Influences of the corticotropic axis and sympathetic activity on neurochemical consequences of 3,4-methylenedioxymethamphetamine (MDMA) administration in Fischer 344 rats

In¯uences of the corticotropic axis and sympatheticactivity on neurochemical consequences of 3,4-methylenedioxymethamphetamine (MDMA) administrationin Fischer 344 rats

Francesca Fernandez, Sylvie Aguerre, Pierre MormeÁde and Francis ChaouloffNeuroGeÂneÂtique et Stress, INSERM U471-INRA, Institut F. Magendie, Rue Camille Saint SaeÈns, 33077 Bordeaux CeÂdex, France

Keywords: adrenalectomy, body temperature, chlorisondamine, hippocampus, serotonergic systems, striatum

Abstract

The respective in¯uences of the corticotropic axis and sympathetic activity on 3,4-methylenedioxymethamphetamine (MDMA,ecstasy) immediate effects on body temperature and long-term neurotoxicity, as assessed by decreases in hippocampal and

striatal [3H]5-hydroxytryptamine ([3H]5-HT) reuptake, [3H]paroxetine binding at 5-HT transporters (5-HTT), and 5-HT and

5-hydroxyindoleacetic acid (5-HIAA) levels, were examined in Fischer 344 rats. On each of the two injections of MDMA (5 or10 mg/kg s.c. once a day for 2 consecutive days) body temperature rapidly increased in a dose-dependent manner. Six days

after the last injection of 10 mg/kg MDMA, [3H]5-HT reuptake, [3H]paroxetine binding and 5-HT and 5-HIAA levels were

decreased in the hippocampus and, to a lower extent, in striatum. Prior adrenalectomy (1 week beforehand), which weakened theimmediate hyperthermic effect of MDMA, prevented the long-term MDMA-elicited reduction in hippocampal and striatal

[3H]paroxetine binding. Supplementation of adrenalectomised Fischer 344 rats with corticosterone almost reinstated the

immediate hyperthermic effect of MDMA and restored MDMA-elicited reduction in hippocampal and striatal [3H]paroxetine

binding. In a ®nal set of experiments, Fischer 344 rats were pretreated (30 min before each of the two injections of 10 mg/kgMDMA) with the ganglionic blocker chlorisondamine (2.5 mg/kg). This pretreatment markedly reduced the amplitudes of the

immediate hyperthermia and long-term declines in hippocampal [3H]5-HT reuptake and [3H]paroxetine binding at 5-HTT, and in

hippocampal and striatal 5-HT and 5-HIAA levels. These results suggest that sympathetic activity (possibly through its control ofbody temperature), but not corticotropic activity, plays a key role in MDMA-elicited neurotoxicity in Fischer 344 rats.

Introduction

Administration of 3,4-methylenedioxymethamphetamine (MDMA,

ècstasy') to rats, guinea pigs and nonhuman primates produces, in

the long term, lasting reductions in markers of serotonergic neurons

(Battaglia et al., 1987; Commins et al., 1987; Battaglia et al., 1988;

Ricaurte et al., 1988; De Souza et al., 1990). These markers include

the concentrations of 5-hydroxytryptamine (serotonin, 5-HT) and its

metabolite 5-hydroxyindoleacetic acid (5-HIAA), tryptophan

hydroxylase activity and the density and the reuptake activity of

plasma membrane 5-HT transporters (5-HTT). Taken with morpho-

logical evidence for an MDMA-elicited degeneration of ®ne 5-HT

nerve terminals (Commins et al., 1987; Molliver et al., 1990), it is

now believed that the reductions in these markers re¯ect neuro-

toxicity (Green et al., 1995; Sprague et al., 1998).

The mechanisms through which MDMA is neurotoxic to

serotonergic systems are only partly identi®ed. There is evidence

for an initial 5-HTT-mediated reuptake of MDMA into serotonergic

nerve terminals followed by 5-HT release from storage vesicles

(Schmidt et al., 1987; Steele et al., 1987; Rudnick & Wall, 1992).

That pretreatment with a 5-HT2A receptor antagonist prevents the

long-term neurotoxic impact of MDMA (Schmidt & Kehne, 1990;

Schmidt et al., 1990) suggests that direct or indirect (through MDMA

metabolites) release of 5-HT and, in turn, 5-HT2A receptor stimulation,

plays a role in neurotoxicity. It has been proposed that MDMA-elicited

release of dopamine (Yamamoto & Spanos, 1988; Nash, 1990;

Schmidt et al., 1994; White et al., 1994) leads to its uptake by

serotonergic nerve terminals where it is deaminated to generate

oxidative processes (Schmidt & Kehne, 1990; Sprague et al., 1998).

Furthermore, the formation of MDMA metabolites which may

generate free radicals may be another mechanism leading to oxidative

stress in serotonergic nerve terminals (Schmidt & Kehne, 1990; Green

et al., 1995; Sprague et al., 1998). This theory has received support

from the measurement of free radicals (Colado et al., 1997), and the

protective effects of spin-trap reagents and metabolic antioxidants

(Schmidt & Kehne, 1990; Colado & Green, 1995; Gudelsky, 1996;

Aguirre et al., 1997; Colado et al., 1997). Lastly, MDMA-elicited

hyperthermia, which may favour the generation of free radicals

(Globus et al., 1995), has also received much attention because

environment- and drug-induced decreases in core temperature prior to

MDMA injection may trigger neuroprotection (Miller & O'Callaghan,

1994; Farfel & Seiden, 1995; Malberg et al., 1996; Malberg & Seiden,

1998; Colado et al., 1999). This result then suggests that drugs

endowed with sympatholytic activity should bear neuroprotective

properties against MDMA-elicited neurotoxicity; however, to our

knowledge, such a possibility has not been tested so far.

Correspondence: Dr Francis Chaouloff, as above.E-mail: [email protected]

Received 13 March 2002, revised 6 June 2002, accepted 10 June 2002

doi:10.1046/j.1460-9568.2002.02110.x

European Journal of Neuroscience, Vol. 16, pp. 607±618, 2002 ã Federation of European Neuroscience Societies

It has been suggested that the corticotropic axis also plays a role in

neurotoxic effects of MDMA. Thus, MDMA stimulates cortico-

sterone release (Nash et al., 1988) and alters corticoid receptor gene

expression, especially in the hippocampus (Yau et al., 1994).

Adrenalectomy, but not adrenalectomy combined with corticosterone

supplementation, prevents or attenuates the long-term decreases in

hippocampal tryptophan hydroxylase activity (and 5-HT and 5-HIAA

levels) elicited, respectively, by single 10 or 20 mg/kg doses of

MDMA (Johnson et al., 1989). This result raises three questions: ®rst,

does the impact of adrenalectomy on these markers extend to other

indices of neurotoxicity (5-HTT binding and/or reuptake activity)?

This question is crucial as corticoids are permissive to 5-HT synthesis

(see Chaouloff, 1993), thus opening the possibility that MDMA-

elicited corticosterone release affects hippocampal 5-HT synthesis

but not the integrity of hippocampal serotonergic neurons. Second, if

corticoids mediate the long-term neurotoxic effects of MDMA, is it

possible to anticipate such a result by the measurement of some

immediate effects of MDMA, e.g. on body temperature? Third, the

study of Johnson et al. (1989) used Sprague-Dawley rats, i.e. rats

endowed with a corticotropic axis thought to be normoresponsive

compared to other strains (Aulakh et al., 1988; Sternberg et al., 1992;

Dhabbar et al., 1993). Thus it would be expected that the

corticotropic axis would impact even more on MDMA-induced

degeneration of hippocampal 5-HT neurons if this axis were

hyperactive and/or hyperreactive. The Fischer 344 (F344) rat strain,

which bears such hyperactivity and hyperesponsiveness of the

corticotropic axis (Sternberg et al., 1989; Calogero et al., 1992;

Sternberg et al., 1992; Dhabbar et al., 1993), should be suitable for

testing the latter hypothesis.

