Prenatal 3, 4-methylenedioxymethamphetamine (ecstasy) exposure induces long-term alterations in the...

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Developmental Brain Resear

Research report

Prenatal 3,4-methylenedioxymethamphetamine (ecstasy) exposure

induces long-term alterations in the dopaminergic and

serotonergic functions in the rat

Laurent Galineaua, Catherine Belzungb, Ercem Kodasa, Sylvie Bodarda,

Denis Guilloteaua, Sylvie Chalona,*

aINSERM U619, IFR135, Laboratoire de Biophysique Medicale and Pharmaceutique, Universite Francois Rabelais des Sciences Pharmaceutiques,

31 avenue Monge, 37200 Tours, FrancebEA3248 Psychobiologie des Emotions, IFR135, Universite Francois Rabelais, Tours, France

Accepted 27 October 2004

Available online 16 December 2004

Abstract

We investigated several aspects of the dopaminergic and serotonergic functions throughout brain development in rats prenatally exposed

to MDMA (becstasyQ). Pregnant rats were treated with MDMA (10 mg/kg s.c.) or saline from the 13th to the 20th day of gestation and studies

were conducted on the progeny from both groups: (i) quantification of whole brain contents of DA, 5-HT and metabolites from the 14th day

of embryonic life (E14) to weaning (21st day of postnatal life, P21); (ii) quantification of DA and 5-HT membrane transporters by

autoradiography from E18 to adult age (P70); (iii) measurement of pharmacologically induced release of DA and 5-HT using microdialysis on

adult (P70) freely moving rats; (iv) measurement of sucrose preference in adults (P70).

Prenatally MDMA-exposed rats showed (i) a two-fold decrease of whole brain levels of 5-HT and 5-HIAA at P0; (ii) no effect on the DAT

and SERT density; (iii) a strongly reduced pharmacologically induced release of DA and 5-HT at P70 in the striatum and hippocampus; and

(iv) a significant 20% decrease in sucrose preference at P70.

This study suggests that a prenatal exposure to MDMA induces transient and long-term neurochemical and behavioural modifications in

dopaminergic and serotonergic functions.

D 2004 Elsevier B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Developmental disorders

Keywords: Brain development; Dopamine; Ecstasy; Microdialysis; Serotonin; Sucrose preference

1. Introduction

The use of the amphetamine derivative 3,4-methylene-

dioxymethamphetamine (MDMA; becstasyQ) is currently

increasing among young adults [29,51,54] but there is still

scarce information on potential long-term consequences on

progeny of mothers who have taken this drug of abuse

during gestation. Acute administration of MDMA is known

0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.devbrainres.2004.10.012

* Corresponding author. Fax: +33 2 47 36 72 24.

E-mail address: [email protected] (S. Chalon).

to affect the peripheral and central nervous system functions

by acting mainly on the serotonergic system (see Refs.

[18,43] for recent reviews). Although the main known

effects of MDMA involve this system, it has been

demonstrated that MDMA also induces the simultaneous

release and reuptake blockade of dopamine (DA), norepi-

nephrine, acetylcholine and histidine [42]. As the seroto-

nergic and dopaminergic systems appear very early in the

embryonic life and have a key role in regulating brain

development [27,41,50,57,60], it can be hypothesized that

exposure to MDMA during foetal life would affect their

ch 154 (2005) 165–176

L. Galineau et al. / Developmental Brain Research 154 (2005) 165–176166

maturation and may have long-term consequences on the

functions regulated by these systems.

Several clinical studies suggest that prenatal MDMA

exposure can be toxic to the developing human foetus

[28,45], whereas few studies conducted on rats had

previously suggested a lack of vulnerability of the brain

following pre or early postnatal treatment with MDMA

[1,10,58]. However, several recent studies have shown that

a perinatal exposure to MDMA in rats led to long-term

learning and memory impairments [7], alterations in brain

glucose utilization [32] and enhanced locomotor activity in

adolescent rats placed in a novel environment [35]. In this

study, we explored the consequences of a chronic admin-

istration of MDMA to pregnant rats on the maturation of the

dopaminergic and serotonergic systems of their progeny

from embryonic to adult life. The administered dose, i.e. 10

mg/kg/day, referred to the method of interspecies scaling

[47] allowing to estimate it equivalent to that used by

humans [30]. We quantified the levels of DA, 5-HT and

their main metabolites in the whole brain from the

embryonic period (foetus aged 14 days) to weaning

(postnatal day 21), and investigated the appearance of the

dopamine and serotonin transporters (DAT and SERT) in

regions containing the cell bodies and projections of DA and

5-HT neurons from embryonic period to adulthood (post-

natal day 70). We used for the DAT and SERT study a

quantitative autoradiographic method with very selective

radioprobes for both targets, (E)-N-(3-iodoprop-2-enyl)-2h-carbomethoxy-3h-(4’-methylphenyl)nortropane, [125I]PE2I,

for the DAT [8] and N,N-dimethyl-2-(2-amino-4-methyl-

phenylthio)benzylamine, [3H]MADAM, for the SERT [9].

Further, the potential long-term effects of prenatal MDMA

exposure have been explored in adult (P70) rats on several

parameters of the DA and 5-HT functions: (i) the levels of

pharmacological-stimulated release of DA and 5-HT using

in vivo cerebral microdialysis, and (ii) a paradigm of

sucrose preference.

