-LRB-

MDMA

-RRB-

neurotoxicity

in

rats

a

reappraisal

of

past

and

present

findings

Rationale

-LRB-

MDMA

-RRB-

is

a

widely

abused

illicit

drug

In

animals

high-dose

administration

of

MDMA

produces

deficits

in

serotonin

-LRB-

-RRB-

neurons

-LRB-

e.g.

depletion

of

forebrain

-RRB-

that

have

been

interpreted

as

neurotoxicity

Whether

such

deficits

reflect

neuronal

damage

is

a

matter

of

ongoing

debate

Introduction

-LRB-

MDMA

or

Ecstasy

-RRB-

is

an

illicit

drug

used

by

young

adults

in

the

US

Europe

and

elsewhere

The

appeal

of

MDMA

is

related

to

its

unique

profile

of

psychotropic

actions

which

includes

amphetamine-like

stimulant

effects

coupled

with

feelings

of

increased

emotional

sensitivity

and

closeness

to

others

-LRB-

Liechti

and

Vollenweider

Vollenweider

et

al.

-RRB-

MDMA

misuse

among

children

and

adolescents

is

widespread

in

the

US

-LRB-

Landry

Yacoubian

-RRB-

a

recent

sampling

of

high

school

students

found

of

graders

reported

using

MDMA

at

least

once

-LRB-

Banken

-RRB-

MDMA-related

medical

complications

have

risen

more

than

in

recent

years

consistent

with

increasing

popularity

of

the

drug

Serious

adverse

effects

of

MDMA

intoxication

include

cardiac

arrhythmias

hypertension

hyperthermia

serotonin

-LRB-

-RRB-

syndrome

hyponatremia

liver

problems

seizures

coma

and

in

rare

cases

death

-LRB-

Schifano

-RRB-

Accumulating

evidence

indicates

that

long-term

MDMA

abuse

is

associated

with

cognitive

impairments

and

mood

disturbances

which

can

last

for

months

after

cessation

of

drug

intake

-LRB-

Morgan

Parrott

-RRB-

Despite

the

risks

of

illicit

MDMA

use

some

clinicians

believe

the

drug

may

have

therapeutic

potential

in

the

treatment

of

psychiatric

disorders

such

as

post-traumatic

stress

disorder

and

clinical

studies

with

MDMA

are

underway

-LRB-

Doblin

-RRB-

It

is

worth

noting

that

MDMA

has

been

administered

to

human

subjects

in

controlled

research

settings

and

few

side

effects

are

observed

under

these

circumstances

-LRB-

Harris

et

al.

Mas

et

al.

-RRB-

These

considerations

provide

compelling

reasons

to

evaluate

the

pharmacology

and

toxicology

of

MDMA

and

related

compounds

In

this

review

we

will

examine

four

topics

related

to

the

hypothesis

of

MDMA-induced

neurotoxicity

in

rats

-LRB-

-RRB-

the

effects

of

MDMA

on

monoamine

neurons

-LRB-

-RRB-

the

use

of

interspecies

scaling

to

extrapolate

doses

of

MDMA

across

species

-LRB-

-RRB-

the

effects

of

MDMA

on

established

markers

of

neurotoxic

damage

and

-LRB-

-RRB-

the

functional

impairments

associated

with

MDMA-induced

depletions

The

review

will

focus

on

data

obtained

from

rats

since

most

preclinical

MDMA

research

has

been

carried

out

in

this

animal

model

Previously

published

and

new

data

from

our

laboratory

at

the

National

Institute

on

Drug

Abuse

-LRB-

NIDA

-RRB-

will

be

included

to

supplement

literature

reports

Clinical

findings

will

be

mentioned

in

specific

instances

to

note

comparisons

between

rats

and

humans

All

experiments

in

our

laboratory

utilized

male

Sprague

Dawley

rats

-LRB-

Wilmington

MA

USA

-RRB-

weighing

g.

Rats

were

maintained

in

facilities

accredited

by

the

American

Association

of

the

Accreditation

of

Laboratory

Animal

Care

and

procedures

were

carried

out

in

accordance

with

the

Animal

Care

and

Use

Committee

of

the

NIDA

Intramural

Research

Program

-LRB-

IRP

-RRB-

Data

from

mice

will

not

be

considered

here

because

this

animal

species

displays

the

unusual

characteristic

of

long-term

DA

depletions

-LRB-

i.e.

DA

neurotoxicity

-RRB-

in

response

to

MDMA

rather

than

long-term

depletions

observed

in

rats

nonhuman

primates

and

most

other

animals

-LSB-

reviewed

by

Colado

et

al.

-LRB-

-RRB-

-RSB-

Finally

the

present

paper

will

not

address

possible

molecular

mechanisms

underlying

MDMA-induced

deficits

as

several

excellent

reviews

have

covered

this

subject

-LRB-

Lyles

and

Cadet

Monks

et

al.

Sprague

et

al.

-RRB-

Effects

of

MDMA

on

monoamine

neurons

To

address

the

topic

of

MDMA-induced

neurotoxicity

the

pharmacology

of

MDMA

must

be

briefly

reviewed

Figure

shows

that

MDMA

is

a

ring-substituted

analog

of

methamphetamine

Ecstasy

tablets

ingested

by

humans

contain

a

racemic

mixture

of

-LRB-

-RRB-

and

-LRB-

-RRB-

isomers

of

MDMA

and

both

stereoisomers

are

known

to

be

bioactive

-LRB-

Johnson

et

al.

Schmidt

et

al.

-RRB-

Ecstasy

tablets

often

contain

other

psychoactive

substances

such

as

substituted

amphetamines

caffeine

or

ketamine

which

can

contribute

to

the

overall

effects

of

the

ingested

preparation

-LRB-

Parrott

-RRB-

Upon

systemic

administration

N-demethylation

of

MDMA

occurs

via

first-pass

metabolism

to

yield

the

ring-substituted

amphetamine

analog

-LRB-

MDA

-RRB-

-LRB-

de

la

Torre

et

al.

-RRB-

Initial

studies

carried

out

in

the

showed

that

MDMA

and

MDA

stimulate

efflux

of

preloaded

-LSB-

-RSB-

and

to

a

lesser

extent

-LSB-

-RSB-

DA

in

nervous

tissue

-LRB-

Johnson

et

al.

Nichols

et

al.

Schmidt

et

al.

-RRB-

Subsequent

findings

revealed

that

MDMA

interacts

with

monoamine

transporter

proteins

to

stimulate

non-exocytotic

release

of

DA

and

norepinephrine

-LRB-

NE

-RRB-

in

rat

brain

-LRB-

Berger

et

al.

Crespi

et

al.

Fitzgerald

and

Reid

-RRB-

Fig.

structures

of

MDMA

and

related

compounds

Table

summarizes

previously

published

data

from

our

laboratory

showing

structure

activity

relationships

for

stereoisomers

of

MDMA

MDA

and

related

drugs

as

monoamine

releasers

in

rat

brain

synaptosomes

-LRB-

Partilla

et

al.

Rothman

et

al.

Setola

et

al.

-RRB-

Like

other

substrate-type

releasers

MDMA

and

MDA

bind

to

plasma

membrane

monoamine

transporters

and

are

translocated

into

the

cytoplasm

The

ensuing

transmitter

release

occurs

by

a

two-pronged

mechanism

-LRB-

-RRB-

transmitter

molecules

exit

the

cell

along

their

concentration

gradients

via

reversal

of

normal

transporter

function

and

-LRB-

-RRB-

cytoplasmic

concentrations

of

transmitter

are

increased

due

to

drug-induced

disruption

of

vesicular

storage

-LSB-

reviewed

by

Rothman

and

Baumann

-LRB-

-RRB-

and

Rudnick

and

Clark

-LRB-

-RRB-

-RSB-

As

shown

in

Table

stereoisomers

of

MDMA

and

MDA

are

substrates

for

transporters

-LRB-

SERT

-RRB-

NE

transporters

-LRB-

NET

-RRB-

and

DA

transporters

-LRB-

DAT

-RRB-

with

-LRB-

-RRB-

isomers

exhibiting

greater

potency

as

releasers

In

particular

-LRB-

-RRB-

isomers

of

MDMA

and

MDA

are

much

more

effective

DA

releasers

than

their

corresponding

-LRB-

-RRB-

isomers

It

is

noteworthy

that

-LRB-

-RRB-

isomers

of

MDMA

and

MDA

are

rather

nonselective

in

their

ability

to

stimulate

monoamine

release

in

vitro

When

compared

to

amphetamine

and

methamphetamine

the

major

effect

of

methylenedioxy

ring

substitution

is

enhanced

potency

for

release

and

reduced

potency

for

DA

release

For

example

-LRB-

-RRB-

MDMA

releases

-LRB-

nM

-RRB-

about

ten

times

more

potently

than

-LRB-

-RRB-

methamphetamine

-LRB-

nM

-RRB-

whereas

-LRB-

-RRB-

MDMA

releases

DA

-LRB-

nM

-RRB-

about

six

times

less

potently

than

-LRB-

-RRB-

methamphetamine

-LRB-

nM

-RRB-

Table

of

MDMA

and

related

compounds

as

monoamine

transporter

substrates

in

rat

brain

release

-LRB-

nM

SD

-RRB-

NE

release

-LRB-

nM

SD

-RRB-

DA

release

-LRB-

nM

SD

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

data

are

taken

from

Partilla

et

al.

Rothman

et

al.

and

Setola

et

al.

Details

concerning

in

vitro

methods

can

be

found

in

these

papers

Substrate

activity

at

SERT

NET

and

DAT

is

reflected

as

release

efficacy

for

the

corresponding

transmitter

Consistent

with

in

vitro

results

in

vivo

microdialysis

experiments

demonstrate

that

MDMA

increases

extracellular

and

DA

in

rat

brain

with

effects

on

being

greater

in

magnitude

-LRB-

Baumann

et

al.

Gudelsky

and

Nash

Kankaanpaa

et

al.

Yamamoto

et

al.

-RRB-

Figure

depicts

new

data

from

our

laboratory

showing

the

stimulatory

effects

of

MDMA

and

MDA

on

extracellular

and

DA

in

rat

nucleus

accumbens

In

these

experiments

i.v.

injections

of

saline

-LRB-

dose

-RRB-

or

drug

were

administered

to

conscious

male

rats

undergoing

in

vivo

microdialysis

Doses

of

and

mg/kg

were

chosen

because

these

doses

of

MDMA

are

self-administered

by

rats

-LRB-

Ratzenboeck

et

al.

Schenk

et

al.

