IMPACTS OF ACUTE AND NEUROTOXIC MDMA EXPOSURE ON OLFACTORY

MEMORY PERFORMANCE IN RATS

Andrew B. Hawkey

A Thesis Submitted to the

University of North Carolina Wilmington in Partial Fulfillment

of the Requirements for the Degree of

Master of Arts

Department of Psychology

University of North Carolina at Wilmington

2012

Approved by

Advisory Committee

Julian Keith Katherine Bruce

Mark Galizio

Chair

Accepted by

Dean, Graduate School

ii

TABLE OF CONTENTS

ABSTRACT .................................................................................................................................. iv

ACKNOWLEDGMENTS ...............................................................................................................v

INTRODUCTION ...........................................................................................................................1

Drug History and Classification ..........................................................................................1

Ecstasy Use in Humans ........................................................................................................2

PHARMACOLOGY OF MDMA ....................................................................................................4

Acute Effects of MDMA .....................................................................................................5

Persistent Effects ..................................................................................................................9

COGNITIVE IMPACTS OF MDMA USE IN HUMANS ...........................................................16

LIMITATIONS OF HUMAN TESTING AND NEED FOR ANIMAL MODELS .....................18

MDMA EFFECTS ON MEMORY ...............................................................................................19

MDMA and Working Memory Tasks in Rats ...............................................................................20

An Alternative Approach to Working Memory Testing ....................................................40

Span....................................................................................................................................41

EXPERIMENT 1: ACUTE EFFECTS OF MDMA ......................................................................45

Method ...............................................................................................................................48

Span Training .....................................................................................................................53

Drug Administration ..........................................................................................................59

Results and Discussion ......................................................................................................59

EXPERIMENT 2: EFFECTS OF BINGE MDMA ADMINISTRATION ...................................67

Methods..............................................................................................................................72

Drug Administration ..........................................................................................................73

iii

Results and Discussion ......................................................................................................74

GENERAL DISCUSSION ............................................................................................................87

REFERENCES ..............................................................................................................................92

iv

LIST OF TABLES

Table Page

1. Acute MDMA Studies of Memory in Rats ........................................................................46

2. Pool of scents for Odor Span and SD Tasks ......................................................................51

3. Training timeline ................................................................................................................56

4. Binge MDMA Studies of Memory Tasks in Rats ..............................................................68

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LIST OF FIGURES

Figure Page

1. Basic Rodent Incrementing Non-Match-to-Sample or Span procedure ............................43

2. Ex.1 Key Dependent Measures ..........................................................................................61

3. Ex.1 Within-Session Analysis ...........................................................................................63

4. Ex.2 Key Dependent Measures ..........................................................................................77

5. Ex.2 Session-by-Session Data ...........................................................................................78

6. Individual Subject Graphs..................................................................................................79

7. Ex.2 Within Session Analysis ............................................................................................81

8. Reversal Data .....................................................................................................................83

iv

ABSTRACT

MDMA is an amphetamine derivative which is taken recreationally in social contexts and

has been linked to disruptions in neurologic and cognitive functioning. In rodents, the effects of

MDMA have been tested on a variety of working memory tasks, but none have measured the

effects of the drug on working memory capacity. The current study used the rodent Odor Span

Task (OST) to assess the impact of two drug administration schedules on load-dependent

learning. Experiment 1 tested the impact of acute doses of MDMA (Saline, 0.3, 1.0, 1.8, 3.0

mg/kg) on the OST. Results from Experiment 1 showed a dose dependent decrease in

performance on the OST and a control task (SD). This is interpreted as a general impairment

which is unrelated to memory. Experiment 2 tested the impact of a binge dosing regimen of

MDMA (10mg/kg, 2 per day x 4 days) or saline on the OST for 4 weeks post-binge. Rats in the

MDMA group scored similarly to controls on all OST measures except omissions, where they

failed to respond more often than controls. This effect was task-independent and appeared to

recover over time. This is interpreted as a general impairment which is unrelated to memory.

MDMA-treated rats did show impaired acquisition on early trials of a simple discrimination

reversal, replicating a previous finding that binge MDMA produces cognitive inflexibility. In

conclusion, the current study suggests that acute MDMA exposure does not produce impairments

on accuracy in the OST or an SD control at any dose. Acute MDMA disrupts olfactory memory

task performance primarily by reducing responding. Furthermore, the current study suggests that

binge or neurotoxic MDMA administration does not produce deficits on olfactory memory, as

accuracies were unaffected in both the OST and SD tasks. Measurable performance disruptions

were related to non-responding (omissions) and reversal learning.

v

ACKNOWLEDGEMENTS

The author would like to thank his Advisor and Thesis Committee Chair, Dr. Mark

Galizio for his support and direction in the design and completion of this project, as well as Dr.

Kate Bruce and Dr. Julian Keith for their support and input. Special thanks are also due to the

members of the Galizio lab at UNC-Wilmington: Brooke April, graduate researchers Melissa

Deal and Christine Hausmann, and undergraduate researcher Kevin Jacobs; for assistance in data

collection. This study was funded by DA029252 through NIDA.

IMPACTS OF ACUTE AND NEUROTOXIC MDMA EXPOSURE ON OLFACTORY

MEMORY PERFORMANCE IN RATS

Drug History and Classification

Methylenedioxymethamphetamine (MDMA) is a ring-substituted amphetamine,

commonly referred to as ecstasy. Merck Corporation first synthesized MDMA in the early 1900s

as a parent compound (McDowell & Kleber, 1994), and it was patented with related compounds

in an effort to develop a new blood clotting drug (Freudenmann, Oxler & Bernschneider-Reif,

2006). Very little is known about the drug for the next several decades beyond development, but

it became publicly available in the United States in the early 1970’s (Freudenmann et al., 2006).

From the mid-1970’s until the drug was given the Schedule 1 designation in 1985, the drug was

sometimes used in psychotherapy, as it was thought to increase openness and aid insight-oriented

therapy (McDowell & Kleber, 1994). Since restrictions were put in place, the licit uses of the

drug have been few, so much of the research on the drug focuses on risks associated with

recreational use.

MDMA is related to a group of compounds with a similar chemical structure (Green,

Mechan, Elliott, O’Shea, & Colado, 2003). In addition to MDMA, this class of compounds

includes amphetamine (AMPH), methamphetamine (MA), 3,4-methylenedioxyamphetamine

(MDA), and 3,4 methylenedioxyethamphetamine (MDEA), among others. MDA, MDEA, and

MDMA have been shown to reduce serotonin reuptake (Green et al., 2003), as well as producing

effects on other neurotransmitter systems. Despite structural similarities, they produce different

effects on an array of cognitive/behavioral tests and tend to produce distinct behavioral profiles,

at least in rodents (Quinteros-Munoz et al., 2010). The MDMA compound has two isoforms, S+

and R-, both of which are pharmacologically active (Karlsen, Spigset & Slordal, 2007). The drug

tends to exhibit stimulant-like effects, as well as a number of socially relevant effects on mood

2

and behavior. These effects complicate the classification of the drug and have led some to refer

to it as an entactogen (e.g. Gouzoulis-Mayfrank and Daumann, 2006; Quinteros-Munos et al.,

2010), rather than a psychostimulant. An entactogen increases social behavior such as

communication or physical contact, and increases subjective feelings of emotional connection or

closeness to others.

In its pure form, MDMA is a white, tasteless powder, but human users typically take the

drug in pill form (Karlsen et al, 2007). It can also be found in other forms, allowing it to be

injected, inhaled, or smoked. Street drugs bearing the name ecstasy may include MDMA or a

number of other drugs with similar properties (Karlsen et al., 2007), but research on the drug has

generally focused on these compounds separately. Within the drug literature, it is often assumed

that the effects of ecstasy are due to the actions of MDMA, but given that a variety of chemicals

may be in ecstasy tablets, there are limits to the validity of this assumption. Within this

discussion, only the illicit use of specific forms of ecstasy, ones which contain solely or

primarily MDMA, can be adequately modeled by studies of MDMA-induced effects.

Ecstasy Use in Humans

Ecstasy users tend to report a wide variety of euphoric and dysphoric symptoms (Karlsen

et al., 2007). Reported physiological effects generally include increased heart rate and body

temperature, dry mouth, nausea, reduced hunger, and insomia. Ecstasy users also tend to report

bruxism (or teeth grinding) and trismus, an involuntary muscular tightening in the jaw. Sensory

symptoms tend to include vestibular abnormalities and altered perception. Subjective emotional

symptoms often include feelings of euphoria, closeness to others, well-being, extroversion and

3

heightened social communication. In some cases, anxiety or panic can also occur. At high doses,

hallucinations or acute psychoses have been reported.

Ecstasy use has been largely associated with adolescents and young adults, particularly

within “rave culture” (McDowell & Kleber, 1994). Raves are parties with loud electronic or

techno music that encourages extended periods of dancing in crowded dance halls. It has been

noted that these conditions encourage the use of stimulants, to assist in sustaining high energy

activities like dancing, and hallucinogens, which alter sensory perception. While raves certainly

contribute to the popularity of the drug, findings from a recent study suggest that ecstasy is often

used in private settings as well (Ramtekkar, Striley & Cottler, 2010). In addition, this study

found that ecstasy is commonly consumed with other substances, most often marijuana or

alcohol. Overall, ecstasy is perceived as being a relatively safe drug and patterns of use appear to

be mediated by a number of different factors (Ramtekkar et al., 2010).

One important factor in the patterns of ecstasy use appears to be based on the rewarding

nature of the context in relation to the drug. Klein, Elifsen and Sterk (2009) investigated

behaviors which occur based on their amplification of the effects of ecstasy. Such behaviors

often include seeking environments with opportunities for intense stimulation. Other behaviors

are exhibited within these environments to provide further stimulation, such as the tendency to

use soft objects to provide tactile stimulation or using certain beverages or lotions to intensify

gustatory and olfactory stimulation. The occurrence of these stimulation-promoting behaviors

was associated with binge consumption of the drug and more adverse effects related to ecstasy

use.

Historically, ecstasy has been considered to be a relatively non-addictive drug, as

compared to other club drugs. Cases of dependence on ecstasy were considered to be relatively

4

rare, as few diagnosed cases of dependence had been reported (e.g. Jansen, 1999). Studies have

found that the frequency of use and occurrence of dependence-related symptoms are greater than

earlier studies suggest (e.g. Cottler, Womack, Compton, & Ben-Abdallah, 2001; Klein, Elifsen &

Sterk, 2009). It has been reported by Abdallah, Scheier, Inciardi, Copeland, & Cottler, (2007)

that among young ecstasy users, nearly sixty-percent of the sample met criteria for dependence at

some point during their lifetimes. As patterns of binge and dependent use among ecstasy-users

become more widely recognized, greater emphasis is placed on the risks associated with ecstasy

use and abuse.

The perceived safety of the drug among young people combined with concern over

lasting effects of the drug has led to growing interest in the acute and persistent neurological and

cognitive effects. Over the last few decades, a substantial literature on these topics has emerged

which seeks to clarify the biological and functional disturbances which accompany recreational

ecstasy use.

PHARMACOLOGY OF MDMA

Immediately after administration of MDMA, a series of neurochemical changes takes

place which impacts a broad range of cellular and chemical systems throughout the central

nervous system. There is still some debate as to which of these diverse systems are directly

influenced by MDMA and its metabolites, and which are indirectly affected by changes in other

systems (Green et al., 2003; Gudelsky & Yamamoto, 2008). Given high or frequent doses of the

MDMA, certain effects have the potential to produce persistent changes well beyond the active

phase of the drug (Gouzoulis-Mayfrank & Daumann, 2006). Much has been learned about the

pharmacology of MDMA, but much is left to be determined about the effects of the drug on a

5

cellular level.

Acute Effects of MDMA

Serotonin

The acute effects of MDMA are neurologically diverse, but in general the drug is most

closely associated with serotonin (5-HT) functioning. The overall effect of acute MDMA

administration on the serotonin system follows a predictable pattern which has been shown in

numerous brain areas (Baumann, Wang & Rothman, 2007; Green et al., 2003; Gudelsky &

Yamamoto, 2008), generally in rats or in isolated brain slices in the lab. Shortly after MDMA

administration, there is a dose-dependent spike in 5-HT release which results in a depletion of

the vesicular stores (e.g. Stone, Merchant, Hanson & Gibb, 1987). Microdialysis studies have

shown that MDMA produces heightened levels of extracellular serotonin in the striatum,

hippocampus and the cortex (Gudelsky & Yamamoto, 2008), which have been shown to be

attenuated by drugs that limit or decrease vesicular stores (Green et al., 2003).

The depletion of serotonin stores appears to stem from two distinct factors. First, MDMA

binds to the serotonin transporter with high affinity (Baumann et al., 2007; Green et al., 2003),

acting as a serotonin reuptake inhibitor. Second, MDMA inhibits the enzymes tryptophan

hydroxylase (TPH) (Stone et al., 1987) and monoamine oxidase (MAO) (Leonardi & Azmitia,

1994). TPH is the rate-limiting enzyme in the production of serotonin, so inhibition of the

enzyme reduces the amount of serotonin available for release during a period of heightened

neuronal firing. MAO is the enzyme responsible for breaking down monoamines (such as

serotonin, dopamine, epinephrine, and norepinephrine) in the terminal button, so inhibition of

this enzyme allows for more serotonin to be packaged and released, contributing to the higher

6

rate of release. As the reuptake of serotonin is inhibited and the production of serotonin is

slowed, serotonergic neurons are unable to maintain serotonin levels during the period of

increased release, eventually producing a depletion of intracellular serotonin.

Dopamine and Contributing Systems

Studies of the acute effects of MDMA have also found significant dose-dependent

effects in the dopamine system. As with serotonin, dopaminergic neurons show a rapid increase

in release until depletion (Green et al., 2003), and a decrease in reuptake (Gudelsky &

Yamamoto, 2008). As MAO is inhibited by MDMA (Leonardi & Azmitia, 1994), dopamine in

the terminal button is broken down at a slower rate, contributing to the increase in dopamine

release. Significant changes in extracellular dopamine have been noted in the striatum, nucleus

accumbens, prefrontal cortex and in the ventral hippocampus (Green et al., 2003). While MDMA

has been shown to have some affinity for dopamine transporter sites, this affinity is relatively

weak (e.g. Fantegrossi et al., 2009) and dopaminergic effects appear to be substantially mediated

by other systems (Gudelsky & Yamamoto, 2008).

Nigrostriatal and cortical dopamine release is thought to be facilitated by activation of 5-

HT2 receptors, as 5-HT2 agonists and antagonists influence levels of dopamine release following

MDMA use (Green et al., 2003; Gudelsky & Yamamoto, 2008). This influence has been related

to PKC, a protein used for intracellular signaling, which stimulates dopamine release in the

striatum, and inhibits DA release in the prefrontal cortex (Gudelsky & Yamamoto, 2008). 5-HT2

is also implicated in decreasing GABA release in the substantia nigra, which likely produces to a

disinhibition of dopamine release in the striatum. A similar increase in GABA activity in the

ventral tegmental area appears to limit the release of dopamine in the nucleus accumbens, though

7

increased release does occur following MDMA (Gudelsky & Yamamoto, 2008). An increase in

dopamine release in the ventral hippocampus appears to be primarily influenced by

noradrenergic inputs.

Despite differences in the mechanism of action, the general effect of MDMA on

dopamine release is similar to the effects of other amphetamines. Some researchers have

emphasized this similarity as a potential source of overlap in the functional effects of these drugs

(e.g. Harper, 2011). Harper (2011) administered MDMA to rats while pharmacologically

blocking D1 and D2 receptors and found an attenuation of the cognitive disturbances associated

with the drug on a delayed match-to-sample task. These effects seem to suggest the importance

of dopamine to functional effects of the drug. However, these results differed from an identical

attempt to block the effects of cocaine, so the relationship between MDMA actions and those of

primarily dopaminergic stimulant drugs remains difficult to explain.

Other Neurochemical Systems

MDMA is most closely associated with the release and depletion of serotonin and

dopamine, but as is demonstrated above, the drug affects multiple systems in the brain and can

produce indirect effects by modifying the activity in influential pathways. Research on the

noradrenergic (NE) system has shown some evidence for norepinephrine release in brain slice

preparations after MDMA administration, but very little is known about these effects in vivo

(Green et al., 2003). There is some evidence that MDMA can interfere with noradrenergic

binding and that these effects are not limited to the central nervous system. More research in this

area is necessary to determine the extent of these effects and the cellular actions of the drug

which produce them.

