A Systematic Review of the MDMA Model to Address Social Impairment in Autism

Abstract

Background

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterised by repetitive behaviours, cognitive rigidity/inflexibility, and social-affective impairment. Unfortunately, no gold-standard treatments exist to alleviate the core socio-behavioural impairments of ASD. Meanwhile, the prosocial empathogen/entactogen 3,4-methylene-dioxy-methamphetamine (MDMA) is known to enhance sociability and empathy in both humans and animal models of psychological disorders.

Objective

We review the evidence obtained from behavioural tests across the current literature, showing how MDMA can induce prosocial effects in animals and humans, where controlled experiments were able to be performed.

Methods

Six electronic databases were consulted. The search strategy was tailored to each database. Only English-language papers were reviewed. Behaviours not screened in this review may have affected the core ASD behaviours studied. Molecular analogues of MDMA have not been investigated.

Results

We find that the social impairments may potentially be alleviated by postnatal administration of MDMA producing prosocial behaviours in mostly the animal model.

Conclusion

MDMA and/or MDMA-like molecules appear to be an effective pharmacological treatment for the social impairments of autism, at least in animal models. Notably, clinical trials based on MDMA use are now in progress. Nevertheless, larger and more extended clinical studies are warranted to prove the assumption that MDMA and MDMA-like molecules have a role in the management of the social impairments of autism.

1. INTRODUCTION

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder manifesting in early childhood [1, 2]. It is characterised by a triad of behavioural symptoms. These include social reciprocal communication and interaction impairment, motor stereotypies (repetitive behaviours), and cognitive rigidity. Each symptom appears to different extents in different individuals [1, 2]. They are the outcome of abnormal brain physiology developed as early as in utero [3]. Briefly, they involve both genetic and environmental risk factors, affecting synaptogenesis and axon motility downstream [4]. The amygdala and nucleus accumbens are purportedly involved [4].

ASD symptoms result in variable impacts on functioning across all domains of life [5-7]. 85% of children with ASD report difficulties at school, with 63% of those having social difficulties, and 52% of those having communication difficulties [8]. These difficulties continue into adulthood, with the majority of individuals with ASD being unemployed or underemployed [9], predictably leading to a considerably lower quality of life [10, 11]. The reported prevalence rates of ASD are rising worldwide [12], implicating an impending increase in disease burden, necessitating the need to develop more effective interventions, as well as treatments to support this population. While it remains unclear what is driving this increased prevalence in ASD [13], factors such as changes in reporting [14], increased awareness and changes in diagnostic criteria [15], among other theories [13], may be contributing factors. Nevertheless, it is imperative to develop more effective interventions and treatments to support this population.

There are currently no FDA-approved pharmacological treatments for the core impairments that define ASD [16]. Two medications are currently FDA-approved for the treatment of accessory traits, irritability and aggressiveness, in ASD: aripiprazole and risperidone. These are controversial for use, due to their adverse effects [16, 17].

A significant number of off-label medications have also been utilised, such as alpha-2 agonists, mood stabilisers, norepinephrine-reuptake inhibitors, serotonin-reuptake inhibitors, antipsychotics and opioid-receptor antagonists [18, 19]. The latter have either insufficient evidence of efficacy, or are only able to address different accessory symptoms of ASD, such as irritability and hyperactivity [18, 19]. In addition, addressing ASD comorbidities can be fraught with complexities. This includes the need to obtain prescriptions for these comorbid affective disorders (such as anxiety and depression) or comorbid neurological disorders (such as epilepsy) [20, 21].

No drugs have been approved for the core impairments in ASD [22, 23]. However, several drugs have shown early-stage evidence for the treatment of these core behaviours, such as bumetanide, pregnenolone, suramin, sulforaphane, folinic acid, propranolol, oxytocin, vasopressin antagonists, and arbaclofen [22, 23]. Furthermore, many non-pharmacological treatments for ASD also exist, though these fail to target core behavioural traits, or do not yet have a sufficient evidence base to be recommended for widespread clinical use [24]. Psychotherapeutic methods include applied behaviour analysis [25-27], meditation/mindfulness [28], early-childhood and parent education [29], cognitive behaviour therapy [30], music therapy [31], and social-skills training [32, 33]. However, these interventions represent a significant financial and time burden for the individuals with ASD and their families, and only address the accompanying traits of ASD (e.g. anxiety).

There have been numerous human studies undertaken on MDMA intake and social interaction, though most of them have been uncontrolled [34]. In contrast, animal models provide a controlled means to examine the potential impact of pharmacological treatments [35]. This means that the model chosen must hold an empirical and theoretical relationship to autism. The behaviours must be unambiguous and homologous between species (construct validity), the model must resemble autism in its clinical features (face validity), and the model must correctly predict clinical treatments for autism (predictive validity) [36].

An animal model of human psychiatric disease is a more ethically accepted way of exploring neurological aberrational mechanisms and drug treatments, where those drugs have not been approved for human use [37-39]. Rodents (mice and rats) have most commonly been used to model ASD pre-clinically, due to construct and face validity, as well as convenience of use in the laboratory [40, 41]. ASD can be environmentally or genetically induced in them, and treatments are then provided to assess alleviation of those induced ASD traits via specially tailored behavioural assays [42].

As far as we are aware, this would be the first systematic review of the studies encompassing animal and human behavioural effects of MDMA, which are relevant to ASD. We conclude that the prosocial behaviours induced by MDMA may counteract at least the social impairments in autism, at least based on support from animal models.

1.1. 3,4-Methylenedioxymethamphetamine (MDMA)

MDMA is more commonly appreciated as the intended-for-consumption agent in ‘ecstasy’, and is known to have prosocial effects in humans [43-46]. In fact, historically it was used in psychotherapy since the early 1960s [47]. Specifically, MDMA increases emotional empathy and sociability [43, 45]. We stipulate, therefore, that various studies in animal models (and also in humans) may provide insights as to the role of MDMA in ameliorating the pathological lack of sociability featured in some individuals on the autism spectrum. MDMA rodent studies have indeed demonstrated that MDMA increases prosocial behaviour and decreases asocial behaviour, similar to MDMA’s effects on humans [43]. Moreover, a recent pilot trial on humans was fruitful in finding social anxiolytic effects of MDMA [48].

The prosocial effect of MDMA is considered to be mediated primarily by serotonin and oxytocin [44, 49-52]. The primary mechanism of action of MDMA is to act as a substrate for serotonin, norepinephrine, and dopamine monoamine transporters, such that their transport is reversed, thereby their respective synapses are saturated [53]. MDMA’s effects on serotonin release seem most relevant to the oxytocin release and prosocial effects [52] because serotoninergic neurons are known to stimulate oxytocin release [54]. MDMA is, however, also a direct agonist at some serotonin receptors. For example, MDMA has a relatively strong affinity for 5-HT1 and 5-HT2 receptors [55], which may also explain its effects on oxytocin and prosocial behaviour, as oxytocin secretion is mediated mostly by 5-HT1A [52, 56], 5-HT2C, and 5-HT4 receptors [56]. In rat models, MDMA activates oxytocinergic neurons in the supraoptic and paraventricular nuclei of the hypothalamus [52]. This induces oxytocin to be synthesised in these areas and released from the posterior pituitary gland into the peripheral blood [56]. Oxytocin, in turn, is likely to decrease amygdala activation and coupling, which normally triggers fear responses, thereby providing a mechanism for reduced social anxiety [57]. This response can vary between individuals, as one study found that variants of the OXTR gene influences the function/structure of oxytocin receptors on the amygdala, thereby modulating downstream impulses to the brainstem to regulate sympathetic and behavioural fear responses [58]. Single-nucleotide polymorphisms in the OXTR gene have even been detected in autistic individuals) [59-61], further supporting the hypothesis that ASD may be at least in part an issue of decreased empathy and increased anxiety (via oxytocin effects on the amygdala and downstream) [59-61]. Whilst MDMA has not been tested on autistic brains, MDMA has shown decreased amygdalar activity in healthy brains [62-64]. Autism is dependent on synaptic plasticity, and there is also plenty of evidence that MDMA has effects on synaptic plasticity [65-67]. We direct the reader to an excellent review on the molecular effects of MDMA [68], as going more into detail is beyond the scope of this RCT-focused review.

