Evidence that brain-derived neurotrophic factor is required for basal neurogenesis and mediates, in part, the enhancement of neurogenesis by dietary restriction in the hippocampus

Evidence that brain-derived neurotrophic factor is required

for basal neurogenesis and mediates, in part, the enhancement

of neurogenesis by dietary restriction in the hippocampus

of adult mice

Jaewon Lee,*,� Wenzhen Duan* and Mark P. Mattson*,�,�

*Laboratory of Neurosciences, National Institute on Aging Gerontology Research Center, Baltimore, Maryland, USA

�Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky, USA

�Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Abstract

To determine the role of brain-derived neurotrophic factor

(BDNF) in the enhancement of hippocampal neurogenesis

resulting from dietary restriction (DR), heterozygous BDNF

knockout (BDNF +/–) mice and wild-type mice were main-

tained for 3 months on DR or ad libitum (AL) diets. Mice were

then injected with bromodeoxyuridine (BrdU) and killed either

1 day or 4 weeks later. Levels of BDNF protein in neurons

throughout the hippocampus were decreased in BDNF +/–

mice, but were increased by DR in wild-type mice and to a

lesser amount in BDNF +/– mice. One day after BrdU injection

the number of BrdU-labeled cells in the dentate gyrus of the

hippocampus was significantly decreased in BDNF +/– mice

maintained on the AL diet, suggesting that BDNF signaling is

important for proliferation of neural stem cells. DR had no

effect on the proliferation of neural stem cells in wild-type or

BDNF +/– mice. Four weeks after BrdU injection, numbers of

surviving labeled cells were decreased in BDNF +/– mice

maintained on either AL or DR diets. DR significantly improved

survival of newly generated cells in wild-type mice, and also

improved their survival in BDNF +/– mice, albeit to a lesser

extent. The majority of BrdU-labeled cells in the dentate gyrus

exhibited a neuronal phenotype at the 4-week time point. The

reduced neurogenesis in BDNF +/– mice was associated with

a significant reduction in the volume of the dentate gyrus.

These findings suggest that BDNF plays an important role in

the regulation of the basal level of neurogenesis in dentate

gyrus of adult mice, and that by promoting the survival of

newly generated neurons BDNF contributes to the enhance-

ment of neurogenesis induced by DR.

Keywords: apoptosis, caloric restriction, dentate gyrus,

neurotrophic factor, stem cells.

J. Neurochem. (2002) 82, 1367–1375.

The adult mammalian brain contains small populations of

neural stem cells that are capable of dividing and differen-

tiating into neurons and glia. This process of neurogenesis

occurs mainly in the subventricular zone adjacent to the

lateral ventricles and in the subgranular zone of the

hippocampal dentate gyrus (Gage 2000). In these two areas,

there appears to be a continuous turnover of interneurons and

granule cells, implying that newborn neurons replace the

dying cells and, indeed, recent evidence suggests that newly

generated neurons form functional synapses (van Praag et al.

2002). This ability of neural progenitor cells to generate

neurons that integrate into functional circuits offers hope for

the development of restorative therapies for ischemic,

traumatic and degenerative brain diseases. However, the

mechanisms that control the proliferation, differentiation and

survival of adult neural progenitor cells are not known. It has

been reported that seizures and ischemic and traumatic brain

injuries can stimulate the proliferation of neural progenitor

Received April 30, 2002; revised manuscript received June 3, 2002;

accepted June 4, 2002.

Address correspondence and reprint requests to Mark P. Mattson,

National Institute on Aging, GRC 4F01, 5600 Nathan Shock Drive,

Baltimore, MD 21224, USA. E-mail: [email protected]

Abbreviations used: AL, ad libitum; BDNF, brain-derived neuro-

trophic factor; BrdU, bromodeoxyuridine; DR, dietary restriction; GFAP,

glial fibrillary acidic protein; NeuN, neuronal nucleus protein;

NGC, newly generated cells; NPC, neural precursor cells; NT-3,

neurotrophin-3; TBS, Tris-buffered saline.