-RRB-

Dialysate

samples

were

collected

every

min

beginning

h

before

injections

until

h

thereafter

samples

were

assayed

for

and

DA

by

high-performance

liquid

chromatography

coupled

to

electrochemical

detection

-LRB-

HPLC

ECD

-RRB-

as

described

elsewhere

-LRB-

Baumann

and

Rutter

-RRB-

Neurochemical

data

were

converted

to

percentage

of

control

values

based

on

three

preinjection

control

samples

The

effects

of

MDMA

and

MDA

are

depicted

as

peak

effects

which

were

observed

in

the

first

min

after

injection

Peak

effect

data

were

analyzed

using

a

one-way

ANOVA

-LRB-

drug

dose

-RRB-

followed

by

Duncan

's

post

hoc

test

MDMA

caused

significant

increases

in

dialysate

-LSB-

P

-RSB-

and

DA

-LSB-

P

-RSB-

MDA

caused

similar

increases

in

-LSB-

P

-RSB-

and

DA

-LSB-

P

-RSB-

but

appeared

to

be

slightly

more

potent

at

releasing

DA

Both

drugs

produced

elevations

in

that

were

greater

than

the

corresponding

effects

on

DA

For

example

the

mg/kg

dose

of

MDMA

produced

a

tenfold

rise

in

and

a

twofold

rise

in

DA

Fig.

of

-LRB-

-RRB-

MDMA

and

-LRB-

-RRB-

MDA

on

extracellular

levels

of

-LRB-

top

panel

-RRB-

and

DA

-LRB-

bottom

panel

-RRB-

in

rat

nucleus

accumbens

Male

rats

undergoing

in

vivo

microdialysis

received

i.v.

injections

of

saline

-LRB-

dose

-RRB-

or

drug

and

dialysate

levels

of

and

DA

were

assayed

by

HPLC

ECD

-LRB-

Baumann

and

Rutter

-RRB-

Data

are

expressed

as

the

percentage

of

three

pre-injection

baseline

samples

each

bar

represents

the

mean

SEM

peak

effect

measured

min

posttreatment

N

rats/group

Baseline

levels

of

and

DA

were

and

μl

respectively

Asterisk

denotes

significance

with

respect

to

zero

dose

control

-LRB-

P

Duncan

's

-RRB-

Acute

central

nervous

system

-LRB-

CNS

-RRB-

effects

of

MDMA

are

mediated

by

the

release

of

monoamine

transmitters

with

the

subsequent

activation

of

presynaptic

and

postsynaptic

receptors

-LSB-

reviewed

by

Cole

and

Sumnall

-LRB-

-RRB-

and

Green

et

al.

-LRB-

-RRB-

-RSB-

As

specific

examples

in

rats

MDMA

suppresses

cell

firing

evokes

neuroendocrine

secretion

and

stimulates

locomotor

activity

MDMA-induced

suppression

of

cell

firing

in

the

dorsal

and

median

raphe

involves

activation

of

presynaptic

autoreceptors

by

endogenous

-LRB-

Gartside

et

al.

Sprouse

et

al.

-RRB-

Neuroendocrine

effects

of

MDMA

include

secretion

of

prolactin

from

the

anterior

pituitary

and

corticosterone

from

the

adrenal

glands

-LRB-

Nash

et

al.

-RRB-

Evidence

indicates

that

these

MDMA-induced

hormonal

effects

are

mediated

via

postsynaptic

receptors

in

the

hypothalamus

which

are

activated

by

released

MDMA

elicits

a

unique

profile

of

locomotor

effects

characterized

by

forward

locomotion

and

elements

of

the

behavioral

syndrome

such

as

forepaw

treading

flattened

body

posture

and

head

weaving

-LRB-

Gold

et

al.

Slikker

et

al.

Spanos

and

Yamamoto

-RRB-

The

complex

motor

effects

of

MDMA

are

dependent

upon

monoamine

release

followed

by

activation

of

multiple

and

DA

receptor

subtypes

in

the

brain

-LSB-

reviewed

by

Bankson

and

Cunningham

-LRB-

-RRB-

Geyer

-LRB-

-RRB-

-RSB-

Adverse

effects

of

acute

MDMA

administration

including

cardiovascular

stimulation

and

elevated

body

temperature

are

thought

to

involve

monoamine

release

from

sympathetic

nerves

in

the

periphery

or

nerve

terminals

in

the

CNS

MDMA

increases

heart

rate

and

mean

arterial

pressure

in

conscious

rats

-LRB-

O'Cain

et

al.

-RRB-

this

cardiovascular

stimulation

is

likely

mediated

by

MDMA-induced

release

of

peripheral

NE

stores

similar

to

the

effects

of

amphetamine

-LRB-

Fitzgerald

and

Reid

-RRB-

MDMA

has

weak

agonist

actions

at

and

receptors

which

might

influence

its

cardiac

and

pressor

effects

-LRB-

Battaglia

and

De

Souza

Lavelle

et

al.

Lyon

et

al.

-RRB-

Moreover

MDA

is

a

potent

agonist

and

this

property

could

contribute

to

adverse

cardiovascular

effects

-LRB-

Setola

et

al.

-RRB-

The

ability

of

MDMA

to

elevate

body

temperature

is

well-characterized

in

rats

-LRB-

Dafters

Dafters

and

Lynch

Nash

et

al.

-RRB-

and

this

response

has

been

considered

a

process

However

a

recent

study

by

Mechan

et

al.

-LRB-

-RRB-

provides

convincing

evidence

that

MDMA-induced

hyperthermia

involves

the

activation

of

postsynaptic

receptors

by

released

DA

The

long-term

adverse

effects

of

MDMA

on

systems

have

attracted

substantial

interest

because

studies

in

rats

and

nonhuman

primates

show

that

high-dose

MDMA

administration

produces

persistent

reductions

in

markers

of

nerve

terminal

integrity

-LSB-

reviewed

by

Lyles

and

Cadet

-LRB-

-RRB-

Sprague

et

al.

-LRB-

-RRB-

-RSB-

Table

summarizes

findings

of

investigators

who

first

demonstrated

that

MDMA

causes

long-term

-LRB-

week

-RRB-

inactivation

of

tryptophan

hydroxylase

activity

depletions

of

brain

tissue

and

reductions

in

SERT

binding

and

function

-LRB-

Battaglia

et

al.

Commins

et

al.

Schmidt

Stone

et

al.

-RRB-

These

serotonergic

deficits

are

observed

in

various

regions

of

rat

forebrain

including

frontal

cortex

striatum

hippocampus

and

hypothalamus

Immunohistochemical

analysis

of

in

cortical

and

subcortical

areas

reveals

an

apparent

loss

of

axons

and

terminals

in

MDMA-treated

rats

especially

the

fine-diameter

projections

arising

from

the

dorsal

raphe

nucleus

-LRB-

O'Hearn

et

al.

-RRB-

Moreover

axons

and

terminals

remaining

after

MDMA

treatment

appear

swollen

and

fragmented

suggesting

structural

damage

Table

effects

of

-LRB-

-RRB-

MDMA

on

neuronal

markers

in

deficitDoseSurvival

intervalReferenceDepletions

of

in

cortex

as

measured

by

HPLC

mg/kg

s.c.

single

weekSchmidt

-LRB-

-RRB-

Depletions

of

in

forebrain

regions

as

measured

by

HPLC

mg/kg

s.c.

twice

daily

weeksCommins

et

al.

-LRB-

-RRB-

Reductions

in

tryptophan

hydroxylase

activity

in

forebrain

mg/kg

s.c.

single

weeksStone

et

al.

-LRB-

-RRB-

Loss

of

-LSB-

-RSB-

paroxetine-labeled

SERT

binding

sites

in

forebrain

mg/kg

s.c.

twice

daily

weeksBattaglia

et

al.

-LRB-

-RRB-

Deceased

immunoreactive

in

fine

axons

and

terminals

in

forebrain

mg/kg

s.c.

twice

daily

weeksO'Hearn

et

al.

-LRB-

-RRB-

Time-course

studies

indicate

that

MDMA-induced

depletion

occurs

in

a

biphasic

manner

with

a

rapid

acute

phase

followed

by

a

delayed

long-term

phase

-LRB-

Schmidt

Stone

et

al.

-RRB-

In

the

acute

phase

which

lasts

for

the

first

few

hours

after

drug

administration

massive

depletion

of

brain

tissue

is

accompanied

by

inactivation

of

tryptophan

hydroxylase

Twenty-four

hours

later

tissue

recovers

to

normal

levels

but

hydroxylase

activity

remains

diminished

In

the

long-term

phase

which

begins

within

week

and

lasts

for

months

marked

depletion

of

is

accompanied

by

sustained

inactivation

of

tryptophan

hydroxylase

and

loss

of

SERT

binding

and

function

-LRB-

Battaglia

et

al.

Scanzello

et

al.

-RRB-

The

findings

in

Table

have

been

replicated

by

many

investigators

and

the

spectrum

of

decrements

is

typically

described

as

neurotoxicity

Most

of

the

studies

designed

to

examine

MDMA

neurotoxicity

in

rats

have

employed

i.p.

or

s.c.

injections

of

mg/kg

or

higher

either

as

single

or

repeated

treatments

These

MDMA

dosing

regimens

are

known

to

produce

significant

hyperthermia

which

can

exacerbate

depletions

caused

by

the

drug

-LRB-

Green

et

al.

Malberg

and

Seiden

-RRB-

All

of

these

experiments

have

involved

administration

of

MDMA

that

is

not

contingent

on

a

specific

behavior

and

this

factor

could

significantly

influence

effects

of

the

drug

There

are

caveats

to

the

hypothesis

that

MDMA

produces

neurotoxicity

O'Hearn

et

al.

-LRB-

-RRB-

showed

that

high-dose

MDMA

administration

has

no

effect

on

cell

bodies

in

the

dorsal

raphe

despite

profound

loss

of

in

forebrain

projection

areas

Thus

the

effects

of

MDMA

on

neurons

are

sometimes

referred

to

as

axotomy

to

account

for

the

fact

that

perikarya

are

not

damaged

-LRB-

Molliver

et

al.

O'Hearn

et

al.

-RRB-

MDMA-induced

reductions

in

levels

and

SERT

binding

eventually

recover

-LRB-

Battaglia

et

al.

Scanzello

et

al.

-RRB-

suggesting

the

possibility

that

terminals

are

not

destroyed

Many

drugs

used

clinically

produce

effects

that

are

similar

to

those

produced

by

MDMA

For

instance

reserpine

causes

sustained

depletions

of

brain

tissue

yet

reserpine

is

not

considered

a

neurotoxin

-LRB-

Carlsson

-RRB-

Chronic

administration

of

selective

reuptake

inhibitors

-LRB-

SSRIs

-RRB-

like

paroxetine

and

sertraline

leads

to

a

marked

loss

of

SERT

binding

and

function

comparable

to

MDMA

but

these

agents

are

therapeutic

drugs

rather

than

neurotoxins

-LRB-

Benmansour

et

al.

Frazer

and

Benmansour

-RRB-

Finally

high-dose

administration

of

SSRIs

produces

swollen

fragmented

and

abnormal

terminals

which

are

indistinguishable

from

the

effects

of

MDMA

and

other

substituted

amphetamines

-LRB-

Kalia

et

al.

