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Developmental exposure to 3,4-methylenedioxymethamphetamine results in downregulation of neurogenesis in the adult mouse hippocampus
Neuroscience 154 (2008) 1034 –1041
DEVELOPMENTAL EXPOSURE TO 3,4-METHYLENEDIOXYMETHAMPHETAMINE RESULTS IN DOWNREGULATION OF NEUROGENESIS IN THE ADULT MOUSE HIPPOCAMPUS K.-O. CHO,a,b G. S. RHEE,c S. J. KWACK,c S. Y. CHUNGc AND S. Y. KIMa,b*
during dance parties called ‘raves’ (Arria et al., 2002). MDMA provides abusers with a sense of closeness to other people and sexual arousal, which can lead to inadvertent pregnancy (Cohen, 1995). Despite the particularly high prevalence of MDMA use among young adults who are of childbearing age (Maxwell, 2003), little is known about the consequences of such use in their offspring. MDMA is known as a 5-HT releaser and its repeated use can cause depletion of monoamines such as 5-HT and dopamine (Aguirre et al., 1997; O’Shea et al., 2001), leading to neurotoxicity in the monoamine system (Sprague et al., 2003) and cognitive deficits (McCann et al., 1999; Sprague et al., 2003). Furthermore, neonatal exposure to MDMA can markedly depress 5-HT and dopamine levels and impair spatial learning and memory function, even when examined at the adult stage (Broening et al., 2001; Crawford et al., 2006). In addition, neonatal MDMA injections appear to promote apoptotic cell death and decreased serotonergic fiber density (Meyer et al., 2004). Therefore, neonatal exposure to MDMA seems to induce long-lasting perturbation of the monoamine system in adults. In the hippocampus, new cells are constantly generated in the subgranular zone of the hippocampal dentate gyrus and differentiate into mature granule neurons (Palmer et al., 2000). Subsequently, these newly generated neurons integrate into existing hippocampal circuits and find appropriate targets (Markakis and Gage, 1999). Among a number of factors, neurotransmitters including 5-HT and dopamine have been shown to play a role in the regulation of hippocampal neurogenesis. A selective 5-HT reuptake inhibitor such as fluoxetine has a positive influence on the proliferation of progenitor cells which contributes to improved memory function, consolidating the stimulating effects of 5-HT on granule cell genesis (Gould, 1999; Malberg et al., 2000; Mowla et al., 2007). In parallel, 5-HT depletion reduced hippocampal neurogenesis (Brezun and Daszuta, 1999). In addition, dopamine depletion led to a decrease of the proliferative activity in the subgranular zone, though the precise effects of dopamine on neurogenesis are still controversial (Hoglinger et al., 2004; Winner et al., 2006). In the present study, we asked whether chronic developmental exposure to MDMA results in any alterations in adult hippocampal neurogenesis. Since the lactational period (postnatal days (PDs) 1–21) in rodents matches the second and third trimester in humans (Clancy et al., 2001),
a
Department of Pharmacology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul, 137-701, Korea
b
Cell Death Disease Research Center, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Korea
c
Reproductive and Developmental Toxicology, National Institute of Toxicological Research, 194 Tongilro, Eunpyeong-gu, Seoul, 122-704, Korea
Abstract—3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a powerful releaser of 5-HT and chronic use of this drug can cause depletion of monoamines. Recently, concerns about the risk of adult brain damage due to fetal exposure to MDMA have been raised. We investigated whether developmental MDMA exposure affected adult neurogenesis in C57 black/6 mice. MDMA (1.25 or 20 mg/kg, p.o.) or vehicle was administered daily to the mother from prenatal 6th day to postnatal 21st day. When the offspring were 11 weeks old, they were injected with 5-bromo-2=-deoxyuridine (BrdU) (120 mg/kg, i.p.) once a day for 4 days. After 24 h or 28 days, the animals were killed to count the BrdU-positive cells in the dentate gyrus. At 24 h after the last BrdU injection, the number of BrdU-positive cells in the offspring developmentally exposed to MDMA was significantly lower than that of the control group. At 28 days post-BrdU labeling, BrdU-positive cells in the dentate gyrus of female offspring with developmental exposure to high dose MDMA were significantly fewer compared with the control group. In addition, most BrdUpositive cells were co-labeled with the mature neuronal marker, neuronal nuclei, while a few BrdU-labeled cells were merged with an astrocyte marker. Our results suggest that developmental exposure to MDMA can result in decreases in the proliferation and survival of mature newborn cells in the adult dentate gyrus. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ecstasy, adult neurogenesis, BrdU, dentate gyrus, hippocampus, abuse.
3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is an amphetamine-like stimulant that is widely abused *Correspondence to: S. Y. Kim, Department of Pharmacology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul, 137-701, Korea. Tel: ⫹82-2-590-1205; fax: ⫹82-2536-2485. E-mail address: [email protected] (S. Y. Kim). Abbreviations: ANOVA, analysis of variance; BrdU, 5-bromo-2=-deoxyuridine; C57BL/6, C57 black/6; FDA, Food and Drug Administration; GFAP, glial fibrillary acidic protein; MDMA, 3,4-methylenedioxymethamphetamine; NeuN, neuronal nuclei; PDs, postnatal days.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.04.040
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MDMA was administered to the mother during the gestational and lactational period. Using 5-bromo-2=-deoxyuridine (BrdU) as a marker of cell division, we examined the difference between controls and developmentally MDMAexposed mice in terms of the rate of cell proliferation and the number of mature newborn cells in the adult dentate gyrus.
