Distinct periods of developmental
sensitivity to the effects of 3,4-(¡)-
(MDMA) on behaviour and monoamines in rats
Matthew R. Skelton, Devon L. Graham, Tori L. Schaefer, Curtis E. Grace,
Amanda A. Braun, Lindsey N. Burns, Robyn M. Amos-Kroohs, Michael T. Williams
and Charles V. Vorhees*
Division of Neurology, Cincinnati Children’s Research Foundation and the Department of Pediatrics, University of Cincinnati
College of Medicine, Cincinnati, OH, USA
Previous findings showed allocentric and egocentric learning deficits in rats after MDMA treatment from
postnatal days (PD) 11–20 but not after treatment from PD 1–10. Shorter treatment periods (PD 1–5, 6–10,
11–15, or 16–20) resulted in allocentric learning deficits averaged across intervals but not for any interval
individually and no egocentric learning deficits individually or collectively. Whether this difference was
attributable to treatment length or age at the start of treatment was unclear. In the present experiment rat
litters were treated on PD 1–10, 6–15, or 11–20 with 0, 10, or 15 mg/kg MDMA q.i.d. at 2-h intervals. Two
male/female pairs/litter received each treatment. One pair/litter received acoustic startle with prepulse
inhibition, straight channel swimming, Cincinnati water maze (CWM), and conditioned fear in a latent
inhibition paradigm. The other pair/litter received locomotor activity, straight channel swimming, Morris
water maze (MWM), and locomotor activity retest with MK-801 challenge. MDMA impaired CWM
learning following PD 6–15 or 11–20 exposure. In MWM acquisition, all MDMA-treated groups showed
impairment. During reversal and shift, the PD 6–15 and PD 11–20 MDMA-treated groups were signifi-
cantly impaired. Reductions in locomotor activity were most evident after PD 6–15 treatment while
increases in acoustic startle were most evident after PD 1–10 treatment. After MK-801 challenge, MDMA-
treated offspring showed less locomotion compared to controls. Region-specific changes in brain
monoamines were also observed but were not significantly correlated with behavioural changes. The
results show that PD 11–20 exposure to MDMA caused the largest long-term cognitive deficits followed by
PD 6–15 exposure with PD 1–10 exposure least affected. Other effects, such as those upon MK-801-
stimulated locomotion showed greatest effects after PD 1–10 MDMA exposure. Hence, each effect has a
different window of developmental susceptibility.
Received 1 March 2011; Revised 16 May 2011; Accepted 19 May 2011; First published online 28 June 2011
Key words: Cincinnati water maze, critical periods, learning and memory, MDMA, Morris water maze.
The abuse of 3,4-methylenedioxymethamphetamine
(MDMA) is an ongoing issue especially in in-
dustrialized countries. As with many other drugs of
abuse, there have been reports of MDMA abuse
during pregnancy (Ho et al. 2001; Moore et al. 2010).
While the effects of MDMA on the adult brain have
been extensively examined, effects on the developing
brain remain poorly understood. It has been shown
that developmental MDMA exposure has long-term
effects on brain development, but many of the studies
do not share a common model to facilitate across-
study comparisons (reviewed in Skelton et al. 2008).
We developed a rat model of late second- and third-
trimester human brain development to examine the
effects of MDMA and related drugs (Broening et al.
2001; Vorhees et al. 2000). The development of the rat
* Address for correspondence: C. V. Vorhees, Ph.D., Division of
Neurology, Cincinnati Children’s Research Foundation, 3333 Burnet
Ave, ML 7044, Cincinnati, OH 45229, USA.
Tel.: (513) 636-8622 Fax: (513) 636-3912
Email: firstname.lastname@example.org [C.V.V.]
Email: email@example.com [M.T.W.]
International Journal of Neuropsychopharmacology (2012), 15, 811–824. f CINP 2011
from postnatal days (PD) 1–20 has been shown to be
analogous to late second- and third-trimester human
brain development (Clancy et al. 2007a,b). Specifically,
structures involved in learning and memory, such as
the hippocampus, are developing during this time
(Clancy et al. 2007b; Rice & Barone, 2000).
Previous studies have shown that MDMA exposure
to the neonatal rat leads to learning and memory defi-
cits. In an initial experiment, rats were exposed from
PD 1–10 or PD 11–20 (Broening et al. 2001). Rats ex-
posed from PD 11–20 showed deficits in route-based
egocentric learning and spatial learning and memory,
while animals exposed from PD 1–10 did not; in-
dicating that MDMA interferes with later stages of
brain development. The MDMA-induced deficits ob-
served from PD 11–20 exposure have been replicated
and have been expanded to show that the deficits
emerge during adolescence and persist until at least
age 1 yr (Cohen et al. 2005; Skelton et al. 2006, 2009;
Vorhees et al. 2004; Williams et al. 2003). In an attempt
to refine the critical period, rats were exposed to
MDMA during shorter 5-d intervals (PD 1–5, 6–10,
11–15, or 16–20) (Vorhees et al. 2009). MDMA treat-
ment caused trends at all intervals but significant
spatial learning deficits were only detected when
summed across all four exposure intervals, suggesting
that 5-d exposure was not sufficient at any one interval
to cause deficits (Vorhees et al. 2009). In the same
experiment, route-based egocentric learning was not
disrupted by any of the 5-d exposure intervals indi-
vidually or collectively. Taken together with previous
studies showing that 10-d exposures caused deficits in
both types of learning, these data reveal that length of
exposure is a critical factor in MDMA-induced learn-
ing and memory deficits.