Journal of Neurochemistry, 2002, 82, 1367–1375

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 1367–1375 1367

cells (Bengzon et al. 1997; Gould and Tanapat 1997). In

addition, more subtle environmental stimuli have been

shown to enhance adult hippocampal neurogenesis including

enriched environments (Kempermann et al. 1997), physical

exercise (van Praag et al. 1999), and dietary restriction (Lee

et al. 2000a, 2002). Presumably, the effects of these envi-

ronmental factors on neurogenesis are mediated by specific

cellular signaling pathways.

The bulk of data concerning signals that control neuro-

genesis has been obtained in studies of neural progenitor

cells cultured from embryonic brains. These studies have

identified basic fibroblast growth factor and epidermal

growth factor as signals that promote proliferation of the

progenitor cells, and brain-derived neurotrophic factor

(BDNF) and neurotrophin-3 as signals that promote their

differentiation into neurons (for review see Cameron et al.

1998; Gage 2000). BDNF is widely expressed in the

developing and adult brain (Conner et al. 1997; Kernie

et al. 2000) and is essential for the survival of many

populations of neurons during development (Linnarsson

et al. 2000). Although the factors that regulate adult

neurogenesis are not known, it has been shown that

environmental stimuli that increase neurogenesis also

increase the production of certain neurotrophic factors. For

example, both environmental enrichment (Ickes et al. 2000)

and dietary restriction (Lee et al. 2000a,b, 2002) have been

shown to increase levels of BDNF and neurotrophin-3 in the

hippocampus. Such neurotrophic factors might promote

neurogenesis by increasing the proliferation of progenitor

cells, by inducing their differentiation into neurons and/or by

increasing the survival of newly generated neurons. BDNF

has been shown to promote the differentiation and survival of

embryonic hippocampal neurons (Ip et al. 1993; Cheng and

Mattson 1994; Lindholm et al. 1996), but its role in adult

neurogenesis has not been established.

Dietary restriction (DR) can increase life span in a wide

variety of species, and can reduce neuronal damage, and

improve behavioral outcome in experimental animal models

relevant to the pathogenesis of several age-related neurolo-

gical disorders (Bruce-Keller et al. 1999; Duan and Mattson

1999; Yu and Mattson 1999; Duan et al. 2001). DR may

promote neuronal survival by stimulating the expression of

genes that encode cytoprotective proteins such as heat-shock

proteins (Duan and Mattson 1999; Yu and Mattson 1999) and

neurotrophic factors (Duan et al. 2001). Similar to the effects

of enriched environments (Kempermann et al. 1997; Nilsson

et al. 1999; Young et al. 1999), DR does not increase the

proliferation of neural stem cells, but does increase survival

of their neuronal progeny (Lee et al. 2000a, 2002). In the

present study we employed mice with reduced levels of

BDNF (Liebl et al. 1997; Lyons et al. 1999) to determine the

role of BDNF signaling in regulating adult neural stem cells,

and to directly test the hypothesis that the enhancement of

neurogenesis in response to DR is mediated by BDNF.

Materials and methods

Mice, diets and BrdU administration

Two-month-old male BDNF +/– mice and wild-type control

littermates were obtained from in-house breeding colonies origin-

ating from heterozygous mutant mice kindly provided by

L. Tessarollo (Liebl et al. 1997). Animals were maintained under

temperature- and light-controlled conditions (20–23�C, 12-h light/12-h dark cycle). Wild-type mice and BDNF +/– mice were divided

into two groups, an ad libitum (AL) group which had continual

access to food, and a DR group which was maintained on an every-

other-day fasting regimen. Previous studies have shown that rats and

mice maintained on such an every-other-day feeding schedule will

consume less calories over time and live longer than animals fed AL

(Goodrick et al. 1983). For evaluations of neurogenesis, 10–16 mice

in each group were given five intraperitoneal injections of

bromodeoxyuridine (BrdU; 100 mg/kg body weight) during a

2-day time window. Half of the mice in each diet group were

killed 1 day after the last BrdU injection and the remaining mice

were killed 4 weeks after the last BrdU injection. An additional six

mice of each genotype/diet group were processed for ELISA

analysis of BDNF protein levels as described below. All procedures

complied with National Institutes of Health guidelines and were

approved by the Institutional Animal Care and Use Committee.

