QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.110510v1 319/3/1172    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shankaran, M. Articles by Hellerstein, M. K. Search for Related Content PubMed PubMed Citation Articles by Shankaran, M. Articles by Hellerstein, M. K. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

NEUROPHARMACOLOGY

Discovery of Novel Hippocampal Neurogenic Agents by Using an in Vivo Stable Isotope Labeling Technique

KineMed, Inc., Emeryville, California (M.S., C.K., J.L., R.B., M.W.); and Department of Nutritional Sciences and Toxicology, University of California Berkeley, Berkeley, California (M.K.H.)

Received July 11, 2006; accepted September 12, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Neurogenesis occurs in discrete regions of adult mammalian brain, including the subgranular zone of the hippocampus. Hippocampal neurogenesis is enhanced by different classes of antidepressants, but screening for neurogenic actions of novel antidepressants has been inefficient because of limitations of 5-bromo-2'-deoxyuridine labeling techniques. We describe an efficient in vivo method for measuring hippocampal neurogenesis involving incorporation of the stable isotope, ²H, into genomic DNA during labeling with ²H₂O (heavy water). Male rodents received 8 to 10% ²H₂O in drinking water; DNA was isolated from hippocampal progenitor cells or neurons. Label incorporation into progenitor cells of Swiss-Webster mice revealed subpopulation kinetics: 16% divided with t_1/2 of 2.7 weeks; the remainder did not divide over 1 year. Progenitor cell proliferation rates in mice were strain-dependent. Chronic antidepressant treatment for 3 weeks, with ²H₂O administered during the final week, increased progenitor cell proliferation across all the strains tested. Fluoxetine treatment increased ²H incorporation into DNA of gradient-enriched neurons or flow-sorted neuronal nuclei 4 weeks after ²H₂O labeling, representing the survival and differentiation of newly divided cells into neurons. By screening 11 approved drugs for effects on progenitor cell proliferation, we detected previously unrecognized, dose-dependent enhancement of hippocampal progenitor cell proliferation by two statins and the anticonvulsant topiramate. We also confirmed stimulatory activity of other anticonvulsants and showed inhibition of progenitor cell proliferation by isotretinoin and prednisolone. In conclusion, stable isotope labeling is an efficient, high-throughput in vivo method for measuring hippocampal progenitor cell proliferation that can be used to screen for novel neurogenic drugs.

Hippocampal neurogenesis has emerged as a central therapeutic target for antidepressant agents based on accumulating evidence supporting the neurogenic theory of depression (Jacobs et al., 2000; Kempermann, 2002). All the known classes of clinical antidepressant drugs, including tricyclics, monoamine oxidase inhibitors, and selective serotonin reuptake inhibitors (SSRI), have been shown to increase cell proliferation in the hippocampus (Malberg et al., 2000; Santarelli et al., 2003). Moreover, Santarelli et al. (2003) provided evidence, by irradiation of the hippocampus, that hippocampal neurogenesis is required to achieve the behavioral effects of antidepressants in animal models. Hippocampal cell proliferation is decreased under conditions of chronic stress, and this effect is reversed by antidepressant treatment (reviewed by Dranovsky and Hen, 2006; Warner-Schmidt and Duman, 2006). Imaging studies have also shown that depressed human subjects exhibit volume loss and atrophy of the hippocampus (Sheline, 2003). In addition to a role in mood disorders, increased neurogenesis may be a repair mechanism in stroke (Lichtenwalner and Parent, 2006) and traumatic brain injury (Emery et al., 2005) and may also contribute to learning and memory (Shors et al., 2001).

A number of factors and intersecting pathways influence the drug-induced stimulation of neurogenesis in the hippocampus. For example, antidepressant drugs activate intracellular second messenger systems, leading to the activation of transcription factors and neurotrophic factors, finally culminating in increased numbers of new neurons in the hippocampus (Warner-Schmidt and Duman, 2006). The complexity of neurogenic regulation opens the possibility that multiple therapeutic targets may exist, on the one hand, but also makes predicting the effect of any agent acting on a particular target difficult and complicates the interpretation of in vitro screening approaches based on receptor binding or modulation of enzyme activities. Therefore, it is important to validate in vivo the neurogenic activity of agents identified through in vitro screens; moreover, in vivo screening may uncover novel mechanisms that contribute to neurogenesis arising from unanticipated connectivity relationships in the whole organism.

Measurement of neurogenesis in vivo has been problematic, however. Early studies used [³H]thymidine to label dividing cells (Altman and Das, 1965; Kaplan and Hinds, 1977), whereas the most commonly used method currently for measuring cell proliferation in the hippocampus involves injecting animals with 5-bromo-2'-deoxyuridine (BrdU) 24 h before sacrifice (Kuhn et al., 1996; Cameron and McKay, 2001). The hippocampus is then serially sectioned for immunohistochemical detection of BrdU, as well as double-immunohistochemical labeling with neuronal markers, to establish the phenotype of BrdU-positive cells. BrdU labeling has limitations, however, such as a short half-life and rapid clearance of BrdU from the brain, variable efficiency of BrdU entry into cellular precursor pools, and the requirement for high doses to accurately estimate the number of proliferating cells (Cameron and McKay, 2001; Gould and Gross, 2002). Moreover, immunohistochemical enumeration of BrdU-labeled cells in the entire hippocampus is labor-intensive, so that throughput with BrdU labeling is not sufficient for use in broad screening or testing of potential neurogenic agents.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Pathways that incorporate 2H into ribose during de novo purine nucleoside synthesis. Potential sites of 2H incorporation from 2H2O into deoxyribose moiety in replicating DNA are shown in color. PPP, pentose phosphate pathway; GNG, gluconeogenesis; DNPS, de novo purine synthesis pathway; DNNS, de novo nucleotide synthesis pathway, RR, ribonucleoside reductase, [3H]dT, tritiated thymidine, and BrdU, bromodeoxyuridine.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Animals. All of the animal studies were carried out within National Institutes of Health guidelines for the care and use of laboratory animals and received approval from the institutional animal use committee. Ten- to 12-week-old male C57Bl/6, Swiss-Webster, or BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA), and 10- to 12-week-old male 129SvEv mice were obtained from Taconic (Oxnard, CA). The outbred Swiss-Webster strain of mice was used for the initial drug-screening experiments. Subsequent experiments were performed in the inbred 129SvEv strain because these showed the least interanimal variability. Male Sprague-Dawley rats (250 g) were obtained from Charles River Laboratories. Animals were housed in a climate-controlled environment and fed standard rodent chow and water ad libitum.