2. Materials and methods

2.1. Animals and treatments

All procedures were carried out in accordance with the

European Community Council Directive 86/609/EEC for

the care of laboratory animals and with the authorization of

the Regional Ethical Committee (INSERM37-003). Ani-

mals were housed in a temperature-controlled room (22 F1 8C) under a 12-h light/dark cycle (lights on at 7:30 a.m.

and off at 7:30 p.m.) and had ad libitum access to food

and water. Timed pregnant Wistar rats purchased from

CERJ (Le Genest, France) were s.c. injected with saline

(control group) or 10 mg/kg MDMA (MDMA group) per

day from gestational days 13 to 20. The progeny was sex

typed from P0; thus, all experiments have been conducted

exclusively on male pups after birth. For monoamine

measurements on whole brains and autoradiographic

studies, dams were sacrificed and embryos were removed

by caesarean section at the age of 14, 16, 18 or 20

embryonic days (E14, E16, E18, E20), or the male progeny

was sacrificed by decapitation at 0, 7, 14, 21, 28 or 70

days of life (P0, P7, P14, P21, P28 or P70).

The quantification of DA, 5-HT and their metabolites on

whole brain was performed on 1 animal for each age (E14,

E16, E18, E20, P0, P7, P14, P21) originated from 3 dams injected

with MDMA and 3 dams injected with saline (representing a

total of 48 litters for this study). The quantification of DAT

and SERT density was performed on brain sections of 1

animal for each age (E18, E20, P0, P7, P14, P21 P28, P70)

originated from 3 dams injected with MDMA and 3 dams

injected with saline (representing a total of 48 litters for this

study). The microdialysis experiments were performed on 1

adult animal (P70) originated from 6 dams injected with

MDMA and 6 dams injected with saline (representing a total

of 12 litters for this study). The sucrose preference test was

performed on 1 adult animal (P70) originated from 6 dams

injected with MDMA and 6 dams injected with saline

(representing a total of 12 litters for this study).

2.2. Drugs

(F)-3,4-Methylenedioxymethamphetamine HCl

(MDMA) and fenfluramine were purchased from Sigma

(St. Quentin-Fallavier, France). 4-Hydroxyphenethylamine

(tyramine) was purchased from Sigma (St. Louis, MO).

Natural cocaine hydrochloride was obtained from Coopera-

tion Pharmaceutique Francaise (Melun, France) and parox-

etine hydrochloride was a gift from GlaxoSmithKline

(Nanterre, France). Radiolabeled N,N-dimethyl-2-(2-amino-

4-methylphenylthio)benzylamine, [3H]MADAM (specific

activity 3.2 TBq/mmol) and (E)-N-(3-iodoprop-2-enyl)-2h-carbomethoxy-3h-(4V-methylphenyl)nortropane, [125I]PE2I

(specific activity 74 TBq/mmol) were prepared as previously

described [9,22].

2.3. Separation and quantification of DA, 5-HT and

metabolites

Brains of animals from E14 to P21 were rapidly removed

on ice without prior perfusion, carefully dried on absorbent

paper and weighed. Each brain was homogenized in 1 mL of

a buffer containing 12 M HClO4, 0.1 mM EDTA, 0.5 mM

Na2S2O5, 3 mM octanesulfonic acid and 3 mM hepta-

nesulfonic acid with an Ultraturrax T25 at 4 8C. After a

30,000 � g centrifugation at 4 8C for 20 min, 100 Al of thesupernatant was kept at �80 8C until use. DA, 5-HT and

metabolites levels were measured in each supernatant by

HPLC with electrochemical detection on a Concorde

apparatus (Waters, St. Quentin-Yvelines, France). Samples

were injected using a Rheodyne 7725i injector valve with a

20-Al injection loop. The mobile phase consisting of 7%

acetonitrile, 3% methanol and 90% 20 mM citric acid, 10

L. Galineau et al. / Developmental Brain Research 154 (2005) 165–176 167

mM monobasic phosphate sodium, 3.25 mM octanesulfonic

acid, 3 mM heptanesulfonic acid, 0.1 mM EDTA, 2 mM

KCl, 6 ml/l o-phosphoric acid and 2 ml/l diethylamine with

pH 3 was pumped at 0.3 ml/min with a Gold 118 system

(Beckman, Fullerton, CA). Separation was performed with a

3-Am C18, 3.2 � 100 mm reversed phase column (LC-22C,

BAS, West Lafayette, IN). A glassy carbon working

electrode set at 610 mV with reference to an in situ Ag/

AgCl reference electrode was used to detect compounds.

Signals were recorded and quantified with a Beckman Gold

118 integrator. Amounts of DA, 5-HT, Dopac, HVA and 5-

HIAA were calculated by comparing peak levels from the

supernatant samples with those of external standards.

2.4. In vitro autoradiographic studies with PE2I and

MADAM

Brains of animals from E18 to P70 were rapidly removed

and immediately frozen at �35 8C in cooled isopentane.

Twenty-micron coronal sections were cut with a cryostat

microtome (Reichert-Jung Cryocut CM3000 Leica, Rueil-

Malmaison, France), thaw mounted on Superfrost slides and

kept at �80 8C until use. Sections were taken from brain

regions corresponding to plates 420, 428, 438, 480, 490,

496, 548, 556, 570 and 574 of the atlas of Altman and Bayer

[3] for embryonic stages and to plates 11, 12, 28, 36, 37 and

48 of the atlas of Paxinos and Watson [52] for the postnatal

stages. Binding studies with [125I]PE2I as DAT ligand and

[3H]MADAM as SERT ligand were performed according to

previous data [8,9]. Sections were incubated for 90 min at

22 8C with 100 pM [125I]PE2I in 100 Al of a pH 7.4

phosphate buffer (10.14 mM NaH2PO4, 137 mM NaCl, 2.7

mM KCl, 1.76 mM KH2PO4, 0.32 M sucrose) or 60 min at

22 8C with 500 pM [3H]MADAM in 100 Al of the same

phosphate buffer. The nonspecific binding was defined on

adjacent sections incubated in the presence of 1 AM cocaine

for [125I]PE2I binding studies or 1 AM paroxetine for

[3H]MADAM binding studies. Six sections were used for

each embryonic or postnatal age for the quantification of the

total binding and three adjacent sections for the nonspecific

binding.