Quantification of newly generated cells

Mice were perfused transcardially with 4% paraformaldehyde and

their brains were removed, postfixed at 4�C overnight, and

transferred to a 30% sucrose solution until they sank. Then brains

were frozen in isopentane and stored at )80�C. The cryoprotectedbrains were sectioned serially at 50 lm in the coronal plane using afreezing microtome. Every section which contained the hippocam-

pal formation was saved. The protocol for immunostaining of brain

sections with BrdU antibody was similar to that described

previously (Lee et al. 2002). Briefly, free-floating sections were

treated with 0.6% H2O2 in Tris-buffered saline (TBS; pH 7.5) to

block endogenous peroxidases, and DNA was denatured by

exposing sections sequentially to heat, acid and base. The sections

were incubated in TBS/0.1% Triton X-100/5% goat serum (TBS-

TS) for 30 min, and then incubated with primary anti-BrdU

antibody (rat monoclonal; Accurate Chemicals, Westbury, NY,

USA; 1 : 400) in TBS-TS overnight at 4�C. Sections were furtherprocessed using a biotinylated secondary goat anti-rat IgG antibody

(Vector Laboratories, Burlingame, CA, USA; 1 : 200), avidin–

peroxidase complex and diaminobenzidine. Stained sections were

mounted onto slides and counter-stained with cresyl violet to

measure granule cell layer volume.

The total number of BrdU-positive cells in the dentate gyrus of

each mouse was estimated using the optical fractionator technique

(West 1993) assisted by a computer-based system, StereologerTM

(SPA, Alexandria, VA, USA) using methods similar to those

described previously (Long et al. 1998). Estimates of region volume

were assessed using the Cavalieri point counting method (Gunder-

sen and Jensen 1987). Cells in every sixth section throughout the

entire rostro-caudal extent of the hippocampus were counted: the

reference space consisted of the granular cell layer of the dentate

gyrus. For each section, the reference space was delineated by

outlining at low power (5· objective; on-screen magnification ¼

1368 J. Lee et al.

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 1367–1375

138·); identification of BrdU-positive cells was accomplished athigh power (63· objective; on-screen magnification ¼ 1714·). Thedimension of the sampling frames was 49.2 lm in length by

49.2 lm in width and 14 lm in depth. The guard height for eachsection was 1 lm. The optical fractionator technique estimates thenumber of cells by multiplying the sum of cells counted by the

reciprocal of the fraction of the region sampled. Volume densities

were calculated by dividing the number of BrdU positive cells

counted by the total volume sampled of the reference space. The

volume of the sampled reference space was the number of dissectors

multiplied by the volume of one dissector. All cell counts were

performed by the same investigator (JL) blind to the group

identification of each section.

Immunohistochemistry

BDNF immunohistochemistry was performed in brain sections

adjacent to those used for BrdU immunostaining. Briefly, free-

floating sections were treated with 0.6% H2O2 in Tris-buffered

saline (TBS; pH 7.5) to block endogenous peroxidase. The sections

were incubated with TBS/0.1% Triton X-100/5% goat serum (TBS-

TS) for 30 min, and incubated with primary anti-BDNF antibody

(polyclonal rabbit; Santa Cruz Biotechnology, Santa Cruz, CA,

USA; 1 : 800) in TBS-TS overnight at 4�C. Sections were furtherprocessed using a biotinylated secondary goat anti-rabbit IgG

antibody (Vector Laboratories, 1 : 200), avidin–peroxidase complex

and diaminobenzidine. The stained sections were mounted onto

slides and cover-slipped. Immunostaining for confocal analysis was

performed on 50 lm coronal brain sections as follows. After DNAdenaturation, sections were incubated for 1 h in a solution

containing 5% normal goat serum, and 0.1% Triton X-100 in

TBS. Primary antibodies were then added and the sections were

incubated overnight at 4�C. The primary antibodies used were a ratmonoclonal antibody against BrdU (Accurate Chemicals, 1 : 200

dilution), rabbit polyclonal antibody against glial fibrillary acidic

protein (Sigma, St Louis, MO, USA; 1 : 500 dilution) and a mouse

monoclonal antibody against the neuron-specific nuclear antigen

NeuN (Chemicon, Temecula, CA, USA; 1 : 500 dilution). Brain

sections were then washed with TBS and incubated for 1 h in the

presence of anti-rat IgG labeled with AlexaFluor-488, anti-rabbit

IgG labeled with AlexaFluor-633 and anti-mouse IgG labeled with

AlexaFluor-568 (Molecular Probes; 1 : 500 dilution). Confocal

images were acquired using a Zeiss 510 CSLM microscope.