²H₂O Labeling. For ²H₂O labeling, animals received a priming i.p. bolus of 49 ml/kg >99% ²H₂O (Spectra Stable Isotopes, Columbia, MD) containing 0.9% NaCl and were maintained on 10% ²H₂O in drinking water for the duration of the labeling period. In previous studies (reviewed in Jones and Leatherdale, 1991), intake of up to 20% ²H₂O has no apparent phenotypic or behavioral effects. For protocols involving label incorporation in hippocampal progenitor cells, mice were labeled continuously for up to 1 year. In a separate study, total hippocampal tissue was isolated after 3, 7, or 14 days of labeling. Animals that received drug treatment were labeled during the final 7 to 10 days of treatment. For studies that assessed proliferation of mature neurons, animals were labeled with 10% ²H₂O for 3 weeks, after which time the label was discontinued, and animals were sacrificed 4 weeks later.

Drug Treatments. Male rats and mice were treated with anti-depressants of different classes, such as an SSRI (fluoxetine), a tricyclic (imipramine), or a serotonin-norepinephrine reuptake inhibitor (venlafaxine), administered in drinking water. The drug solutions were dissolved at a concentration of 100, 200, and 100 mg/l for fluoxetine, imipramine, and venlafaxine, respectively. These concentrations were calculated to achieve a dose of 10 mg/kg/day for fluoxetine and venlafaxine and 20 mg/kg/day for imipramine, based on the average cage consumption of water (3 ml/30-g mouse or 25 ml/250-g rat). Treatment with antidepressant drugs was continued for 3 to 5 weeks.

Various approved drugs were screened for neurogenic activity (Table 1). These agents were selected based on long-standing use in humans and on the recognition of having several therapeutic actions (i.e., pleiotropic effects). This effort was intended to be a proof-of-concept study to show "indications discovery," i.e., ability to find unexpected actions of agents with potential use in a new indication. The doses selected were based on use in published preclinical studies with these agents, and the concentrations in food or water were calculated based on the average cage consumption of food (3 g/30-g mouse) and water (3 ml/30-g mouse). As a follow-up study after initial screening, a potential "class" effect for one of the agents (topiramate) tested in the initial screen was investigated by treating mice p.o. for 3 weeks with 1 of 11 other anticonvulsants: valproate (1 g/kg), clonazepam (3 mg/kg), gabapentin (100 mg/kg), carbamazepine (30 mg/kg), ethosuximide (300 mg/kg), levetiracetam (30 mg/kg), oxcarbazepine (100 mg/kg), phenytoin (100 mg/kg), primidone (100 mg/kg), tiagabine (30 mg/kg), or zonisamide (50 mg/kg).

Dose-response studies were also done for topiramate (10, 30, 100, and 150 mg/kg p.o.), atorvastatin (1, 3, 10, and 30 mg/kg in diet), and simvastatin (1, 3, 10, and 30 mg/kg p.o.). In addition, mice received chronic treatment with potential inhibitors of neurogenesis, such as isotretinoin (1 and 3 mg/kg i.p.) or prednisolone (5 and 40 mg/kg in diet).

Isolation of Hippocampal Progenitor Cells and Neurons. Animals were euthanized by CO₂ asphyxiation; brains were immediately removed; and the hippocampus was dissected out. Progenitor cells and neurons were isolated by a modification of methods described previously (Palmer et al., 1999). In brief, tissues were finely minced and digested in a solution of papain (4 U/ml) (Worthington Biochemical Corporation, Lakewood, NJ) and DNase (250 U/ml) (Roche Applied Science, Indianapolis, IN) dissolved in Hibernate-A (BrainBits LLC, Springfield, IL). The digested tissue was then mechanically triturated and thoroughly mixed with an equal volume of Percoll solution, made by mixing nine parts of Percoll (Amersham Biosciences, Piscataway, NJ) with one part 10x phosphate-buffered saline (PBS). The cell suspension was fractionated by centrifugation for 30 min, 18°C at 20,000g. Density beads were run in parallel, and the progenitor cells were fractionated between 1.064 and 1.075 g/ml, whereas neuronal cells fractionated at densities 1.035 g/ml. The progenitor and neuronal cell fractions were collected, washed free of Percoll, and stored frozen at –20°C until isolation of DNA. A flow chart of the method is shown in Fig. 1A.

Characterization of Progenitor Cells and Neurons by Flow Cytometry. The gradient-purified progenitor cells were immunofluorescently stained for intracellular markers, nestin and vimentin, and analyzed by flow cytometry. The cells were fixed and permeabilized with IntraCyte buffers (Orion Biosolutions, Vista, CA) and incubated overnight at 4°C with mouse antinestin (1:50, Rat401; BD Pharmingen, San Diego, CA) or mouse antivimentin (1:50; BD Pharmingen) primary antibodies. Gradient-purified hippocampal neurons were stained for tetanus toxin C fragment (TTX), a cell surface marker for neurons. The cells were fixed with 4% paraformaldehyde and incubated with TTX followed by anti-TTX mouse monoclonal antibody (Roche Applied Science). After washing, cells were incubated with Alexa 488-conjugated goat anti-mouse IgG secondary antibody (Molecular Probes, Eugene, OR). Propidium iodide (PI) was used to stain DNA, and Alexa 488 staining of PI-positive single cells (gated on plots of forward scatter peak area versus peak height) was analyzed on a Coulter EpicsXL cytometer (Beckman Coulter, Fullerton, CA).