Sections were then washed twice for 10 min in the buffer

at 4 8C and rinsed in distilled water. After drying, sections

were exposed to sensitive films (Biomax MR, Kodak,

France) with standards (125I-microscales or 3H-microscales,

Amersham Bioscience AB) in X-ray cassettes for 1 day or 3

months, respectively, for [125I]PE2I and [3H]MADAM

binding. After revelation and fixation, the films were

analyzed using an image analyzer (Biocom, Les Ulis,

France) after identifying anatomical regions of interest.

The absorbance obtained was converted into apparent tissue

ligand concentration with reference to standard and specific

activity of the radioligand. The intensity of [125I]PE2I and

[3H]MADAM binding was thus expressed in fmol/mg of

equivalent tissue. The studied structures containing the cell

bodies of the dopaminergic and serotonergic neurons were

respectively the substantia nigra, ventral tegmental area and

raphe nuclei. The brain regions containing the dopaminergic

projections were the striatum and nucleus accumbens. The

studied regions containing the serotonergic projections were

the hypothalamus, thalamic nuclei, hippocampus and

somatosensory areas.

2.5. Surgery, microdialysis dual-probe implantation and

microdialysis procedure

Rats were anesthetized with ketamine (150 mg/kg i.p.,

Imalgene, Rhone Merieux, France) and placed in a stereo-

taxic apparatus (Stoelting, USA). Body temperature was

maintained at 37F 1 8C throughout the surgery time using a

thermostatically controlled heating blanket (CMA 150,

CMA/Microdialysis, Stockholm, Sweden). The skull was

exposed and two holes were drilled. One guide cannula

(MAB 2 14 G, CMA/Microdialysis) was implanted into the

striatum (coordinates AP +1.2 mm, L +3.0 mm, DV �4.4

mm from Bregma) [52] and the second (MAB 2 20 G,

CMA/Microdialysis) was implanted into the hippocampus

(coordinates AP �4.8 mm, L �5.0 mm, DV �5.2 mm from

Bregma) [52]. Guides were then anchored to the skull with a

stainless-steel screw and dental cement. Microdialysis

probes with a 3-mm membrane length (MAB 6 14 3, 15

kDa molecular mass cut-off) in the striatum and a 1-mm

membrane length (MAB 6 20 1, 15 kDa molecular mass cut-

off) in the hippocampus were slowly lowered through the

guide cannula. Animals were housed in cylindrical Plexiglas

cages (diameter 40 cm, height 32 cm), which served as

home cages during the entire microdialysis session, with a

counterbalance arm holding a liquid swivel. They were

allowed to recover post-operatively overnight and given ad

libitum access to water and food. After implantation, the

probes were immediately and continuously perfused with

Dulbecco buffer modified liquid (ICN, USA) supplemented

with 2.2 mM CaCl2 and 1.1 mM MgCl2 (pH 7.4) at 0.8 Al/min using a microsyringe pump (Harvard Apparatus, South

Natick, USA).

After a postoperative recovery period (22 h), the flow

rate was increased to 1.2 Al/min (striatum) or 1 Al/min

(hippocampus) for 1 h to reach equilibrium before experi-

ments. Dialysates were collected at 20- or 25-min intervals,

respectively, for the striatum and hippocampus, into vials

that were preloaded with 5 Al of 0.1 M perchloric acid. The

first day after surgery was devoted to the tyramine

stimulation protocol in the striatum. The microinjection

pump was mounted with two syringes, one containing

perfusion buffer alone and one containing perfusion buffer

supplemented with 800 AM tyramine (Sigma, St. Louis,

MO) freshly dissolved in Dulbecco buffer before use.

During the first 80 min, the dialysis probe was infused

with perfusion buffer alone, and then with the tyramine

solution for 40 min by switching syringe. Perfusion was

then continued with buffer alone until the end of experi-

ment. The second day after surgery was devoted to the


fenfluramine stimulation protocol in the hippocampus. The

microinjection pump was mounted with two syringes, one

containing perfusion buffer alone and one containing

perfusion buffer supplemented with 1.2 mM fenfluramine

(Sigma, St. Louis, MO) freshly dissolved in Dulbecco buffer

before use. During the first 100 min, the dialysis probe was

infused with perfusion buffer alone, and then with the

fenfluramine solution for 50 min by switching syringe.

Perfusion was then continued with buffer alone until the end

of experiment.

The baseline values of extracellular DA and 5-HT were

obtained by averaging the first four dialysate samples, and

values obtained in subsequent samples were expressed as

percentage of each baseline. Animals were sacrificed after

the experiment by ether inhalation and the localization of

the dialysis probe was macroscopically checked on coronal

brain sections.

