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Keywords:

  • apoptosis;
  • caloric restriction;
  • dentate gyrus;
  • neurotrophic factor;
  • stem cells

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

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 maintained 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 enhancement of neurogenesis induced by DR.

Abbreviations used
AL

ad libitum

BDNF

brain-derived neurotrophic 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.

The adult mammalian brain contains small populations of neural stem cells that are capable of dividing and differentiating 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 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 environmental factors on neurogenesis are mediated by specific cellular signaling pathways.

The bulk of data concerning signals that control neurogenesis 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 neurological 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

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Mice, diets and BrdU administration

Two-month-old male BDNF +/– mice and wild-type control littermates were obtained from in-house breeding colonies originating 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 cryoprotected brains were sectioned serially at 50 µm 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 (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 further processed 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 (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 (5× objective; on-screen magnification = 138×); identification of BrdU-positive cells was accomplished at high power (63× objective; on-screen magnification = 1714×). The dimension of the sampling frames was 49.2 µm in length by 49.2 µm in width and 14 µm in depth. The guard height for each section was 1 µm. 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 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 further processed 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 µm coronal brain sections as follows. After DNA denaturation, 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 rat monoclonal 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 µg/mL aprotinin, 1 µg/mL leupeptin and 0.5 mm sodium orthovanadate; pH 7.2) at 4°C. Homogenates were centrifuged at 2000 g for 20 min (4°C), and the supernatant 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 neutralization. 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 µL 1 m phosphoric acid, and absorbance was measured at 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

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

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 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.

image

Figure 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).

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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 miceBDNF +/– mice DFF-value
ALDRALDR
  1. 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 = 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.

Proliferation, 1 day3533 ± 177.63226 ± 105.82789 ± 189.2a3146 ± 264.63 3.230
Survival, 4 week 967 ± 84.71496 ± 80.9d 626 ± 48.0ab1028 ± 88.8c320.287
Survival (%), 4 week  27 ± 2.4  46 ± 2.5d  22 ± 1.7b  33 ± 2.8c318.768
Regional volume (mm3), 1 day0.174 ± 0.0130.175 ± 0.0100.148 ± 0.0050.162 ± 0.0103 1.398
Regional volume (mm3), 4 week0.201 ± 0.0110.190 ± 0.0110.147 ± 0.008ab0.175 ± 0.0163 3.731

At the 4-week post-BrdU time point there were significantly 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 immunohistochemical 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 cytoplasmic 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 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 immunoreactivity 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 = 6 mice 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.

image

Figure 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.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

The hippocampal subgranular zone is a region where new neurons and glia are generated in the adult brain; neurogenesis 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 hippocampal 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 synaptic 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 immunoreactive 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 differentiation into neurons and long-term survival. We conclude that BDNF mediates, at least in part, the enhancement 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 neuronal 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 hippocampal plasticity and resistance to age-related neuronal degeneration. 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 transplantation 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

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

We thank L. Tessarollo for providing initial breeding pairs of BDNF +/– mice.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
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