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4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed and post-fixed for 4 h in the same fixative and, then, embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan). For the detection of BrdU immunoreactivity, DNA denaturation was conducted by incubation in 50% formamide and 2⫻ sodium citrate solution for 2 h at 65 °C. Subsequently, sections (20 m thick) were incubated for 1 h in 2 N HCl at 37 °C and then for 10 min in boric acid. The sections were then incubated with anti-mouse BrdU (1:100; DAKO, Glostrup, Denmark) overnight at 4 °C. After that, the sections were put into solutions containing secondary antibody (biotinylated horse anti-mouse; Vector Laboratories, Burlingame, CA, USA) for 2 h, followed by amplification with an avidin– biotin complex (Vector Laboratories) for 1 h. Between each step, washing with 0.01 M phosphate-buffered saline was carried out for 15 min. Finally, the brain sections were visualized with 0.05% 3,3=-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide, and were later counterstained with hematoxylin. After hydration, the slides were incubated with hematoxylin solution for 2 min. Then, excessive hematoxylin was washed by submerging the slides in 1% HCl, which is followed by rinsing with distilled water and dehydrating with xylene. For the double labeling experiments, the sections were incubated with anti-rat BrdU (1:200; Accurate, Westbury, NY, USA) overnight at 4 °C, then incubated with carbocyanine 3– conjugated goat anti-rat IgG (1:500; Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. After rinsing with 0.1 M phosphate buffer (pH 7.4), the sections were incubated with antimouse neuronal nuclei (NeuN) (1:400; Chemicon International, Temecula, CA, USA) or glial fibrillary acidic protein (GFAP) (1: 400; Chemicon International) overnight at 4 °C. The next day, fluorescein isothiocyanate– conjugated goat anti-mouse IgG (1: 25; Jackson ImmunoResearch) was then applied for 2 h at room temperature and the sections were observed using a confocal microscope (MRC-1024; Bio-Rad Laboratories, Hercules, CA, USA) after mounting with prolong gold antifade reagents (Invitrogen, Carlsbad, CA, USA). The images were generated using Image-Pro Plus software (MediaCybernetics, Silver Spring, MD, USA) and they were not modified other than relative adjustments of levels, brightness, and contrast.
EXPERIMENTAL PROCEDURES Animals and MDMA schedule All animal procedures were approved by the Ethics Committee of the Catholic University of Korea and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Efforts were made to try to minimize animal suffering and to reduce the number of animals used. Male and female C57 black/6 (C57BL/6) mice (Orientbio, Kyungki-do, Korea) weighing 22–25 g were housed three per cage at a standard temperature (22⫾1 °C) and in a light-controlled environment (lights on from 8:00 a.m. to 8:00 p.m.) with ad libitum access to food and water. They were mated in a separate cage and recognized as pregnant on the day when vaginal plugs were detected. The day mice gave birth was designated as PD 0. Eighteen female C57BL/6 mice received either ⫾3,4-MDMA HCl (Sigma, St. Louis, MO, USA) dissolved in saline or saline once a day from day 6 of pregnancy to day 21 after delivery, according to the U.S. Food and Drug Administration (U.S. FDA) guideline for detection of toxicity to reproduction of medicinal products (FDA, 1994). MDMA (1.25 mg/kg, 20 mg/kg) or saline was administered to the mother by oral gavage (12 mL/kg). The dose of MDMA was selected based on our previous study showing the effects of chronic MDMA on cell proliferation in the adult dentate gyrus (Cho et al., 2007). After delivery, maternal behavior was observed daily in the cage of each mother and her litter between PD 1 and PD 28. On PD 28, they were housed in groups by sex.
Schedule of BrdU injections When offspring were 11 weeks old, five or six mice per group were allocated. After measuring the body weight, BrdU (120 mg/10 mL/ kg, i.p.) dissolved in 0.007 M NaOH solution was injected into these mice once a day for 4 days in order to label DNA of cells in the S-phase of mitosis and follow the labeled cells. For evaluation of proliferative activities, 18 female and 15 male mice born to 18 different dams were killed using 15% chloral hydrate at 24 h after the last BrdU injection. For assessment of the survival of precursor cells, 15 female mice were cared for in six cages until they were killed using 15% chloral hydrate at 28 days after the last BrdU administration.
Cell counting and statistics Quantification was done blind to the experimental treatments. Every fifth section (i.e. a total of three) was processed for BrdU labeling. The area investigated in the present study was ⫺1.5 to ⫺1.7 mm from the bregma according to the atlas of Paxinos and Franklin (2001). For the cell proliferation study, BrdU-labeled cells in the subgranular zone of the dentate gyrus were counted bilaterally in each section using a light microscope (BX51; Olympus, Tokyo, Japan) equipped with a charge-coupled device camera (DP70; Olympus). The BrdU-immunoreactive cells in a cluster were differentially counted at 400⫻ magnification by adjusting the focus. BrdU-positive cells in the dentate gyrus used to evaluate the survival of newborn cells were also counted in the same way. Only cells located in the subgranular zone or the granule cell layer
Immunohistochemistry Animals (n⫽5– 6 from each group) were transcardially perfused with 30 mL of saline, followed by 150 mL of a fixative containing
Table 1. Physical parameters of dams and litter developmentally exposed to MDMA Dose of MDMA (mg/kg)
Maternal weight at GD 18 (g)
No. of pups per litter
M:F ratio per litter
0.00 1.25 20.00
35.49⫾1.78 33.82⫾1.31 32.93⫾0.69
8.33⫾0.67 8.50⫾0.43 8.67⫾1.09
1.43⫾0.40 1.39⫾0.43 1.07⫾0.29
Neonatal weight at PD 4 (g)
Postnatal weight at PD 21 (g)
Postnatal weight at PD 77 (g)
Male
Female
Male
Female
Male
Female
2.36⫾0.08 2.36⫾0.08 2.40⫾0.05
2.48⫾0.10 2.38⫾0.10 2.22⫾0.09
8.36⫾0.25 8.70⫾0.29 8.47⫾0.09
8.97⫾0.28 8.26⫾0.38 7.99⫾0.17
23.77⫾0.49 23.82⫾0.61 23.78⫾0.26
20.54⫾0.41 21.69⫾0.22 21.83⫾0.59
Values are means⫾S.E.M. of six dams, 11 female or five male litters per group. There was no statistical significance among MDMA-administered groups and control. GD, gestational day; M, male; F, female.
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K.-O. Cho et al. / Neuroscience 154 (2008) 1034 –1041 tions of treatment⫻sex; accordingly, results from males and females are presented together. Cell survival and the body weight were evaluated by one-way ANOVA and post hoc Scheffe’s test. Differences were assumed to be statistically significant when P⬍0.05.