2.6. Separation and quantification of monoamines after

microdialysis

DA and 5-HTwere measured in dialysates by HPLC with

electrochemical detection on a Concorde apparatus

(Waters). Samples were injected using a Rheodyne 7725i

injector valve with a 20-Al injection loop. The mobile phase

consisted of 7% acetonitrile, 3% methanol and 90% 20 mM

citric acid, 10 mM monobasic phosphate sodium, 3.25 mM

octanesulfonic acid, 3 mM heptanesulfonic acid, 0.1 mM

EDTA, 2 mM KCl, 6 ml/l o-phosphoric acid and 2 ml/l

diethylamine (pH 3) for DA and 90 mM monobasic

phosphate sodium, 0.45 mM octanesulfonic acid, 0.14

mM EDTA, 2 mM KCl and 18% methanol (pH 4) for 5-

HT. Mobile phases were pumped at 0.3 ml/min with a Gold

118 system (Beckman). Separations were performed with a

3-Am C18, 3.2 � 100 mm reverse phase column (LC-22C,

BAS). A glassy carbon working electrode set at 610 mV for

DA and 800 mV for 5-HT with reference to an in situ Ag/

AgCl reference electrode was used to detect compounds.

Signals were recorded and quantified with a Beckman Gold

118 integrator. Amounts of monoamines were calculated by

comparing peak levels from the microdialysis samples with

those of external standards. Under these conditions, the limit

of detection was 1 fmol/Al for DA and 0.1 fmol/Al for 5-HT.

2.7. Sucrose preference protocol

The protocol consisted first in a habituation phase during

which 2–3-month-old rats were allowed to drink a 2%

sucrose (Sigma, St. Quentin-Fallavier, France) solution.

During this period, animals were housed in individual cages

with an exclusive access to the sucrose solution in 10-ml

pipettes, during 1 h for 5 consecutive days. The experiments

started at 6 a.m. or 9 p.m., i.e. 1.5 h before or after lights

turned on or off in the rat room. The challenge between the

sucrose solution and water was then conducted during 30 min

for 3 consecutive days. At 6 a.m. or 9 p.m., rats were given

access to both the sucrose solution pipettes and the pipettes

containing water and their fluid intake was measured over 30

min. The position of the pipettes was alternated at each

session to prevent position biased drinking. This allowed to

determine the percentage of preference, i.e. (sucrose intake/

total intake)*100. The mean preference during these six

sessions was then calculated for each subject.

2.8. Statistical analysis

For the quantification study of DA, 5-HT and their

metabolites on whole brains, the amounts obtained for each

stage of development were compared to all other stages

using a one-way analysis of variance (ANOVA) followed by

post-hoc Bonferroni’s test. For the quantification study of

DA, 5-HT and their metabolites on whole brains and the

autoradiographic study of DAT and SERT density, differ-

ence between groups (MDMA and saline) were analysed

using a one-way analysis of variance followed by post-hoc

Student’s t-test.

Data obtained from microdialysis experiments were

analysed using a one-way analysis of variance (ANOVA)

followed by post-hoc Dunnet’s test.

Data from the sucrose preference test were analysed

using a non parametric Mann–Whitney test.

3. Results

3.1. Quantification of DA, 5-HT and their metabolites

The tissue levels of DA from embryonic age (E14) to

weaning (P21) were not significantly different in MDMA

and saline groups (Fig. 1A).

The profiles obtained for both groups showed a significant

increase of DA levels from E14 to E18 (from 0.025 F 0.001

to 0.214 F 0.012 nM/g tissue for the saline group, df = 1,

F = 282.13, p b 0.0001; from 0.030 F 0.005 to 0.267 F0.029 nM/g tissue for the MDMA group, df = 1, F = 96.72,

p = 0.0006). DA levels remained stable between E18 and E20

and then significantly increased to a maximum peak at P7(from 0.230 F 0.013 to 0.642 F 0.037 nM/g tissue for the

saline group, df = 1, F = 160.12, p = 0.0002; from 0.243 F0.009 to 0.573 F 0.031 nM/g tissue for the MDMA group,

df = 1, F = 153.03, p = 0.0002). The DA contents thereafter

significantly decreased from P7 to P14 (from 0.642 F 0.037

to 0.191F 0.038 nM/g tissue for the saline group, df = 1, F =

64.53, p = 0.0013; from 0.573F 0.031 to 0.297F 0.146 nM/

g tissue for the MDMA group, df = 1, F = 96.49, p = 0.0006),

and underwent an increase until P21 (from 0.191 F 0.038 to

0.453 F 0.064 nM/g tissue for the saline group, df = 1, F =

7.11, p = 0.0560; from 0.297F 0.146 to 0.437F 0.026 nM/g

tissue for the MDMA group, df = 1, F = 34.25, p = 0.0043).

As for DA, there was no significant difference in the

developmental profiles of Dopac and HVA between the

MDMA and saline groups (Fig. 1B and C).

Fig. 2. Levels of 5-HT (A) and 5-HIAA (B) in whole rat brains from

embryonic age (E14) to weaning (P21) in animals prenatally exposed to

MDMA or saline. n = 3 pups for each age. Results are expressed as

means F S.E.M. nmol/g tissue. Levels of 5-HT and 5-HIAA obtained for

each stage of development were compared to all other stages using a one-

way analysis of variance (ANOVA) followed by post-hoc Bonferroni’s

test (+p b 0.05, ++p b 0.01, +++p b 0.001). Values between groups

(MDMA and saline) were analysed using a one-way analysis of variance

followed by post-hoc Student’s t-test (*p b 0.05).

