Effects of MDMA (“ecstasy”) during adolescence on place conditioning and hippocampal neurogenesis

European Journal of Pharmacology 628 (2010) 96–103

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Behavioural Pharmacology

Effects of MDMA (“ecstasy”) during adolescence on place conditioning and hippocampal neurogenesis Briony J. Catlow a,b,c, Kimberly A. Badanich d, Ashley E. Sponaugle c, Amanda R. Rowe b,c, Shijie Song b,c, Igor Rafalovich b,c, Vasyl Sava b,c, Cheryl L. Kirstein a, Juan Sanchez-Ramos b,c,⁎ a

Department of Psychology, University of South Florida, Tampa, FL 33620, USA Department of Neurology, University of South Florida, Tampa, FL 33620, USA James Haley VA Medical Center, Tampa, FL 33613, USA d Center for Drug and Alcohol Programs, Medical University of South Carolina, Charleston, SC, 29425, USA b c

a r t i c l e

i n f o

Article history: Received 20 May 2009 Received in revised form 30 October 2009 Accepted 10 November 2009 Available online 20 November 2009 Keywords: Adolescent MDMA Conditioned place preference Hippocampus Proliferation Neurogenesis Flow cytometry Development

a b s t r a c t The use of 3,4,methylenedioxymethamphetamine (MDMA), the active agent in ecstasy, during adolescence is widespread yet the effects on adolescent behavior and brain development are unknown. The aim of the present study was 1) to evaluate effects of MDMA in adolescent rats using the conditioned place preference (CPP) paradigm to measure MDMA-induced reward and 2) assess effects of MDMA administration on cellular proliferation, survival and neurogenesis in the dentate gyrus of the hippocampus. During the adolescent period, MDMA CPP was measured in adolescents [postnatal day (PND) 28–39] by training rats to associate 1.25, 2.5, 5.0 mg/kg MDMA or saline administration with environmental cues. After CPP ended, bromodeoxyuridine (BrdU) was injected and rats were euthanized either 24 h (to evaluate cell proliferation) or 2 weeks (to assess neurogenesis) after the last MDMA injection. Adolescents expressed a CPP for 2.5 mg/ kg MDMA. Repeated exposure to 5.0 mg/kg MDMA during adolescence increased cell proliferation, yet diminished neurogenesis, an effect that was replicated using flow cytometry. These findings suggest differential dose effects of adolescent MDMA exposure on reward related behaviors and hippocampal neurogenesis. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Hippocampal neurogenesis in the adult brain can be influenced by stress, odors, electroconvulsive therapy, physical activity, learning, hormones, age and psychoactive drugs (Kempermann et al., 1998; Malberg and Duman, 2003; Malberg et al., 2000; van et al., 1999; Tanapat et al., 2001; Eisch et al., 2000; Nixon and Crews, 2002; Yamaguchi et al., 2004; Hernandez-Rabaza et al., 2006; Cameron et al., 1993). Proserotonergic drugs have been shown to upregulate hippocampal neurogenesis (Malberg and Duman, 2003) while some drugs such as alcohol, opioids, and MDMA negatively regulate neurogenesis (Eisch et al., 2000; Yamaguchi et al., 2004; Nixon and Crews, 2002; Hernandez-Rabaza et al., 2006; Cho et al., 2008) by altering the proliferation and survival of neural progenitor within the dentate gyrus (Aberg et al., 2005). Neurogenesis in the adolescent rodent and its role in brain remodeling have been sparsely investigated. Recent studies have shown elevations in adolescent hippocampal neurogenesis (He and Crews, 2007) and reductions ⁎ Corresponding author. Department of Neurology MDC 55, University of South Florida, 12901 Bruce B Downs Blvd, Tampa, FL 33612, USA. Tel.: +1 813 974 5841; fax: +1 813 974 7200. E-mail address: [email protected] (J. Sanchez-Ramos). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.11.017

resulting from acute ethanol administration (Crews et al., 2006) however the effects of drugs of abuse such as 3,4,methylenedioxymethamphetamine (MDMA) on cellular proliferation and neurogenesis in the adolescent rodent have not yet been investigated. MDMA not only decreases neurogenesis but has also been shown to possess positive reinforcing (rewarding) effects as shown via the conditioned place preference (CPP) paradigm (Bilsky et al., 1990; Marona-Lewicka et al., 1996; Meyer et al., 2002; Salzmann et al., 2003; Robledo et al., 2004; Braida et al., 2005; Herzig et al., 2005; Tourino et al., 2008; Robledo et al., 2007; Marie-Claire et al., 2008). Place conditioning measures the rewarding/aversive effects of drugs of abuse by classically conditioning subjects to associate the unconditioned stimulus properties of a drug with contextual cues (Tzschentke, 2007; Bardo and Bevins, 2000). Adult rodents express CPP for MDMA (Bilsky et al., 1990; Marona-Lewicka et al., 1996; Meyer et al., 2002; Salzmann et al., 2003; Robledo et al., 2004; Braida et al., 2005; Herzig et al., 2005; Tourino et al., 2008; Robledo et al., 2007; Marie-Claire et al, 2008). MDMA-induced place preferences have also been shown in rodents as young as PND 38 (Daza-Losada et al., 2007) indicating adolescent rodents learn to associate the unconditioned stimulus properties of MDMA with contextual cues. However, only relatively high doses of MDMA have been tested in adolescent rodents (5.0–20.0 mg/kg MDMA; (Daza-Losada et al.,