With these questions in mind, we ®rst analysed the immediate body

temperature and long-term neurochemical effects of MDMA in F344

rats. To address the issue of the long-term neurochemical conse-

quences of MDMA administration, we focused our attention on

hippocampal [3H]5-HT reuptake, hippocampal and striatal

[3H]paroxetine binding at 5-HTT, and hippocampal and striatal

5-HT and 5-HIAA levels. Then, to investigate the role of the

corticotropic axis in the long-term neurochemical effects of MDMA,

we measured the respective impacts of adrenalectomy with and

without corticosterone supplementation on the aforementioned

neurochemical variables to which we added striatal [3H]5-HT

reuptake analyses. In keeping with the observation that all these

manipulations had consequences on the immediate body temperature

responses to MDMA, we took advantage of the lack of information as

to the putative neuroprotective properties of sympatholytics (see

above) to analyse, by means of the ganglionic blocker chlorisond-

amine, the extent to which sympathetic activity had also an in¯uence

on the long-term neurochemical effects of MDMA.

Materials and methods

Animals and housing conditions

Inbred male F344 rats (IFFA CREDO, Les Oncins, France), aged

5 weeks on arrival, were housed four per cage under constant

temperature (22 6 1 °C) and a 12-h light : 12-h dark cycle (lights on

at 07.00 h), with food and water ad libitum. All rats were tested at

least 2 weeks after their arrival, except for rats undergoing sham

surgery or adrenalectomy, which were tested at least 3 weeks after

their arrival (1 week after surgery). All efforts were made to

minimize the number of animals used, and procedures were as

established by the French legislation on animal welfare (O.J. no. 87±

848).

MDMA administration

After weighing, rats were injected s.c. with either saline or MDMA,

this protocol being repeated 24 h later. Seven days after the ®rst of

the two injections, the rats were killed by cervical dislocation, the

brains removed and the hippocampi and the striata rapidly dissected

out. In the ®rst series of experiments, 5 and 10 mg/kg doses of

MDMA were used, whereas only the highest dose was used

thereafter. Note that in the last series of experiments, rats were

pretreated 30 min beforehand with saline or chlorisondamine

(2.5 mg/kg i.p.) before each of the two injections of saline or

MDMA. This dose of chlorisondamine has been shown to fully block

sympathetic activity (McCarty & Kopin, 1979) without altering the

vigilance state of the animal (Chaouloff et al., 1994).

Surgery and plasma corticosterone analyses

In one series of experiments, rats were anaesthetized with a 60-mg/kg

dose of pentobarbitone sodium, and the adrenals visualized (sham

rats) or removed (adrenalectomised rats) through bilateral incision in

the ¯anks. In a second series of experiments, a similar procedure was

followed, except that half of the adrenalectomised rats were at the

same time implanted with subcutaneous corticosterone pellets. For

this purpose, powdered corticosterone was mixed with cholesterol

(1 : 1), melted and poured into wax moulds designed for 2-week

release, as previously reported (Cador et al., 1993; Kulikov et al.,

1997). The weight of the pellet contents was adjusted to 100 mg

(50 mg corticosterone). Following surgery, the animals were returned

to their home cages with tap water containing (adrenalectomised

rats) or not (sham rats) 0.9% NaCl (saline). In the two series of

experiments, rats were injected twice with saline or MDMA 7 and

8 days after surgery, and killed another week after the ®rst injection.

In the second series of experiments, thymus weights as well as

circulating levels of corticosterone were analysed at the time of

dissection. For corticosterone analyses, trunk blood was collected in

EDTA-coated tubes and centrifuged. Plasma corticosterone levels

were assessed by competitive protein binding following extraction

with absolute ethanol using rhesus monkey serum as the source of

transcortin and [3H]corticosterone as the radioligand (Cador et al.,

1993; Kulikov et al., 1997).

Drugs and chemicals

MDMA hydrochloride was generously supplied by the National

Institute on Drug Abuse (Bethesda, MD, USA). Corticosterone,

cholesterol, pargyline hydrochloride, 5-HT hydrochloride and

5-HIAA were purchased from Sigma Chemicals (St Quentin

Fallavier, France). Fluoxetine hydrochloride and citalopram hydro-

chloride were, respectively, obtained from Mediat (Milan, Italy) and

H. Lundbeck A/S (Copenhagen, Denmark); chlorisondamine hydro-

chloride was obtained from Ciba-Geigy (now Novartis, Basel,

Switzerland). [3H]5-HT creatinine sulphate (21.4 Ci/mmol),

[3H]paroxetine (19.7 Ci/mmol) and [3H]corticosterone (76.5 Ci/

mmol) were all purchased from New England Nuclear (Paris,

France).

Neurochemical assays

[3H]5-HT reuptake assays were performed as previously described

(Pollier et al., 2000). Thus, fresh hippocampi and/or striata were

homogenized in ice-cold 0.32 M sucrose and centrifuged at 1000 g

(10 min, 4 °C). The supernatants were removed and centrifuged at

12 000 g (30 min, 4 °C): the resulting P2 pellets were then

resuspended in ice-cold 0.32 M sucrose and used for reuptake studies.

Sample aliquots of 50 mL were preincubated (5 min, 37 °C) in the

608 F. Fernandez et al.

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 16, 607±618

presence of 350 mL of oxygenated (5% CO2/95% O2 for 30 min)

Krebs buffer containing (in mM) NaCl, 120; NaHCO3, 25; glucose,

10; KCl, 5; MgCl2, 1.2; EDTA, 0.05; CaCl2, 1.3; NaH2PO4, 1;

ascorbic acid, 0.1; and pargyline, 0.06 (glucose, ascorbic acid and

pargyline were made fresh every day). After preincubation, 50 mL of

10 nM [3H]5-HT were added, and the tubes left for further incubation

under weak agitation (10 min, 37 °C). For inhibition studies, 10 nM

[3H]5-HT were added to the buffer that contained (or not: baseline

level) 50 mL of MDMA (10-9±10-3M). All uptake assays, performed

in duplicate, were stopped by the addition of cold buffer through

Whatman GF/C glass ®bre ®lters (Poly Labo, Strasbourg, France).

The tubes and the ®lters were then washed with the buffer, and

entrapped radioactivity counted by liquid scintillation. Nonspeci®c

uptake was determined with 10 mM ¯uoxetine. Protein concentrations

were assessed using bovine serum albumin as standard (Bradford,

1976).

[3H]Paroxetine binding assays were performed as described

previously (Pollier et al., 2000), except that [3H]paroxetine was

used instead of [3H]citalopram. Following dissection, hippocampi

and striata were put on dry ice before storage at ±80 °C. Tissues were

then homogenized in 40 volumes ice-cold Tris-HCl buffer (pH 7.4),

and centrifuged (20 000 g for 10 min at 4 °C). The resulting pellets

were washed twice by resuspension in 40 volumes Tris-HCl, the last

washing lasting 1 h (35 °C) to remove endogenous 5-HT. The pellets

resulting from the third centrifugation were then stored at ±80 °C

until binding experiments. At that time, the pellets were suspended in

a 50-mM Tris±HCl buffer (pH 7.4) containing 5 mM KCl and 120 mM

NaCl, and transferred to glass tubes. The reaction was carried out for

90 min at 25 °C in the presence of 1 nM [3H]paroxetine with/without

10 mM ¯uoxetine for estimation of nonspeci®c binding. All assays

were stopped by the addition of cold buffer followed by a rapid

®ltration through Whatman GF/B glass ®bre ®lters. The ®lters were

washed twice with the buffer, and entrapped radioactivity counted by

liquid scintillation. Protein concentrations were assessed using bovine

serum albumin as standard (Bradford, 1976).