8

Though MDMA is not usually associated with the cholinergic (ACh) system, studies that

analyzed acetylcholine levels have shown that acute MDMA stimulates cholinergic activity in

the striatum and the prefrontal cortex (Gudelsky & Yamamoto, 2008). Increases in the release of

acetylcholine have been shown to be the product of direct MDMA action on the H1 histamine

receptor (Gudelsky & Yamamoto, 2008), though the drug has a much greater affinity for

serotonergic and dopaminergic receptors (Green et al., 2003).

Internal Temperature

In a recent published chapter on temperature regulation, Kiyatkin (2010) noted that in

general, psychostimulants can produce hyperthermia in human and non-human users. He also

noted that such drugs are very likely to produce hyperthermia when taken in warm settings and

during physical activity, both of which are considered to be relevant to recreational ecstasy use.

He also noted that MDMA has a distinct dose-dependent relationship with internal body

temperature which differs from other amphetamines like methamphetamine (MA). Low doses of

MDMA actually tend to decrease body temperature, while higher doses will tend to increase it.

While MDMA produces lesser stimulant and hyperthermic effects than MA does at the same

dose, vasoconstrictive effects which contribute to increased body and brain temperature are

stronger and last longer. Kiyatkin expressed a general concern for increased potential for toxicity

and for dangerously high body temperatures given the right conditions.

Rodent studies which rely on consistent physiological effects at a given dose have also

expressed concern over body temperature (e.g. Marston, Reid, Lawrence, Olverman & Butcher,

1999), and some researchers have manipulated body temperature to determine the role that

environmental heat might have on neurochemical changes (e.g. Fedduccia, Kongovy &

9

Duvauchelle, 2010; Johnson & Yamamoto, 2010). Others have expressed concerns that increased

body heat could potentially contribute to lasting damage by promoting oxidative processes which

produce free radicals (Gouzoulis-Mayfrank & Daumann, 2006). Overall, it has been reported

(see review, Green et al., 2003) that minimizing hyperthermia in the acute phase tends to

decrease the risk of long term serotonergic disturbances associated with MDMA.

Other Physiological Effects

MDMA produces a broad range of other physiological effects that have been studied,

though they receive less attention than the primary neurochemical effects have. MDMA affects

cardiovascular functioning, typically raising blood pressure and heart rate, constricting blood

vessels, and otherwise decreasing parasympathetic functions (Green et al., 2003; Kiyatkin,

2010). The drug impacts the neuroendocrine system, increasing the release of a number of

hormones, such as corticosterone, prolactin, oxytocin, and vassopressin. Acutely, the drug

produces a general suppression of immune system functioning. In addition, growing attention has

also been drawn to metabolic factors and free radical production (Green et al., 2003; Gouzoulis-

Mayfrank & Daumann, 2006), as this may be relevant to persistent effects.

Persistent Effects

The widespread effects of the acute phase have been well documented and future

neurobiological research will seek to answer many remaining questions about the systemic,

cellular and molecular mechanisms which MDMA acts on. As the subjective effects of the drug

are seemingly short lived, many consider the drug to be relatively safe, but concern over

potential residual or neurotoxic effects began to form relatively quickly after the drug gained

10

notoriety as a recreational drug (McDowell & Kleber, 1994). Research on residual biological

effects of the drug generally studies the effects of binge or chronic use of the drug (Gouzoulis-

Mayfrank & Daumann, 2006).

Persistence of Serotonergic Effects

Most of the varied effects of MDMA disappear within hours of drug administration, but

many scientists have worked to determine which effects can last beyond the acute phase of drug

activity, and how long the recovery process might take. Though a number of neurotransmitter

systems are altered in the acute drug phase, evidence for neurotoxicity is absent in virtually all

except in the serotonergic system (Gouzoulis-Mayfrank & Daumann, 2006; Green et al., 2003;

Gudelsky & Yamamoto, 2008). Most serotonergic neurons are found in the raphe nucleus with

axons extending into other regions of the brain, which could allow alterations in this system to

produce effects in a number of structures with very different functional roles (Gouzoulis-

Mayfrank & Daumann, 2006).

In general, large or chronic doses of MDMA reduce levels of serotonin (5-HT) and the

metabolite 5-HIAA (e.g. Battaglia et al., 1987; Byrne, Baker & Poling, 2000; Ricaurte et al.,

1993). Ricaurte et al. (1993) tested 5-HT and 5-HIAA levels in the rat hippocampus and

neocortex one week following MDMA administration (2 x 20mg/kg x 4 days). This analysis

revealed a 71% reduction in hippocampal 5-HT levels, and significant reductions in the

neocortex. A second group of subjects received the administration schedule twice, producing

significant reductions in 5-HT in various brain regions (hippocampus, neocortex, striatum, and

thalamus) five months after the second administration. Battaglia et al. (1987) found that two

weeks after chronic MDMA administration (2 x 20mg/kg x 4 days), levels of 5-HT and 5-HIAA

11

were greatly reduced in the rat cortex and hypothalamus. A smaller, but significant, reduction in

5-HT was found in the striatum. Byrne et al. (2000) performed neurochemical tests on a group of

rats 17-18 days following chronic MDMA administration (2 x 20mg/kg x 4 days). This analysis

found a significant reduction in 5-HT and 5-HIAA levels in the prefrontal cortex and the

striatum. The regions showing lasting depletion seem to vary despite receiving the same doses (2

x 20mg/kg x 4 days); however, this may be due to differences in which regions were analyzed or

the time frame when biochemical tests took place.

Large or repeated doses of MDMA have also been shown to produce a loss in the

serotonin transporter (SERT) protein (Battaglia et al., 1987; Biezonski & Meyer, 2009; Sabol,

Lew, Richards, Vosmer & Seiden, 1996; Xie et al., 2006). Levels of SERT binding in

biochemical tests are understood as indicating the availability of reuptake sites on serotonergic

neurons. Battaglia et al. (1987) tested for binding at serotonin uptake sites two weeks after drug

administration. Reductions in SERT were evident in the cerebral cortex, hippocampus, striatum,

hypothalamus, and midbrain. Biezonski and Meyer (2006) found similar reductions in SERT

binding two weeks after administration, though they only tested the hippocampus. In addition,

they found evidence for reduced gene expression of the SERT (discussed in detail later). Xie et

al. (2006) found similar reductions in binding, as well as reductions in the SERT protein in the

midbrain, striatum, neocortex, and cerebellum. Sabol et al. (1996) conducted a study on the

recovery of 5-HT functioning, and found that the reduction in SERT binding lasted at least eight

weeks following drug administration, based on a single administration schedule (2 x 20mg/kg x

4 days), in rats.

Reductions in tryptophan hydroxylase (TPH) activity have been shown to be persistent

for a period of several weeks or months after binge MDMA administration (Stone et al., 1987).

12

In a study by Stone et al. (1987) TPH activity was assessed after a binge dosing schedule of

MDMA (5 or 10 mg/kg x 5 injections x 1 day). Assessments took place 18 hours, 30 days, or 110

days later. The 5 mg/kg condition showed significant reductions of TPH activity in the

hippocampus 30 days after binge dosing. The 10 mg/kg condition showed persistent reductions

in the striatum, hippocampus, and cortex at the 30 and 110 days after administration, and

reductions in the hypothalamus at the 30 day assessment only.

MDMA Toxicity

The persistent cellular and neurochemical effects that follow large or repeated doses of

MDMA are often referred to as evidence for neurotoxicity, but the connection between MDMA-

induced changes and neurotoxicity, as it is broadly defined, has rarely been considered carefully.

Baumann et al. (2007) reviewed the literature on persistent effects of MDMA administration, and

attempted to relate this literature to a definition of neurotoxicity which is typical in

neurodegenerative research. By this definition, evidence for neurotoxicity would include

neuronal death, glial cell reactivity which is typical of neurodegeneration, and silver staining of

cellular damage, rather than more indirect measures of structural change.

Silver stains fail to show widespread neuronal damage, even at doses well above those

which parallel recreational use (4 x 25-150 mg/kg) (Jensen et al., 1993). Jensen et al. (1993)

found dose dependent damage to axons in the frontal parietal cortex, and some staining in the

thalamus at higher doses, a pattern which is in contrast to the broad distribution of reductions in

extracellular serotonin described previously (e.g. Battaglia et al., 1987; Byrne, Baker & Poling,

2000; Ricaurte et al., 1993).

13

In addition, glial reactivity following MDMA use is not well correlated with serotonin

depletion, as evidenced by expression of glial fibrillary acidic protein (GFAP), OX-6, or

heat/shock protein (Baumann et al., 2007; Biezonski and Meyer, 2010). In a discussion of an

alternative explanation for persistent biological effects, Biezonski and Meyer (2010) note that

glial responses can occur at high doses, but again, these doses are too large to indicate risks

associated with recreational use. These authors also note that certain regions, such as the

hippocampus, cerebellum, and cortex, may be more sensitive to the potential toxicity of the drug,

as these regions are most likely to show this reactivity. An earlier analysis used these studies to

support the toxicity hypothesis, though the distribution of staining or glial reactivity was not

directly compared with 5-HT deficits in the discussion of those findings (Green et al, 2003)

Despite a general lack of evidence for neurodegeneration at behaviorally relevant doses,

the characterization of MDMA as potentially toxic to neurons has persisted. This fact is reflected

in a review by Gouzoulis-Mayfrank and Daumann (2006), in which the authors stated that region

dependent recovery may indicate different distances which regrowing axons must travel during

neuroplastic recovery. While profound cellular damage would certainly produce losses in a

number of biochemical assays, this is not the only explanation for these results.

In a recent paper, Biezonski and Meyer (2010) argue that these results could also be

explained by a down-regulation in protein expression. The authors conducted a study to assess

gene expression as relates to SERT and vesicular monoamine transporter 2 (VMAT2). VMAT2

is a membrane bound protein which is not generally considered in MDMA research, but would

be expected to be significantly reduced if structural membrane damage were to occur. As

expected, this study found significant reductions in SERT, but surprisingly found equal levels of

VMAT2 binding between control and drug conditions, suggesting an alternative cause for

14

protein reduction. In a second experiment, tissues in the dorsal and medial raphe nucleus were

tested for levels of transcripts for SERT and VMAT2 production following chronic MDMA

administration. These levels were found to be somewhat reduced for both proteins, but much

more so for SERT. This suggests that a down-regulation in gene expression occurs after chronic

doses of MDMA, and that this may contribute to lower levels of proteins such as SERT.

A more nuanced explanation for persistent cellular changes might be that MDMA

significantly reduces the expression of relevant proteins at doses too low to produce cell death or

major structural damage, and that at proper doses such damage is dose-dependent and region-

dependent. A more fitting definition of neurotoxicity, and the one which will be used from this

point on, is that MDMA-induced neurotoxicity consists of alterations to the neurochemical and

enzymatic activity of affected neurons, particularly in the serotonin system. Whether these

effects are selective or evidence for more general structural damage is up for debate.

Replications and further research will be necessary to fully explain the lasting serotonergic

deficits involved in persistent MDMA effects.

Loss and Recovery of Neurobiological Markers

The loss and recovery of cellular markers has been studied extensively in rats, and has

been shown to be highly dose- dependent and region-dependent (Green et al., 2003). Battaglia et

al. (1991) found that two weeks after MDMA administrations, several regions of the brain were

in very different stages of loss and recovery. At this point, processes in the dorsal caudate were

beginning to be expressed, persistent deficits were in place in the cortex, and recovery had begun

in the substantia nigra. Sabol et al. (1996) administered chronic doses of MDMA (2 x 20 mg/kg

x 4 days) and studied the recovery of serotonin levels and binding sites in several relevant brain

15

regions. Extracellular serotonin levels were lower in all brain regions tested except the septum,

and tests at a limited number of times during recovery showed evidence for region-dependent

recovery trends. Serotonin binding was also measured, and had largely recovered by eight weeks

after administration.

While studies in rodents tend to show recovery over the first weeks and months following

drug administration, there are studies with primates which show effects which appear to be much

more resistant to recovery (e.g. Hatzidimitriou, McCann & Ricaurte, 1999). Hatzidimitriou and

colleagues (1999) notably found persistent serotonergic dysfunction in squirrel monkeys seven

years after MDMA administration (5 mg/kg, twice daily x 4 days). Immunocytochemical

staining techniques were used and imaging techniques allowed serotonergic axon density to be

assessed. Persistent differences were found in the neocortex, hippocampus, striatum, amygdala,

hypothalamus, and thalamus, although substructures within these areas showed a broad range of

recovery or lack thereof. This study does suggest the possibility that certain effects could be

semi-permanent or at least last several years in primates, depending on the structure. It is

difficult to explain the these findings in the context of rodent data, but at minimum, they provide

some added concern for the potential for effects to last for extended periods of time following

MDMA administration.

Implications of Neurobiological Effects of MDMA

The literature investigating the neurobiology of MDMA has demonstrated diverse, but

short lived, acute effects, as well as a few lasting deficits, generally limited to the serotonin

system. While the molecular and cellular biology of MDMA-induced effects have drawn a great

deal of attention, interest in the drug extends much further. The functional significance of these

16

disturbances must be considered to evaluate the impact of the drug on recreational MDMA users,

as well as conceptualizing the usefulness of the drug as a tool for studying the neurobiology of

cognition. The field of behavioral pharmacology has considered both of these issues while

evaluating the drug and some evidence has accumulated showing that the drug and relevant

neurotransmitters are related to cognitive and executive functioning in humans and non-humans.

COGNITIVE IMPACTS OF MDMA IN HUMANS

Ecstasy, and MDMA by implication, has been most closely associated with alterations in

emotion and social behavior acutely and when used chronically with abnormalities in emotional

and behavioral regulation. Increases in risk for issues such as depression, anxiety, psychosis,

impulsivity and aggression have been reported in a number of studies (see review, Karlsen et al.,

2007). Cognitive testing with recreational ecstasy users has also implicated the drug in producing

abnormalities in learning and memory (Gouzoulis-Mayfrank & Daumann, 2006) and executive

functioning (e.g. Montgomery & Fisk, 2008; Murphy, Wareing, Fisk & Montgomery, 2009).

These findings have attracted the attention of many to the subtle cognitive effects of recreational

ecstasy use.

Some researchers have investigated the cognitive impact of the drug by studying

performance under the influence of the drug. This is both a scientifically interesting question and

one which applies to public safety. For instance, Downey and colleagues, (Downey et al., 2012),

administered methamphetamine and MDMA to abstinent recreational drug users in a double-

blind experimental study, then tested them in a standardized sobriety test. The tests used

included: eye tracking while gaze followed objects moving vertically and horizontally, a motor

coordination test consisting of walking heel to toe and turning, and the one-leg stand testing

balance. These tests are used to assess cognitive and motor capabilities and are thought to reflect

17

the readiness of the individual to engage in complex tasks like driving. It was found that nearly a

quarter of subjects failed the sobriety test four hours after a moderate dose (100mg) of MDMA

was given. These results do not indicate the source of impairments related to driving, but they

suggest that at early time points after use, MDMA can produce performance deficits which are

measurable on relatively simple motor and attention tasks. Other studies have found somewhat

more mixed results, indicating that complex tasks such as navigating high traffic areas and

predicting the movements of objects are impaired by moderate MDMA use (e.g. Lamers et al.,

2003, Stough et al., 2012), while other types of performance are enhanced (Lamers et al., 2003).

The tendency to task-dependently produce both cognitive enhancement and impairment has been

found with other drugs as well, and it is acknowledged by some that these may be characteristic

effects of amphetamine-like drugs (e.g. Harper, 2011).

Parrott and Lasky (1998) investigated the impacts of MDMA exposure over a more

extended period of time. This study compared heavy, light and non-MDMA users over four time

points around a night of partying. Drug use was recorded and cognitive tests were performed

before the party, during the party, two days after, and seven days after. Results showed a

reduction in performance on verbal recall and visual search tasks while under the influence of

MDMA, although controls also showed reductions in recall. These impairments showed full

recovery by the two day post-test. Drug use and memory performance was consistent with group

membership designations.