1.2. Aims/rationale

We aim to show that, throughout the animal and human literature, MDMA has been shown to have prosocial effects at certain doses (subject to mode of administration and timing of dosing). Hence, clinical studies using MDMA to alleviate the social impairment in ASD are warranted to help establish MDMA as a clinically approved drug in ASD patients. Of note, MDMA could be able to manage a core impairment constituting ASD, which has never been directly addressed by an approved drug before. We also aim to investigate the animal and human literature to see whether MDMA has had effects on the other two core impairments in ASD, namely stereotypy and cognitive rigidity. This latter goal could shed light on the dose adjustments required to optimally reduce these other impairments in future human clinical studies, as well as acting on the social impairment we focus on in this review. We refer to both acute and chronic administration. Acute means a singular dose that is administered to the test animal or human, whereas chronic means several doses administered to the test animal or human over time. The exact timings of these doses are specified in the tables, alongside their respective study. Testing itself may occur between or after the chronic doses, and this is also specified for each study.

2. METHODS

2.1. Study Design

A systematic review of the literature exploring how MDMA influences the presentation of ASD-like characteristics, in particular social behaviours, in animals and humans, was conducted. Specifically, these are studies in rodent strains without autism-salient mutations or exposures, and humans without autism, as no preceding papers have tested MDMA in organisms with ASD.

2.2. Search Strategy

Six electronic databases including ProQuest, PsychInfo, Scopus, Medline, Web of Science and PubMed (search cut-off date: 21/05/20 inclusive) were consulted. The search strategy, tailored to each database, is detailed in Fig. (2). Only English-language studies were used. The study process is outlined via the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, illustrated in Fig. (1).

Fig. (2).

Search strategy, in each database, for both animal and human studies pertaining to the effects of MDMA on ASD-related behaviour.

Fig. (1).

The study numbers used in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines used for this study, for the animal behavioural tests.

2.3. Study Inclusion and Exclusion Criteria

This systematic review identifies literature examining the effects of postnatal MDMA administrations on the core behaviours affected in ASD, in rats and mice, other animals and humans. This includes MDMA treatment at any age past birth. These include social impairment, repetitive behaviour and cognitive rigidity [1, 2]. These diagnostic criteria are taken from DSM-5 and ICD-11, the current gold-standard reference texts for psychological disorders [1, 2] . To this end, the following behavioural tests have been included for rodents: 1) ultrasonic-vocalisation; 2) social-preference; 3) social-novelty-preference; 4) social-interaction; 5) open-field; 6) T/Y-maze; 7) marble-burying; and 8) novel-object-recognition. This review places no limits on age, species or cognitive abilities of the subjects. The inclusion criteria were behaviours core to ASD, behavioural experiments on animals, the use of MDMA to affect the behavioural results, and placebo-controlled studies. The exclusion criteria were applied to papers that only assessed the accessory behaviours to ASD and behaviours not related to ASD, drug self-administration or reinforcement studies, prenatal MDMA administration, or behaviours associated with disorders other than ASD (including Rett syndrome), drugs other than MDMA, uncontrolled studies, and serotonin syndrome. With rodents, approximately 3573 studies were screened, and 127 studies finally reviewed. With other animals, approximately 3456 studies were screened, and 16 studies finally reviewed. With humans, approximately 6205 studies were screened, and 49 studies finally reviewed. “Healthy” human subjects refer to non-autistic patients, whether MDMA-experienced or -naïve, and whether diagnosed with other psychological conditions or not. All were given singular or chronic MDMA doses.

2.4. Data Extraction and Synthesis

2.4.1. Rodent and Non-rodent Animals

From animal studies, the data extracted were as follows: species and sex tested, sample size, dosage timing and frequency, dosage mass and route, treatment and testing ages, ambient temperature (as temperature was shown to influence behavioural outcomes with MDMA) [69], and results of the experimental animals as compared to the control group. The findings are summarised in Tables 1-9. In this review, dosage routes have been categorised as singular (only one dose for the subjects) and chronic (multiple doses for a subject). It is worth noting that some papers use the term, “sub-chronic”, to indicate multiple doses. Initial extraction of the data revealed that MDMA generally serves to increase intra-species sociable behaviours in rodents.

2.4.2. Humans

From human studies, the data extracted were as follows: the method of recruitment of the participants, the population type the participants were selected from, number of participants, sex ratio, mean age of the participants, the oral dose of MDMA administered (this was the only administration route used in humans), timing of doses administered, the time between MDMA intake and the first core-ASD-relevant measurement made, the relevant tests undertaken, the relevant results obtained, and the exact parameters taken into account for each relevant test. The findings are summarised in Tables 10 and 11. Initial extraction of the data revealed that MDMA generally serves to increase social behaviour and altruistic feelings in humans.

2.4.3. Interspecies Focus

We take an inter-species perspective in investigating these behavioural effects of MDMA, in the form of a systematic literature review. This is because it is our intention to attempt to translate laboratory-controlled animal studies to their potential impact in humans, where such rigorous techniques cannot be trialled in humans directly [70].

3. RESULTS

Below, we summarise the general trends of behavioural effect that MDMA has had on animals and humans in the laboratory (placebo-controlled) experiments. Where we state “chronic” doses, these are doses delivered more than once. The timings of these dosages are specified for each study in the tables below. The act of repetitive grooming, whether self- or allo-grooming, is classed as repetitive behaviour in these studies, both by the respective study authors and ourselves, but we consider them also as possible signs of cognitive rigidity.

3.1. Core ASD Behaviour 1: Social Impairment

3.1.1. Rodents

There are 4 major tests, currently in the literature, testing social and communication behaviour among mice and rats of different strains. These are the ultrasonic-vocalisation test, the social-preference test, the social-novelty-preference test and the social-interaction test [71-73]. We have also included a novel-object-recognition test here, to see whether the behavioural effects seen in the social-novelty-preference test are dependent on social or general novelty. In the studies reviewed, a “conspecific” will be mentioned, which is a member of the same species (in these cases, a mouse or rat). Below, we summarise each of these tests along with the MDMA-induced alterations on the respective induced social impairments in rodents.

Each study is shown alongside the species, sex and sample size of the rodents tested (control and relevant treatment groups), whether the study used singular or chronic (multiple) dosing, and if they used chronic dosing, the timing of the doses; the dose and route (mode of injection) used, the ages the rodents were treated and tested at, the temperature of the testing environment, and the results obtained from the experiment in the treated rodents (where it is different from the results in the corresponding control rodents). s.c. = subcutaneous mode of injection.

Each study is shown alongside the species, sex and sample size of the rodents tested (control and relevant treatment groups), whether the study used singular or chronic (multiple) dosing, and if they used chronic dosing, the timing of the doses; the dose and route (mode of injection) used, the ages the rodents were treated and tested at, the temperature of the testing environment, and the results obtained from the experiment in the treated rodents (where it is different from the results in the corresponding control rodents). The studies used singular dosing. i.p. = intraperitoneal mode of injection.

Each study is shown alongside the species, sex and sample size of the rodents tested (control and relevant treatment groups), whether the study used singular or chronic (multiple) dosing, and since they used chronic dosing, the timing of the doses; the dose and route (mode of injection) used, the ages the rodents were treated and tested at, the temperature of the testing environment, and the results obtained from the experiment in the treated rodents (where it is different from the results in the corresponding control rodents). i.p. = intraperitoneal mode of injection.

3.1.1.1. Ultrasonic-vocalisation Tests

The ultrasonic-vocalisation test is performed prior to weaning of the pup, and is a measure of the pup’s ultrasonic vocalisation (USV) ability in response to being separated from its mother [74]. It is believed that the purpose of these distress calls is to induce the mother to retrieve the pup back to its home litter [75]. The normal USV emission by maternally separated rodents is shown to be increased in the rate of calls on PD 6-12, which persists till PD 15 and then declines [76]. Teratogenic agents administered in utero may result in deviations from this pattern as well as the sounds therein. In ASD rodents, MDMA caused the abnormal calls to return to having the normal decrease and subsequent increase in call rate that is observed in healthy rodents within a day [76].