ELISA analysis

Hippocampal and cortical tissues were homogenized in lysis buffer

(137 mM NaCl, 20 mM Tris, 1% NP-40 detergent, 10% glycerol,

1 mM phenylmethyl sulfonyl fluoride, 10 lg/mL aprotinin, 1 lg/mLleupeptin and 0.5 mM sodium orthovanadate; pH 7.2) at 4�C.Homogenates were centrifuged at 2000 g for 20 min (4�C), and thesupernatant was used for ELISA analysis. BDNF protein levels were

quantified using a commercially available kit (Promega, Madison,

WI, USA) according to the manufacturer’s protocol. Briefly,

samples were processed by acidification and subsequent neutraliza-

tion. Ninety-six-well plates were coated with monoclonal BDNF

antibody, incubated in the presence of block and sample buffer, and

washed in TBS/oil% Triton X-100 (TBST). Samples were added to

triplicate wells in each plate, and serial dilutions of recombinant

BDNF standard (0–500 pg/mL) were added to duplicate wells in

each plate in order to generate a standard curve. Plates were

incubated for 2 h, washed five times in TBST, and incubated in a

solution containing either HRP conjugated polyclonal BDNF

antibody. Wells were washed five times with TBST, and a hydrogen

peroxide solution was added together with a peroxidase substrate,

and plates were incubated for 10 min. Reactions were stopped by

adding 100 lL 1 M phosphoric acid, and absorbance was measuredat 450 nm using a plate reader. Triplicate determinations for each

sample were averaged, and the level of BDNF protein in each

sample was determined using the standard curve.

Statistical analyses

Data were analyzed using a one-way analysis of variance (ANOVA) and

post-hoc comparison of means were based on Fisher’s protected least

significant differences (PLSD) procedure. p-Values less than 0.05

were considered statistically significant. Analyses were performed

using STATVIEW� software (SAS Institute, Cary, NC, USA).

Results

Dietary restriction enhances neurogenesis

in both wild-type and BDNF +/– mice

Wild-type and BDNF +/– mice were maintained on either an

AL or a DR feeding regimen in which they were fed every

other day; their body weights after 3 months on the diets

(5 months of age) were: wild-type-AL, 32.0 ± 1.1 g; wild-

type-DR, 26.5 ± 1.0 g (p < 0.01 compared with wild-type –

AL), BDNF +/– AL, 41.9 ± 1.9 g (p < 0.001 compared with

wild-type AL); BDNF +/– DR, 31.0 ± 0.4 g (p < 0.001

compared with BDNF +/– AL). As expected, based on

previous studies (Lyons et al. 1999; Kernie et al. 2000),

BDNF +/– mice exhibited increased body weight. However,

the DR regimen decreased the body weights of the BDNF

+/– mice and the wild-type mice.

