Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice

Dietary restriction enhances neurotrophin expression

and neurogenesis in the hippocampus of adult mice

Jaewon Lee,*, Kim B. Seroogy and Mark P. Mattson*, ,à

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

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

àDepartment of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Abstract

The adult brain contains small populations of neural precursor

cells (NPC) that can give rise to new neurons and glia, and

may play important roles in learning and memory, and

recovery from injury. Growth factors can in¯uence the proli-

feration, differentiation and survival of NPC, and may mediate

responses of NPC to injury and environmental stimuli such as

enriched environments and physical activity. We now report

that neurotrophin expression and neurogenesis can be mod-

i®ed by a change in diet. When adult mice are maintained on a

dietary restriction (DR) feeding regimen, numbers of newly

generated cells in the dentate gyrus of the hippocampus are

increased, apparently as the result of increased cell survival.

The new cells exhibit phenotypes of neurons and astrocytes.

Levels of expression of brain-derived neurotrophic factor

(BDNF) and neurotrophin-3 (NT-3) are increased by DR, while

levels of expression of high-af®nity receptors for these

neurotrophins (trkB and trkC) are unchanged. In addition, DR

increases the ratio of full-length trkB to truncated trkB in the

hippocampus. The ability of a change in diet to stimulate

neurotrophin expression and enhance neurogenesis has

important implications for dietary modi®cation of neuroplas-

ticity and responses of the brain to injury and disease.

Keywords: brain-derived neurotrophic factor, caloric restric-

tion, neurotrophin-3, stem cells, trkB, tyrosine kinase.

J. Neurochem. (2002) 80, 539±547.

The brain of adult mammals, including humans, contains

populations of cells that can divide and differentiate into

neurons and glia (Gage 2000). These neural precursor cells

(NPC) are present in the subventricular zone and in the

dentate gyrus of the hippocampus, Neurogenesis may allow

the brain to respond to environmental demands such as

increased intellectual challenge and brain injury. Indeed,

studies of rodents have shown that the proliferation of NPC

is reduced in association with age-related cognitive decline

(Kuhn et al. 1996), and that suppression of NPC prolifera-

tion can impair learning and memory (Shors et al. 2001). In

addition, ischemic and excitotoxic brain injuries (Parent

et al. 1997; Liu et al. 1998), exposure to enriched environ-

ments (Kempermann et al. 1997; Nilsson et al. 1999; Young

et al. 1999) and physical activity (van Praag et al. 1999) can

increase the production and/or survival of new neural cells

in the dentate gyrus of the hippocampus. The signaling

mechanisms that mediate the effects of environmental stimuli

on NPC proliferation, differentiation and survival are not yet

established, but appear to involve neurotrophic factors

(Cameron et al. 1998). Growth factors that have been shown

to affect NPC include basic ®broblast growth factor,

epidermal growth factor, and members of the neurotrophin

family including brain-derived neurotrophic factor (BDNF)

and neurotrophin-3 (NT-3). Data suggest that BDNF and

NT-3 can affect the proliferation, differentiation and/or

survival of NPC from different brain regions including the

subventricular zone and hippocampus (Vicario-Abejon et al.

1995; Lachyankar et al. 1997; Shetty and Turner 1998;

Zigova et al. 1998; Benoit et al. 2001).

The impact of diet on brain function and susceptibility

to neuropsychiatric and neurodegenerative disorders is

increasingly appreciated (Young 1993). Dietary restriction

Received October 24, 2001; revised manuscript received November 28,

2001; accepted November 29, 2001.

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, bromo-deoxyuridine; DR, dietary restriction;

GFAP, glial ®brillary acidic protein; MAP-2, microtubule-associated

protein-2; NPC, neural precursor cells; NT-3, neurotrophin-3; TBS, Tris-

buffered saline.

Journal of Neurochemistry, 2002, 80, 539±547

Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 539±547 539

(DR) can increase the lifespan of rodents and may ward off

many different age-related diseases (Sohal and Weindruch

1996) including neurodegenerative disorders (Mattson 2000).

Increasing numbers of reports have documented ÔantiagingÕeffects of DR on the brain. Epidemiological data suggest that

individuals with a low calorie intake are at reduced risk for

Parkinson's (Logroscino et al. 1996) and Alzheimer's (May-

eux et al. 1999) diseases. In addition, rodents maintained on

DR perform better on learning and memory tasks than do rats

fed ad libitum (Idrobo et al. 1987; Ingram et al. 1987;

Stewart et al. 1989). DR increases the resistance of neurons

to degeneration and improves behavioral outcome in exper-

imental animal models of Alzheimer's disease (Bruce-Keller

et al. 1999; Zhu et al. 1999), Parkinson's disease (Duan and

Mattson 1999), Huntington's disease (Bruce-Keller et al.

1999) and stroke (Yu and Mattson 1999). It was recently

reported that levels of BDNF are increased in the hippo-

campus and cerebral cortex of rats maintained on a dietary

restriction feeding regimen (Lee et al. 2000; Duan et al.

2001). Here we show that DR can enhance neurogenesis in

the hippocampus of adult mice, and that this effect of DR on

NPC is associated with increased production of BDNF and

NT-3. Our ®ndings suggest a contribution of enhanced

neurogenesis to the bene®cial effects of DR on hippocampal

plasticity and resistance to neurodegenerative disorders.