Isolation of Nuclei from Mature Neurons and Sorting NeuN-Positive Nuclei by Flow Cytometry. Neuronal nuclei were isolated from frozen hippocampal tissue by a modification of a recently described method (Spalding et al., 2005). In brief, tissue was homogenized in 1 ml of lysis buffer (0.32 M sucrose, 5 mM CaCl₂, 3 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, and 1 mM DTT). Homogenized samples were gently suspended in 1.8 ml of sucrose solution (1.8 M sucrose, 3 mM magnesium acetate, 1 mM DTT, and 10 mM Tris-HCl, pH 8.0), layered onto a cushion of 1 ml of sucrose solution, and centrifuged at 30,000g for 2.5 h at 4°C. The isolated nuclei were resuspended in 1 ml of PBS and stored overnight at 4°C.

To identify neuronal nuclei, anti-NeuN antibodies were directly conjugated with Zenon mouse IgG labeling reagent (Alexa 488; Molecular Probes) by mixing 10 µl of Alexa 488 conjugate in 100 µl of blocking buffer (PBS/0.5% bovine serum albumin/10% normal goat serum) with 300 µl of NeuN antibody (1 mg/ml; diluted 1:250 in PBS) and incubating at room temperature for 5 min. The suspension of nuclei (1 ml) was added and incubated at 4°C for 1 h. The nuclei were then washed twice with 3 ml of PBS by centrifuging at 1000g for 10 min. PI was used to stain DNA, and neuronal nuclei were sorted as a homogeneous population of NeuN^bright cells using a Coulter Epics Elite sorter (Beckman Coulter) with gates set for PI-positive single nuclei.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Phenotype and proliferative characteristics of hippocampal progenitor cells. A, schematic illustration of Percoll gradient isolation method. B, flow cytometric analysis of fixed and permeabilized progenitor cells incubated with primary antibodies for nestin and vimentin, followed by Alexa 488 conjugated secondary antibody; PI was used to label nuclei. C, fractional synthesis of hippocampal progenitor cell DNA in male Swiss-Webster mice labeled for either 3 days, 1, 2, 4, 16, 28, or 52 weeks with 10% 2H2O. D, progenitor cell proliferation rate in male C57/Bl6, Swiss-Webster, BALB/c, or 129SvEv mice labeled with 10% 2H2O for 1 week. *, p < 0.05 for comparisons between C57Bl/6 and BALB/c, as well as Swiss-Webster and 129SvEv; **, p < 0.01 for comparison between C57Bl/6 and Swiss-Webster; ***, p < 0.001 for comparisons of 129SvEv with C57Bl/6 or BALB/c. Data represent mean ± S.E.M. of six animals per group.

The fraction of newly labeled cells was calculated as the ratio of excess ²H enrichment (EM1) in isolated cells to the corresponding enrichment in bone marrow DNA (an essentially fully turned-over tissue after 7–10 days of labeling, thereby representing an asymptotic enrichment value for comparison with other tissues) as described previously (Neese et al., 2002). For studies involving shorter labeling periods than 7 to 10 days, ²H incorporation in fully turned-over tissue DNA was estimated from body water ²H enrichments at sacrifice based on previously established relationships between body water ²H enrichments and asymptotic labeling in tissues (Neese et al., 2002).

Statistical Analysis. For label incorporation curves, the data were fit by nonlinear regression analysis (SigmaPlot). Student's t test was used with a 95% confidence interval for comparison between two groups. For comparison between multiple groups, one-way analysis of variance was used with a post-hoc Student-Newman-Keul test for all the pairwise multiple comparisons or Dunnett's test for comparisons with a negative control (SigmaStat). Data were considered significant at p < 0.05.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Flow Cytometric Analysis of Gradient-Enriched Hippocampal Progenitor Cells. Measurement of ²H incorporation into the DNA of hippocampal progenitor cells requires isolation of this cell population from intact tissue. To this end, progenitor cells were isolated from the hippocampus of Swiss-Webster mice by Percoll gradient fractionation (Fig. 2A); the isolated cells were fixed and permeabilized, stained for specific intracellular markers of progenitor cells (nestin and vimentin), and analyzed by flow cytometry after gating on nucleated (PI-stained) cells and excluding doublets. A majority of cells stained positively for either nestin (65% positive in Fig. 2B) or vimentin (72% positive) compared with isotype controls (2.1% positive). A broad distribution of fluorescence intensity was observed, and the cells remaining in the negative region of the dot plot appeared to be weakly stained.

Label Incorporation Kinetics in Progenitor Cells. To characterize the proliferation kinetics of gradient-enriched hippocampal progenitor cells, Swiss-Webster mice were labeled continuously with 10% ²H₂O in drinking water starting at 10 weeks of age; hippocampal progenitor cells were isolated after various labeling times, and ²H incorporation into DNA, analyzed by GC/MS, was used to determine the fraction of cells that had incorporated the label through cell division (Fig. 2C). Approximately 16% of cells incorporated label at plateau, with a half-life within this dividing population of approximately 2.7 weeks; the majority of cells continued to be unlabeled over the course of 1 year. This is consistent with a "kinetic subpopulation" pattern wherein a preponderance of nondividing precursors is present with a subset of actively dividing cells that enters and exits the pool (by differentiation or death). The baseline rate of progenitor proliferation was strain-dependent (Fig. 2D). The initial rates of labeling, measured after 1 week of ²H₂O intake, varied approximately 3-fold among four mouse strains tested and were significantly (p < 0.001) different from each other. The highest proliferation rate was observed in C57Bl/6 mice, whereas the 129SvEv mice had the lowest rate of proliferation.