We found one study where MDMA was given post-natally, and the pups were measured for vocalisation ability upon maternal separation (Table 1). The study shows that when 10 mg/kg MDMA is injected subcutaneously singularly to pups pre-weaning, call frequency (the number of calls emitted) decreases initially, then increases later within a day [76]. When the same is given multiple times at an earlier age, later ages show decreased frequency in proportion to dose [76].

Table 1.

Ultrasonic vocalisations (upon maternal separation) from postnatally MDMA-treated rodents, compared with control rodents.

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Males	Females	Both	No. Pups per Mother
Winslow 1990 [76]	Rat	Both	Combined	42	Singular	NA	0.5-10	s.c.	9-11	9-11	24	NA	NA	Decreased rate of pup calls, increased locomotion. 10 mg/kg: call frequency decreased at 0.5 and 3 hours post-injection, increased at 10 and 24 hours post-injection.	9-12
Winslow 1990 [76]	Rat	Both	Combined	36	Chronic	7 injections in total	10	s.c.	1-4	6, 9, 12, 15	24	NA	NA	PD 9-15: dose-dependent long-lasting decreased call rate	9-12

3.1.1.2. Social-preference Tests

The social-preference test assesses a rodent’s preference to spend time with a conspecific vs. an inanimate object. Thus, the apparatus for the test consists of three chambers: a “home base” and two flanking chambers containing either a caged unknown sex- and age-matched conspecific or an “object”. Control rodents usually spend more time in and make more entries into, the chamber with the caged conspecific than the chamber with the empty cage, indicating social preference. Studies directly take this as a social-preference index, being the ratio of time spent in the conspecific chamber over either the time spent in the object chamber or the total time spent in all chambers. We found three studies where 3-15 mg/kg MDMA, given once intra-peritoneally approximately on PD 56-84, acutely increased rodent social preference (Table 2).

Table 2.

Social preferences in postnatally MDMA-treated rodents, compared with control rodents.

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Males	Females	Both
Heifets 2019 [187]	Mouse	Both	NA	36-80	Singular	NA	3, 7.5, 15	i.p.	NA	NA	NA	NA	NA	7.5 and 15 mg/kg: dose-dependently increased
Kuteykin-Teplyakov 2014 [188]	Mouse	Males	NA	59	Singular	NA	2, 3, 4, 6	i.p.	56-84	56-84	21	3 mg/kg: increased	NA	NA
Ramos 2016 [189]	Rat	Males	NA	64	Singular	NA	2.5, 5	i.p.	“Adult” (250-300g)	“Adult” (250-300g)	21	Well-handled, 5 mg/kg: increased. Minimally handled, 5 mg/kg: no effect. 2.5 mg/kg: no effect.	NA	NA

3.1.1.3. Social-novelty-preference Tests

The social-novelty-preference test assesses a rodent’s preference to spend time with an unfamiliar conspecific over a familiar conspecific. The test uses the same set-up as the social-preference test, but the object (empty cage) is replaced by a new sex- and age-matched conspecific (the unfamiliar). The familiar is the previously encountered conspecific in the opposite flanking chamber. Studies directly take this as a social-novelty-preference index, being the ratio of time spent in the unfamiliar chamber over either the time spent in the familiar chamber or the total time spent in all chambers. We found one study where 5 or 10 mg/kg MDMA, given chronically intraperitoneally on PD 28-52, increased mouse social-novelty preference when tested later on PD 120 (Table 3). This therefore, also shows a long-lasting prosocial effect of the MDMA, which would increase the value of MDMA as a treatment.

Table 3.

Social-novelty preferences in postnatally MDMA-treated rodents, compared with control rodents.

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Tempera- ture (°C)	Males	Females	Both
Morley-Fletcher 2002 [190]	Mouse	Both	Separated	NA	Chronic	Once on each of 3 days, 2 days apart for each	5, 10	i.p.	28, 38, 52	120	21	Increased	Increased	NA

3.1.1.4. Novel-object-recognition Tests

The novel-object-recognition test assesses novelty preference and memory for a new object replacing an existing familiar object. We have classed this in the “social impairment” tests in order to compare with the “social-novelty preference” tests. In control rodents, the norm is to spend more time with the unfamiliar than the familiar object, indicating a natural curiosity to explore the unexplored, and is indicative of an intact representation that the existing environment has changed.

We found 33 studies where MDMA was given mostly chronically postnatally, and the rodent offspring were measured for their tendency to spend more time exploring the unfamiliar/novel object than the familiar (Table 4). The studies show that giving mostly chronic 5-10 mg/kg MDMA, intraperitoneally or subcutaneously, actually exacerbates this loss in the rodent’s exploratory discrimination between the two objects, which is in contrast to our expected effect on social impairment (see social-novelty-preference test). This may confirm that if there is disinterest in both social and non-social novelty, this may indicate cognitive rigidity as opposed to social impairment, because MDMA appears to exacerbate cognitive rigidity (see below). Interestingly, one study shows that a higher temperature (28°C) exacerbates this supposed memory deficit more than a cooler temperature (16^oC) [77].

Table 4.

Novel-object preferences in postnatally MDMA-treated rodents, compared with control rodents.

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Males	Females	Both
Abad 2014 [191]	Rat	Males	NA	14-18	Chronic	Twice daily for 4 days	20	s.c.	35	7 days after treatment ended	26	No effect	NA	NA
Abad 2016 [192]	Rat	Males	NA	14-18	Chronic	3 times a day, every 3 hours, once a week for 2 weeks	5	NA	28-49	28-49	26	Reduced	NA	NA
Abad 2016 [192]	Rat	Males	NA	14-18	Chronic	3 times a day, every 3 hours, once a week for 2 weeks for another 3 weeks	7.5	NA	49-70	49-70	26	Reduced	NA	NA
Abad 2016 [192]	Rat	Males	NA	14-18	Chronic	3 times a day, every 3 hours, once a week for 2 weeks for another 3 weeks	10	NA	70-91	70-91	26	Reduced	NA	NA
Able 2006 [193]	Rat	Males	NA	40	Chronic	Once every 2 hours, for 4 doses on 1 day	15	s.c.	225-250g	5 days later	22	No effect	NA	NA
Adeniyi 2016 [194]	Mouse	Males	NA	10	Chronic	5 times over 10 days, at 2-day intervals	2	s.c.	21	31, 32	NA	Reduced	NA	NA
Bubenikova-Valesova 2010 [195]	Rat	Males	NA	NA	Chronic	4 days	2.5, 5	s.c.	NA	NA	NA	No effect	NA	NA
Clemens 2007 [136]	Rat	Females	NA	16	Chronic	1 injection per week, for 16 weeks	8	i.p.	238g	120 days later	28	NA	No effect	NA
Cohen 2005 [196]	Rat	Males	NA	30	Chronic	Twice a day (8 hours apart)	20	s.c.	11-20	40-49	21	Reduced	NA	NA
Cohen 2005 [196]	Rat	Males	NA	30	Chronic	4 times, at 2 intervals	15	s.c.	82-100	131-129	21	No effect	NA	NA
Edut 2011 [197]	Mouse	Males	NA	34	Singular	NA	10	i.p.	25-30g	“Juvenile”	23	No effect	NA	NA
Edut 2014 [198]	Mouse	Males	NA	18-26	Singular	NA	10	i.p.	25-30g	7, 30 days later	23	No effect	NA	NA
García-Pardo 2017 [94]	Mouse	Males	NA	45	Singular	NA	5, 10	i.p.	42	63, 64	35-37	Reduced	NA	NA
Llorente-Berzal 2013 [199]	Rat	Both	Separated	55	Chronic	Every 5 days, twice daily (4 hours apart)	10	s.c.	30-45	75	22	No effect	No effect	NA
Ludwig 2008 [200]	Rat	Males	NA	39+	Singular	Single injection	5	s.c.	“Adult” (242-275g)	7 days later	NA	No effect	NA	NA
Ludwig 2008 [200]	Rat	Males	NA	39+	Chronic	5 daily injections	5	s.c.	“Adult” (242-275g)	7 days later	NA	No effect. Multiple doses: increased	NA	NA