RESULTS Developmental exposure to MDMA had no influence on the physical parameters of the dams and offspring
Fig. 1. Schematic representation of MDMA and BrdU administration. MDMA (1.25 or 20 mg/kg) or saline was administered to dams orally once a day from the 6th day after pregnancy (E6) to the 21st day after delivery (P21). When female and male offspring were 11 weeks old, BrdU (120 mg/kg) was injected intraperitoneally once a day for 4 days. For female mice, 1 day or 28 days after the last BrdU injection (0d), the animals were killed to evaluate cell proliferation or survival of mature newborn cells, respectively. For male mice, 1 day after the last BrdU injection (0d), the animals were killed to evaluate cell proliferation. (A) MDMA- or saline-administered dams, (B) male offspring born of MDMAor saline-administered dams. (C) Female offspring born of MDMA- or saline-administered dams.
were counted, and cells located more than two cells away from the granule cell layer were classified as being in the hilus. Numbers of BrdU-positive cells and the body weight were expressed as means⫾S.E.M. Since we selected one male and one female from each dam, cell proliferation was evaluated by two-way analysis of variance (ANOVA) which did not identify any significant interac-
Chronic developmental exposure to MDMA by our regimen did not produce any significant alterations in maternal care of pups or alterations in physical parameters of the dams and offspring (Table 1). In an observational test during the lactational period, there were no significant differences in nursing, grooming or carrying pups among MDMA-administered dams and controls. Moreover, our preliminary study showed that the fetal survival rate was not different between vehicle- and 20 mg/kg MDMA-administrated groups (data not shown). With regard to the physical data, maternal MDMA administration did not alter the body weight of the dams or the litter. In addition, there was no significant difference in the litter compositions or the number of pups per litter among controls and MDMA-administered groups. Developmental MDMA exposure decreased the rate of cell proliferation in the adult subgranular zone Female and male mice exposed to MDMA during their entire developmental stage were examined at 11 weeks to
Fig. 2. BrdU-stained cells in the subgranular zone of 11-week-old female offspring who were born of control or MDMA-treated dams at 24 h post-BrdU labeling. (A–C) Controls; (D–F) mice with developmental exposure to 20 mg/kg of MDMA. Note that BrdU-positive cells in the female dentate gyrus are significantly lower in the groups developmentally exposed to MDMA (D–F) than in the controls (A–C). Each rectangle represents the area magnified on the right side of the figure. Scale bar⫽100 m (A, D); 50 m (B, E); 20 m (C, F).
K.-O. Cho et al. / Neuroscience 154 (2008) 1034 –1041
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Fig. 3. BrdU-stained cells in the subgranular zone of 11-week-old male offspring who were born of control or MDMA-treated dams at 24 h post-BrdU labeling. (A–C) Controls; (D–F) mice with developmental exposure to 20 mg/kg of MDMA. Note that BrdU-positive cells in the male dentate gyrus are significantly lower in the groups developmentally exposed to MDMA (D–F) than in the controls (A–C). Each rectangle represents the area magnified on the right side of the figure. Scale bar⫽100 m (A, D); 50 m (B, E); 20 m (C, F).
determine the proliferative activities in the subgranular zone of the dentate gyrus by BrdU-immunolabeling (Fig. 1). Cells stained for BrdU were usually clustered and located just beneath the granule cell layer both in female and male dentate gyrus (Figs. 2 and 3). The number of BrdUpositive cells was significantly downregulated in offspring of MDMA-administered dams, compared with controls, when examined 24 h after the last BrdU injection (F⫽12.99, P⬍0.05; Fig. 4A). Specifically, offspring of dams administered either 1.25 mg/kg or 20 mg/kg MDMA showed fewer BrdU-labeled cells than control mice, although there was no significant difference between these two groups (Fig. 4A). Developmental exposure to high dose MDMA decreased the number of mature newborn cells in the adult dentate gyrus At 28 days after the BrdU pulse-labeling, the number of BrdU-positive cells was examined to check the effect of developmental MDMA exposure on the survival of newly generated postmitotic cells in the adult hippocampus (Fig. 1). BrdU-stained cells in the dentate gyrus of female offspring were found inside the granule cell layer as well as in the subgranular zones. Most of them were as round as mature granule neurons. In addition, the size of BrdUlabeled cells was large enough to be compatible with surrounding hematoxylin-stained granule cells (Fig. 5). Female offspring born of high dose MDMA-administered dams showed markedly fewer BrdU-positive cells at 28
days post-BrdU labeling (F2,12⫽8.61, P⬍0.05; Fig. 4B). Specifically, 20 mg/kg MDMA administration reduced the number of BrdU-positive cells in the dentate gyrus of female offspring by 60%. However, low dose maternal MDMA administration had no effect on the number of cells surviving in the dentate gyrus of the second generation (Fig. 4B). In order to examine the phenotype of surviving newly generated cells, double immunofluorescence was carried out (Fig. 6). BrdU-labeled cells were co-localized with the mature neuron-specific marker, NeuN, as well as the marker for astrocytes, GFAP.
DISCUSSION The results of this study, for the first time, link developmental MDMA exposure to adult hippocampal neurogenesis. Chronic maternal MDMA administration markedly reduced the number of BrdU-positive newly generated cells in the subgranular zone of offspring. In addition, mice exposed to high dose MDMA during their developmental period had fewer mature newborn cells in the adult dentate gyrus than those born of saline-treated dams. As a prerequisite to discussing the influences of developmental MDMA exposure on adult neurogenesis, it is necessary not only to validate BrdU as a marker of cell division but also to confirm the maternal–fetal kinetics of MDMA. There is a growing concern that BrdU may mark DNA repair in dying cells rather than DNA replication in
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Fig. 4. Bar charts showing the number of BrdU-positive cells among the control and developmentally MDMA-exposed groups at 1 day or 28 days after the last BrdU injection. BrdU-labeling was carried out when the animals were 11 weeks old. (A) Rate of cell proliferation. Note that BrdU-positive cells in the adult dentate gyrus are significantly fewer in groups that were developmentally exposed to MDMA (1.25 and 20 mg/ kg) than in the controls. * P⬍0.05 by two-way ANOVA. (B) Survival of mature newborn cells. Note that developmental exposure to high dose (20 mg/kg) MDMA decreased the number of BrdU-labeled cells in the female adult dentate gyrus. Data are presented as means⫾S.E.M. * P⬍0.05 by one-way ANOVA.