Materials and methods

Mice, diets and BrdU administration

Fifty-six adult (8 weeks old) male C57BL/6 mice obtained from

the National Cancer Institute were maintained under temperature-

and light-controlled conditions (20±23°C, 12-h light/12-h dark

cycle). Mice were divided into two groups (28 mice/group), an

ad libitum (AL) group which had continual access to food, and a

DR group which was provided food on alternate days. Previous

studies have shown that rats and mice maintained on such an

alternate day feeding schedule will consume less calories over time

and live longer than animals fed AL (Goodrick et al. 1983). For

evaluations of neurogenesis, 12 mice in each group were given a

daily intraperitoneal injection of bromodeoxyuridine (BrdU;

50 mg/kg body weight) for 12 days. Half of the mice in each

diet group were killed 1 day after the last BrdU injection and half

were killed 1 month after the last BrdU injection. The remaining

16 mice in each diet group were processed for analyses of

neurotrophin and neurotrophin receptor expression as described

below. All procedures complied with National Institutes of Health

guidelines and were approved by the Institutional Animal Care and

Use Committee.

Quanti®cation of newly produced neural cells

Mice were perfused transcardially with 4% paraformaldehyde and

their brains were removed, post®xed at 4°C overnight, and

transferred to a 30% sucrose solution. The cryoprotected brains

were sectioned serially at 40 lm in the coronal plane using a

freezing microtome. Every section which contained the hippocampal

formation was saved. The protocol for immunostaining of brain

sections with BrdU antibody was similar to that described previously

(Nilsson et al. 1999). Brie¯y, free ¯oating 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-100/3% goat serum (TBS-TS) for

30 min, and incubated with primary anti-BrdU antibody (rat

monoclonal, 1 : 400; Accurate Chemicals, Westbury, NY, USA) in

TBS-TS overnight at 4°C. Sections were further processed using a

biotinylated secondary goat anti-rat IgG antibody (Vector Labora-

tories, 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 (Gundersen

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 (10 ´ objective; on-screen magni®cation � 271 ´); identi®-

cation of BrdU-positive cells was accomplished at high power

(63 ´ objective; on-screen magni®cation � 1714 ´). The dimension

of the sampling frames were 92.2 lm in length by 92.2 lm in width

and 12 lm in depth. The guard height for each section was 1 lm.

The optical fractionator technique estimates the number 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 disectors multiplied by the volume of one

disector. All cell counts were performed by the same investigator (JL)

blind to the group identi®cation of each section.

Immunohistochemistry

Immunostaining for confocal analysis was performed on 40 lm

coronal brain sections as follows. Sections were incubated for 1 h

in a solution containing 2.5% normal horse serum, 2.5% normal

goat serum, and 0.1% Triton X-100 in TBS. Primary antibodies

were then added and the cultures were incubated overnight at

4°C. The primary antibodies used were a rat monoclonal antibody

against BrdU (Accurate Chemicals, 1 : 200 dilution), rabbit

polyclonal antibody against GFAP (Sigma, St Louis, MO, USA,

1 : 500 dilution) and a mouse monoclonal antibody against the

neuron-speci®c nuclear antigen NeuN (Chemicon, Temecula, CA,

USA, 1 : 500 dilution) and mature neuron-speci®c cytoskeletal

antigen MAP2ab (Chemicon, 1 : 500 dilution). Cultures 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-543 (Molecular Probes, Eugene, OR, USA; 1: 2000

dilution). Confocal images were acquired using a Zeiss 510

CSLM microscope.

540 J. Lee et al.

Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 539±547

In situ hybridization

Adjacent, coronal sections through the hippocampus were processed

for the in situ hybridization detection of BDNF, NT-3, trkB, and

trkC mRNAs by using 35S-labeled cRNA probes as described

previously (Seroogy et al. 1994; Seroogy and Herman 1997; Numan

and Seroogy 1999). Brie¯y, the slide-mounted sections were

brought to room temperature, placed in 4% paraformaldehyde for

10 min, and washed sequentially in 0.1 M phosphate buffer (PB),

0.1 M PB/0.2% glycine, and 0.25% acetic anhydride in 0.1 M

triethanolamine. The sections were then dehydrated with increasing

concentrations of ethanol, delipidated in chloroform, and air-dried.

Sections were hybridized at 60°C overnight in a hybridization

solution consisting of 50% formamide, 10% dextran sulfate, 1 ´Denhardt's solution, 0.15 mg/mL yeast tRNA, 0.33 mg/mL dena-

tured salmon sperm DNA, 40 mM dithiothreitol, 1 mM EDTA,

20 mM Tris-HCl and the 35S-labeled cRNA probe at a concentration

of 1.0 ´ 106 cpm/50 lL/slide. Both sense and antisense cRNA

probes for each neurotrophin and trk receptor were prepared by

in vitro transcription using linearized DNA constructs in the

presence of RNA polymerase (T3, T7 or SP6) and [35S]UTP

(New England Nuclear; Boston, MA, USA). BDNF and NT-3

cDNA constructs (generous gifts from C. Gall and J. Lauterborn,

University of California at Irvine) resulted in antisense transcripts

that were 540 and 550 bases long, respectively. The cDNA

constructs for trkB and trkC (kindly supplied by D. McKinnon,

State University of New York at Stony Brook) resulted in antisense

RNA transcripts that were 196 and 300 bases long, respectively. The

trkB cRNA probe detects only the kinase-speci®c, full-length

catalytic form of the receptor mRNA (Klein et al. 1990; Middlemas

et al. 1991; Sternini et al. 1996), whereas the trkC cRNA probe

recognizes mRNA transcripts for both the catalytic and non-catalytic

isoforms of the receptor (Valenzuela et al. 1993; Dixon and

McKinnon 1994; Albers et al. 1996). For posthybridization treat-

ment, sections were washed several times in 4 x saline sodium

citrate buffer (SSC; 1 ´ SSC � 0.15 M sodium chloride, 0.015 M

sodium citrate, pH 7.0) containing 10 mM sodium thiosulfate,

at 37°C. The sections were then incubated in ribonuclease A

(0.05 mg/mL) for 30 min at 45°C, followed by several washes in

decreasing concentrations of SSC (2 ´, 0.5 ´ and 0.1 ´) at 37°C.