-RRB-

The

caveats

mentioned

above

raise

a

number

of

questions

with

respect

to

MDMA

neurotoxicity

Of

course

the

most

important

question

is

whether

MDMA

abuse

causes

neurotoxic

damage

in

humans

This

complex

issue

is

a

matter

of

ongoing

debate

which

has

been

addressed

by

a

number

of

recent

papers

-LRB-

Gouzoulis-Mayfrank

et

al.

Kish

Reneman

-RRB-

Clinical

studies

designed

to

critically

evaluate

the

long-term

effects

of

MDMA

are

hampered

by

a

number

of

factors

including

comorbid

psychopathology

and

polydrug

abuse

among

MDMA

users

Animal

models

afford

the

unique

opportunity

to

evaluate

the

effects

of

MDMA

without

many

of

these

complicating

factors

and

the

main

focus

here

will

be

to

review

the

evidence

pertaining

to

MDMA-induced

neurotoxicity

in

rats

Interspecies

scaling

and

MDMA

dosing

regimens

A

major

point

of

controversy

relates

to

the

relevance

of

MDMA

doses

administered

to

rats

when

compared

to

doses

taken

by

humans

-LSB-

see

-LRB-

Cole

and

Sumnall

-LRB-

-RRB-

-RSB-

As

noted

above

MDMA

regimens

that

produce

depletions

in

rats

involve

administration

of

single

or

multiple

injections

of

mg/kg

whereas

the

typical

amount

of

MDMA

abused

by

humans

is

one

or

two

tablets

of

mg

or

mg/kg

administered

orally

-LRB-

Green

et

al.

Schifano

-RRB-

Based

on

principles

of

interspecies

scaling

some

investigators

have

proposed

that

neurotoxic

doses

of

MDMA

in

rats

correspond

to

recreational

doses

in

humans

-LRB-

Ricaurte

et

al.

-RRB-

To

critically

evaluate

this

claim

a

brief

discussion

of

interspecies

scaling

is

warranted

The

concept

of

interspecies

scaling

is

based

upon

shared

biochemical

mechanisms

among

eukaryotic

cells

-LRB-

e.g.

aerobic

respiration

-RRB-

and

it

was

initially

developed

to

describe

variations

in

basal

metabolic

rate

-LRB-

BMR

-RRB-

between

animal

species

of

different

sizes

-LSB-

reviewed

by

White

and

Seymour

-LRB-

-RRB-

-RSB-

In

the

Kleiber

-LRB-

-RRB-

derived

what

is

now

called

the

allometric

equation

to

describe

the

relationship

between

body

mass

and

BMR

The

generic

form

of

the

allometric

equation

is

where

Y

is

the

variable

of

interest

W

is

body

weight

a

is

the

allometric

coefficient

and

b

is

the

allometric

exponent

In

the

case

where

Y

is

BMR

b

is

accepted

to

be

West

et

al.

-LRB-

-RRB-

have

shown

that

most

biological

phenomena

scale

according

to

a

universal

quarter

power

law

as

illustrated

by

the

space-filling

fractal

networks

of

branching

tubes

used

by

the

circulatory

system

Given

that

the

allometric

equation

is

grounded

in

fundamental

commonalities

across

organisms

it

is

not

surprising

this

equation

can

describe

the

relationship

between

body

mass

and

physiological

variables

such

as

BMR

heart

rate

and

circulation

time

-LSB-

e.g.

Noujaim

et

al.

-LRB-

-RRB-

-RSB-

Because

circulation

time

and

organ

blood

flow

strongly

influence

drug

pharmacokinetics

the

allometric

equation

has

been

used

in

the

medication

development

process

to

scale-up

dosages

from

animal

models

to

man

-LSB-

reviewed

by

Mahmood

-LRB-

-RRB-

-RSB-

In

general

smaller

animals

have

faster

heart

rates

and

circulation

times

leading

to

faster

clearance

of

exogenous

drugs

However

this

relationship

does

not

hold

true

for

all

classes

of

drugs

especially

those

that

are

extensively

metabolized

-LRB-

Lin

-RRB-

The

most

important

variable

to

consider

when

examining

therapeutic

or

adverse

effects

of

any

drug

is

the

concentration

of

bioactive

compound

reaching

target

tissues

Tissue

drug

concentrations

are

governed

by

drug

absorption

distribution

metabolism

and

elimination

-LRB-

ADME

-RRB-

in

a

complex

manner

Many

factors

such

as

dose

route

species

strain

age

and

gender

can

affect

the

ADME

profile

of

a

given

drug

thereby

affecting

tissue

concentrations

Campbell

-LRB-

-RRB-

has

described

numerous

limitations

of

interspecies

scaling

to

predict

pharmacokinetic

parameters

in

different

species

In

particular

the

allometric

equation

does

not

account

for

species-specific

variability

in

tissue

uptake

of

substituted

amphetamines

as

illustrated

by

the

fact

that

brain-to-plasma

ratios

of

the

releaser

fenfluramine

are

for

the

rat

yet

for

humans

No

studies

have

compared

the

brain

tissue

uptake

of

MDMA

in

various

animal

models

Perhaps

the

most

problematic

issue

in

extrapolating

doses

across

species

is

the

unpredictable

nature

of

drug

metabolism

which

occurs

chiefly

in

the

liver

-LSB-

see

Campbell

-LRB-

-RRB-

-RSB-

Marked

species

differences

in

hepatic

drug

metabolism

are

due

to

variations

in

expression

and

activity

of

cytochrome

enzymes

which

catalyze

biotransformation

reactions

-LRB-

Lin

-RRB-

MDMA

is

extensively

metabolized

in

humans

as

depicted

in

Fig.

and

the

major

pathway

of

biotransformation

involves

-LRB-

-RRB-

O-demethylenation

catalyzed

by

cytochrome

-LRB-

-RRB-

and

-LRB-

-RRB-

O-methylation

catalyzed

by

catechol-O-methyltransferase

-LRB-

COMT

-RRB-

-LSB-

reviewed

by

de

la

Torre

et

al.

-LRB-

-RRB-

-RSB-

and

COMT

are

both

polymorphic

in

humans

and

differential

expression

of

isoforms

leads

to

interindividual

variations

in

the

metabolism

of

serotonergic

medications

-LRB-

e.g.

SSRIs

-RRB-

-LRB-

Charlier

et

al.

-RRB-

Interestingly

is

not

present

in

rats

which

express

a

homologous

but

functionally

distinct

cytochrome

-LRB-

Malpass

et

al.

Maurer

et

al.

-RRB-

A

minor

pathway

of

MDMA

biotransformation

in

humans

involves

N-demethylation

of

MDMA

to

form

MDA

which

is

subsequently

O-demethylenated

and

O-methylated

N-demethylation

of

MDMA

represents

a

more

important

pathway

for

rats

when

compared

to

humans

-LRB-

de

la

Torre

and

Farre

-RRB-

The

metabolism

of

MDMA

and

MDA

generates

a

number

of

metabolites

some

of

which

may

be

active

-LSB-

e.g.

Escobedo

et

al.

-LRB-

-RRB-

Forsling

et

al.

-LRB-

-RRB-

-RSB-

Determining

the

potential

neurotoxic

properties

of

the

various

metabolites

of

MDMA

is

an

important

area

of

research

-LSB-

reviewed

by

Baumgarten

and

Lachenmayer

-LRB-

-RRB-

Monks

et

al.

-LRB-

-RRB-

-RSB-

Fig.

of

MDMA

in

man

Cytochrome

cytochrome

COMT

catechol-O-methyltransferase

This

is

adapted

from

de

la

Torre

et

al.

-LRB-

-RRB-

To

complicate

matters

further

de

la

Torre

et

al.

-LRB-

-RRB-

have

shown

that

MDMA

displays

nonlinear

kinetics

in

humans

such

that

administration

of

increasing

doses

or

multiple

doses

leads

to

unexpectedly

high

plasma

levels

of

the

drug

Enhanced

plasma

and

tissue

levels

of

MDMA

are

most

likely

related

to

auto-inhibition

of

MDMA

metabolism

mediated

via

formation

of

a

metabolite

enzyme

complex

that

irreversibly

inactivates

-LRB-

Wu

et

al.

-RRB-

Because

MDMA

displays

nonlinear

kinetics

repeated

drug

dosing

could

produce

serious

adverse

consequences

due

to

unusually

high

blood

and

tissue

levels

of

the

drug

-LRB-

Parrott

Schifano

-RRB-

The

existing

database

of

MDMA

pharmacokinetic

studies

represents

a

curious

situation

where

clinical

findings

are

well-documented

while

preclinical

data

are

lacking

Specifically

few

studies

in

animals

have

assessed

the

relationship

between

pharmacodynamic

effects

and

pharmacokinetics

of

MDMA

after

administration

of

single

or

repeated

doses

-LSB-

but

see

Chu

et

al.

-LRB-

-RRB-

-RSB-

No

studies

have

systematically

characterized

the

nonlinear

kinetics

of

MDMA

in

animal

models

Collectively

the

available

data

demonstrate

that

potential

species

differences

in

tissue

drug

uptake

variations

in

metabolic

enzymes

and

their

activities

and

the

phenomenon

of

nonlinear

kinetics

preclude

the

use

of

interspecies

scaling

to

extrapolate

MDMA

doses

between

animals

and

humans

-LSB-

reviewed

by

de

la

Torre

and

Farre

-LRB-

-RRB-

-RSB-

The

uncertainties

and

limitations

of

allometric

scaling

led

us

to

investigate

the

method

of

effect

scaling

as

an

alternative

strategy

for

matching

equivalent

doses

of

MDMA

in

rats

and

humans

-LSB-

see

Winneke

and

Lilienthal

-LRB-

-RRB-

-RSB-

In

this

approach

the

lowest

dose

of

drug

that

produces

a

specific

pharmacological

response

is

determined

for

rats

and

humans

and

subsequent

dosing

regimens

in

rats

are

calculated

with

reference

to

the

predetermined

threshold

dose

In

the

case

of

MDMA

this

strategy

is

simplified

because

CNS

drug

effects

such

as

neuroendocrine

and

behavioral

changes

have

already

been

investigated

in

different

species

Theoretically

equivalent

drug

effects

in

vivo

should

reflect

similar

drug

concentrations

reaching

active

sites

in

tissue

suggesting

that

the

method

of

effect

scaling

can

account

for

differences

in

ADME

across

species

-LRB-

at

least

for

low

drug

doses

-RRB-

Table

shows

the

doses

of

MDMA

that

produce

comparable

CNS

effects

in

rats

and

humans

Remarkably

the

findings

reveal

that

doses

of

MDMA

in

the

range

of

mg/kg

produce

pharmacological

effects

that

are

equivalent

in

both

species

It

is

noteworthy

that

MDMA

is

typically

administered

to

rats

via

the

i.p.

or

s.c.

route

whereas

humans

take

the

drug

orally

Given

the

similar

effects

of

MDMA

in

rats

and

humans

at

the

same

doses

it

appears

that

drug

bioavailability

is

comparable

after

i.p.

s.c.

or

oral

administration

-LSB-

e.g.