proliferating cells (Yang et al., 2001). However, careful validation of BrdU-labeled cells revealed that BrdU was found in less than 1% of NeuN-positive or apoptotic cells, but rather in most of the proliferative cells (Palmer et al., 2000; Katchanov et al., 2001; Cooper-Kuhn and Kuhn, 2002). As for the transmission of MDMA from mother to fetus, Campbell et al. (2006) reported that when MDMA was administered during pregnancy, it was efficiently transferred to the fetal brain through the amniotic fluid. Although there are no available data confirming the presence of MDMA in breast milk, it can be inferred from several factors that MDMA may be transferred to postnatal pups by lactation. Considering that MDMA is a low-molecular-weight and hydrophobic molecule, its passage across capillary endothelial membrane in the breast would be favored. In addition, MDMA is a basic drug with a pKa of 10.4, and basic drugs can accumulate in breast milk because it is typically more acidic than blood (Rasmussen,
1983). Similarly, amphetamine, which has a pKa of 9.8, was reported to have a high milk/plasma concentration ratio (Steiner et al., 1984). Moreover, the target areas of MDMA in the brain included the cerebral cortex and the hippocampus, which are important for learning and memory functions (De Letter et al., 2003). Accordingly, our data regarding impaired adult neurogenesis evoked by developmental MDMA exposure can be considered to be the result of direct action of MDMA on the offspring hippocampus during development. The mechanisms underlying the deleterious influences of developmental MDMA exposure on adult neurogenesis in the hippocampus remain to be determined. Among them, however, one possible factor affecting the outcome may be the disruption of the monoamine system. To date, a number of investigations have confirmed that MDMA produces long-lasting neurotoxicity in the 5-HT as well as the dopamine systems of adult mice (Itzhak et al., 2003; Achat-Mendes et al., 2005). In addition, neonatal MDMA exposure caused persistent decreases in monoamine levels in neonatal and adult rats (Broening et al., 2001; Koprich et al., 2003; Crawford et al., 2006), although prenatal exposure did not alter monoamine concentrations in pups (Green et al., 2003). Considering the positive roles of 5-HT and dopamine in neurogenesis via the activation of the 5-hydroxytryptamine1A receptor and dopamine D2 receptor, respectively (Brezun and Daszuta, 1999; Banasr et al., 2004; Hoglinger et al., 2004; Hiramoto et al., 2007), MDMA administration can be expected to downregulate cell proliferation and/or survival. Indeed, a couple of studies showed that repeated MDMA injections in adult rats were proved to decrease 5-hydroxytryptamine1A receptor density (Aguirre et al., 1997) and MDMA injections impaired the survival of postmitotic newborn cells in the adult hippocampus (Hernandez-Rabaza et al., 2006). Consistent with these data, the present study showed that developmental exposure to MDMA produced fewer new cells in the adult dentate gyrus. Interestingly, our data showed that chronic exposure to MDMA during developmental periods significantly reduced the proliferative activity in the hippocampal dentate gyrus with doses as low as 1.25 mg/kg, which can be regarded as equivalent to a dose of 7 mg in a 60-kg human according to the interspecies scaling relationship, Dhuman⫽ Danimal(Whuman/Wanimal)0.7, where D denotes the dose of MDMA in milligrams and W denotes body weight in kilograms (Mordenti and Chappell, 1989). Moreover, 20 mg/kg of MDMA in a 25-g rodent equates to a dose of 116 mg in a 60-kg human. Considering the fact that ecstasy tablets have been reported to generally contain between 100 and 150 mg of MDMA (Schifano, 1991), the proliferative activity in the adult dentate gyrus seems to be highly vulnerable to developmental MDMA exposure. In addition, since the body weight of adult offspring was not significantly affected by MDMA exposure, reduced cell proliferation could be released from concerns about a secondary effect of the mice born of MDMA-administered dams being smaller than the controls (St Omer et al., 1991). In accordance with our data on cell proliferation, repeated administration of psy-
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Fig. 5. BrdU-stained cells in the dentate gyrus of the female offspring born of control or MDMA-administered dams at 28 days post-BrdU labeling. (A–C) Control; (D–F) mice with developmental exposure to 20 mg/kg of MDMA. Note that BrdU-positive cells in the female dentate gyrus are significantly fewer in the groups developmentally exposed to MDMA (D–F) than in the controls (A–C). Each rectangle represents the area magnified on the right side of the figure. Scale bar⫽100 m (A, D); 50 m (B, E); 20 m (C, F).
chostimulants such as MDMA, cocaine or methamphetamine decreased the number of dividing adult subgranular
zone cells (Teuchert-Noodt et al., 2000; Yamaguchi et al., 2005; Cho et al., 2007). However, contrary to our data,
Fig. 6. Newly formed neurons and astrocytes in the dentate gyrus. On day 28 after the last BrdU injection, brain sections were immunostained with BrdU (red) and NeuN or GFAP (green). Note that newly generated neurons (A–C) and astrocytes (D–F) are located in the granule cell layer. Scale bar⫽20 m.
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binge administration of MDMA four times a day for 2 days did not show any effects on the proliferation of precursor cells in the adult hippocampus (Hernandez-Rabaza et al., 2006). This discrepancy seems to be attributable to the different duration, route and schedule of MDMA administration and the schedule of BrdU injections, as well as the different species used. Although a few studies have provided insight into the impact of psychostimulant drugs on neurogenesis, the survival of postmitotic newborn cells has not been studied extensively with drugs such as cocaine, amphetamine, methamphetamine or MDMA. Recently, chronic exposure to cocaine showed no difference in terms of cell survival (Dominguez-Escriba et al., 2006). On the other hand, binge injections with MDMA decreased the number of mature newborn cells in the adult dentate gyrus (Hernandez-Rabaza et al., 2006), which supported our survival data for chronic high dose exposure to MDMA. However, because of the limited amount of evidence, more rigorous testing is needed to determine the clear consequences of psychostimulants on cell survival. Finally, our data demonstrated a decrease in cell proliferation by both low- and high-dose MDMA, but a reduction in cell survival only in the presence of highdose MDMA. Although a definite reduction in cell proliferation was noted at both doses, intact cell survival in the presence of low-dose MDMA is unexplained. It has been shown that the rate of cell proliferation in the hippocampus does not always predict the rate of cell survival (Lehmann et al., 2005). In addition, one study showing that chronic cocaine treatment decreased the proliferative activity of progenitors without affecting survival of neural precursors, supports the plausibility of our data (Dominguez-Escriba et al., 2006). Therefore, our findings, which show the difference in adult neurogenesis caused by low- and high-dose MDMA, may be an intriguing subject for further investigation.
CONCLUSION In summary, our findings revealed that exposure to MDMA for the entire developmental period caused a negative influence on hippocampal cell division as well as survival of postmitotic newborn cells, even when the animals grew up. Furthermore, cell proliferation decreased regardless of the dose of MDMA, whereas the survival of mature newborn cells was reduced only by high dose exposure to MDMA. Collectively, our results can shed light upon the irreversible adverse effects of developmental MDMA exposure on adult neurogenesis. Further studies are required to explain the mechanisms and functional significance of decreased neurogenesis. Acknowledgement—This research was equally supported by a grant (06132485) from the Korea Food and Drug Administration in 2006 and by Korea Science and Engineering Foundation grant R13-2002-005-02002-0.