The sections were then brie¯y rinsed in dH20, dipped in 95%

ethanol, and air-dried. To generate ®lm autoradiograms the sections

were exposed to b-Max Hyper®lm (Amersham; Arlington Heights,

IL, USA) for 11 days (BDNF and NT-3) or 7 days (trkB and trkC).

In control procedures, prehybridization treatment of tissue with

ribonuclease A (0.05 mg/mL; 45°C for 30 min), processing tissue

with 35S-labeled sense strand transcripts for each probe, and

processing tissue with no probe at all (positive chemography

control), resulted in no speci®c hybridization signal. Film autora-

diograms were analyzed with NIH Image public domain software

(Image 1.62) to compare the densities of hybridization (mean

corrected gray level) of each probe in various hippocampal sub®elds

(dentate gyrus, CA1 and CA3) and in parietal cortex in each

treatment paradigm. We did not attempt to quantify hybridization

levels in speci®c subpopulations of cortical neurons; the analysis

was made on the entire thickness of the cortex to provide a measure

of overall levels of mRNA in the cortex. At least six measurements

were taken for each probe from each animal. Statistical analysis

included Student's unpaired t-test, and analysis of variance (ANOVA)

followed by Fisher's protected least signi®cant differences proce-

dure where appropriate. The NIH image software was also used to

acquire images of representative sections from ®lm autoradiograms.

Immunoblot analyses

Hippocampal and cerebral cortical tissues were homogenized in a

sample buffer (62 mM Tris, 2 mM EDTA, 2 mM EGTA, 2% SDS,

10% glycerol and a protease inhibitor cocktail; pH 6.0). Solubilized

proteins were separated by electrophoresis on a 7.5% SDS±

acrylamide gel, and transferred to a nitrocellulose sheet. Following

incubation of the membrane in blocking solution (5% non-fat milk

in TTBS), the membrane was incubated overnight at 4°C in TTBS

containing a primary antibody. The primary antibody was a rabbit

polyclonal antibody that recognizes both full-length and truncated

forms of trkB (Santa Cruz Biotechnology, Santa Cruz, CA, USA;

1 : 500 dilution). The membrane was then incubated for 1 h in

TTBS containing HRP-conjugated secondary antibody (1 : 3000;

Jackson Immunological Research Laborites Inc., West Grove, PA,

USA) and immunoreactive proteins were visualized using a

chemiluminescence-based detection kit according to the manufac-

ture's protocol (ECL kit; Amersham Corp., Arlington Heights, IL,

USA). Bands were quanti®ed by densitometric scanning.

Results

Mice were maintained on either an ad libitum diet (AL) or a

dietary restriction (DR) feeding regimen in which they were

fed every other day; their body weights after 3 months on the

diets were: AL, 32.9 � 0.46 g; DR, 27.2 � 0.35 g (n � 24;

p < 0.0001; paired t-test). Mice were then administered

bromodeoxyuridine (BrdU) and killed either 1 day or

4 weeks later. BrdU-immunoreactive cells in the dentate

gyrus of the hippocampus were quanti®ed using unbiased

stereological methods. At the 1 day time point the numbers

of BrdU positive cells in the dentate gyrus were not

signi®cantly different in groups AL and DR (Figs 1a,b;

Table 1). At the 4-week time point there were signi®cantly

more BrdU-positive cells in the dentate gyrus of mice in

group DR compared to rats in the control group (Figs 1c,d;

Table 1). The volume of the dentate gyrus was not signi®-

cantly different in mice that had been maintained on AL and

DR diets (data not shown).

In order to determine the phenotypes of the newly

generated cells, we performed either triple or double label

confocal immunohistochemical analysis of hippocampi using

antibodies against the astrocyte protein (GFAP) and the

mature neuron-speci®c protein (NeuN or MAP2ab), in

combination with the BrdU antibody. At one day after BrdU

administration the vast majority of BrdU-positive cells were

con®ned to the subgranular zone of the dentate gyrus and

were not immunoreactive with either the GFAP or NeuN

antibodies (Figs 2a and b). At 4 weeks after BrdU admini-

stration, BrdU-positive cells were scattered throughout the

dentate gyrus. Essentially all BrdU-positive cells that were

located in the granule cell layer were also NeuN and

Dietary restriction, neurotrophins and neurogenesis 541


MAP2ab positive (Figs 2c, d, e, g and h, arrow). BrdU-

positive cells located in the molecular layers of the dentate

gyrus were mostly GFAP-positive (Fig. 2f, arrowhead),

although some cells in the molecular layers were MAP2ab-

positive (Fig. 2h).