Finnegan

et

al.

-LRB-

-RRB-

-RSB-

but

verification

of

this

hypothesis

awaits

further

investigation

Table

neurobiological

effects

of

-LRB-

-RRB-

MDMA

administration

in

rats

and

humansCNS

effectDose

in

ratsDose

in

humansIn

vivo

release

of

and

mg/kg

i.p.

-LRB-

Gudelsky

and

Nash

-RRB-

mg/kg

s.c.

-LRB-

Kankaanpaa

et

al.

-RRB-

mg/kg

p.o.a

-LRB-

Liechti

et

al.

Liechti

and

Vollenweider

-RRB-

Secretion

of

prolactin

and

mg/kg

i.p.

-LRB-

Nash

et

al.

-RRB-

mg/kg

p.o.

-LRB-

Mas

et

al.

-RRB-

mg/kg

p.o.

-LRB-

Harris

et

al.

-RRB-

Drug

mg/kg

i.p.

-LRB-

Oberlender

and

Nichols

Schechter

-RRB-

mg/kg

p.o.

-LRB-

Johanson

et

al.

-RRB-

Drug

mg/kg

i.v.

-LRB-

Wakonigg

et

al.

-RRB-

mg/kg

p.o.b

-LRB-

Tancer

and

Johanson

-RRB-

aSubjective

effects

were

attenuated

by

uptake

blockers

suggesting

the

involvement

of

transporter-mediated

release

bReinforcing

effects

were

determined

based

on

a

multiple

choice

procedure

Administration

of

MDMA

at

i.p.

doses

of

mg/kg

causes

marked

elevations

in

extracellular

and

DA

in

rat

brain

as

determined

by

in

vivo

microdialysis

-LRB-

Baumann

et

al.

Gudelsky

and

Nash

Kankaanpaa

et

al.

-RRB-

The

data

from

Fig.

illustrate

that

doses

of

MDMA

as

low

as

mg/kg

i.v.

stimulate

a

significant

rise

in

extracellular

in

rat

nucleus

accumbens

Although

it

is

impossible

to

directly

measure

and

DA

release

in

living

human

brain

clinical

studies

indicate

that

subjective

effects

of

recreational

doses

of

MDMA

-LRB-

mg/kg

p.o.

-RRB-

involve

transporter-mediated

release

of

-LRB-

Liechti

et

al.

Liechti

and

Vollenweider

-RRB-

Nash

et

al.

-LRB-

-RRB-

showed

that

i.p.

injections

of

mg/kg

of

MDMA

stimulate

prolactin

and

corticosterone

secretion

in

rats

and

similar

oral

doses

increase

plasma

prolactin

and

cortisol

in

human

drug

users

-LRB-

Harris

et

al.

Mas

et

al.

-RRB-

The

dose

of

MDMA

discriminated

by

rats

and

humans

is

identical

mg/kg

i.p.

for

rats

-LRB-

Glennon

and

Higgs

Oberlender

and

Nichols

Schechter

-RRB-

and

mg/kg

p.o.

for

humans

-LRB-

Johanson

et

al.

-RRB-

A

few

studies

have

shown

that

rats

will

self-administer

MDMA

at

doses

ranging

from

mg/kg

i.v.

indicating

these

doses

possess

reinforcing

efficacy

-LRB-

Ratzenboeck

et

al.

Schenk

et

al.

-RRB-

Wakonigg

et

al.

-LRB-

-RRB-

demonstrated

that

a

single

i.v.

injection

of

mg/kg

MDMA

serves

a

powerful

reinforcer

in

an

operant

runway

procedure

and

MDMA

displays

similar

reinforcing

potency

in

Sprague

Dawley

and

Long

Evans

rat

strains

Tancer

and

Johanson

-LRB-

-RRB-

reported

that

and

mg/kg

of

MDMA

have

reinforcing

properties

in

humans

that

resemble

those

of

-LRB-

-RRB-

amphetamine

The

findings

summarized

in

Table

suggest

that

there

is

no

scientific

justification

for

using

interspecies

scaling

to

adjust

MDMA

doses

between

rats

and

humans

Based

on

this

analysis

we

devised

an

MDMA

dosing

regimen

in

rats

which

attempts

to

mimic

binge

use

of

MDMA

in

humans

Male

Sprague

Dawley

rats

weighing

g

were

double-housed

in

plastic

cages

under

conditions

of

constant

ambient

temperature

-LRB-

°C

-RRB-

and

humidity

-LRB-

-RRB-

in

a

vivarium

In

our

initial

studies

three

i.p.

injections

of

or

mg/kg

MDMA

were

administered

one

dose

every

h

to

yield

cumulative

doses

of

or

mg/kg

Control

rats

received

saline

vehicle

according

to

the

same

schedule

Rats

were

removed

from

their

cages

to

receive

i.p.

injections

but

were

otherwise

confined

to

their

home

cages

The

kg

dose

was

used

as

a

low

behavioral

dose

whereas

the

kg

dose

was

used

as

a

high

noxious

dose

-LRB-

i.e.

a

dose

fivefold

greater

than

threshold

-RRB-

Our

repeated

dosing

regimen

was

designed

to

account

for

the

common

practice

of

sequential

dosing

-LRB-

i.e.

bumping

-RRB-

used

by

human

subjects

during

rave

parties

-LRB-

Parrott

-RRB-

During

the

binge

dosing

procedure

body

temperatures

were

measured

by

insertion

of

a

thermometer

probe

into

the

rectum

and

behaviors

were

scored

every

hour

Rats

were

decapitated

weeks

after

dosing

brain

regions

were

dissected

and

tissue

levels

of

and

DA

were

determined

by

HPLC

ECD

as

described

previously

-LRB-

Baumann

et

al.

-RRB-

Neurochemical

data

were

normalized

to

percentage

of

saline

control

values

for

each

brain

region

examined

Data

were

analyzed

using

a

one-way

ANOVA

-LRB-

MDMA

dose

-RRB-

followed

by

Duncan

's

post

hoc

test

Figure

illustrates

new

data

showing

that

our

binge

MDMA

dosing

regimen

increases

core

body

temperature

in

rats

-LRB-

P

-RRB-

Specifically

repeated

i.p.

doses

of

mg/kg

MDMA

elicited

persistent

hyperthermia

on

the

day

of

treatment

whereas

doses

of

mg/kg

did

not

The

kg

dose

caused

temperature

increases

that

were

about

°C

greater

than

control

treatment

The

data

in

Fig.

demonstrate

that

binge

MDMA

treatment

significantly

decreases

tissue

levels

in

the

frontal

cortex

-LRB-

P

-RRB-

striatum

-LRB-

P

-RRB-

and

olfactory

tubercles

-LRB-

P

-RRB-

when

assessed

weeks

later

Post

hoc

tests

revealed

that

high-dose

MDMA

produced

long-term

depletions

of

tissue

-LRB-

reductions

-RRB-

in

all

three

regions

examined

but

the

low-dose

group

had

concentrations

similar

to

saline

controls

Transmitter

depletion

was

selective

for

neurons

since

tissue

DA

levels

were

unaffected

The

magnitude

of

depletions

depicted

in

Fig.

is

similar

to

that

observed

by

others

-LRB-

Battaglia

et

al.

Schmidt

Stone

et

al.

-RRB-

Our

findings

demonstrate

that

repeated

treatment

with

behaviorally

relevant

doses

of

MDMA

does

not

cause

acute

hyperthermia

or

long-term

depletions

In

contrast

repeated

administration

of

MDMA

at

a

dose

that

is

fivefold

higher

than

the

behavioral

dose

causes

both

of

these

adverse

effects

The

data

are

consistent

with

those

of

O'Shea

et

al.

-LRB-

-RRB-

who

reported

that

high-dose

MDMA

-LRB-

or

mg/kg

i.p.

-RRB-

but

not

low-dose

MDMA

-LRB-

mg/kg

i.p.

-RRB-

causes

acute

hyperthermia

and

long-term

depletion

in

Dark

Agouti

rats

Fig.

effects

of

-LRB-

-RRB-

MDMA

on

core

body

temperature

in

rats

Male

rats

received

three

i.p.

injections

of

or

mg/kg

MDMA

one

dose

every

h

-LRB-

i.e.

injections

at

and

h

-RRB-

Saline

was

administered

on

the

same

schedule

Core

temperature

was

recorded

via

insertion

of

a

rectal

thermometer

probe

every

h.

Data

are

mean

SEM

for

N

rats/group

Asterisk

denotes

significance

with

respect

to

saline-injected

control

at

each

time

point

-LRB-

P

Duncan

's

-RRB-

Fig.

effects

of

-LRB-

-RRB-

MDMA

on

tissue

levels

of

-LRB-

top

panel

-RRB-

and

DA

-LRB-

bottom

panel

-RRB-

in

brain

regions

Male

rats

received

three

i.p.

injections

of

or

mg/kg

MDMA

one

dose

every

h.

Saline

was

administered

on

the

same

schedule

Rats

were

killed

weeks

after

injections

brain

regions

were

dissected

and

tissue

and

DA

were

assayed

by

HPLC

ECD

-LRB-

Baumann

et

al.

-RRB-

Data

are

mean

SEM

expressed

as

the

percentage

of

saline-treated

control

values

for

each

region

N

rats/group

Control

values

of

and

DA

were

and

pg/mg

tissue

for

frontal

cortex

-LRB-

CTX

-RRB-

and

pg/mg

tissue

for

striatum

-LRB-

STR

-RRB-

and

and

pg/mg

tissue

for

olfactory

tubercle

-LRB-

OT

-RRB-

Asterisk

denotes

significance

compared

to

saline-injected

control

for

each

region

-LRB-

P

Duncan

's

-RRB-

Effects

of

MDMA

on

established

markers

of

neurotoxicity

Hallmark

indicators

of

neurotoxicity

include

cell

death

silver

positive

staining

-LRB-

i.e.

argyrophilia

-RRB-

and

glial

cell

hypertrophy

-LRB-

Baumgarten

and

Lachenmayer

O'Callaghan

and

Sriram

Switzer

-RRB-

Despite

the

abundance

of

evidence

showing

that

MDMA

causes

depletions

few

studies

have

examined

the

effects

of

MDMA

on

established

markers

of

neurotoxic

damage

Indeed

some

investigators

have

argued

that

based

on

abnormalities

alone

one

can

not

infer

the

presence

of

neurotoxicity

-LRB-

O'Callaghan

and

Miller

Wang

et

al.