For 5-HT and 5-HIAA determinations, tissues were assayed as

previously described (Pollier et al., 2000). Except for the column and

the electrochemical detector (see below), all other materials (auto-

matic injector, cooled autosampler, pump, peak integrator, software)

were from Hewlett-Packard (series 1100; Hewlett-Packard France,

Evry, France). Tissues were stored at ±80 °C before being weighed,

sonicated in 0.4 N perchloric acid, and centrifuged (15 000 g for

10 min at 4 °C). Ten microlitres were injected in a C18 reversed-

phase Spherisorb column (150 3 4.6 mm, 5-mm spheres; Touzart et

Matignon, Courtaboeuf, France) with a mobile phase consisting of

0.05 M sodium acetate, 0.045 M citrate buffer (pH 3.8) containing

0.001% octane sulphonic acid and 15% methanol. Hippocampal and

striatal 5-HT and 5-HIAA concentrations were assessed using a

Decade electrochemical detector with capillary cell design (Antec,

Leiden, The Netherlands) set at 0.67 V (20 nA) against a Ag/AgCl

reference electrode.

Body temperature recordings

Rats were gently restrained and temperature measured by insertion of

a thermocouple, connected to a digital readout (Thermalert TH-5,

Physitemp; Phymep, Paris, France), into the rectum. Body tempera-

tures were measured three times, each separated by a 30-min interval,

before the s.c. administration of saline or MDMA. For technical

reasons, body temperature recordings in the ®rst series of experiments

(dose±responses to MDMA) lasted only for the 2 h (four recordings)

that followed MDMA injection. Note, however, that MDMA effects

on temperature are already maximal at 2 h (Dafters, 1994; Malberg

et al., 1996; Colado et al., 1997; Malberg & Seiden, 1998), including

in F344 rats (see Results). In the other series of experiments, body

temperatures were recorded up to 3 h after MDMA injection, except

for some chlorisondamine-pretreated rats which were recorded up to

7 h after MDMA injection (see Results). All these procedures were

repeated 24 h later. In all series of experiments, the ambient

temperature ranged between 22 and 23 °C and stable body tempera-

ture was reached within the 30 s that followed the probe insertion.

Statistics

All data are given as means 6 SEM. When necessary, data were log-

transformed to achieve homogeneity of variances before comparison

through one-way and multivariate analyses of variance (ANOVA)

followed, if signi®cant, by Tukey's multiple comparison test. One-

way ANOVAs included one between-factor (Dose of MDMA) whereas

multivariate ANOVAs included two between-factors, namely

Pretreatment (sham or adrenalectomy; sham, adrenalectomy or

adrenalectomy plus corticosterone; saline or chlorisondamine) and

Treatment (saline or MDMA) to which was added one within-factor

(i.e. Time) for body temperature data analyses.

Results

Immediate effects of MDMA on body temperature in F344 rats

On the ®rst injection, the pretreatment (F2,21 = 45.21, P < 0.0001),

the latency between drug injection and body temperature recording

(F4,84 = 131.01, P < 0.0001) and the interaction between these

variables (F8,84 = 55.84, P < 0.0001), all affected rectal temperature.

MDMA administration was found to elicit dose-dependent, albeit

delayed, increases in body temperature in F344 rats (Fig. 1A). As

observed on the ®rst injection, the pretreatment (F2,12 = 124.13,

P < 0.0001) and the latency between drug injection and body

temperature recording (F4,48 = 119.6, P < 0.0001) affected rectal

temperature on the second day, as did the interaction between these

variables (F8,48 = 37.75, P < 0.0001). Indeed, it was found that the

second injection of MDMA, as opposed to the ®rst injection, elicited

immediate dose-dependent increases in body temperature in F344 rats

(Fig. 1B).

Long-term neurochemical effects of MDMA in F344 rats

Table 1 reports the effects of MDMA (last of the two injections

6 days beforehand) on hippocampal [3H]5-HT reuptake rates, and on

hippocampal and striatal [3H]paroxetine binding at 5-HTT and 5-HT

and 5-HIAA levels in F344 rats (striatal [3H]5-HT reuptake was not

assayed in this ®rst series of experiments). Hippocampal [3H]5-HT

reuptake and [3H]paroxetine binding were in¯uenced by the pretreat-

ment (F2,20 = 16.13, P < 0.0001 and F2,14 = 12.49, P = 0.0012,

respectively) with MDMA decreasing [3H]5-HT reuptake in a dose-

dependent manner whereas only the highest dose proved effective on

[3H]paroxetine binding (Table 1). Similarly, hippocampal 5-HT and

5-HIAA levels differred with regard to the pretreatment

(F2,7 = 22.58, P = 0.0009 and F2,6 = 25.09, P = 0.0012, respect-

ively), with the 10 mg/kg dose of MDMA decreasing these levels

(Table 1).

In striatum, [3H]paroxetine binding measurements and 5-HT

levels varied with the pretreatment (F2,9 = 5.24, P = 0.031, and

F2,15 = 4.22, P = 0.039, respectively), with the 10 but not the

5 mg/kg dose having signi®cant impacts (Table 1).

As these results indicated that F344 rats were sensitive to the long-

term neurochemical effects of MDMA, we next examined whether

the corticotropic axis mediated such a sensitivity. To do so, we only

Stress hormones, MDMA and serotonergic systems 609


used the highest dose of MDMA (10 mg/kg) but included striatal

[3H]5-HT reuptake analyses.

Impact of adrenalectomy on MDMA immediate effects on bodytemperature, and long-term neurochemical effects in F344 rats

On the ®rst injection of saline or MDMA (10 mg/kg), adrenalectomy

(F1,20 = 10.5, P = 0.004), MDMA (F1,20 = 28.13, P < 0.0001) and

the latency between drug injection and body temperature recording

(F5,100 = 47.92, P < 0.0001) all affected rectal temperature, as did

the interactions between these variables (P < 0.0063 for each

interaction; data not shown). As shown in Fig. 2A, MDMA injection

promoted hyperthermia during the last two hours of temperature

analysis in sham animals whereas it ®rst triggered hypothermia and

then hyperthermia (the amplitude of which was weaker than in sham

rats) in adrenalectomised animals. Twenty-four hours later, adrena-

lectomy (F1,12 = 5.4, P = 0.039), MDMA (F1,12 = 35.87, P <

0.0001), the latency between drug injection and body temperature

recording (F5,60 = 21.61, P < 0.0001), and the interaction between

the two last variables (F5,60 = 39.95, P < 0.0001) affected rectal

temperature. Actually, the overall pattern of body temperature

responses to MDMA injection in sham and adrenalectomised rats

was similar to that observed on the ®rst injection, except that the

amplitude of MDMA-elicited hyperthermia in adrenalectomised rats

was much larger on the second injection (Fig. 2B).