Previous studies have shown that environmental stimuli

that increase neurogenesis in the dentate gyrus of the

hippocampus also stimulate expression of the neurotrophins

BDNF and NT-3 (Ballarin et al. 1991; Lee et al. 1997;

Young et al. 1999). We therefore performed in situ hybrid-

ization analyses of brain sections from AL and DR mice to

determine whether DR affects the expression of BDNF, NT-3

and/or their high-af®nity receptors trkB and trkC. Examin-

ation of the pseudocolor densitometric images of autoradio-

grams revealed that the overall cellular pattern of expression

of BDNF, NT-3, trkB and trkC was unchanged in mice that

had been maintained on DR (Fig. 3). BDNF and trkB were

expressed in pyramidal neurons in all regions of the

hippocampus and in dentate granule cells. The BDNF

mRNA hybridization signal appeared to be increased in CA1

and CA3 pyramidal neurons. NT-3 expression was con®ned

to dentate granule cells and a small population of pyramidal

neurons in region CA2, while trkC was expressed in all

pyramidal neurons in all regions of the hippocampus and in

dentate granule cells. The pattern of hybridization with each

of the probes also appeared similar in the cerebral cortex of

AL and DR mice (Fig. 3). Quantitative comparisons of

levels of mRNAs encoding BDNF and NT-3 revealed

signi®cant effects of DR. Levels of BDNF mRNA were

signi®cantly increased by approximately 20% in CA1 and

CA3 pyramidal neurons in hippocampi of DR mice

compared to AL mice (Fig. 4). Levels of BDNF mRNA in

dentate granule cells and cerebral cortical cells were

unaffected by DR. Levels of NT-3 mRNA were signi®cantly

increased by approximately 30% in dentate granule cells of

DR mice compared to AL mice (Fig. 4). There were no

signi®cant differences in trkB or trkC mRNA levels in

hippocampal pyramidal cells, dentate granule cells or

cortical cells in AL and DR mice.

The BDNF receptor trkB exists in cells in full-length and

truncated forms; the full-length form is a functional receptor

tyrosine kinase, while the truncated form may serve to

negatively regulate trkB by sequestering BDNF. In order for

the DR-induced increase of BDNF expression to enhance

BDNF signaling in target neurons, it is essential that levels of

functional trkB are maintained. We therefore determined

Table 1 Proliferation, survival and survival rate of cells in the dentate

gyrus of mice fed ad libitum in comparison with mice maintained on

dietary restriction

Ad libitum Dietary restriction

Proliferation, 1 day 3579 � 222.3 3188 � 116.8

Survival, 4 weeks 992 � 113.5 1404 � 73.7*

Survival (%), 4 weeks 28 � 3.2 44 � 2.3*

All mice received BrdU (50 mg per kg) for 12 days. Cell proliferation

was assessed on 1 day after last injection. Survival of BrdU-labeled

cells in the dentate gyrus were determined 4 weeks after last injection

(n � 6 per group). All data presented as means � standard error.

*Signi®cantly different from ad libitum group (p < 0.02).

Fig. 1 Dietary restriction increases neuro-

genesis in the dentate gyrus of adult mice.

Photomicrographs of BrdU-positive cells in

the dentate gyrus 1 day (a and b) and

4 weeks (c and d) after BrdU administration

in mice that had been maintained fed on

either ad libitum (a and c) or dietary

restriction (b and d) for 3 months prior

to BrdU administration. Quanti®cation of

labeled cells is shown in Table 1.

542 J. Lee et al.


relative levels of full-length and truncated trkB in hippo-

campal and cortical tissue from AL and DR mice (Fig. 5a).

The ratio of full-length trkB to truncated trkB was signi®-

cantly increased by approximately 25% in the hippocampus

of DR mice compared to AL mice (Fig. 5b). Levels of full-

length and truncated trkB in the cerebral cortex were not

different in AL and DR mice.

AL DR

Fig. 3 Pseudocolor densitometric images of ®lm autoradiograms

showing hybridization levels of BDNF, NT-3, trkB and trkC mRNAs in

hippocampi from a control mouse fed ad-libitum (AL) and a mouse that

had been maintained for 3 months on dietary restriction (DR). Arrows

indicate regions of increased mRNA expression in the DR mice.

Fig. 2 Confocal images documenting the phenotypes of newly gen-

erated cells in the dentate gyrus of mice that had been maintained on a

dietary restriction feeding regimen. Sections were triple-labeled with

either antibodies against BrdU (green, newborn cells), GFAP (white,

astrocyte antigen) and NeuN (red, neuronal antigen) (a±d), or BrdU

(green), GFAP (white) and MAP2ab (red) (g and h). (f) shows the

molecular layer of the dentate gyrus in a section double labeled with

antibodies against BrdU (green) and GFAP (red) (the arrowhead

points to a double-labeled cell). Most of the BrdU-positive cells in the

dentate gyrus did not exhibit a neuronal or astrocyte antigen 1 day

after BrdU administration (a and b). Four weeks after BrdU adminis-

tration, the majority of cells labeled with BrdU were also immunore-

active with the NeuN antibody (arrows in c and e). The neuronal

phenotype of newborn cells on 4 weeks after BrdU administration was

con®rmed by immuno¯uorescence using antibodies to another neu-

ronal marker MAP2, which labeled cell bodies and dendrites of granule

neurons (arrows in g and h).

Fig. 4 Dietary restriction increases BDNF mRNA levels in CA1-3

pyramidal cells and NT-3 levels in dentate gyrus granule cells.

Densitometric analyses of hybridization levels of BDNF, NT-3, trkB

and trkC mRNAs were performed on autoradiograms of hippocampal

brain sections from mice that had been maintained for 3 months on AL

or DR diets. Values are the mean and SE of determinations made in

six mice per group. *p < 0.02, **p < 0.01 compared to corresponding

value for mice fed AL [ANOVA with Fisher's protected least signi®cant

difference procedure (PLSD)].