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(Accepted 20 April 2008) (Available online 2 May 2008)
DEVELOPMENTAL EXPOSURE TO 3,4-METHYLENEDIOXYMETHAMPHETAMINE RESULTS IN DOWNREGULATION OF NEUROGENESIS IN THE ADULT MOUSE HIPPOCAMPUS K.-O. CHO,a,b G. S. RHEE,c S. J. KWACK,c S. Y. CHUNGc AND S. Y. KIMa,b*
during dance parties called ‘raves’ (Arria et al., 2002). MDMA provides abusers with a sense of closeness to other people and sexual arousal, which can lead to inadvertent pregnancy (Cohen, 1995). Despite the particularly high prevalence of MDMA use among young adults who are of childbearing age (Maxwell, 2003), little is known about the consequences of such use in their offspring. MDMA is known as a 5-HT releaser and its repeated use can cause depletion of monoamines such as 5-HT and dopamine (Aguirre et al., 1997; O’Shea et al., 2001), leading to neurotoxicity in the monoamine system (Sprague et al., 2003) and cognitive deficits (McCann et al., 1999; Sprague et al., 2003). Furthermore, neonatal exposure to MDMA can markedly depress 5-HT and dopamine levels and impair spatial learning and memory function, even when examined at the adult stage (Broening et al., 2001; Crawford et al., 2006). In addition, neonatal MDMA injections appear to promote apoptotic cell death and decreased serotonergic fiber density (Meyer et al., 2004). Therefore, neonatal exposure to MDMA seems to induce long-lasting perturbation of the monoamine system in adults. In the hippocampus, new cells are constantly generated in the subgranular zone of the hippocampal dentate gyrus and differentiate into mature granule neurons (Palmer et al., 2000). Subsequently, these newly generated neurons integrate into existing hippocampal circuits and find appropriate targets (Markakis and Gage, 1999). Among a number of factors, neurotransmitters including 5-HT and dopamine have been shown to play a role in the regulation of hippocampal neurogenesis. A selective 5-HT reuptake inhibitor such as fluoxetine has a positive influence on the proliferation of progenitor cells which contributes to improved memory function, consolidating the stimulating effects of 5-HT on granule cell genesis (Gould, 1999; Malberg et al., 2000; Mowla et al., 2007). In parallel, 5-HT depletion reduced hippocampal neurogenesis (Brezun and Daszuta, 1999). In addition, dopamine depletion led to a decrease of the proliferative activity in the subgranular zone, though the precise effects of dopamine on neurogenesis are still controversial (Hoglinger et al., 2004; Winner et al., 2006). In the present study, we asked whether chronic developmental exposure to MDMA results in any alterations in adult hippocampal neurogenesis. Since the lactational period (postnatal days (PDs) 1–21) in rodents matches the second and third trimester in humans (Clancy et al., 2001),
a
Department of Pharmacology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul, 137-701, Korea
b
Cell Death Disease Research Center, College of Medicine, The Catholic University of Korea, Seoul, 137-701, Korea
c
Reproductive and Developmental Toxicology, National Institute of Toxicological Research, 194 Tongilro, Eunpyeong-gu, Seoul, 122-704, Korea
Abstract—3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a powerful releaser of 5-HT and chronic use of this drug can cause depletion of monoamines. Recently, concerns about the risk of adult brain damage due to fetal exposure to MDMA have been raised. We investigated whether developmental MDMA exposure affected adult neurogenesis in C57 black/6 mice. MDMA (1.25 or 20 mg/kg, p.o.) or vehicle was administered daily to the mother from prenatal 6th day to postnatal 21st day. When the offspring were 11 weeks old, they were injected with 5-bromo-2=-deoxyuridine (BrdU) (120 mg/kg, i.p.) once a day for 4 days. After 24 h or 28 days, the animals were killed to count the BrdU-positive cells in the dentate gyrus. At 24 h after the last BrdU injection, the number of BrdU-positive cells in the offspring developmentally exposed to MDMA was significantly lower than that of the control group. At 28 days post-BrdU labeling, BrdU-positive cells in the dentate gyrus of female offspring with developmental exposure to high dose MDMA were significantly fewer compared with the control group. In addition, most BrdUpositive cells were co-labeled with the mature neuronal marker, neuronal nuclei, while a few BrdU-labeled cells were merged with an astrocyte marker. Our results suggest that developmental exposure to MDMA can result in decreases in the proliferation and survival of mature newborn cells in the adult dentate gyrus. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ecstasy, adult neurogenesis, BrdU, dentate gyrus, hippocampus, abuse.
3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is an amphetamine-like stimulant that is widely abused *Correspondence to: S. Y. Kim, Department of Pharmacology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul, 137-701, Korea. Tel: ⫹82-2-590-1205; fax: ⫹82-2536-2485. E-mail address: [email protected] (S. Y. Kim). Abbreviations: ANOVA, analysis of variance; BrdU, 5-bromo-2=-deoxyuridine; C57BL/6, C57 black/6; FDA, Food and Drug Administration; GFAP, glial fibrillary acidic protein; MDMA, 3,4-methylenedioxymethamphetamine; NeuN, neuronal nuclei; PDs, postnatal days.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.04.040
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MDMA was administered to the mother during the gestational and lactational period. Using 5-bromo-2=-deoxyuridine (BrdU) as a marker of cell division, we examined the difference between controls and developmentally MDMAexposed mice in terms of the rate of cell proliferation and the number of mature newborn cells in the adult dentate gyrus.