Mice were given a total of five intraperitoneal injections of

BrdU during a 2-day period to label newly generated cells,

and were killed either 1 day or 4 weeks later to study the

proliferation and survival of neural precursor cells in the

dentate gyrus of the hippocampus. BrdU-immunoreactive

cells in the dentate gyrus of the hippocampus were quantified

using unbiased stereological methods described previously

(Lee et al. 2002). At the 1-day time point the numbers of

BrdU-positive cells in the dentate gyrus of wild-type mice

maintained on AL and DR diets were not significantly

different (Figs 1a and b; Table 1). However, the number of

BrdU-positive in the BDNF +/– mice maintained on the AL

diet was significantly lower than that of wild-type mice on

the AL diet (Figs 1a and c; Table 1), suggesting that BDNF

signaling is required for maintenance of the basal level of

proliferation of neural stem cells. Numbers of BrdU-labeled

cells at the 1-day time point in BDNF +/– mice maintained

on DR were greater than in BDNF +/– mice on AL, and were

not significantly different than in wild-type mice on either

BDNF, dietary restriction and neurogenesis 1369


Fig. 1 Confocal images showing the phenotypes of newly generated

cells in the brains of mice that had been maintained on either an ad

libitum (AL) or a dietary restriction (DR) feeding regimen. Sections

were triple-labeled with antibodies against BrdU (green), glial fibrillary

acidic protein (white) and NeuN (red) (a–h) or BrdU (green), BDNF

(white) and NeuN (red) (I–m). One day after the last BrdU injection

(a–d, and l), decreased numbers of BrdU-labeled cells were present in

the dentate gyrus of BDNF +/– AL mice (c) compared with wild-type AL

mice (a), whereas similar numbers of BrdU-labeled cells were present

in the dentate gyrus of wild-type DR (b) and BDNF +/– DR (d) mice.

Four weeks after BrdU injection the majority of BrdU-positive cells

expressed the neuron-specific marker, NeuN in the dentate gyrus

(e–g) but not in CA3 (k) and neocortex (m). Very small numbers of

BrdU-labeled cells remained undifferentiated in the subgranular zone

of dentate gyrus even at the 4-week time point (f and h). Essentially all

neurons in the hippocampus and cerebral cortex exhibited BDNF

immunoreactivity (i–m). Newly generated neurons in the dentate gyrus

expressed BDNF (arrow in i); undifferentiated BrdU-labeled cells in

other brain regions including hippocampal regions CA1 (j) and CA3 (k)

and neocortex (m) lacked BDNF immunoreactivity. However, a few

NeuN-negative cells (presumptive glial cells) in several different brain

regions expressed BDNF (arrows in k and l). Many BDNF-positive

cells were seen in periventricular regions, but most of them were

BrdU-negative (l).

1370 J. Lee et al.


AL or DR diets (Table 1). These findings suggest that DR

can counteract an adverse effect of reduced levels of BDNF

on neural stem cell proliferation.

At the 4-week post-BrdU time point there were signifi-

cantly fewer BrdU-positive cells present in the dentate gyrus

in each of the four groups of mice (Table 1). However, the

magnitude of the decrease was significantly less in wild-type

and BDNF +/– mice maintained on DR compared with the

corresponding genotypes of mice fed AL (Table 1). The

numbers of labeled cells in BDNF +/– mice maintained on

DR were greater than in AL BDNF +/– mice, but were

significantly lower than in DR wild-type mice. In order to

provide a measure of cell survival during the 4-week post-

BrdU time period, we expressed the number of BrdU-labeled

cells at the 4-week time point as a percentage of the number

present at the 1-day time point. This analysis revealed that DR

significantly increased the survival of newly generated cells in

both wild-type and BDNF +/– mice, but was significantly

more effective in increasing survival of cells in wild-type

mice as compared with BDNF +/– mice (Table 1). There were

no differences in the regional volume of the dentate gyrus at

the 1-week time point. Interestingly, however, at the 4-week

time point the regional volume had increased in all groups

except the BDNF +/– mice fed AL (Table 1), suggesting a

reduction in neurogenesis in these mice.

In order to determine the phenotypes of the BrdU-labeled

cells, we performed triple-labeling confocal immunohisto-

chemical analysis of brain sections using an antibody against

the astrocyte protein GFAP, an antibody against the mature

neuron-specific protein NeuN, and an antibody against BrdU.

One day after last BrdU administration the vast majority of

BrdU-positive cells were confined to the subgranular zone of

the dentate gyrus and were not immunoreactive with either

the GFAP or NeuN antibodies (Figs 1a–d). At 4 weeks after

BrdU administration, BrdU-positive cells were scattered

throughout the dentate gyrus (Figs 1e and f). The vast

majority of BrdU-positive cells that were located within the

granule cell layer were also NeuN positive (Figs 1e–g).

Using a series of Z-step section scans we were able to

confirm that BrdU-positive cells located in the granular cell

layer of the dentate gyrus showed a neuronal phenotype

(Fig. 1g). BrdU-labeled cells were seen not only in the

dentate gyrus, but also in several other brain areas including

hippocampal regions CA1 and CA3 and cerebral cortex, but

these cells were not immunoreactive with the NeuN or GFAP

antibodies (data not shown). A small number of BrdU

positive cells that did not label with either NeuN or GFAP

antibodies were also detected in the dentate gyrus at the

4-week time point (Figs 1f and h).