As shown in Table 2, MDMA pretreatment (6 and 7 days

beforehand) had inhibitory consequences on all the variables

examined in the hippocampus, namely [3H]5-HT reuptake

(F1,18 = 49.08, P < 0.0001), [3H]paroxetine binding (F1,18 = 17.19,

P = 0.0006) and 5-HT (F1,16 = 112.14, P < 0.0001) and 5-HIAA

(F1,16 = 29.74, P < 0.0001) levels. These effects were associated

with a stimulatory in¯uence of adrenalectomy on [3H]5-HT reuptake

(F1,18 = 10.19, P = 0.005) and a signi®cant MDMA 3 adrenal-

ectomy interaction (F1,18 = 5.77, P = 0.027) on [3H]paroxetine

binding at 5-HTT. Thus, adrenalectomy prevented the MDMA-

elicited decrease in [3H]paroxetine binding through a reduction in

that variable in saline-injected rats (Table 2). With regard to

the striatum, [3H]5-HT reuptake (F1,16 = 30.23, P < 0.0001),

[3H]paroxetine binding at 5-HTT (F1,20 = 9.8, P = 0.0053, and

5-HT (F1,20 = 26.19, P < 0.0001) and 5-HIAA (F1,20 = 5.62,

P = 0.0028) levels, were all under the inhibitory in¯uence of

MDMA, such an in¯uence being associated with a stimulatory

in¯uence of adrenalectomy on 5-HIAA (F1,20 = 5.62, P = 0.0028)

and MDMA 3 adrenalectomy interactions on [3H]paroxetine binding

at 5-HTT (F1,20 = 13.02, P = 0.002) and on 5-HT levels (F1,20 =

6.95, P = 0.0158). As reported for the hippocampus, adrenalectomy

prevented the MDMA-elicited decrease in [3H]paroxetine binding

through a reduction in that variable in saline-injected rats.

Furthermore, the amplitude of the MDMA-induced decrease in

striatal 5-HT and 5-HIAA observed in sham rats was markedly

weakened by prior adrenalectomy (Table 2).

In keeping with these results indicating that adrenalectomy had

signi®cant impacts on MDMA-elicited hyperthermia on the one

hand, and several long-term neurochemical responses to MDMA

TABLE 1. Long-term effects of MDMA on hippocampal and striatal

serotonergic systems in F344 rats

MDMA (mg/kg/day, twice)²

0 5 10

Hippocampus[3H]5-HT reuptake

(pmol/min/mg protein)0.59 6 0.02 0.46 6 0.03** 0.35 6 0.02**

[3H]Paroxetine binding(fmol/mg protein)

483 6 44 562 6 45 271 6 38**

5-HT (nmol/g) 1.63 6 0.14 1.60 6 0.03 0.73 6 0.07**5-HIAA (nmol/g) 1.42 6 0.04 1.30 6 0.06 0.83 6 0.09**

Striatum[3H]Paroxetine binding

(fmol/mg protein)528 6 53 420 6 21 375 6 24*

5-HT (nmol/g) 2.58 6 0.08 2.40 6 0.23 1.75 6 0.22**5-HIAA (nmol/g) 1.86 6 0.16 1.76 6 0.14 1.44 6 0.17

²All rats were injected twice (1-day interval) either with saline or MDMA (5or 10 mg/kg, s.c.) and tissues assayed one week after the ®rst injection. Valuesare given as the mean 6 SEM of 7±8 ([3H]5-HT reuptake), 5±6([3H]paroxetine binding) and 3±6 (5-HT and 5-HIAA concentrations)determinations. *P < 0.05, **P < 0.01 for the in¯uence of MDMA vs. saline.

FIG. 1. Effects of (A) the ®rst and (B) the second injections of saline orMDMA (5 or 10 mg/kg, s.c. at time 0) on body temperature in F344 rats.The two injections were separated by a 1-day interval and bodytemperatures recorded every 30 min for 2 h. Rats were given a 1-hhabituation period (three body temperature recordings) to the experimentalset up before each injection. Each value is the mean 6 SEM of eight(injection 1) and ®ve (injection 2) determinations. *P < 0.05, **P < 0.01for the in¯uence of MDMA vs. saline.



administration on the other hand, we next performed a series of

experiments aimed at determining the role of the adrenal cortex in

these responses.

Impact of corticosterone supplementation on MDMAimmediate effects on body temperature, and long-termneurochemical effects in adrenalectomised F344 rats

Table 3 depicts the respective in¯uences of MDMA on the one hand,

and adrenalectomy with or without corticosterone supplementation on

the other, on thymus weights and circulating corticosterone levels.

Thymus absolute (F2,82 = 27.08, P < 0.0001) and relative (F2,82 =

76.47, P < 0.0001) weights were increased by adrenalectomy, and

restored to sham values by corticosterone supplementation. Plasma

corticosterone levels were affected by the initial pretreatment

(F2,52 = 31.35, P < 0.0001) and a pretreatment 3 MDMA inter-

action (F2,52 = 3.65, P = 0.033); corticosterone supplementation

almost reversed the adrenalectomy-elicited fall in hormone levels

of saline-treated rats whilst MDMA increased plasma corticosterone

levels only in sham animals (Table 3).

Figure 3 reports the immediate body temperature responses to the

two injections of MDMA (10 mg/kg) in sham and adrenalectomised

(supplemented or not with corticosterone) rats. Administration of

MDMA (F1,42 = 66.9, P < 0.0001, and F1,42 = 49.5, P < 0.0001, for

the ®rst and second injection, respectively), and the latency between

drug injection and body temperature recording (F5,210 = 93.86,

P < 0.0001, and F5,210 = 60.85, P < 0.0001, for the ®rst and second

series of experiments, respectively) affected rectal temperature, as

did the interactions between all variables (P < 0.022 for each

interaction; data not shown). As observed earlier (see above), each

injection of MDMA elicited a rapid hypothermia in adrenal-

ectomised rats that was followed by hyperthermia, the amplitude of

which was weaker than in sham animals. Corticosterone supple-

mentation prevented the hypothermia consecutive to the second but

not the ®rst injection of MDMA, whilst restoring the maximal

hyperthermic effect of MDMA to the level reached in sham animals

(Fig. 3).

Table 4 shows the long-term neurochemical in¯uences of MDMA

in the hippocampus and striatum of sham and adrenalectomised

(supplemented or not with corticosterone) rats. As far as [3H]5-HT

reuptake was concerned, it was affected in hippocampus by the initial

pretreatment (F1,28 = 6.58, P = 0.0045), with adrenalectomy increas-

ing that variable. In addition, MDMA administration was endowed

with inhibitory in¯uences on hippocampal (F1,28 = 111.09,

P < 0.0001) and, albeit to a much lower extent (as illustrated by

the lack of post hoc differences), on striatal (F1,26 = 7.36, P = 0.012)

[3H]5-HT reuptake. With regard to [3H]paroxetine binding at 5-HTT,

the initial pretreatment had an in¯uence on hippocampal

(F2,27 = 3.99, P = 0.03) and striatal (F2,39 = 3.69, P = 0.034)

5-HTT binding whilst MDMA decreased such a binding in both

regions (hippocampus: F1,27 = 34.73, P < 0.0001; striatum:

F1,39 = 9.71, P = 0.003). Indeed, post hoc analyses revealed that,

whatever the region, MDMA-elicited decreases in [3H]paroxetine

binding reached signi®cance only in sham rats and in adrenalectom-

ised rats supplemented with corticosterone (Table 4). In slight

contrast with the results mentioned above (Table 2), the post hoc

comparison between sham and adrenalectomised (saline-injected) rats

for hippocampal [3H]paroxetine binding did not reach signi®cance in

the present series of experiments (Table 4). Administration of

MDMA, but not the pretreatment, had signi®cant (inhibitory) effects

on hippocampal 5-HT and 5-HIAA levels (F1,24 = 63.01, P < 0.0001

and F1,24 = 63.74, P < 0.0001, respectively). Striatal 5-HT and

5-HIAA levels were also decreased by MDMA (F1,26 = 28.24,

P < 0.0001 and F1,26 = 18.46, P = 0.0002, respectively) but, as

opposed to the experiments mentioned under Table 2, the amplitudes

of MDMA inhibitory effects were strong enough to observe

signi®cant post hoc differences in adrenalectomised rats (Table 4).