Fig. 1. Levels of DA (A), Dopac (B) and HVA (C) in whole rat brains

from embryonic age (E14) to weaning (P21) in animals prenatally exposed

to MDMA or saline. n = 3 pups for each age. Results are expressed as

means F S.E.M. nmol/g tissue. Levels of DA, Dopac and HVA obtained

for each stage of development were compared to all other stages using a

one-way analysis of variance (ANOVA) followed by post-hoc Bonferro-

ni’s test (+p b 0.05, ++p b 0.01, +++p b 0.001). Values between groups

(MDMA and saline) were analysed using a one-way analysis of variance

followed by post-hoc Student’s t-test.


The Dopac levels remained stable between E14 and E16

and then significantly increased between E16 and E18 (from

0.020 F 0.005 to 0.087 F 0.006 nM/g tissue for the saline

group, df = 1, F = 374.71, p b 0.0001; from 0.020 F 0.021

to 0.098 F 0.008 nM/g tissue for the MDMA group, df = 1,

F = 32.29, p = 0.0045). Dopac levels remained stable

between E18 and E20 were and then increased to a maximum

peak obtained at P7 for the saline group (from 0.101 F0.006 to 0.210 F 0.012 nM/g tissue, df = 1, F = 112.09, p =

0.0005) and at P0 for the MDMA group (from 0.101 F0.002 to 0.143 F 0.012 nM/g tissue, df = 1, F = 18.52, p =

0.0126). This was followed by a significant decline between

P7 and P14 (from 0.202 F 0.012 to 0.033 F 0.003 nM/g

tissue for the saline group, df = 1, F = 298.80, p = 0.0001;

from 0.162 F 0.008 to 0.038 F 0.002 nM/g tissue for the

MDMA group, df = 1, F = 346.79, p b 0.0001). Thereafter,

Dopac levels underwent a decline from P14 to P21 (from


group, df = 1, F = 50.42, p = 0.0021; from 0.034 F 0.002 to

0.024 F 0.002 nM/g tissue for the MDMA group, df = 1,

F = 230.37, p = 0.0001).

The HVA levels remained stable between E14 and E16 and

underwent a significant increase from E16 to a maximum

peak at P7 (from 0.014F 0.011 to 0.330F 0.032 nM/g tissue

for the saline group, df = 1, F = 21.99, p = 0.0094; from

0.021F 0.044 to 0.287F 0.023 nM/g tissue for the MDMA

group, df = 1, F = 14.46, p = 0.0191). This was followed by a

significant decrease to P14 (from 0.330 F 0.032 to 0.175 F0.042 nM/g tissue for the saline group, df = 1, F = 13.07, p =

0.0224; from 0.287 F 0.023 to 0.102 F 0.024 nM/g tissue

for the MDMA group, df = 1, F = 21.98, p = 0.0094). HVA

levels were then stable between P14 and P21.

The tissue levels of 5-HT from embryonic age (E14) to

weaning (P21) showed different profiles in MDMA and

saline groups (Fig. 2A). In the saline group, it increased

Fig. 3. Representative autoradiographic image showing [125I]PE2I binding to the DAT on coronal brain sections of adult (P70) male rats prenatally exposed to

MDMA or saline.


significantly from E14 to a maximum peak obtained at P0(from 0.357 F 0.075 to 6.193 F 0.588 nM/g tissue, df = 1,

F = 65.02, p = 0.0013). This was followed by a decline to

reach stable levels at P14 (from 6.193 F 0.588 to 1.286 F0.266 nM/g tissue, df = 1, F = 38.83, p = 0.0034). In the

MDMA group, 5-HT levels also showed a significant in-

crease from E14 to a maximum peak at P0 (from 0.473 F0.135 to 2.939 F 0.073 nM/g tissue, df = 1, F = 101.15, p =

0.0005). The P0 value was significantly lower in the MDMA

compared to the saline group (2.939 F 0.073 vs. 6.193 F0.158 nmol/g tissue, respectively, df = 1, F = 45.25, p =

0.009). The 5-HT levels remained stable between P0 and P7,

and thereafter significantly decreased to reach a stable value

at P14 (from 3.412F 0.230 to 0.958F 0.033 nM/g tissue, df

= 1, F = 166.94, p = 0.0002) that was similar to that

obtained for the saline group.

Table 1

Effects of prenatal exposure to MDMA on the density of [125I]PE2I binding to the

nerve endings of dopaminergic neurons

E18 E20 P0 P7

SN/VTA Saline 0.42 F 0.04 0.54 F 0.05 0.84 F 0.29 1.32

MDMA 0.47 F 0.05 0.65 F 0.19 0.82 F 0.20 1.35

Str Saline 0.10 F 0.01 0.30 F 0.02 0.47 F 0.06 2.13

MDMA 0.14 F 0.01 0.33 F 0.03 0.40 F 0.10 2.64

Nac Saline 0.04 F 0.01 0.08 F 0.01 0.15 F 0.04 0.96

MDMA 0.03 F 0.01 0.10 F 0.01 0.14 F 0.04 1.24

Results are expressed as mean F S.E.M. fmol/mg tissue of [125I]PE2I specifica

substantia nigra; VTA, ventral tegmental area) and nerve endings (Str, striatum; Na

females exposed to MDMA or saline between the 13th and 20th day of gestation (n

3 exposed to saline).