The fraction of new cells in whole hippocampal tissue of C57Bl/6 mice after 3, 7, or 14 days of labeling was 0.7 ± 0.01, 1.3 ± 0.3, and 2.9 ± 0.6%, respectively. Thus, gradient isolation enriched for proliferating cells, and the consistency of the labeling results indicated that the cell isolation method was highly consistent in this regard.

Effects of Antidepressant Drugs on Progenitor Cell Proliferation in Rodent Hippocampus. Male Swiss-Webster mice (Fig. 3A) or Sprague-Dawley rats (Fig. 3B) were treated for 3 weeks with antidepressant drugs of different classes: an SSRI (fluoxetine, 10 mg/kg/day), a tricyclic (imipramine, 20 mg/kg/day), or a serotonin-norepinephrine reuptake inhibitor (venlafaxine, 10 mg/kg/day). After labeling with ²H₂O during the last week of treatment, antidepressant-treated animals from all the groups showed a significant increase in the progenitor cell proliferation rate in the hippocampus (Fig. 3, A and B). Both baseline proliferation and the magnitude of the drug effects were somewhat different between mice and rats, however. Fluoxetine treatment produced a significant (p < 0.01) increase in the hippocampal progenitor cell proliferation in C57Bl/6, 129SvEv, Swiss-Webster, and BALB/c mice (Fig. 3C). The percent stimulation of progenitor cell proliferation by fluoxetine was similar across strains.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Antidepressants increase hippocampal progenitor cell proliferation. A, fractional synthesis of hippocampal progenitor cell DNA in male Swiss-Webster mice treated with vehicle, fluoxetine (10 mg/kg/day), imipramine (20 mg/kg/day), or venlafaxine (10 mg/kg/day) in drinking water for 3 weeks and labeled with 10% 2H2O during the final week of treatment. *, p < 0.05 compared with "Vehicle"; data represent mean ± S.E.M. of eight mice per group. B, fractional synthesis of hippocampal progenitor cell DNA in male Sprague-Dawley rats treated with vehicle, fluoxetine (10 mg/kg/day), imipramine (20 mg/kg/day), or venlafaxine (10 mg/kg/day) in drinking water for 3 weeks and labeled with 10% 2H2O during the final week of treatment. *, p < 0.05 compared with "Vehicle"; data represent mean ± S.E.M. of six rats per group. C, fractional synthesis of hippocampal progenitor cell DNA in male C57Bl/6, Swiss-Webster, BALB/c, or 129SvEv mice treated with fluoxetine (10 mg/kg/day) in drinking water for 3 weeks and labeled with 10% 2H2O in the final week of treatment. **, p < 0.01 compared with "Vehicle" for each of the strain; data represent mean ± S.E.M. of six to eight mice per group.

Effects of Antidepressant Treatment on Survival and Differentiation of Newly Divided Cells. A "pulse-chase" protocol was used to assess the survival and differentiation of progenitor cells into neurons in vehicle- and fluoxetine-treated animals. Male 129SvEv mice were treated with fluoxetine (10 mg/kg/day) or vehicle for 5 weeks and labeled with 10% ²H₂O for the last 3 weeks of treatment. A longer labeling time was used in this protocol to allow greater numbers of newly divided progenitor cells to be labeled, allowing for some label loss as the result of death of labeled cells and differentiation into non-neuronal progeny. The antidepressant treatment and ²H₂O intake were then discontinued, and the animals were followed for another 4 weeks, after which time intact neurons or neuronal nuclei were isolated for analysis of DNA labeling. Figure 4A shows that >80% of gradient-enriched hippocampal neurons stained positively for TTX, a surface marker for neuronal cells. However, further purification of neurons by flow cytometric sorting proved difficult because of the large size range of neuronal cell bodies and large amount of axonal debris present in these gradient fractions (data not shown). Successful resolution of NeuN^bright and NeuN^dim nuclei obtained from frozen mouse brain was possible (Fig. 4B), allowing sorting of brightly stained nuclei of mature neurons to >98% purity on reanalysis (not shown). GC/MS analysis of extracted DNA from both preparations showed that fluoxetine treatment produced a significant increase in the fraction of ²H-labeled neurons 4 weeks after the end of a 3-week labeling period (Fig. 4, A and B). ²H incorporation into DNA of gradient-enriched neurons exceeded that of NeuN^bright neuronal nuclei; although Percoll gradients efficiently resolve proliferating neural progenitors from neuronal cells, the gradient-enriched population may include a greater proportion of immature neurons derived from dividing precursors that do not yet express high levels of NeuN. Taken together, these results confirm that differentiation of recently divided precursors into mature neurons is enhanced by fluoxetine and show that this antidepressant drug effect is detectable by ²H₂O labeling.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Flow cytometric analysis and label incorporation in neuronal DNA. Male 129SvEv mice were treated with fluoxetine (10 mg/kg/day) in drinking water for 5 weeks with 10% 2H2O labeling for the last 3 weeks of treatment and then sacrificed 4 weeks after the end of treatment and label. A, (left) hippocampal neuronal cells isolated by Percoll gradient fractionation were fixed and incubated with TTX and anti-TTX primary antibody, followed by Alexa 488 conjugated secondary antibody; PI was used to identify nuclei. Right, fractional synthesis of neuronal cell DNA from fluoxetine-treated animals was significantly (**, p < 0.01) different from vehicle-treated animals; data represent mean ± S.E.M. of eight mice per group. B, (left) hippocampal nuclei were isolated by ultracentrifugation, incubated with Alexa 488 conjugated anti-NeuN primary antibody and sorted after gating on PI+ve events. Right, fractional synthesis of neuronal nuclear DNA from fluoxetine-treated animals was significantly (***, p < 0.001) different from vehicle-treated animals; data represent mean ± S.E.M. of eight mice per group.