Cognitive disturbances have also been found in a number of studies studying cognition

in abstinent ecstasy users. Such effects have been found in a variety of cognitive tasks, such as

visuospatial recall and manipulation tasks (For review, see Murphy et al., 2012) or verbal

memory (e.g. Montgomery & Fisk, 2008). It has been noted that performance deficits are most

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likely to be found when tasks require particularly complex forms of cognition are required, like

those which require the manipulation of information (Murphy et al., 2009). It should be

mentioned however, that it has been difficult to isolate the effects of MDMA use from

confounding variables related to recreational use. Some researchers have attempted to control

these variables and subsequently failed to replicate previous drug effects on measures of

cognitive function (e.g. Halpern et al., 2011).

LIMITATIONS TO HUMAN TESTING AND NEED FOR ANIMAL MODELS

Human research on residual cognitive effects of MDMA has shown some interesting

effects, but certain limitations have consistently weakened the evidence. A number of

problematic factors in human drug research have been identified, and these have proven to be

difficult to control for experimentally (e.g. Gouzoulis-Mayfrank & Daumann, 2006; Jager et al.,

2008; Montgomery & Fisk, 2008). Perhaps the top concern related to drug effects is the fact that

those who use ecstasy recreationally tend to use other psychoactive substances as well. It is very

possible that common substances such as alcohol and marijuana contribute to the memory

deficits found in human cognitive studies. To compound the issue of polydrug use, pills

distributed as ecstasy may contain a number of compounds in a range of concentrations, so the

subjects in the study may be oblivious to their true drug history (Montgomery & Fisk, 2008). It

has also been noted that using non-drug users as controls may not control for factors which are

unique to drug users, but unrelated to the use of MDMA. In addition, assessing the connections

between drug use history on impairment could be impacted by social desirability or intentional

deception, as they could be with any self report study.

These limitations do not reduce the need to human pharmacological research, but they do

suggest that models should be developed which can be tested in a more controlled setting.

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Pharmacological models of serotonergic deficits using laboratory animals may help to explain

the cognitive impairments which are implied by the human literature, and control for or

manipulate the factors which may be at play in human populations. The neurochemical and

physiological effects of MDMA have been reproduced rather convincingly in rats, so developing

experimental models of cognitive processing and impairment is a logical next step in

understanding the functional significance of MDMA use. It has been a challenge to adequately

tie non-human memory tasks and models to their equivalents in the human literature, but much

progress has been made in developing behavioral tasks which incorporate relevant aspects of

human cognitive memory models.

MDMA EFFECTS ON MEMORY

Over the years, cognitive scientists have designed and tested models which aim to

identify and describe subtypes of memory and to investigate the storage and processing of

information through these subtypes. The earliest version of this model included stores for

sensory information, short term memory, and long term memory (Atkinson and Schiffrin, 1968),

and over the following decades, a number of modifications have been proposed and tested. The

first model to distinguish working memory from other types of short term memory was proposed

by Baddeley and Hitch (1974). At its most basic level, this model describes a form of memory

which simultaneously retains and manipulates information, relying on a number of distinct

functional systems (Baddeley, 1986).

While storage models have been widely used within human memory research, their

influence on rodent models of working memory have been somewhat limited. Distinguishing

working memory from other subtypes of memory has proven difficult in behavioral tasks,

20

leading many scientists in animal cognition to seek alternative definitions which can be more

easily applied non-humans (For review, see Dudchenko, 2004). One such alternative defines

working memory as “a short term memory for an object, stimulus, or location that is used within

a testing session, but not typically between sessions” (Dudchenko, 2004, pg.700). This definition

allows the label of working memory to be applied based on task requirements rather than the

function of internal processes, so while cognitive explanations will vary, relevant tasks can be

consistently classified behaviorally. All further discussion of working memory in rodents will be

based on this simple, yet useful, operational definition. This definition should allow for a closer

analysis of the potential relationship between MDMA and working memory.

MDMA and Working Memory Tasks in Rats

As the neurobiological effects of MDMA have been uncovered, a literature has formed

investigating MDMA-induced effects using working memory tasks in rodents. These tasks have

often been used to investigate the acute and residual effects of the drug on spatial working

memory. The effects of MDMA on common working memory tasks in rodents are reviewed

below, and include studies utilizing the radial arm maze (Braida, Pozzi, Cavallini, & Sala., 2002;

Hernandez-Rabaza et al., 2010; Kay et al., 2010, 2011), the Complex maze (Slikker et al., 1989,

the Morris swim task (Arias-Cavieres et al., 2010; Camarasa, Marimon, Rodrigo, Escubedo &

Pubill, 2008, Galizio, Byrd, Robinson, Hawkey, Rayburn-Reeves & April, In Review; Robinson,

Castaneda & Whishaw, 1993), repeated acquisition in the operant chamber (Galizio, McKinney.

Cerutti & Pitts, 2009), T-maze spatial alternation (Ricaurte et al., 1993), delayed match-to-

sample (Harper, Wisnewski, Hunt & Schenk, 2005; Harper, Hunt & Shenk, 2006), and delayed

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non-match to place (Marston et al., 1999).

Response Acquisition

Before considering working memory tasks, there is a procedural aspect of many studies

in rodents which should be considered, as it is likely to impact their results. Many researchers

have preferred not to pre-train their subjects on a task prior to administering drugs. This means

that drug effects on initial acquisition of an operant response would likely impact the results of

those studies in a way which is unique to this type of design. To investigate the impact of

MDMA on operant response acquisition, Byrne, Baker and Poling (2000) tested the effects of

acute and chronic administrations of MDMA on the acquisition of lever pressing to receive water

deliveries.

In experiment 1, Byrne et al. (1999) administered experimentally-naïve, water -deprived

rats MDMA (0, 1.0, 3.2 or 5.6 mg/kg) prior to an eight hour acquisition session, where the rats

had the opportunity to press a lever to receive a water reward. Rats were also placed in a delay

condition, where a delay of 0, 10 or 20 seconds was inserted between the response and

subsequent reinforcer delivery. The response rate was tracked over time for each of the rats and

was analyzed based on drug and delay condition. The results of this analysis showed that there

was a dose-dependent relationship between MDMA and the total number of lever presses. As the

dose of MDMA increased, the total number of lever presses increased. This appeared to be

mediated by delay, as the 0 second delay condition showed this effect, but 10 and 20 second

delay conditions did not. However, as the dose of MDMA increased, the time prior to responding

was much greater. In the 5.6 MDMA condition, responding did not typically begin until 100

minutes into the session. This is understood as evidence for a disruption in acquisition, in that

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MDMA-treated rats did not respond during the early part of the session. The source of this

disruption is unknown, but could clearly be related to a variety of sensory, cognitive, and

motivational factors.

In experiment 2, Byrne et al. (2000) investigated the potential for disruptions in

acquisition following chronic, potentially neurotoxic, administrations of MDMA. Neurochemical

analyses were later done to verify the toxicity of the dosing regimen used. Rats were

administered MDMA (20 mg/kg, 2 per day x 4 days) or saline, and were given a lever-press

acquisition session, as described in experiment 1. This acquisition session took place fourteen

days after the completion of the drug administrations. Response patterns during the acquisition

session were indistinguishable between MDMA and control rats for the fourteen subjects which

acquired the task (6 MDMA, 8 control). Two MDMA rats did not acquire the task, though the

reasons for this are uncertain.

This study generally showed that acute doses of MDMA can interrupt the acquisition of a

response, in the sense that subjects are much slower to begin exhibiting the response. Once

responding begins, they appear to be equivalent to controls. This may be related to one of a

number of effects of the drug, perhaps on attention, motivation or memory, but it is unclear

exactly how the drug affects initial learning. Based on this, it may be expected that acute doses

of MDMA will interrupt learning during short sessions. Experiment 2 showed that this effect

tends to disappear after the drug is no longer active, even after undergoing a neurotoxic drug

administration schedule. Based on this, the acquisition of a response is not strongly influenced by

the type of serotonergic disruptions which follow chronic schedules of MDMA administration.

At minimum, this type of task is not sensitive to the cognitive impairments which may be related

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to MDMA toxicity. Other tasks, such as working memory tasks, may be more sensitive to the

residual effects of the drug.

Radial Arm Maze

The eight-arm radial arm maze (RAM) is popularly considered a working memory task,

so much so that its developer, David Olton, defined working memory as memory which allows

rats to remember previously visited locations in the RAM (Dudchenko, 2004). The RAM

apparatus consists of a central platform with eight arms radiating out at equal angles, bordered by

walls to restrict movement within the maze (Braida et al., 2002; Dudchenko, 2004; Hernandez-

Rabaza et al., 2010; Kay et al., 2010; Kay et al., 2011). RAM sessions generally consist of

placing a rat in the center of the maze and allowing it to explore the arms, retrieving food

rewards which have been placed at the distal ends of the arms. The experimenter notes which

arms the rat explores and how many times each arm is explored. The RAM fits Dudchenko’s

definition of a working memory task because a typical session requires the subject to remember

which arms were previously visited within that session, independently of other sessions.

Braida et al. (2002) used the standard RAM to test the acute effects of MDMA on short

and long term working memory. In Experiment 1, subjects were allowed to visit one arm per trial

for eight trials per session. Experimenters took note of the total number of errors (times revisiting

an arm), the number of trials prior to the first error, and the percentage of subjects making more

than one error. Subjects received IP injections of MDMA (1.0, 2.0, or 3.0 mg/kg) 20 minutes

prior to testing. The two lowest doses (1.0, 2.0 mg/kg) did not produce measurable effects, but

the percentage of subjects making more than one error was significantly higher at the highest

dose (3.0 mg/kg) than in saline controls. In Experiment 2, a two hour delay was inserted between

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the first and second four trials, and access to four of the arms was restricted prior to the delay.

MDMA was administered as described above prior to the post-delay testing.

All drug doses increased the percentage of rats with more than one error compared to

controls and the 3.0mg/kg dose significantly increased the number of total errors in post-delay

testing. This study showed that a dose of 3.0 mg/kg MDMA increases errors on both short and

long term retention tasks, while lower doses selectively impair performance on the long term

retention task. This provides evidence for some differential sensitivity between short and long

term memory tasks.

Kay, Harper and Hunt (2010) were also interested in the acute effects of MDMA on

memory performance, but unlike Braida et al. (2002), Kay et al. was able to test MDMA-induced

impairments in reference and working memory simultaneously. The modified RAM contained

four baited and four unbaited arms, held in constant positions throughout all trials. All eight arms

were accessible at all times, but only four positions were baited. This task requires rats to

remember which arms were visited during a session, as well as which arms were baited in

previous training, generally defined as reference memory. Rats entered four arms on each trial

before being removed, with arms rebaited between trials. MDMA (0, .75, 3.0, 4.0 mg/kg) was

administered 20 minutes prior to training. MDMA produced dose dependent impairments on

overall accuracy and increases in working and reference memory, though reference memory

errors were significantly more frequent than working memory errors. While impairments were

found on the working memory task, Kay et al. argue that the reference memory task was more

sensitive to the effects of the drug. Furthermore, Kay et al. noted an increase in trial times,

suggesting other behavioral effects of MDMA which could potentially influence the subtypes of

memory differently.

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Both Braida et al. (2002) and Kay et al. (2010) found impairments on the RAM following

acute administration of MDMA. During the acute phase of drug administration, rats are more

likely to make working memory errors. While the authors may disagree on the significance of

this finding, both acknowledge a dose dependent effect on performance on the RAM under acute

drug conditions. Both authors also utilized a two component design which incorporated short and

long term memory tests. While the short/long-term memory and working/reference memory

distinctions are not perfectly aligned, the results of these two experiments map onto one another

quite closely. Results indicate that memory tests which incorporate substantial delays between

initial experience and current testing are more sensitive to the disruptions produced by acute

MDMA exposure than those with short delays.

Hernandez-Rabaza et al. (2010) performed a study to determine whether these acute

deficits translate to any residual effects on performance in the RAM. In addition, MDMA effects

were compared with deficits induced by alcohol and co-administered MDMA and alcohol.

Subjects received ten injections of MDMA (10 mg/kg), 20% ethanol (1.5 mg/kg), or both

(MDMA 10 mg/kg, 20% ethanol 1.5 mg/kg) six hours apart. Ten days after treatments, subjects

were allowed to explore the RAM on two days, then run for five days on a RAM procedure very

similar to that used by Kay et al. (2010).While some evidence for toxicity was found in all drug

groups, a significant increase in reference and working memory errors was found only when

ethanol and MDMA were administered together. This demonstrates that there are no measurable

deficits in the radial arm maze 12-16 days after exposure to MDMA, at least at the dose used in

this study.

In a follow-up to their 2010 study of acute effects, Kay, Harper & Hunt (2011) tested

residual memory effects over the approximately 70 days following a binge dosing schedule of

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MDMA (10 mg/kg x 4 doses in 8 hours). Acquisition of the RAM with four baited and four

unbaited arms (as in Kay et al., 2010) took place over the first 32 days following drug

administration, followed by an acute drug test and a reversal. Unlike in the acute study, Kay et

al. found that there were no lasting effects on working memory errors from the doses used, as

measured by revisiting arms in the maze. Results showed increases in reference memory errors,

as measured by visiting arms which were not baited in previous sessions. An increase in

reference memory errors without an increase in working memory errors suggests that the subject

may be using a simple strategy which does not involve memory but prevents re-entry of arms,

such as visiting the arms in a clockwise order. Despite comparable performance to controls on

working memory performance, drug group rats acquired the task and the reversal more slowly.

The reversal consisted of switching the locations of baited and unbaited arms, so that the animal

must adapt its arm visiting behavior to match the new arrangement. The slower rate of

acquisition for the reversal suggests cognitive inflexibility due to MDMA administration. The

acute effect test consisted of a single dose of MDMA (4.0 mg/kg) and a session of the RAM

presented above. Performance on this acute test was significantly higher than in control rats,

indicating tolerance effects 32 days after administration.

Studies using the radial arm maze (RAM) (Braida et al., 2002; Kay et al., 2010, 2011;

Hernandez-Rabaza et al. 2010) show different cognitive effects of MDMA during the acute drug

phase and the residual period. More specifically, they show that acute MDMA administration

significantly increases both working memory errors and reference memory errors. Chronic or

binge dosing schedules, on the other hand, have not produced measurable effects on working

memory errors, but have produced increases in reference memory errors. These studies

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emphasize the fact that subtypes of memory are differentially relevant to performance on this

task, based on small changes in experimental design.

Complex Maze

Aside from the standard RAM, other complex mazes have been used to assess drug

effects on memory. One such maze is generally referred to simply as the Complex Maze, and has

been repeatedly used by R.R. Holson and Colleagues (Holson et al., 1989) to study memory

under a variety of pharmacological and neurological states. Slikker et al. (1989) used this

procedure to assess memory deficits 2-4 weeks following repeated doses of MDMA (0, 5.0 or 10

mg/kg, 1 per day x 4 days). This maze consists of 24 connected arms with two designated arms

(a start arm and a baited finish arm) and 22 unbaited distracter arms. For each trial, the rat was

placed in the start arm and allowed to explore the maze until it retrieved the reinforcer in the

finish arm. The location of the finish arm remained the same from trial to trial and from day to

day. Slikker conducted 15 minute sessions for three consecutive days and subjects were given as

many trials as were completed within 15 minutes of testing. Dependent measures included

number of reinforcers retrieved, number of arms entered per trial, latency, etc.

This study failed to find significant effects on any of the dependent measures used for the

three drug conditions included. This is in spite of the fact that neurological testing found

significant reductions of 5-HT (5.0 mg/kg: frontal cortex, 10mg/kg: frontal cortex/hippocampus)

and 5-HIAA (10mg/kg: hippocampus). The failure to find significant effects was likely related to

a few notable shortcomings of the study. The dosing regimen selected was relatively low for a

study of chronic MDMA effects, so the reduction in serotonin may have been too small to be

behaviorally relevant. Slikker et al. (1989) point out that at lower levels of depletion, the brain

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may have mechanisms in place that prevented any effects from being measurable. It is worth

noting that testing began two to four weeks after administration, so substantial recovery may

have occurred by the time testing began. This study seems to suggest that the doses selected and

the timing of the testing period are important considerations in the design of such a study and

that the relative level of neurological alterations may be a more important consideration than the

statistical significance of the alteration.