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2007)). It is important to determine whether adolescent rodents can be conditioned to the rewarding effects of MDMA at lower doses given that adolescents are particularly sensitive to the rewarding effects of low dose psychostimulants (Badanich et al., 2006; Badanich and Kirstein, 2007). Furthermore, doses of MDMA examined in the rodent (∼10–20 mg/kg MDMA Daza-Losada et al., 2007) may be greater than the typical dose consumed by humans (∼ 1–2 mg/kg MDMA; Pizarro et al., 2004) especially due to the presence of substances other than MDMA in ecstasy pills. Determining the effects of MDMA at lower doses than previously used on place conditioning and neurogenesis would make the model of MDMA adolescent exposure more relevant to the human adolescent population. Moreover, this study provides an opportunity to explore the relationship between CPP and hippocampal neurogenesis. Some forms of classical conditioning “trace eyeblink conditioning” and “fear conditioning” have been shown to require hippocampal neurogenesis (Shors et al., 2002). Since CPP is a form of classical conditioning linking mesolimbic DA-mediated behavior to temporal and spatial environmental cues, it can be hypothesized to also require hippocampal neurogenesis as demonstrated by others in fear conditioning paradigms. The present study aimed to evaluate the effects of repeated MDMA exposure during adolescence on 1) MDMA-induced CPP and locomotor activity and 2) cellular proliferation, survival and neurogenesis in the hippocampus. The hippocampus and associated circuitry undergo critical developmental changes during adolescence which may result in heightened susceptibility to MDMA (Andersen and Teicher, 2004). It was hypothesized that exposure to MDMA during adolescence would induce a place preference associated with normal or increased hippocampal neurogenesis. If hippocampal neurogenesis is decreased in the presence of MDMA-induced CPP then it is unlikely hippocampal neurogenesis is important for this form of classical conditioning. 2. Materials and methods 2.1. Subjects Fourty-seven male Sprague-Dawley rats, offspring of established breeding pairs (Harlan Laboratories, IN, USA) in the laboratory at the University of South Florida, Tampa, were used in the present study. Date of birth was designated as postnatal day (PND) 0 and litters were sexed and culled to 8–10 pups per litter on PND 1. Pups remained housed with their respective dams in a temperature (64–79 F) and humidity (30–70%) — controlled vivarium on a 12:12-hour light/dark cycle (lights on from 0700 to 1900 h). On PND 21, pups were weaned and housed in groups of two or three. As in humans, the adolescent period for rodents overlaps with sexual maturation (Odell and Swerdloff, 1976). Adolescence in rodents begins at approximately PND 28, extends to PND 46, and is marked by several developmental events including the onset of puberty and changes in neuroendocrine systems in addition to increased socialization and exploratory behaviors (Spear, 2000; Tirelli et al., 2003). For place conditioning, all rats were tested at PND 28–39. The same rats from the place conditioning experiments were used to evaluate cell proliferation (at PND 40) and the phenotypic fate of surviving cells (at PND 54). To eliminate the potential confound of litter effects, no more than one pup per litter was used for any given condition and remaining pups were used for other ongoing laboratory experiments. In all respects, the maintenance and treatment of rats were within guidelines for animal care as approved by the University of South Florida's Institutional Animal Care and Use Committee, the Principles of Laboratory Animal Care and the National Institutes of Health. 2.2. Conditioning apparatus The conditioning apparatus was a single runway comprised of black Plexiglas (Rohm and Haas Company, Philadelphia, Pennsylvania) that

