does Not Rescue the Obesity Phenotype
of BDNF Conditional Mutant Mice
Serotonin has been associated previously with BDNF regulation of
food intake (4). To examine this possibility, we treated
control and BDNF conditional mutants with fluoxetine, a serotonin
reuptake inhibitor (5 mg/kg of body weight) once daily for 20 d.
After 7 d of treatment, mutants appeared less hyperactive than
vehicle-treated mutants when handled, indicating that the fluoxetine
treatment was effective. Monitoring of food intake and weight revealed
that fluoxetine treatment did not significantly decrease the amount of
food consumed by conditional mutants after 20 d of treatment (Fig. 6A
). Furthermore, the weight of
vehicle-treated mutants increased by 4.0% during the course of the
treatment and that of the fluoxetine-treated mutants increased by 6.6%
(Fig. 6B
). These results suggest that factors other than, or in
addition to, serotonin are components of the mechanism through which
BDNF regulates weight.

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Figure 6. Treatment of Control and BDNF Conditional Mutant Mice
with Fluoxetine
A, Measurement of daily food intake of control and conditional mutant
mice that were treated with vehicle or with fluoxetine (5 mg/kg of body
weight) once daily for 20 days (n = 4). B, Measurement of body
weight of control (open circles) and mutant (open
squares) treated with vehicle and control (solid
circles) and mutant (solid squares) treated with
fluoxetine at days 0, 10, and 20 of treatment (n = 4).
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The Starvation Response in BDNF Conditional Mutants Is Abnormal
The starvation response triggered by extended fasting is
characterized by a decrease in serum levels of leptin and insulin in
normal animals (21, 23, 24). Fasting also elicits a
dramatic elevation in the levels of NPY, an orexigenic factor, and a
decrease in the levels of POMC, the precursor for the anorexigenic
factor
-MSH, in the normal hypothalamus (25, 26). All
of these responses signal the animal to eat. Because BDNF appeared to
have an anorexigenic effect, we decided to examine whether its
expression levels were reduced during the starvation response. To
accomplish this, the levels of expression of BDNF mRNA were measured in
hypothalami obtained from wild-type mice fasted for 68 h and
compared with those of control mice fed ad libitum. We found
that fasting did not dramatically alter the hypothalamic levels of BDNF
(data not shown), suggesting that changes in the expression of this
neurotrophin were not involved in the starvation response.
We also investigated whether in the absence of BDNF in the brain, the
starvation response was normal. Wild-type and BDNF conditional mutant
mice were fasted for 48 h, and the serum levels of leptin,
insulin, and glucose and the hypothalamic levels of NPY and POMC mRNA
were measured. Whereas fasting serum levels of insulin in the mutants
were similar to those of the controls, their levels of leptin remained
elevated (data not shown). In addition, glucose levels were reduced to
3 mg/dl (97% reduction) and 86 mg/dl (60% reduction) in control and
mutant mice, respectively. Basal levels of NPY mRNA in the hypothalamus
were comparable in the controls and the conditional mutants, confirming
the findings of the immunohistochemical analysis (Fig. 7
). As expected, fasting induced a 3-fold
increase in the levels of NPY mRNA in the control hypothalamus. In
contrast, conditional mutants had an attenuated response to fasting,
their levels of NPY mRNA being increased only by 30% (Fig. 7
).
However, the feeding behavior of the mutants after fasting was
comparable to that of the controls, albeit containing lower fasting
levels of hypothalamic NPY mRNA (data not shown). These data show that
BDNF is an important regulatory signal for NPY expression during the
starvation response.

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Figure 7. The Starvation Response Is Not Normal in BDNF
Conditional Mutants
Representative Northern blot showing levels of hypothalamic NPY and
POMC mRNA in mice fed ad libitum or fasted for 48
h. C, Control; CM, conditional mutant.
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POMC mRNA levels in the mutant hypothalamus were also altered. A
50100% increase in basal levels of POMC mRNA was detected in 75% of
the BDNF conditional mutants examined when compared with controls (Fig. 7
). As in wild-type mice, POMC levels were slightly reduced after a
48-h fast in the conditional mutants. The anorexigenic effect of POMC
is mediated by its processed derivative,
-MSH, acting through the
melanocortin-4 receptor (MC4-R) (27). BDNF conditional
mutants are hyperphagic in spite of an increase in POMC, suggesting
that MC4-R signaling might be compromised. To investigate whether POMC
mRNA levels were elevated in the mutants due to a reduction in the
expression levels of MC4-R, hypothalamic samples obtained from control
and mutant mice were subjected to Western blot analysis. We found that
expression levels of the melanocortic receptor in the hypothalamus were
not dramatically changed in the conditional mutants (data not shown).