Morris Swim Task

The Morris Swim Task (MST), also known as the Morris Water Maze, is based on a

somewhat different premise than other maze designs, but is generally considered to be a working

memory task (Dudchenko, 2004). Rodents are placed in a circular pool of opaque water which

has a platform concealed just below the surface of the liquid (Arias-Cavieres et al., 2010;

Camarasa et al., 2008; Galizio et al., In Review; Robinson et al., 1993). It is widely held that

swimming in a pool is aversive for rats, leading them to locate the platform and escape the pool.

Experimenters test how well the animals learn and remember the location of the platform

over successive trials. Visuospatial cues are used to guide the swim path to the platform.

Experimenters often record the latency of escape, distance traveled to the platform or the amount

of time spent swimming where an absent platform was previously. The MST fits the operational

definition of working memory (Dudchenko, 2004) when it follows a repeated acquisition or

match-to-sample design, randomizing the location of the platform for each session, but holding it

constant within sessions.

Arias-Cavieres et al. (2010) were interested in how the acute effects of repeated low

doses of MDMA might affect MST performance. To test this, researchers trained young rats on a

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MST procedure in which the location of the submerged platform never changed. Training took

place over six days, during which time they received injections of MDMA (0.2 or 2.0 mg/kg) or

saline twice per day. This low chronic dosing schedule was meant to prevent neurotoxic effects,

an aim confirmed by later biochemical testing. Results from training and platform-absent probes

revealed that the 2.0 mg/kg condition increased escape latencies overall as compared to saline

controls, while the 0.2 mg/kg condition only differed from controls late in the chronic dosing

schedule. This demonstrates a dose dependent and progressive effect of chronic low doses of

MDMA on learning in the MST. In addition, MDMA treated rats spent less time swimming in

the target quadrant than saline controls.

Galizio et al. (In Review) was interested in the effects of both acute and neurotoxic

MDMA administration on the MST. Both experiments utilized a modified version of the MST.

This modified procedure included two components: repeated acquisition and performance

(Thomspon & Moerschbaecher, 1979). A repeated acquisition design varies the learning

requirement from day to day so that learning takes place within session. In this case, the location

of the platform shifted from session to session. The performance component is based on across

session learning. In this case, the platform location does not change from session to session.

Including this second trial type provides a very unique form of performance control. These

control trials serve to measure effects which are unrelated to within-session learning, such as

sensory, motor and motivational drug effects (Galizio et al., 2009), but would impact reference

memory performance.

Experiment 1 produced within-subject dose-response curves for acute MDMA (0, 0.3,

1.0, 1.7, 3.0, 5.6 mg/kg) using this two-task design. Swimming speed in the MST was unaffected

by any dose of MDMA used on both the repeated acquisition and control tasks. Latency to the

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platform increased dose-dependently as MDMA dose increased. This impairment was

nonselective, as both tasks exhibited this effect equally at each dose given. There was a

significant effect for path ratio at the 3.0 dose but given the failure to find effects on swim speed

and the non-selective nature of the effect on latency, it is not believed that this demonstrates a

deficit which is relevant to learning processes. Acute MDMA appears to produce some altered

strategies or behavioral tendencies which could interfere with swim task performance in general,

like thigmotaxis.

Experiment 2 used a similar design to study the persistence of impairments produced by a

binge schedule of MDMA administration (20 mg/kg, twice per day x 4 days). Testing began

three days after the end of the binge administration. While drug treated animals did not differ

significantly from controls, their path ratios were elevated during the first few post-binge

sessions were elevated relative to baseline. This is a somewhat complicated finding as this

impairment was evident on the acquisition task but did not impact other measures such as escape

latency. The authors interpreted this as a weak impairment of working memory which was

measurable early in post-binge testing but disappeared quickly.

Camarasa et al. (2008) used a reference memory version of the MST to study the residual

effects of MDMA, as well as the neuroprotective effects of the NMDA antagonist memantine.

The effect of memantine is not relevant to this review, but the MDMA effects formed a baseline

for assessing the drug’s effectiveness. Rats were given a dose of saline or MDMA (1.0-15mg/kg)

twice per day for four days. One week following treatment, these rats completed eight MST

sessions over two days, learning the constant spatial location of a platform based on proximal

spatial cues. MDMA treated rats did not differ from saline controls with respect to escape

latency. They were then tested for learning by removing the platform and observing how long

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they swam in the quadrant where the platform had been previously. Rats in the MDMA

conditions spent significantly less time in the target quadrant in the test phase than saline or

memantine treatment conditions. This indicates a reduction in memory performance during the

test phase which was limited to the platform-absent task.

Robinson, Castaneda & Whishaw (1993) also used the MST to study the residual effects

of MDMA exposure, but unlike Camarasa (2008) and like Galizio et al (In Review), the location

of the platform changed from session to session. Therefore, the subjects were required to learn a

new platform location on each session. Robinson et al. administered MDMA (10mg/kg, twice

per day x 4 days) or an equal volume of saline and began testing on the MST on the third post-

binge day. Animals were given one eight trial session per day for three consecutive days.

Subjects in the MDMA group showed significantly higher latencies for the first trial within each

session, but performed normally on all other trials. This suggests that what impairments were

produced by binge MDMA exposure were related to the initial discovery of the platform rather

than retention of the location. While this is an interesting effect, it does not appear to be

consistent with the conclusion that within session learning is impaired. Robinson refers to this as

impairment in strategy selection, which could be a non-cognitive effect related to stereotypic

responses which are relevant to the drug.

In assessing the impact of acute and neurotoxic MDMA exposure, two distinct versions

of the MST have been used. The first is a reference memory design, in which the location of the

platform remains the same throughout testing. The second is a working memory design, in which

the platform location varied from session to session.

Arias-Cavieres et al. (2010) and Experiment 1 of Galzio et al (In Review) tested the acute

effects of MDMA on the MST. Arias-Cavieres et al. (2010) used a reference memory design and

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found that repeated low doses of MDMA produced an acute impairment of MST performance.

Galizio et al. (In Review) tested both reference and working memory designs and failed to find

acute selective impairments, meaning that impairments on the two tasks occurred at the same

doses. The authors argued that since the effects of the drug cannot be differentiated, then the

findings indicated a global impairment rather than specific cognitive effects. Based on these

studies, acute MDMA produces impairments on both reference and working memory designs,

but since these effects cannot be meaningfully differentiated from one another, a general

disruption of performance is the best explanation for existing data on acute MDMA exposure in

the MST.

Experiment 2 of Galizio et al. (In Review), Camarasa et al. (2008), and Robinson et al.

(1993) tested the effects of neurotoxic MDMA exposure on the MST. Camarasa et al (2008) used

a reference memory design and found that repeated doses of MDMA (ranging from 1-15 mg/kg)

produced a deficit in performance on the platform-absent test. This effect appears to be

somewhat selective, as performance on the platform-present test was not impaired. Robinson et

al (1993) used a working memory design to test the effects of neurotoxic MDMA exposure and

found longer escape latencies on the first trial, but not elsewhere in the session. This indicates

that neurotoxic MDMA exposure disrupts the initial search for the platform, but not performance

which requires memory for the platform location. This is interpreted as a non-mnemonic effect

of the drug. Experiment 2 of Galizio et al. (In Review) found a weak selective impairment that

appeared to recover quickly. These studies suggest that working memory deficits following

neurotoxic exposure to MDMA are weak or difficult to detect. Galizio et al. (In Review) note that

their findings and those of Robinson et al (1993) are not as dramatic or persistent as the deficits

noted by some other studies (e.g. Voorhees, Reed, Skelton & Williams, 2004) but suggests that

33

this may be due to the fact that Galizio and Robinson pre-trained their animals on the MST

before drugs were given. Pre-training increases the precision of the measures used, preventing

deficits related to initial acquisition from being measured and obscuring the nature of other

effects of interest.

T-Maze Spatial Alternation

The spatial alternation T-Maze (Ricaurte et al., 1993) is an established procedure for

testing working memory. The maze is a single straight pathway that leads to two arms leading

out from it to create a simple T-shaped maze. The subject is introduced to the maze and explores

one of the arms to retrieve a reinforcer. In the alternation T-Maze, the location of the reinforcer

switches for every trial, so the optimal behavioral strategy is to visit the arm not visited on the

previous trial, if it was reinforced. This requires the subject to remember which arm was visited

on the previous trial. The subject remains in a holding chamber prior to entering the maze, so the

ITI can be manipulated to measure the effect of delay on accuracy in the task. This task fits the

operational definition of working memory (Dudchenko, 2004) as presented in Ricaurte et al.

(1993), because accurate responding is based solely on within-session remembering.

Ricaurte et al. (1993) used the spatial alternation T-Maze to test the residual effects of

chronic MDMA use. Rats received two chronic schedules of MDMA administration (20 mg/kg,

twice a day x 4 days) with a one week break in between. Four weeks after the second chronic

administration was completed (beginning post-treatment week 7), shaping in the T-maze began,

and continued for three weeks. After shaping, each subject was tested on a basic T-Maze design

for four weeks, then underwent testing with various delays (5, 30, 60, 120, or 180 seconds)

inserted. Though evidence for persistent toxicity was found as late as five months after the

34

second administration, MDMA rats did not differ from saline controls on accuracy in the basic or

delay phases of the experiment.

The results of this study emphasize the fact that significant reductions in serotonin are not

evidence for impairment. A near complete depletion produced by 5/7-DHT/DMI produced

significant impairment on the T-maze, but the selected MDMA administration schedule did not.

While the connection between serotonin functioning and T-maze performance is supported by

this result, it is very unclear with this design what type of impairment is produced by dramatic

reductions in extracellular serotonin. Ricaurte (1993) did not include sufficient controls to claim

that working memory was impaired as opposed to more global functions. Generally speaking

though, these results suggest that binge administration of MDMA does not produce impairment

of T-maze alternation while identical dosing regimens can be shown to produce impairments in

within session learning in more complex mazes (e.g Kay et al., 2011). As suggested by human

research, relatively simple tasks may be relatively insensitive to disruption by behaviorally

relevant doses of MDMA.

Repeated Acquisition and Performance in the Operant Chamber

As noted previously, a repeated acquisition and performance (RAP) design can be used to

test within-session learning while using a performance control to measure non-mnemonic

impairments which might be present. This design was used by Galizio et al. (In Review) in the

Morris Swim Task, but could also be adapted to the operant chamber. In the operant chamber,

working memory tasks can use positive reinforcement rather than escape contingencies, as in the

MST, and can assess learning impairment based on choice accuracy rather than latency of a

35

correct response. It also requires less movement than mazes and should be less sensitive to

interference by certain stereotypic behavioral patterns produced by the drug (e.g. thigmotaxis).

Galizio et al. (2009) used a RAP procedure to test acute MA, MPD, and MDMA-induced

working memory deficits in the operant chamber. The chamber contained touchscreen panels

which recorded nose pokes. During a session, these panels displayed colored shapes to indicate

whether each trial was acquisition or performance. During acquisition trials, nosepokes to one of

the six panels were reinforced. This position was randomized for each session, but remained

constant during the session. On performance trials, responses to one panel were reinforced, and

this position remained constant throughout training.

Prior to drug phase sessions, the subjects received MDMA (0.3–10 mg/kg), MA (0.1–3

mg/kg) or MPD (1–17 mg/kg). Each of the three drugs produced dose-dependent impairments in

acquisition, but none of the doses of MA or MPD met the criteria for working memory

impairment. At the 3 mg/kg dose of MDMA, there was a significant loss of accuracy on

acquisition without a loss in accuracy on performance, suggesting a selective effect of MDMA

on the repeated acquisition or working memory component.

It is interesting that this study was able to find a dose of MDMA which impacts working

memory, given the strict criteria required by the experimenters. Given the findings of Kay et al.

(2010), long term or reference memory should have been more sensitive to the effects of MDMA

than working memory and thus the performance task should have been impacted. The failure to

replicate this finding in the operant chamber may indicate that other design considerations

beyond working and reference memory classifications are relevant in determining impairment on

these tasks.

36

The tasks utilized by Kay et al. (2010) and Galizio et al. (2009) are generally similar but

they differ in a few ways that may address their inconsistent findings. First, the RAM requires

the exploration of an environment rather than selection from a fixed array. The RAM should be a

more difficult spatial discrimination than the operant chamber task because navigational cues are

outside of the maze, the locations of the cues shift as the animal moves around, and all possible

selections cannot be viewed simultaneously. In the operant chamber, all panels are clustered

together, the cues that separate them are relatively fixed, and all stimuli are visible prior to

selection. Second, the RAM requires multiple responses per trial while separate trials were

arranged in the operant chamber. Third, working and reference memory components are

presented simultaneously in the RAM and on a multiple schedule in the operant chamber. Taken

together, these three factors may suggest why the reference memory tasks chosen differ in

sensitivity to MDMA-induced-disruption. They suggest that the RAM presents a more complex

set of discriminations than is present in the operant chamber design. Particular attention is drawn

to the third consideration, as it is not known whether reference memory impairments in the RAM

were due solely to the reference memory requirement or to the combination of requirements.

Delayed Match/Non-Match-to-Place

Delayed Match-to-Place (DMTP) is a working memory procedure which can be

performed in a number of ways, but is generally used for non-humans in an operant chamber

(Harper et al., 2005; Harper et al., 2006). In this procedure, a trial consists of a few basic parts. A

sample lever is inserted into the chamber and the subject makes an observing response by

pressing it. The sample is then removed and a delay period begins. After the delay, at least two

levers are inserted, only one of which is in the same spatial location as the sample was.

37

Responses to the comparison lever which matches the sample result in reinforcer delivery.

Delayed Non-Match-to-Place (NMTP) is a working memory task which adapts the DMTS

procedure to measure the ability to remember the location of a previously presented stimulus and

to respond to a stimulus in a different spatial location (Marston et al., 1999). This basic

procedure meets the operational definition of working memory (Dudchenko, 2004), as accuracy

depends on remembering stimuli from within a trial, but not across trials or sessions.

Harper and his colleagues have used a DMTP procedure in two related studies of acute

MDMA-induced deficits in working memory (Harper et al., 2005; Harper et al., 2006). In 2005,

Harper et al. assessed acute impairments in working memory following the administration of a

range of doses for MDMA (0-1.0 mg/kg), d-Amphetamine (0-20.0 mg/kg), and cocaine (0-3.0

mg/kg). One of four delays (1, 3, 9, or 18 sec) was inserted between sample lever presentation

and the presentation of two comparison levers. These three drugs produced a delay-independent

reduction in accuracy on the DMTS task. This means that the reduction in accuracy was equal in

all delay conditions. Further analysis revealed that the response made in the previous trial played

a significant role in the accuracy of responding for the drug conditions. More specifically,

subjects were more likely to press the correct comparison lever when this response was the same

as in the previous trial than if it was different on the previous trial. The influence of a previous

trial on current memory performance is referred to as proactive interference. This interference

between trials is greater for moderate and large doses of these three drugs than it is under saline

or low dose conditions. This also appeared to interact with delay, such that previous responses

were more influential at higher delays. The interference account of DMTS disruption is that the

drugs reduce the discriminability of responses on successive trials, an account that was addressed

in a follow-up study.

38

Harper et al. (2006) replicated these findings with MDMA only, and showed that by

increasing the inter-trial interval, the disruptive effect of MDMA on DMTP was significantly

attenuated. This suggests that, at least for DMTP, disruptions produced by acute MDMA are

related to interaction between trials that are close together in time. Furthermore, this impairment

can be resolved under the same pharmacological conditions, using only procedural

modifications. This does not fit perfectly with the delay-independent reductions in accuracy

which were found in the 2005 study, so some questions remain about the sources of impairment

in low delay conditions which are less sensitive to proactive interference. The type of

interference described by Harper et al. is unique to the working memory tasks with separate trials

where delay is manipulated in this way. Other designs, such as non-match to sample tasks, are

also likely subject to these types of effects.