No difference was found between the MDMA and saline groups for each age (one

The 5-HIAA content (Fig. 2B) significantly increased

between E14 and P0 in both groups (from 0.227 F 0.008 to

12.537F 2.295 nM/g tissue for the saline group, df = 1, F =

57.67, p = 0.0016; from 0.211 F 0.023 to 2.791 F 0.355

nM/g tissue for the MDMA group, df = 1, F = 42.96, p =

0.0028). However, a 4.5-fold reduced peak was observed in

the MDMA compared to the saline group (2.791 F 0.355

vs. 12.537 F 2.295 nM/g tissue, respectively, df = 1, F =

26.41, p = 0.018). The 5-HIAA levels of the saline group

then significantly decreased from P0 to P7, at which values

in both groups were similar. Finally, a steady decline in 5-

HIAA levels occurred from P7 to P21 in both groups (from


group, df = 1, F = 86.89, p = 0.0007; from 2.929 F 0.069

to 1.736 F 0.264 nM/g tissue for the MDMA group, df = 1,

F = 28.64, p = 0.0059).

DAT from embryonic (E18) to adult (P70) age in the region of cell bodies and

P14 P21 P28 P70

F 0.15 1.66 F 0.17 2.12 F 0.10 2.29 F 0.11 2.56 F 0.19

F 0.44 1.58 F 0.32 2.24 F 0.18 2.53 F 0.24 2.68 F 0.27

F 0.38 4.23 F 0.23 5.72 F 0.29 6.24 F 0.19 7.22 F 0.35

F 0.34 3.45 F 0.76 5.11 F 0.43 5.66 F 0.68 7.33 F 0.40

F 0.14 2.05 F 0.10 3.70 F 0.08 3.93 F 0.07 4.55 F 0.18

F 0.17 2.13 F 0.17 3.79 F 0.40 4.57 F 0.40 4.21 F 0.16

lly bound on coronal rat brain sections in the region of cell bodies (SN,

c, nucleus accumbens) of dopaminergic neurons. Animals are the progeny of

= 3 animals for each age originated from 3 females exposed to MDMA and

-way analysis of variance, ANOVA, followed by post-hoc Student’s t-test).

Fig. 4. Representative autoradiographic image showing [3H]MADAM binding to the SERT on coronal brain sections of adult (P14) male rats prenatally exposed

to MDMA or saline.


3.2. Dopamine transporter density in the region of cell

bodies and nerve endings of dopaminergic neurons

In this study, the density of [125I]PE2I binding sites (Fig.

3) was assimilated to DAT levels. The images obtained from

[125I]PE2I autoradiographs first detected the presence of

DAT in the regions of cell bodies (substantia nigra, ventral

tegmental area) and nerve endings (striatum, nucleus

accumbens) at E18. As shown in Table 1, no significant

difference was observed between groups (MDMA and

saline) at each developmental stage both in the regions of

cell bodies and nerve endings.

Table 2

Effects of prenatal exposure to MDMA on the density of [3H]MADAM binding to the

nerve endings of serotonergic neurons

E18 E20 P0 P7

Raphe Saline 141.20 F 4.94 197.18 F 3.65 268.59 F 9.06 247.41 F 7

MDMA 146.91 F 14.24 198.12 F 17.65 262.71 F 12.12 231.13 F 9

Hyp Saline 95.06 F 12.24 94.353 F 10.71 76.33 F 5.18 80.15 F 1

MDMA 104.09 F 4.59 107.71 F 16.12 87.06 F 7.18 74.32 F 2

Thal Saline ND ND ND 50.94 F 6

MDMA ND ND ND 56.28 F 9

Hip Saline ND ND ND 29.88 F 2

MDMA ND ND ND 32.24 F 6

Cx Saline ND ND 65.75 F 3.18 197.05 F 1

MDMA ND ND 71.95 F 1.65 163.73 F 1

Results are expressed as mean F S.E.M. fmol/mg tissue of [3H]MADAM specifically b

nerve endings (Hyp, hypothalamus; Thal, thalamus; Hip, hippocampus; Cx, somatose

Animals are the progeny of females exposed to MDMA or saline between the 13th an

exposed to MDMA and 3 exposed to saline).

No difference was found between the MDMA and saline groups for each age (one-wa

3.3. Serotonin transporter density in the region of cell

bodies and nerve endings of serotonergic neurons

In this study, the density of [3H]MADAM binding sites

(Fig. 4) was assimilated to SERT levels.

In the regions of cell bodies (raphe nuclei), the SERTwas

first detected at E18 in both MDMA and saline groups, and

no significant difference was observed in the SERT density

of both groups whatever the stage of development (Table 2).

In other brain regions, the SERTwas detected from E18 in

the hypothalamus, from P0 in the somatosensory cortical

areas and from P7 in the thalamus and hippocampus (Table 2).

SERT from embryonic (E18) to adult (P70) age in the region of cell bodies and

P14 P21 P28 P70

.58 221.77 F 27.77 174.94 F 17.29 99.88 F 21.41 54.24 F 5.53

.39 226.47 F 4.24 188.20 F 26.47 112.12 F 14.12 61.41 F 10.94

.41 84.53 F 4.00 139.52 F 10.94 121.41 F 12.59 106.31 F 5.09

0.12 103.42 F 10.19 129.358 F 35.73 139.33 F 5.85 107.18 F 9.33

.48 102.82 F 10.07 98.31 F 8.69 79.74 F 7.54 58.00 F 3.71

.68 101.62 F 6.69 84.33 F 8.91 96.86 F 2.73 55.53 F 5.52

.36 40.35 F 1.47 51.41 F 0.89 35.53 F 1.88 26.71 F 2.53

.00 51.88 F 5.41 46.23 F 6.00 45.82 F 6.01 36.91 F 4.57

4.59 162.52 F 31.53 112.93 F 2.14 49.02 F 7.77 40.54 F 6.59

0.59 188.80 F 18.82 91.01 F 5.18 55.38 F 10.47 66.69 F 10.94

ound on coronal rat brain sections in the region of cell bodies (Raphe nuclei) and

nsory cortical areas) of serotonergic neurons. ND, under the limit of detection.

d 20th day of gestation (n = 3 animals for each age originated from 3 females

y analysis of variance, ANOVA, followed by post-hoc Student’s t-test).