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was divided into two equal sized sections: each (21 × 24.5 × 20.5 cm) with visual and tactile cues of either black and white horizontal striped (1 in. thick) walls with a grey sandpaper floor or black and white vertical striped walls (1 in. thick) with a wire-mesh floor. A removable Plexiglas door separated each chamber. A two-chamber apparatus, rather than a three-chamber, was used to eliminate age-related differences in novelty-induced exploration (Stansfield and Kirstein, 2006) that may likely be induced by a less familiar central choice chamber which is typically incorporated in the three-chamber place conditioning paradigm. A two-chamber apparatus is commonly used in adult place conditioning studies (Tzschentke and Schmidt, 1998). 2.3. Place conditioning procedure A biased place conditioning design was used evaluate and control for any chamber biases prior to treatment and to ensure that time spent in the paired chamber was not biased by an initial baseline preference for the paired chamber. Place conditioning consisted of four phases: handling (days one and two), baseline (day three), drug conditioning (days four-eleven) and test (day twelve). On days one and two (PND 28–29), rats were wheeled on a cart into the room where conditioning would take place and gently handled, weighed and placed in the injection position. Handling occurred once a day so that rats would become used to the experimenter. On day three (PND 30), a biased design was used to determine baseline chamber preferences. Naïve-rats were placed in the center of the two conditioning chambers in a dimly lit room and given free access to the entire apparatus for fifteen minutes. A camera was suspended above the conditioning apparatus to record behavior. Time (seconds) spent in each chamber and total distance moved (cm) were recorded. The camera signal was digitized and sent to a computer (Dell OptiPlex GX110) for analysis. Once data were received, movement was analyzed by distinguishing the tracked object (i.e.., Sprague-Dawley rat) from the black background (Ethovision video tracking system, Noldus, Netherlands). The chamber in which each animal spent the least amount of time was designated as the paired chamber on subsequent conditioning days whereas the alternate chamber was designated the unpaired chamber. On day four (PND 31), rats were injected with either saline or 1.25, 2.5 or 5.0 mg/kg MDMA (SigmaAldrich) dissolved in saline solution intraperitoneally (i.p.) and confined to the paired chamber for thirty minutes. The following day (PND 32), rats were injected with saline and confined to the unpaired chamber for thirty minutes. Lower doses of MDMA (1.25, 2.5 mg/kg MDMA) were used in the present study to investigate the effects of MDMA on reward at doses lower than what has been previously investigated in rodents (Daza-Losada et al., 2007). Control rats in the present study received saline injections in both chambers. Conditioning occurred once a day over eight consecutive days (PND 31–38) for a total of four paired and four unpaired chamber exposures. The apparatus was cleaned with Quatricide (Pharmacal Research Laboratories Incorporated) and ethanol prior to each trial to remove odors. On day twelve (PND 39), the conditioned effects of MDMA were tested. Rats were tested drug-free in the same manner as at baseline (day three). A total of thirty-eight rats were used for place conditioning (saline n = 7, 1.25 mg/kg MDMA n = 10; 2.5 mg/kg MDMA n = 10; 5.0 mg/kg MDMA n = 11). A place preference was defined as rats spending more time in the paired chamber relative to saline controls. 2.4. BrdU administration and tissue preparation After place conditioning, rats received two injections of 200 mg/kg bromodeoxyuridine (BrdU, Sigma-Adlrich) 18 h apart, to label the birth date of cells. Rats were euthanized either 24 h (to evaluate cell proliferation on PND 40) or 2 weeks (to assess the phenotypic fate on PND 54) after the last MDMA injection. Rats were anesthetized with

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pentobarbital, then transcardially perfused with heparinized 0.9% saline followed by 4% paraformaldehyde. Brains were fixed in 4% paraformaldehyde for 24 h, then transferred to 20% sucrose solution and sectioned coronally throughout the hippocampus using a cryostat (Leica, Germany) at 30 µM in a 1:6 series and stored in 24-well plates in cryoprotectant at − 20 °C. Neurogenesis in the dentate gyrus of the hippocampus was measured via immunofluorescence and stereological estimates of positive cell counts. 2.5. Histology and quantification For BrdU immunohistochemistry, serial sections throughout the hippocampus were processed so that each section is 180 µM apart. Free-floating sections were denatured using 2 N HCl and neutralized in 0.15 M borate buffer then washed in phosphate buffered saline (PBS). Sections were treated in 3% H2O2 solution to block endogenous peroxidase, washed in PBS then blocked in PBS + (PBS, 10% normal goat serum, 1% 100x Triton X, 10% BSA) for 1 h at 4 °C. Tissue was incubated overnight at 4 °C in mouse anti-BrdU (Chemicon MAB3424) diluted 0.25 µg/ml in PBS. Sections were washed in PBS, incubated in goat anti-mouse secondary antibody followed by ABC reaction (Vector Laboratories). BrdU immunoreactivity was visualized by metal enhanced DAB as a chromagen (Pierce). Tissue was mounted then dehydrated with xylene and coverslipped. For the double labeling of progenitor cells in the dentate gyrus free-floating sections were denatured using 2 N HCl and neutralized in 0.15 M borate buffer then washed in PBS. Tissue was blocked in PBS + (PBS, 10% normal goat serum, 1% 100x Triton X, 10% BSA) for 1 h at 4 °C and incubated for 48 h at 4 °C in an antibody cocktail of rat monoclonal anti-BrdU (AbD Serotec, Raleigh NC, #OBT0030G, 1:100) plus mouse anti-NeuN (Chemicon) in PBS. Sections were washed in PBS and incubated in a secondary antibody cocktail of goat anti-rat IgG Alexa Fluor 594 (Invitrogen, Eugene OR, #A11007) plus goat anti-mouse IgG Alexa Fluor 488 (Invitrogen) and coated with vectorshield mounting medium (Invitrogen). For BrdU immunohistochemistry cell counts were estimated based on sectioning and counting positively labeled cells in every 6th section of tissue (180 µm). A modification to the optical dissector method was used so that cells on the upper and lower planes were not counted to avoid counting partial cells. The number of BrdU+ cells counted in every 6th section was multiplied by 6 to get the total number of BrdU +cells in the dentate gyrus (Shors et al., 2002). For the quantification of double labeled cells using immunofluroescence, the number of BrdU and BrdU +NeuN labeled cells were estimated using every 12th section taken throughout the dentate gyrus. Positive labeling was verified by confocal microscopy (Zeiss) and was determined by excluding cells in the outermost focal plane to avoid counting partial cells. Cells determined to be BrdU positive and BrdU + NeuN positive were tallied and multiplied by the number of intervening sections. 2.6. Preparation of samples for flow cytometry To assess the feasibility of using an alternative and time efficient method for assessment of neurogenesis, a separate group of rats were pretreated with 5.0 mg/kg MDMA (n = 5) or saline (n = 4) using the same MDMA exposure protocol. Rats were euthanized with pentobarbital (100 mg/kg, BW). Prior to brain harvesting, rats were perfused transcardially with PBS (pH 7.4) for 5 min under pentobarbital anesthesia. Removed brain was dissected according to the protocol previously described (Bilsland et al., 2006). In short, hippocampal lobes were dissected from the brain into ice cold Hibernate-A solution and kept on ice (BrainBits, Springfields, IL). Once hippocampi were dissected from all animals, the white matter was scraped from the surface of the hippocampus using a flat spatula and samples were minced with the razor blade following the dissociation into a single cell suspension using MACS cell dissociation kit (Miltenyi