Marston et al. (1999) used a DNMTP task to test the acute and residual effects of a

chronic schedule of MDMA administration. Stimuli consisted of two levers located on one wall

of the operant chamber. After training the rats to performance criterion on the DNMTP, subjects

were given doses of MDMA twice per day over a span of three days (10 mg/kg x 2 doses, 15

mg/kg x 2 doses, 20 mg/kg x 2 doses) or saline, and continued testing for the next sixteen days.

Subjects were tested in the DNMTP each day just after the first dose and just before the second

dose. One of a range of delays (.3-30 sec) was inserted before the presentation of comparisons.

Marston et al. (1999) found disrupted performance on the DNMTP task in the acute

phase, immediately following morning administration. Accuracy on the DNMTP and the number

of trials completed were reduced during acute testing and appeared to improve over the three

acute testing sessions. This effect was accompanied by major disruptions of behavioral and

motor functions. By the afternoon testing session, performance on all measures had returned to

39

baseline levels. Following the end of drug administrations, the MDMA group did not differ from

saline controls in accuracy on the DNMTP task. This suggests that chronic MDMA exposure did

not reduce the accuracy of working memory performance relative to controls. However, the

MDMA group failed to improve in accuracy in long delay conditions over the following weeks,

and was significantly different from controls 12 days after treatment. By that point, the MDMA

group also showed significant biases in responding at high delays. Generally, as accuracy on a

task improves, bias scores decrease, reflecting that response patterns approach the distribution of

the correct choices. The failure to reduce the bias score over time may reflect one of a few

potential effects of the drug. Resistance to altering existing strategies is referred to as cognitive

inflexibility and it has been suggested by Kay et al. (2011) that such inflexibility results from

binge exposure to MDMA. Alternatively, weak but systematic variations in the response patterns

of drug-treated rats could produce consistent error rates in the face of added practice. Examples

of such variations would include proactive interference and lever preference.

It is difficult to say if the acute effects found in this study are primarily cognitive in

nature, given the reduction in responding and the disruption of motor and other behavioral

measures. Most forms of disruption disappear within a matter of hours, and by afternoon,

performance has returned to baseline.

After the end of the chronic treatment, no significant differences in performance existed

between drug and saline treated animals. However, with added practice, residual differences

between the groups emerged. While the saline group improved accuracy on DNMTP in long

delay conditions (20-30 sec), the MDMA treated group failed to improve within the time frame

tested. Biased patterns of responding persisted with added practice. The observed differences

were not initially present so it is not fair to conclude that working memory performance was

40

impaired by the binge drug administration. Differences emerged over time, reflecting a

difference in sensitivity to practice effects at long delays in the DNMTP.

An Alternative Approach to Working Memory Testing

The rodent paradigms reviewed above have taken a variety of approaches to memory

testing and substantially added to current knowledge of the effects of MDMA on behavior.

However, despite the innovative use of these tools, the literature on working memory tasks in

rodents is somewhat lacking in its ability to address the varied concerns which are investigated in

human studies of working memory performance. Each of these paradigms assesses the accuracy

of memory for a single item or location over a length of time. Duration is a key variable in

human and non-human memory alike, but human research also addresses how the number of

items to be remembered (memory load or capacity) impacts memory accuracy. The capacity of

working memory is addressed in tasks where multiple items must be remembered for a short

time and quite often these tasks require the stored information to be updated or manipulated in

some way. In a review, Nulsen, Fox and Hammond (2010) noted that these tasks tend to be

sensitive to the impairments experienced by recreational ecstasy users. A related position is that

MDMA impairs tasks that are particularly demanding to executive functioning (e.g. Murphy et

al., 2009). Given these conclusions, it is problematic that capacity-based deficits have been

overlooked in cognitive testing of rodent models of MDMA use. Relatively recently, rodent

analogues to the popular human span paradigms have emerged which manipulate memory load

in rodents, filling an important need within the field. As a baseline for pharmacological

procedures, these tasks offer a solution to the current disconnect between approaches to working

memory testing in humans and rodents.

41

Span

In the cognitive literature, it is understood that humans, and presumably non-humans, are

limited in the amount of information which can be retained and processed at any one time. The

capacity of human working memory has generally been characterized as seven items of

information, plus or minus two items (Miller, 1956), although it has been argued that the true

capacity may be lower (e.g. Cowan, Chen & Rouder, 2004).Without rehearsal, this information

is only retained for a brief time. This limited capacity for working memory is understood

primarily by studying span. Span tasks in humans consist of a list of items which must be used

after a retention period, generally by verbally repeating the presented items. In humans, the

number of items which can be correctly retained and repeated or written is taken as a measure of

working memory span. The reliance on numerical or verbal skills in human tasks offers a

conceptual problem for non-human researchers, but a clever task design for rodents has been

developed.

The first published studies using rodent span tasks appeared in 2000, introduced by

Dudchenko, Wood & Eichenbaum (2000) and by Turchi and Sarter (2000). These studies used a

novel paradigm for rodent working memory, described elsewhere as an incrementing non-match

to sample (INMS) procedure (MacQueen, Bullard & Galizio, 2011). The INMS procedure adapts

the non-match to sample task to allow a list of items to be non-matched against, as opposed to a

single sample item (See Figure 1, from Dudchenko et al., 2000).

On the first trial, a single stimulus is presented and a response to this stimulus is

reinforced. On the second trial, the stimulus from trial one is presented, as is a novel stimulus.

Only responses to the novel stimulus are reinforced. On the third trial, the two previously

42

presented stimuli are presented, as is a novel stimulus. Again, only responses to the stimulus not

presented on the previous trials is reinforced. This progression continues until the end of the

session, presenting the previously encountered stimuli with a novel stimulus on each additional

trial. This design requires the animal to recall which scents have been presented previously in

order to identify and respond to the novel stimulus. Since the number of items to remember

increments throughout the session, the experimenter can measure within-session changes in

accuracy as a function of memory load. This basic design forms the basis for olfactory span

designs ( Dudchenko et al., 2000; MacQueen et al, 2011; Rushforth et al., 2010; Young, Sharkey

& Finlayson, 2009; Young, Crawford, Kelly, Kerr, Marston, Spratt, Finlayson, and Sharkey,

2007; Young, Kerr, Kelly, Marston, Spratt, Finlayson, and Sharkey, 2007).

43

Figure 1. Basic rodent incrementing non-match-to-sample or span procedure.

Source Dudchenko et al. (2000).

44

Odor Span

The basic span procedure can be adapted to use olfactory stimuli fairly simply

(Dudchenko et al., 2000). This can be accomplished by presenting cups which contain sand

mixed with distinct scents ( Dudchenko et al., 2000, Rushforth et al., 2010; Young, Sharkey &

Finlayson, 2009; Young, Crawford et al., 2007; Young, Kerr et al., 2007) or by using scented

lids to mark individual cups (MacQueen, Bullard & Galizio, 2011). On each trial, the subject

must identify and respond to the stimulus which has not been presented on any previous trial. In

order to respond to the correct stimulus and receive a reinforcer, the subject must recall which

odors were presented on previous trials. Like the previously described working memory tasks,

this relies on memory for items presented in previous trials, with the addition of repeated

updating of the memory set throughout the session. This factor is unique to span tasks, and

allows memory effects to be measured which cannot currently be measured otherwise. The Odor

Span Task (OST) parallels key characteristics of human working memory models (e.g. Baddeley

& Hitch, 1974; Nulsen et al., 2010). The span task requires the subject to respond to a stimulus and to

inhibit responding to that stimulus on every subsequent presentation of that stimulus. However, all of the

scents used in the session must be designated as novel on beginning the next session. These two aspects

of the odor span task appear to require an updating of the appropriate response cued by a familiar stimulus

based on contextual factors and reinforcement history. To the extent that this memory task is demanding,

as described by Murphy et al. (2009), it may be expected that this task would be relatively sensitive to

disruption by MDMA exposure.

The current study addressed the impact of MDMA on performance in the OST under two

different administration conditions. Experiment 1 tested OST performance under a range of acute doses of

MDMA to generate a dose-response function for the task. Experiment 2 tested OST performance

following exposure to repeated doses of MDMA which are sufficient to produce lasting neurotoxicity.

45

EXPERIMENT 1: ACUTE EFFECTS OF MDMA

A variety of procedures and designs are available for studying cognitive functions which

are relevant to learning and memory. It seems likely that procedural differences could determine

the sensitivity of a measure for MDMA-induced effects, and a number of possible design

features may play a role. The acute studies above and their relevant features are listed in Table 1.

It displays the pharmacological procedure used, the paradigm and design (working, reference or

both) used, the presence of pre-training on a task prior to drug administration, and the result

found. The designs and measures have each been explained in detail previously. Importantly,

certain authors found measurable impairment following administrations of MDMA, while others

did not, which implies that not all designs are equally likely to be sensitive to the effects

produced by MDMA.

46

Authors Dose(s) Paradigm WM/RM Pretrained Results Found

Braida et al.

2002

0, 1.0,

2.0, 3.0

RAM WM short

vs

long delay

No WM errors- elevated at 3.0

WM post-long delay-

elevated at all doses.

Galizio et al.

2009

0, 0.3–

10

RAP- Op

chamber

Both Yes WM- Signif. effect on

accuracy at 3.0

RM- No signif. effects at 3.0

WM/RM- Equal dose-

dependent effects on

other measures

Galizio et al. In

Review Ex 1

0, 0.3-

5.6

MST Both Yes WM/RM- Equal dose

dependent effects on

all measures

Path ratio signif. effect at

3.0

Harper et al.

2005

0-1.0 DMTS WM Yes -Delay independent effects

on accuracy

-Previous trial influential

Harper et al

2006

0-1.0 DMTS WM Yes -Replication

-Longer ITI eliminated

previous-trial effect

Kay et al. 2010 0, 0.75,

3.0, 4.0

RAM Both Yes WM errors- Signif. elevated

RM errors- Signif. elevated,

>WM impairment

Marston et al.

1999

10, 15,

20

DNMTP WM Yes -Morning- all measures

impaired at all doses

-Afternoon- all measures

equal to baseline

Table 1. Acute MDMA studies of memory in rats. Note: WM= Working Memory, RM=

Reference Memory

47

It has generally been shown that MDMA acutely disrupts performance on memory tasks

but at present, the ability to characterize those impairments is not well developed. Braida et al.

(2002) and Kay et al. (2011) both found impairments of performance in both short and long term

memory versions of the RAM. The generality of the effect in both cases makes it difficult to

argue that cognitive effects are specific to short term or working memory. In addition to the

general loss in performance noted by Braida (2002) and Kay et al (2010), evidence for specific

disruptions has been described by Galizio et al (In Review) in the MST and Harper et al. (2005,

2006) in DMTS. Galizio and Harper make a strong case for impairments of non-working

memory functioning, producing disruptive behavioral tendencies and proactive interference

respectively. Marston (1999) showed no initial impairment relative to controls on DNMTP but

did find that drug-treated rats failed to improve long delay performance with added practice.

Kay et al. (2010) and Braida et al. (2002) argue that when the duration of memory was

extended in time, the sensitivity to disruption by acute MDMA was amplified. Of the three time

frames included, the short term working memory was the least sensitive to disruption. The

overlapping amounts of impairment on the three tasks suggest that effects on a continuous or

short-delay working memory measure may be the result of a general or mutual source of

disruption by MDMA rather than something specific to such requirements.

The unique finding in this analysis is from Galizio et al. (2009), who found effects on

working memory performance which were separable from more general disruptions in

performance. Rats could obtain reinforcers by nosepoking a particular panel on one wall of the

operant chamber. The location of this panel varied based on trial type. The procedure included

repeated acquisition and control trials, and so was conceptually similar to the one used by

Galizio (In Review) in the MST. Surprisingly, the dose (3.0 mg/kg) which produced selective

48

effects in the operant chamber produced only nonselective impairments in the swim task. This

particular procedure does have some distinct advantages given the types of impairments outlined

by other studies. Incompatible behaviors produced by the drug in an open field or maze are

unlikely to interfere with performance in the operant chamber as the motor requirements are

relatively low. In addition, trials consisting of single presentations of stimuli rather than sample-

comparison pairs provided highly discriminable trials and likely reduced interference between

responses on previous and current trials, as described by Harper (2005, 2006). This study

demonstrates that selective effects of MDMA on working memory can be shown under proper

conditions but that considerable experimental control is necessary.

One strength of the available studies on the acute effects of MDMA on rodent memory

tasks is the use of multiple measures and doses to allow the selectivity of memory deficits to be

assessed to some degree (Braida et al., 2002, Harper et al., 2005, Harper et al. 2006, Galizio et

al., 2009, Galizio et al., In Review, Kay et al., 2010, Marston et al., 1999). Of these, the RAP

designs used by Galizio et al. (In Review, 2009) utilize the strictest controls. These studies

included both within- and across-session learning tasks independently on a multiple schedule.

These measures are identical to one another except for the memory requirement, so differences

in the effects of the drug are more convincingly attributable to deficits in working memory as

opposed to more general cognitive or behavioral alterations.

Currently, all available studies of the acute effects of MDMA on working memory in rodents

pertain to the duration of memory. Prior to the current study, nothing was known about capacity-based

memory performance in rats under the influence of acute MDMA. Experiment 1 tested OST performance

under a range of acute doses of MDMA to generate dose-response functions for measures on the OST.

Method

49

Subjects

Subjects in this study consisted of six adult male Holtzman Sprague–Dawley albino rats.

All rats were individually housed on a 12 hour light–dark cycle. Subjects were given free access

to water and food was limited such that animals maintain stable body weights of approximately

85% of their free-feeding body weight.

Apparatus

Olfactory span training and testing took place in a circular open-field apparatus. This

span arena consists of a circular table 29.2 cm above the floor and 94 cm in diameter, bordered

by a 32 cm high wall of sheet metal baffling. The surface of the table contains eighteen holes, 5.5

cm in diameter, positioned in two concentric circles. Twelve evenly spaced holes form the outer

ring, 2.5 cm from the arena wall. Six evenly spaced holes form the inner ring, 21.5 cm from the

arena wall (Fig. 1). Plastic cups (2 oz.) are placed in each hole during session trials. Speakers

adjacent to the span arena will provide white noise during all sessions. A web cam (Logitech,

Inc.) was used to digitally record each session.

Stimuli

All stimuli consisted of plastic cups (2 oz) half filled with fine grained, white, play sand

covered by scented plastic lids. The sand served to weight the cups into the holes in the arena

surface, preventing them from being displaced during responses. Plastic lids were scented by

storing them in airtight plastic containers containing household spices and flavorings (see Table

2- list of scents). These scented lids were placed lightly on the stimulus cups for each trial and

were exchanged for unused lids prior to each presentation of a given scent. This ensured that lids

50

were easily removable and that any scent traces from previous responses did not influence

responses. Prior to drug or saline administrations, all subjects progressed through a shaping

procedure and four training phases.

51

Span Scents SD Scents Alternates Bay Mustard Almond Allspice

Caraway Nutmeg Banana Anise

Celery Onion Bubble Gum Carob

Cinnamon Oregano Cherry Ginger

Clove Paprika Grape Spinach

Coriander Rosemary Peach

Cumin Sage Pineapple

Dill Savory Strawberry

Fennel Sumac Vanilla

Fenugreek Turmeric

Garlic Thyme

Marjoram Worcestershire

Table 2. Pool of scents for Odor Span and SD tasks.

52

General Procedure and Measures

All sessions consisted of 24-30 experimental trials separated by an ITI of approximately

one minute. During the ITI, subjects were kept in a temporary holding cage and the experimenter

placed stimuli in the arena for the next trial. Each trial began when the subject was placed in the

arena and was completed when a correct response is made and the pellet consumed or after 2 min

without a response A response was defined as the removal of a lid from a stimulus cup using the

paws or snout of the rat. In trials using scented lids, a correction procedure was used, such that

the trial continued until the correct lid is removed, and all errors prior to this were recorded. If

two minutes elapsed without a response, an omission was recorded and the rat was given a single

trial where only the S+ of an omitted trial was present in the arena. This gave the animal two

minutes to respond to the S+ without comparisons present. If the animal failed to respond within

two minutes again, the experimenter manually placed the animal at the S+ and allowed it to

sample the S+ odor. If the two minute trial termination criterion was met six times within the

session, the session was ended prematurely and all remaining trials were scored as omissions.