These results suggest that if melanocortin signaling is abnormal in the
mutants, the defect is not due to an absence of MC4-R protein.
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DISCUSSION
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BDNF conditional mutants are hyperaggressive and hyperphagic, have
increased levels of anxiety, and are substantially more obese than
previously described for Bdnf +/-
mice. As BDNF was removed after birth, subsequent to the migration and
differentiation of most neurons in the central nervous system, we
conclude that BDNF has a maintenance function in the modulation of
anxiety-related behavior and of food intake. In addition, because BDNF
was present at normal levels in all tissues except the brains of the
mutants, we can infer that the obesity arose from the lack of a central
function of this neurotrophin.
BDNF has been shown previously to affect behavior. Bdnf+/- and BDNF conditional mutant males have
increased intermale aggression (Ref. 4 and our unpublished
observations) that can be attenuated with fluoxetine, a serotonin
reuptake inhibitor. Locomotor behavior was also examined in Bdnf+/- mice but the results reported are
conflicting. One group found no differences in horizontal activity
between control and Bdnf +/- mutant
mice and a significant reduction in vertical activity in the latter
(4). Another group reported that only a subset of
Bdnf +/- mice displayed hyperactivity
that inversely correlated with obesity (3). BDNF
conditional mutants examined in this study had a substantial increase
in total locomotor activity when stressed but not at baseline,
suggesting a role for this neurotrophin in the regulation of
anxiety-related behavior. This finding is supported by results obtained
from the light/dark exploration test, which demonstrated that the
mutants were anxiety prone. As BDNF has been suggested to be important
for proper serotonergic neurotransmission (4, 28, 29), a
careful examination of this system in the mutants could help explain
the behavior observed.
Unraveling the mechanism through which BDNF regulates food intake
has proven challenging. Previous studies examining factors relevant in
weight regulation found that expression levels of cocaine and
amphetamine-related transcript and leptin receptors were normal in
Bdnf +/- mice (3).
Another report suggested that a reduction in serotonin in Bdnf+/- mice induced obesity (4).
However, reduction in serotonin levels was not detected until 18 months
of age even though weight increase was already detected by 8 wk of age.
The authors argued that serotonergic neurotransmission was possibly
defective in the mutants, preceding a substantial decrease in serotonin
levels. Here, we show that treatment with fluoxetine, a serotonin
reuptake inhibitor, did not significantly reduce food intake or body
weight in the conditional mutants. The treatment, however, was
effective in curtailing the hyperactivity of the mutants. Our results
suggest that a reduction in serotonin could be partly responsible for
the obesity observed in the mutants but that other factors must also be
involved.
It has been suggested that BDNF acts peripherally to regulate weight
(30). Moreover, in addition to being present in the
central nervous system, BDNF and Trk B expression have been detected in
peripheral tissues involved in metabolic function such as exocrine and
endocrine pancreas (31) and adrenal gland
(32). Previous results based upon Bdnf+/- mutant mice could not distinguish
between a peripheral and a central effect of BDNF on obesity. Because
BDNF depletion was restricted to the brain in the conditional mutant
mice described here, we can definitively conclude that the dramatic
increase in body weight observed was due to the disruption of a central
function of BDNF.
A thorough study of the BDNF conditional mutant hypothalamus
determined that expression levels of NPY, AGRP, TRH,
-MSH, MCH,
orexin, and serotonin, all factors involved in regulation of food
intake and metabolic function, were not dramatically changed. Although
expression of some of these factors is regulated by leptin and insulin
and BDNF conditional mutants had elevated levels of these hormones,
previous reports show that changes in expression of these factors are
not obligatory under conditions of leptin and insulin resistance
(33, 34).