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4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed and post-fixed for 4 h in the same fixative and, then, embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan). For the detection of BrdU immunoreactivity, DNA denaturation was conducted by incubation in 50% formamide and 2⫻ sodium citrate solution for 2 h at 65 °C. Subsequently, sections (20 m thick) were incubated for 1 h in 2 N HCl at 37 °C and then for 10 min in boric acid. The sections were then incubated with anti-mouse BrdU (1:100; DAKO, Glostrup, Denmark) overnight at 4 °C. After that, the sections were put into solutions containing secondary antibody (biotinylated horse anti-mouse; Vector Laboratories, Burlingame, CA, USA) for 2 h, followed by amplification with an avidin– biotin complex (Vector Laboratories) for 1 h. Between each step, washing with 0.01 M phosphate-buffered saline was carried out for 15 min. Finally, the brain sections were visualized with 0.05% 3,3=-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide, and were later counterstained with hematoxylin. After hydration, the slides were incubated with hematoxylin solution for 2 min. Then, excessive hematoxylin was washed by submerging the slides in 1% HCl, which is followed by rinsing with distilled water and dehydrating with xylene. For the double labeling experiments, the sections were incubated with anti-rat BrdU (1:200; Accurate, Westbury, NY, USA) overnight at 4 °C, then incubated with carbocyanine 3– conjugated goat anti-rat IgG (1:500; Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. After rinsing with 0.1 M phosphate buffer (pH 7.4), the sections were incubated with antimouse neuronal nuclei (NeuN) (1:400; Chemicon International, Temecula, CA, USA) or glial fibrillary acidic protein (GFAP) (1: 400; Chemicon International) overnight at 4 °C. The next day, fluorescein isothiocyanate– conjugated goat anti-mouse IgG (1: 25; Jackson ImmunoResearch) was then applied for 2 h at room temperature and the sections were observed using a confocal microscope (MRC-1024; Bio-Rad Laboratories, Hercules, CA, USA) after mounting with prolong gold antifade reagents (Invitrogen, Carlsbad, CA, USA). The images were generated using Image-Pro Plus software (MediaCybernetics, Silver Spring, MD, USA) and they were not modified other than relative adjustments of levels, brightness, and contrast.
EXPERIMENTAL PROCEDURES Animals and MDMA schedule All animal procedures were approved by the Ethics Committee of the Catholic University of Korea and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Efforts were made to try to minimize animal suffering and to reduce the number of animals used. Male and female C57 black/6 (C57BL/6) mice (Orientbio, Kyungki-do, Korea) weighing 22–25 g were housed three per cage at a standard temperature (22⫾1 °C) and in a light-controlled environment (lights on from 8:00 a.m. to 8:00 p.m.) with ad libitum access to food and water. They were mated in a separate cage and recognized as pregnant on the day when vaginal plugs were detected. The day mice gave birth was designated as PD 0. Eighteen female C57BL/6 mice received either ⫾3,4-MDMA HCl (Sigma, St. Louis, MO, USA) dissolved in saline or saline once a day from day 6 of pregnancy to day 21 after delivery, according to the U.S. Food and Drug Administration (U.S. FDA) guideline for detection of toxicity to reproduction of medicinal products (FDA, 1994). MDMA (1.25 mg/kg, 20 mg/kg) or saline was administered to the mother by oral gavage (12 mL/kg). The dose of MDMA was selected based on our previous study showing the effects of chronic MDMA on cell proliferation in the adult dentate gyrus (Cho et al., 2007). After delivery, maternal behavior was observed daily in the cage of each mother and her litter between PD 1 and PD 28. On PD 28, they were housed in groups by sex.
Schedule of BrdU injections When offspring were 11 weeks old, five or six mice per group were allocated. After measuring the body weight, BrdU (120 mg/10 mL/ kg, i.p.) dissolved in 0.007 M NaOH solution was injected into these mice once a day for 4 days in order to label DNA of cells in the S-phase of mitosis and follow the labeled cells. For evaluation of proliferative activities, 18 female and 15 male mice born to 18 different dams were killed using 15% chloral hydrate at 24 h after the last BrdU injection. For assessment of the survival of precursor cells, 15 female mice were cared for in six cages until they were killed using 15% chloral hydrate at 28 days after the last BrdU administration.
Cell counting and statistics Quantification was done blind to the experimental treatments. Every fifth section (i.e. a total of three) was processed for BrdU labeling. The area investigated in the present study was ⫺1.5 to ⫺1.7 mm from the bregma according to the atlas of Paxinos and Franklin (2001). For the cell proliferation study, BrdU-labeled cells in the subgranular zone of the dentate gyrus were counted bilaterally in each section using a light microscope (BX51; Olympus, Tokyo, Japan) equipped with a charge-coupled device camera (DP70; Olympus). The BrdU-immunoreactive cells in a cluster were differentially counted at 400⫻ magnification by adjusting the focus. BrdU-positive cells in the dentate gyrus used to evaluate the survival of newborn cells were also counted in the same way. Only cells located in the subgranular zone or the granule cell layer
Immunohistochemistry Animals (n⫽5– 6 from each group) were transcardially perfused with 30 mL of saline, followed by 150 mL of a fixative containing
Table 1. Physical parameters of dams and litter developmentally exposed to MDMA Dose of MDMA (mg/kg)
Maternal weight at GD 18 (g)
No. of pups per litter
M:F ratio per litter
0.00 1.25 20.00
35.49⫾1.78 33.82⫾1.31 32.93⫾0.69
8.33⫾0.67 8.50⫾0.43 8.67⫾1.09
1.43⫾0.40 1.39⫾0.43 1.07⫾0.29
Neonatal weight at PD 4 (g)
Postnatal weight at PD 21 (g)
Postnatal weight at PD 77 (g)
Male
Female
Male
Female
Male
Female
2.36⫾0.08 2.36⫾0.08 2.40⫾0.05
2.48⫾0.10 2.38⫾0.10 2.22⫾0.09
8.36⫾0.25 8.70⫾0.29 8.47⫾0.09
8.97⫾0.28 8.26⫾0.38 7.99⫾0.17
23.77⫾0.49 23.82⫾0.61 23.78⫾0.26
20.54⫾0.41 21.69⫾0.22 21.83⫾0.59
Values are means⫾S.E.M. of six dams, 11 female or five male litters per group. There was no statistical significance among MDMA-administered groups and control. GD, gestational day; M, male; F, female.
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K.-O. Cho et al. / Neuroscience 154 (2008) 1034 –1041 tions of treatment⫻sex; accordingly, results from males and females are presented together. Cell survival and the body weight were evaluated by one-way ANOVA and post hoc Scheffe’s test. Differences were assumed to be statistically significant when P⬍0.05.