Discussion

The present ®ndings establish an effect of diet on the

expression of neurotrophins and on neurogenesis in the

hippocampus of adult mice. DR had no signi®cant effect on

the proliferation of NPC; instead, DR enhanced neurogenesis

by increasing the survival of newly generated cells. Some of

the newly generated cells were localized to the dentate

granule cell layer and expressed NeuN suggesting that they

had differentiated into granule neurons, in agreement with

previous reports that the majority of newly generated cells in

the dentate gyrus migrate into the granule cell layer and

display neuron-like properties (Parent et al. 1997; Young

et al. 1999). A previous study showed that mice maintained

in an enriched environment exhibit increased neurogenesis in

the dentate gyrus, and a signi®cant increase in the total

number of dentate granule neurons, compared with litter-

mates housed in standard cages (Kempermann et al. 1997).

Another study showed that rats raised in an enriched

environment exhibit increased neurogenesis (Nilsson et al.

1999). Similar to the effect of DR, the enriched environment

did not increase NPC proliferation but did increase survival

of the NPC progeny. This suggests that environmental

enrichment and DR share a common mechanism of action in

increasing the number of newly generated dentate cells. The

progeny of many NPC may undergo apoptosis, as indicated

by a decrease in the number of BrdU-positive cells with

increasing time postlabeling. Consistent with this interpreta-

tion, environmental enrichment reduces spontaneous death of

newly generated neural cells in the hippocampus (Young

et al. 1999). We did not observe a signi®cant effect of DR on

the volume of the dentate gyrus, despite a signi®cant increase

in the survival of BrdU-labeled cells. This result is similar to

that observed in animals maintained for several months in an

enriched environment (van Praag et al. 1999). Perhaps a

more prolonged period of DR would result in a measurable

increase in hippocampal volume. On the other hand, it is

possible that DR also affects the rate of loss of granule

neurons or their size, or DR might affect gliogenesis or

activation of glial cells (activated astrocytes increase in size).

Indeed, it has been reported that DR can reduce activation of

astrocytes (Major et al. 1997).

Our ®ndings suggest a role for neurotrophins in mediating

the positive effects of DR on neurogenesis. The expression of

BDNF was increased in CA1 and CA3 pyramidal neurons,

and the expression of NT-3 was increased in granule neurons

of the dentate gyrus, in mice maintained on DR. The ratio of

full-length to truncated trkB was increased in hippocampus

which, in the presence of increased BDNF, would be

expected to result in increased signaling via trkB. BDNF

can promote the survival and differentiation of hippocampal

NPC in culture (Lowenstein and Arsenault 1996; Shetty and

Turner 1998) and of newly generated embryonic hippocam-

pal and cortical neurons (Cheng and Mattson 1994; Mattson

et al. 1995; Cheng et al. 1997; Hetman et al. 1999). Further

evidence that BDNF mediates the effects of DR on

neurogenesis comes from studies showing that stimuli that

increase neurogenesis in the dentate gyrus also increase

BDNF expression including seizure activity (Parent et al.

1997; Lowenstein and Arsenault 1996; Lee et al. 1997),

ischemia (Lindvall et al. 1992; Liu et al. 1998) and an

enriched environment (Cameron et al. 1998; Young et al.

1999). A study of primary hippocampal progenitor cells in

culture showed that NT-3 and BDNF promote neuronal

differentiation as indicated by increased expression of

glutamate receptors (Sah et al. 1997). NT-3 promotes the

maturation of the immature cells in the embryonic striatum

into neurons that produce one or more neurotransmitters and

(Vicario-Abejon et al. 1995), and also promotes differenti-

ation of adult hippocampal NPC (Takahashi et al. 1999).

Studies of mice lacking NT-3 revealed a requirement for this

neurotrophin for the survival of certain populations of NPC

CORTEX

HIPPOCAMPUS

(a) (b)

Fig. 5 The ratio of full-length to truncated trkB is increased in the

hippocampus of mice maintained on DR. (a) Proteins in homogenates

of hippocampus and cerebral cortex from AL and DR mice were

subjected to immunoblot analysis using antibodies that selectively

recognize either full-length (145 kDa) or truncated (95 kDa) trkB. Each

lane was loaded with a sample (50 lg) from a different mouse.

(b) Densitometric analyses of full-length and truncated trkB levels were

performed on immunoblots. Values are the mean and SE of determi-

nations made in six mice per group. **p < 0.01 compared to corre-

sponding AL value (ANOVA with PLSD test).

544 J. Lee et al.


and their neuronal and glial progeny (El Shamy et al. 1998;

Kahn et al. 1999).

It has been reported that the proliferation of NPC is rapidly

increased in response to kainic acid-induced seizures and

ischemic stroke in the adult brain; these insults also increase

the production of several different neurotrophic factors

and cytokines including BDNF (Mattson and Lindvall

1997). Under basal conditions, NT-3 is expressed in the

hippocampus predominately in dentate granule neurons and

CA2 pyramidal neurons, and is down-regulated in dentate

neurons following seizures (Rocamora et al. 1992; Lowen-

stein and Arsenault 1996). In contrast, we found that NT-3

expression was increased in dentate neurons in response to

DR. Because BDNF and NT-3 can promote differentiation

and survival of granule neurons, the DR-induced increases in

BDNF and NT-3 levels may enhance the production of new

granule neurons.

The mechanism whereby DR up-regulates BDNF and

NT-3 production and enhances neurogenesis is not known.

One possibility is that DR induces a mild metabolic stress

response in neurons, a possibility supported by data showing

that levels of the stress protein chaperones HSP-70 and

GRP-78 are increased in neurons in the brains of rats

and mice maintained on DR (Duan and Mattson 1999; Yu and

Mattson 1999), and that more severe metabolic (ischemic)

stress can induce neurogenesis (Liu et al. 1998). On the

other hand, it is reasonable to consider that the effects of DR

on neurotrophin expression and neurogenesis in the hippo-

campus are secondary to an effect of DR on behavior. For

example, if the DR mice are more active such that they

receive more exercise and more environmental exposure, this

might account for our ®ndings. Indeed, previous studies have

suggested that motor activity is increased in rodents main-

tained on DR (Duffy et al. 1997), and enriched environments

can induce increased expression of BDNF and NT-3 in the

hippocampus (Torasdotter et al. 1996). However, increased

physical activity is unlikely to account for the effect of DR

documented in the present study because exercise increases

both the proliferation and survival of NPC (van Praag et al.