-RRB-

Support

for

the

hypothesis

of

MDMA-induced

axotomy

relies

heavily

upon

immunohistochemical

analysis

of

levels

and

the

use

of

semiquantitative

methods

could

produce

misleading

results

if

not

validated

by

other

means

For

example

the

MDMA-induced

disappearance

of

immunoreactive

in

specific

brain

regions

could

reflect

depletion

of

transmitter

to

a

low

level

-LRB-

i.e.

below

the

level

of

detection

-RRB-

in

intact

axons

and

nerve

terminals

Thus

an

alternative

hypothesis

consistent

with

available

data

is

that

MDMA-induced

deficits

in

systems

are

due

to

persistent

adaptive

changes

in

gene

expression

or

protein

function

and

these

changes

reflect

a

state

of

metabolic

quiescence

or

exhaustion

rather

than

neurotoxic

damage

Table

summarizes

the

findings

from

studies

that

have

examined

the

effects

of

MDMA

on

established

indicators

of

neurotoxic

damage

All

of

the

studies

employed

large

doses

of

MDMA

either

as

single

or

repeated

injections

which

cause

hyperthermia

and

its

associated

complications

Table

of

-LRB-

-RRB-

MDMA

administration

on

markers

of

neuronal

degeneration

in

ratsCNS

markerDosing

regimenSurvival

intervalReferenceIncreased

silver-positive

staining

in

degenerating

mg/kg

s.c.

twice

daily

hCommins

et

al.

-LRB-

-RRB-

mg/kg

s.c.

twice

daily

daysJensen

et

al.

-LRB-

-RRB-

Increased

Fluoro-Jade

B

staining

of

degenerating

neurons

mg/kg

i.p.

single

daysSchmued

-LRB-

-RRB-

Reactive

astrogliosis

as

measured

by

elevations

in

mg/kg

s.c.

twice

daily

daysO'Callaghan

and

Miller

-LRB-

-RRB-

a

mg/kg

i.p.

single

weekAguirre

et

al.

-LRB-

-RRB-

mg/kg

s.c.

twice

daily

days

days

weekPubill

et

al.

-LRB-

-RRB-

a

mg/kg

i.p.

three

weeksWang

et

al.

-LRB-

-RRB-

a

aThese

investigators

found

no

effect

of

MDMA

on

GFAP

at

doses

which

significantly

depleted

levels

in

brain

tissue

Silver

staining

methods

are

commonly

utilized

to

identify

degenerating

neuronal

elements

in

the

CNS

-LSB-

reviewed

by

Switzer

-LRB-

-RRB-

-RSB-

In

particular

silver

stains

are

sensitive

indicators

of

damage

caused

by

neurotoxic

chemicals

Commins

et

al.

-LRB-

-RRB-

examined

the

neurotoxic

potential

of

MDMA

by

using

the

Fink

Heimer

procedure

to

detect

argyrophilic

neuronal

structures

In

their

study

male

Sprague

Dawley

rats

received

single

or

multiple

s.c.

injections

of

mg/kg

MDMA

and

were

killed

to

h

later

Single

doses

of

MDMA

increased

silver-positive

staining

only

in

the

frontoparietal

cortex

Multiple

doses

caused

more

extensive

damage

degenerating

nerve

terminals

were

found

in

the

striatum

while

degenerating

terminals

axons

and

cell

bodies

were

observed

throughout

layers

III

and

IV

of

the

parietal

cortex

In

a

comparable

study

Jensen

et

al.

-LRB-

-RRB-

used

the

de

Olmos

cupric

silver

procedure

to

delineate

areas

of

the

brain

damaged

by

MDMA

Male

Long

Evans

rats

received

twice

daily

s.c.

injections

of

mg/kg

MDMA

for

days

and

were

sacrificed

h

thereafter

In

this

case

MDMA

produced

dose-dependent

increases

in

silver-positive

staining

in

the

parietal

cortex

with

some

irregular

staining

in

the

striatum

and

thalamus

at

higher

doses

Most

of

the

staining

in

the

cortex

was

associated

with

degenerating

axons

and

terminals

but

cell

bodies

were

also

stained

The

collective

results

show

that

administration

of

MDMA

at

sufficient

doses

-LRB-

i.e.

mg/kg

-RRB-

can

cause

neuronal

damage

as

indicated

by

sensitive

silver

staining

methods

There

are

several

key

factors

to

consider

when

interpreting

the

effects

of

MDMA

on

silver

staining

in

rat

brain

Most

importantly

both

of

the

cited

studies

employed

massive

cumulative

doses

of

MDMA

ranging

from

to

mg/kg

Based

on

the

concept

of

effect

scaling

described

previously

-LRB-

see

Table

-RRB-

the

single

injections

of

MDMA

administered

by

Commins

et

al.

and

Jensen

et

al.

are

times

higher

than

a

behaviorally

relevant

dose

of

mg/kg

At

such

extraordinary

doses

of

MDMA

excessive

sympathetic

activation

and

hyperthermia

could

cause

physiological

dysregulation

sufficient

to

elicit

nonspecific

neuropathy

The

idea

that

high-dose

MDMA

causes

nonspecific

neuronal

damage

is

supported

by

the

observation

that

increases

in

silver

staining

are

not

confined

to

cells

Both

studies

noted

the

presence

of

argyrophilic

cell

bodies

in

the

cortex

of

MDMA-treated

rats

yet

cell

bodies

are

not

present

in

the

cortex

-LRB-

Steinbusch

-RRB-

Furthermore

the

pattern

of

MDMA-induced

silver

staining

which

is

largely

confined

to

the

frontoparietal

cortex

does

not

correspond

to

the

pattern

of

innervation

or

the

pattern

of

depletions

Given

these

findings

it

seems

feasible

that

increases

in

silver-positive

staining

produced

by

high-dose

MDMA

do

not

reflect

neurotoxicity

per

se

Schmued

-LRB-

-RRB-

used

a

novel

histological

stain

Fluoro-Jade

B

to

examine

neuronal

damage

produced

by

MDMA

administration

in

rats

Fluoro-Jade

B

is

an

anionic

fluorescein

derivative

that

selectively

stains

degenerating

terminals

axons

and

cell

bodies

-LRB-

Schmued

and

Hopkins

-RRB-

Male

Sprague

Dawley

rats

received

single

i.p.

injections

of

mg/kg

MDMA

and

were

killed

h

later

After

mg/kg

MDMA

only

one

of

four

rats

exhibited

Fluoro-Jade

staining

in

the

cortex

and

this

rat

experienced

significant

hyperthermia

at

the

time

of

treatment

At

higher

doses

of

MDMA

most

rats

displayed

Fluoro-Jade

staining

in

a

variety

of

brain

areas

For

example

degenerating

pyramidal

and

nonpyramidal

cells

were

stained

in

the

parietal

cortex

while

degenerating

multipolar

cells

were

stained

in

the

ventral

thalamus

The

findings

with

Fluoro-Jade

B

indicate

that

sufficient

doses

of

MDMA

-LRB-

i.e.

mg/kg

-RRB-

can

produce

neuronal

degeneration

However

analogous

to

the

findings

with

silver

staining

methods

many

of

the

damaged

cells

identified

by

Fluoro-Jade

B

are

nonserotonergic

and

the

pattern

of

staining

does

not

overlap

with

the

pattern

of

deficits

Additionally

neuronal

degeneration

was

only

found

in

rats

that

experienced

hyperthermia

of

°C

again

suggesting

an

important

link

between

elevated

body

temperature

and

adverse

effects

A

universal

reaction

to

damage

in

the

CNS

is

hypertrophy

of

astrocytes

or

reactive

gliosis

-LSB-

reviewed

by

O'Callaghan

and

Sriram

-LRB-

-RRB-

-RSB-

This

response

is

accompanied

by

enhanced

expression

of

glial-specific

structural

proteins

such

as

glial

fibrillary

acidic

protein

-LRB-

GFAP

-RRB-

O'Callaghan

et

al.

-LRB-

-RRB-

have

verified

that

a

wide

range

of

neurotoxic

chemicals

increase

the

levels

of

GFAP

in

rodent

CNS

indicating

this

protein

can

be

used

as

a

sensitive

marker

of

neuronal

damage

These

investigators

-LRB-

O'Callaghan

and

Miller

-RRB-

carried

out

extensive

studies

to

evaluate

the

effects

of

MDMA

administration

on

GFAP

expression

in

rat

brain

Male

Long

Evans

rats

were

treated

twice

daily

with

s.c.

injections

of

mg/kg

MDMA

for

days

and

were

sacrificed

h

after

the

last

dose

This

high-dose

regimen

of

MDMA

did

not

alter

the

expression

of

GFAP

even

though

levels

were

markedly

reduced

in

the

cortex

hippocampus

and

striatum

A

separate

group

of

rats

received

twice

daily

s.c.

injections

of

mg/kg

MDMA

for

days

and

these

rats

were

killed

h

later

In

response

to

these

massive

doses

of

MDMA

levels

of

GFAP

were

significantly

elevated

in

various

brain

regions

but

these

increases

did

not

correlate

with

the

degree

of

depletions

Finally

the

effects

of

MDMA

were

compared

to

the

effects

of

the

neurotoxin

-LRB-

-RRB-

At

doses

of

MDMA

and

which

caused

comparable

depletions

only

increased

GFAP

In

a

more

recent

study

Pubill

et

al.

-LRB-

-RRB-

compared

the

effects

of

MDMA

and

methamphetamine

on

SERT

binding

and

a

number

of

glial-specific

markers

Male

Sprague

Dawley

rats

received

twice

daily

s.c.

injections

of

mg/kg

MDMA

for

days

and

were

killed

at

and

days

posttreatment

MDMA

caused

marked

decreases

in

-LSB-

-RSB-

paroxetine-labeled

SERT

binding

but

had

no

effect

on

any

measures

of

reactive

gliosis

In

particular

MDMA

treatment

failed

to

induce

astroglial

activation

as

indicated

by

the

lack

of

changes

in

immunoreactive

GFAP

MDMA

had

no

effect

on

microglial

markers

such

as

peripheral

benzodiazepine

receptors

and

protein

Interestingly

methamphetamine

produced

significant

increases

in

astroglial

and

microglial

markers

in

this

study

indicating

an

important

distinction

between

methamphetamine

and

MDMA

Consistent

with

the

findings

of

Pubill

and

colleagues

Bai

et

al.

-LRB-

-RRB-

found

that

mg/kg

s.c.

MDA

failed

to

increase

GFAP

expression

in

Sprague

Dawley

rats

In

contrast

Aguirre

et

al.

-LRB-

-RRB-

reported

that

a

single

i.p.

dose

of

mg/kg

MDMA

enhanced

histochemical

staining

for

GFAP

in

the

hippocampus

of

Wistar

rats

The

reasons

for

the

discrepancies

between

the

findings

of

Aguirre

et

al.

vs

the

findings

of

others

are

not

readily

apparent

but

could

be

due

to

differences

in

experimental

methods

across

studies

-LRB-

i.e.

different

rat

strains

different

brain

regions

examined

etc

-RRB-

The

majority

of

data

indicate

that

doses

of

MDMA

causing

significant

depletions

-LRB-

i.e.

single

or

repeated

doses

of

mg/kg

-RRB-

do

not

reliably

induce

silver

staining

or

glial

activation

Recent

results

from

our

laboratory

support

this

assertion

-LRB-

Rothman

et

al.