Fig. 5. Effect of tyramine infusion on the dopamine release in the striatum

of MDMA and saline groups of adult awake animals (P70). bStimulationQcorresponds to the period of infusion in each group (n = 6 for each group).

Results are expressed as the mean percentage of basal levelsFS.E.M.

Differences between groups were compared using a one-way analysis of

variance (ANOVA) followed by post-hoc Dunnet’s test (*p b 0.05, **p b

0.01, ***p b 0.001).

Fig. 7. Sucrose preference test in male adult rats (P70) prenatally exposed to

MDMA or saline (n = 6 for each group). Results are expressed as individual

percentage of sucrose preference with the median in each group. Differ-

ences between groups were compared using a non parametric Mann–

Whitney test (***p b 0.001).


No significant difference was observed between groups

(MDMA and saline) at each developmental stage whatever

the brain region.

3.4. Microdialysis study of dopamine and serotonin release

under pharmacological stimulation

Tyramine and fenfluramine infusions via microdialysis

probes in P70 animals induced consistent release of DA in

the striatum (Fig. 5) and of 5-HT in the hippocampus (Fig. 6)

of both saline and MDMA groups. However, the release of

DA in the striatum and of 5-HT in the hippocampus were

significantly lower in the MDMA compared to saline group.

At the maximal effect of stimulation, DA levels were

Fig. 6. Effect of fenfluramine infusion on the serotonin release in the

hippocampus of MDMA and saline groups of adult awake animals (P70).

bStimulationQ corresponds to the period of infusion in each group (n = 6 for

each group). Results are expressed as the mean percentage of basal levelsFS.E.M. Differences between groups were compared using a one-way

analysis of variance (ANOVA) followed by post-hoc Dunnet’s test (*p b

0.05, **p b 0.01, ***p b 0.001).

approximately 11-fold lower in the MDMA than in the

saline group (2378 F 616% in the MDMA vs. 26397 F8184% in the saline group, p b 0.01) and 5-HT levels were

approximately 4-fold lower in the MDMA than in the saline

group (168.4 F 30.9% in the MDMA vs. 717.6 F 200% in

the saline group, p b 0.01).

3.5. Sucrose preference test

Adult (P70) rats from the saline group exhibited a strong

sucrose preference (94.0 F 1.7%) (Fig. 7). Rats from the

MDMA group displayed a mean significantly lower

preference (69.3 F 4.1%, U = 34, p b 0.001) (Fig. 7).

4. Discussion

The effects of prenatal exposure to MDMA on several

aspects of the maturation of the dopamine and serotonin

systems and on a behavioural test involving these systems

were studied in the model of rat. The interspecies scaling

technique was used in order to administrate MDMA doses

equivalent to those used by humans: a dose of 10 mg/kg/day

was administered to rats weighing around 300 mg; this

corresponds to a dose of 136 mg in a 70-kg human, according

to the relationship Dhuman = Danimal(Whuman/Wanimal)0.7,

where D = dose of drug in mg and W = body weight in kg

[47]. It is known that neurotoxicity observed in adult rats

after an MDMA exposure is exacerbated by hyperthermia

[49] and that MDMA induces a dysregulation of the animal

body temperature, this being highly dependant to the ambient

temperature [2,44,46]. Although our experiments have been

conducted in a temperature-controlled room (22 F 1 8C), it


cannot be excluded that a variation in the core temperature

may have occurred in animals exposed to the drug. Exposure

to MDMA is also known to reduce food intake, and in

agreement with this we observed that the maternal body

weight gain between the 13th and the 20th day of gestation

was 40% decreased in comparison to the saline-injected

females. Although we also found that the number of pups at

birth, their body and brain weights and the male/female ratio

were not modified (data not shown), effects of the maternal

low weight on the progeny can not be excluded and would

need more investigation.