In order to localize the sensitive period, we treated
rats with MDMA during one of three overlapping 10-d
intervals: PD 1–10, 6–15, or 11–20. The PD 1–10 ex-
posure was included to test for egocentric learning
using a new test paradigm that more specifically as-
sesses route-based learning. When MDMA-treated
animals were tested previously in this test [Cincinnati
water maze (CWM)] they were tested under various
levels of light, which allowed for use of distal cues. To
eliminate distal cues herein we tested under infrared
lighting (Herring et al. 2008). In addition, rats were
tested for fear conditioning with a latent inhibition (LI)
paradigm in order to assess emotional, fear-based
memory in MDMA-treated animals. Acoustic startle
response/prepulse inhibition (ASR/PPI) was assessed
in order to determine if sensory gating was disrupted
in MDMA-treated animals. The PD 11–20 exposure
group was included for comparison and because LI
learning had not been previously tested. In addition
to examining different periods of MDMA exposure,
two doses of MDMA were evaluated (10 and 15 mg/
kg) to determine if there were dose–response effects.
Finally, this study examined NMDA receptor function
by measuring locomotor response to the NMDA
receptor agonist MK-801. The NMDA receptor was
selected for examination due to its role in spatial
learning and memory (Morris, 1989; Morris et al.
Materials and methods
Subjects and treatments
Nulliparous male and female Sprague–Dawley CD
Laboratories (USA). Food (Purina 5006) and filtered
water were available ad libitum. Litters were culled to
12 pups on PD 1 (birth was designated PD 0) balancing
for sex. Litters were randomly assigned to one of three
treatment ages (regimens): (1) PD 1–10, (2) PD 6–15, or
(3) PD 11–20. Two males and two females per litter
were subcutaneously injected four times daily (2 h
inter-dose interval) with 0 (saline; Sal), 10 or 15 mg/kg
MDMA (MDMA-10 and MDMA-15 respectively; ex-
pressed as free base). The 10 mg/kg dose of MDMA
(95% pure; NIH, USA) was chosen because it has
consistently been shown to induce learning and
memory deficits after neonatal treatment (Broening
et al. 2001; Skelton et al. 2006; Vorhees et al. 2004, 2007;
Williams et al. 2003). Dose extrapolation to humans
has been discussed previously showing that this regi-
men produces similar plasma MDMA levels in rats as
achieved by humans (Skelton et al. 2008). On PD 28,
litters were separated from the dam and divided by
sex and treatment group and placed in one of two
testing arms. Body weights were recorded daily dur-
ing treatment and weekly thereafter. Twenty litters
were used per regimen; hence there were 20 males
and 20 females in each treatment/regimen/testing
arm. The vivarium is accredited by the Association
for the Assessment and Accreditation of Laboratory
Animal Care and protocols were approved by the
Institutional Animal Care and Use Committee.
Within a litter, one male/female pair/treatment group
was assigned to one of two testing arms with the first
consisting of ASR/PPI, straight channel swimming,
CWM, and conditioned fear/LI. The other male/
female pair/treatment group/litter was assigned to:
locomotor activity, straight channel swimming, Morris
812 M. R. Skelton et al.
developmental MDMA exposure in some regions and
these data do not rule out the possibility of NMDA
changes in the hippocampus. Further studies should
investigate the role of MDMA exposure on NMDA
receptor structure and function in hippocampus as
well as in the striatum and the role of NMDA re-
ceptors during different exposure periods.
This work was supported by NIH project grant
DA021394 and training grant T32 ES007051. We
gratefully acknowledge the assistance of Mary Moran
and Holly Johnson for technical assistance.
Statement of Interest
Broening HW, Morford LL, Inman-Wood SL, Fukumura M,
et al. (2001). 3,4-methylenedioxymethamphetamine
(ecstasy)-induced learning and memory impairments
depend on the age of exposure during early development.
Journal of Neuroscience 21, 3228–3235.
Bronson ME, Jiang W, Clark CR, DeRuiter J (1994). Effects of
designer drugs on the chicken embryo and 1-day-old
chicken. Brain Research Bulletin 34, 143–150.
Cain DP (1997). Prior non-spatial pretraining eliminates
sensorimotor disturbances and impairments in water maze
learning caused by diazepam. Psychopharmacology (Berlin)
Clancy B, Finlay BL, Darlington RB, Anand KJ (2007a).
Extrapolating brain development from experimental
species to humans. Neurotoxicology 28, 931–937.
Clancy B, Kersh B, Hyde J, Darlington RB, et al. (2007b).
Web-based method for translating neurodevelopment
from laboratory species to humans. Neuroinformatics 5,
Cohen MA, Skelton MR, Schaefer TL, Gudelsky GA, et al.