Effect of Retinoid or Glucocorticoid Administration on Progenitor Cell Proliferation. We also evaluated the activity of a retinoid and a glucocorticoid on hippocampal progenitor cell proliferation. Male 129SvEv mice were treated with isotretinoin (1 or 3 mg/kg i.p.) for 3 weeks and labeled with 10% ²H₂O in the final 10 days of treatment. There was a dose-dependent decrease in hippocampal progenitor cell proliferation following retinoid treatment (Fig. 5A). Treatment with a 3-mg/kg dose of isotretinoin produced a 38% reduction over baseline, which was statistically significant (p < 0.05) compared with vehicle-treated controls.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Isotretinoin and prednisolone decrease hippocampal progenitor cell proliferation. A, fractional synthesis of hippocampal progenitor cell DNA in male 129SvEv mice treated with either vehicle or isotretinoin (1 and 3 mg/kg/day i.p.) for 3 weeks and labeled with 10% 2H2O in the final week of treatment. *, p < 0.05 compared with "Vehicle"; data represent mean ± S.E.M. of six mice per group. B, fractional synthesis of hippocampal progenitor cell DNA in male Swiss-Webster mice treated with either vehicle or prednisolone (5 and 40 mg/kg/day) in diet and labeled with 10% 2H2O for 4 weeks. **, p < 0.01 compared with "Vehicle"; data represent mean ± S.E.M. of six mice per group.

Use of the Hippocampal Progenitor Cell Proliferation Assay to Screen Drugs. Next, we explored the feasibility of using hippocampal progenitor cell proliferation as a screen for candidate neurogenic agents. To this end, a panel of approved drugs with known or suspected pleiotropic actions (Table 1), but not previously known to have neurogenic activity in normal murine hippocampus, was tested for their effects on proliferation of hippocampal progenitor cells in Swiss-Webster mice (Fig. 6). Two of these agents were found to increase the rate of progenitor cell proliferation. The hydroxymethyl-glutaryl CoA reductase inhibitor (statin) atorvastatin (10 mg/kg p.o.) and the anticonvulsant topiramate (100 mg/kg in diet), each given daily for 3 weeks, significantly increased progenitor cell proliferation as measured by ²H₂O labeling during the 3rd week. The increases were of a similar magnitude as observed with the positive controls, fluoxetine and imipramine (Fig. 6). No stimulatory effects were seen for nine other drugs given by various routes (diet, drinking water, oral gavage, or i.p.) at daily doses reported in the literature to be pharmacologically active for other actions (Table 1).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Screening of pluripotent drugs for hippocampal progenitor cell proliferation. Fractional synthesis of hippocampal progenitor cell DNA in male Swiss-Webster mice treated for 3 weeks with various drugs (details are given in Table 1) and labeled with 10% 2H2O in the final week of treatment. *, p < 0.05 compared with "Vehicle"; data represent mean ± S.E.M. of six mice per group.

View larger version (28K): [in this window] [in a new window]   Fig. 7. A, dose response of topiramate for hippocampal progenitor cell proliferation. Male 129SvEv mice were treated with vehicle or topiramate (10, 30, 100, 150 mg/kg/day p.o.) for 3 weeks and labeled with 10% 2H2O in the final week of treatment. *, p < 0.05 compared with "Vehicle"; data represent mean ± S.E.M. of six mice per group. B, screening of anticonvulsants for hippocampal progenitor cell proliferation. Male Swiss-Webster mice were treated with different anticonvulsants (details given under Materials and Methods) for 3 weeks and labeled with 10% 2H2Ointhe final week of treatment. *, p < 0.05 compared with "Vehicle"; data represent mean ± S.E.M. of six mice per group.

View larger version (26K): [in this window] [in a new window]   Fig. 8. Dose response of statins for hippocampal progenitor cell proliferation. A, male 129SvEv mice were treated with atorvastatin (1, 3, 10, or 30 mg/kg/day) in diet for 3 weeks and labeled with 10% 2H2O in the final week of treatment. *, p < 0.05 compared with "Vehicle"; data represent mean ± S.E.M. of six mice per group. B, male 129SvEv mice were treated with simvastatin (1, 3, 10, or 30 mg/kg/day p.o.) for 3 weeks and labeled with 10% 2H2O in the final week of treatment. *, p < 0.05 compared with "Vehicle"; data represent mean ± S.E.M. of six mice per group.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Overall, our ²H₂O labeling results are consistent with measurements of hippocampal neurogenesis by BrdU labeling. Assuming that the hippocampus has 2 x 10⁶ total cells (Abusaad et al., 1999), the labeling rate of 0.2% per day in total C57Bl/6 hippocampus that we observed with ²H₂O would be equivalent to 4000 labeled cells/day, a value comparable with estimates by 12- to 24-h saturation labeling with BrdU (Hayes and Nowakowski, 2002). Palmer et al. (1999) reported that 0.7% of progenitor cells were labeled after daily BrdU injection for 6 days in a confocal analysis of BrdU incorporation into gradient-isolated progenitor cells from rat hippocampus, a similar value as we measured by ²H₂O labeling on similarly isolated rat hippocampal progenitors (1% per week). In mice, the hierarchy of baseline progenitor cell proliferation across different mouse strains in our study agreed well with strain effects on total hippocampal cell proliferation obtained by BrdU labeling (C57Bl/6 > BALB/c > Swiss-Webster > 129/Sv) (Kempermann et al., 1997; Hayes and Nowakowski, 2002) and further supports the view that genetic background strongly influences hippocampal neurogenesis. Interestingly, genetic differences in hippocampal neurogenesis have been shown to correlate with hippocampal function (Kempermann et al., 1998), and recently, using recombinant inbred strain of mice, Kempermann et al. (2006) have identified several genes that control adult neurogenesis. In terms of survival and differentiation of proliferated cells, 1% new NeuN^+ve neurons were observed in the hippocampus of 129SvEv mice 1 month after a 3-week ²H₂O labeling protocol, similar to the estimation of BrdU-labeled hippocampal granule cells in the closely related 129/SvJ strain of mice (Kempermann et al., 1997). Most importantly for drug studies, ²H₂O labeling qualitatively and quantitatively reproduced the known enhancing effects of antidepressants of various classes on hippocampal progenitor cell proliferation (Malberg et al., 2000; Santarelli et al., 2003), as well as the known inhibitory effect of isotretinoin on hippocampal progenitor cell proliferation (Crandall et al., 2004), and the proneurogenic activity of valproate (Hao et al., 2004). We conclude that ²H₂O labeling generates similar results and reveals the same neurogenic drug actions as BrdU labeling.