1999), whereas DR increases survival only. Identi®cation of

the speci®c mechanism of action of DR on neurotrophic

signaling pathways will require considerable further work.

Neurogenesis in the adult brain may be important for

maintenance of certain neuronal populations in the face of

continual cell loss, and may play critical roles in learning and

memory (Shors et al. 2001) and the brain's response to injury

(Parent et al. 1997; Liu et al. 1998). In this regard it is of

considerable interest that DR can increase lifespan, can

enhance learning and memory (Sohal and Weindruch 1996;

Ingram et al. 1987), and can improve outcome following

brain injury (Duan and Mattson 1999; Yu and Mattson 1999).

Enhanced neurotrophin signaling may be the mechanism

underlying these different bene®cial effects of DR on the

brain. Indeed, data from in vivo studies and analyses of

synaptic plasticity in hippocampal slices have shown that

BDNF plays an important role in learning and memory

(Levine et al. 1995; Minichiello et al. 1999). The ability of a

change in diet to affect neurotrophin expression and neuro-

genesis therefore has important implications for brain

function in humans. For example, it may be possible

to establish dietary regimens that enhance learning and

memory, increase resistance of neurons to neurodegenerative

conditions, and improve outcome following brain injury. In

this view, brain healthspan might be increased through

dietary manipulations.

Acknowledgements

We thank K. Lundgren for technical assistance, and M. Rao and

D. Ingram for valuable discussions. Supported by the NIA and

a grant to KBS from the NINDS (NS39128).

References

Albers K. M., Perrone T. N., Goodness T. P., Jones M. E., Green M. A.

and Davis B. M. (1996) Cutaneous overexpression of NT-3

increases sensory and sympathetic neuron number and enhances

touch dome and hair follicle innervation. J. Cell Biol. 134,

487±497.

Ballarin M., Ernfors P., Lindefors N. and Persson H. (1991) Hippo-

campal damage and kainic acid injection induce a rapid increase in

mRNA for BDNF and NGF in the rat brain. Exp. Neurol. 114,

35±43.

Benoit B. O., Savarese T., Joly M., Engstrom C. M., Pang L., Reilly J.,

Recht L. D., Ross A. H. and Quesenberry P. J. (2001) Neurotrophin

channeling of neural progenitor cell differentiation. J. Neurobiol.

46, 265±280.

Bruce-Keller A. J., Umberger G., McFall R. and Mattson M. P. (1999)

Food restriction reduces brain damage and improves behavioral

outcome following excitotoxic and metabolic insults. Ann. Neurol.

45, 8±15.

Cameron H. A., Hazel T. G. and McKay R. D. (1998) Regulation of

neurogenesis by growth factors and neurotransmitters. J. Neuro-

biol. 36, 287±306.

Cheng B. and Mattson M. P. (1994) NT-3 and BDNF protect CNS

neurons against metabolic/excitotoxic insults. Brain Res. 640,

56±67.

Cheng Y., Gidday J. M., Yan Q., Shah A. R. and Holtzman D. M. (1997)

Marked age-dependent neuroprotection by brain-derived neuro-

trophic factor against neonatal hypoxic-ischemic brain injury. Ann.

Neurol. 41, 521±529.

Dixon J. E. and McKinnon D. (1994) Expression of the trk gene family

of neurotrophin receptors in prevertebral sympathetic ganglia. Dev.

Brain Res. 77, 177±182.

Duan W. and Mattson M. P. (1999) Dietary restriction and 2-deoxy-

glucose administration improve behavioral outcome and reduce

degeneration of dopaminergic neurons in models of Parkinson's

disease. J. Neurosci. Res. 57, 195±206.

Duan W., Lee J., Guo Z. and Mattson M. P. (2001) Dietary restriction

stimulates BDNF production in the brain and thereby protects

neurons against excitotoxic injury. J. Mol. Neurosci. 16, 1±12.

Duffy P. H., Leakey J. E., Pipkin J. L., Turturro A. and Hart R. W. (1997)

The physiologic, neurologic, and behavioral effects of caloric

restriction related to aging, disease, and environmental factors.

Environ. Res. 73, 242±248.



El Shamy W. M., Fridvall L. K. and Ernfors P. (1998) Growth arrest

failure, G1 restriction potential override, and S phase death of

sensory precursor cells in the absence of neurotrophin-3. Neuron

21, 1003±1015.

Gage F. H. (2000) Mammalian neural stem cells. Science 287, 1433±

1438.

Goodrick C. L., Ingram D. K., Reynolds M. A., Freeman J. R. and Cider

N. L. (1983) Differential effects of intermittent feeding and

voluntary exercise on body weight and lifespan in adult rats.

J. Gerontol. 38, 36±45.

Gundersen H. J. G. and Jensen E. B. (1987) The ef®ciency of systematic

sampling in stereology and its prediction. J. Micros. 147, 229±263.

Hetman M., Kanning K., Cavanaugh J. E. and Xia Z. (1999) Neuro-

protection by brain-derived neurotrophic factor is mediated by

extracellular signal-regulated kinase and phosphatidylinositol

3-kinase. J. Biol. Chem. 274, 22569±22580.