Newly generated neurons in the dentate

gyrus contain BDNF

To determine whether any of the newly generated neuronal

cells in the dentate gyrus expressed BDNF, we performed

triple-label confocal analysis using an antibody against

BDNF in combination with NeuN and BrdU antibodies in

sections from mice killed 4 weeks after BrdU administration.

An example of a BrdU-positive cell (green) which also

exhibited nuclear NeuN immunoreactivity (red) and cyto-

plasmic BDNF immunoreactivity (white) in the dentate gyrus

is shown in Fig. 1(i) (arrow). BDNF immunoreactivity was

present in CA1 and CA3 pyramidal neurons of hippocampus

and in cortical neurons; however, none of the BrdU-labeled

cells in CA1, CA3 and cortex were colocalized with either

NeuN or BDNF (Figs 1j–m). A few BDNF immunoreactive

cells were seen in NeuN-negative cells in hippocampus and

periventricular regions suggesting that glial cells are also a

source of BDNF (Figs 1j–m). We also performed double-

labeling of sections from mice killed 1 d after BrdU

administration using BDNF and BrdU antibodies. We were

unable to detect double-labeled cells suggesting that majority

of BrdU-labeled cells at this time point are undifferentiated

Table 1 Proliferation, survival and survival rate of cells in the dentate gyrus of mice fed ad libitum (AL) in comparison with mice maintained on

dietary restriction (DR)

Wild-type mice BDNF +/– mice

DF F-valueAL DR AL DR

Proliferation, 1 day 3533 ± 177.6 3226 ± 105.8 2789 ± 189.2a 3146 ± 264.6 3 3.230

Survival, 4 week 967 ± 84.7 1496 ± 80.9d 626 ± 48.0ab 1028 ± 88.8c 3 20.287

Survival (%), 4 week 27 ± 2.4 46 ± 2.5d 22 ± 1.7b 33 ± 2.8c 3 18.768

Regional volume (mm3), 1 day 0.174 ± 0.013 0.175 ± 0.010 0.148 ± 0.005 0.162 ± 0.010 3 1.398

Regional volume (mm3), 4 week 0.201 ± 0.011 0.190 ± 0.011 0.147 ± 0.008ab 0.175 ± 0.016 3 3.731

BDNF, brain-derived neurotrophic factor. All mice received bromodeoxyuridine (BrdU; 100 mg per kg; five injections during a 2-day time period).

Cell proliferation was assessed on 1 day after last injection. Survival of BrdU-labeled cells in the dentate gyrus were determined 4 weeks after the

last injection (n ¼ 4–7 per group). All data are presented as means ± standard error. ap < 0.02 compared with the wild-type AL value, bp < 0.02

compared with the wild-type DR value, cp < 0.02 compared with the BDNF +/– AL value, dp < 0.01 compared with each of the other values. DF,

degrees of freedom.



neural precursor cells that did not express BDNF, although

they may express BDNF receptors and respond to BDNF

(Lachyankar et al. 1997).

Dietary restriction increases BDNF protein levels

to a lesser amount in BDNF +/– mice

BDNF immunohistochemistry and ELISA analyses were

performed to determine the levels of BDNF protein in brains

taken from each of the four groups of mice (Fig. 2). To

examine non-specific staining, brain sections were processed

without primary antibody, and no peroxidase reaction

product was observed in those sections (data not shown).

Incubation of brain section with preabsorbed BDNF antibody

with an excess of blocking peptide dramatically decreased

the intensity of immunostaining indicating that the BDNF

antibody used in the present study is highly specific (Fig. 2,

bottom left panel). It was previously shown that the BDNF

antibody used in our studies does not to cross-react with

nerve growth factor (NGF) or NT-3 (Inoue et al. 1998).

BDNF immunoreactivity was observed in all hippocampal

regions and in the cerebral cortex, with a cellular expression

pattern similar to that previously described (Inoue et al.