For screening purposes, we chose to analyze progenitor cells rather than mature neurons, even though labeling of both was increased by antidepressants. Progenitor cells comprise the majority of proliferating cells in the hippocampus, and sufficient ²H label for quantification is incorporated into DNA of gradient-enriched progenitors after only 7 to 10 days of ²H₂O exposure, whereas label detection in mature neurons takes an additional 4 weeks, and the new neurons are diluted into a much larger number of nondividing cells. Because standard antidepressants exhibit a lag period of approximately 10 days before stimulating neurogenesis in rodent hippocampus (Santarelli et al., 2003), we designed our screening experiments for 3 weeks of treatment, with ²H₂O labeling for the last 7 to 10 days. The lag between drug treatment and labeling can be shortened for detecting early onset of action of novel agents.

Using ²H₂O labeling, we were able to explore aspects of hippocampal cell dynamics that would not have been readily accessible by BrdU labeling. First, analysis of gradient-enriched hippocampal progenitors after up to 1 year of in vivo ²H₂O labeling revealed kinetic heterogeneity in this population: only 16% of these cells turned over during this time. This finding suggests that high-density hippocampal gradient fractions harbor a mixture of proliferating progenitors and resting cells. ²H₂O labeling may be valuable in searching for cellular markers associated with the proliferating progenitor cell subset.

Second, we were able to exploit the increased throughput of the ²H₂O progenitor cell proliferation assay to screen a sizable panel of approved drugs for previously unknown neurogenic effects in the hippocampus. Two of the 11 agents tested initially, atorvastatin and topiramate, increased progenitor cell proliferation, and these findings were robust in follow-up dose-response studies. Moreover, the proliferative activity of these agents appeared to be shared by other drugs of their class: a second statin, simvastatin, showed stimulatory activity, as did another 2 of 12 structurally diverse anticonvulsants tested, oxcarbazepine and valproate. This surprisingly high "hit" rate supports our notion, discussed in detail elsewhere (Turner and Hellerstein, 2005), that unanticipated cross-talk of drug actions on apparently off-target pathways in vivo might be more common than previously thought because of unappreciated connectivity relationships in complex metabolic networks. In the case of neurogenesis, this makes sense in view of the recognized complex regulation of neurogenesis through sensory input, neurotransmitters, hormones, and neurotrophic factors such as brain-derived neurotrophic factor, vascular endothelial growth factor, insulin growth factor (Warner-Schmidt and Duman, 2006), and several other known and unknown pathways.

The drug activities that we uncovered also seem plausible, after the fact, based on published literature. Atorvastatin has been reported to boost neurogenesis in the hippocampus and subventricular zone after middle cerebral artery occlusion, a model for stroke (Chen et al., 2003), but drug effects in uninjured brain were not measured in that study. Increased expression of vascular endothelial growth factor and brain-derived neurotrophic factor may mediate the neurogenic effect of atorvastatin in both stroke-induced (Chen et al., 2005) and in unmanipulated animals. Moreover, statins also alter expression of genes associated with apoptosis, cell growth, and signaling (Johnson-Anuna et al., 2005). The proliferation-stimulating activities of oxcarbazepine and topiramate are plausible, given published results with valproate, although these results would have been difficult to predict because many anticonvulsants did not increase progenitor cell proliferation. Although the exact mechanism of neurogenic activation by some anticonvulsants remains unknown, these drugs influence several neurotransmitter systems, as well as intracellular signaling cascades, such as the extracellular signal regulated kinase pathway, which has been shown to be activated by the neurogenic anticonvulsant valproate (Hao et al., 2004). The inhibition of hippocampal progenitor cell proliferation by the synthetic corticosteroid analog, prednisolone, confirms the suppressive effect of glucocorticoids because similar effects have been reported for corticosterone and dexamethasone (Cameron and Gould, 1994; Kim et al., 2004).

Intriguingly, the novel proneurogenic and antineurogenic drug effects that we discovered closely parallel the known behavioral effects of these drugs. Retinoid therapy may cause depression in humans as a side effect of treatment (Hull and D'Arcy, 2003). Likewise, conditions exhibiting hypercortisolism (Cushing's syndrome, stress, glucocorticoid therapy) are also associated with clinical depression (Mitchell and O'Keane, 1998). Conversely, anticonvulsants have been used as augmentation therapy in depression (Hantouche et al., 2005); differential neurogenic activity may guide the choice of anticonvulsants for this therapeutic use. Moreover, there is increasing evidence that statins may have utility in the treatment of neurological diseases (Menge et al., 2005). Long-term use of statins has been shown to be associated with reduced risk of anxiety, depression, and hostility (Young-Xu et al., 2003). In a trial for Alzheimer's disease, patients treated with atorvastatin for 1 year showed significant improvement of depression (Sparks et al., 2005). Together with our observations of progenitor cell stimulatory activity of these drugs, these findings provide further support for the neurogenic theory of depression and indicate that in vivo screens for neurogenesis may be useful in the discovery and preclinical validation of novel antidepressants.