Even though
-MSH expression appeared normal in the mutants, mRNA
levels of its precursor, POMC, were increased by 50100% in most of
the mutants examined. This discrepancy could be due to the fact that
-MSH expression was examined by immunohistochemistry, which is not a
quantitative assay, and subtle differences in
-MSH expression could
have gone undetected. The elevated serum levels of leptin, an inducer
of POMC (26), are unlikely to be the cause of the POMC
mRNA up-regulation observed in the mutants because increased levels of
this anorexigenic factor were also detected in lean mutants with normal
levels of leptin. It is also improbable that BDNF acts directly to
inhibit expression of POMC, as this would result in an increase in food
intake, and this and other reports show that BDNF inhibits eating
(3, 4, 35). Alternatively, the elevated levels of POMC
mRNA in the mutant hypothalamus could be indicative of defective
melanocortin signaling. In such a scenario, where factors downstream of
POMC could potentially be absent or defective, increased levels of POMC
would not be expected to antagonize the hyperphagic behavior induced by
the absence of BDNF. Curiously, linear growth was significantly
increased in BDNF conditional mutants, reminiscent of other obesity
models involving melanocortin signaling such as the POMC and MC4-R-null
mice (12, 36). Those mutants have a 511% increase in
body length, which is not characteristic of other obesity models such
as the leptin and leptin receptor-deficient mice (37, 38).
In addition, like POMC and MC4-R-null mice, BDNF conditional mutants
display late-onset obesity. The fact that expression levels of MC4-R
protein in the mutant hypothalamus appeared normal suggests that if
melanocortin signaling was perturbed, it was not at the level of
receptor expression but rather somewhere downstream of MC4-R
activation.
BDNF conditional mutants also failed to exhibit the dramatic elevation
in hypothalamic NPY normally induced by extended fasting. Leptin, a
negative regulator of NPY, was substantially elevated in the mutants
even under fasting conditions and may have prevented the up-regulation
of NPY or other orexigenic factors. However, this seems unlikely as
conditional mutants appeared to be leptin resistant, as demonstrated by
the fact that their NPY basal levels remained normal in the presence of
a 15-fold excess of leptin. An alternative explanation is that BDNF is
a required inductive signal for increased NPY expression during the
starvation response. The ability of BDNF to induce NPY expression in
other neuronal cell populations during development is well documented
(39, 40). Thus, together these data show that in addition
to having a developmental role in the differentiation of certain
NPY-containing neurons, BDNF has a maintenance role in the
hypothalamus, facilitating expression of NPY during fasting.
In conclusion, our results show that BDNF is an essential factor in the
central regulation of locomotor behavior and food intake. As BDNF
conditional mutants are viable and have a postnatal depletion of BDNF
exclusively in the brain, they provide a genetic model with which to
investigate mood disorders and central mediators of obesity. Because
the phenotype of the mutants is easy to assess, these animals should
provide a valuable tool by which to examine the efficacy of treatments
aimed at activating the TrkB receptor signaling pathway. Such
strategies may be beneficial in ameliorating these debilitating
conditions.
 |
MATERIALS AND METHODS
|
---|
Generation of Bdnf 2lox Allele
and of Conditional Mutants
For the generation of floxed BDNF mice, a targeting construct
was designed in which exon 5, the single coding exon in the BDNF gene,
was flanked by lox P sites (Fig. 1a
). A
cytomegalovirus-hygromycin-thymidine kinase selection cassette flanked
by lox P sites was also introduced downstream of exon 5. This targeting
construct was introduced into J1 embryonic stem (ES) cells by
electroporation, and selected homologous integrant clones were
transiently transfected with a cre recombinase-containing
vector to remove the selection cassette and generate Bdnf2lox/+ ES cell clones. These were used for
the generation of Bdnf 2lox allele
carrier mice. Bdnf2lox/2lox and
Bdnf 2lox/- mice were crossed to mice
expressing the cre recombinase under the direction of cam kinase-cre
promoter. All the animals used in these studies were of mixed
background, and all studies were conducted in accord with the
principles outlined in "Guidelines for Care and Use of Experimental
Animals."
Southern and Northern Blot Analysis
To determine cre-mediated recombination levels of the floxed
BDNF allele, DNA was extracted from cerebral cortex, cerebellum,
hypothalamus, hippocampus, kidney, and heart from Bdnf2lox/2lox/93 and Bdnf2lox/2lox/159 mice. DNA samples were
digested with BglII and subjected to Southern blot analysis
using a 3'-probe (Fig. 1
). Bands 12, 9, and 7.5 kb in size were
detected from Bdnf +, Bdnf2lox, and Bdnf1lox alleles, respectively. For Northern
blot analysis, hypothalamic tissue samples were obtained from
control and Bdnf 2lox/2lox/93
mice that were fed ad libitum or fasted for 48 or 68 h.