1998). In wild-type mice on the AL diet hilar cells and

pyramidal neurons throughout Ammon’s horn were stained

moderately, and granule cells were more faintly stained. As

expected (Ernfors et al. 1994; Korte et al. 1995; Lyons et al.

1999), BDNF immunoreactivity was decreased in AL-fed

BDNF +/– mice in all regions of the brain, and the difference

was most distinct in CA3 of hippocampus and in the cerebral

cortex (Fig. 2). DR increased the level of BDNF immunore-

activity in wild-type mice and BDNF +/– mice; however, the

level of BDNF immunoreactivity in hippocampal cells of

BDNF +/– mice on DR was clearly lower than in wild-type

mice on DR (Fig. 2). Increased levels of BDNF protein in

wild-type and BDNF +/– mice maintained on DR were

confirmed by ELISA analysis of hippocampal homogenates.

BDNF protein levels were (pg/mg protein; mean ± SE, n ¼ 6mice per group): wild-type AL, 26.0 ± 4.6; wild-type DR,

76.8 ± 5.5 (p < 0.001 compared with wild-type AL); BDNF

+/– AL, 10.0 ± 0.8 (p < 0.001 compared with wild-type

AL); BDNF +/– DR, 26.9 ± 3.5 (p < 0.001 compared with

BDNF +/– AL; p < 0.001 compared with wild-type DR).

The fold increase of BDNF protein levels in BDNF +/– mice

induced by DR (2.7) being similar to the fold increase in the

wild-type mice on DR (2.9), suggesting that the mechanism

whereby DR induces BDNF production is not affected by

BDNF haploinsufficiency.

Discussion

The hippocampal subgranular zone is a region where new

neurons and glia are generated in the adult brain; neurogen-

esis in this area can be enhanced by several environmental

manipulations including enriched environments, physical

activity and dietary restriction (Kempermann et al. 1997;

Nilsson et al. 1999; van Praag et al. 1999; Lee et al. 2000a;

Lee et al. 2002). The present findings suggest that BDNF

signaling plays important roles in regulating adult hippo-

campal neurogenesis under basal conditions and in response

to DR. BDNF null mutant mice typically do not survive

beyond 21 days of age and exhibit widespread neuronal

deficits (Conover et al. 1995; Conover and Yancopoulos

1997). While BDNF +/– mice survive and reproduce well,

they exhibit several phenotypes including increased food

intake and weight gain, aggressiveness, alterations in brain

serotonergic and dopaminergic systems, and impaired syn-

aptic plasticity (Korte et al. 1995; Dluzen et al. 1999; Lyons

et al. 1999; Kernie et al. 2000; Olofsdotter et al. 2000). Our

data identify impaired neurogenesis as a novel phenotype in

BDNF +/– mice. Analyses of brains of mice killed 1 day

after BrdU administration revealed significantly fewer BrdU-

labeled cells in the dentate gyrus of BDNF +/– AL mice

compared with wild-type AL mice. The latter results suggests

that there is a decreased pool of neural stem cells present in

the dentate gyrus of BDNF +/– mice and/or that the stem

cells that are present have a decreased proliferation rate. We

conclude that BDNF signaling plays an important role in

maintenance of the basal rate of neural stem cell proliferation

and/or survival in the dentate gyrus. This conclusion is

consistent with results of analyses of BDNF-/– mouse

embryos with provided evidence that BDNF plays a role in

proliferation of neural precursor cells (Linnarsson et al. 2000).