RESULTS Developmental exposure to MDMA had no influence on the physical parameters of the dams and offspring
Fig. 1. Schematic representation of MDMA and BrdU administration. MDMA (1.25 or 20 mg/kg) or saline was administered to dams orally once a day from the 6th day after pregnancy (E6) to the 21st day after delivery (P21). When female and male offspring were 11 weeks old, BrdU (120 mg/kg) was injected intraperitoneally once a day for 4 days. For female mice, 1 day or 28 days after the last BrdU injection (0d), the animals were killed to evaluate cell proliferation or survival of mature newborn cells, respectively. For male mice, 1 day after the last BrdU injection (0d), the animals were killed to evaluate cell proliferation. (A) MDMA- or saline-administered dams, (B) male offspring born of MDMAor saline-administered dams. (C) Female offspring born of MDMA- or saline-administered dams.
were counted, and cells located more than two cells away from the granule cell layer were classified as being in the hilus. Numbers of BrdU-positive cells and the body weight were expressed as means⫾S.E.M. Since we selected one male and one female from each dam, cell proliferation was evaluated by two-way analysis of variance (ANOVA) which did not identify any significant interac-
Chronic developmental exposure to MDMA by our regimen did not produce any significant alterations in maternal care of pups or alterations in physical parameters of the dams and offspring (Table 1). In an observational test during the lactational period, there were no significant differences in nursing, grooming or carrying pups among MDMA-administered dams and controls. Moreover, our preliminary study showed that the fetal survival rate was not different between vehicle- and 20 mg/kg MDMA-administrated groups (data not shown). With regard to the physical data, maternal MDMA administration did not alter the body weight of the dams or the litter. In addition, there was no significant difference in the litter compositions or the number of pups per litter among controls and MDMA-administered groups. Developmental MDMA exposure decreased the rate of cell proliferation in the adult subgranular zone Female and male mice exposed to MDMA during their entire developmental stage were examined at 11 weeks to
Fig. 2. BrdU-stained cells in the subgranular zone of 11-week-old female offspring who were born of control or MDMA-treated dams at 24 h post-BrdU labeling. (A–C) Controls; (D–F) mice with developmental exposure to 20 mg/kg of MDMA. Note that BrdU-positive cells in the female dentate gyrus are significantly lower in the groups developmentally exposed to MDMA (D–F) than in the controls (A–C). Each rectangle represents the area magnified on the right side of the figure. Scale bar⫽100 m (A, D); 50 m (B, E); 20 m (C, F).
K.-O. Cho et al. / Neuroscience 154 (2008) 1034 –1041
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Fig. 3. BrdU-stained cells in the subgranular zone of 11-week-old male offspring who were born of control or MDMA-treated dams at 24 h post-BrdU labeling. (A–C) Controls; (D–F) mice with developmental exposure to 20 mg/kg of MDMA. Note that BrdU-positive cells in the male dentate gyrus are significantly lower in the groups developmentally exposed to MDMA (D–F) than in the controls (A–C). Each rectangle represents the area magnified on the right side of the figure. Scale bar⫽100 m (A, D); 50 m (B, E); 20 m (C, F).
determine the proliferative activities in the subgranular zone of the dentate gyrus by BrdU-immunolabeling (Fig. 1). Cells stained for BrdU were usually clustered and located just beneath the granule cell layer both in female and male dentate gyrus (Figs. 2 and 3). The number of BrdUpositive cells was significantly downregulated in offspring of MDMA-administered dams, compared with controls, when examined 24 h after the last BrdU injection (F⫽12.99, P⬍0.05; Fig. 4A). Specifically, offspring of dams administered either 1.25 mg/kg or 20 mg/kg MDMA showed fewer BrdU-labeled cells than control mice, although there was no significant difference between these two groups (Fig. 4A). Developmental exposure to high dose MDMA decreased the number of mature newborn cells in the adult dentate gyrus At 28 days after the BrdU pulse-labeling, the number of BrdU-positive cells was examined to check the effect of developmental MDMA exposure on the survival of newly generated postmitotic cells in the adult hippocampus (Fig. 1). BrdU-stained cells in the dentate gyrus of female offspring were found inside the granule cell layer as well as in the subgranular zones. Most of them were as round as mature granule neurons. In addition, the size of BrdUlabeled cells was large enough to be compatible with surrounding hematoxylin-stained granule cells (Fig. 5). Female offspring born of high dose MDMA-administered dams showed markedly fewer BrdU-positive cells at 28
days post-BrdU labeling (F2,12⫽8.61, P⬍0.05; Fig. 4B). Specifically, 20 mg/kg MDMA administration reduced the number of BrdU-positive cells in the dentate gyrus of female offspring by 60%. However, low dose maternal MDMA administration had no effect on the number of cells surviving in the dentate gyrus of the second generation (Fig. 4B). In order to examine the phenotype of surviving newly generated cells, double immunofluorescence was carried out (Fig. 6). BrdU-labeled cells were co-localized with the mature neuron-specific marker, NeuN, as well as the marker for astrocytes, GFAP.
DISCUSSION The results of this study, for the first time, link developmental MDMA exposure to adult hippocampal neurogenesis. Chronic maternal MDMA administration markedly reduced the number of BrdU-positive newly generated cells in the subgranular zone of offspring. In addition, mice exposed to high dose MDMA during their developmental period had fewer mature newborn cells in the adult dentate gyrus than those born of saline-treated dams. As a prerequisite to discussing the influences of developmental MDMA exposure on adult neurogenesis, it is necessary not only to validate BrdU as a marker of cell division but also to confirm the maternal–fetal kinetics of MDMA. There is a growing concern that BrdU may mark DNA repair in dying cells rather than DNA replication in
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Fig. 4. Bar charts showing the number of BrdU-positive cells among the control and developmentally MDMA-exposed groups at 1 day or 28 days after the last BrdU injection. BrdU-labeling was carried out when the animals were 11 weeks old. (A) Rate of cell proliferation. Note that BrdU-positive cells in the adult dentate gyrus are significantly fewer in groups that were developmentally exposed to MDMA (1.25 and 20 mg/ kg) than in the controls. * P⬍0.05 by two-way ANOVA. (B) Survival of mature newborn cells. Note that developmental exposure to high dose (20 mg/kg) MDMA decreased the number of BrdU-labeled cells in the female adult dentate gyrus. Data are presented as means⫾S.E.M. * P⬍0.05 by one-way ANOVA.