We observed no modification in the levels of DA, Dopac

or HVA in the whole brain of pups from MDMA-treated

dams. In contrast, the brain 5-HT and 5-HIAA contents were

two-fold decreased in the MDMA group at P0, a devel-

opmental stage characterized by the occurrence of 5-HT and

5-HIAA peaks. These reductions were no more observed at

P7 and P14, in agreement with previous data [1,10]. As the

time of MDMA administration to pregnant dams ended at

the 20th day of gestation, it cannot be excluded that such a

decrease in P0 pups could be related to a subacute effect of

MDMA. In addition, a more precise quantification of

monoamine levels throughout the brain, i.e. related to the

measurement of protein concentration in specific cerebral

regions, would be of great value in the interpretation of

these results. However, as 5-HT plays a key role in cerebral

maturation due to its involvement in neurogenesis, neuronal

differentiation, neuropil formation, axon myelinisation and

synaptogenesis [36–39], it can be hypothesized that the

decrease we observed in 5-HT brain content during this

critical period may have long-term consequences on brain

maturation and functions. In agreement with this, in vivo

microdialysis studies in adult rats prenatally exposed to

MDMA showed strong decreased levels of pharmacologi-

cally stimulated release of DA and 5-HT in the striatum and

hippocampus, respectively. As tyramine [14,40] and fenflur-

amine [4] have been demonstrated to induce the massive

release of vesicular DA and 5-HT, respectively, these

reductions probably reflect a reduced storage pool of DA

and 5-HT. This reduction could be due to several processes

among them a reduced number of projection fibres in the

striatum and hippocampus, a decreased activity of the

tyrosine and tryptophan hydroxylase, or alterations in the

density or function of the vesicular monoamine transporter

(VMAT2). We studied here the potential effects of prenatal

MDMA exposure on the maturation of dopaminergic and

serotonergic systems by the quantitative analysis of the DAT

and SERT from embryonic to adult ages, as these trans-

porters are relevant indices of the density and functionality

of monoaminergic neurons [16]. The SERT level was also

studied in the barrel-fields of the somatosensory cortical

areas whose development is regulated by thalamic non-

serotonergic neurons transiently expressing 5-HT, as it has

been demonstrated that the experimental depletion of 5-HT

between P0 and P4 causes shrinkage of barrels [5]. The

developmental profiles of the DAT and SERT in this report

were in agreement with several previous data [11,15,59]. No

effect of the prenatal exposure to MDMA was observed in

these profiles in both cell bodies and nerve endings. This

result suggests that no alteration occurred in the dopami-

nergic and serotonergic fibres. However, a reduced activity

had previously been demonstrated for the tryptophan

hydroxylase after an acute administration of MDMA [53],

while an increase in tyrosine hydroxylase positive fibres has

been described in the striatum after a prenatal treatment with

MDMA [35]. It can be proposed that the reduced DA and 5-

HT stimulated release could be consecutive to alterations in

the VMAT2 density or function as already observed in a

model of chronic nutritional deficit during development

[34,64]. In addition, it has already been demonstrated that

an acute administration of MDMA was able to induce a

decrease in the vesicular DA and 5-HT transport [6,24].

However, this hypothesis needs additional experimental

results to be confirmed.

The modifications of dopaminergic and serotonergic

functions observed in adult prenatally MDMA exposed rats

put the question of the occurrence of alterations in

behaviours related to these functions. It had already been

demonstrated that exposition to prenatal stressors could

induce symptoms of depression [61]. One of the core

symptoms of depression is anhedonia which mechanism

seems to implicate DA and 5-HT, and which can be studied

in rodents via a sucrose preference paradigm. We used this

behavioural test in adult rats and observed a decrease of

sucrose preference in the prenatally MDMA exposed group.

This cannot be due to a trivial modification of sucrose

consumption [31,62,33], as we assessed preference for a

sucrose solution rather than sucrose intake [21]. The two-

bottle procedure was used, measuring amounts of both

water and sucrose consumed [12,13,19,26], rather than the

simply monitoring of water intake periodically during the

experiment [48,63]. Moreover, there were no weight differ-

ences between saline and MDMA group (results not shown)

that could explain the altered sucrose consumption. Differ-

ent interpretations can be suggested as to this modification

in sucrose preference such as a decrease in the sensory

perception of sweet, anhedonia, reduction in the incentive

salience or deficit in goal directed behaviours. The decrease

in sucrose preference and the alterations in monoaminergic

neurotransmission observed in our study could be linked.

Indeed, a decrease in sucrose preference has already been

associated with dopaminergic [23,55,56] as well as seroto-

nergic [17,25] functions. In addition, changes in brain

maturation related to the modification of 5-HT during early

post-natal period could also be involved in the result of

behavioural test. In agreement with this, anxiety-like

behaviours in adult mice seem to be related to the

expression of the 5-HT1A receptor during the early postnatal

period rather than at adult age [20].

Several studies had already showed consequences of

acute or chronic MDMA exposure on both the serotonergic

and dopaminergic functions (see Refs. [18,43] for review),


but few data were available on the effects of prenatal

exposure to this drug. We demonstrated in rats that this

exposure induced alteration of 5-HT and 5-HIAA cerebral

levels at P0 but no alteration in DA and its metabolites levels

from E14 to P21. The serotonergic system appeared therefore

to be more vulnerable to an MDMA exposure in the rat

developing brain than the dopaminergic system. However, if

no long-term consequences have been observed in DAT and

SERT levels at P70 (our study) as well as on tyrosine and

tryptophan hydroxylase activity [1,10,35], we found severe

decreases in DA and 5-HT storage pools in adult rats

prenatally exposed to MDMA and altered sucrose prefer-

ence. These long-term consequences could be related to the

effect of MDMA on decreased 5-HT cerebral levels during

brain development, particularly during the occurrence of the

maximum peak of 5-HT. It can also be suggested that

MDMA could interact with other components of the

monoaminergic function during brain development such as

the VMAT2.

This report thus reinforces and extends the recent study of

Koprich et al. [35] showing that prenatal exposure to MDMA

is able to induce short and long-term neurochemical and

behavioural alterations. Our rat model was little different

from the previous (for example we used a dose of 10 mg/kg

instead or 2 � 15 mg/kg/day during the same gestational

period), but both studies demonstrated alterations in the

monoamine brain tissue levels, and in behaviour, even if the

precise modifications were different, probably due to differ-

ent protocols (for example developmental stages and

cerebral region in neurochemical studies, type of tests in

behavioural studies). The present study suggests that

prenatal MDMA exposure acts on anhedonia, which appears

as a major symptom of several psychiatric disorders, raising

the possibility that prenatal MDMA exposure in rat could be

an interesting model to study developmental disturbances

underlying such disorders.

Acknowledgments

This work was supported by Inserm, MRT and the

Programme Interdisciplinaire 2001–2004 bImagerie du Petit

AnimalQ. We thank Mary-Christine Furon for animal care.

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Prenatal 3, 4-methylenedioxymethamphetamine (ecstasy) exposure induces long-term alterations in the...

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