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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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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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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, 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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). 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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). 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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). 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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-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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). 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. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 50–100% 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 5–11% 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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 2–6 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), 8–10 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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D. Tsao, H. K. Thomsen, J. Chou, J. Stratton, M. Hagen, C. Loo, C. Garcia, D. L. Sloane, A. Rosenthal, and J. C. Lin TrkB Agonists Ameliorate Obesity and Associated Metabolic Conditions in Mice Endocrinology, March 1, 2008; 149(3): 1038 - 1048. [Abstract] [Full Text] [PDF]
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L. M. Flanagan-Cato, S. J. Fluharty, E. B. Weinreb, and D. R. LaBelle Food Restriction Alters Neuronal Morphology in the Hypothalamic Ventromedial Nucleus of Male Rats Endocrinology, January 1, 2008; 149(1): 93 - 99. [Abstract] [Full Text] [PDF]
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 T. J. Unger, G. A. Calderon, L. C. Bradley, M. Sena-Esteves, and M. Rios Selective Deletion of Bdnf in the Ventromedial and Dorsomedial Hypothalamus of Adult Mice Results in Hyperphagic Behavior and Obesity J. Neurosci., December 26, 2007; 27(52): 14265 - 14274. [Abstract] [Full Text] [PDF]
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D. M. Kurrasch, C. C. Cheung, F. Y. Lee, P. V. Tran, K. Hata, and H. A. Ingraham The Neonatal Ventromedial Hypothalamus Transcriptome Reveals Novel Markers with Spatially Distinct Patterning J. Neurosci., December 12, 2007; 27(50): 13624 - 13634. [Abstract] [Full Text] [PDF]
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 H. Sha, J. Xu, J. Tang, J. Ding, J. Gong, X. Ge, D. Kong, and X. Gao Disruption of a novel regulatory locus results in decreased Bdnf expression, obesity, and type 2 diabetes in mice Physiol Genomics, October 19, 2007; 31(2): 252 - 263. [Abstract] [Full Text] [PDF]
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 C. Wang, E. Bomberg, C. Billington, A. Levine, and C. M. Kotz Brain-derived neurotrophic factor in the hypothalamic paraventricular nucleus reduces energy intake Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1003 - R1012. [Abstract] [Full Text] [PDF]
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K. Kohara, H. Yasuda, Y. Huang, N. Adachi, K. Sohya, and T. Tsumoto A Local Reduction in Cortical GABAergic Synapses after a Loss of Endogenous Brain-Derived Neurotrophic Factor, as Revealed by Single-Cell Gene Knock-Out Method J. Neurosci., July 4, 2007; 27(27): 7234 - 7244. [Abstract] [Full Text] [PDF]
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 D. Ize-Ludlow, J. A. Gray, M. A. Sperling, E. M. Berry-Kravis, J. M. Milunsky, I. S. Farooqi, C. M. Rand, and D. E. Weese-Mayer Rapid-Onset Obesity With Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation Presenting in Childhood Pediatrics, July 1, 2007; 120(1): e179 - e188. [Abstract] [Full Text] [PDF]
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