RNA extracted from those samples was subjected to Northern blot
analysis to examine NPY, POMC, and BDNF mRNA expression. Two or three
independent experiments were performed for NPY and POMC mRNA
measurements, respectively. A total of five individual hypothalamic
samples from each experimental group were examined for NPY mRNA
expression and eight for POMC mRNA expression. Quantification of the
Southern and Northern blots was done using a BAS 2000 phosphoimager
(Fuji Photo Film Co., Ltd.) and the Image Gauge (Fuji
Photo Film Co., Ltd.) v3.3 computer software.
X-gal Staining
To examine cre-mediated recombination in the brain, CamK-cre93
and 159 mice were crossed to the ROSA26-lacZ reporter mice
(16). Brains obtained from their progeny were processed
for X-gal staining, which was performed as described previously
(16).
Measurement of Locomotor Activity
Differences in locomotor activity were assessed during the light
cycle by placing mutants 26 months of age and sex and age-matched
controls individually into cages and monitoring locomotor activity at
baseline and after exposure to a novel chamber. Some of the mutants
used in this study were not yet obese. Baseline activity was measured
for 1 h subsequent to allowing animals to habituate to the
activity monitor for 3 h. Activity was also monitored for 1 h
(habituation period) immediately after placement into a fresh cage and
for 1 h subsequent to habituation. Exposure to a novel cage has
been used previously as a mild stressor (41). Total
activity was quantified using the Opto-Varimex-Mini infrared photocell
activity monitor (Columbus Instruments, Columbus, OH). Statistical
significance was determined using a unpaired t test and
values represent mean ± SEM.
Light/Dark Exploration Test
The light/dark exploration test is an accepted and frequently
used anxiety test (42, 43). To test anxiety behavior,
control and BDNF conditional mutant mice (n = 12), 810 wk of
age, were placed in a box (20 x 20 x 45 cm) containing a
light and dark chamber. The light chamber constructed of clear plastic
material was two-thirds the size of the box and was brightly
illuminated by a 150-W lamp. The dark compartment occupied the
remaining third part of the box and was constructed of black plastic
material that prevented the entrance of light. The two chambers were
separated by a black plastic wall with a doorway (7 x 7 cm) to
allow passage from one chamber to the other. Animals were placed in the
dark compartment, and the latency for the first transition to the light
compartment, total time spent in the light compartment, and number of
transitions from the dark compartment to the light compartment were
monitored for a period of 5 min. The box was cleaned after testing each
animal. Statistical significance was determined using an unpaired
t test and values represent mean ±
SEM.
Body Weight, Monitoring of Food Intake, and Linear
Growth
Control and mutant mice were maintained in a 12-h light/12-h
dark cycle and fed a standard chow diet and water ad
libitum. Growth curves for males and females were obtained by
measuring body weight at 6, 8, 10, 12, 14, 16, and 30 wk of age. Food
intake was determined by individually caging animals between 12 and 16
wk of age that were fed ad libitum and weighing their food
every 3 d for a total of 9 d. Food restriction experiments
were performed by feeding individually caged mice 4 g of food
daily and measuring their body weight every 3 d. For determination
of linear growth, mice that were 20 wk old were fully extended in order
to measure the naso-anal distance. Statistical significance was
determined using a paired t test and all values represent
mean ± SEM.
Analysis of Serum
For determination of insulin, glucose, cholesterol, and
triglyceride levels, blood samples were collected at 1100 h from
conditional mutant and control mice that had been fasted for the
previous 17 h. Leptin levels were measured in serum samples
obtained at 1100 h from animals fed ad libitum. For
analysis of the serum, RIAs were performed in duplicates using leptin
and insulin RIA kits (Linco Research, Inc., St. Charles,
MO). To examine levels of glucose, cholesterol, and triglycerides,
colorimetric kit assays were performed and analyzed using a 747
spectrophotometer (Hitachi, Mountain View, CA) Statistical
significance was determined using a paired t test, and all
values represent mean ± SEM.
