Journal of Neurochemistry, 2002, 80, 539±547
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 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,
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 539±547
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.
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: mattsonm@grc.nia.nih.gov
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.
539
540 J. Lee et al.
(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 ÔantiagingÕ
effects 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
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 539±547
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 ˆ 271 ´); identi®-
cation of BrdU-positive cells was accomplished at high power
(63 ´ objective; on-screen magni®cation ˆ 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.

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 ˆ 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)
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 539±547
Dietary restriction, neurotrophins and neurogenesis 541
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 ˆ 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
542 J. Lee et al.
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 ˆ 6 per group). All data presented as means ‹ standard error.
*Signi®cantly different from ad libitum group (p < 0.02).
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
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 539±547
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.
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
 Dietary restriction, neurotrophins and neurogenesis 543

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 Fig. 4 Dietary restriction increases BDNF mRNA levels in CA1-3
after BrdU administration (a and b). Four weeks after BrdU adminis- pyramidal cells and NT-3 levels in dentate gyrus granule cells.
tration, the majority of cells labeled with BrdU were also immunore- Densitometric analyses of hybridization levels of BDNF, NT-3, trkB
active with the NeuN antibody (arrows in c and e). The neuronal and trkC mRNAs were performed on autoradiograms of hippocampal
phenotype of newborn cells on 4 weeks after BrdU administration was brain sections from mice that had been maintained for 3 months on AL
con®rmed by immuno¯uorescence using antibodies to another neu- or DR diets. Values are the mean and SE of determinations made in
ronal marker MAP2, which labeled cell bodies and dendrites of granule six mice per group. *p < 0.02, **p < 0.01 compared to corresponding
neurons (arrows in g and h). value for mice fed AL [ANOVA with Fisher's protected least signi®cant
difference procedure (PLSD)].

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

campal and cortical tissue from AL and DR mice (Fig. 5a). of DR mice compared to AL mice (Fig. 5b). Levels of full-
The ratio of full-length trkB to truncated trkB was signi®- length and truncated trkB in the cerebral cortex were not
cantly increased by approximately 25% in the hippocampus different in AL and DR mice.

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

544 J. Lee et al.
(a) CORTEX
HIPPOCAMPUS
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
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
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 539±547
(b)
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).
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

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
Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 539±547
Dietary restriction, neurotrophins and neurogenesis 545
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).
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