Finally, hippocampal neurogenesis is involved in other biological processes besides depression. Because neurogenesis plays a significant role in synaptic plasticity, disorders of learning and memory may be amenable to discovery efforts using this as a biomarker. Neurogenesis may also be a therapeutic target for other conditions, such as traumatic brain injury, stroke, and Alzheimer's disease.

In conclusion, ²H₂O labeling represents a quantitative, reproducible, and relatively high-throughput in vivo method for measuring hippocampal progenitor cell proliferation, which is closely linked to neurogenesis and is useful for screening and discovering novel neurogenic stimulatory drugs. Statins and certain anticonvulsant agents were discovered to have potent stimulating activity and may have therapeutic uses based on this activity.

	Acknowledgements

 
We thank Michael Swenson, Kerstin Kiefer, Michelle Goldfinger, Chancy Fessler, and Holly Turner for technical assistance.

	Footnotes

 
This study was supported in part by KineMed, Inc. and University of California-Industry Discovery Program (BioStar Grant No. Bio04-10445 to M.K.H.).

Part of this work was presented at the Annual Society of Neuroscience Meeting: Shankaran M, King C, Busch R, Gee T, Keifer K, Mahsut A, and Hellerstein M (2005) In vivo heavy water (²H₂O) labeling: a novel method for measuring hippocampal cell proliferation and neurogenesis. Program 1024.9, in 2005 Abstract Viewer/Itinerary Planner; 2005 Nov 12–16; Washington, DC. Online, Society for Neuroscience, Washington, DC.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.110510.

ABBREVIATIONS: SSRI, selective serotonin reuptake inhibitor(s); BrdU, 5-bromo-2'-deoxyuridine; PBS, phosphate-buffered saline; TTX, tetanus toxin C fragment; PI, propidium iodide; GC/MS, gas chromatography/mass spectrometry.

Address correspondence to: Dr. Mahalakshmi Shankaran, KineMed, Inc., 5980 Horton Street, Suite 400, Emeryville, CA 94608. E-mail: mshankaran{at}kinemed.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Abusaad I, MacKay D, Zhao J, Stanford P, Collier DA, and Everall IP (1999) Stereological estimation of the total number of neurons in the murine hippocampus using the optical dissector. J Comp Neurol 408: 560–566.[CrossRef][Medline]

Altman J and Das GD (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124: 319–335.[CrossRef][Medline]

Busch R, Cesar D, Higuera-Alhino D, Gee T, Hellerstein MK, and McCune JM (2004) Isolation of peripheral blood CD4(+) T cells using RosetteSep and MACS for studies of DNA turnover by deuterium labeling. J Immunol Methods 286: 97–109.[CrossRef][Medline]

Cameron HA and Gould E (1994) Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neuroscience 61: 203–209.[CrossRef][Medline]

Cameron HA and McKay RD (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435: 406–417.[CrossRef][Medline]

Chen J, Zhang C, Jiang H, Li Y, Zhang L, Robin A, Katakowski M, Lu M, and Chopp M (2005) Atorvastatin induction of VEGF and BDNF promotes brain plasticity after stroke in mice. J Cereb Blood Flow Metab 25: 281–290.[CrossRef][Medline]

Chen J, Zhang ZG, Li Y, Wang Y, Wang L, Jiang H, Zhang C, Lu M, Katakowski M, Feldkamp CS, et al. (2003) Statins induce angiogenesis, neurogenesis, and synaptogenesis after stroke. Ann Neurol 53: 743–751.[CrossRef][Medline]

Crandall J, Sakai Y, Zhang J, Koul O, Mineur Y, Crusio WE, and McCaffery P (2004) 13-cis-retinoic acid suppresses hippocampal cell division and hippocampal-dependent learning in mice. Proc Natl Acad Sci USA 101: 5111–5116.[Abstract/Free Full Text]

Dranovsky A and Hen R (2006) Hippocampal neurogenesis: regulation by stress and antidepressants. Biol Psychiatry 59: 1136–1143.[CrossRef][Medline]

Emery DL, Fulp CT, Saatman KE, Schutz C, Neugebauer E, and McIntosh TK (2005) Newly born granule cells in the dentate gyrus rapidly extend axons into the hippocampal CA3 region following experimental brain injury. J Neurotrauma 22: 978–988.[CrossRef][Medline]

Gage FH, Kempermann G, Palmer TD, Peterson DA, and Ray J (1998) Multipotent progenitor cells in the adult dentate gyrus. J Neurobiol 36: 249–266.[CrossRef][Medline]

Gould E and Gross CG (2002) Neurogenesis in adult mammals: some progress and problems. J Neurosci 22: 619–623.[Free Full Text]

Hantouche EG, Akiskal HS, Lancrenon S, and Chatenet-Duchene L (2005) Mood stabilizer augmentation in apparently "unipolar" MDD: predictors of response in the naturalistic French national EPIDEP study. J Affect Disord 84: 243–249.[CrossRef][Medline]

Hao Y, Crecon T, Zhang L, Li P, Du F, Yuan P, Gould TD, Manji HK, and Chen G (2004) Mood stabilizer valproate promotes ERK pathway-dependent cortical neuronal growth and neurogenesis. J Neurosci 24: 6590–6599.[Abstract/Free Full Text]

Hayes NL and Nowakowski RS (2002) Dynamics of cell proliferation in the adult dentate gyrus of two inbred strains of mice. Brain Res Dev Brain Res 134: 77–85.[CrossRef][Medline]