Biotech, Auburn, CA) according to manufacturer protocol. Samples were washed with Dulbecco's PBS and centrifuged at 3000 g. Supernatant was aspirated and samples were resuspended in 1 ml Dulbecco's PBS with light trituration following the addition of 3 ml of absolute ethanol to fix cells overnight in 75% ethanol. The fixed cells were subjected to standard procedure of antigen retrieval using 2 N HCl/ 5% Triton-X following neutralization with 0.1 M borate buffer (Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy By M. A. Hayat Published by Springer, 2002). Cells were double labeled using BrdU and NeuN antibodies. FITC-pre-conjugated mouse anti-BrdU from BD Pharmingen was used (San Diego, CA). The mouse anti-NeuN antibody (Millipore, Billerica, MA) was conjugated with Alexa Fluor 405 just before the analysis using DyLight Microscale Anitbody Labeling kit (Pierce, Rockford, IL). Samples were incubated overnight in the dark at 4 °C in labeling cocktail comprised of 20 µl anti-BrdU, 4 µl antiNeuN, 10 µl of 100 mg/ml RNase, and 46 µl of 1% BSA/0.5% Tween-20/ PBS. After incubation, samples were washed with Dulbecco's PBS, then resuspended in 1 ml of 50 µl/ml of the DNA intercalating agent propidium iodide (PI) and analyzed on the flow cytometer. 2.7. Flow cytometry protocol The analysis was performed using FACS Diva flow cytometry system (BD Biosciences, San Jose, CA). Cells were first gated according to DNA content represented by PI intensity. A histogram was used to isolate the events representing cells with diploid DNA content. The gated subpopulations of PI expressing cells were “back-gated” on the forward/side scatter plot to eliminate debris and reduce auto fluorescence prior to analysis. To measure amount of BrdU + cells, the secondary analysis plot was created using intensity of FITC fluorescence versus PI fluorescence. The subpopulation of FITC + cells was gated to discriminate cells not labeled with BrdU. At least twenty thousand PI expressing events were collected and the number of BrdU bearing cells counted. Data were expressed as number of BrdU + cells per 100 PI + cells. To measure amount of double labeled BrdU + NeuN positive cells, the Alexa-405 fluorescence was plotted versus FITC fluorescence and subpopulation of BrdU + NeuN cells was clustered in quadrant representing double positive signal. Negative controls were used to separate Alexa-405 positive cells. Data were expressed as number of NeuN + cells per 100 BrdU + cells. 2.8. Design and analyses It was our aim to investigate whether adolescent rats express a place preference for MDMA and whether these same doses alter neurogenesis in the hippocampus. A two-way mixed model analyses of variance (ANOVA), with Dose (saline, 1.25, 2.5, 5.0 mg/kg MDMA) as the between subjects factor and Trial (baseline, test) as the repeated measure, was used to determine the effects of MDMA on place conditioning and locomotor activity. Time spent in the paired chamber (sec in paired − sec in unpaired) was the dependent measure for place conditioning and total distance moved (cm) was the dependent measure for locomotor activity. It should be noted that saline control rodents tend to spend more time in the designated paired chamber during the post-test when a biased CPP experimental design is used. This is to be expected given the chamber were rodents spend the least amount of time at baseline is designated the paired chamber during conditioned trials. Rodents repeatedly given saline in both chambers come to spend equal amounts of time in both chambers and spend more time in the paired chamber. Therefore it is imperative treatment groups are compared to saline controls when determining if place conditioning has occurred. Because of these observations, a place preference for MDMA exposed rodents was defined as rats spending more time in the paired chamber relative to saline controls. For BrdU + and BrdU + NeuN cell counts, separate

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one-way analyses of variance (ANOVA) were used to determine the effects of Dose (saline, 1.25, 2.5, 5.0 mg/kg) for proliferation and/or survival. When appropriate, post hoc analyses such as simple effects and Fisher's least significant difference were used to isolate Dose and Time effects. Simple effects were used if a significant interaction was revealed. For analyses of double labeled cells from flow cytometry, a t-test was performed to detect differences between saline and 5.0 mg/kg MDMA. Statistical significance was denoted as alpha less than or equal to 0.05.

3. Results 3.1. MDMA-induced place conditioning in adolescent rats Chamber biases differed as a function of both Trial and Dose groups. Fig. 1A illustrates a significant Trial × Dose interaction for adolescents [F(3,68) = 2.69, P = 0.05] for time spent in the paired chamber at baseline or test expressed as a difference score (time in paired − time in unpaired). There were no significant effects of Dose at baseline (F(3,34) = 0.32, P N 0.05]; however, simple effects at expression revealed a significant effect of Dose (F(3,34) = 4.96, P b 0.05]. Subsequent post hoc analyses of Dose at expression revealed adolescents treated with 1.25 and 2.5 mg/kg MDMA spent more time in the paired chamber during the expression test than at baseline [Fisher's LSD, P b 0.05; indicated by * in Fig. 1A]. Only rats treated with 2.5 mg/kg MDMA spent more time in the paired chamber at expression than saline controls indicating only 2.5 mg kg MDMA

Fig. 1. Adolescents show MDMA place conditioning but no changes in locomotor activity. Adolescent rats received 4 injections of MDMA (1.25 mg/kg, 2.5 mg/kg, 5.0 mg/kg) or 0.9% saline solution every other day from PND 31–39. A) A preference for the paired chamber at 2.5 mg/kg MDMA was found. Saline n = 7; 1.25 mg/kg MDMA n = 10; 2.5 mg/kg MDMA n = 10; 5.0 mg/kg MDMA n = 11. Each bar represents mean and S.E.M. ⁎ = differs from baseline. # = differs from saline. A place preference was defined as being statistically different from saline controls. B) There were no Trial or Dose effects for locomotor activity. Same animals from panel A.

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produced a MDMA-induced place preference [Fisher's LSD, P b 0.05; indicated by # in Fig. 1A]. There were no Trial or Dose effects on locomotor activity in the place conditioning apparatus [Fig. 1B; Trial × Dose (F(3, 60) = 0.28, P N 0.05]. Together these data indicate that adolescents express a place preference at a relatively low dose of MDMA. 3.2. MDMA-induced increase in cell proliferation To determine the effects of MDMA on progenitor cell proliferation BrdU was administered 24 h before brain removal and the resulting number of BrdU positive cells in the dentate gyrus was estimated. A one way ANOVA revealed a significant effect of dose on the number of BrdU + cells in the dentate gyrus [F(3,11) = 3.59, P b 0.05]. As can be seen in Fig. 2A, the highest dose of MDMA (5.0 mg/kg) produced a significant increase in the number of BrdU positive cells in the subgranular zone compared to saline, 1.25 mg/kg MDMA and 2.5 mg/ kg MDMA (indicated by *). These results show that repeated exposure to high doses of MDMA during adolescence significantly elevates hippocampal progenitor cell proliferation in adolescence. A photomicrograph of a representative section of BrdU + staining in the dentate gyrus is shown in low magnification in Fig. 2C. 3.3. MDMA decreases cell survival and neurogenesis in the hippocampus In order to investigate the effects of MDMA administration of cell survival, BrdU was administered 2 weeks before brain removal and the number of BrdU positive cells in the dentate gyrus was quantified. A one way ANOVA revealed a significant effect of dose on the number of surviving BrdU positive cells in the granular cell layer of the dentate gyrus [F(3,15) = 3.76, P b 0.034]. Fig. 2B illustrates that 5.0 mg/kg MDMA significantly reduced the number of BrdU positive cells compared to saline (P b 0.05; indicated by *). These data indicate that exposure to high doses of MDMA during adolescence results in a significant reduction in the number of surviving progenitor cells in the hippocampus. High magnification confocal micrographs of NeuN (A) BrdU (B) and BrdU + NeuN (C) in the granular layer of dentate gyrus 2 weeks after BrdU administration are shown in Fig. 3. The phenotypic fate of surviving cells was first determined by immunofluroescent double-labeling of BrdU and NeuN. A one way ANOVA revealed a significant effect of dose on the number of double labeled neurons in the dentate gyrus [F(3,15) = 3.46, P b 0.043]. As can be seen in Fig. 3D, 5.0 mg/kg MDMA significantly diminished the number of BrdU+ NeuN positive cells in comparison to saline (P b 0.05), suggesting that repeated exposure to high doses of MDMA during adolescence significantly reduces hippocampal neurogenesis. In addition to the immunofluroescent assessment of neurogenesis, a subset of rats that received either 5.0 mg/kg MDMA or saline were used to assess the feasibility of using an alternative and time efficient method for measuring neurogenesis via flow cytometry. A similar approach has been used to assess neurogenesis by labeling cells with BrdU and 7Amino-actinomycin D (7-AAD) (Bilsland et al., 2006; Balu et al., 2009). Analysis of the total number of BrdU labeled cells in the hippocampus did not differ between saline (mean= 9.0 S.E.M.= ±2.74) and MDMA (mean= 6.2 S.E.M.= ±0.49) [t(7)= 1.1, P = 0.15]. It is important to note that the quantitation of BrdU + cells obtained from frozen sections were selectively counted only in the granular cell layer of the dentate gyrus, whereas FACS analysis counts all BrdU+ cells in the hippocampus (since samples obtained for FACS come from complete hippocampal dissection). Since Brdu + cells are often reported in other subregions of the hippocampus a replication of Brdu+ cell counts using stereological methods and FACS is not meaningful. For the assessment of neurogenesis, FACS analysis is comparable to stereologic counts since new BrdU + NeuN cells are only located in the dentate gyrus and not other subregions of the hippocampus. As can be seen in Fig. 4 (panel l), the number of BrdU + NeuN double labeled neurons significantly decreased

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peaks with greater PI fluorescence that represent clusters of cells unsuccessfully dissociated. Events within the first gate on a forward/side scatter plot are shown in panels b and g and excludes events with low forward and side scatter excludes debris. Panels c and h show scatter plots of PI fluorescence (x-axis) vs. FITC fluorescence (y-axis) with a cluster of gated events correspond to BrdU positive cells. Alexa-405 fluorescence (y-axis) vs. FITC fluorescence (x-axis) are shown in panels d and i, which allowed for the visualization of Alexa-405 (NeuN positive) events. Quantification of each gate is shown for saline (panel e) and MDMA (panel j). Panel k shows a sample not stained with Alexa405 conjugated anti-NeuN which was used as a negative control. 4. Discussion

Fig. 2. Effect of adolescent MDMA administration on proliferation and survival of progenitor cells in the dentate gyrus. A) Effect of MDMA during adolescence on cellular proliferation in the dentate gyrus. After MDMA administration ended, half of the rats from each condition in Fig. 1 received BrdU and were euthanized 24 h later. The number of BrdU positive cells in the subgranular zone of the dentate gyrus was estimated. A significant effect of MDMA dose on the number of BrdU + cells in the dentate gyrus was detected [P b 0.03]. The highest dose (5.0 mg/kg) of MDMA produced a significant increase in the number of BrdU positive cells in the subgranular zone. ⁎ indicates significant difference from saline, 1.25 and 2.5 mg/kg MDMA. B) Effect of MDMA during adolescence on cell survival in the dentate gyrus. Following MDMA administration, the other half of the rats received BrdU and were euthanized 2 weeks later. The number of surviving BrdU positive cells were estimated in the granule cell layer of the dentate gyrus. 5.0 mg/kg MDMA significantly reduced the number of BrdU positive cells compared to saline (P b 0.05; indicated by *), indicating that exposure to high doses of MDMA during adolescence results in a significant reduction in the number of surviving progenitor cells in the hippocampus. Each bar represents mean and S.E.M. C) Photomicrograph of a representative section of BrdU + staining in the dentate gyrus. BrdU + cells are shown in low magnification, scale = 100 μM.

by 32% in adolescent rats that received MDMA [t(7) = 3.5, P b 0.005], results which confirm our initial findings that MDMA decreases hippocampal neurogenesis in adolescent rats. Details of analysis by flow cytometry are shown in Fig. 4 comparing a saline treated (a–e) to a 5.0 mg/kg MDMA treated (panels f–j) animal. Panels a and f show the histograms of PI fluorescence. The gated peak represents events corresponding to a homogenous amount of DNA which excludes

The present investigation illustrates the effects of MDMA exposure during adolescence on place conditioning and cell proliferation/ neurogenesis in the hippocampus. Exposure to a moderate dose of MDMA (2.5 mg/kg) during adolescence produced a CPP (Fig. 1A) and did not alter locomotor activity, progenitor cell proliferation or neurogenesis (Figs. 1B, 2 and 3). This is in contrast to high doses of MDMA (5.0 mg/kg) in which no CPP was demonstrated (Fig. 1A) yet significant increases in proliferation and diminished cell survival and neurogenesis in the dentate gyrus were observed (Figs. 2 and 3). Although there was a significant increase in time spent in the paired chamber for adolescent rats treated with 1.25 mg/kg MDMA, this effect was not significantly different from saline controls and indicated rats did not find this particular low dose of MDMA rewarding enough to produce a place preference. There were no effects of 1.25 mg/kg MDMA on cell proliferation/neurogenesis (Figs. 2 and 3). These data are the first to investigate the effects of lower doses of MDMA in adolescent rats on place conditioning and cell proliferation/neurogenesis. It is important to note that lower doses of MDMA administered in the current study in which adolescent rats express a CPP (2.5 mg/kg) may be comparable to those typically used by humans (Pizarro et al., 2004). Interestingly, only the highest dose of MDMA (5.0 mg/kg) reduced progenitor cell survival and neurogenesis which is markedly higher than doses typically used by humans (Pizarro et al., 2004). Although it is difficult to compare the exact absorption, metabolism and excretion rate of MDMA in rodents and humans, especially in the less investigated adolescent populations, it is interesting that effects of place conditioning are exhibited at relatively low (Fig. 1A) while inhibitory effects of MDMA on neurogenesis were only observed at higher doses. These data suggest that MDMA alters cell proliferation and cell survival in the dentate gyrus of the hippocampus in a dose dependent manner. Chronic exposure to MDMA during adolescence significantly increased proliferative activity in the subgranular zone of the dentate gyrus. This increase in proliferation varied with the dose of MDMA administered where saline, 1.25 mg/kg and 2.5 mg/kg had similar levels of proliferation and the highest dose of MDMA (5.0 mg/kg) administered significantly elevated proliferative activity (see Fig. 2A). Binge administration of MDMA in adult rodents was not found to alter BrdU and Ki67 immunolabeling, suggesting that the proliferative capacity of hippocampal progenitors is intact in the adult animal (Hernandez-Rabaza et al., 2006). It has recently been demonstrated that chronic maternal MDMA (1.25 mg/kg and 20.0 mg/kg) administration reduced progenitor cell proliferation in the dentate gyrus of offspring (Cho et al., 2008) demonstrating the unique vulnerability of the developing brain to MDMA. Many studies provide evidence demonstrating that drugs of abuse influence neural progenitor cell proliferation, survival and differentiation into neuronal phenotype in the granular cell layer of the dentate gyrus (Eisch et al., 2000; Yamaguchi et al., 2004; Nixon and Crews, 2002). However, few studies have investigated the effects of adolescent drug exposure on hippocampal neurogenesis (Crews et al., 2006). The present data indicate that exposure to MDMA during adolescence results in a significant reduction in the number of

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Fig. 3. Effect of adolescent MDMA administration on hippocampal neurogenesis. Immunofluroescent confocal micrographs of A) NeuN B) BrdU and C) BrdU + NeuN merge in the granular layer of dentate gyrus 2 weeks after BrdU. D) The phenotypic fate of surviving cells was determined by immunofluroescent double-labeling of BrdU and NeuN. A one way ANOVA found a significant effect of dose on the number of double labeled neurons in the dentate gyrus. 5.0 mg/kg MDMA significantly diminished the number of BrdU + NeuN positive cells in comparison to saline (P b 0.05), suggesting that repeated exposure to high doses of MDMA during adolescence significantly reduces hippocampal neurogenesis. Each bar represents mean and S.E.M.

surviving progenitor cells in the dentate gyrus of the hippocampus (Fig. 2B). Cho and colleagues demonstrated that chronic maternal exposure to a high dose of MDMA (20.0 mg/kg) decreased the number of BrdU positive cells 28 days post BrdU labeling in the dentate gyrus of female offspring, an effect which was not observed with low doses of MDMA (Cho et al., 2008). Additionally, binge administration of MDMA in adult rodents significantly diminished the survival rate of new born granular cells in the dentate gyrus two weeks after BrdU administration (Hernandez-Rabaza et al., 2006). In order to assess the phenotype of surviving cells, a quantitative analysis of the number of cells expressing both BrdU and NeuN, a marker of mature neurons, was performed using confocal microscopy and FACS. Fig. 3D illustrates the significant reduction in the number of BrdU + NeuN positive cells after exposure to 5.0 mg/kg MDMA, suggesting that repeated exposure to high doses of MDMA during adolescence significantly reduces hippocampal neurogenesis, a finding which was replicated via flow cytometry using a separate group of rats. The use of flow cytometry to assess neurogenesis has been demonstrated previously (Bilsland et al., 2006; Balu et al., 2009). Mechanisms central to the reduction observed in hippocampal neurogenesis after adolescent exposure to high doses of MDMA are presently unknown. Neurochemical effects of MDMA include reversed functioning of monoamine transporters and subsequent enhanced

release of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) (Gudelsky and Yamamoto, 2008). MDMA elevates DA, NE and acetylcholine in the HPC (Fitzgerald and Reid, 1990; Shankaran and Gudelsky, 1998; Nair and Gudelsky, 2006) and 5-HT in rat striatum, prefrontal cortex and HPC (Gudelsky and Nash, 1996; Mechan et al., 2002). The administration MDMA has complex pharmacological actions which may affect hippocampal proliferation and neurogenesis in a number of ways. For example, dopaminergic fibers have been observed in the subgranular zone and treatment with the neurotoxin 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) decreased progenitor cell proliferation (Hoglinger et al., 2004). In addition, serotonergic input to the hippocampus and subventricular zone upregulates adult neurogenesis (Brezun and Daszuta, 1999) and proserotonergic drugs like selective 5-HT reuptake inhibitors increase hippocampal neurogenesis (Malberg and Duman, 2003). Furthermore, MDMA is known to produce a depletion in 5-HT and 5-HIAA in the cortex, HPC and striatum of rats 7 days after exposure (O'Shea et al., 1998) and depletion of 5-HT via 5,7-HT reduced cell survival in the dentate gyrus (Brezun and Daszuta, 2000). In sum, these two experiments provide results suggesting exposure to high doses of MDMA via the place conditioning procedure during adolescence alters neural progenitor cell proliferation and diminishes hippocampal neurogenesis in the developing brain. Low

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Fig. 4. Gating for Flow Cytometry Analysis of Neurogenesis. Panels F–J correspond to animals treated with 5.0 mg/kg MDMA while panels A–E correspond to saline-treated animals. A and F show histograms of PI fluorescence. The gated peak represents events corresponding to a homogenous amount of DNA. This gate excludes peaks with greater PI fluorescence that represent clusters of cells unsuccessfully dissociated. Panels B and G show events within the first gate on a forward/side scatter plot. Exclusion of events with low forward and side scatter excludes debris. C and H show scatter plots of PI fluorescence vs. FITC fluorescence. Cluster of gated events correspond to BrdU positive cells. D and I show a scatter plot of Alexa-405 fluorescence vs. FITC fluorescence. This allows for the visualization of Alexa-405 (NeuN positive) events. Quantification of each gate is shown in E and J. K shows a sample not stained with Alexa-405 conjugated anti-NeuN which was used as a negative control. L Illustrates a significant decrease in the number of BrdU+ NeuN double labeled neurons by 32% in adolescent rats that received MDMA compared to saline (P b 0.005), confirming our initial findings using immunofluroescence that MDMA decreases hippocampal neurogenesis in adolescent rats.

dose MDMA (2.5 mg/kg) was found to be rewarding but did not affect locomotor activity, proliferation, survival or differentiation of neural progenitor cells, while high doses of MDMA increased proliferation and diminished neurogenesis. It should be noted the results of the place conditioning experiment should be attributed to drug wanting/ seeking behaviors elicited by drug-associated salient stimuli in the MDMA user's environment rather than in comparison to drug-taking behaviors. This caveat is key for interpretation of the present data given that place conditioning measures the ability of drug-associated environmental cues to elicit drug-seeking behaviors. It would be interesting to see if similar interactions between MDMA, behavior and neurogenesis would be revealed using measures of drug-taking behaviors such as self-administration.

It is expected the present data will provoke interest in examining more comprehensive studies examining the effects of early MDMA use on drug wanting/seeking behaviors as well as the effects of repeated MDMA use during adolescence on the development of the brain. For example, it may seem counterintuitive that MDMA could be both rewarding and also detrimental to neurogenesis. It is important to note that the rewarding effects on behavior and detrimental effects on neurogenesis are dose dependent in adolescent rats, effects that could be exacerbated (or abolished) in adult rodents. The moderate dose produced a place preference while the highest dose decreased neurogenesis. These data suggest the rewarding and detrimental effects of MDMA produced different dose response curves with peak effects being shifted to the right for neurogenesis in comparison to place

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preferences. Frequently, place conditioning has been used to condition the stimulus properties of drugs of abuse to distinct contextual cues via classical conditioning (Tzschentke, 2007), however, it is not known whether place conditioning is dependent on generation of new neurons. Data in the present experiments suggest that place conditioning is not dependent on the birth of new neurons. Future studies should examine if place preferences can be abolished by inhibiting neurogenesis with the chemotherapeutic agent methylazoxymethanol acetate (MAM). It should also be noted that neurogenesis is important for some but not all hippocampal dependent learning (i.e., trace conditioning) and particularly if temporal encoding of episodic memory is required to learn the task (Shors et al., 2002). 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