Immunohistochemistry
Mice between 14 and 16 wk of age were anesthetized with Nembutal
(50 mg/kg), and blood was taken from the inferior vena cava and
perfused transcardially with 10 ml 0.01 M PBS, pH 7.4,
containing 15,000 U/liter heparin sulfate, followed by 30 ml 2%
paraformaldehyde/4% acrolein in 0.1 M phosphate buffer
(PS), pH 7.4, and 10 ml 2% paraformaldehyde in the same buffer. Brains
were cryoprotected in a 20% sucrose solution, snap frozen on dry ice,
and sectioned in a cryostat. Free floating sections (20-µm) through
the rostral-caudal extent of the hypothalamus were preincubated with
1% sodium borohydride in distilled water followed by 0.5%
H2O2 in PBS for 15 min, and
then permeabilized with 0.5% Triton X-100 in PBS for 20 min. To reduce
nonspecific antibody binding, the sections were treated with 2.5%
normal horse serum in PBS for 20 min. Every fourth section through the
hypothalamus was incubated for 2 d at 4 C in one of the following
antibodies: rabbit anti-NPY (1:10,000, Peninsula Laboratories, Inc. Belmont, CA), rabbit anti-AGRP (1:16,000, Phoenix Pharmaceuticals, Inc., Mountain View, CA), rabbit anti-TRH
(1:25,000) (44), sheep anti-
-MSH (1:40,000)
(45), rabbit anti-MCH (1:12,000, a gift of E. Flier)
(9), orexin (1:6,000, a gift of M. Yanagisawa)
(8), and serotonin (1:1000, DiaSorin, Inc.,
Stillwater, MN). After washes in PBS, the sections were incubated in
biotinylated donkey antirabbit IgG or biotinylated donkey antisheep IgG
(Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA) at 1:500 for 2 h at room temperature and developed using
Vectastain detection system (Vector Elite Kit,
Vector Laboratories, Inc., Burlingame, CA) using
3',3-diamino benzidine HCl as the chromagen.
Fluoxetine Treatment
Control and conditional mutant mice between 18 and 20 wk of age
that were individually caged, received ip injections of fluoxetine at a
dose of 5 mg/kg of body weight once daily for 20 d. Food intake
and weight were monitored during the course of the treatment.
 |
ACKNOWLEDGMENTS
|
---|
We thank Jessica Dausman, Ruth Flannery, and Jeanne Reis for
technical support, S. Akbarian and G. Kemske for review of the
manuscript, and P. Soriano for generously providing the ROSA26 cre
reporter mice.
 |
FOOTNOTES
|
---|
This work was supported by the Fidelity Non-profit Management
Foundation and NIH/NCI grant 5-R35-CA44339 to R.J.
1 Present address: Genetics Institute, Inc., 35
Cambridge Park Drive, Cambridge, Massachusetts 02140. 
Abbreviations: AGRP, Agouti-related protein; BDNF,
brain-derived neurotrophic factor; CamK,
-calcium/calmodulin-dependent protein kinase II; ES, embryonic stem;
MC4-R, melanocortin 4 receptor; MCH, melanin-concentrating
hormone.
Received for publication March 23, 2001.
Accepted for publication June 18, 2001.
 |
REFERENCES
|
---|
-
Ernfors P, Lee KF, Jaenisch R 1994 Mice lacking
brain-derived neurotrophic factor develop with sensory deficits. Nature 368:147150[CrossRef][Medline]
-
Jones KR, Farinas I, Backus C, Reichardt LF 1994 Targeted
disruption of the BDNF gene perturbs brain and sensory neuron
development but not motor neuron development. Cell 76:989999[Medline]
-
Kernie SG, Liebl DJ, Parada LF 2000 BDNF regulates eating
behavior and locomotor activity in mice. EMBO J 19:12901300[Abstract/Free Full Text]
-
Lyons WE, Mamounas LA, Ricaurte GA, et al. 1999 Brain-derived
neurotrophic factor-deficient mice develop aggressiveness and
hyperphagia in conjunction with brain serotonergic abnormalities. Proc
Natl Acad Sci USA 96:1523915244[Abstract/Free Full Text]
-
Kristensen P, Judge ME, Thim L, et al. 1998 Hypothalamic CART
is a new anorectic peptide regulated by leptin. Nature 393:7276[CrossRef][Medline]
-
Ollmann MM, Wilson BD, Yang YK, et al. 1997 Antagonism of
central melanocortin receptors in vitro and in
vivo by agouti-related protein. Science 278:135138[Abstract/Free Full Text]
-
Pollock JD, Rowland N 1981 Peripherally administered
serotonin decreases food intake in rats. Pharmacol Biochem Behav 15:179183[CrossRef][Medline]
-
Sakurai T, Amemiya A, Ishii M, et al. 1998 Orexins and orexin
receptors: a family of hypothalamic neuropeptides and G protein-coupled
receptors that regulate feeding behavior. Cell 92:573585[Medline]
-
Shimada M, Tritos NA, Lowell BB, Flier JS, Maratos-Flier E 1998 Mice lacking melanin-concentrating hormone are hypophagic and
lean. Nature 396:670674[CrossRef][Medline]
-
Stanley BG, Leibowitz SF 1985 Neuropeptide Y injected in the
paraventricular hypothalamus: a powerful stimulant of feeding behavior.