Wang

et

al.

-RRB-

We

examined

the

possible

neurotoxic

effects

of

MDMA

and

two

other

substituted

amphetamines

-LRB-

-RRB-

fenfluramine

and

p-chloroamphetamine

-LRB-

PCA

-RRB-

Specifically

the

effects

of

drug

treatments

on

the

expression

of

SERT

and

GFAP

were

evaluated

using

Western

blot

methods

In

the

first

study

male

Sprague

Dawley

rats

received

four

i.p.

injections

of

mg/kg

-LRB-

-RRB-

fenfluramine

or

one

i.p.

injection

of

mg/kg

PCA

and

rats

were

killed

days

or

weeks

thereafter

-LRB-

-RRB-

Fenfluramine

and

PCA

decreased

cortical

levels

-LRB-

-RRB-

but

neither

drug

caused

long-term

changes

in

the

expression

of

SERT

or

GFAP

-LRB-

Rothman

et

al.

-RRB-

Similar

results

were

found

when

three

i.p.

injections

of

mg/kg

MDMA

were

administered

to

rats

according

to

the

binge

dosing

regimen

described

above

-LRB-

see

Figs.

and

-RRB-

MDMA

did

not

alter

levels

of

immunoreactive

SERT

or

GFAP

when

examined

weeks

after

treatment

even

though

the

same

rats

displayed

substantial

depletions

of

in

the

cortex

striatum

and

hippocampus

-LRB-

Wang

et

al.

-RRB-

Thus

doses

of

MDMA

that

deplete

tissue

do

not

alter

expression

of

SERT

or

GFAP

The

SERT

data

are

especially

surprising

given

that

MDMA

administration

is

known

to

reduce

the

number

of

SERT

binding

sites

thereby

decreasing

-LSB-

-RSB-

uptake

capability

-LRB-

Battaglia

et

al.

Commins

et

al.

Schmidt

-RRB-

To

explore

this

phenomenon

further

we

compared

the

effects

of

MDMA

and

on

the

expression

of

SERT

GFAP

and

other

markers

of

gliosis

-LRB-

Wang

et

al.

-RRB-

In

all

cases

proteins

were

measured

by

Western

blot

analysis

using

sensitive

polyclonal

antibodies

directed

against

the

protein

of

interest

Sprague

Dawley

rats

received

three

i.p.

injections

of

mg/kg

MDMA

or

saline

vehicle

and

were

killed

at

days

and

weeks

later

A

separate

group

of

rats

received

single

intracerebroventricular

-LRB-

i.c.v.

-RRB-

infusions

of

μg

or

ascorbic

acid

vehicle

Both

MDMA

and

caused

depletions

of

in

the

frontal

cortex

at

days

and

weeks

after

treatments

Figure

depicts

previously

published

data

from

our

laboratory

showing

that

MDMA

does

not

affect

tissue

levels

of

SERT

at

weeks

posttreatment

whereas

causes

significant

decreases

in

this

protein

-LRB-

Wang

et

al.

-RRB-

The

data

in

Fig.

reveal

that

binge

MDMA

treatment

does

not

affect

GFAP

while

increases

levels

of

this

protein

It

should

be

mentioned

that

treatment

with

but

not

MDMA

produces

persistent

increases

in

another

astrocyte

marker

heat

shock

protein

On-going

studies

in

our

laboratory

indicate

that

MDMA

does

not

alter

the

intracellular

trafficking

of

SERT

proteins

from

endosomes

to

the

plasma

membrane

-LRB-

Wang

et

al.

-RRB-

Rather

it

seems

that

MDMA

renders

SERT

proteins

nonfunctional

though

the

mechanism

responsible

for

this

effect

is

not

known

Our

findings

provide

evidence

that

decreases

SERT

and

increases

GFAP

whereas

MDMA

does

not

alter

expression

of

either

protein

Accordingly

at

the

doses

tested

in

these

studies

displays

the

profile

of

a

neurotoxin

whereas

MDMA

does

not

Fig.

effects

of

-LRB-

-RRB-

MDMA

-LRB-

top

panel

-RRB-

and

-LRB-

bottom

panel

-RRB-

on

SERT

protein

expression

in

dissected

brain

regions

One

group

of

rats

received

three

i.p.

injections

of

saline

or

mg/kg

MDMA

one

dose

every

h.

Another

group

received

single

i.c.v.

infusions

of

or

vehicle

Rats

were

killed

weeks

later

and

brain

regions

were

dissected

Western

blot

analysis

of

SERT

immunoreactivity

in

the

frontal

cortex

-LRB-

CTX

-RRB-

striatum

-LRB-

STR

-RRB-

and

hippocampus

-LRB-

HIPP

-RRB-

was

carried

out

as

described

-LRB-

Wang

et

al.

-RRB-

Blots

were

digitized

and

quantified

using

densitometry

-LRB-

NIH

IMAGE

software

-RRB-

Changes

in

immunoreactivity

are

expressed

relative

to

their

corresponding

control

-LRB-

defined

as

value

-RRB-

Each

value

is

the

mean

SEM

for

N

rats/group

Asteriskdenotes

significance

with

respect

to

control

in

each

region

-LRB-

P

Student

's

t

test

-RRB-

Data

taken

from

Wang

et

al.

Fig.

effects

of

-LRB-

-RRB-

MDMA

-LRB-

top

panel

-RRB-

and

-LRB-

bottom

panel

-RRB-

on

GFAP

expression

in

dissected

brain

regions

One

group

of

rats

received

three

i.p.

injections

of

saline

or

mg/kg

MDMA

one

dose

every

h.

Another

group

received

single

i.c.v.

infusions

of

or

vehicle

Rats

were

killed

weeks

later

and

brain

regions

were

dissected

Western

blot

analysis

of

GFAP

immunoreactivity

in

the

frontal

cortex

-LRB-

CTX

-RRB-

striatum

-LRB-

STR

-RRB-

and

hippocampus

-LRB-

HIPP

-RRB-

was

carried

out

as

described

-LRB-

Wang

et

al.

-RRB-

Blots

were

digitized

and

quantified

using

densitometry

-LRB-

NIH

IMAGE

software

-RRB-

Changes

in

immunoreactivity

are

expressed

relative

to

their

corresponding

control

-LRB-

defined

as

value

-RRB-

Each

value

is

the

mean

SEM

for

N

rats/group

Asterisk

denotes

significance

with

respect

to

control

in

each

region

-LRB-

P

Student

's

t

test

-RRB-

Data

were

taken

from

Wang

et

al.

Functional

consequences

of

MDMA-induced

depletion

Any

operational

definition

of

neurotoxicity

must

include

the

precept

that

functional

impairments

accompany

neuronal

damage

-LRB-

Moser

Winneke

and

Lilienthal

-RRB-

As

noted

previously

high-dose

MDMA

causes

persistent

inactivation

of

tryptophan

hydroxylase

which

leads

to

inhibition

of

synthesis

and

loss

of

-LRB-

O'Hearn

et

al.

Stone

et

al.

-RRB-

Moreover

MDMA-induced

reduction

in

the

density

of

SERT

binding

sites

leads

to

decreased

capacity

for

uptake

in

nervous

tissue

-LRB-

Battaglia

et

al.

Schmidt

-RRB-

Regardless

of

whether

these

deficits

reflect

neurotoxic

damage

or

long-term

adaptation

such

changes

would

be

expected

to

have

discernible

in

vivo

correlates

Many

investigators

have

examined

functional

consequences

of

high-dose

MDMA

administration

and

a

comprehensive

review

of

this

subject

is

beyond

the

scope

of

the

present

paper

-LSB-

reviewed

by

Cole

and

Sumnall

Green

et

al.

-LRB-

-RRB-

-RSB-

The

following

discussion

will

consider

long-term

effects

of

MDMA

-LRB-

i.e.

week

-RRB-

on

in

vivo

indicators

of

function

in

rats

as

measured

by

electrophysiological

recording

microdialysis

sampling

neuroendocrine

secretion

and

specific

aspects

of

behavior

A

number

of

key

findings

are

summarized

in

Table

In

general

few

published

studies

have

been

able

to

relate

the

magnitude

of

MDMA-induced

depletion

to

the

degree

of

specific

functional

impairment

Furthermore

MDMA

administration

rarely

causes

persistent

changes

in

baseline

measures

of

neural

function

and

deficits

are

most

readily

demonstrated

by

provocation

of

the

system

by

pharmacological

-LRB-

e.g.

drug

challenge

-RRB-

or

physiological

means

-LRB-

e.g.

environmental

stress

-RRB-

Table

of

-LRB-

-RRB-

MDMA

administration

on

functional

indices

of

transmission

in

ratsCNS

effectDosing

regimenSurvival

intervalReferenceNo

change

in

cell

mg/kg

s.c.

twice

daily

weeksGartside

et

al.

-LRB-

-RRB-

Reductions

in

evoked

release

in

mg/kg

s.c.

twice

daily

weeksSeries

et

al.

-LRB-

-RRB-

mg/kg

i.p.

twice

daily

weekShankaran

and

Gudelsky

-LRB-

-RRB-

Changes

in

corticosterone

and

prolactin

mg/kg

s.c.

single

weeksPoland

et

al.

-LRB-

-RRB-

mg/kg

s.c.

twice

daily

days

and

monthsIncreased

anxiety-like

mg/kg

s.c.

or

four

injections

monthsMcGregor

et

al.

-LRB-

-RRB-

a

and

Morley

et

al.

-LRB-

-RRB-

mg/kg

sc

twice

daily

weeksFone

et

al.

-LRB-

-RRB-

a

aThese

investigators

noted

marked

increases

in

anxiogenic

behaviors

in

the

absence

of

significant

MDMA-induced

depletion

in

brain

projections

innervating

the

rat

forebrain

have

cell

bodies

residing

in

the

raphe

nuclei

-LRB-

Steinbusch

-RRB-

These

neurons

exhibit

pacemaker-like

electrical

activity

which

can

be

recorded

using

electrophysiological

techniques

-LRB-

Aghajanian

et

al.

Sprouse

et

al.

-RRB-

Gartside

et

al.

-LRB-

-RRB-

used

extracellular

recording

methods

to

examine

cell

firing

in

the

dorsal

raphe

of

rats

previously

treated

with

MDMA

Rats

received

two

daily

injections

of

mg/kg

s.c.