A set of data and notes were gathered by the experimenter throughout each session. The

accuracy (correct/incorrect) of the first response made during a trial and the latency of all

responses were recorded, whether correct or incorrect. The odor identity of scented lids which

were removed in error were also recorded, to allow the experimenter to identify and eliminate

scents which are chosen incorrectly at higher frequency than other scents. It is sometimes the

case in this procedure that a given rat may consistently refuse to respond to stimuli of a certain

scent or select it in error with relatively high frequency. These types of errors are considered to

be non-mnemonic and can be avoided by removing those scents from the pool of scents.

53

Across a session, accuracy, span, longest run, latency and omissions are the primary

measures. The accuracy of the first response in each trial was noted and compiled into an overall

accuracy measure (e.g. 18 correct first-responses/24 total trials = 75% Accuracy). As noted

previously, span refers to the number of trials completed before the first incorrect response.

Results from a recent unpublished study from our lab have suggested that a useful alternative to

the span measure might be longest run, identified as the longest series of correct responses within

a session. Latency was measured as the time prior to the first response within each trial.

Omissions occurred when two minutes elapsed within a trial without a response. These errors

were excluded from accuracy calculations and are used to generate a percent omission score,

reflecting the percentage of trials which are omitted.

Shaping

In pre-training, subjects were habituated to the arena and allowed to obtain 45 mg sucrose

pellets from stimulus cups until sugar pellets are consistently retrieved. Subjects were then

shaped to remove unscented lids from the stimulus cups. Shaping trials consisted of a single

stimulus cup placed in a pseudo-random location in the arena, with a lid partially covering the

opening of the cup. On successive shaping trials, the lid was positioned to more fully cover the

opening of the cup until subjects consistently remove lids from fully covered cups. Shaping was

completed when lids are removed from fully covered cups in least 23 of the 24 trials in a session.

Span training

Phase 1

54

In Phase 1 of span training, subjects removed scented lids from incrementing number of

cups (see Figure 1). In the first trial, a single stimulus (A+) cup baited with a 45 mg sucrose

pellet was placed in the arena. As with all stimuli used in this procedure, the location and odor

identity of each stimulus was assigned pseudo-randomly. The subject was then placed in the

arena and allowed to locate the stimulus cup, remove the lid, and obtain the sucrose pellet.

During the ITI, the stimulus from the previous trial was removed and two stimuli were placed in

randomized locations for the second trial. One of these stimuli was covered by a lid of the same

scent used on the previous trial (A-), while the other was covered by a novel scented lid (B+). As

this is a non-match procedure, a sucrose pellet was placed in the cup with the novel scented lid

only. A response to the novel stimulus allowed access to a sucrose pellet.

On the third trial, there was a novel scent stimulus (C+) and two stimuli with previously

presented scents (A-, B-) in the arena. A response to the novel scent stimulus was reinforced,

while responses to the two old scent stimuli were not. For all following trials, a response to the

novel stimulus was reinforced, and the number of previously reinforced scents continued to

increment by one from the last trial. Since there are twenty-four trials with novel scents and only

eighteen locations to place stimuli, all trials with more than seventeen old scents present in the

arena will use seventeen scents randomly selected from those presented previously.

The total number of stimuli in the arena incremented by one on each trial, as long as the

subject continued to respond to the novel scented stimulus first. When the first response was to a

stimulus of a previously presented scent, the trial continued until the correct response is made.

However, the following trial removes all scents used in previous trials, resetting the incrementing

non-match to sample to a single novel scent stimulus, as in trial one. Resetting the incrementing

scent set also resets the number of stimuli to remember to zero. The length of these incrementing

55

spans can be used to measure how well the incrementing non-match to sample relations were

learned.

Subjects must demonstrate a level of proficiency prior to advancing to Phase 2. The

criterion for advancement was one session with a longest run of ten or two consecutive sessions

with longest runs of five. Subjects typically completed Phase 1 in ~8 sessions (see Table 3).

56

Phase Criterion for advancement Number of sessions in phase

1 Longest run of 10 or 2 consecutive sessions with

longest run of 5

7.75 (range 6-10)

2 2 consecutive sessions with 70.83% accuracy or better 5 (range 2-8)

3 One session with >70.83% on OST and 100% on SD 7.33 (range 5-12)

4 10 day stability: days {(mean of sessions 1-5) - (mean

of sessions 6-10) < .15 x (mean sessions 1-10)

16 (range 10-35)

Drug 36.6 (range 22-48)

Table 3. Training timeline. Advancement criteria and average number of sessions

required to advance to drug phase testing.

57

Phase 2

In Phase 2 of span training, sessions used a five-comparison olfactory span design.

Unlike Phase 1, the number of stimuli in the arena incremented in the first five trials of the

session, then was held constant at five for all remaining trials. For trials five through twenty-four,

one novel stimulus was presented as well as four scent stimuli randomly selected from those

which have already been presented. As in phase one, only a response to the novel scent stimulus

allowed access to a sucrose pellet. Holding the number of stimuli in the arena constant is a

procedural control which eliminates a potentially problematic confound. In a typical span design,

as the number of previously presented scents increases, the size of the array the novel stimulus is

presented in increases as well. Using this method, the experimenter can ensure that losses in

accuracy in later trials are due to increased memory load rather than the average number of non-

match choices to make per trial. The criterion for advancement to Phase 3 was two consecutive

sessions with overall accuracy scores of 17/24 or better (70.83%). Subjects typically completed

phase 2 in 5 sessions (see Table 3).

Phase 3

In Phase 3 of span training, sessions consisted of a five-comparison olfactory span task,

as described in phase 2, followed by six simple discrimination (SD) control trials. Simple

discriminations used five scents which were not included in the pool for span trials, one of which

was designated as S+ and four are designated as S-. Responses to S+ were always reinforced and

responses to S- were never reinforced. These SD trials required the same level of motor function,

olfactory perception, and motivation to complete as span trials, but are not influenced by

working memory function, and so are sensible controls to measure for potential non-mnemonic

58

effects of a drug, such as MDMA. The criterion for advancement to Phase 4 was one session

with overall accuracy of 17/24 (70.83%) or better on span trials and correct responses on all six

(100%) SD control trials. Subjects typically completed Phase 3 in ~8 sessions (see Table 3).

Phase 4

In Phase 4, the twenty-four trial olfactory span trials were presented as in Phase 3, but the

six SD control trials were placed pseudo-randomly throughout the session. Testing during this

phase provided a baseline for later drug testing. Before drug administration begins, all subjects

must meet a ten day stability criterion. Percent accuracy for span trials and SD controls must be

stable over the previous ten sessions, such that the difference between the average accuracy of

the first five and second five sessions are no more than fifteen percent of the average accuracy

over the ten days {(mean of sessions 1-5) - (mean of sessions 6-10) < .15 x (mean sessions 1-

10)} (Perone, 1991). Stability was calculated for span and SD separately, and both met the

criterion prior to beginning the drug administration. Subjects typically completed Phase 4 in 16

sessions (see Table 3).

Pellet Detection Controls

In order to control for possible olfactory detection of pellets during testing, unbaited

control trials were conducted as well. During one baseline training session per week, six

randomly selected span trials were conducted without sucrose pellets present in the correct

stimulus cup (S+). Instead the experimenter manually placed a pellet in the correct stimulus cup

after the trial is completed, before the subject has been removed from the arena. This control

allows the experiment to measure what, if any, influence the scent of the reward pellet may have

59

on the selection of stimuli. In previous studies (e.g. MacQueen et al., 2011), such pellet detection

controls have shown that scented lids adequately mask the odor of the pellet.

Drug Administration

Once stability criteria are met in phase 4 of training, the following testing sequence

began. On Mondays and Thursdays, sessions were conducted as in phase 4 to produce an

ongoing baseline for performance on the span and SD tasks. Each Tuesday, an IP injection of

saline was given prior to testing to control for any effects of the injection procedures on

performance. Active drug administration (MDMA .3, 1.0, 1.8, or 3.0 mg/kg) occurred on

Wednesdays and Fridays. Saline-based drug solutions were injected IP at a volume of 1ml/kg

fifteen minutes prior to the beginning of testing. Each dose of acute MDMA was determined 2-4

times, as necessary. Subjects typically completed drug phase testing in ~36 sessions (see Table

3).

Results & Discussion

Results

Figure 2 shows the effects of MDMA on the key dependent variables in this study:

percent correct, omissions, span, longest run and latency. The top panel shows percent correct as

a function of MDMA dose for the OST (black circles) and SD (white circles) tasks. Under

baseline, saline and all MDMA conditions accuracies in both tasks were quite high, approaching

100% on SD trials and only slightly lower, nearly 90%, on the OST. Percent correct was

calculated according to all trials in which a response occurred, so errors of omission, or a failure

to respond within 2 minutes, were excluded. The percentage of trials which were omitted due to

60

non-responding is indicated by the bars in Figure 2. Response omissions were quite infrequent

during baseline, saline and low dose conditions. At the 1.8mg/kg dose, omissions became more

frequent on both OST and SD trials, and at the highest MDMA dose (3.0 mg/kg) omissions

accounted for the majority of trials. A dose X task analysis was performed excluding the 3.0 dose

due to omissions. A significant effect was found for task [F(1,5)=9.361, p<.05], and post hoc

tests confirmed that accuracy was higher on the SD task than the OST (p<.05). No significant

effect was found for dose [F (4,20)=0.162, p>.05], and no significant interaction was found

[F(4,20)=0.387, p>.05].

The middle panel of Figure 2 plots two measures of consecutive correct responses: span

(black circles) and longest run (white circles). Under baseline and control conditions, mean spans

averaged 9-10 odors, whereas longest runs were somewhat higher with runs of 11-12. Separate

univariate ANOVAs were performed on span and longest run. MDMA produced dose dependent

reductions in both span [F (5, 25) = 7.33, p < .05] and longest run [F (5, 25) = 10.591, p < .05].

Post hoc tests confirmed that span was significantly below saline levels at doses of 3.0 mg/kg

(p<.05), as are longest runs (p<.05), but not at lower doses.

Latency data for the OST (black circles) and SD (gray circles) are shown in the bottom

panel of Figure 2. Omissions were removed from this analysis. MDMA failed to produce

significant effects in response latencies, as evidenced by non-significant effects of dose [F (4,

20) = 1.343, p > .05]. Latencies for responding in the OST and SD tasks were significantly

different [F (1,5) = 9.704, p < .05] and post-hoc tests verified that latencies in the OST were

longer than the SD (p<.05). No significant interaction was found [F (4, 20) = 1.629, p >.05)].

61

0

20

40

60

80

100

0

20

40

60

80

100

Omissions SD

Omissions OST

OST

SD

Perc

en

t C

orr

ect

Perc

en

t Om

issio

ns

0

5

10

15

20

Span

Longest Run

Co

nsecu

tive C

orr

ect

BL Saline 0.3 1.0 1.8 3.00

5

10

15

20

OST

SD

MDMA mg/kg

Late

ncy (

s)

Figure 2. Ex.1 Key dependent measures. (Top) Percent correct on the OST and SD

task by dose. Bars indicate percent omissions by task. (Middle)Span and longest run

on the OST by dose. (Bottom) Latency on the OST and SD task by dose. All error

bars indicate SEM.

* *

n

62

Figure 3 shows a comparison of accuracy as a function of number of stimuli to remember

(memory load) in the saline (circles) and 1.8 mg/kg MDMA (triangles) conditions, on the OST

(black symbols) and SD (gray symbols) tasks. Separate dose X memory load analyses were

performed for the OST and SD. In the OST, no significant effects were observed for dose

[F(1,5)=.027, p<.05], a significant effect was found for memory load [F(5,25)= 3.440, p<.05],

and no interaction was found [F(5,25)=.425, p>.05]. Post hoc testing (LSD) showed that

accuracy on the OST declined as the number of stimuli increased. In the SD task, a significant

result was found for dose [F(1,5)=10.311, p<.05]. Post hoc tests (LSD) showed that SD

performance in the 1.8 MDMA condition was higher than in the saline condition. However, the

difference in averages for the SD condition (see top panel of Figure 2) was not significant, and

the significant finding in the within-session data is based on abnormally low variability in the 1.8

MDMA data, as the mean approached the accuracy ceiling (100%). A significant effect was also

observed for order of presentation[F(5,25)=4.094, p<.05]. Post hoc testing (LSD) showed that

accuracy improved later into the session. No significant interaction between dose and order was

found[F(5,25)=1.349, p>.05].

63

1-4 5-8 9-12 13-16 17-20 21-240

20

40

60

80

100

Saline OST

Saline SD

1.8 MDMA OST

1.8 MDMA SD

Number of Stimuli to Remember

Perc

en

t C

orr

ect

Figure 3. Ex.1 within-session analysis. Percent correct as a function of memory

load (number of stimuli to remember). Error bars indicate SEM.

64

Non-baited control trials were inserted weekly to control for sugar pellet detection.

Accuracies for these trials were consistent with average accuracies for subjects in this

experiment. A number of sessions were selected and scored by a second experimenter to control

for between-experimenter differences. Inter-rater agreement was high (99%).

In summary, the main effect of MDMA was to produce response omissions when

relatively high doses (1.8 and 3.0 mg/kg) were administered. MDMA failed to produce effects on

OST accuracy or latency on trials where a response occurred (errors of omission removed).

Measures of consecutive correct responding were reduced, but only at doses which also produced

large numbers of omitted trials.

Discussion

In the current study, rats were trained to stability criteria on an olfactory incrementing

non-match to sample task, the OST, and a simple discrimination (SD) performance control.

Meeting these criteria, they entered the drug phase, where they were tested under a baseline and

saline conditions, as well as a range of acute doses of MDMA. Data from these various testing

conditions was used to produce dose-response functions for each of the dependent measures. In

each case, the patterns of preservation or disruption of baseline performance was consistent

between the OST and SD control. As calculated, the dose-response functions for accuracy and

latency were flat, meaning that the drug did not impact those measures at any dose. Dose x

memory load analyses also showed no impairment by MDMA at the dose of interest (1.8

MDMA). Dose-response functions for span and longest run were curved, in that as the dose

increased, consecutive correct responses decreased. Available data suggests that reductions in

consecutive correct responses do not constitute a selective working memory impairment, as

65

reductions in these two measures coincided with gains in the rate of omissions. Errors of

omission were the only error type to be produced by acute MDMA in this paradigm.

It was known from existing studies (see Table 2) that MDMA produces disruptions in

performance on a variety of learning tasks and that these impairments can be directly related to

learning and memory (e.g. Galizio et al., 2009), or can be general, producing a global alteration

in behavior (e.g. Marston et al., 1999). In dose-response curves produced by these studies, as the

dose of MDMA increases, the size of the disruption tends to increase. The current study used a

sophisticated set of controls which aimed to assist in defining the nature of the disruptions that

are produced in this yet untested memory task. The most crucial control to defining the

disruptions produced in Experiment 1 was the distinction between two error types: commission

and omission. Errors of commission were errors produced by flipping an incorrect lid, based on

the rules of the task. These errors constituted the error rate in percent correct calculations. Errors

of omission were produced when latencies exceeded two minutes, thereby terminating the trial as

a timeout. These errors were removed from accuracy and latency calculations.

The observation that acute MDMA reduces responding in this study is an interesting, if

somewhat surprising, finding. Marston et al. (1999) also reported a reduction in the number of

trials completed, but the doses used in that study (10-20 mg/kg) were substantially higher than

those used in the current study (0.3-3.0 mg/kg). This is also surprising because the majority of

trials were removed due to omissions at the 3.0 dose, and disruptions of this kind have not been

reported in other studies using this dose (e.g. Galizio et al., 2009, Kay et al., 2010).

Upon closer inspection of the video recordings of omitted trials, an interesting series of

events unfolds. Under the influence of 3.0 MDMA, trials frequently occurred where two minutes

passed without a response. While the animals did not respond, they were quite active. Upon

66

entering the arena, the animals explored the space as they normally did and sniffed the olfactory

stimuli as they reached them. Upon arriving at the correct cup for the trial, the animal would

often sniff the lid and continue circling the arena without flipping it. The exact behavioral

patterns occurring during omitted and completed trials have not been identified and it appears

that such an analysis would be necessary and beneficial. A more systematic analysis of these

videos may yield other clues as to what aspects of within-arena behavior are altered on omitted

trials.