Proc Natl Acad Sci USA 82:39403943[Abstract]
-
Vijayan E, McCann SM 1977 Supression of feeding and drinking
activity in rats following intraventricular injection of thyrotropin
releasing hormone (TRH). Endocrinology 100:17271730[Abstract]
-
Yaswen L, Diehl N, Brennan MB, Hochgeschwender U 1999 Obesity
in the mouse model of pro-opiomelanocortin deficiency responds to
peripheral melanocortin. Nat Med 5:10661070[CrossRef][Medline]
-
Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K 1994 Deletion
of a DNA polymerase ß gene segment in T cells using cell
type-specific gene targeting. Science 265:103106[Medline]
-
Capecchi MR 1989 Altering the genome by homologous
recombination. Science 244:12881292[Medline]
-
Minichiello L, Korte M, Wolfer D, et al. 1999 Essential role
for TrkB receptors in hippocampus-mediated learning. Neuron 24:401414[Medline]
-
Soriano P 1999 Generalized lacZ expression with the ROSA26 Cre
reporter strain. Nat Genet 21:7071[CrossRef][Medline]
-
Fletcher JM, Haggarty P, Wahle KW, Reeds PJ 1986 Hormonal
studies of young lean and obese Zucker rats. Horm Metab Res 18:290295[Medline]
-
Malewiak MI, Griglio S, Mackay S, Lemonnier D, Rosselin G 1977 Nutritionally induced variations in insulinaemia, blood ketone bodies
and plasma and liver triglycerides in genetically obese rats. Diabete
Metab 3:8189[Medline]
-
Saladin R, De Vos P, Guerre-Millo M, et al. 1995 Transient
increase in obese gene expression after food intake or insulin
administration. Nature 377:527529[CrossRef][Medline]
-
Seino Y, Seino S, Takemura J, et al. 1984 Changes in insulin,
somatostatin, and glucagon secretion during the development of obesity
in ventromedial hypothalamic-lesioned rats. Endocrinology 114:457461[Abstract]
-
Trayhurn P, Thomas ME, Duncan JS, Rayner DV 1995 Effects of
fasting and refeeding on ob gene expression in white adipose tissue of
lean and obese (oblob) mice. FEBS Lett 368:488490[CrossRef][Medline]
-
Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG 2000 Central nervous system control of food intake. Nature 404:661671[Medline]
-
Boden G, Baile CA, McLaughlin CL, Matschinsky FM 1981 Effects
of starvation and obesity on somatostatin, insulin, and glucagon
release from an isolated perfused organ system. Am J Physiol
241:E215220
-
Triscari J, Bryce GF, Sullivan AC 1980 Metabolic consequences
of fasting in old lean and obese Zucker rats. Metabolism 29:377385[Medline]
-
Mizuno TM, Makimura H, Silverstein J, Roberts JL, Lopingco T,
Mobbs CV 1999 Fasting regulates hypothalamic neuropeptide Y,
agouti-related peptide, and proopiomelanocortin in diabetic mice
independent of changes in leptin or insulin. Endocrinology 140:45514557[Abstract/Free Full Text]
-
Schwartz MW, Seeley RJ, Woods SC, et al. 1997 Leptin increases
hypothalamic pro-opiomelanocortin mRNA expression in the rostral
arcuate nucleus. Diabetes. 46:21192123
-
Mountjoy KG, Mortrud MT, Low MJ, Simerly RB, Cone RD 1994 Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine
and autonomic control circuits in the brain. Mol Endocrinol 8:12981308[Abstract]
-
Mamounas LA, Blue ME, Siuciak JA, Altar CA 1995 Brain-derived
neurotrophic factor promotes the survival and sprouting of serotonergic
axons in rat brain. J Neurosci 15:79297939[Abstract]
-
Siuciak JA, Clark MS, Rind HB, Whittemore SR, Russo AF 1998 BDNF induction of tryptophan hydroxylase mRNA levels in the rat brain.