(2005). Learning and memory after neonatal exposure to
3,4-methylenedioxymethamphetamine (ecstasy) in rats:
Interaction with exposure in adulthood. Synapse 57,
Grace CE, Schaefer TL, Graham DL, Skelton MR, et al.
(2010). Effects of inhibiting neonatal methamphetamine-
induced corticosterone release in rats by adrenal
autotransplantation on later learning, memory, and plasma
corticosterone levels. International Journal of Developmental
Neuroscience 28, 331–342.
Herring NR, Schaefer TL, Gudelsky GA, Vorhees CV,
et al. (2008). Effect of (+)-methamphetamine on path
integration learning, novel object recognition, and
neurotoxicity in rats. Psychopharmacology (Berlin) 199,
Ho E, Karimi-Tabesh L, Koren G (2001). Characteristics
of pregnant women who use ecstasy
(3, 4-methylenedioxymethamphetamine). Neurotoxicology
and Teratology 23, 561–567.
Kirk RE (1995). Experimental Design, 3rd edn. Pacific Grove:
California Brooks/Cole Publishing Company.
Moore DG, Turner JD, Parrott AC, Goodwin JE, et al. (2010).
During pregnancy, recreational drug-using women stop
taking ecstasy (3,4-methylenedioxy-N-
methylamphetamine) and reduce alcohol consumption,
but continue to smoke tobacco and cannabis: initial
findings from the Development and Infancy Study. Journal
of Psychopharmacology 24, 1403–1410.
Morris RG (1989). Synaptic plasticity and learning: selective
impairment of learning rats and blockade of long-term
potentiation in vivo by the N-methyl-D-aspartate
receptor antagonist AP5. Journal of Neuroscience 9,
Morris RG, Anderson E, Lynch GS, Baudry M (1986).
Selective impairment of learning and blockade of long-
term potentiation by an N-methyl-D-aspartate receptor
antagonist, AP5. Nature 319, 774–776.
Rice D, Barone S (2000). Critical periods of vulnerability for
the developing nervous system: evidence from humans
and animal models. Environmental Health Perspectives 108
(Suppl. 3), 511–533.
Saucier D, Hargreaves EL, Boon F, Vanderwolf CH, et al.
(1996). Detailed behavioral analysis of water maze
acquisition under systemic NMDA or muscarinic
antagonism: nonspatial pretraining eliminates
spatial learning deficits. Behavioral Neuroscience 110,
Skelton MR, Schaefer TL, Herring NR, Grace CE,
et al. (2009). Comparison of the developmental effects of
5-methoxy-N,N-diisopropyltryptamine (Foxy) to
in rats. Psychopharmacology (Berlin) 204, 287–297.
Skelton MR, Williams MT, Vorhees CV (2006). Treatment
with MDMA from P11–20 disrupts spatial learning and
path integration learning in adolescent rats but only spatial
learning in older rats. Psychopharmacology (Berlin) 189,
Skelton MR, Williams MT, Vorhees CV (2008).
Developmental effects of 3,4-
methylenedioxymethamphetamine: a review. Behavioral
Pharmacology 19, 91–111.
Sodhi MS, Sanders-Bush E (2004). Serotonin and brain
development. International Reviews of Neurobiology 59,
Vorhees CV (1987). Maze learning in rats: a comparison of
performance in two water mazes in progeny prenatally
exposed to different doses of phenytoin. Neurotoxicology
and Teratology 9, 235–241.
Vorhees CV, Inman-Wood SL, Morford LL, Broening HW,
et al. (2000).Adult learning deficits after neonatal exposure
to D-methamphetamine: selective effects on spatial
navigation and memory. Journal of Neuroscience 20,
Critical periods of MDMA exposure 823
Vorhees CV, Reed TM, Skelton MR, Williams MT (2004).
Exposure to 3,4-methylenedioxymethamphetamine
(MDMA) on postnatal days 11–20 induces reference but
not working memory deficits in the Morris water maze in
rats: implications of prior learning. International Journal of
Developmental Neuroscience 22, 247–259.
Vorhees CV, Schaefer TL, Skelton MR, Grace CE, et al.
(MDMA) dose-dependently impairs spatial learning in
the morris water maze after exposure of rats to different
five-day intervals from birth to postnatal day twenty.
Developmental Neuroscience 31, 107–120.
Vorhees CV, Schaefer TL, Williams MT (2007).
Developmental effects of¡-methylenedioxy-
methamphetamine on spatial vs. path integration learning:
effects of dose distribution. Synapse 61, 488–499.
Vorhees CV, Williams MT (2006). Morris water
maze: procedures for assessing spatial and
related forms of learning and memory. Nature Protocols
Williams MT, Morford LL, Wood SL, Rock SL, et al.
3,4-methylenedioxymethamphetamine (MDMA) impairs
sequential and spatial but not cued learning independent
of growth, litter effects or injection stress. Brain Research
Williams MT, Vorhees CV, Boon F, Saber AJ,
et al. (2002). Methamphetamine exposure from
postnatal day 11 to 20 causes impairments
in both behavioral strategies and spatial
learning in adult rats. Brain Research 958,
824 M. R. Skelton et al.