MDMA

for

days

and

were

tested

under

chloral

hydrate

anesthesia

weeks

later

MDMA

pretreatment

had

no

effect

on

the

number

of

classical

or

burst-firing

cells

encountered

during

recording

Additionally

cell

firing

rates

and

action

potential

characteristics

were

not

different

between

MDMA

and

saline-pretreated

groups

These

data

show

that

neurons

and

their

firing

properties

are

not

altered

after

MDMA

administration

and

this

agrees

with

immunohistochemical

evidence

demonstrating

that

MDMA

does

not

destroy

perikarya

The

electrophysiological

data

from

MDMA-pretreated

rats

differ

from

the

findings

reported

with

In

rats

treated

with

i.c.v.

the

number

of

classical

and

burst-firing

neurons

is

dramatically

decreased

in

the

dorsal

raphe

in

conjunction

with

a

loss

of

fluorescence

-LRB-

Aghajanian

et

al.

Hajos

and

Sharp

-RRB-

Thus

produces

reductions

in

cell

firing

that

are

attributable

to

cell

death

but

MDMA

does

not

In

vivo

microdialysis

allows

continuous

sampling

of

extracellular

fluid

from

intact

brain

and

this

method

has

been

used

to

evaluate

persistent

neurochemical

consequences

of

MDMA

exposure

-LRB-

Gartside

et

al.

Matuszewich

et

al.

Series

et

al.

Shankaran

and

Gudelsky

-RRB-

Series

et

al.

carried

out

microdialysis

in

rat

frontal

cortex

to

examine

long-term

effects

of

MDMA

administration

Male

Sprague

Dawley

rats

received

twice

daily

s.c.

injections

of

mg/kg

MDMA

for

days

and

were

tested

under

chloral

hydrate

anesthesia

weeks

later

Prior

MDMA

exposure

did

not

affect

baseline

extracellular

levels

of

but

levels

of

the

metabolite

acid

-LRB-

-RRB-

were

decreased

to

of

control

Moreover

the

ability

of

-LRB-

-RRB-

fenfluramine

-LRB-

mg/kg

i.p.

-RRB-

to

evoke

release

was

markedly

blunted

in

MDMA-pretreated

rats

when

compared

to

saline-pretreated

controls

In

an

analogous

investigation

Shankaran

and

Gudelsky

-LRB-

-RRB-

assessed

neurochemical

effects

of

acute

MDMA

challenge

in

rats

that

had

previously

received

four

i.p.

injections

of

mg/kg

MDMA

In

vivo

microdialysis

was

performed

in

the

striatum

of

conscious

rats

week

after

high-dose

MDMA

treatment

Baseline

levels

of

dialysate

were

not

altered

by

prior

MDMA

exposure

even

though

tissue

levels

of

in

striatum

were

depleted

by

The

ability

of

MDMA

to

evoke

release

was

severely

impaired

in

MDMA-pretreated

rats

while

the

concurrent

DA

response

was

normal

In

this

same

study

effects

of

MDMA

on

body

temperature

and

syndrome

were

attenuated

in

MDMA-pretreated

rats

suggesting

the

development

of

tolerance

Other

investigations

using

in

vivo

microdialysis

methods

have

shown

that

release

in

response

to

physiological

or

stressful

stimuli

is

impaired

in

rats

pretreated

with

high-dose

MDMA

-LRB-

Gartside

et

al.

Matuszewich

et

al.

-RRB-

Taken

together

the

microdialysis

studies

reveal

important

long-term

consequences

of

MDMA

administration

-LRB-

-RRB-

baseline

levels

of

dialysate

are

not

altered

despite

depletions

of

tissue

transmitter

-LRB-

-RRB-

baseline

levels

of

dialysate

are

consistently

decreased

and

-LRB-

-RRB-

stimulated

release

of

is

blunted

in

response

to

pharmacological

or

physiological

provocation

The

microdialysis

findings

with

MDMA

resemble

those

obtained

from

rats

treated

with

in

which

lesioned

rats

display

normal

baseline

dialysate

but

decreased

-LRB-

Hall

et

al.

Kirby

et

al.

Romero

et

al.

-RRB-

In

a

representative

study

Kirby

et

al.

-LRB-

-RRB-

used

microdialysis

in

rat

striatum

to

evaluate

the

long-term

neurochemical

effects

of

i.c.v.

these

investigators

showed

that

reductions

in

dialysate

and

impairments

in

stimulated

release

are

highly

correlated

with

loss

of

tissue

whereas

baseline

dialysate

is

not

In

fact

depletions

of

brain

tissue

up

to

did

not

affect

baseline

levels

of

dialysate

-LRB-

Kirby

et

al.

-RRB-

It

seems

that

adaptive

mechanisms

serve

to

maintain

normal

concentrations

of

synaptic

even

under

conditions

of

severe

transmitter

depletion

A

comparable

situation

exists

after

lesions

of

the

nigrostriatal

DA

system

where

baseline

levels

of

extracellular

DA

are

maintained

in

the

physiological

range

despite

substantial

loss

of

tissue

DA

-LSB-

see

Zigmond

et

al.

-LRB-

-RRB-

-RSB-

In

the

case

of

high-dose

MDMA

treatment

it

seems

feasible

that

reductions

in

uptake

-LRB-

e.g.

less

functional

SERT

protein

-RRB-

and

metabolism

-LRB-

e.g.

decreased

monoamine

oxidase

activity

-RRB-

compensate

for

depletions

to

keep

optimal

concentrations

of

bathing

nerve

cells

On

the

other

hand

deficits

in

release

are

readily

demonstrated

in

MDMA-pretreated

rats

when

systems

are

taxed

by

drug

challenge

or

environmental

stressors

neurons

projecting

to

the

hypothalamus

provide

stimulatory

input

for

the

secretion

of

adrenocorticotropin

-LRB-

ACTH

-RRB-

and

prolactin

from

the

anterior

pituitary

-LRB-

Van

de

Kar

-RRB-

Accordingly

releasers

-LRB-

e.g.

fenfluramine

-RRB-

and

receptor

agonists

increase

plasma

levels

of

these

hormones

in

rats

and

humans

-LSB-

reviewed

by

Levy

et

al.

-LRB-

-RRB-

-RSB-

Neuroendocrine

challenge

experiments

have

been

used

to

demonstrate

changes

in

serotonergic

responsiveness

in

rats

treated

with

MDMA

-LRB-

Poland

Poland

et

al.

Series

et

al.

-RRB-

In

the

most

comprehensive

study

Poland

et

al.

-LRB-

-RRB-

examined

effects

of

high-dose

MDMA

on

hormone

responses

elicited

by

acute

fenfluramine

challenge

Male

Sprague

Dawley

rats

received

single

s.c.

injections

of

mg/kg

MDMA

and

were

tested

weeks

later

Prior

MDMA

exposure

did

not

alter

baseline

levels

of

circulating

ACTH

or

prolactin

However

in

MDMA-pretreated

rats

fenfluramine-induced

ACTH

secretion

was

reduced

while

prolactin

secretion

was

enhanced

The

MDMA

dosing

regimen

caused

significant

depletions

of

tissue

in

various

brain

regions

including

hypothalamus

In

a

follow-up

time-course

study

rats

received

twice

daily

s.c.

injections

of

mg/kg

MDMA

for

days

and

were

challenged

with

fenfluramine

-LRB-

mg/kg

s.c.

-RRB-

at

and

months

thereafter

As

observed

in

the

single

dose

MDMA

study

rats

exposed

to

multiple

MDMA

doses

displayed

blunted

ACTH

responses

and

augmented

prolactin

responses

to

fenfluramine

Interestingly

the

impaired

ACTH

response

persisted

for

months

in

MDMA-pretreated

rats

even

though

tissue

levels

of

were

not

depleted

at

this

time

point

The

data

show

that

high-dose

MDMA

can

cause

functional

abnormalities

for

up

to

year

and

changes

in

responsiveness

do

not

necessarily

parallel

the

extent

of

recovery

from

depletions

in

brain

In

our

laboratory

we

wished

to

further

explore

the

long-term

neuroendocrine

consequences

of

MDMA

administration

Utilizing

the

binge

MDMA

dosing

regimen

described

previously

-LRB-

see

Figs.

and

-RRB-

male

Sprague

Dawley

rats

received

three

i.p.

injections

of

or

mg/kg

MDMA

one

dose

every

h.

Control

rats

received

saline

vehicle

according

to

the

same

schedule

One

week

after

MDMA

treatment

rats

were

fitted

with

indwelling

jugular

catheters

under

pentobarbital

anesthesia

After

week

of

recovery

from

surgery

-LRB-

i.e.

weeks

after

MDMA

or

saline

-RRB-

rats

were

brought

into

the

testing

room

and

i.v.

catheters

were

connected

to

extension

tubes

Each

rat

received

an

i.v.

injection

of

mg/kg

MDMA

at

time

zero

followed

by

an

injection

of

mg/kg

MDMA

min

later

Blood

samples

were

withdrawn

via

the

catheters

immediately

before

and

min

after

each

dose

of

MDMA

Plasma

levels

of

corticosterone

and

prolactin

were

measured

by

radioimmunoassay

methods

-LRB-

Baumann

et

al.

-RRB-

Hormone

data

were

normalized

to

percentage

of

preinjection

control

and

analyzed

using

a

two-way

ANOVA

-LRB-

pretreatment

acute

treatment

-RRB-

followed

by

Duncan

's

post

hoc

test

Figure

shows

new

data

from

our

laboratory

demonstrating

the

effects

of

binge

MDMA

pretreatment

on

hormone

responses

examined

weeks

later

MDMA

pretreatment

did

not

alter

baseline

levels

of

either

hormone

Acute

administration

of

MDMA

elicited

dose-related

elevations

in

circulating

corticosterone

-LRB-

P

-RRB-

and

prolactin

-LRB-

P

-RRB-

as

shown

by

others

-LRB-

Nash

et

al.

-RRB-

Importantly

MDMA

pretreatment

significantly

attenuated

MDMA-induced

corticosterone

-LRB-

P

-RRB-

and

prolactin

-LRB-

P

-RRB-

responses

Post

hoc

tests

revealed

that

rats

exposed

to

high-dose

MDMA

pretreatment

displayed

reductions

in

corticosterone

and

prolactin

secretion

in

response

to

acute

MDMA

challenge

whereas

hormone

responses

in

the

low-dose

MDMA

rats

were

indistinguishable

from

controls

Fig.

of

-LRB-

-RRB-

MDMA

pretreatment

on

secretion

of

corticosterone

-LRB-

top

panel

-RRB-

and

prolactin

-LRB-

bottom

panel

-RRB-

evoked

by

acute

-LRB-

-RRB-

MDMA

challenge

Male

rats

received

three

i.p.

injections

of

or

mg/kg

MDMA

one

dose

every

h.

Saline

was

administered

on

the

same

schedule

Two

weeks

later

each

rat

received

a

single

i.v.

injection

of

mg/kg

MDMA

at

time

zero

followed

by

a

single

injection

of

mg/kg

MDMA

min

later

Blood

samples

were

drawn

via

indwelling

catheters

immediately

before

injections

and

min

after

each

injection

plasma

corticosterone

and

prolactin

were

measured

by

RIA

-LRB-

Baumann

et

al.