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Males	Females	Both
Ludwig 2008 [200]	Rat	Males	NA	39+	Both	Chronic, then single	5	s.c.	“Adult” (242-275g)	7 days later	NA	Multiple doses: increased	NA	NA
McGregor 2003 [77]	Rat	Males	NA	32	Chronic	2 consecutive days, every hour for 4 hours	5	i.p.	60-75	70-84 days later	28, 16	28°C: reduced	NA	NA
Meyer 2008 [201]	Rat	Males	NA	NA	Chronic	2 doses, 4 hours apart	10	s.c.	35, 40, 45, 50, 55, 60	35, 40, 45, 50, 55, 60	22-23	Reduced	NA	NA
Nawata 2010 [202]	Mouse	Males	NA	10-30	Singular	NA	10	i.p.	30-35g	30-35g	23	No effect	NA	NA
Nawata 2010 [202]	Mouse	Males	NA	10-30	Chronic	Once daily for 7 days	10	i.p.	30-35g	30-35g	23	Reduced	NA	NA
Piper 2004 [203]	Rat	Males	NA	16	Chronic	On every 5th day, twice daily, intervals of 4 hours	10	s.c.	35-60	65	22	Reduced	NA	NA
Piper 2005 [204]	Rat	Males	NA	20	Chronic	Hourly intervals over 4 hours, once every 5 days	5	s.c.	35-60	67, 68, 69	22	Reduced	NA	NA
Piper 2008 [205]	Rat	Males	NA	20-24	Chronic	4 doses, 1 each hour	10	s.c.	Young adult (307.7g)	Young adult	23	Reduced	NA	NA
Rodsiri 2011 [206]	Rat	Males	NA	21-24	Chronic	Every 2 hours over 6 hours (3 injections)	3, 6	i.p.	100-130g	14 days later	21	3 × 6 mg/kg: reduced. 6 mg/kg: no effect	NA	NA
Ros-Simo 2013 [207]	Mouse	Males	NA	20-24	Chronic	Twice, 6 hours apart	20	i.p.	25	28	22	Reduced, long-term	NA	NA
Schulz 2013 [208]	Rat	Males	NA	24	Chronic	Once daily for 10 days, twice daily (4 hours apart) for 5 days	7.5	s.c.	“Adult” (230-300g)	40-65, 80-105	22	Reduced	NA	NA
Shortall 2012 [209]	Rat	Both	Combined	16	Chronic	Once daily for 7 days	5	i.p.	170-205g	7 days later	NA	NA	NA	No effect
Shortall 2013 [210]	Rat	Males	NA	12-16	Chronic	2 consecutive days a week, for 3 weeks	10	i.p.	Young adult	Young adult	21	Reduced	NA	NA
Skelton 2008 [211]	Rat	Males	NA	63	Chronic	4 per day (2 hours apart), 1 day per week, 5 weeks (1-week intervals)	15	s.c.	225-250g	35-39 days later	22	No effect	NA	NA
van Nieuwenhuijzen 2010 [212]	Rat	Males	NA	24	Chronic	Daily over 10 days	5	i.p.	220-300g	220-300g	21	Reduced	NA	NA
Vorhees 2007 [213]	Rat	Both	Separated	160	Chronic	Every 2 hours, each day	40 once a day, 20 twice a day, or 10 four times a day	s.c.	11-20	64-68	21	No effect	No effect	NA
Vorhees 2009 [214]	Rat	Both	Separated	NA	Chronic	4 doses, every 2 hours each day	10, 15, 20, 25	s.c.	1-20	60	21	No effect	No effect	NA

3.1.1.5. Social-interaction Tests

The social-interaction test is a simple and direct test of social interaction. The test rodent and a conspecific of the same treatment group are placed in an open arena and their behaviour monitored. Different behavioural acts are measured, and specialist terms are listed in Table 5.1.

We found 59 studies where MDMA was given singularly or chronically postnatally, and the rodents observed for their inter-conspecific social interactions (Table 5). The studies show that singular doses of 5-10 mg/kg MDMA, given intraperitoneally, increases prosocial behaviour and decreases asocial behaviour. Interestingly, at these same doses, chronic dosing has the opposite effect: decreasing prosocial behaviour and increasing asocial behaviour (Table 5). One study was an exception to this [78], where 5-20 mg/kg MDMA given twice daily over 3 days actually increased the duration of social investigation. On either side of the dosage window, 5-10 mg/kg MDMA, this opposite effect is also seen: decreased prosocial behaviour and increased asocial behaviour (Table 5).

Table 5.

Social-interaction behaviours in postnatally MDMA-treated rodents, compared with control rodents.

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Social Restriction	Males	Females	Both
Ando 2006 [87]	Rat	Males	NA	34	Either	1 or 2 days (49 days apart)	15	i.p.	49-56	49-56	21	Nil	Decreased regular total social-interaction time, locomotion, self-grooming, rearing. Increased adjacent-lying time (more than social-interaction decrease).	NA	NA
Ando 2006 [87]	Rat	Males	NA	34	Either	1 or 2 days (49 days apart)	15	i.p.	49-56	70-77	21	Nil	3 weeks later, dosed on 1st day: increased aggression, rearing and line crossings. Acute, dosed on 2nd day: increased total time of social behaviour (adjacent lying), total number of non-social activities and line crossings.	NA	NA
Ando 2010 [215]	Rat	Males	NA	24	Singular	NA	15, 30	i.p.	42-49	222-229	21	Nil	Fewer line crossings. No change in duration or number of social interactions, aggressive behaviours.	NA	NA
Bull 2003 [133]	Rat	Males	NA	24	Chronic	Twice daily	15	i.p.	28-30	50	21	Nil	Reduced total social interaction.	NA	NA
Bull 2003 [216]	Rat	Both	NA	NA	Chronic	Four times daily, for 2 consecutive days	5	i.p.	28	84	NA	Nil	After 54 days' abstinence, social interaction decreased by 27%	NA	NA
Bull 2004 [132]	Rat	Males	NA	32	Chronic	Each of 4 hours	5	i.p.	28	84	21	Nil	Increased social anxiety, decreased total social interaction.	NA	NA
Cagiano 2008 [84]	Rat	Males	NA	90	Singular	NA	0.3, 1, 3	i.p.	“Adult”	“Adult”	20-22	Nil	3 mg/kg: increased time till intromission and ejaculation, decreased copulatory activity.	NA	NA
Clemens 2004 [217]	Rat	Males	NA	24-30	Chronic	4 injections in 1 day, 2 hours apart	2.5, 5	i.p.	376g	28 days later	28	Nil	Increased total locomotor activity. 5 mg/kg: reduced social-interaction time.	NA	NA
Clemens 2005 [218]	Rat	Females	NA	16-18	Chronic	Every 2 hours in 1 day, 4 times	4	i.p.	281g	42 days later	28	Nil	NA	Less social interaction.	NA
Clemens 2007 [136]	Rat	Females	NA	32	Chronic	1 injection per week for 16 weeks	8	i.p.	238g	49 days later	28	Nil	NA	Decreased total social-interaction time.	NA
Clemens 2007 [136]	Rat	Females	NA	16	Chronic	1 injection per week for 16 weeks	8	i.p.	238g	113 days later	28	Nil	NA	Reduced social-interaction time.	NA

Interestingly, one study saw a difference in MDMA’s effects on aggressive vs. timid mice: whilst MDMA increased timidity in both types of mice, MDMA increased social behaviour in aggressive mice, and decreased social behaviour in timid mice [79]. Timidity was determined in a preliminary interaction test where mice would be defined as “timid” if they showed no attack, but significant defensive-escape behavior, even in the absence of partner aggression, when placed in a neutral arena with a conspecific [79]. If timidity is a factor in influencing sociability, this finding would make sense, since excessively timid mice would not be inclined to socialise, and aggressive mice without tempering with timidity would not be inclined to socialise. However, timid rats were more likely to become aggressive when a young intruder is introduced into the cage, when given MDMA chronically at 6 mg/kg [80].