DR did not affect the number of BrdU-labeled cells in

wild-type or BDNF +/– mice killed 1 day after BrdU

injection suggesting that DR has no major impact on

proliferation of neural stem cells. Instead, DR enhanced

neurogenesis in wild-type mice by increasing the survival of

newly generated cells with no significant effect on the

proliferation of neural progenitor cells. Although there was

enhanced survival of newly generated cells in BDNF +/–

mice maintained on DR compared with AL-fed BDNF +/–

mice, the survival rate was significantly lower than that of

wild-type mice maintained on DR. In agreement with

previous reports (Parent et al. 1997; Young et al. 1999; Lee

et al. 2002), we found that the majority of newly generated

cells in the dentate gyrus migrate into the granule cell layer

and display neuron-like properties; in our study a neuronal

phenotype was inferred by their expression of NeuN. Triple-

labeled confocal images showed that BrdU-positive cells that

had differentiated into dentate granule neurons were immu-

noreactive with a BDNF antibody, providing further evidence

of their neuronal phenotype. Indeed, we never observed

BDNF immunoreactivity in NeuN-negative BrdU-labeled

cells in hippocampus. Although the neural progenitor cells

may not produce BDNF, they do express the BDNF receptor

trkB and can respond to BDNF (Lachyankar et al. 1997). Our

observations suggest that BDNF produced by mature neurons

may act upon neural progenitor cells to promote their

1372 J. Lee et al.


Fig. 2 Localization and expression level of BDNF protein in dentate

gyrus, CA3 and neocortex of wild-type and BDNF +/– mice that had

been maintained for 3 months on either AL or DR diets. BDNF

immunoreactivity was decreased in hippocampal and cortical neurons

of BDNF +/– AL mice. DR increased the level of BDNF protein in

hippocampal and cortical neurons of both wild-type and BDNF +/–

mice compared with AL-fed mice. The lower left panel shows the

dentate gyrus of a brain section of a wild-type AL mice stained with

BDNF antibody that had been preincubated with an excess of the

BDNF peptide antigen.



differentiation into neurons and long-term survival. We

conclude that BDNF mediates, at least in part, the enhance-

ment of neurogenesis induced by DR.

We found that the regional volume of the dentate gyrus

was decreased in BDNF +/– mice compared with wild-type

mice, while the dentate volume of BDNF +/– mice

maintained on DR was between that of BDNF +/– AL and

wild-type AL or DR mice. Comparison of dentate volumes in

the four groups of mice killed 4 weeks after BrdU injection

with those killed 1 day after injection showed that the dentate

volume increased during this 1 month time period in all

groups except the BDNF +/– AL mice. These results suggest

that the decreased neurogenesis resulting from reduced

BDNF levels may contribute to a reduced size of the dentate

gyrus. Nevertheless, DR was able to significantly enhance

neurogenesis and increase dentate volume in BDNF +/–

mice. Because DR increased levels of BDNF in BDNF +/–

mice it is possible that BDNF also mediates the enhanced

neurogenesis in BDNF +/– mice.

DR has been shown to have several beneficial effects on

the brain including amelioration of age-related deficits in

learning and memory (Ingram et al. 1987), increased neuro-

nal survival and improved behavioral outcome in rodent

models of severe epileptic seizures (Bruce-Keller et al.

1999), Parkinson’s disease (Duan and Mattson 1999) and

focal ischemic stroke (Yu and Mattson 1999). Some of these

beneficial effects of DR might be the result of increased

production of BDNF and its direct actions on mature

neurons. For example, it has been shown that BDNF can

enhance long-term potentiation of synaptic transmission in

the hippocampus, a cellular correlate of learning and memory

(Kovalchuk et al. 2002; Ying et al. 2002). In addition,

BDNF can protect neurons in culture (Cheng and Mattson

1994; Nakao et al. 1995) and in vivo (Bemelmans et al.

1999; Duan et al. 2001) against excitotoxic and oxidative

injury. Our findings suggest that, in addition to direct actions

on mature neurons, enhancement of neurogenesis by BDNF

may contribute to the beneficial effects of DR on hippocam-

pal plasticity and resistance to age-related neuronal degen-

eration. Consistent with the latter possibility, recent findings

suggest that neurogenesis may be required for the formation

of trace memories (Shors et al. 2001), and that transplanta-

tion of neural stem cells into the hippocampus can ameliorate

learning and memory deficits induced by ischemia and aging

(Hodges et al. 2000; Toda et al. 2001). The ability of DR to

up-regulate BDNF expression and enhance neurogenesis in

rodents suggests that it may also be possible to enhance brain

function and resistance to injury and disease in humans by

controlling food intake.

Acknowledgement

We thank L. Tessarollo for providing initial breeding pairs of BDNF

+/– mice.

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Evidence that brain-derived neurotrophic factor is required for basal neurogenesis and mediates, in part, the enhancement of neurogenesis by dietary restriction in the hippocampus