Idrobo F., Nandy K., Mostofsky D. I., Blatt L. and Nandy L. (1987)

Dietary restriction: effects on radial maze learning and lipofuscin

pigment deposition in the hippocampus and frontal cortex. Arch.

Gerontol. Geriatr. 6, 355±362.

Ingram D. K., Weindruch R., Spangler E. L., Freeman J. R. and Walford

R. L. (1987) Dietary restriction bene®ts learning and motor

performance of aged mice. J. Gerontol. 42, 78±81.

Kahn M. A., Kumar S., Liehl D., Chang R., Parada L. F. and De Vellis J.

(1999) Mice lacking NT-3, and its receptor trkC, exhibit profound

de®ciencies in CNS glial cells. Glia 26, 153±165.

Kempermann G., Kuhn H. G. and Gage F. H. (1997) More hippocampal

neurons in adult mice living in an enriched environment. Nature

386, 493±495.

Klein R., Conway D., Parada L. F. and Barbacid M. (1990) The trkB

tyrosine protein kinase gene codes for a second neurogenic

receptor that lacks the catalytic kinase domain. Cell 61, 647±656.

Kuhn H. G., Dickinson-Anson H. and Gage F. H. (1996) Neurogenesis

in the dentate gyrus of the adult rat: age-related decrease of

neuronal progenitor proliferation. J. Neurosci. 16, 2027±2033.

Lachyankar M. B., Condon P. J., Quesenberry P. J., Litofsky N. S., Recht

L. D. and Ross A. H. (1997) Embryonic precursor cells that

express Trk receptors: induction of different cell fates by NGF,

BDNF, NT-3, and CNTF. Exp. Neurol. 144, 350±360.

Lee J., Duan W., Long J. M., Ingram D. K. and Mattson M. P. (2000)

Dietary restriction increases the number of newly generated neural

cells, and induces BDNF expression, in the dentate gyrus of rats.

J. Mol. Neurosci. 15, 99±108.

Lee S., Williamson J., Lothman E. W., Szele F. G., Chesselet M. F., Von

Hagen S., Sapolsky R. M., Mattson M. P. and Christakos S. (1997)

Early induction of mRNA for calbindin-D28k and BDNF but not

NT-3 in rat hippocampus after kainic acid treatment. Mol. Brain.

Res. 47, 183±194.

Levine E. S., Dreyfus C. F., Black I. B. and Plummer M. R. (1995)

Brain-derived neurotrophic factor rapidly enhances synaptic

transmission in hippocampal neurons via postsynaptic tyrosine

kinase receptors. Proc. Natl Acad. Sci. USA 92, 8074±8077.

Lindvall O., Ernfors P., Bengzon J., Kokaia Z., Smith M. L., Siesjo B. K.

and Persson H. (1992) Differential regulation of mRNAs for nerve

growth factor, brain-derived neurotrophic factor, and neurotrophin

3 in the adult rat brain following cerebral ischemia and hypogly-

cemic coma. Proc. Natl Acad. Sci. USA 89, 648±652.

Liu J., Solway K., Messing R. O. and Sharp F. R. (1998) Increased

neurogenesis in the dentate gyrus after transient global ischemia in

gerbils. J. Neurosci. 18, 7768±7778.

Logroscino G., Marder K., Cote L., Tang M. X., Shea S. and Mayeux R.

(1996) Dietary lipids and antioxidants in Parkinson's disease: a

population-based, case-control study. Ann. Neurol. 39, 89±94.

Long J. M., Kalehua A. N., Muth N. J., Hengemihle J. M., Jucker M.,

Calhoun M. E., Ingram D. and Mouton P. R. (1998) Stereological

estimation of total microglia number in mouse hippocampus.

J. Neurosci. Methods 84, 101±108.

Lowenstein D. H. and Arsenault L. (1996) The effects of growth factors

on the survival and differentiation of cultured dentate gyrus

neurons. J. Neurosci. 16, 1759±1769.

Major D. E., Kesslak J. P., Cotman C. W., Finch C. E. and Day J. R.

(1997) Life-long dietary restriction attenuates age-related increases

in hippocampal glial ®brillary acidic protein mRNA. Neurobiol.

Aging 18, 523±526.

Mattson M. P. (2000) Impact of dietary restriction on brain aging and

neurodegenerative disorders: emerging ®ndings from experimental

and epidemiological studies. Anti-Aging Med. 2, 331±336.

Mattson M. P. and Lindvall O. (1997) Neurotrophic factor and cytokine

signaling in the aging brain, in The Aging Brain, Advances in Cell

Aging Gerontology 2 (Mattson M. P. and Geddes J. W., eds),

pp 299±345. JAI Press, Greenwich, CT, USA.

Mattson M. P., Lovell M. A., Furukawa K. and Markesbery W. R. (1995)

Neurotrophic factors attenuate glutamate-induced accumulation of

peroxides, elevation of intracellular Ca2+ concentration, and

neurotoxicity and increase antioxidant enzyme activities in

hippocampal neurons. J. Neurochem. 65, 1740±1751.

Mayeux R., Costa R., Bell K., Merchant C., Tung M. X. and Jacobs D.

(1999) Reduced risk of Alzheimer's disease among individuals

with low calorie intake. Neurology 59, S296±S297.

Middlemas D. S., Lindberg R. A. and Hunter T. (1991) trkB, a neural

receptor protein-tyrosine kinase: evidence for a full-length and two

truncated receptors. Mol. Cell. Biol. 11, 143±153.

Minichiello L., Korte M., Wolfer D., Kuhn R., Unsicker K., Cestari V.,

Rossi-Arnaud C., Lipp H. P., Bonhoeffer T. and Klein R. (1999)

Essential role for TrkB receptors in hippocampus-mediated

learning. Neuron 24, 401±414.