Beside indicating that corticosterone release may have differential

in¯uences on the neurochemical consequences of MDMA adminis-

tration (see Discussion), the results from this series of experiments

also suggested that the adrenomedulla plays a role in the immediate

body temperature response to MDMA. In keeping with this obser-

vation, we then addressed the question of the role of the entire

sympathetic nervous system on body temperature responses to

MDMA, and its long-term consequences on hippocampal and striatal

serotonergic systems.

FIG. 2. Effects of (A) the ®rst and (B) the second injections of saline orMDMA (10 mg/kg, s.c. at time 0) on body temperature in 7-day sham andadrenalectomised F344 rats. The two injections were separated by a 1-dayinterval and body temperatures recorded every 30 min for 3 h. Rats weregiven a 1-h habituation period (three body temperature recordings) to theexperimental setup before each injection. Each value is the mean 6 SEM ofsix (injection 1) and four (injection 2) determinations. *P < 0.05,**P < 0.01 for the in¯uence of MDMA against saline. For clarity, thedifferences between sham and adrenalectomy are not shown.



Impact of ganglionic blockade on MDMA immediate effects onbody temperature, and long-term neurochemical effects inF344 rats

When examined up to 3 h after saline or MDMA injections (Fig. 4),

body temperature was found to be altered by chlorisondamine

pretreatment (F1,20 = 233.6, P < 0.0001 and F1,20 = 54.5,

P < 0.0001, for the ®rst and second series of injections), by

MDMA (F1,20 = 128.8, P < 0.0001 and F1,20 = 20.2, P = 0.0002,

for the ®rst and second series of injections), and by the latency

between MDMA injection and body temperature recording

(F6,120 = 23.68, P < 0.0001, and F6,120 = 11.57, P < 0.0001, for the

®rst and second series of injections). Furthermore, the interactions

between variables were all found to exert in¯uences on body

temperature (P < 0.0008 for each interaction; data not shown). Thus,

chlorisondamine and MDMA displayed, respectively, hypothermic

and hyperthermic properties throughout the two series of experiments

(Fig. 4). In addition, compared to saline administration, MDMA

administration to chlorisondamine-pretreated rats triggered a time-

dependent pattern of temperature responses, i.e. an ampli®cation of

chlorisondamine-elicited hypothermia followed by a progressive rise

in body temperature, with the amplitude of that rise decreasing on the

second MDMA injection (compared to the ®rst MDMA injection).

Because MDMA-elicited hyperthermia in chlorisondamine-pretreated

rats did not reach a plateau level at the end of the 3 h of analysis,

another experiment was set up where body temperature was

registered up to 7 h after MDMA injection (n = 4 rats/group). The

hyperthermia elicited by the ®rst injection of MDMA in chlorison-

damine-pretreated rats reached its maximum 4 h after MDMA

administration, this level remaining constant for an additional 2 h

and decreasing thereafter (MDMA-elicited hyperthermia was equiva-

lent in saline- and chlorisondamine-pretreated rats during the last 3 h

of analysis; data not shown). With regard to the second injection of

MDMA, its hyperthermic consequences in chlorisondamine-pre-

treated rats reached their maximum 3 h after MDMA administration

and vanished thereafter; indeed, neither chlorisondamine nor MDMA

had any in¯uence on body temperature for the last 3 h of analysis

(data not shown).

Table 5 shows the long-term neurochemical in¯uences of MDMA

in the hippocampus and striatum of saline- and chlorisondamine-

pretreated rats. In the hippocampus, [3H]5-HT reuptake was

in¯uenced by chlorisondamine (F1,19 = 5.72, P = 0.027) and

MDMA (F1,19 = 18.35, P = 0.0004) administration, post hoc analy-

ses showing that MDMA was endowed with a signi®cant effect in

saline-treated animals only. [3H]Paroxetine binding data con®rmed

[3H]5-HT reuptake data, as chlorisondamine (F1,27 = 13.68,

P = 0.001) and MDMA (F1,27 = 8.76, P = 0.0063) exerted strong

in¯uences whilst post hoc tests showed that the inhibitory impact of

MDMA lacked signi®cance in chlorisondamine-pretreated rats.

Hippocampal 5-HT levels were affected by chlorisondamine

(F1,20 = 4.71, P = 0.042), MDMA (F1,20 = 45.72, P < 0.0001), and

by the interaction between each treatment (F1,20 = 7.56, P = 0.012).

In addition, 5-HIAA levels were sensitive to MDMA administration

(F1,20 = 12.03, P = 0.0024) and to a chlorisondamine 3 MDMA

interaction (F1,20 = 6.04, P = 0.023). As far as striatum was

TABLE 2. Long-term effects of MDMA on hippocampal and striatal serotonergic systems in sham and adrenalectomised F344 rats

Sham Adrenalectomy

Saline MDMA (10 mg/kg) Saline MDMA (10 mg/kg)

Hippocampus[3H]5-HT reuptake (pmol/min/mg protein) 0.54 6 0.03 0.27 6 0.02** 0.60 6 0.03 0.39 6 0.04**,+

[3H]Paroxetine binding (fmol/mg protein) 354 6 10 210 6 26** 275 6 25+ 237 6 215-HT (nmol/g) 2.18 6 0.07 1.38 6 0.05** 2.12 6 0.05 1.35 6 0.10**5-HIAA (nmol/g) 1.00 6 0.05 0.75 6 0.02** 1.03 6 0.04 0.77 6 0.06**

Striatum[3H]5-HT reuptake (pmol/min/mg protein) 0.64 6 0.01 0.50 6 0.03** 0.58 6 0.03 0.50 6 0.01*[3H]Paroxetine binding (fmol/mg protein) 426 6 19 317 6 17** 355 6 16++ 363 6 105-HT (nmol/g) 2.47 6 0.11 1.73 6 0.11** 2.33 6 0.10 2.09 6 0.06+

5-HIAA (nmol/g) 1.41 6 0.07 1.07 6 0.04** 1.46 6 0.10 1.41 6 0.10++

All rats were injected twice (1-day interval) either with saline or MDMA (10 mg/kg, s.c.) and tissues assayed one week after the ®rst injection. Sham surgery oradrenalectomy were performed one week before the ®rst saline or MDMA injection. Values are given as the mean 6 SEM of 5±6 ([3H]5-HT reuptake,[3H]paroxetine binding) or 4±6 (5-HT and 5-HIAA concentrations) determinations. *P < 0.05, **P < 0.01 for the in¯uence of MDMA vs. saline. +P < 0.05,++P < 0.01 for the in¯uence of adrenalectomy.

TABLE 3. Long-term effects of MDMA on thymus weights and plasma corticosterone levels in sham, adrenalectomised and corticosterone-treated

adrenalectomised F344 rats

Sham Adrenalectomy Adrenalectomy + Corticosterone

Saline MDMA (10 mg/kg) Saline MDMA (10 mg/kg) Saline MDMA (10 mg/kg)

Thymus (mg) 420 6 13 415 6 10 524 6 13++ 511 6 13++ 433 6 23 400 6 18Thymus (mg/100 g b.w.) 201 6 6 197 6 5 298 6 10++ 309 6 10++ 221 6 12 214 6 8Plasma corticosterone (ng/mL) 45 6 10 73 6 13** 9 6 2++ 5 6 1++ 28 6 4 27 6 3++

All rats were injected twice (1-day interval) either with saline or MDMA (10 mg/kg, s.c.) and the animals killed for thymus weight and plasma corticosteroneanalyses. Sham surgery, adrenalectomy and the s.c. implantation of Cort. (50 mg) pellets were all performed 1 week before the ®rst saline or MDMA injection.Values are given as the mean 6 SEM of 14±16 (thymus weights) and 9±10 (plasma corticosterone) determinations. **P < 0.01 for the in¯uence of MDMA vs.saline; ++P < 0.01 vs. sham animals.



concerned, neither [3H]5-HT reuptake nor [3H]paroxetine binding

proved sensitive to chlorisondamine and/or MDMA in these series of

experiments. However, 5-HT and 5-HIAA were in¯uenced by

MDMA treatment (5-HT: F1,20 = 22.25, P < 0.0001; 5-HIAA:

F1,26 = 17.62, P = 0.0004), and were sensitive to a chlorisond-

amine 3 MDMA interaction (5-HT: F1,20 = 4.45, P = 0.0475; 5-

HIAA: F1,20 = 4.36, P = 0.0499). Indeed, post hoc tests showed that

MDMA-elicited reductions in hippocampal and striatal 5-HT and

5-HIAA levels were either markedly reduced or prevented by

chlorisondamine pretreatment (Table 5).