proliferating cells (Yang et al., 2001). However, careful validation of BrdU-labeled cells revealed that BrdU was found in less than 1% of NeuN-positive or apoptotic cells, but rather in most of the proliferative cells (Palmer et al., 2000; Katchanov et al., 2001; Cooper-Kuhn and Kuhn, 2002). As for the transmission of MDMA from mother to fetus, Campbell et al. (2006) reported that when MDMA was administered during pregnancy, it was efficiently transferred to the fetal brain through the amniotic fluid. Although there are no available data confirming the presence of MDMA in breast milk, it can be inferred from several factors that MDMA may be transferred to postnatal pups by lactation. Considering that MDMA is a low-molecular-weight and hydrophobic molecule, its passage across capillary endothelial membrane in the breast would be favored. In addition, MDMA is a basic drug with a pKa of 10.4, and basic drugs can accumulate in breast milk because it is typically more acidic than blood (Rasmussen,
1983). Similarly, amphetamine, which has a pKa of 9.8, was reported to have a high milk/plasma concentration ratio (Steiner et al., 1984). Moreover, the target areas of MDMA in the brain included the cerebral cortex and the hippocampus, which are important for learning and memory functions (De Letter et al., 2003). Accordingly, our data regarding impaired adult neurogenesis evoked by developmental MDMA exposure can be considered to be the result of direct action of MDMA on the offspring hippocampus during development. The mechanisms underlying the deleterious influences of developmental MDMA exposure on adult neurogenesis in the hippocampus remain to be determined. Among them, however, one possible factor affecting the outcome may be the disruption of the monoamine system. To date, a number of investigations have confirmed that MDMA produces long-lasting neurotoxicity in the 5-HT as well as the dopamine systems of adult mice (Itzhak et al., 2003; Achat-Mendes et al., 2005). In addition, neonatal MDMA exposure caused persistent decreases in monoamine levels in neonatal and adult rats (Broening et al., 2001; Koprich et al., 2003; Crawford et al., 2006), although prenatal exposure did not alter monoamine concentrations in pups (Green et al., 2003). Considering the positive roles of 5-HT and dopamine in neurogenesis via the activation of the 5-hydroxytryptamine1A receptor and dopamine D2 receptor, respectively (Brezun and Daszuta, 1999; Banasr et al., 2004; Hoglinger et al., 2004; Hiramoto et al., 2007), MDMA administration can be expected to downregulate cell proliferation and/or survival. Indeed, a couple of studies showed that repeated MDMA injections in adult rats were proved to decrease 5-hydroxytryptamine1A receptor density (Aguirre et al., 1997) and MDMA injections impaired the survival of postmitotic newborn cells in the adult hippocampus (Hernandez-Rabaza et al., 2006). Consistent with these data, the present study showed that developmental exposure to MDMA produced fewer new cells in the adult dentate gyrus. Interestingly, our data showed that chronic exposure to MDMA during developmental periods significantly reduced the proliferative activity in the hippocampal dentate gyrus with doses as low as 1.25 mg/kg, which can be regarded as equivalent to a dose of 7 mg in a 60-kg human according to the interspecies scaling relationship, Dhuman⫽ Danimal(Whuman/Wanimal)0.7, where D denotes the dose of MDMA in milligrams and W denotes body weight in kilograms (Mordenti and Chappell, 1989). Moreover, 20 mg/kg of MDMA in a 25-g rodent equates to a dose of 116 mg in a 60-kg human. Considering the fact that ecstasy tablets have been reported to generally contain between 100 and 150 mg of MDMA (Schifano, 1991), the proliferative activity in the adult dentate gyrus seems to be highly vulnerable to developmental MDMA exposure. In addition, since the body weight of adult offspring was not significantly affected by MDMA exposure, reduced cell proliferation could be released from concerns about a secondary effect of the mice born of MDMA-administered dams being smaller than the controls (St Omer et al., 1991). In accordance with our data on cell proliferation, repeated administration of psy-
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Fig. 5. BrdU-stained cells in the dentate gyrus of the female offspring born of control or MDMA-administered dams at 28 days post-BrdU labeling. (A–C) Control; (D–F) mice with developmental exposure to 20 mg/kg of MDMA. Note that BrdU-positive cells in the female dentate gyrus are significantly fewer in the groups developmentally exposed to MDMA (D–F) than in the controls (A–C). Each rectangle represents the area magnified on the right side of the figure. Scale bar⫽100 m (A, D); 50 m (B, E); 20 m (C, F).
chostimulants such as MDMA, cocaine or methamphetamine decreased the number of dividing adult subgranular
zone cells (Teuchert-Noodt et al., 2000; Yamaguchi et al., 2005; Cho et al., 2007). However, contrary to our data,
Fig. 6. Newly formed neurons and astrocytes in the dentate gyrus. On day 28 after the last BrdU injection, brain sections were immunostained with BrdU (red) and NeuN or GFAP (green). Note that newly generated neurons (A–C) and astrocytes (D–F) are located in the granule cell layer. Scale bar⫽20 m.
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binge administration of MDMA four times a day for 2 days did not show any effects on the proliferation of precursor cells in the adult hippocampus (Hernandez-Rabaza et al., 2006). This discrepancy seems to be attributable to the different duration, route and schedule of MDMA administration and the schedule of BrdU injections, as well as the different species used. Although a few studies have provided insight into the impact of psychostimulant drugs on neurogenesis, the survival of postmitotic newborn cells has not been studied extensively with drugs such as cocaine, amphetamine, methamphetamine or MDMA. Recently, chronic exposure to cocaine showed no difference in terms of cell survival (Dominguez-Escriba et al., 2006). On the other hand, binge injections with MDMA decreased the number of mature newborn cells in the adult dentate gyrus (Hernandez-Rabaza et al., 2006), which supported our survival data for chronic high dose exposure to MDMA. However, because of the limited amount of evidence, more rigorous testing is needed to determine the clear consequences of psychostimulants on cell survival. Finally, our data demonstrated a decrease in cell proliferation by both low- and high-dose MDMA, but a reduction in cell survival only in the presence of highdose MDMA. Although a definite reduction in cell proliferation was noted at both doses, intact cell survival in the presence of low-dose MDMA is unexplained. It has been shown that the rate of cell proliferation in the hippocampus does not always predict the rate of cell survival (Lehmann et al., 2005). In addition, one study showing that chronic cocaine treatment decreased the proliferative activity of progenitors without affecting survival of neural precursors, supports the plausibility of our data (Dominguez-Escriba et al., 2006). Therefore, our findings, which show the difference in adult neurogenesis caused by low- and high-dose MDMA, may be an intriguing subject for further investigation.
CONCLUSION In summary, our findings revealed that exposure to MDMA for the entire developmental period caused a negative influence on hippocampal cell division as well as survival of postmitotic newborn cells, even when the animals grew up. Furthermore, cell proliferation decreased regardless of the dose of MDMA, whereas the survival of mature newborn cells was reduced only by high dose exposure to MDMA. Collectively, our results can shed light upon the irreversible adverse effects of developmental MDMA exposure on adult neurogenesis. Further studies are required to explain the mechanisms and functional significance of decreased neurogenesis. Acknowledgement—This research was equally supported by a grant (06132485) from the Korea Food and Drug Administration in 2006 and by Korea Science and Engineering Foundation grant R13-2002-005-02002-0.
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(Accepted 20 April 2008) (Available online 2 May 2008)