Hull PR and D'Arcy C (2003) Isotretinoin use and subsequent depression and suicide: presenting the evidence. Am J Clin Dermatol 4: 493–505.[CrossRef][Medline]

Jacobs BL, Praag H, and Gage FH (2000) Adult brain neurogenesis and psychiatry: a novel theory of depression. Mol Psychiatry 5: 262–269.[CrossRef][Medline]

Johnson-Anuna LN, Eckert GP, Keller JH, Igbavboa U, Franke C, Fechner T, Schubert-Zsilavecz M, Karas M, Muller WE, and Wood WG (2005) Chronic administration of statins alters multiple gene expression patterns in mouse cerebral cortex. J Pharmacol Exp Ther 312: 786–793.[Abstract/Free Full Text]

Jones PJ and Leatherdale ST (1991) Stable isotopes in clinical research: safety reaffirmed. Clin Sci (Lond) 80: 277–280.[Medline]

Kaplan MS and Hinds JW (1977) Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs. Science (Wash DC) 197: 1092–1094.[Abstract/Free Full Text]

Kempermann G (2002) Regulation of adult hippocampal neurogenesis—implications for novel theories of major depression. Bipolar Disord 4: 17–33.[CrossRef][Medline]

Kempermann G, Brandon EP, and Gage FH (1998) Environmental stimulation of 129/SvJ mice causes increased cell proliferation and neurogenesis in the adult dentate gyrus. Curr Biol 8: 939–942.[CrossRef][Medline]

Kempermann G, Chesler EJ, Lu L, Williams RW, and Gage FH (2006) Natural variation and genetic covariance in adult hippocampal neurogenesis. Proc Natl Acad Sci USA 103: 780–785.[Abstract/Free Full Text]

Kempermann G, Kuhn HG, and Gage FH (1997) Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad Sci USA 94: 10409–10414.[Abstract/Free Full Text]

Kim JB, Ju JY, Kim JH, Kim TY, Yang BH, Lee YS, and Son H (2004) Dexamethasone inhibits proliferation of adult hippocampal neurogenesis in vivo and in vitro. Brain Res 1027: 1–10.[CrossRef][Medline]

Kim SJ, Turner S, Killion S, and Hellerstein MK (2005) In vivo measurement of DNA synthesis rates of colon epithelial cells in carcinogenesis. Biochem Biophys Res Commun 331: 203–209.[CrossRef][Medline]

Kuhn HG, Dickinson-Anson H, and Gage FH (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 16: 2027–2033.[Abstract/Free Full Text]

Lichtenwalner RJ and Parent JM (2006) Adult neurogenesis and the ischemic forebrain. J Cereb Blood Flow Metab 26: 1–20.[CrossRef][Medline]

Malberg JE, Eisch AJ, Nestler EJ, and Duman RS (2000) Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci 20: 9104–9110.[Abstract/Free Full Text]

Menge T, Hartung HP, and Stuve O (2005) Statins—a cure-all for the brain? Nat Rev Neurosci 6: 325–331.[CrossRef][Medline]

Mitchell A and O'Keane V (1998) Steroids and depression. BMJ 316: 244–245.[Free Full Text]

Neese RA, Misell LM, Turner S, Chu A, Kim J, Cesar D, Hoh R, Antelo F, Strawford A, McCune JM, et al. (2002) Measurement in vivo of proliferation rates of slow turnover cells by ²H₂O labeling of the deoxyribose moiety of DNA. Proc Natl Acad Sci USA 99: 15345–15350.[Abstract/Free Full Text]

Palmer TD, Markakis EA, Willhoite AR, Safar F, and Gage FH (1999) Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 19: 8487–8497.[Abstract/Free Full Text]

Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, Weisstaub N, Lee J, Duman R, Arancio O, et al. (2003) Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science (Wash DC) 301: 805–809.[Abstract/Free Full Text]

Sheline YI (2003) Neuroimaging studies of mood disorder effects on the brain. Biol Psychiatry 54: 338–352.[CrossRef][Medline]

Shors TJ, 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 (Lond) 410: 372–376.[CrossRef][Medline]

Spalding KL, Bhardwaj RD, Buchholz BA, Druid H, and Frisen J (2005) Retrospective birth dating of cells in humans. Cell 122: 133–143.[CrossRef][Medline]

Sparks DL, Sabbagh MN, Connor DJ, Lopez J, Launer LJ, Browne P, Wasser D, Johnson-Traver S, Lochhead J, and Ziolwolski C (2005) Atorvastatin for the treatment of mild to moderate Alzheimer disease: preliminary results. Arch Neurol 62: 753–757.[Abstract/Free Full Text]

Turner SM and Hellerstein MK (2005) Emerging applications of kinetic biomarkers in preclinical and clinical drug development. Curr Opin Drug Discov Dev 8: 115–126.[Medline]

van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, and Gage FH (2002) Functional neurogenesis in the adult hippocampus. Nature (Lond) 415: 1030–1034.[CrossRef][Medline]

Warner-Schmidt JL and Duman RS (2006) Hippocampal neurogenesis: opposing effects of stress and antidepressant treatment. Hippocampus 16: 239–249.[CrossRef][Medline]

Young-Xu Y, Chan KA, Liao JK, Ravid S, and Blatt CM (2003) Long-term statin use and psychological well-being. J Am Coll Cardiol 42: 690–697.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. P. Keller, Y. Choi, P. Wang, D. Belt Davis, M. E. Rabaglia, A. T. Oler, D. S. Stapleton, C. Argmann, K. L. Schueler, S. Edwards, et al. A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility Genome Res., May 1, 2008; 18(5): 706 - 716. [Abstract] [Full Text] [PDF]

				M. K. Hellerstein Exploiting Complexity and the Robustness of Network Architecture for Drug Discovery J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 1 - 9. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.110510v1 319/3/1172    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shankaran, M.