Each study is shown alongside the species, sex and sample size of the rodents tested (control and relevant treatment groups), whether the study used singular or chronic (multiple) dosing, and if they used chronic dosing, the timing of the doses; the dose and route (mode of injection) used, the ages the rodents were treated and tested at, the temperature of the testing environment, and the results obtained from the experiment in the treated rodents (where it is different from the results in the corresponding control rodents). i.p. = intraperitoneal mode of injection; s.c. = subcutaneous mode of injection.

Each study is shown alongside the species, sex and sample size of the rodents tested (control and relevant treatment groups), whether the study used singular or chronic (multiple) dosing, and if they used chronic dosing, the timing of the doses; the dose and route (mode of injection) used, the ages the rodents were treated and tested at, the temperature of the testing environment, the duration of social restriction (isolation) enforced before testing, and the results obtained from the experiment in the treated rodents (where it is different from the results in the corresponding control rodents). i.p. = intraperitoneal mode of injection; s.c. = subcutaneous mode of injection.

There have also been temperature, inter-sex-interaction and group effects seen. Similar to human studies with MDMA, a study found that the higher temperature increased social interaction more than the lower temperature [69]. In fact, some studies deliberately increased the testing temperature to 28^oC, in order to amplify these social effects [81-83]. Only one study investigated inter-sex interactions, and found that singular 3 mg/kg MDMA decreased the inclination to copulate [84], similar to the doses below 5 mg/kg MDMA, which decreased prosocial behaviour. A study also found that group-housed mice became more physically active, more so than single-housed mice, when given MDMA; and that social interaction duration was increased in the group-housed and not the single-housed group [85]. This is similar to human studies where prosocial effects of MDMA appear enhanced when taken in groups of people [86].

Some interesting effects are noted as follows: one study found a difference between acute and chronic effects of MDMA: when tested on the same day, prosocial behaviour was increased in MDMA-treated rats; when tested 3 weeks later, asocial behaviour was increased a day later, but when dosed again, it acutely increased prosocial behaviour [87]. One study’s singular 8 mg/kg dose caused decreased social exploration [88], another study’s singular 5-10 mg/kg dose also decreased social exploration [89], and another’s singular 5 mg/kg increased asocial behaviours [90]. Notably, these studies socially isolated their animals for 30 days prior to testing, to increase aggressive behaviour [88-90]. Perhaps, in these cases, MDMA’s effects could not entirely overcome that increased aggressive behaviour. Some studies had a significant time delay between testing and treating: 65, 70 and 21 days [91-93], with surprising results of decreased prosocial behaviour [91-93] and increased asocial behavior [93]. One study used social defeat as a variable to influence social interaction, where they used 30 mins. of isolating the test rodent before testing, to induce aggression during testing [94]. Three other studies gave surprising results: social and prosocial behaviours were decreased when MDMA was given at a singular 5-10 mg/kg [95-97].

To summarise the social behavioural tests in rodents, USV calls increased gradually in emission rate when 10 mg/kg MDMA is given singularly or chronically postnatally (Table 1). Rodents’ preference to spend time with a conspecific over an object, or an unfamiliar conspecific over a familiar, increases when given 5-10 mg/kg MDMA singularly or chronically postnatally (Tables 2, 3). Social behavioural results are mixed between the studies, when 5-10 mg/kg MDMA is given singularly or chronically postnatally (Table 5). The literature also shows reports of chronic effects, whereby MDMA-treated rodents retain these prosocial effects (Tables 3, 4, 5).

3.1.2 Other Animals

Most of the non-rodent animal studies focused on social behaviour after MDMA treatment. Since dosage effects are different between species, we will observe each animal model in turn. Four studies tested the behavioural effects of MDMA on monkeys [98-101]. They found that chronic administration of 1.5 mg/kg s.c. [98], or singular dose of 0.03-3 mg/kg i.m. [100] increased prosocial behaviour. Interestingly, Pitts et al. (2017) showed that with lower doses, it was the S(+) MDMA enantiomer that increased this gregariousness more, and at higher doses, the R(−) MDMA enantiomer increased affiliative behaviour more. Chronic 1.5 mg/kg (p.o. and especially i.m.) increased vigilance, indicating perhaps decreased social trust [101]. But the studies also found that chronic 12 mg/kg p.o. decreased vocalisations [99], and that both 12 mg/kg p.o. and 20 mg/kg i.g. [102] induced serotonin behaviour, indicating this range was an unnecessarily high dose.

Four studies examined ASD-specific behaviour in MDMA-treated fish, in particular social behaviour [103-106]. They found that when 1 or 5 mg/kg MDMA was singularly intramuscularly injected into electric fish, asocial behaviour decreased and prosocial behaviour increased [103]. Interestingly, in zebrafish, singularly immersing the animals in 80 mg/L actually increased the distance between adjacent fish and shortened the duration spent near each other [104]. This may be explained by the fact that in one study, which tested for general anxiety in zebrafish, immersing the animals in 0.25-120 mg/L increased the chance of the fish entering and dwelling at the top of the fish tank [107], indicating reduced anxiety. This is because fish tend to become anxious towards the water’s surface as there is a greater risk of predation there [108, 109], and shoaling behaviour is generally a result of alarm pheromones dissipated among the fish [104, 110]. Ponzoni et al. (2016, 2017) found that a singular intramuscular injection of 0.1-10 mg/kg MDMA increased social preference [105, 106]. Two studies looked at social behaviour in singularly MDMA-immersed octopodes [111, 112]. They found that when octopodes were doused in 0.5-0.005 mg/kg MDMA solution, not only did social preference increase [111, 112], but so did voluntary body contact between the animals [112].

3.1.3 Humans

The human studies tested MDMA effects on non-autistic subjects, except for one study which looked specifically at the effects of MDMA on social anxiety in autistic adults [48]. In this study, autism was diagnosed if the subject fulfilled Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders- Fourth Edition Axis I Research Version (SCID-I-RV) or Autism Diagnostic Observation Schedule (ADOS-2 Module 4) criteria. The assessments in all studies were between subjects, where one group were given MDMA and the other placebo. Most studies assessed mostly chronically given 1.5 mg/kg (often by an absolute dose of 100 mg) which may increase the likelihood of finding positive results. We assume a linear positive relationship (gradient: 1.5 mg/kg per 100 mg; intersect: 0 mg/kg = 0 mg) between mg/kg and mg, and convert absolute doses to per kg of participant body weight accordingly. Each study had its own method of measuring social outcomes, therefore classifying into types of tests (as able to be done for the rodent model) would be inaccurate. Specific details of these tests are therefore listed in the tables for each respective study.

As shown in Table 10, all doses increased prosocial behaviour. This includes mostly self-perceptions of extroversion, openness, sociability, talkativeness, thoughtfulness or caring, sensitivity, friendliness, insightfulness of others, gregariousness or desire to be with others, empathy, lovingness, playfulness, closeness to others. Interestingly, social interaction increased more at 0.5 mg/kg than 1 mg/kg in one study [86], and playfulness and lovingness increased more at 1.5 than 0.75 mg/kg (chronic dosing).

Table 10.

Social behaviour in postnatally MDMA-treated subjects, compared with placebo-treated subjects.