Nilsson M., Per®lieva E., Johansson U., Orwar O. and Eriksson P. S.

(1999) Enriched environment increases neurogenesis in the adult

rat dentate gyrus and improves spatial memory. J. Neurobiol. 39,

569±578.

Numan S. and Seroogy K. B. (1999) Expression of trkB and trkC

mRNAs by adult midbrain dopamine neurons: a double-label

in situ hybridization study. J. Comp. Neurol. 403, 295±308.

Parent J. M., Yu T. W., Leibowitz R. T., Geschwind D. H., Sloviter R.

S. and Lowenstein D. H. (1997) Dentate granule cell neurogen-

esis is increased by seizures and contributes to aberrant network

reorganization in the adult rat hippocampus. J. Neurosci. 17,

3727±3738.

van Praag H., Kempermann G. and Gage F. H. (1999) Running increases

cell proliferation and neurogenesis in the adult mouse dentate

gyrus. Nat. Neurosci. 2, 266±270.

Rocamora N., Palacios J. M. and Mengod G. (1992) Limbic seizures

induce a differential regulation of the expression of nerve growth

factor, brain-derived neurotrophic factor and neurotrophin-3, in the

rat hippocampus. Mol. Brain Res. 13, 27±33.

Sah D. W., Ray J. and Gage F. H. (1997) Regulation of voltage- and

ligand-gated currents in rat hippocampal progenitor cells in vitro.

J. Neurobiol. 32, 95±110.

Seroogy K. B. and Herman J. P. (1997) In situ hybridization approaches

to the study of the nervous system, in Neurochemistry: a Practical

Approach, 2nd edn. (Turner, A. J. and Bachelard, H. S., eds),

pp 121±150. Oxfod University Press, Oxford.

Seroogy K. B., Lundgren K. H., Tran T. M. D., Guthrie K. M., Isackson

P. J. and Gall C. M. (1994) Dopaminergic neurons in the rat ventral

midbrain express brain-derived neurotrophic factor and neurotro-

phin-3 mRNAs. J. Comp. Neurol. 342, 321±334.

546 J. Lee et al.


Shetty A. K. and Turner D. A. (1998) In vitro survival and differentiation

of neurons derived from epidermal growth factor-responsive

postnatal hippocampal stem cells: inducing effects of brain-derived

neurotrophic factor. J. Neurobiol. 35, 395±425.

Shors T. J., Miesegaes G., Beylin A., Zhao M., Rydel T. and Gould E.

(2001) Neurogenesis in the adult is involved in the formation of

trace memories. Nature 410, 372±376.

Sohal R. S. and Weindruch R. (1996) Oxidative stress, caloric restriction,

and aging. Science 273, 59±63.

Sternini C., Su D., Arakawa J., Giorgio R. D., Rickman D. W., Davis B.

M., Albers K. M. and Brecha N. C. (1996) Cellular localization of

pan-trk immunoreactivity and trkC mRNA in the enteric nervous

system. J. Comp. Neurol. 368, 597±607.

Stewart J., Mitchell J. and Kalant N. (1989) The effects of life-long food

restriction on spatial memory in young and aged Fischer 344 rats

measured in the eight-arm radial and the Morris water mazes.

Neurobiol. Aging 10, 669±675.

Takahashi J., Palmer T. D. and Gage F. H. (1999) Retinoic acid and

neurotrophins collaborate to regulate neurogenesis in adult-derived

neural stem cell cultures. J. Neurobiol. 38, 65±81.

Torasdotter M., Metsis M., Henriksson B. G., Winblad B. and

Mohammed A. H. (1996) Expression of neurotrophin-3 mRNA in

the rat visual cortex and hippocampus is in¯uenced by environ-

mental conditions. Neurosci. Lett. 218, 107±110.

Valenzuela D. M., Maisonpierre P. C., Glass D. J., Rojas E., Nunez L.,

Kong L., Gies D. R., Stitt T. N., Ip N. Y. and Yancopoulos G. D.

(1993) Alternative forms of rat trkC with different functional

capabilities. Neuron 10, 963±974.

Vicario-Abejon C., Johe K. K., Hazel T. G., Collazo D. and McKay R.

D. (1995) Functions of basic ®broblast growth factor and

neurotrophins in the differentiation of hippocampal neurons.

Neuron 15, 105±114.

West M. J. (1993) New stereologial methods for counting neurons.

Neurobiol. Aging 14, 275±285.

Young S. N. (1993) The use of diet and dietary components in the study

of factors controlling affect in humans: a review. J. Psychiatry

Neurosci. 18, 235±244.

Young D., Lawlor P. A., Leone P., Dragunow M. and During M. J.

(1999) Environmental enrichment inhibits spontaneous apop-

tosis, prevents seizures and is neuroprotective. Nat. Med. 5,

448±453.

Yu Z. F. and Mattson M. P. (1999) Dietary restriction and 2-deoxyglu-

cose administration reduce focal ischemic brain damage and

improve behavioral outcome: evidence for a preconditioning

mechanism. J. Neurosci. Res. 57, 830±839.

Zhu H., Guo Q. and Mattson M. P. (1999) Dietary restriction protects

hippocampal neurons against the death-promoting action of a

presenilin-1 mutation. Brain Res. 842, 224±229.

Zigova T., Pencea V., Wiegand S. J. and Luskin M. B. (1998)

Intraventricular administration of BDNF increases the number of

newly generated neurons in the adult olfactory bulb. Mol. Cell.

Neurosci. 11, 234±245.



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Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice