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. Author manuscript; available in PMC: 2019 Jul 13.
Published in final edited form as: Prog Neuropsychopharmacol Biol Psychiatry. 2018 Mar 28;85:46–53. doi: 10.1016/j.pnpbp.2018.03.023

Chronic administration of amphetamines disturbs development of neural progenitor cells in young adult nonhuman primates

Rahul R Dutta 1, Michael A Taffe 1, Chitra D Mandyam 1,2,3
PMCID: PMC5962428  NIHMSID: NIHMS962093  PMID: 29601895

Abstract

The detrimental effects of amphetamines on developmental stages of NPCs are limited to rodent brain and it is not known if these effects occur in nonhuman primates which are the focus of the current investigation. Young adult rhesus macaques either experienced MDMA only, a combination of amphetamines (MDMA, MDA and methamphetamine) or no amphetamines (controls) and hippocampal tissue was processed for immunohistochemical analysis. Quantitative stereological analysis showed that intermittent exposure to MDMA or the three amphetamines over 9.6 months causes > 80% decrease in the number of Ki-67 cells (actively dividing NPCs) and > 50% decrease in the number of NeuroD1 cells (NPCs that have attained a neuronal phenotype). Co-labeling analysis revealed distinct, actively dividing hippocampal NPCs in the subgranular zone of the dentate gyrus that were in transition from stem-like radial glia-like cells (type-1) to immature transiently amplifying neuroblasts (type-2a, type-2b, and type-3). MDMA-alone and the combination reduced the number of dividing type-1 and type-3 NPCs and cells that were not NPCs. These data indicate that amphetamines interfere with the division and migration of NPCs. Notably, the reduction in the number of NPCs and immature neurons were not associated with changes in cell death (via apoptosis) or granule cell neuron numbers, indicating that amphetamines selectively affected the generation and maturation of newly born granule cell neurons. In sum, our findings suggest that alterations in the cellular composition in the dentate gyrus during chronic exposure to amphetamines can effect neuroplasticity in the hippocampus and influence functional properties of hippocampal neurons.

Keywords: Granule Cell Neurons, Hippocampus, Ki-67, NeuroD1, MDMA, Methamphetamine

Introduction

Illicit drugs have posed serious political, social, and economic problems to countries around the world. Of these drugs, (±) 3, 4- methylenedioxymethamphetamine (MDMA), (±) 3,4- methylenedioxyamphetamine (MDA) and (+) methamphetamine (METH) all belong to a class of illicit substances known as amphetamine type psychostimulants. Although some amphetamines (e.g., d-amphetamine, d-methamphetamine), can be used therapeutically by clinicians to help treat disorders such as attention deficit hyperactive disorder and narcolepsy, most other amphetamines are only used recreationally (Teixeira-Gomes et al., 2015). MDMA, MDA and METH all are psychoactive substances that can act as stimulants, euphorics, anorectics, entatogenics, and/or hallucinogenic agents (Carvalho et al., 2012).

In the brain, a shared feature of MDMA, MDA, and METH is that they all act to release monoamines (dopamine, norepinephrine, serotonin) into the synaptic cleft through a multitude of pharmacological mechanisms (Lewin et al., 2011; Miller, 2011). In addition these drugs can act as direct agonists to the alpha adrenergic and several serotoninergic receptors, interactions that are part of the basis underlying their physiological and psychological effects (Ray, 2010). Repeated use of amphetamines can have lasting detrimental consequences. For example, heavy MDMA use has been associated with changes in mental processing speed, impulsivity, mood and working memory (Wareing et al., 2000; Parrott, 2012). METH is also known to impair both spatial and non-spatial working memory in humans and nonhuman primates, which suggests potential hippocampal neurotoxicity (Camarasa et al., 2012; Groman et al., 2012; Sofuoglu et al., 2016; Zhong et al., 2016).

Neurotoxicity by amphetamines as a function of decreased adult neurogenesis (a phenomenon that continuously generates newly born neurons in the subgranular zone of the dentate gyrus of the hippocampus (Altman, 1969)) has been reported in rodent studies. However, only a small body of research has been devoted to understanding the effect of these amphetamines on the development of neural progenitor cells (NPCs) that contribute to adult neurogenesis. Although it is poorly understood at present, cognitive changes associated with amphetamine use could be due in part to disruption of adult neurogenesis. New neurons come about as a result of either the division of neural stem cells or the division of early neural progenitor cells (NPCs; (Enikolopov et al., 2015; Goncalves et al., 2016)). Not all NPCs survive and mature into new neurons (Dayer et al., 2003), but the ones that survive and mature form functional connections with pre-existing neural circuits (Toni et al., 2008). Even though no functional significance can be attributed to individual neurons as they are newly incorporated into circuits of the dentate gyrus, much research has gone into establishing correlations between the role of new neurons, such as increased neuronal survival in the dentate gyrus and learning and memory behaviors dependent on the hippocampus (Sahay et al., 2011; Galinato et al., 2017). Understanding how prevalently used amphetamines like MDMA, MDA and METH impact NPCs and their development could help further understanding of the neural basis of the cognitive deficits associated with heavy use.

In this context, METH exposure in rodents produces a hostile cellular environment in the progenitor pool (Yuan et al., 2011). For example, METH reduces net proliferation of NPCs and number of immature neurons by reducing the number of proliferating preneuronal neuroblasts (type-2a cells; (Kronenberg et al., 2003)) and increasing the number of proliferating preneuronal progenitor cells (type-3 cells; (Kronenberg et al., 2003; Yuan et al., 2011)), suggesting that a decrease in the number of progenitors and immature neurons, to a large degree, is attributable to the decrease in the ability of neuroblasts to divide and produce stable NPCs that survive as immature neurons (Teuchert-Noodt et al., 2000; Mandyam et al., 2008; Schaefers et al., 2009; Venkatesan et al., 2011; Yuan et al., 2011; Baptista et al., 2014). Parallel work in rodents demonstrates that MDMA affects net proliferation and survival of NPCs, suggesting that similar hostile cellular environment is observed in rodents after either METH or MDMA exposure (Hernandez-Rabaza et al., 2006; Cho et al., 2008; Garcia-Cabrerizo & Garcia-Fuster, 2016). However, these studies on toxic effects of amphetamines on NPCs are limited to rodent models. Therefore, the current study tested the hypothesis that MDMA, MDA and METH reduce net proliferation and survival of NPCs in the dentate gyrus of nonhuman primates, and these effects are attributed to altered division and migration of NPCs.

Materials and Methods

Animals

Fourteen male rhesus monkeys (Macaca mulatta; Chinese origin) participated in this study. Animals were ages 7–10 years old when euthanized. Daily chow (Lab Diet 5038, PMI Nutrition International; 3.22 kcal of metabolizable energy (ME) per gram) was modified individually by the veterinary weight management plan and ranged from 160 to 230 g per day for the animals in this study. Animals were individually housed in a controlled temperature environment (23.5°C), on a 12 h: 12 h light:dark cycle and fed in the home cage. The animals’ normal diet was supplemented with fruit or vegetables seven days per week and water was available ad libitum in the home cage at all times. Animals on this study had previously been immobilized with ketamine (5–20mg/kg) no less than semiannually for purposes of routine care and some experimental procedures. Eleven animals also had various acute exposures to scopolamine, raclopride, methylphenidate, SCH23390, Δ9-THC, tetrahydrocannabinol nicotine, and mecamylamine in behavioral pharmacological studies similar to those described in (Taffe et al., 1999; Taffe et al., 2002; Katner et al., 2004; Von Huben et al., 2006; Taffe, 2012). These experimental drug treatments had been administered a minimum of one year prior to the start of MDMA, MDA and METH investigations, involved doses, substances and regimens not reported to have lasting effects on the measures used here, and thus were not anticipated to have any bearing on the results of the current study. All protocols were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute (La Jolla). The United States National Institutes of Health guidelines for laboratory animal care were followed (Clark et al., 1997).

Experimental Design

Drug naïve group

Hippocampal tissue from three animals that did not experience any amphetamine or drug treatment were used for analysis. Two left hemispheres and one right hemisphere were used for the current study. Brain tissue from the other hemispheres were used for another study published elsewhere (Taffe et al., 2010).

Drug challenge group

For these studies, doses of MDMA (0.0, 0.56, 1.0, 1.78, or 2.4 mg/kg), MDA (0.56, 1.0, 1.78, 2.4 mg/kg) or METH (0.1, 0.32, 0.56, 1.0 mg/kg) were administered intramuscularly in a volume of 0.1 ml/kg saline. MDMA, MDA and METH were provided by the National Institute on Drug Abuse. Treatment was pseudorandomized within compound to the extent possible with the small sample size to minimize the impact of any potential order effects. Generally, the MDMA studies were conducted first, MDA second, and METH last; however, there was some degree of overlap of the schedule across compounds. Animals were injected via brief physical restraint using the moveable back of the home cage, a procedure to which they are well accustomed. All animals remained in home cage for the duration of the study. The dose range was based on pill content analyses suggesting ~75–125 mg MDMA per “Ecstasy” pill thus 1–1.78 mg/kg MDMA for a single pill taken by the standard 70kg person but as much as 2.5 mg/kg in a 50 kg woman or as little as 0.83 mg/kg in a 90 kg man. Relevant dose ranges for MDA and meth were determined initially by reference to MDMA:MDA and MDMA:meth ratios in pills analyzed by ecstasydata.org. These ranges were further refined based on pilot studies conducted for this and other projects and taking into consideration the minimum dose threshold for lasting or neurotoxic effects. All challenges were administered in the middle of the light cycle, with active doses separated by 1–2 weeks. Animals were visually observed for a period of two hours following injections and efforts were made to minimize noise and excitement in rooms during these intervals. Normal daily activity such as afternoon feedings and interactions with other animals not on the study resumed after the two hour interval. Details on the amphetamine regimen and behavioral studies conducted on the animals used in the current study are reported elsewhere (Crean et al., 2006; Taffe et al., 2006; Crean et al., 2007; Von Huben et al., 2007).

Tissue Preparation

After completion of the studies, monkeys were immobilized with 10 mg/kg ketamine/5 mg/kg xylazine and euthanized by 10 mg/kg pentobarbital followed by transcardial perfusion with ice-cold PBS. Brains were hemisectioned, transported on dry ice, and frozen at −80°C. Left or right hemispheres were used for histological analysis (control group: two left hemispheres and one right hemisphere; amphetamine groups: all left hemispheres). The frozen brain hemisphere was thawed on ice to −20°C, and blocks of brain tissue containing the hippocampal formation were dissected and processed for immunohistochemistry (Taffe et al., 2010). Hippocampal-enriched blocks were immediately immersed in cold, freshly prepared paraformaldehyde solution (pH 7.4) and left to post-fix for 5–7 days at 4°C. Blocks of brain tissue were then cryoprotected in 30% sucrose, after which they were sectioned coronally on a freezing microtome into 40 μm sections. Hippocampal sections (about 250 sections in total for each monkey) were serially collected in six wells and stored in PBS containing sodium azide (0.1%). Nine sections per animal (approximately every 30th section through the hippocampus), each containing different depths of the hippocampus were mounted on slides and were air dried and processed for immunohistochemistry (Perera et al., 2007; Taffe et al., 2010). Slide mounted sections were processed for each of 3 biomarkers, Ki-67, NeuroD, and activated caspase 3 (AC-3) and for the two sets of co-labeling analysis. All sections were mounted and stained by an observer blinded to the study.

Antibodies and Immunohistochemistry

The following primary antibodies were used for immunohistochemistry (IHC): rabbit anti-Ki-67 (1:500, Neo Markers), goat anti-NeuroD1 (1:500, SantaCruz Biotechnology), rabbit anti-AC-3 (1:500, Cell Signaling), goat anti-Sox-2 (SRY (sex determining region Y)-box 2, 1:50; Santa Cruz Biotechnology), chicken polyclonal anti-GFAP (glial fibrillary acidic protein, 1:500; Abcam). The sections used for IHC were pretreated, blocked and incubated with the aforementioned antibodies followed by biotin-tagged secondary antibodies (Taffe et al., 2010).

Microscopic Analysis

Immunoreactive cells in the SGZ (i.e., cells that touched and were within three cell widths inside and outside the hippocampal granule cell- hilus border) were quantified by an observer blinded to the animal groups with a Zeiss Axiophot photomicroscope (x60 magnification) using manual counting for positive cells. Area measurements were done via contouring in the optical fractionator setting of SteroInvestigator. These techniques were both used to quantify the amount of cells per mm2 for Ki-67, Neuro D1, and AC-3.

For type-1, type-2a, type-2b, and type-3 phenotype analysis, five sections through the hippocampus were triple-labeled with one of the combinations GFAP/Sox2/Ki-67 or Sox2/NeuroD1/Ki-67. All Ki-67-IR cells in the SGZ, approximately 15 Ki-67-IR cells from each monkey, were scanned and analyzed for Ki-67+/Sox2+/GFAP+ (type-1), Ki-67+/Sox2+/NeuroD1− (type-2a), Ki-67+/Sox2+/NeuroD1+ (type-2b), Ki-67+/Sox2−/NeuroD1+ (type-3), or Ki-67-alone labeling (Taffe et al., 2010). All labeling was visualized and analyzed using a confocal microscope (LaserSharp 2000, version 5.2, emission wavelengths 488, 568, and 647 nm; Bio-Rad Laboratories).

Measurement of hippocampal granule cell number was performed as previously described (Taffe et al., 2010). Quantitative analysis to obtain unbiased estimates of the total number of hippocampal granule layer cell bodies was performed on a Zeiss AxioImagerA2 Microscope equipped with MicroBrightField (Colchester, VT) Stereo Investigator software, a three-axis Mac 5000 motorized stage (Ludl Electronics Products, Hawthorne, NY), a digital MRc video camera (Zeiss, Thornwood, NY), PCI color frame grabber, and personal computer workstation. All 40 μm sections from the hippocampus were saved in strict anatomical order. Systematic random sampling of the hippocampus consisted of a one-in-thirty section analysis, and nine sections were analyzed per monkey. All sections for quantitative analysis were counterstained with Nuclear Fast Red, and all portions of the granule cell layer were examined. Live video images were used to draw contours delineating the dentate gyrus granule cell layer. All contours were drawn at low magnification using a Zeiss Neoflaur 5x objective, numerical aperture 0.15. Granule cell layer area was measured. After determination of mounted section thickness, z-plane values, and selection of contours, an optical fractionator analysis was used to determine hippocampal granule cell neuron number. A counting frame of appropriate dimensions denoting forbidden and nonforbidden zones (10 μm × 10 μm × 2 μm counting grid, and a 2 μm top and bottom guard zone) was superimposed on the video monitor, and the optical fractionator analysis was performed using a Zeiss Plan Apochromat 63x oil objective, numerical aperture 1.4 and a 1.4 auxillary condenser lens. Cells were identified as neurons based on standard morphology, and only neurons with a focused nucleus within the nonforbidden regions of the counting frame were counted. Over 500 cells per animal were counted. The total number of granule cells is presented as the average density of cells (cells/μm3). Granule cell number estimates were made by an observer blind to the study.

Data Analysis

One way randomized block analysis of variance (ANOVA) was employed to evaluate significance in differential expression of Ki-67, NeuroD1, and AC-3, between controls and either MDMA only exposure group or controls and MDA/MDMA/METH exposure group. Total granule cell neuron differences between controls and drug exposure groups also were analyzed by one way ANOVA. Dunnett’s post hoc test was applied after each one way ANOVA analysis. Values of p< 0.05 were considered statistically significant. Two way ANOVAs with treatment and cell type as a between-subject factor was performed to determine effects of amphetamine groups on co-labeling analysis. Following significant interaction (p<0.05), post hoc comparisons were performed to determine individual differences. All analysis was done on GraphPad Prism (v. 6; GraphPad Software, Inc, San Diego CA).

Results

Doses of amphetamines administered to subjects do not differ significantly

The amount of amphetamines administered to each subject in the study did not significantly differ, and the average amount of MDMA administered to all eleven subjects (in both amphetamine exposure groups) was 17.08 (SEM: 1.75) mg/kg (Table 1). The average amount of MDMA administered to five MDMA only exposed animals was 15.02 (1.72) mg/kg (Table 1). The average amount of MDMA administered to six MDA/MDMA/METH animals was 18.80 (2.81) mg/kg (Table 1). The average amount of MDA administered to six MDA/MDMA/METH animals was 7.54 (1.25) mg/kg (Table 1). The average amount of METH administered to six MDA/MDMA/METH animals was 1.55 (0.37) mg/kg (Table 1).

Table 1.

Experimental Design and Average Dose Administration per Experimental Group

Total amphetamine doses over 9.6 months
Treatment MDA MDMA METH Citations
MDMA ONLY - 15.6 mg/kg - (Taffe et al., 2006; Von Huben et al., 2007)
MDMA ONLY - 15.6 mg/kg -
MDMA ONLY - 20.6 mg/kg -
MDMA ONLY - 10.1 mg/kg -
MDMA ONLY - 13.2 mg/kg -
AVERAGE: N/A 15.02 mg/kg N/A
MDA, MDMA, METH 6.78 mg/kg 12.12 mg/kg 2.84 mg/kg (Crean et al., 2006; Crean et al., 2007)
MDA, MDMA, METH 5 mg/kg 11.78 mg/kg 0.32 mg/kg
MDA, MDMA, METH 5 mg/kg 15.27 mg/kg 1.94 mg/kg
MDA, MDMA, METH 10 mg/kg 21.55 mg/kg 1.30 mg/kg
MDA, MDMA, METH 12.52 mg/kg 29.06 mg/kg 2.04 mg/kg
MDA, MDMA, METH 5.96 mg/kg 23.06 mg/kg 0.84 mg/kg
AVERAGE: 7.54 mg/kg 18.80 mg/kg 1.55 mg/kg
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A total of 14 young adult rhesus macaque monkeys, age 7–10 years, were used in this experiment. Three of the monkeys were used as controls, five were exposed to doses of MDMA, and six were exposed to doses of MDA, MDMA, and METH. Over the span of 9.6 months, 11 monkeys were given doses of either MDMA only or MDA, MDMA, and methamphetamine according to their respective experimental group. Monkeys given only MDMA received an average dose of 15.02 mg/kg over the 9.6 month period while monkeys given MDA, MDMA, and METH were given doses of 7.54 mg/kg, 18.80 mg/kg, and 1.55 mg/kg respectively in a 9.6 month period. MDMA was administered intramuscularly and doses of 0.00, 0.56, 1.0, 1.78, or 2.4mg/kg in an injection volume of 0.1 ml/kg with saline were separated by 1–2 weeks in a pseudorandomized order. MDMA, MDA, METH were administered intramuscularly and doses of MDMA (0.00, 0.56, 1.0, 1.78, or 2.4mg/kg), MDA (0.00, 0.56, 1.0, 1.78, or 2.4mg/kg) and METH (0.1, 0.32, 0.56, 1.0 mg/kg) were given in an injection volume of 0.1 ml/kg with saline. Similar to the MDMA only group, treatment order was pseudorandomized in the MDMA/MDA/METH group and randomized within compound to the extent possible to minimize the impact of any potential order effects. Generally, the MDMA studies were conducted first, MDA second and the METH last; however, there was some degree of overlap of the schedule across compounds.

Amphetamines significantly decrease proliferation of NPCs in the dentate gyrus

Ki-67 cell density (cells/mm2) was calculated by dividing the total number of Ki-67 immunoreactive cells per animal by total granule cell layer area per animal. One way ANOVA analysis confirmed that animals exposed to MDMA and animals exposed to MDA/MDMA/METH had significantly lower number of Ki-67 cells (F(2,11)= 15.03, p=0.0007) compared to drug naïve controls. MDMA exposure caused an 82% decrease in the number of Ki-67 cells in comparison with controls while MDA/MDMA/METH exposure caused an 87% decrease in the number of Ki-67 cells (Figure 1). Dunnett’s post hoc analysis further confirmed a significant effect of MDMA and MDA/MDMA/METH (p<0.01, and p<0.002 respectively).

Figure 1.

Figure 1

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(a–d) Photomicrographs of immunoreactive cells labeled with Ki-67 (a), NeuroD1 (b), AC-3 (c), and nuclear staining FastRed (d). Scale bar in d is 20μm, applies a–d. Hil, hilus; Mol, molecular layer; GCL, granule cell layer; GCNs, granule cell neurons. (e–h) Quantitative analysis of immunoreactive cells stained for Ki-67 (e), NeuroD1 (f), AC-3 (g) and granule cell neurons (h). Data is expressed as mean cells/mm2 ± SEM (control n=3, MDMA only n=5, MDA/MDMA/METH n=6). *p<0.05 compared with drug naïve controls.

Amphetamines significantly decrease maturation of NPCs in the dentate gyrus

NeuroD1 cell density (cells/mm2) was calculated by dividing the total number of NeuroD1 immunoreactive cells per animal by total granule cell layer area per animal. One way ANOVA analysis confirmed that animals exposed to MDMA and animals exposed to MDA/MDMA/METH had significantly lower number of NeuroD1 cells (F(2,11)= 5.594, p=0.0211). MDMA exposure caused a 55% decrease in NeuroD1 expression in comparison with controls while MDA/MDMA/METH exposure caused a 63% decrease in NeuroD1 expression (Figure 1). Dunnett’s post hoc analysis further confirmed a significant effect of MDMA and MDA/MDMA/METH exposure on the number of NeuroD1 cells when compared to controls (ps<0.05).

Amphetamines do not significantly increase apoptosis in the dentate gyrus

Cell density of AC-3 (cells/mm2) was calculated and data from each animal was averaged amongst its treatment group (Figure 1). One way ANOVA analysis confirmed that animals exposed to MDMA and animals exposed to MDA/MDMA/METH did not have significantly different levels of caspase activation (F(2,11)= 0.3246, p=0.7295).

Amphetamines do not significantly alter area of the granule cell layer and total number of granule cell neurons in the dentate gyrus

Area of the granule cell layer was determined for each section used for quantification of granule cell neurons. One way ANOVA did not detect a significant difference in the area between groups (area in mm2: Controls, 5.8 ± 0.28; MDMA only, 6.8 ± 0.64; MDMA/MDA/METH 6.2 ± 0.42; (F(2,11)= 0.84, p=0.45). Total granule cell neurons in the dentate gyrus was calculated and data from each animal was averaged among its treatment group (Figure 1). One way ANOVA analysis did not confirm significantly different numbers of total granule cells in animals exposed to MDMA and animals exposed to MDA/MDMA/METH (F(2,11)= 0.9815, p=0.4053).

Decreased number of NPCs is attributable to distinct effects on types of actively dividing hippocampal NPCs during their later phases of neuronal development

Using endogenous markers of radial glia-like precursors, proliferating progenitors, and differentiating progenitors, the types of actively dividing NPCs through the phases of neuronal development were identified and quantified (Taffe et al., 2010). For example, Ki-67 was used to mark actively dividing type-1, type-2a, type-2b, and type-3 NPCs. GFAP was used as a stem-like marker to distinguish between type-1 and type-2a NPCs (Steiner et al., 2006; Taffe et al., 2010; Yuan et al., 2011), in which type-1 cells express GFAP and type-2 do not. Sox2 was used as a marker to distinguish between preneuronal type-2 and neuronal type-3 NPCs (Steiner et al., 2006; Taffe et al., 2010), in which type-2 cells express Sox2 and type-3 cells do not. NeuroD1 was used to distinguish type-2a and type-2b NPCs (Steiner et al., 2006; Taffe et al., 2010), in which type-2b express NeuroD1 and type-2a do not. Therefore, using a novel combination of endogenous markers—GFAP, Sox2, Ki-67, and NeuroD1—and the current neurogenesis model for the initial phases of neuronal development of dividing NPCs (Steiner et al., 2006; Taffe et al., 2010), progenitors were distinctly labeled as type-1, type-2a, type-2b, and type-3. Specifically, actively dividing type-1 progenitors were identified as GFAP+/Sox2+/Ki-67+ cells. Actively dividing type-2a progenitors were identified as Sox2+/Ki-67+/NeuroD1− cells. Actively dividing type-2b progenitors were identified as Sox2+/Ki-67+/NeuroD1+ cells. Actively dividing type-3 progenitors were identified as Sox2−/Ki-67+/NeuroD1+ cells (Figure 2). A total of 62 ± 7 Ki-67-IR cells from controls, 18 ± 2 cells from MDMA group and 19 ± 3 cells from MDA/MDMA/METH group were analyzed for triple labeling analysis (Figure 2). Two-way ANOVA indicated a significant amphetamine treatment x cell type interaction F (8, 55) = 3.382, p = 0.0032; main effect of amphetamines F (2, 55) = 23.45, p <0.001; and main effect of cell types F (4, 55) = 25.22, p < 0.001. Post hoc analysis revealed significant reduction in the number of Ki-67 cells that were type-1 and type-3 NPCs and non NPCs in the amphetamine groups compared with controls (Figure 2). Analysis of the proportion of Ki-67-IR cells within each experimental groups indicated that a significant number of Ki-67-IR cells were type-1, -2, and -3 NPCs (56.4% in controls vs. 47.5% in the MDMA group and 43.6% in the MDA/MDMA/METH group; p > 0.05, one way ANOVA; Figure 2).

Figure 2.

Figure 2

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(a–h) Photomicrographs of confocal images of two sets of triple labeling analysis. (a–d) Ki-67/Sox-2/GFAP triple labeling, with Ki-67 in green (a, FITC), Sox-2 in red (b, CY3), GFAP in blue (c, CY5) and merged orthogonal view of the cell (d). (e–h) Ki-67/Sox-2/GFAP triple labeling, with Ki-67 in green (e, FITC), Sox-2 in red (f, CY3), NeuroD1 in blue (g, CY5) and merged view of the cell (h). (i–k) Pie charts of distribution of types of NPCs labeled with Ki-67. (l) Quantitative analysis of the number of Ki-67 cells that were either type-1, type-2a, type-2b, type-3 cells or neither cell type. Data is expressed as mean ± SEM (control n=3, MDMA only n=5, MDA/MDMA/METH n=6). *p<0.05 compared with drug naïve controls.

Discussion

The goal of the present study was to confirm the findings of decreased NPCs and neurogenesis in the hippocampus resulting from amphetamine insult reported in rodents in a higher order organism (Teuchert-Noodt et al., 2000; Hernandez-Rabaza et al., 2006; Cho et al., 2008; Mandyam et al., 2008; Schaefers et al., 2009; Venkatesan et al., 2011; Yuan et al., 2011; Baptista et al., 2014; Garcia-Cabrerizo & Garcia-Fuster, 2016). Many studies in the field have focused on understanding the effects of amphetamines on neurotransmitters and neurotransmitter induced neurotoxicity, but very few have sought to understand neurotoxicity as a function of altered neurogenesis. Furthermore, neurogenesis in the adult mammalian hippocampus is an important mediator of relapse to methamphetamine seeking behavior (Galinato et al., 2017; Galinato et al., 2018). Therefore, the purpose of the current investigation of hippocampal NPCs was to extend earlier research to organisms that are closer evolutionarily to human as well as to further explicate the factors involved in hippocampal plasticity subsequent to stimulant exposure.

By using model organisms in the adolescent and young adult developmental stages (i.e., age matched with humans who typically take MDA/MDMA/METH), a close model for understanding the effects of psychostimulants on the human hippocampus can be established (Crean et al., 2006; Crean et al., 2007). Furthermore, the doses of amphetamines administered to the macaques in each of their respective experimental groups mimicked some aspects of human consumption. For example, the average MDMA dose administered to all animals in both of the experimental groups over a period of 9 to 10 months was 17.08 + 1.75 mg/kg. Ecstasy pills seized and analyzed by ecstasydata.org showed that ~75 to 125mg of MDMA existed in an individual pill. This corresponds to 1–1.78 mg/kg for the average 70kg human user (Tanner-Smith, 2006; Von Huben et al., 2007). The amount of MDMA administered to subjects in the present study ranged from 1.7–5mg/kg, with changes in dose after each couple administrations. This range of MDMA doses is comparable to those generated in humans after consuming ecstasy pills and, combined with the fact that they were given to animals over a 9.6 month period, this represents a model for club-going recreational use rather than addiction (Rothe et al., 1997; Huang et al., 2003; Parrott, 2004). The experimental group exposed to MDA, MDMA, and METH was critical to include since human club drug users often (knowingly or unknowingly) use different amphetamines either in conjunction (see ecstasydata.org) or separately over a period of time (Fendrich et al., 2008; Halkitis et al., 2008; Daskalopoulou et al., 2014; Hunter et al., 2014). The inclusion of both types of exposure groups, MDMA only and MDA/MDMA/METH, permits a broader inference about the effects of repeated amphetamine use and subsequent effects on NPCs in the hippocampus to help us better understand effects on the human hippocampus.

The majority of prior related studies in nonhuman primates have focused on neurotransmitter-related neurotoxicity and have found that both MDMA and METH are neurotoxic to serotonergic and dopaminergic nerve terminals (Ricaurte et al., 2002; Madden et al., 2005; Ricaurte et al., 2005; Castner & Williams, 2007; Fantegrossi, 2007). The present study provides the first evidence of neurotoxicity by amphetamines in the hippocampus in nonhuman primates in confirmation of recent rodent studies that have found altered neurogenesis in the hippocampus of adult animals (Teuchert-Noodt et al., 2000; Hernandez-Rabaza et al., 2006; Cho et al., 2008; Mandyam et al., 2008; Schaefers et al., 2009; Venkatesan et al., 2011; Yuan et al., 2011; Baptista et al., 2014; Garcia-Cabrerizo & Garcia-Fuster, 2016). For example, in the present study it was found that amphetamines reduce NPCs expressing Ki-67 and NeuroD1 by greater than 80 and 50%. Ki-67 is a transcription factor expressed during multiple phases of the cell cycle, and is absent in quiescent cells (Scholzen & Gerdes, 2000). Therefore, reduction in the number of Ki-67 labeled NPCs suggests depletion of the pool of progenitors available for cell division. Mechanistically, several pathways may be involved in amphetamine’s effect on Ki-67 labeled NPCs. For example, amphetamines could alter how proliferating progenitors cycle through the cell cycle (e.g. kill NPCs by producing cell cycle arrest). Cell cycle kinetic studies in adult rats that experienced chronic METH does not support this hypothesis as METH did not alter the synthesis phase of the cell cycle (Yuan et al., 2011). While the limitation of the current study is that this was not accessible, the current study determined whether a particular stage of NPC development was altered by amphetamines. For instance, Ki-67 labeled NPCs in the postnatal hippocampus are not homogeneous, and represent cells in developmental stages that progress in parallel, including cycling cells that are radial glia-like (type 1), preneuronal (type-2a), intermediate (type-2b), and early neuronal (type-3) (Kronenberg et al., 2003; Steiner et al., 2006; Taffe et al., 2010; Yuan et al., 2011). Therefore, amphetamines could be altering a distinct type of NPC. Study conducted in rats revealed that METH reduced the percent of NPCs that were type-2a and enhanced the percent of NPCs that were type-3, indicating an alteration in the population of NPCs in the dentate gyrus (Yuan et al., 2011). Our findings in the nonhuman primate hippocampus demonstrates that MDMA and MDMA/MDA/METH reduced the number of Ki-67 cells that were type-1 and type-3 NPCs (almost wiped out these cells) and non NPCs. Our findings also indicate that amphetamines did not alter the number of Ki-67 cells that were type-2 NPCs in the dentate gyrus. A potential limitation in the interpretation of these results is that triple labeling studies were conducted with markers expressed in distinct stages of development for examining type-2 cells. While we are confident with the proportion of proliferating cells in each stage of development examined, quadruple labeling of these markers may have been optimal for examining type-2a and type-2b progenitors. Nevertheless, these findings suggest that a combination of amphetamines produced a higher neurotoxic environment in the dentate gyrus by depleting radial glia-like stem cells and early neuronal cells. Overall the reduction in the number of NPCs, in certain cell types contributed to the reduced number of NeuroD1 cells in the dentate gyrus. Future studies would be required to determine any detrimental effects of the depletion of early stem-like precursor cells and immature neurons in the hippocampus on hippocampus dependent behaviors. Both MDMA and MDMA/MDA/METH significantly reduced the number of Ki-67 cells that were non NPC (i.e., neither of the cell types examined). The phenotype of these cells could be endothelial, oligodendroglial and astrocytic cell types (Perera et al., 2007), and a reduction in these cells could alter the cellular composition of the dentate gyrus and perturb the neurogenic niche (Morrens et al., 2012; Ehret et al., 2015). Altogether, the present data indicate that chronic amphetamine exposure decreases hippocampal neurogenesis by initially altering the precursor cell pool, followed by altering the transition of developmental stages of proliferating progenitors from NPCs to immature neurons and altering the neurogenic niche. Whether the altered type-1 and -3 cells of proliferating progenitors harbor specific receptors or neurochemicals that are exclusive to amphetamine’s neuropharmacology is yet to be determined and will be a highly productive pursuit for future studies.

Studies in rodent models have indicated that METH reduces proliferation and neurogenesis in the dentate gyrus through increased cell death (Yuan et al., 2011). Active programmed cell death (apoptosis) was analyzed in the present study. There was no increase in apoptosis found in either amphetamine-exposed group in this study at the time point examined. These data indicate that changes in apoptosis may have occurred at an earlier time point during amphetamine experience, or are short-lived, and NPCs may die through other cell death pathways. We also investigated whether the alteration in the pool of NPCs produces changes in the number of granule cell neurons mostly generated during development in the dentate gyrus. Our results do not show a significant change in the number of preexisting granule cell neurons in the amphetamine groups, suggesting that the effect of amphetamines were more potent on newly born granule cell neurons. Few reasons could contribute to a loss in newly born granule cells without significantly altering the preexisting population of granule cells. First, studies from rodent models show that the number of newly born granule cells is about 6% of the total population of granule cells in the dentate gyrus (Cameron & McKay, 2001). This relationship may be true in nonhuman primates (Gould et al., 2001), and therefore a loss in cell numbers in newly born neurons may not entirely translate to loss in cell numbers of preexisting granule cell neurons. Second, the hippocampus is sensitive to changes in levels of glucocorticoids, and increased levels of glucocorticoids has been associated with reduction in newly born and preexisting granule cell neurons in rodents and nonhuman primates (Sapolsky et al., 1985; Gould et al., 1991; Coe et al., 2003; Cinini et al., 2014). These studies suggest that factors other than the drug itself could produce profound effects on newly born and preexisting granule cell neurons in the dentate gyrus. Nevertheless, given that emerging studies are indicating functional roles for newly born neurons in behaviors dependent on the hippocampus (McAvoy et al., 2015; Galinato et al., 2017; Galinato et al., 2018), it will be critical to determine if the reduction in NPCs and neurogenesis in nonhuman primates will correlate with hippocampus dependent memory deficits in the context of chronic amphetamine experience.

Collectively, this study proposes that administering repeated intermittent doses of amphetamines to young adult nonhuman primates results in significant reductions in both proliferating NPCs and immature neurons, to an extent that the integrity of the hippocampus may be compromised. It was also shown that decreases in neurogenesis were not directly associated with increased apoptosis at the time point examined; so it is most likely that other forms of cell death play a role in regulating amphetamine-induced depletion of progenitor pool.

Highlights.

  • Amphetamines reduce proliferation and immature neurons in the nonhuman primate hippocampus

  • Reduced number of progenitors is due to alterations in the proportion of neural progenitor cells undergoing cell division

  • Reduced number of progenitors is not related to enhanced apoptosis or loss of granule cell neurons in the hippocampus

Acknowledgments

This work was supported by National Institutes of Health grants from the National Institute on Drug Abuse, USA (DA018418 to M.A.T., DA034140 to C.D.M.) and National Institute of Alcoholism and Alcohol Abuse, USA (AA016807 to M.A.T.; AA020098, AA06420 to C.D.M.). The authors would like to thank Christopher C. Lay, Stefani N. Von Huben and Sophia A. Vandewater for their expert technical assistance with the animal studies, McKenzie Fannon and Elena Crawford for technical assistance with histology. A significant proportion of this work was submitted in part as Master’s Thesis to the Division of Biological Sciences, University of California, San Diego.

Footnotes

CDM, RRD and MAT designed the research and RRD and CDM wrote the manuscript. CDM and RRD performed the research. CDM and MAT contributed new reagents and analytic tools. CDM and RRD analyzed the data.

Ethical statement:

All animal experiments comply with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and is indicated in the manuscript that such guidelines have been followed.

The authors have no financial and personal relationships with other people or organizations that could inappropriately influence (bias) the current work.

The current work described here has not been published previously (except in the form of an academic thesis), and is not under consideration for publication elsewhere, and is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out.

Author contribution: CDM and RRD designed and performed the study. MAT and CDM contribute to the reagents and MAT contributed the animals. CDM and RRD performed the statistical analysis and CDM, MAT and RRD wrote the manuscript.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Altman J. Autoradiographic and histological studies of postnatal neurogenesis. 3. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J Comp Neurol. 1969;136:269–293. doi: 10.1002/cne.901360303. [DOI] [PubMed] [Google Scholar]
  2. Baptista S, Lasgi C, Benstaali C, Milhazes N, Borges F, Fontes-Ribeiro C, Agasse F, Silva AP. Methamphetamine decreases dentate gyrus stem cell self-renewal and shifts the differentiation towards neuronal fate. Stem cell research. 2014;13:329–341. doi: 10.1016/j.scr.2014.08.003. [DOI] [PubMed] [Google Scholar]
  3. Camarasa J, Rodrigo T, Pubill D, Escubedo E. Memory impairment induced by amphetamine derivatives in laboratory animals and in humans: a review. Biomolecular concepts. 2012;3:1–12. doi: 10.1515/bmc.2011.048. [DOI] [PubMed] [Google Scholar]
  4. Cameron HA, McKay RD. Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. Journal of Comparative Neurology. 2001;435:406–417. doi: 10.1002/cne.1040. [DOI] [PubMed] [Google Scholar]
  5. Carvalho M, Carmo H, Costa VM, Capela JP, Pontes H, Remiao F, Carvalho F, de Bastos ML. Toxicity of amphetamines: an update. Archives of toxicology. 2012;86:1167–1231. doi: 10.1007/s00204-012-0815-5. [DOI] [PubMed] [Google Scholar]
  6. Castner SA, Williams GV. From vice to virtue: insights from sensitization in the nonhuman primate. Prog Neuropsychopharmacol Biol Psychiatry. 2007;31:1572–1592. doi: 10.1016/j.pnpbp.2007.08.026. [DOI] [PubMed] [Google Scholar]
  7. Cho KO, Rhee GS, Kwack SJ, Chung SY, Kim SY. Developmental exposure to 3,4-methylenedioxymethamphetamine results in downregulation of neurogenesis in the adult mouse hippocampus. Neuroscience. 2008;154:1034–1041. doi: 10.1016/j.neuroscience.2008.04.040. [DOI] [PubMed] [Google Scholar]
  8. Cinini SM, Barnabe GF, Galvao-Coelho N, de Medeiros MA, Perez-Mendes P, Sousa MB, Covolan L, Mello LE. Social isolation disrupts hippocampal neurogenesis in young non-human primates. Front Neurosci. 2014;8:45. doi: 10.3389/fnins.2014.00045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clark JD, Gebhart GF, Gonder JC, Keeling ME, Kohn DF. Special Report: The 1996 Guide for the Care and Use of Laboratory Animals. ILAR J. 1997;38:41–48. doi: 10.1093/ilar.38.1.41. [DOI] [PubMed] [Google Scholar]
  10. Coe CL, Kramer M, Czeh B, Gould E, Reeves AJ, Kirschbaum C, Fuchs E. Prenatal stress diminishes neurogenesis in the dentate gyrus of juvenile rhesus monkeys. Biol Psychiatry. 2003;54:1025–1034. doi: 10.1016/s0006-3223(03)00698-x. [DOI] [PubMed] [Google Scholar]
  11. Crean RD, Davis SA, Taffe MA. Oral administration of (+/−)3,4-methylenedioxymethamphetamine and (+)methamphetamine alters temperature and activity in rhesus macaques. Pharmacol Biochem Behav. 2007;87:11–19. doi: 10.1016/j.pbb.2007.03.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crean RD, Davis SA, Von Huben SN, Lay CC, Katner SN, Taffe MA. Effects of (+/−)3,4-methylenedioxymethamphetamine, (+/−)3,4-methylenedioxyamphetamine and methamphetamine on temperature and activity in rhesus macaques. Neuroscience. 2006;142:515–525. doi: 10.1016/j.neuroscience.2006.06.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Daskalopoulou M, Rodger A, Phillips AN, Sherr L, Speakman A, Collins S, Elford J, Johnson MA, Gilson R, Fisher M, Wilkins E, Anderson J, McDonnell J, Edwards S, Perry N, O’Connell R, Lascar M, Jones M, Johnson AM, Hart G, Miners A, Geretti AM, Burman WJ, Lampe FC. Recreational drug use, polydrug use, and sexual behaviour in HIV-diagnosed men who have sex with men in the UK: results from the cross-sectional ASTRA study. The lancet. HIV. 2014;1:e22–31. doi: 10.1016/S2352-3018(14)70001-3. [DOI] [PubMed] [Google Scholar]
  14. Dayer AG, Ford AA, Cleaver KM, Yassaee M, Cameron HA. Short-term and long-term survival of new neurons in the rat dentate gyrus. J Comp Neurol. 2003;460:563–572. doi: 10.1002/cne.10675. [DOI] [PubMed] [Google Scholar]
  15. Ehret F, Vogler S, Kempermann G. A co-culture model of the hippocampal neurogenic niche reveals differential effects of astrocytes, endothelial cells and pericytes on proliferation and differentiation of adult murine precursor cells. Stem cell research. 2015;15:514–521. doi: 10.1016/j.scr.2015.09.010. [DOI] [PubMed] [Google Scholar]
  16. Enikolopov G, Overstreet-Wadiche L, Ge S. Viral and transgenic reporters and genetic analysis of adult neurogenesis. Cold Spring Harbor perspectives in biology. 2015;7:a018804. doi: 10.1101/cshperspect.a018804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fantegrossi WE. Reinforcing effects of methylenedioxy amphetamine congeners in rhesus monkeys: are intravenous self-administration experiments relevant to MDMA neurotoxicity? Psychopharmacology (Berl) 2007;189:471–482. doi: 10.1007/s00213-006-0320-8. [DOI] [PubMed] [Google Scholar]
  18. Fendrich M, Mackesy-Amiti ME, Johnson TP. Validity of self-reported substance use in men who have sex with men: comparisons with a general population sample. Annals of epidemiology. 2008;18:752–759. doi: 10.1016/j.annepidem.2008.06.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Galinato MH, Lockner JW, Fannon-Pavlich MJ, Sobieraj JC, Staples MC, Somkuwar SS, Ghofranian A, Chaing S, Navarro AI, Joea A, Luikart BW, Janda KD, Heyser C, Koob GF, Mandyam CD. A synthetic small-molecule Isoxazole-9 protects against methamphetamine relapse. Mol Psychiatry. 2017 doi: 10.1038/mp.2017.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Galinato MH, Takashima Y, Fannon MJ, Quach LW, Morales Silva RJ, Mysore KK, Terranova MJ, Dutta RR, Ostrom RW, Somkuwar SS, Mandyam CD. Neurogenesis during abstinence is necessary for context-driven methamphetamine-related memory. J Neurosci. 2018 doi: 10.1523/JNEUROSCI.2011-17.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Garcia-Cabrerizo R, Garcia-Fuster MJ. Comparative effects of amphetamine-like psychostimulants on rat hippocampal cell genesis at different developmental ages. Neurotoxicology. 2016;56:29–39. doi: 10.1016/j.neuro.2016.06.014. [DOI] [PubMed] [Google Scholar]
  22. Goncalves JT, Schafer ST, Gage FH. Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior. Cell. 2016;167:897–914. doi: 10.1016/j.cell.2016.10.021. [DOI] [PubMed] [Google Scholar]
  23. Gould E, Vail N, Wagers M, Gross CG. Adult-generated hippocampal and neocortical neurons in macaques have a transient existence. Proc Natl Acad Sci U S A. 2001;98:10910–10917. doi: 10.1073/pnas.181354698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gould E, Woolley CS, Cameron HA, Daniels DC, McEwen BS. Adrenal steroids regulate postnatal development of the rat dentate gyrus: II. Effects of glucocorticoids and mineralocorticoids on cell birth. J Comp Neurol. 1991;313:486–493. doi: 10.1002/cne.903130309. [DOI] [PubMed] [Google Scholar]
  25. Groman SM, Lee B, Seu E, James AS, Feiler K, Mandelkern MA, London ED, Jentsch JD. Dysregulation of D(2)-mediated dopamine transmission in monkeys after chronic escalating methamphetamine exposure. J Neurosci. 2012;32:5843–5852. doi: 10.1523/JNEUROSCI.0029-12.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Halkitis PN, Moeller RW, Siconolfi DE, Jerome RC, Rogers M, Schillinger J. Methamphetamine and poly-substance use among gym-attending men who have sex with men in New York City. Annals of behavioral medicine: a publication of the Society of Behavioral Medicine. 2008;35:41–48. doi: 10.1007/s12160-007-9005-8. [DOI] [PubMed] [Google Scholar]
  27. Hernandez-Rabaza V, Dominguez-Escriba L, Barcia JA, Rosel JF, Romero FJ, Garcia-Verdugo JM, Canales JJ. Binge administration of 3,4-methylenedioxymethamphetamine (“ecstasy”) impairs the survival of neural precursors in adult rat dentate gyrus. Neuropharmacology. 2006;51:967–973. doi: 10.1016/j.neuropharm.2006.06.019. [DOI] [PubMed] [Google Scholar]
  28. Huang YS, Liu JT, Lin LC, Lin CH. Chiral separation of 3,4-methylenedioxymeth- amphetamine and related compounds in clandestine tablets and urine samples by capillary electrophoresis/fluorescence spectroscopy. Electrophoresis. 2003;24:1097–1104. doi: 10.1002/elps.200390128. [DOI] [PubMed] [Google Scholar]
  29. Hunter LJ, Dargan PI, Benzie A, White JA, Wood DM. Recreational drug use in men who have sex with men (MSM) attending UK sexual health services is significantly higher than in non-MSM. Postgrad Med J. 2014;90:133–138. doi: 10.1136/postgradmedj-2012-131428. [DOI] [PubMed] [Google Scholar]
  30. Katner SN, Davis SA, Kirsten AJ, Taffe MA. Effects of nicotine and mecamylamine on cognition in rhesus monkeys. Psychopharmacology (Berl) 2004;175:225–240. doi: 10.1007/s00213-004-1804-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kronenberg G, Reuter K, Steiner B, Brandt MD, Jessberger S, Yamaguchi M, Kempermann G. Subpopulations of proliferating cells of the adult hippocampus respond differently to physiologic neurogenic stimuli. J Comp Neurol. 2003;467:455–463. doi: 10.1002/cne.10945. [DOI] [PubMed] [Google Scholar]
  32. Lewin AH, Miller GM, Gilmour B. Trace amine-associated receptor 1 is a stereoselective binding site for compounds in the amphetamine class. Bioorg Med Chem. 2011;19:7044–7048. doi: 10.1016/j.bmc.2011.10.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Madden LJ, Flynn CT, Zandonatti MA, May M, Parsons LH, Katner SN, Henriksen SJ, Fox HS. Modeling human methamphetamine exposure in nonhuman primates: chronic dosing in the rhesus macaque leads to behavioral and physiological abnormalities. Neuropsychopharmacology. 2005;30:350–359. doi: 10.1038/sj.npp.1300575. [DOI] [PubMed] [Google Scholar]
  34. Mandyam CD, Wee S, Crawford EF, Eisch AJ, Richardson HN, Koob GF. Varied access to intravenous methamphetamine self-administration differentially alters adult hippocampal neurogenesis. Biol Psychiatry. 2008;64:958–965. doi: 10.1016/j.biopsych.2008.04.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McAvoy K, Besnard A, Sahay A. Adult hippocampal neurogenesis and pattern separation in DG: a role for feedback inhibition in modulating sparseness to govern population-based coding. Front Syst Neurosci. 2015;9:120. doi: 10.3389/fnsys.2015.00120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miller GM. The emerging role of trace amine-associated receptor 1 in the functional regulation of monoamine transporters and dopaminergic activity. J Neurochem. 2011;116:164–176. doi: 10.1111/j.1471-4159.2010.07109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Morrens J, Van Den Broeck W, Kempermann G. Glial cells in adult neurogenesis. Glia. 2012;60:159–174. doi: 10.1002/glia.21247. [DOI] [PubMed] [Google Scholar]
  38. Parrott AC. Is ecstasy MDMA? A review of the proportion of ecstasy tablets containing MDMA, their dosage levels, and the changing perceptions of purity. Psychopharmacology (Berl) 2004;173:234–241. doi: 10.1007/s00213-003-1712-7. [DOI] [PubMed] [Google Scholar]
  39. Parrott AC. MDMA and 5-HT neurotoxicity: the empirical evidence for its adverse effects in humans - no need for translation. Br J Pharmacol. 2012;166:1518–1520. doi: 10.1111/j.1476-5381.2012.01941.x. discussion 1521–1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Perera TD, Coplan JD, Lisanby SH, Lipira CM, Arif M, Carpio C, Spitzer G, Santarelli L, Scharf B, Hen R, Rosoklija G, Sackeim HA, Dwork AJ. Antidepressant-induced neurogenesis in the hippocampus of adult nonhuman primates. J Neurosci. 2007;27:4894–4901. doi: 10.1523/JNEUROSCI.0237-07.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ray TS. Psychedelics and the human receptorome. PLoS One. 2010;5:e9019. doi: 10.1371/journal.pone.0009019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ricaurte GA, Mechan AO, Yuan J, Hatzidimitriou G, Xie T, Mayne AH, McCann UD. Amphetamine treatment similar to that used in the treatment of adult attention-deficit/hyperactivity disorder damages dopaminergic nerve endings in the striatum of adult nonhuman primates. J Pharmacol Exp Ther. 2005;315:91–98. doi: 10.1124/jpet.105.087916. [DOI] [PubMed] [Google Scholar]
  43. Ricaurte GA, Yuan J, Hatzidimitriou G, Cord BJ, McCann UD. Severe dopaminergic neurotoxicity in primates after a common recreational dose regimen of MDMA (“ecstasy”) Science. 2002;297:2260–2263. doi: 10.1126/science.1074501. [DOI] [PubMed] [Google Scholar]
  44. Rothe M, Pragst F, Spiegel K, Harrach T, Fischer K, Kunkel J. Hair concentrations and self-reported abuse history of 20 amphetamine and ecstasy users. Forensic science international. 1997;89:111–128. doi: 10.1016/s0379-0738(97)00123-0. [DOI] [PubMed] [Google Scholar]
  45. Sahay A, Scobie KN, Hill AS, O’Carroll CM, Kheirbek MA, Burghardt NS, Fenton AA, Dranovsky A, Hen R. Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation. Nature. 2011;472:466–470. doi: 10.1038/nature09817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sapolsky RM, Krey LC, McEwen BS. Prolonged glucocorticoid exposure reduces hippocampal neuron number: implications for aging. J Neurosci. 1985;5:1222–1227. doi: 10.1523/JNEUROSCI.05-05-01222.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schaefers AT, Teuchert-Noodt G, Bagorda F, Brummelte S. Effect of postnatal methamphetamine trauma and adolescent methylphenidate treatment on adult hippocampal neurogenesis in gerbils. Eur J Pharmacol. 2009;616:86–90. doi: 10.1016/j.ejphar.2009.06.006. [DOI] [PubMed] [Google Scholar]
  48. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182:311–322. doi: 10.1002/(SICI)1097-4652(200003)182:3<311::AID-JCP1>3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
  49. Sofuoglu M, DeVito EE, Waters AJ, Carroll KM. Cognitive Function as a Transdiagnostic Treatment Target in Stimulant Use Disorders. J Dual Diagn. 2016;12:90–106. doi: 10.1080/15504263.2016.1146383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Steiner B, Klempin F, Wang L, Kott M, Kettenmann H, Kempermann G. Type-2 cells as link between glial and neuronal lineage in adult hippocampal neurogenesis. Glia. 2006;54:805–814. doi: 10.1002/glia.20407. [DOI] [PubMed] [Google Scholar]
  51. Taffe MA. Delta(9)Tetrahydrocannabinol impairs visuo-spatial associative learning and spatial working memory in rhesus macaques. J Psychopharmacol. 2012;26:1299–1306. doi: 10.1177/0269881112443743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Taffe MA, Davis SA, Yuan J, Schroeder R, Hatzidimitriou G, Parsons LH, Ricaurte GA, Gold LH. Cognitive performance of MDMA-treated rhesus monkeys: sensitivity to serotonergic challenge. Neuropsychopharmacology. 2002;27:993–1005. doi: 10.1016/S0893-133X(02)00380-9. [DOI] [PubMed] [Google Scholar]
  53. Taffe MA, Kotzebue RW, Crean RD, Crawford EF, Edwards S, Mandyam CD. Long-lasting reduction in hippocampal neurogenesis by alcohol consumption in adolescent nonhuman primates. Proc Natl Acad Sci U S A. 2010;107:11104–11109. doi: 10.1073/pnas.0912810107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Taffe MA, Lay CC, Von Huben SN, Davis SA, Crean RD, Katner SN. Hyperthermia induced by 3,4-methylenedioxymethamphetamine in unrestrained rhesus monkeys. Drug Alcohol Depend. 2006;82:276–281. doi: 10.1016/j.drugalcdep.2005.09.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Taffe MA, Weed MR, Gold LH. Scopolamine alters rhesus monkey performance on a novel neuropsychological test battery. Brain Res Cogn Brain Res. 1999;8:203–212. doi: 10.1016/s0926-6410(99)00021-x. [DOI] [PubMed] [Google Scholar]
  56. Tanner-Smith EE. Pharmacological content of tablets sold as “ecstasy”: results from an online testing service. Drug Alcohol Depend. 2006;83:247–254. doi: 10.1016/j.drugalcdep.2005.11.016. [DOI] [PubMed] [Google Scholar]
  57. Teixeira-Gomes A, Costa VM, Feio-Azevedo R, de Bastos ML, Carvalho F, Capela JP. The neurotoxicity of amphetamines during the adolescent period. Int J Dev Neurosci. 2015;41:44–62. doi: 10.1016/j.ijdevneu.2014.12.001. [DOI] [PubMed] [Google Scholar]
  58. Teuchert-Noodt G, Dawirs RR, Hildebrandt K. Adult treatment with methamphetamine transiently decreases dentate granule cell proliferation in the gerbil hippocampus. Journal of neural transmission (Vienna, Austria: 1996) 2000;107:133–143. doi: 10.1007/s007020050012. [DOI] [PubMed] [Google Scholar]
  59. Toni N, Laplagne DA, Zhao C, Lombardi G, Ribak CE, Gage FH, Schinder AF. Neurons born in the adult dentate gyrus form functional synapses with target cells. Nat Neurosci. 2008;11:901–907. doi: 10.1038/nn.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Venkatesan A, Uzasci L, Chen Z, Rajbhandari L, Anderson C, Lee MH, Bianchet MA, Cotter R, Song H, Nath A. Impairment of adult hippocampal neural progenitor proliferation by methamphetamine: role for nitrotyrosination. Mol Brain. 2011;4:28. doi: 10.1186/1756-6606-4-28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Von Huben SN, Davis SA, Lay CC, Katner SN, Crean RD, Taffe MA. Differential contributions of dopaminergic D1- and D2-like receptors to cognitive function in rhesus monkeys. Psychopharmacology (Berl) 2006;188:586–596. doi: 10.1007/s00213-006-0347-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Von Huben SN, Lay CC, Crean RD, Davis SA, Katner SN, Taffe MA. Impact of ambient temperature on hyperthermia induced by (+/−)3,4-methylenedioxymethamphetamine in rhesus macaques. Neuropsychopharmacology. 2007;32:673–681. doi: 10.1038/sj.npp.1301078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wareing M, Fisk JE, Murphy PN. Working memory deficits in current and previous users of MDMA (‘ecstasy’) British journal of psychology (London, England: 1953) 2000;91(Pt 2):181–188. doi: 10.1348/000712600161772. [DOI] [PubMed] [Google Scholar]
  64. Yuan CJ, Quiocho JM, Kim A, Wee S, Mandyam CD. Extended access methamphetamine decreases immature neurons in the hippocampus which results from loss and altered development of neural progenitors without altered dynamics of the S-phase of the cell cycle. Pharmacol Biochem Behav. 2011;100:98–108. doi: 10.1016/j.pbb.2011.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhong N, Jiang H, Du J, Zhao Y, Sun H, Xu D, Li C, Zhuang W, Li X, Hashimoto K, Zhao M. The cognitive impairments and psychological wellbeing of methamphetamine dependent patients compared with health controls. Prog Neuropsychopharmacol Biol Psychiatry. 2016;69:31–37. doi: 10.1016/j.pnpbp.2016.04.005. [DOI] [PubMed] [Google Scholar]

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