Study	Recruitment		Population Sampled		No. Participants		Sexes		Mean Ages (years)			Oral MDMA Dose		Dosage Timing		Time till First Test (after baseline)	Relevant Tests	Relevant Results	Relevant Parameters Tested
Baggott 2016 [284]	Newspaper, online adverts, word of mouth		Healthy, MDMA-experienced		12		6 female, 6 male		29			1.5 mg/kg		Singular		1.5 hours	Self-reports of social anxiety and interpersonal functioning	Decreased social anxiety; increased affiliative (nurturance/communion) feelings	Apprehension towards being judged; dominance/agency and nurturance/communion
Baggott 2015 [285]	Newspaper, community bulletin board, online adverts		Healthy, MDMA-experienced, Caucasian		35		12 female, 23 male		24.3			1.5 mg/kg		Singular		4 hours	Verbal recounts of significant others and own emotions	Increased words with social/sexual content. No sex differences. Increased words with factual, decreased words with psychological, content about target person. No changes in phrases referring to relationship to target person, decreased words relating to body of target person and increased words related to cognition and insight	Names of 3 personally important people, altered use of words, discussion topics, number of words spoken, isolated descriptions of target person, proportion and number of phrases describing relationship to target person, words relating to target person's body vs. cognition and insight, drug-related emotions (including social); words with social, negative and positive valence
Bajger 2015 [286]	Word of mouth, online and newspaper adverts, poster flyers		Black, Hispanic, white		12		3 female, 9 male		28.9			50, 100 mg (~0.75, 1.5 mg/kg)		Twice daily over 5 days		60 mins.	Self-reports of affect	Repeated dose: increased gregariousness, decreased preference for solitude, 100 mg: increased time spent verbally communicating with others	Private and social (talking and silence) time, mood, preference for solitude or gregariousness, effects of singular vs. repeated doses
Bedi 2014 [287]	Adverts		Healthy, ecstasy-experienced, mostly Caucasian		13		4 female, 9 male		24.5			0.75, 1.5 mg/kg		Singular		Mood: 65 mins.; speech: 130 mins.	Verbal recounts of significant others	1.5 mg/kg: words had increased proximity to “friend”, “support”, “intimacy”, “rapport”. 0.75 mg/kg: words had increased proximity to “empathy”	Degrees of “compassion”, “empathy”, “forgive”, “friend”, “intimacy”, “love”, “rapport”, “support”, “talk”, “confident”
Bedi 2010 [288]	Internet adverts, word of mouth		Healthy, ecstasy-experienced, mostly Caucasian		21		9 female, 12 male		24.4			0.75, 1.5 mg/kg		4 times, 5/+-day intervals		Mood: 0; emotion recognition: 65 mins.	Self-reports of mood, emotion-recognition tasks	1.5 mg/kg: increased ratings of “loving”, “friendly”, “playfulness”, decreased accuracy of facial fear recognition in others, increased likelihood of misclassifying emotional expressions as neutral. 0.75 mg/kg: increased ratings of “loneliness”	Moods: “Sociable”, “Playful”, “Loving”, “Lonely”. Friendliness: “‘friendly”, “agreeable”, “helpful”, “forgiving”, “good-natured”, “warm-hearted”, “good-tempered”, “kindly”. Facial cues: anger, fear, happiness, sadness Vocal tones: happy, sad, angry, fearful; with high/low emotional intensities
Bedi 2009 [64]	Online adverts, word of mouth		Healthy, right-handed, ecstasy-experienced, mostly Caucasian		9		2 female, 7 male		24			0.75, 1.5 mg/kg		3 sessions, 6/+-day intervals, doses in ascending order		Mood: 0; emotion recognition: 45 mins.	Mood, facial emotion recognition	Increased ratings of sociability, no effect on accuracy of emotion recognition	Moods: sociability and friendliness. Facial emotion recognition (happy, neutral, angry, fearful) for “pleasant”, “neutral”, “unpleasant”
Study	Recruitment		Population Sampled		No. Participants		Sexes		Mean Ages (years)			Oral MDMA Dose		Dosage Timing		Time till First Test (after baseline)	Relevant Tests	Relevant Results	Relevant Parameters Tested
Bershad 2017 [289]	Newspaper, bulletin board, online adverts		Healthy, ecstasy-experienced		39		9 female, 30 male		24.1			0.5, 1 mg/kg		Twice (same dose), 7 days apart		60 mins.	Self-reports of moods, public-speaking task	1 mg/kg: increased stress, tension, insecurity. 0.5, 1 mg/kg: increased perceptions of public speaking being challenging and threatening	Self-ratings of stress, tension, insecurity; stressfulness and challenge of public speaking
Bershad 2016 [290]	Flyers, online adverts, word of mouth		Healthy, Caucasian, occasional MDMA users		68		29 female, 39 male		23.8			0.75, 1.5 mg/kg		3 sessions, 5/+-day intervals		30 mins.	Self-reports of mood	Increased sociability	Degrees of “lonely”, “sociable,” “loving,” “playful,” “friendly”, “confident”
Bershad 2019 [291]	University campus and surrounds		Healthy, MDMA-experienced		36		18 female, 18 male		24.8			0.75, 1.5 mg/kg		4 sessions, 2/+ days apart		30 mins.	Subjective ratings of mood states and touch stimuli, psychophysiological responses, visual attention to emotional faces	Increased feelings of being “insightful”, “playful”; dose-dependently increased ratings of pleasantness of experienced affective touch; 1.5mg/kg: MDMA increased total number of times participants looked toward happy faces; increased zygomatic activity (smiling when looking at others affectively touching)	“Sociable,” “Confident,” “Lonely,” “Playful,” “Loving,” “Friendly,” and ‘Restless”; “pleasantness” upon being physically touched or observation of physical touching between others; activity of zygomatic (smiling) and corrugator (frowning) muscles; eye movements for attention bias
Cami 2000 [292]	Word of mouth		MDMA-experienced, healthy, mostly smokers		8		Male		26.5			75, 125 mg (~ 1.1, 1.9 mg/kg)		4 sessions, 1/+-week intervals		1 hour	Mood states	No effect on friendliness	Self-ratings of friendliness
Clark 2015 [148]	Flyers, posters, word of mouth		Mostly Caucasian		33		16 female, 17 male		24.5			0.75, 1.5 mg/kg		Singular		30 mins.	Subjective drug effects	Increased prosocial feelings of “loving” and “insightful”	Self-ratings of “playful,” “loving”, “insightful”
Corey 2016 [114]	Psychotherapy patients		Caucasian, PTSD patients		20		18 female, 5 male		40.5			125 mg (~1.9 mg/kg), 62.5 mg/kg		1-3 times		Large dose, then smaller dose after 2 hours	Talk sessions	Increased ensuic, empathic, entactic utterances	Utterances where patients initiated empathic, entactic or ensuic topics
Danforth 2018 [48]	Internet adverts, word of mouth, clinician referrals		Healthy, MDMA-naïve		12		16.7% female (all MDMA), 83.3% male		31.3			75, 100, 125 mg (~ 1.1, 1.5, 1.9 mg/kg)		Twice, 1-month interval		1 day	Social anxiety	Decreased social anxiety, also at 6-month follow-up	Change from baseline, of social anxiety scores, over time
de Wit 2011 [293]	Online adverts, word of mouth		MDMA-experienced polydrug users, mostly Caucasian smokers		9		2 female, 7 male		24			0.75, 1.5 mg/kg		Ascending order, 6 days apart		0	Mood states, emotion recognition	1.5 mg/kg: increased sociability (and insignificant trend towards friendliness). No effect on emotion-recognition time or accuracy	Sociability and friendliness, emotion-recognition time and accuracy
Dolder 2018 [294]	University		Mostly MDMA-naïve		24		12 female, 12 male		22.6			125 mg (~ 1.9 mg/kg)		4 sessions, 7/+-day intervals		Mood 0; emotion recognition: 2.5 hours	Mood states, facial emotion recognition	Increased “happiness”, “open”, “trust”, “feeling close to others”, “I want to be with other people”, “I want to hug someone”, “well-being”, “emotional	“Happy,” “concentration,” “open,” “trust,” “feeling close to others,” “I want to be with other people,” “I want to hug someone”,
Study	Recruitment		Population Sampled		No. Participants		Sexes		Mean Ages (years)			Oral MDMA Dose		Dosage Timing		Time till First Test (after baseline)	Relevant Tests	Relevant Results	Relevant Parameters Tested