While it is interesting that errors of omission become frequent at the highest dose

administered in this study, it is equally important that errors of commission do not occur. This

means that even at high doses, when the animals respond, they do so with high accuracy. Given

this pattern of accuracy, it appears that memory performance is spared in both of these two tasks.

It also seems to suggest that abnormalities in olfactory perception or discrimination are not

viable explanations for the impairments observed.

Based on available data, it cannot be conclusively stated what the origins of the mutual

disruption produced by acute MDMA in the OST and SD task are, but a dose dependent change

in omissions combined with stable rates of commissions is consistent with some form of

competitive behavioral interference. This means that another effect of the drug, perhaps a

stereotypic alteration of exploratory behavior, has reduced task completion while preserving the

efficiency and accuracy are preserved when they occur.

None of the rodent acute MDMA studies included in the prior review reported any

enhancements in learning or cognitive function, as has sometimes been reported in human

pharmacology studies (e.g. Lamers et al., 2003). The current study also failed to find convincing

evidence of cognitive enhancement. While a statistical enhancement effect was found in the

67

within-session analysis of SD accuracy, this is not interpreted as evidence for an enhancement.

Percent correct for SD in baseline conditions was over 90% and the accuracy ceiling (100%)

prevents sizeable enhancements from occurring on the SD task. The significant result is due to

reductions in variability in the within-session analysis. Multiple memory load scores in the 1.8

MDMA condition had standard deviations of zero. Greater variability was present in the overall

accuracy score, which did not produce a significant result. In the current study, span and longest

run measures were assessed in addition of percent correct, as they are not subject to the low

ceiling effect that accuracy scores meet.

Based on data from Experiment 1, evidence for disruption on olfactory span and simple

discrimination tasks consisted of increases in non-responding as the dose of MDMA increases.

Despite this disruption, accuracy of responding was preserved. No cognitive enhancements were

observed at any dose.

EXPERIMENT 2: EFFECTS OF BINGE MDMA ADMINISTRATION

A number of studies of the effects of binge or neurotoxic MDMA administration on common

working memory tasks have been discussed in detail in this review. A summary of these studies

is found in Table 4. This table displays a selection of relevant studies studying the effect of binge

administration of MDMA in rodents. It displays the pharmacological procedure used, the

paradigm and design (working memory, reference memory or both) used, the presence of pre-

training on a task prior to drug administration, the post-treatment time frame when testing

occurred, the dependent measures chosen, and the result of each test.

68

Authors Dosing Testing start Task/Design Pretrained Results Found

Camarasa

et al 2008

1-15mg/kg, 2

per day x 4

days

7 days post-

binge

MST-RM No Latency-No signif. diff.

Platform absent test- Signif.

effect

Galizio et

al. In

Review Ex2

20mg/kg, 2

per day x 4

days

3 days post-

binge

MST- WM,

RM

Yes WM- Elevated path ratios,

recovered quickly

Other- No Signif. effect

Hernandez-

Rabaza et

al 2010

10mg/kg, 10

doses in 2.5

days

12 days post-

binge

RAM- WM,

RM

No All measures- No effect

Kay et al

2011

10mg/kg, 4

doses in 8

hours

2 days post-

binge

RAM- WM,

RM

Yes WM- No effect, RM- Signif.

effect, Reversal effect

Marston et

al 1999

10mg/kg x 2,

15mg/kg x 2,

20 mg/kg x 2

During acute

phase

NNMTP-

WM

Yes No effect on accuracy post-

binge, MDMA group failed to

improve long delay accuracy

Ricaurte et

al 1993

20mg/kg, 2

per day x 4

days

7 weeks post-

binge

T-Maze-

WM

No No effects on alternation

Robinson et

al 1998

10mg/kg, 2

per day x 4

days

3 days post-

binge

MST- WM Yes Elevated latencies- first trial

per session only

Slikker et

al 1989 0, 5.0 or 10

mg/kg, 1 per

day x 4 days

14-28 days

post-binge

Complex

Maze- RM

No No effects on acquisition

Table 4. Binge MDMA studies of memory tasks in rats. Note: WM= Working Memory, RM=

Reference Memory

69

While the paradigms included in this analysis have been associated with working

memory in the past, different designs and measures used with these paradigms do not reasonably

measure performance of the same subtypes of memory. In accordance with the definition offered

by Dudchenko (2004), those which assess memory for events within a testing session are

referred to as working memory tasks/measures. Those which assess memory for events in

previous sessions are referred to as reference memory tasks/measures. Of those reviewed above,

two studies (Camarasa et al., 2008, Slikker et al., 1989) fail to include measures which meet the

definition for working memory. These designs meet the definition of reference memory instead.

The remaining studies include either working memory measures or both working and

reference memory measures. Both Hernandez-Rabaza et al. (2010) and Kay et al. (2011) used

RAM designs with both working and reference memory components. Hernandez-Rabaza et al.

(2010) failed to find impairments on either measure. Kay et al. found selective impairments on

the reference memory component and differences in acquisition of a contingency reversal.

Robinson et al. (1998) used a working memory version of the MST and Experiment 2 of Galizio

et al. (In Review) used both working and reference memory designs of the MST. Both studies

found evidence for a weak disruption of MST performance. Robinson observed that MDMA pre-

treated animals had longer escape latencies on the first trial than controls, but that all trials

demonstrating memory for the location were equal to controls. Galizio et al. found elevated path

ratios for the acquisition or working memory task which disappeared within the first several days

of testing. This elevation was selective to working memory, but was weak and recovered

quickly. Marston et al. (1999) used the DNMTP and found no differences between drug-treated

animals and controls during the early stages of post-binge testing. Rather, it was found that drug-

treated animals failed to improve on long-delay NMTP with added training, while controls did.

70

Ricaurte et al. (1993) tested rats in T-maze spatial alternation and failed to find differences in

performance between drug-treated animals and controls. These studies utilizing working memory

measures have generally failed to find deficits which are consistent with the claim that MDMA-

induced neurotoxicity produces impairments that are specific to working memory tasks. Rather,

disruptions that are produced seem to indicate other types of impairment such as reference

memory deficits (Kay et al., 2011), cognitive inflexibility (Kay et al., 2011, Marston et al., 1999)

or general behavioral alterations which are specific to the task (Robinson et al., 1993). The only

study examined here which showed evidence for specific impairments in working memory was

Galizio et al. (In Review) but the observed impairments were not profound or persistent. These

working memory designs have been able to detect a range of impairments produced by

neurotoxic administration of MDMA but previous procedural approaches have some weaknesses

which may have prevented the weak, transient effects described by Galizio et al. from being

detected by other designs and paradigms.

One procedural issue is the timing of testing relative to the binge administration. Only

one study of residual effects began testing on the measures of interest immediately in the acute

phases (Marston et al., 1999) and continuously measured them. There does appear to be evidence

for physiological recovery (Sabol et al., 1996), so it is logical that if there are measurable effects,

they should be most prominent in the first few weeks of testing. Even so, the available studies

greatly vary in the time frame under which testing began. Ricaurte et al. (1993) did not begin T-

Maze testing until seven weeks after drug. Hernandez-Rabaza et al. (2010) began RAM testing

after twelve days. Kay, Harper & Hunt (2011) began testing on the RAM two days after

administration and Galizio et al. (In Review) and Robinson et al. (1993) began testing just three

days post-binge. It is worth nothing that those who began testing immediately (Marston, 1999) or

71

within a few days (Galizio et al., In Review, Kay, Harper & Hunt, 2011, Robinson et al., 1993)

did find evidence for disruptions in behavior, though these would not be described as working

memory deficits.

Another issue in the design of these procedures is whether drug administration occurred

before or after acquisition of the task. The procedural advantage to using a working memory task

should be that the underlying cognitive process is equally relevant in each session, allowing the

researcher to train the subjects to stability prior to administering drugs. That option has been

adopted by researchers such as Galizio et al. (In Review), and Marston et al. (1999) and allows

for a more complete picture of the period following large or repeated doses. This design not only

removes the issue of timing, but it removes confounded procedural requirements associated with

initial acquisition (e.g. Byrne et al., 2000) which might otherwise be interpreted as memory-

based. Future studies that are not focused on initial acquisition should consider training prior to

drug administration and beginning testing relatively early in order to maximize detection and

characterization of any measurable effects there may be.

Beyond these testing considerations, it is also possible that in general these tasks are

simply not sensitive enough to measure any relevant effects that do exist at the doses used and

during the periods when they have been measured. All rodent working memory tasks share

similar memory requirements, usually meaning that they measure the speed and/or accuracy of

remembering the location of a single stimulus within a session. However, as Nulsen, Fox and

Hammond (2010) noted, differences in more specific requirements which contribute to the

cognitive demand of the tasks used are also relevant to determining what impact a drug will have

on performance. Generally, more demanding tasks are more sensitive to disruption and will be

better able to detect impairments that do exist. More complex tasks may be beneficial to

72

understanding the effects of the drug, as humans perform under a broader range of simple and

complex forms of stimulus control than are utilized experimentally with rodents. Testing with

complex designs, novel manipulations and more difficult discriminations may increase detection

and characterization of specific working memory deficits in MDMA-treated rats.

As in studies of acute MDMA exposure, research on the effects of neurotoxic exposure to

MDMA on working memory tasks in rats has focused on performance in duration-based memory

tasks. Prior to the current study, nothing was known about capacity-based memory performance in

rats following MDMA. Experiment 2 tested performance in the rodent OST following a binge dosing

regimen of MDMA or saline.

Methods

Subjects

Subjects in this study consisted of eight adult male Holtzman Sprague–Dawley albino

rats. All subjects were experimentally naïve prior to entering this study and housing conditions

were identical to those described in Experiment 1.

Training

All training and testing procedures from initial shaping until stability criteria are met at

the end of phase four are identical in Experiments 1 and 2.

Contingency Reversal

In an attempt to replicate the findings of Kay et al (2011), a reversal was added to the

simple discrimination control trials. During initial post-binge testing, five scents were designated

for these trials, one of which always indicated the location of the reinforcer (ex. S+ almond),

73

while the other four never indicated the location of the reinforcer (ex. S- sage, cumin, oregano,

vanilla). In the reversed condition, the previous S+ was designated as the S-, and one of the four

previous S- (ex. sage) was designated as the new S+. Thus, on performance trials in the reversed

phase, two stimuli were presented, the previous S+ (now the S-), and the new S+ (previously an

S-). This contingency reversal was put in place on the 11th

post-treatment testing session.

Acquisition of the reversal was then monitored for 10 sessions to determine a reversal acquisition

curve for each subject.

Temperature

Electronic thermometers were used to measure environmental temperature, and the

vivarium temperature was noted for each administration the subjects received. Average

temperature was 72.6ºf, or 22.55ºC.

Drug Administration

Once subjects met the ten day stability criterion in Phase 4, subjects continued testing in

Phase 4 until the current week of testing was over. Then chronic administrations of MDMA or

saline began the following Monday. Subjects in the drug group received an IP injection of 10

mg/kg MDMA twice per day for four days, once in the morning (~8-9am) and again in the

evening (~4-5pm). Subjects in the control group were administered an equal volume of saline

solution under the same injection schedule. No span testing occurred occur during this phase.

After the chronic administrations were complete, a 72 hour recovery period (Friday-

Sunday) went into effect prior to span testing resuming. This recovery period allowed enough

time for the drug to run its course, ensuring that all acute effects of the drug were excluded from

74

analysis of the MDMA group. Once testing resumed on Monday, subjects returned to Phase 4

sessions and established a post-treatment baseline over the following four weeks. During the first

two weeks of this baseline, the SD performance control was identical to that used prior to the

binge administration or MDMA or saline. During the final two weeks, a contingency reversal

was placed on the performance component, as described below.

A heavier dosing regimen (20 mg/kg, 2 per day x 4 days) has been used frequently and

shown to produce dramatic serotonergic effects but for practical reasons, the more moderate

regimen used by Robinson, Castaneda and Whishaw (1993) was selected for the current study.

Larger doses carry a greater risk, as evidenced by the fact that the first subject tested in this study

was administered the first dose of the heavier dosing regimen (20 mg/kg, as in Galizio et al., In

Review) and subsequently overdosed. As testing with the span task requires a high investment of

training prior to drug administration, this risk had to be minimized to allow the project to be

timely and efficiently completed.

Robinson, Castaneda and Whishaw (1993) demonstrated significant serotonergic

dysfunction following the binge dosing regimen of MDMA used in the current study (10mg/kg,

twice per day x 4 days). Thirty-five to forty days after the binge was completed, tissue

concentrations of serotonin were decreased by 72% in the neocortex, with less dramatic

reductions in the caudate nucleus. As expected, levels of dopamine were not reduced. Based on

this study, the current pharmacological procedure produces serotonergic deficits which persist

for the first several weeks following administration.

Results & Discussion

Results

75

Figure 4 shows the effects of MDMA on the key dependent variables in this study:

percent correct, omissions, span, longest run, and latency. The top panel shows percent correct as

a function of testing phase for OST (circles) and SD (triangles) tasks for saline (black symbols)

and MDMA (white symbols) groups. In the baseline and post-binge phases, accuracies in both

tasks were high (80-100%). In the reversal phase, accuracy on the OST remained high and as

expected, SD performance was reduced. A group X phase analysis was performed for OST

performance and showed no main effects for group [F(1,6)=0.7, p>.5], phase [F(2,12)=0.09,

p>.5] or an interaction of the two [F(2,12)=0.25, p>.5]. Group X phase analysis was also

performed for SD performance, resulting in no significant effect for group [F(1,6)= 0.3, p>.05], a

significant effect for phase [F(2,12)= 228.01, p<.05], and no significant interaction[F(2,12)=

0.68, p>.05]. Due to low power with the current sample size, post hoc tests were not performed.

However, means for each phase are consistent with the a priori hypothesis that SD accuracy in

the reversal phase would be lower than baseline values. Percent correct was calculated according

to all trials in which a response occurred, so any trials where a subject failed to respond within

two minutes were excluded. The percentage of trials which were omitted due to non-responding

is indicated by the bars in the top panel. Response omissions were quite infrequent during

baseline for both groups, and in both tasks. After binge administrations, omissions accounted for

ten percent of trials for the MDMA group on both tasks, while the control group continued to

complete almost all trials. During the reversal phase, omissions were relatively infrequent for

both groups in both tasks. Accuracy and omission data is shown in Figure 5 on a session by

session basis. The top panel displays data for the OST. The bottom panel shows data for the SD

task. Accuracy is relatively consistent between the two groups under all conditions tested.

Omissions occur early in the post-binge period, but attenuate over successive sessions. Figure 6

76

shows individual subject graphs for OST percent correct and omissions for the four subjects in

the MDMA group (D6, E3, E18, F13). Accuracies are relatively consistent across rats. Individual

rats in this group show great variability in the tendency to produce omissions following binge

MDMA exposure.

The middle panel of Figure 4 plots two measures of consecutive correct responses: span

(black circles) and longest run (white circles). Under baseline and control conditions, mean spans

averaged 9-10 odors, whereas longest runs were somewhat higher with runs of 11-12. Separate

group X phase ANOVAs were performed on span and longest run. There were no significant

effects of group or phase on span [F (1,6)=3.38, p > .05)] [F(2,12)=1.14, p > .05)] or on longest

run [F (1,6) = 1.44, p > .05)][F (2,12) = 1.5, p > .05)]. There were no significant interactions.

Latency data for the OST (black circles) and SD (white circles) are shown in the bottom

panel of Figure 4. Omissions were removed from this analysis. MDMA failed to produce

significant effects in response latencies, as evidenced by non-significant effects of group [F (1,6)

= 0.89, p > .05)] and phase[F (2,12) = 3.38, p > .05)]. No significant interaction was found [F

(2,12) = 0.8, p >.05)].