-RRB-

Data

are

mean

SEM

expressed

as

the

percentage

of

baseline

hormone

levels

for

N

rats/group

Baseline

corticosterone

and

prolactin

levels

were

and

ng/ml

of

plasma

respectively

Asterisk

denotes

significance

compared

to

saline-pretreated

control

group

-LRB-

P

Duncan

's

-RRB-

Our

neuroendocrine

results

are

consistent

with

the

development

of

tolerance

to

hormonal

effects

of

MDMA

These

findings

do

not

agree

completely

with

the

data

of

Poland

et

al.

-LRB-

-RRB-

discussed

above

however

our

findings

do

agree

with

previous

data

showing

blunted

hormonal

responses

to

fenfluramine

in

rats

with

fenfluramine-induced

depletions

-LRB-

Baumann

et

al.

-RRB-

Perhaps

more

importantly

the

data

shown

in

Fig.

are

strikingly

similar

to

clinical

findings

in

which

cortisol

and

prolactin

responses

to

acute

-LRB-

-RRB-

fenfluramine

administration

are

reduced

in

human

MDMA

users

-LRB-

Gerra

et

al.

Gouzoulis-Mayfrank

et

al.

-RRB-

Indeed

Gerra

et

al.

-LRB-

-RRB-

reported

that

-LRB-

-RRB-

fenfluramine-induced

prolactin

secretion

is

blunted

in

abstinent

MDMA

users

for

up

to

year

after

cessation

of

drug

use

The

mechanism

-LRB-

s

-RRB-

underlying

reduced

sensitivity

to

-LRB-

-RRB-

fenfluramine

and

MDMA

are

not

known

but

it

is

tempting

to

speculate

that

MDMA-induced

impairments

in

evoked

release

are

involved

as

shown

by

in

vivo

microdialysis

studies

While

some

investigators

have

cited

neuroendocrine

changes

in

human

MDMA

users

as

evidence

for

neurotoxicity

Gouzoulis-Mayfrank

et

al.

-LRB-

-RRB-

provide

a

compelling

argument

that

endocrine

abnormalities

in

MDMA

users

could

be

related

to

cannabis

use

rather

than

MDMA

Further

experiments

will

be

required

to

resolve

the

precise

nature

of

neuroendocrine

changes

in

MDMA

users

One

of

the

more

serious

and

disturbing

clinical

findings

is

that

MDMA

causes

persistent

cognitive

deficits

in

some

users

-LRB-

Morgan

Reneman

-RRB-

Numerous

research

groups

have

examined

the

effects

of

MDMA

treatment

on

learning

and

memory

processes

in

rats

yet

most

studies

failed

to

identify

persistent

impairments

even

when

extensive

depletions

were

present

-LRB-

Byrne

et

al.

McNamara

et

al.

Ricaurte

et

al.

Robinson

et

al.

Seiden

et

al.

Slikker

et

al.

-RRB-

While

an

exhaustive

review

of

this

literature

is

not

possible

here

representative

findings

will

be

mentioned

In

an

extensive

series

of

experiments

Seiden

et

al.

-LRB-

-RRB-

evaluated

the

effects

of

high-dose

MDMA

on

a

battery

of

tests

including

open-field

behavior

schedule-controlled

behavior

one-way

avoidance

discriminated

two-way

avoidance

forced

swim

and

radial

maze

performance

Male

Sprague

Dawley

rats

received

twice

daily

s.c.

injections

of

mg/kg

MDMA

for

days

and

were

tested

beginning

weeks

after

treatment

Despite

large

depletions

of

brain

tissue

MDMA-pretreated

rats

exhibited

normal

behaviors

in

all

paradigms

Likewise

Robinson

et

al.

-LRB-

-RRB-

found

that

MDMA-induced

depletion

of

cortical

up

to

did

not

alter

spatial

navigation

skilled

forelimb

use

or

foraging

behavior

in

rats

On

the

other

hand

Marston

et

al.

-LRB-

-RRB-

reported

that

MDMA

administration

produces

persistent

deficits

in

a

delayed

nonmatch

to

performance

-LRB-

DNMTP

-RRB-

procedure

when

long

delay

intervals

are

employed

-LRB-

i.e.

s

-RRB-

Specifically

saline-pretreated

rats

exhibited

progressive

improvement

in

task

performance

over

successive

days

of

testing

whereas

MDMA-pretreated

rats

did

not

The

authors

theorized

that

delay-dependent

impairments

in

the

DNMTP

procedure

reflect

MDMA-induced

deficits

in

short-term

memory

possibly

attributable

to

depletion

With

the

exception

of

the

findings

of

Marston

et

al.

the

collective

behavioral

data

in

rats

indicate

that

MDMA-induced

depletions

of

brain

have

little

effect

on

cognitive

processes

There

are

several

potential

explanations

for

this

apparent

paradox

First

high-dose

MDMA

administration

produces

only

partial

depletion

of

in

the

range

of

in

most

brain

areas

This

level

of

loss

may

not

be

sufficient

to

elicit

behavioral

alterations

as

compensatory

adaptations

in

neurons

could

maintain

normal

physiological

function

Second

MDMA

appears

to

selectively

affect

fine

diameter

fibers

arising

from

the

dorsal

raphe

and

it

is

possible

that

these

circuits

may

not

subserve

the

behaviors

being

monitored

Third

the

behavioral

tests

utilized

in

rat

studies

might

not

be

sensitive

enough

to

detect

subtle

changes

in

learning

and

memory

processes

Finally

the

functional

reserve

capacity

in

the

CNS

might

be

sufficient

to

compensate

for

even

large

depletions

of

a

single

transmitter

While

MDMA

appears

to

have

few

long-term

effects

on

cognition

in

rats

a

growing

body

of

evidence

demonstrates

that

MDMA

administration

can

cause

persistent

anxiety-like

behaviors

in

this

species

-LRB-

Fone

et

al.

Gurtman

et

al.

Morley

et

al.

-RRB-

Morley

et

al.

-LRB-

-RRB-

first

reported

that

MDMA

exposure

induces

long-term

anxiety

in

rats

These

investigators

gave

male

Wistar

rats

one

or

four

i.p.

injections

of

mg/kg

MDMA

for

two

consecutive

days

Subjects

were

tested

months

later

in

a

battery

of

anxiety-related

paradigms

including

elevated

plus

maze

emergence

and

social

interaction

tests

Rats

receiving

either

single

or

multiple

MDMA

injections

displayed

marked

increases

in

anxiogenic

behaviors

in

all

three

tests

when

compared

to

control

rats

Because

no

endpoints

were

examined

it

was

impossible

to

relate

changes

in

behavior

to

changes

in

transmission

In

a

follow-up

study

Gurtman

et

al.

-LRB-

-RRB-

replicated

the

original

findings

of

Morley

et

al.

using

rats

pretreated

with

four

i.p.

injections

of

mg/kg

MDMA

for

days

persistent

anxiogenic

effects

of

MDMA

were

associated

with

depletions

of

in

the

amygdala

hippocampus

and

striatum

Interestingly

Fone

et

al.

-LRB-

-RRB-

showed

that

twice

daily

injections

of

mg/kg

MDMA

for

days

caused

impairments

in

social

interaction

in

adolescent

Lister

rats

even

in

the

absence

of

depletions

or

reductions

in

-LSB-

-RSB-

paroxetine-labeled

SERT

binding

sites

These

data

suggested

the

possibility

that

MDMA-induced

anxiety

does

not

require

deficits

In

an

attempt

to

determine

potential

mechanisms

underlying

MDMA-induced

anxiety

McGregor

et

al.

-LRB-

-RRB-

evaluated

effects

of

the

drug

on

anxiety-related

behaviors

and

a

number

of

postmortem

parameters

including

autoradiography

for

SERT

and

receptor

subtypes

Rats

received

moderate

-LRB-

mg/kg

i.p.

days

-RRB-

or

high

-LRB-

mg/kg

i.p.

four

injections

days

-RRB-

doses

of

MDMA

and

tests

were

conducted

weeks

later

This

study

confirmed

that

moderate

doses

of

MDMA

could

cause

protracted

increases

in

anxiety-like

behaviors

without

significant

depletions

Furthermore

the

autoradiographic

analysis

revealed

that

anxiogenic

effects

of

MDMA

may

involve

long-term

reductions

in

receptors

rather

than

reductions

in

SERT

binding

Additional

work

by

Bull

et

al.

-LRB-

-RRB-

suggests

that

decreases

in

the

sensitivity

of

receptors

but

not

receptors

could

underlie

MDMA-associated

anxiety

Clearly

more

investigation

into

this

important

area

of

research

is

warranted

Summary

The

findings

reviewed

in

this

paper

allow

a

number

of

conclusions

to

be

drawn

with

regard

to

MDMA-induced

neurotoxicity

First

there

is

little

doubt

that

MDMA

targets

monoamine

transporters

and

transporter-mediated

release

of

DA

and

NE

underlies

pharmacological

effects

of

the

drug

While

MDMA

has

been

considered

a

predominately

serotonergic

agent

certain

adverse

effects

including

cardiovascular

stimulation

and

hyperthermia

likely

involve

NE

and

DA

mechanisms

respectively

There

seems

to

be

no

scientific

rationale

for

using

allometric

scaling

to

adjust

doses

of

MDMA

between

rats

and

humans

because

the

pharmacologically

relevant

doses

are

similar

in

both

species

-LRB-

e.g.

mg/kg

-RRB-

Nonetheless

the

complex

metabolism

of

MDMA

needs

to

be

examined

in

various

animal

species

to

permit

comparison

with

clinical

literature

and

to

validate

appropriate

preclinical

models

With

regard

to

MDMA-induced

neurotoxicity

it

seems

that

deficits

are

not

always

synonymous

with

axonal

death

because

doses

of

MDMA

which

cause

marked

depletions

of

brain

tissue

in

rats

-LRB-

e.g.

mg/kg

-RRB-

are

not

associated

with

silver-positive

staining

reactive

gliosis

or

loss

of

SERT

protein

Like

many

other

psychotropic

drugs

MDMA

is

capable

of

producing

bona

fide

neurotoxicity

at

sufficient

doses

-LRB-

e.g.

mg/kg

-RRB-

and

damage

is

not

confined

to

neurons

Many

aspects

of

function

appear

to

be

normal

in

MDMA-pretreated

rats

despite

significant

loss

of

brain

perhaps

illustrating

the

profound

adaptive

capability

of

the

CNS

On

the

other

hand

MDMA-induced

depletions

are

accompanied

by

impairments

in

evoked

release

and

neuroendocrine

secretion

that

suggest

tolerance

development

The

clinical

relevance

of

preclinical

findings

is

often

uncertain

but

the

fact

that

MDMA

can

produce

persistent

increases

in

anxiety-like

behaviors

without

measurable

deficits

suggests

that

even

moderate

doses

may

pose

risks

mdma serotonin depletion neurotoxicity release hormones behavior