-	-		-		-		-		-			-		-		-	-	excitation”, “extraversion”, “introversion”, impaired recognition of fearful faces, increased misclassification of emotions as happy, increased sexual measures - “tingly all over,” “sensitive to touch,” “enthusiastic,” “warm all over,” “flushed,” “heart beats faster,” “seductive”, “enthusiastic,” “warm all over,” “passionate,” “sensual,” “pleasure,” “heart beats faster,” “happy,” “powerful,” “forget about all else”, “anticipatory”	sexual - “tempted”, “passionate”, “seductive”, “attractive”, “sensitive to touch”, “stimulated”, “excited”, “heart beats faster”, “anxious”, “displeasure”, “repulsion”, “angry”, “driven”, “urge to satisfy”, “horny”, “impatient”. Accuracy for degrees of happiness, sadness, anger, and fear facial expressions, misclassification of expressions as neutral
Dumont 2009 [44]	Internet and drug-testing service adverts		Healthy, MDMA-experienced		14		3 female, 12 male		21.1			100 mg (~1.5 mg/kg)		7-day interval		0	Subjective amicability and gregariousness	Subjective amicability were positively correlated with MDMA concentrations, but subjective gregariousness was not. (NB: Both subjective amicability and subjective gregariousness were positively correlated with oxytocin concentrations; measures had stronger correlations with oxytocin than MDMA).	Measures of antagonistic/amicable, withdrawn/gregarious
Frye 2014 [145]	Flyers, online adverts		Healthy, MDMA-experienced, mostly Caucasian		36		18 female, 18 male		24.6			0.75, 1.5 mg/kg		3 sessions, 96/+-hour intervals		Mood: 30 mins.; social rejection: 2.75 hours	Mood effects, reactions to social-rejection simulation, correct perceptions of social-exclusion manipulations	Increased “loving” (before and after social-rejection paradigm). Reduced decreasing effect of rejection on mood and self-esteem. Increased perceived percentage of inclusive throws under rejection condition	Self-ratings of ‘Insightful’, ‘Sociable’, ‘Confident’, ‘Lonely’, ‘Playful’, ‘Loving’, and ‘Friendly’, special emphasis on ‘Loving’. Reactions to social rejection: “I felt sad”, “I felt somewhat inadequate during the game”, “I felt like an outsider during the game”, with recollection of number of ball throws (social inclusions) received
Gabay 2018 [146]	Community		MDMA-experienced		20		Male		24.8			100 mg (~ 1.5 mg/kg)		2 sessions, 1/+ weeks apart		95 mins.	Preferences of resource distribution amongst others, social-reward measures	Lower probability of rejecting unfair offers in the first-person condition, but not the third-party condition. Increased average percentage offer, prosocial interaction	Preference measures: “unfair” (selfish), “fair” (equal), “hyper-fair” (altruistic) offers. Social-reward measures: admiration, negative social potency, passivity, prosocial interactions, sexual relationships, sociability.
Gabay 2019 [147]	Community		MDMA-experienced		20		Male		24.8			100 mg (~ 1.5 mg/kg)		2 sessions, 1/+ weeks apart		95 mins.	Mood states, Prisoner's Dilemma (cooperation vs.	Reduced accuracy in identifying fear and anger. Increased cooperation on the second run of the Prisoner's Dilemma.	Social decision-making and changes in trust (during social interaction), facial emotion recognition (happy, sad, fear, or anger - with intensities; identifying and degree of empathising with emotion)
Study	Recruitment		Population Sampled		No. Participants		Sexes		Mean Ages (years)			Oral MDMA Dose		Dosage Timing		Time till First Test (after baseline)	Relevant Tests	Relevant Results	Relevant Parameters Tested
-	-		-		-		-		-			-		-		-	betrayal, when forced to choose), emotion recognition, cognitive and affective empathy	Increased probability of a cooperative decision with trustworthy opponent, but not untrustworthy opponent or game server (neutral). Increased proportion of participants continuing to cooperate after first decision to compete by opponent. Maintained overall level of cooperation (declined with placebo)
Harris 2002 [295]	NA		Healthy, MDMA-experienced, Caucasian		8		3 female, 5 male		24-39			0.5, 1.5 mg/kg		3 sessions, 7/+days apart		30 mins.	Mood states	1.5 mg/kg: increased yes responses to “Have you had a greater feeling of love for others?” and “Have you liked having people around more?”, insignificant trend towards increased “friendly” self-ratings and “closeness to others”	Degrees of Closeness to Others, Energetic, Talkative, Friendly, Confident, Insightful, Anxious. “Have you had a greater feeling of love for others?” and “Have you liked having people around more?”
Holze 2020 [296]	University campus		Healthy, over-25-year-olds		28		14 females, 14 males		28			125 mg		Singular		30 mins.	Mood states	Increased prosocial feelings of “talkative”, “open”, “extraversion”, and general positive feelings of “well-being”, “blissful state”, “positive mood”, “ineffability”	“Talkative”, “open”, “ego dissolution”
Hysek 2012 [51]	University campus		Healthy, mostly MDMA-naïve		48		24 female, 24 male		26			125 mg (~ 1.9 mg/kg)		3 sessions, 10/+ days apart		Eye-reading: 90 mins.; mood: 0	Identifying emotions/thoughts from eye regions of others; mood states	Increased accuracy in reading positive emotions from the eye region, decreased accuracy in reading negative. No change in neutral emotions or total score. Increased “closeness,” “open,” and “talkative” mood self-reports	Total number of correct discriminations of eye-reading test; subscores computed for positive, negative, neutral emotional valences. Prosocial effects: degrees of “closeness to others,” “open,” and “talkative.”
Hysek 2014 [45]	University campus		Healthy, mostly MDMA-naïve		32		16 female, 16 male		25			125 mg (~ 1.9 mg/kg)		10/+ days apart		Empathy test: 3 hours	Mood states, cognitive and emotional empathy, interpersonal reactivity, social value orientation, facial affect recognition	Increased self-ratings of “happy”, “open” and “close to others”, implicit and explicit emotional empathy for positive emotions in men only, increased empathy and prosocial behaviour in men to become comparable to that in women.	Prosociality: ‘happy’, ‘open’, ‘close to others’, inferring mental states (cognitive empathy) degree to which participant felt for individual in picture (explicit emotional empathy), and degree to which participant was aroused by the scene (implicit empathy), trait empathy, social behaviour by resource allocation (degree of maximising allocation for other person) with inequality aversion and joint gain maximisation, accuracy in identifying emotions from facial expressions
Study	Recruitment		Population Sampled		No. Participants		Sexes		Mean Ages (years)			Oral MDMA Dose		Dosage Timing		Time till First Test (after baseline)	Relevant Tests	Relevant Results	Relevant Parameters Tested
-	-		-		-		-		-			-		-		-	-	Increased prosociality in men but not women (resource allocation). Promoted shift from joint gain maximization to inequality aversion. Impaired accuracy of emotion recognition, especially in women (insignificant in men), impaired recognition of fearful, angry, disgusted and surprised faces. Impaired recognition accuracy for fearful, angry and sad faces, only in women. Increased detection threshold for fearful faces
Hysek 2012 [297]	NA		Healthy, ecstasy-naïve		16		8 female, 8 male		26.1			125 mg (~ 1.9 mg/kg)		10/+ days apart		0	Mood states	Increased “open,” “closer to others,” and more “talkative”, “extroversion”	Degrees of “closeness to others,” “talkative,” and “open”, “extroversion” and “introversion”
Kirkpatrick 2015 [46]	Newspaper, community bulletin board, online adverts		Healthy, MDMA-experienced, mostly Caucasian		32		9 female, 23 male, mostly Caucasian		24.9			0.5, 1 mg/kg		3 sessions, 5/+ days apart		60 mins.	Generosity towards stranger	1.0 mg/kg: increased generosity toward friend, but not stranger. 0.5 mg/kg: increased generosity toward a stranger, in females	Point at which participant switches from decision to monetarily benefit selves vs. others
Kirkpatrick 2014 [298]	Different institutes		Healthy, mostly MDMA-experienced		220		44% female, 66% male		25.4			1.5 mg/kg, 125 mg (~ 1.9 mg/kg)		NA		NA	Mood states	Increased closeness to others	Degrees of “closeness to others”