Discussion

The goal of this series of experiments was to explore the roles of the

corticotropic axis and sympathetic activity in MDMA-elicited

neurotoxicity in the hippocampus and the striatum, the extent of

this neurotoxic effect being assessed by the simultaneous measure-

ment of [3H]5-HT reuptake, [3H]paroxetine binding at 5-HTT, and

5-HT and 5-HIAA levels (Molliver et al., 1990; Green et al., 1995;

Sprague et al., 1998). Furthermore, the possibility that corticotropic

activity also affects some immediate responses to MDMA, e.g.

hyperthermia, thought to be predictive of its neurotoxic impact (see

below), led us to monitor body temperature. However, because we

chose to address our questions in F344 rats, which bear a hyperactive

and hyperesponsive corticotropic axis (Sternberg et al., 1989;

Calogero et al., 1992; Sternberg et al., 1992; Dhabbar et al., 1993),

we ®rst checked by means of two doses of MDMA that this strain

displays hyperthermia and signi®cant long-term reductions in the

aforementioned neurochemical variables.

Corticosterone, sympathetic activity and MDMA-elicitedhyperthermia in F344 rats

Acute administration of MDMA has a rapid effect on the

thermoregulatory system, the amplitude of which depends on ambient

temperature and is independent of MDMA-induced changes in

locomotion (Dafters, 1994). Indeed, MDMA elicits hypothermia and

hyperthermia when ambient temperatures are, respectively, below

and above 20±22 °C (Gordon et al., 1991; Dafters, 1994; Malberg &

Seiden, 1998), the value of that `break point' depending on the rat

strain (Malpass et al., 1999) and the rat environment (familiar vs.

unfamiliar: Miller & O'Callaghan, 1994). In our hands, a dose-

dependent hyperthermia was observed on both injections in F344 rats.

However, such a hyperthermic effect of MDMA was preceded by a

hypothermic phasis if the rats were either adrenalectomised or

pretreated with the ganglionic blocker chlorisondamine. Furthermore,

corticosterone implants, at doses that reversed adrenalectomy-elicited

thymus hypertrophy (see below), proved ineffective on MDMA-

induced hypothermia in adrenalectomised F344 rats, thus indicating

that the lack of adrenomedulla, rather than the lack of adrenal cortex,

was responsible for the immediate hypothermia in adrenalectomised

rats. This result, which indicates that MDMA may rapidly stimulate

the adrenomedulla in intact animals, is in keeping with past evidence

for adrenaline-releasing effects of 5-HT indirect (5-HT reuptake

inhibitors, 5-HT releasers) and direct (5-HT1A and 5-HT2A receptor)

agonists (Chaouloff, 1993). Lastly, with regard to the long-lasting

hyperthermic effects of MDMA in F344 rats, our experiments suggest

that the sympathetic system (as shown by chlorisondamine pretreat-

ment) and, to a lesser extent, corticoids (as shown by the respective

effects of adrenalectomy and corticosterone supplementation) medi-

ate such a hyperthermia, the effects of corticoids being possibly

indirect through their control of sympathetic activity (Axelrod &

Reisine, 1984).

Neurochemical effects of MDMA in F344 rats

Administration of MDMA triggered long-term dose-dependent

decreases in hippocampal [3H]5-HT reuptake. Although MDMA

bears intrinsic inhibitory properties on [3H]5-HT reuptake (Steele

et al., 1987; Rudnick & Wall, 1992; Crespi et al., 1997), including in

F344 rats (IC50 = 0.93 6 0.08 mM, n = 4), it is unlikely that the

long-term decrease in hippocampal [3H]5-HT reuptake in MDMA-

treated rats is due to such an intrinsic effect. Thus, other variables

examined in the hippocampus, such as [3H]paroxetine binding at

5-HTT and 5-HT and/or 5-HIAA levels, also proved sensitive in the

long term to MDMA administration. However, this was true for the

FIG. 3. Effects of (A) the ®rst and (B) the second injections of saline orMDMA (10 mg/kg, s.c. at time 0) on body temperature in F344 rats bearinga sham surgery, adrenalectomy, or adrenalectomy supplemented withcorticosterone (CORT.) pellets. All pretreatments were performed 7 daysbefore the ®rst injection of saline or MDMA. The two injections of salineor MDMA were separated by a 1-day interval and body temperaturesrecorded every 30 min for 3 h. Rats were given a 1-h habituation period (3body temperature recordings) to the experimental set up before eachinjection. Each value is the mean 6 SEM of 8 determinations. *P < 0.05and **P < 0.01 for the in¯uence of MDMA against saline. For clarity, thedifferences between pretreatments are not shown.



highest dose of MDMA used whereas the 5 mg/kg dose decreased

neither 5-HTT densities nor 5-HT and 5-HIAA levels although it

diminished [3H]5-HT reuptake. Such a dissociation between MDMA

respective effects on 5-HTT density and function has also been

recently observed in animals treated with another substituted

amphetamine (namely methamphetamine: Haughey et al., 2000).

The analysis of 5-HTT densities and indole concentrations in the

striatum revealed a pattern of decreases in MDMA-treated animals

that were almost consistent with the aforementioned data in the

hippocampus. However, these decreases (taken as percentages of the

values measured in saline-treated rats) were weaker in striatum than

in hippocampus. Indeed, the high sensitivity of hippocampal, as

compared to striatal, serotonergic neurons to the long-term effects of

MDMA was observed throughout the whole study, including in the

last series of experiments (chlorisondamine pretreatments; see

below).