J Neurosci Res 52:149158[CrossRef][Medline]
-
Tonra, JR, Ono M, Liu X, et al. 1999 Brain-derived
neurotrophic factor improves blood glucose control and alleviates
fasting hyperglycemia in C57BLKS-Lepr(db)/lepr(db) mice. Diabetes 48:588594[Abstract]
-
Miknyoczki SJ, Lang D, Huang L, Klein-Szanto AJ, Dionne CA,
Ruggeri BA 1999 Neurotrophins and Trk receptors in human pancreatic
ductal adenocarcinoma: expression patterns and effects on in
vitro invasive behavior. Int J Cancer 81:417427[CrossRef][Medline]
-
Suter-Crazzolara C, A Lachmund, Arab SF, Unsicker K 1996 Expression of neurotrophins and their receptors in the developing and
adult rat adrenal gland. Brain Res Mol Brain Res 43:351355[Medline]
-
Lauterio TJ, Davies MJ, DeAngelo M, Peyser M, Lee J 1999 Neuropeptide Y expression and endogenous leptin concentrations in a
dietary model of obesity. Obes Res 7:498505[Abstract]
-
Tritos NA, Elmquist JK, Mastaitis JW, Flier JS, Maratos-Flier
E 1998 Characterization of expression of hypothalamic
appetite-regulating peptides in obese hyperleptinemic brown adipose
tissue-deficient (uncoupling protein-promoter-driven diphtheria toxin
A) mice. Endocrinology 139:46344641[Abstract/Free Full Text]
-
Pelleymounter MA, Cullen MJ, Wellman CL 1995 Characteristics
of BDNF-induced weight loss. Exp Neurol 131:229238[Medline]
-
Huszar D, Lynch CA, Fairchild-Huntress V, et al. 1997 Targeted
disruption of the melanocortin-4 receptor results in obesity in mice.
Cell 88:131141[Medline]
-
Dubuc PU 1976 The development of obesity, hyperinsulinemia,
and hyperglycemia in ob/ob mice. Metabolism 25:15671574[Medline]
-
Joosten HF, van der Kroon PH 1974 Growth pattern and
behavioral traits associated with the development of the
obese-hyperglycemic syndrome in mice (ob-ob). Metabolism 23:11411147[Medline]
-
Barnea A, Cho G, Lu G, Mathis M 1995 Brain-derived
neurotrophic factor induces functional expression and phenotypic
differentiation of cultured fetal neuropeptide Y-producing neurons.
J Neurosci Res 42:638647[Medline]
-
Nawa H, Pelleymounter MA, Carnahan J 1994 Intraventricular
administration of BDNF increases neuropeptide expression in newborn rat
brain. J Neurosci 14:37513765[Abstract]
-
Baumgartner A, Hiedra L, Pinna G, Eravci M, Prengel H,
Meinhold H 1998 Rat brain type II 5'-iodothyronine deiodinase activity
is extremely sensitive to stress. J Neurochem 71:817826[Medline]
-
Ragnauth A, Schuller A, Morgan M, et al. 2001 Female
preproenkephalin-knockout mice display altered emotional responses.
Proc Natl Acad Sci USA 98:19581963[Abstract/Free Full Text]
-
van Gaalen MM, Steckler T 2000 Behavioural analysis of four
mouse strains in an anxiety test battery. Behav Brain Res 115:95106[CrossRef][Medline]
-
Legradi G, Lechan RM 1999 Agouti-related protein containing
nerve terminals innervate thyrotropin-releasing hormone neurons in the
hypothalamic paraventricular nucleus. Endocrinology 140:36433652[Abstract/Free Full Text]
-
Fekete C, Legradi G, Mihaly E, et al. 2000
-Melanocyte-stimulating hormone is contained in nerve terminals
innervating thyrotropin-releasing hormone-synthesizing neurons in the
hypothalamic paraventricular nucleus and prevents fasting-induced
suppression of prothyrotropin-releasing hormone gene expression. J
Neurosci 20:15501558[Abstract/Free Full Text]