77

BL Post-Binge Reversal0

20

40

60

80

100

0

20

40

60

80

100Saline OST

Saline SD

MDMA OST

MDMA SD

Omissions OST-MDMA

Omissions SD-MDMA

Omissions OST-Saline

Omissions SD-Saline

Perc

en

t C

orr

ect

Perc

en

t Om

issio

ns

BL Post-Binge Reversal0

5

10

15

20

Span- MDMA

Span- Saline

LR- MDMA

LR- Saline

Co

nsecu

tive C

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ect

BL Post-Binge Reversal0

5

10

15

20

MDMA- OST

MDMA- SD

Saline- OST

Saline- SD

Testing Phase

Late

ncy (

s)

Figure 4. Ex.2 Key dependent measures. (Top) Percent correct for each group and

task over the testing phases. Bars indicate percent omissions. (Middle) Span and

longest run for each group and task over the testing phases. (Bottom) Latency for each

group and task over the testing phases. All error bars indicate SEM.

78

Figure 5. Ex.2 Session-by-session data. (Top) Accuracy in the OST presented by

individual sessions, by group. Bars indicate percent omissions. (Bottom ) Accuracy in

the SD task presented by individual sessions, by group. All error bars indicate SEM.

79

BL Post-Binge Reversal0

20

40

60

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100

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100

Percent Correct

OmissionsD6

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60

80

100

0

20

40

60

80

100

E3

Perc

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ect

Perc

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BL Post-Binge Reversal0

20

40

60

80

100

0

20

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60

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E18

Testing Phase

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F13

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Figure 6. Individual subject graphs. Accuracy and omissions data for individual

subjects in the MDMA group.

80

In Figure 7, the top panel shows a comparison of accuracy as a function of number of

stimuli to remember for the MDMA group in each phase of testing on the OST. No significant

effects were observed for phase [F(2,6)=.113, p>.05], a significant effect was found on memory

load [F(5,15)=6.378, p>.05], and no interaction was found [F(10,30)=1.428, p>.05]. Post hoc

(LSD) testing showed that accuracy declined as a function of memory load. The bottom panel

shows accuracy as a function of the order of presentation within the session in the baseline and

post-binge phases in the SD (bottom panel) task. No significant effects were observed for

phase[F(1,3)=1.857, p>.05] or order of presentation[F(5,15)=1.848, p>.05] in the SD task, an no

significant interaction was found[F(5,15)=2.353, p>.05].

81

1-4 5-8 9-12 13-16 17-20 21-240

20

40

60

80

100

Baseline

Post-Binge

Reversal

OST

Number of Stimuli to Remember

Perc

en

t C

orr

ect

0 1 2 3 4 5 60

20

40

60

80

100

Baseline

Post-Binge

SD

Presentation Order

Perc

en

t C

orr

ect

Figure 7. Ex.2 within-session analysis. (Top) Within session accuracy across

memory load conditions. (Bottom) Accuracy on SD trials as a function of

presentation order. All error bars indicate SEM.

82

Figure 8 shows the acquisition curves for each group in the SD reversal phase. A group X

session analysis showed no significant difference between groups [F(1,3)=.728, p>.05] and a

significant effect of session [F(9,27)=30.077, p<.05], but no significant interaction

[F(9,27)=1.677, p>.05]. Post hoc analysis of the session effect showed that accuracy improved

between the first and sixth days of testing in the reversed SD. 95% Confidence interval

calculations indicated that saline and MDMA groups differed in accuracy on the reversed SD

during the first two sessions only. On the first two sessions, the MDMA group was less accurate.

83

0 1 2 3 4 5 6 7 8 9 10 110

20

40

60

80

100

Saline Group

MDMA Group

Session

Accu

racy

Figure 8. Reversal data. Acquisition of SD reversal over ten sessions. Asterisks indicate

significant differences between corresponding groups.

*

*

84

Non-baited control trials were inserted weekly to control for sugar pellet detection.

Accuracies for these trials were consistent with average accuracies for subjects in this

experiment. A number of sessions were selected and scored by a second experimenter to control

for between-experimenter differences. Inter-rater agreement was high (98.2%).

In summary, binge administration of MDMA failed to produce effects on percent correct,

span, longest run, or latency in either task. Within session analyses showed that binge MDMA

produced no effects on accuracy as a function of the number of stimuli to remember. Binge

MDMA and saline groups did not differ on the acquisition of the reversed SD overall, but the

MDMA group was less accurate than controls on the first two sessions of reversal testing.

Discussion

In the current study, rats were trained to stability criteria on an olfactory incrementing

non-match to sample task, the OST, and a simple discrimination (SD) performance control.

Meeting these criteria, they entered the drug phase, where they were administered a binge dosing

regimen of saline or MDMA (10mg/kg, 2 per day x 4 days), shown by Robinson, Castaneda and

Whishaw (1993) to produce persistent serotonergic dysfunction. After a washout period, all rats

were tested in the OST for an additional 20 sessions (over ~four weeks) following the end of

binge administration. No differences were observed between the MDMA group and controls

with respect to percent correct, span, longest run or latency during post-binge testing. The pre-

trained SD task was administered for the first 10 post-binge sessions, followed by 10 sessions in

a reversed contingency SD task. No differences were observed between the MDMA group and

controls during the first 10 sessions of post-binge testing. Memory performance, as measured in

the current study, appeared to be spared during the post-binge phase. The only measure which

was impacted by the MDMA binge was omissions. Omissions were elevated for the MDMA

85

group early in post- binge testing, and this effect appeared to recover over time. Omissions for

the control group remained low throughout testing. The SD reversal did not produce significant

group differences overall, but accuracy for the MDMA group was reduced during the first two

sessions post-reversal relative to controls.

The current study did not find evidence for MDMA-induced disruptions in performance

on either of the olfactory memory tasks used. Generally speaking, binge MDMA exposure did

not alter performance in any measurable way. Binge MDMA exposure did not produce any

measurable alterations of the impact of memory load on performance in the OST relative to

baseline. The only measure which indicated notable impairment was omissions, which peaked in

the early sessions post-binge and attenuated over time. As accuracy of responding was preserved,

an increase in omissions indicates a persistent disruption of task completion which is

independent of the memory requirements found in the OST and SD tasks. The tendency to

commit errors of omission varied greatly across rats in the MDMA group, indicating a

differential sensitivity to this effect. One subject (E18) is responsible for most of the errors of

omission observed in this study. Though only a single subject showed dramatic gains in

omissions, the magnitude of this effect is such that the potential for non-responding following

binge MDMA administration may be considerable. Future replications of this finding will be

necessary to support such a conclusion.

A small impairment in reversal acquisition was observed in the MDMA group relative to

controls. Omissions remained low for these sessions, indicating that these low accuracy values

are the result of selecting the old S+ (current S-) more often than controls during the first twelve

presentations (6 per session) of the reversed condition. On the first session, none of the MDMA-

treated rats flipped the correct lid first, while the saline group averaged close to one out of six.

86

On the second session, the MDMA group averaged close to one out of six, while the saline group

approached chance (~3 out of 6). By the third session, accuracy on the reversed condition was

identical between the two groups and improved similarly, reaching peak values on session six or

seven.

The delay and low frequency of correct responses early in the reversal phase is evidence

for cognitive inflexibility or rigidity. In order to reach chance levels of responding on a reversal,

subjects must respond to the new S+ when they encounter it. This means that subjects must begin

responding to an olfactory stimulus which has been under extinction for all but the most recent

training conditions. This could be described as a breakdown in behavioral inhibition for

responses to the stimulus. In order to reach high levels of accuracy, the subject must also inhibit

responses to the previous S+ (current S-). An appropriate analysis of patterns of responding in

the reversal has not yet been performed, so it is unclear whether this early reversal effect reflects

deficits in one or both of these behavioral shifts. It is also unclear whether these occur

successively or simultaneously. Such an analysis would include comparing the two groups with

respect to: the first correct response in the reversal phase, the rate of S+ selection on a trial by

trial basis, the first non-response (sniff-only) to the S-, and the rate of S- selection on a trial by

trial basis.

Experiment 2 generally demonstrates that binge MDMA exposure does not produce

deficits in performance of the two well-learned olfactory memory tasks included in this study:

OST and SD. In a subset of subjects, it produces an elevated tendency for errors of omission.

Binge MDMA exposure did produce a measurable difference in reversal learning relative to

controls. This difference consisted of reduced accuracy in responding during the first two

sessions of reversal training or the first twelve presentations of the reversed contingency.

87

GENERAL DISCUSSION

The OST is a novel procedure for testing the impact of MDMA exposure on within-

session learning. Once acquired, all rats in this study performed the OST with high accuracy

while completing all requirements of pre-drug training. These rats also acquired the SD task

easily and performed the two tasks on a multiple schedule with high accuracy. This demonstrates

that despite to relative complexity of the stimulus control which was established, rats are well

equipped for complex olfactory discriminations and are not challenged by these requirements

under baseline conditions. Prior to the current study, it was not known whether acute or

neurotoxic MDMA administration produced effects which were relevant to capacity-dependent

performance in rats.

In Experiment 1, performance on the OST and SD tasks, as measured by accuracy of

responding, appeared to be insensitive to the disruptions produced by acute MDMA. Dose

dependent increases in errors of omission (non-response) were produced without any effect on

errors of commission (incorrect response). The failure to observe elevated rates of incorrect

responses was unexpected given that previous studies utilizing measures of accuracy (Braida et

al. 2002, Galizio et al., 2009, Harper et al., 2005, Kay et al., 2010, Marston et al., 1999) each

observed acute reductions of accuracy in within- or across-session learning, or both. The current

results appear to be inconsistent with previous results that specific impairments in reference (e.g.

Kay et al., 2010) or working memory performance (Galizio et al., 2009) are produced by

MDMA. The differences between the current paradigm and these previous tasks may offer an

explanation for this inconsistency.

At first glance, it would appear that the OST is a more difficult task than a more typical

rodent working memory task. After all, span tasks are designed as NMTS sessions where each

stimulus presented in a session serves as a sample for all subsequent trials. However, it must not

88

be ignored that the OST and SD task are olfactory memory tasks, and as such rely on the

rodent’s most developed discriminative modality. Differences in the salience of the stimuli

involved, as well as the relevant brain regions involved, may account for the relative insensitivity

of memory performance to the effects of acute MDMA, relative to other behavioral processes

which may impact task completion.

Such an account would explain why acute MDMA failed to produce impacts on accuracy

in the current study, but the current results are not strikingly different than all existing data.

Current results are relatively similar to those produced in the MST by Galizio et al (In Review).

In the MST, acute MDMA did not produce impairments of key measures until both within- and

across-session versions of the swim task were impaired. While Galizio et al. could not rule out a

profound cognitive impairment accounting for these results, the parsimonious conclusion was

that MDMA produced effects that interfered with task completion. Examples for the MST would

include alterations in search path (ex. thigmotaxis) or the discriminability of trial types based on

distal cues. At high doses disturbances also involved motor impairments, as swimming and

climbing onto the platform were impaired. The pattern of errors in the current study also seems

to suggest interruption of task completion. It is possible that the swim and span tasks may share

certain behavioral disruptions which are unique to testing in the open field apparatus. Given that

MDMA-treated rats in the OST and SD failed to respond once reaching the stimulus, other

aspects of the tasks are likely relevant. In general, previous studies suggest that the exact form of

impairments and behavioral alterations which are produced by acute MDMA is relatively

paradigm dependent. Further experimentation with MDMA in olfactory tasks and with span

tasks relying upon other modalities may help to explain the insensitivity of the current task

89

designs to performance impairments which are readily observed in other rodent memory

paradigms.

In Experiment 2, dosing and temperature conditions were favorable to produce

neurotoxicity. The dosing regimen was shown by Robinson et al. (1993) to produce persistent

serotonergic dysfunction and environmental temperatures were generally above threshold to

produce hyperthermia (20-22ºC).

All measures of OST and pre-reversal SD performance were indistinguishable between

saline and MDMA-treated rats after binge administration. This suggests that the OST and SD

tasks are not impacted by impairments produced by binge MDMA exposure. With respect to the

OST, this finding fits well with existing studies of MDMA effects on working memory measures

in adult rats, particularly those which also pre-trained their subjects (Galizio et al., In Review,

Kay, Harper and Hunt, 2011, Marston et al., 1999, Robinson et al., 1993). Of those, only Galizio

et al. (In Review) found any evidence for impairment on a working memory or within-session

learning task. The effect was weak and short in duration, suggesting that any specific effects of

the drug on working memory measures are transient and difficult to measure. Regardless of the

within-session manipulation, whether based on the duration or capacity of memory, these

measures are insensitive to the effects produced by neurotoxic MDMA exposure in adult rats.

With respect to the SD task, the current results are inconsistent with certain previous studies

which found impairment on reference memory tasks (e.g. Camarasa et al., 2008, Kay, Harper and

Hunt, 2011) but are consistent with others which failed to find effects (e.g. Galizio et al., In

Review). As with the results of Experiment 1, results seem to be more consistent with those

shown in the MST than in the RAM. It could be argued that the current tasks are insensitive to

the persistent effects of the drug due to reliance on the olfactory modality or due to other

90

procedural factors in the olfactory arena. Further testing with similar designs may help to

determine if such factors are necessary to produce these results or whether the results generalize

to other span tasks in rodents.

While task performance was unaffected on trials which were completed, omissions

occurred in a subset of the binge MDMA-treated group at greater frequency after binge

administration. One of the four rats produced large numbers of omissions early after the binge

and recovered baseline patterns of responding over the following sessions. Given the small

sample size (N=4 per group) and the amount of this effect which is attributable to a single

subject, more data must be gathered before it can be convincingly determined whether the

elevation of omissions is a general effect of the drug or due to the abnormal response of a single

rat to the drug. Further testing in this line of research will expand the current sample size to

verify the current findings.

The reversal phase was included in the current study to replicate a finding from Kay,

Harper and Hunt (2011) which suggested that binge MDMA exposure produces impaired

acquisition of contingency reversals. Kay, Harper and Hunt interpreted this finding as indicating

cognitive inflexibility, a view that is generally compatible with the observation that binge

MDMA tends to alter behavior related to initial acquisition (e.g. Byrne et al., 2000). In the

current study, significant group differences in reversal accuracy were observed during the first

two post-reversal sessions, providing further evidence for cognitive inflexibility. It is worth

noting that the form and timing of this difference is somewhat different than what Kay, Harper

and Hunt found in the RAM. RAM performance was initially identical between the two groups.

Between session four and five of the reversal, accuracy nearly doubled (~30-60%) for the saline

group, while the MDMA group never made gains of more than a few percent between any two

91

sessions. In contrast, all rats in the current SD reversal made rapid gains in accuracy during early

sessions of the reversal phase, but the first session accuracy for drug-treated animals was lower

than controls and the most rapid gains occurred on later sessions than controls. In other words, it

took more presentations of the reversed SD before responding began to shift in favor of the new

S+.

Future research on the cognitive deficits produced by acute and neurotoxic MDMA in

rats will help to expand upon the conclusions made by the current study and to address the

questions which have been unanswered by it. A limitation of the current study is in the ability to

adequately compare results in the olfactory arena to studies in more researched paradigms. A

number of procedural and conceptual differences exist and based on the current use of available

measures, it is not possible to fully clarify the sources of differentiation between current study

and existing studies. Research with variations of the current procedures will allow the relevant

variables to be more systematically assessed. Such variations might include instituting delays

inserted to increase odor task difficulty or presenting and testing odor lists in a different way (ex.

non-incrementing presentations, match to sample, etc.). Alternatively, incrementing-non match

paradigms could be instituted requiring memory for other modalities such as memory for spatial

locations (as in Dudchenko et al., 2000) or for geometric shapes.

A second limitation of the current study is its relation to human cognitive functioning.

While the OST is loosely based on the digit span task in humans, the accuracy of memory as a

function of memory load is strikingly different between the two tasks. Accuracy in the OST has a

shallow slope which fails to show dramatic losses in accuracy, even with twenty-four stimuli to

remember. This suggests that the performance being measured is relatively stable over long

durations and does not have a clear upper limit. Digit span, by contrast, produces a function with

92

a flat slope followed by a dramatic loss in accuracy around a specific number of items (e.g.

Cowan et al., 2004), dipping to close to and zero remaining there for additional items. While it is

tempting to interpret the current results as evidence that acute and neurotoxic MDMA does not

impact the capacity of working memory, such a conclusion would be premature and perhaps not

defensible. More research on the characteristics of learning in the OST is necessary. For now, the

current study suggests that acute and neurotoxic MDMA exposure does not impair the accuracy

or load-dependence of olfactory discrimination for multiple familiar objects in rats.

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