Role of the corticotropic axis in MDMA neurochemical effectsin F344 rats

In keeping with the results depicted above, we next examined

whether the corticotropic axis partly or totally mediated the long-term

neurochemical in¯uences of MDMA in F344 rats. One support for

such a hypothesis would have been the preliminary demonstration

that the corticotropic axis of F344 rats responds positively to MDMA

administration. Indeed, such an experiment was not set up for the

following two reasons: (i) a correct assessment of this responsiveness

would have required dose- and time-dependent responses of plasma

corticosterone to MDMA administration, an experiment rendered

impossible by the limited amount of MDMA available, and (ii) a

previous study has shown that the acute stimulation of 5-HT2A,C

receptors, i.e. the receptors that mediate MDMA-elicited cortico-

sterone release (Nash et al., 1988), is able to increase plasma

corticosterone in F344 rats (Calogero et al., 1992). Instead, we

thought that a correct estimation of the role of the corticotropic axis

in the long-term effects of MDMA should rely on the demonstration

that adrenalectomy attenuates or prevents such long-term effects in

F344 rats. In the hippocampus, MDMA-induced reductions in [3H]5-

HT reuptake and, in tissue, 5-HT and 5-HIAA levels, proved

insensitive to adrenalectomy. In striatum, an almost similar pattern of

responses could be observed although the weak in¯uence of MDMA

in that region and some heterogeneity in the data between the two

series of experiments cannot be dismissed. As concerns hippocampal

and striatal [3H]paroxetine binding, the amplitudes of MDMA-

elicited reductions in that variable were markedly weakened, if not

removed, by prior adrenalectomy. Indeed, the overall analysis of

[3H]paroxetine binding in the two series of experiments strongly

suggest that such an impact of adrenalectomy was due to intrinsic

inhibitory effects of adrenalectomy on baseline (i.e. saline-treated

rats) [3H]paroxetine binding at 5-HTT. Thus, adrenalectomy reduced,

respectively, hippocampal and striatal baseline [3H]paroxetine bind-

ing by 22% and 17% in the ®rst series of experiments, and by 14%

(trend only) and 17% in the second series of experiments although,

for an unknown reason, baseline [3H]paroxetine binding differred

between the two series of experiments. Furthermore, it was found that

corticosterone supplementation reinstated the inhibitory impact of

MDMA on hippocampal and striatal [3H]paroxetine binding, but not

the adrenalectomy-induced reduction in baseline [3H]paroxetine

binding. One explanation could be that the adrenomedulla, rather

than the adrenal cortex, controls baseline hippocampal and striatal

[3H]paroxetine binding, but the results from the chlorisondamine

experiments partly rule out such an hypothesis (see below).

Alternatively, MDMA-elicited reductions in [3H]paroxetine binding

and baseline [3H]paroxetine binding might be sensitive to low and

high corticosterone concentrations, respectively, with these high

concentrations being above those provided by our supplementation

regimen. At ®rst glance, this could be supported by the observation

that our corticosterone regimen provided circulating corticosterone

levels that did not reach those measured in sham animals. However, it

is likely that the higher circulating corticosterone levels in sham rats

are fully accounted for by a rapid release of corticosterone as they

were killed. Furthermore, in keeping with the high and low af®nities

of, respectively, mineralocorticoid receptors and glucocorticoid

receptors for corticosterone (Reul & De Kloet, 1985), and past

evidence for the exclusive presence of glucocorticoid receptors in the

TABLE 4. Long-term effects of MDMA on hippocampal and striatal serotonergic systems in sham, adrenalectomised and corticosterone-treated

adrenalectomised F344 rats

Sham Adrenalectomy Adrenalectomy + Corticosterone

Saline MDMA (10 mg/kg) Saline MDMA (10 mg/kg) Saline MDMA (10 mg/kg)

Hippocampus[3H]5-HT reuptake 0.53 6 0.02 0.31 6 0.02** 0.61 6 0.02+ 0.38 6 0.02** 0.62 6 0.04+ 0.38 6 0.02**(pmol/min/mg protein)[3H]Paroxetine binding 520 6 26 378 6 31** 449 6 19 398 6 24 465 6 27 291 6 21**,+

(fmol/mg protein)5-HT (nmol/g) 1.93 6 0.15 1.08 6 0.16** 2.08 6 0.21 1.24 6 0.07** 2.07 6 0.09 1.04 6 0.10**5-HIAA (nmol/g) 1.16 6 0.04 0.75 6 0.07** 1.31 6 0.08 0.91 6 0.04** 1.17 6 0.05 0.82 6 0.06**

Striatum[3H]5-HT reuptake 0.82 6 0.04 0.74 6 0.05 0.80 6 0.03 0.73 6 0.06 0.96 6 0.07 0.71 6 0.09**(pmol/min/mg protein)[3H]Paroxetine binding 715 6 31 550 6 34** 593 6 60+ 571 6 26 579 6 34+ 483 6 23*(fmol/mg protein)5-HT (nmol/g) 2.44 6 0.08 1.87 6 0.09** 2.55 6 0.19 1.94 6 0.15** 2.30 6 0.07 1.90 6 0.07*5-HIAA (nmol/g) 1.40 6 0.06 1.11 6 0.09* 1.59 6 0.08 1.19 6 0.08** 1.33 6 0.11 1.16 6 0.06

All rats were injected twice (1-day interval) either with saline or MDMA (10 mg/kg, s.c.) and tissues assayed one week after the ®rst injection. Sham surgery,adrenalectomy and the s.c. implantation of corticosterone (Cort., 50 mg) pellets were all performed one week before the ®rst saline or MDMA injection. Values aregiven as the mean 6 SEM of 5±6 ([3H]5-HT reuptake), 5±8 ([3H]paroxetine binding) and 5±6 (5-HT and 5-HIAA concentrations) determinations. *P < 0.05,**P < 0.01 for the in¯uence of MDMA vs. saline. +P < 0.05 vs. sham rats.



thymus (Miller et al., 1990), the fact that our corticosterone

supplementation regimen fully prevented the adrenalectomy-elicited

increase in thymus weights (thus con®rming previous data: Cador

et al., 1993) may be taken as evidence for an effective reinstatement

of adequate corticosterone concentrations. Furthermore, plasma

corticosterone levels were increased by MDMA pretreatment in

sham rats, an observation either shared (Yau et al., 1994) or not

(McNamara et al., 1995; Aguirre et al., 1999) by others; actually, the

ineffectiveness of MDMA in altering thymus weights (and adrenal

weights; data not shown), and which con®rms previous data (Yau

et al., 1994), rather suggests increased fear at the time of killing and/

or increased activity of the corticotropic axis, such an increase being,

however, not sustained enough to alter target organs.

The observation that corticosteroid removal by adrenalectomy

prevented MDMA-elicited reductions in [3H]paroxetine binding, but

not in [3H]5-HT reuptake and 5-HT and 5-HIAA levels in the

hippocampus, raises the question of the net impact of adrenalectomy

on the MDMA neurotoxic effect. Although most, if not all, studies

consider the neurochemical variables used herein as valuable indices

of neurotoxicity (see Introduction), the lack of morphological and/or

immunocytochemical tools in this study impedes any clearcut answer

to that point. Glial ®brillary acidic protein overexpression may be the

simplest tool to show neurotoxicity (O'Callaghan et al., 1995),

including following MDMA injection (O'Callaghan et al., 1995;

Aguirre et al., 1997); unfortunately, adrenalectomy per se also

increases glial ®brillary acidic protein expression (O'Callaghan et al.,

1989), thus rendering inadequate that paradigm in the present study.

At the present stage, we can only underline the ®nding that the

MDMA-elicited reduction in [3H]5-HT reuptake, i.e. a measure of

membrane-bound 5-HTT, was insensitive to adrenalectomy, as were

5-HT and 5-HIAA levels, thereby suggesting that adrenalectomy

affected only cytosolic 5-HTT (assuming that [3H]paroxetine

measures both cytosolic and membrane 5-HTTs) without any

interaction with the MDMA neurodegenerative effects. Such a

statement is supported by the chlorisondamine experiments where

MDMA-elicited reductions in hippocampal [3H]5-HT reuptake,

[3H]paroxetine binding and 5-HT and 5-HIAA levels all proved

sensitive to the pretreatment (see below).

Role of sympathetic activity in MDMA neurochemical effects inF344 rats

As indicated earlier, the body temperature responses to MDMA in

adrenalectomised F344 rats, complemented or not with cortico-

sterone, point to a permissive effect of the adrenomedulla in the early

(up to 60 min) hyperthermic effects, but not the long-term

neurochemical effects, of MDMA. Furthermore, the possibility that

the mechanisms that allow MDMA to promote hyperthermia at later

times (i.e. after 60 min) also play a role in its long-term neuro-

chemical effects remained, however, open. This was supported by

pharmacological and physiological studies (Miller & O'Callaghan,

1994; Farfel & Seiden, 1995; Malberg et al., 1996; Malberg &

Seiden, 1998; Colado et al., 1999) which have suggested a link

between the hyperthermic and the neurotoxic properties of MDMA.

However, some results either do not support a direct link between

hyperthermia and neurotoxicity or suggest that hyperthermia only

partially explains neurotoxicity (Malberg et al., 1996; Aguirre et al.,

1997; Sprague et al., 1998). Moreover, the challenges (e.g. cold

exposure, amino acid and monoamine receptor ligands, catechol-

amine synthesis inhibitors) that have allowed the establishment of

that link are also endowed with peripheral and central actions which

are beyond the sole control of body temperature. Indeed, there is

evidence for an immediate stimulatory in¯uence of MDMA upon

sympathetic activity (O'Cain et al., 2000; Pedersen & Blessing,

2001), which is in keeping with the acute sympathoexcitatory

properties of 5-HT reuptake inhibitors/releasers (Chaouloff et al.,

1992) and 5-HT2A receptor agonists (Chaouloff, 1993). Con®rmingly,

the vasoconstrictor properties of MDMA have been shown to

contribute to its hyperthermic effect (Pedersen & Blessing, 2001).

In the present study, we chose to selectively address the role of

sympathetic activity by measuring the impacts of the ganglionic

blocker chlorisondamine (Gosling & Lu, 1969; Clarke et al., 1994) on

the hyperthermic and long-term neurochemical effects of MDMA. In

the hippocampus, the amplitudes of MDMA-elicited decreases in

[3H]5-HT reuptake, [3H]paroxetine binding and 5-HT and 5-HIAA

levels were markedly reduced (by 56, 63, 56 and 81%, respectively)

FIG. 4. Effects of (A) the ®rst and (B) the second injections of saline orMDMA (10 mg/kg, s.c. at time 0) on body temperature in F344 ratspretreated 30 min beforehand with saline or chlorisondamine (`CHLOR.';2.5 mg/kg i.p). The two injections of saline or MDMA were separated by a1-day interval and body temperatures recorded every 30 min for 3 h. Ratswere given a 30-min habituation period (two body temperature recordings)to the experimental set up before saline or chlorisondamine pretreatments.Each value is the mean 6 SEM of six determinations. *P < 0.05,**P < 0.01 for the in¯uence of MDMA against saline. For clarity, thedifferences between pretreatments are not shown (see text for bodytemperature data up to 7 h after MDMA injection).



by chlorisondamine pretreatment. In striatum, for an unknown reason,

neither [3H]5-HT reuptake nor [3H]paroxetine binding were

decreased to signi®cant extents by MDMA in these series of

experiments; however, con®rming the hippocampal data mentioned

above, it was observed that MDMA-elicited reductions in 5-HT and

5-HIAA levels were, respectively, diminished by 60 and 67% in

chlorisondamine-pretreated rats. Surprisingly, we also observed that

chlorisondamine pretreatment was endowed with slight but signi®-

cant intrinsic stimulatory in¯uences on baseline hippocampal [3H]5-

HT reuptake and more evidently on [3H]paroxetine binding. The

reason for such an increase is unknown, as sympathetic activity has

never been reported to affect 5-HTT binding, especially in the long

term. Indeed, one possibility would be that although chlorisondamine

is a preferential peripheral nicotinic receptor antagonist (Gosling &

Lu, 1969; Clarke et al., 1994), it also blocked central (e.g. in

hippocampus) nicotinic receptors, thereby altering, in the long-term,

[3H]paroxetine binding. This is, however, unlikely because (i) central

nicotinic receptor blockade by chlorisondamine has been shown to

only occur with high doses (10 mg/kg s.c.: Clarke, 1984; Clarke et al.,

1994) (ii) such a high dosage proved, however, ineffective on long-

term nicotinic receptor binding (Clarke et al., 1994), and (iii) acute

hippocampal nicotinic receptor blockade does not alter, per se, 5-HT

transmission as indicated by in vitro release experiments (Kenny

et al., 2000; Reuben & Clarke, 2000). Although our results show that

the ganglionic blocker chlorisondamine markedly weakened the long-

term consequences of MDMA on hippocampal and striatal seroto-

nergic neurons, we believe that the mechanisms underlying its

intrinsic effects on serotonergic systems should be investigated.

Conclusions

By means of neurochemical tools thought to provide a good estimate

of the integrity of serotonergic nerve terminals, this study strongly

suggests that sympathetic blockade, but not corticosterone removal,

diminishes the neurotoxic impact of MDMA, especially in the

hippocampus. Future work should, however, assess the extent to

which this result is supported by morphological ®ndings and how it

extends to other rat strains, including standard strains. We believe

that future work should also analyse whether the protective effect of

chlorisondamine reported above can be further increased by a longer-

lasting ganglionic blockade (indicating that the neurotoxic effect of

MDMA is fully accounted for by its hyperthermic effect) or is already

maximal (indicating that body temperature-independent mechanisms

may further protect against neurotoxicity).

Acknowledgements

This work was supported by la Mission InterministeÂrielle de Lutte contre laDrogue et la Toxicomanie (grant 99D05/AH003D to F.C), INSERM, INRA,and le Conseil ReÂgional d'Aquitaine. The authors wish to thank the NationalInstitute on Drug Abuse (Bethesda, MD, USA) for the kind gift of MDMA,and Aline Foury for corticosterone assays.

Abbreviations

5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, 5-hydroxytryptamine/serotonin;5-HTT, 5-hydroxytryptamine/serotonin transporter; F344, Fischer 344;MDMA, 3,4-methylenedioxymethamphetamine.

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TABLE 5. Long-term effects of MDMA on hippocampal and striatal serotonergic systems in saline and chlorisondamine-treated F344 rats

Saline-treated Chlorisondamine-treated

Saline MDMA (10 mg/kg) Saline MDMA (10 mg/kg)

Hippocampus[3H]5-HT reuptake (pmol/min/mg protein) 0.39 6 0.02 0.24 6 0.03** 0.41 6 0.03 0.34 6 0.02+

[3H]Paroxetine binding (fmol/mg protein) 377 6 18 244 6 28* 427 6 35 398 6 26++

5-HT (nmol/g) 1.78 6 0.09 1.01 6 0.08** 1.73 6 0.08 1.41 6 0.07*,++

5-HIAA (nmol/g) 0.95 6 0.03 0.65 6 0.06** 0.89 6 0.06 0.84 6 0.05+

Striatum[3H]5-HT reuptake (pmol/min/mg protein) 0.57 6 0.01 0.51 6 0.02 0.57 6 0.02 0.54 6 0.02[3H]Paroxetine binding (fmol/mg protein) 499 6 36 501 6 61 543 6 46 602 6 475-HT (nmol/g) 2.53 6 0.08 1.84 6 0.08** 2.41 6 0.14 2.15 6 0.09+

5-HIAA (nmol/g) 1.44 6 0.08 1.00 6 0.04** 1.28 6 0.09 1.13 6 0.05

All rats were injected twice (1-day interval) either with saline or MDMA (10 mg/kg, s.c) and tissues assayed one week after the ®rst injection. Saline orchlorisondamine (2.5 mg/kg i.p.) pretreatments were performed 30 min before each of the two saline or MDMA injections. Values are given as the mean 6 SEMof 5±7 ([3H]5-HT reuptake), 7±8 ([3H]paroxetine binding) and 6 (5-HT and 5-HIAA concentrations) determinations. *P < 0.05, **P < 0.01 for the in¯uence ofMDMA vs. saline. +P < 0.05, ++P < 0.01 for the in¯uence of chlorisondamine.



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Influences of the corticotropic axis and sympathetic activity on neurochemical consequences of 3,4-methylenedioxymethamphetamine (MDMA) administration in Fischer 344 rats

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