Kirkpatrick 2015 [86]	Posters, print, internet adverts, word of mouth		Healthy, MDMA-experienced		33		9 female, 24 male		Early 20s			0.5, 1 mg/kg		3 sessions		1.5 mins.	Mood states, social interaction, perceptions of others and selves	Increased social interaction (mostly by talking), especially 0.5 mg/kg. 1 mg/kg: rated another as more socially attractive	Degrees of prosocial effects (“confident,” “friendly,” “insightful,” “loving,” “lonely,” “playful,” “sociable”). Proportion of 1.5 min intervals interacting or talking. Level of erceived social attractiveness and physical attractiveness of another person. Level of attention, interest, understanding, and empathy of another person, participant's own level of responsiveness toward another. Perception of their own levels of social affiliation and social power or status.
Kirkpatrick 2012 [299]	Word of mouth, newspaper and online adverts		Healthy, MDMA-experienced, mostly polydrug users		11		2 female, 9 male		29.3			100 mg (~1.5 mg/kg)		4 sessions, 2 days' washout period		7.5 hours	Video recordings of social interaction, with 2 films played	No effect on social interaction	Private (time spent in bathroom/bedroom) and social (time spent in the recreational area). Social time: time spent talking and time spent in silence. Total minutes spent engaging in each behavior per day.
Study	Recruitment		Population Sampled		No. Participants		Sexes		Mean Ages (years)			Oral MDMA Dose		Dosage Timing		Time till First Test (after baseline)	Relevant Tests	Relevant Results	Relevant Parameters Tested
Kirkpatrick 2014 [300]	Newspaper, community bulletin board, online adverts		Healthy, MDMA-experienced, Caucasian		65		25 female, 40 male		23.8			0.75, 1.5 mg/kg		4 sessions, 5/+ days apart		25 mins.	Mood states, social and emotional processing	Increased “friendly” (dose-dependently) and “lonely”. 1.5 mg/kg: reduced accuracy in identifying angry and fearful faces. Increased likelihood rating socialising as more desirable	Prosocial effects (‘Confident,' ‘Friendly,' ‘Insightful,' ‘Loving', ‘Lonely,' ‘Playful,' ‘Sociable'). Accuracy of identifying facial expressions of anger, fear, happiness, sadness. Perceived degrees of attractiveness, friendliness, and trustworthiness of facial pictures. Desire to engage in chatting with another, solving word probems or sitting quietly alone
Kolbrich 2008 [301]	TV, radio, newspaper adverts, flyers, word of mouth		MDMA-experienced, mostly African-Americans		8		2 females, 6 males		21.1			1, 1.6 mg/kg		3 sessions, 7 days between sessions		0	Mood states	Insignificantly increased feelings of closeness to others	Degrees of feelings of closeness to others
Kuypers 2018 [153]	University and website adverts, word of mouth		Healthy, MDMA-experienced polydrug users		20		8 female, 12 male		21.2			75 mg (~1.1 mg/kg)		7-day washouts		90 mins.	Mood states; processing of affective sounds (recorded from strangers); approach-avoidance behaviour to social, threat and trust stimuli	Equally aroused by positive and negative sounds (controls: negative sounds produced more arousal), insignificant avoidance bias to threat faces, insignificant approach bias to trust faces; increased friendliness	Friendliness scale; sounds of happy, sad, fear, disgust, anger; joystick manipulation to indicate approach/avoidance
Kuypers 2014 [154]	University and website adverts, word of mouth		Healthy, MDMA-experienced polydrug users		20		NA		18-26			75 mg (~1.1 mg/kg)		4 sessions, 7-day washouts		1 hour, 25 mins.	Empathy: reading emotions from eye region, cognitive and emotional empathy, interpersonal reactivity. Social interaction: trust, social ball-tossing. Mood states	More concerned and more aroused by the emotional content of the pictures	Empathy: identifying emotions as “negative”, “positive”, “neutral”; identifying emotion, rating how concerned (explicit emotional empathy) and aroused (implicit emotional empathy) for the person; tendency to imaginatively transpose oneself into fictional social situations, tendency to spontaneously adopt the psychological viewpoint of others, feelings of warmth, compassion and concern for others, self-oriented feelings of anxiety and discomfort resulting from tense interpersonal settings. Social interaction: inferring mental state and choosing to cooperate, social reciprocation. Mood state: friendliness scale
Study	Recruitment		Population Sampled		No. Participants		Sexes		Mean Ages (years)			Oral MDMA Dose		Dosage Timing		Time till First Test (after baseline)	Relevant Tests	Relevant Results	Relevant Parameters Tested
Kuypers 2017 [155]	NA		Healthy		118		55 female, 63 male		21.2-25.75			75, 125 mg (~ 1.1, 1.9 mg/kg)		7/+-day washouts		120 mins.	Cognitive, implict and explicit emotional empathy; interpersonal reactivity	More concern (especially for positive stimuli) and arousal (for both positive and negative (as opposed to just positive, as in controls) stimuli) for people depicting emotions	Inferring emotional state, rating how aroused/concerned they felt for the other person; tendency to imaginatively transpose oneself into fictional social situations, tendency to spontaneously adopt the psychological viewpoint of others, feelings of warmth, compassion and concern for others, self-oriented feelings of anxiety and discomfort resulting from tense interpersonal settings.
Kuypers 2008 [302]	Newspaper adverts, snowballing		Polydrug users, MDMA-experienced, healthy		14		7 female, 7 male		22.93			50, 75 mg (~ 0.75, 1.1 mg/kg)		7/+-day washouts		30 mins.	Mood states	Increased friendliness	Friendliness scale
Liechti 2000 [303]	University hospital, medical school		Healthy, mostly unversity students and physicians		16		4 female, 12 male		27.4			1.5 mg/kg		4 sessions, 14/+-day intervals		120 mins.	Mood states	Increased “self-confidence”, “extroversion”, “introversion”	Scales of “extroversion”, “introversion”, “aggression-anger”, “self-confidence”
Liechti 2001 [115]	University hospital staff, medical school		Healthy, mostly unversity students and physicians, mostly MDMA-naive		74		20 female, 54 male		27			10, 50 mg (~0.2, 0.75 mg/kg)		2 sessions, 2/+-week interval		30 mins.	Mood and consciousness states	Increased “comprehensive love”, self-confidence, extroversion, openness, sociability, talkativeness. Increased thoughtfulness and sensitivity in women	Self-confidence, extroversion, introversion, aggression-anger, thoughtfulness, sensitivity. Changes in mood, perception, experience of the self and of the environment
Liechti 2000 [149]	University hospital, medical school		Healthy, mostly MDMA-naïve		14		1 female, 13 male		26			1.5 mg/kg		4 sessions, 10/+-day intervals		75 mins.	Mood states	Decreased vigilance; increased self-confidence, sensitivity and extroversion	Self-confidence, extroversion, introversion, aggression-anger, vigilance
Liechti 2000 [304]	University hospital, medical school		Healthy, mostly unversity students and physicians, mostly MDMA-naïve, mostly non-smokers		14		5 female, 9 male		26			1.5 mg/kg		4 sessions, 10/+-day intervals		75 mins.	Mood states	Increased extroversion and sociability	Introversion, extroversion, sociability
Liechti 2001 [55]	University hospital, medical school		Mostly university students or physicians, healthy, mostly MDMA-naïve		44		10 female, 34 male		26, 27			1.5 mg/kg		4 sessions		120 mins.	Mood states	Increased self-confidence, extraversion	Self-confidence, extroversion, introversion, sensitivity, aggression/anger
Study	Recruitment		Population Sampled		No. Participants		Sexes		Mean Ages (years)			Oral MDMA Dose		Dosage Timing		Time till First Test (after baseline)	Relevant Tests	Relevant Results	Relevant Parameters Tested
Schmid 2014 [150]	University		Mostly MDMA-naïve, healthy		30		15 female, 15 male		24			75 mg (~ 1.1 mg/kg)		7/+-day intervals		Emotion recognition: 75 mins.; mood: 90 mins.; moral judgement: 2 hours; cognitive and emotional empathy: 3 hours; prosociality: 4 hours; mood: 0, 1.25, 5 hours	Facial emotion recognition; cognitive (also in social scenarios) and emotional empathy; prosociality; social decision-making (moral judgement); mood states	Impaired identification of sad, angry and fearful faces; increased misclassification of emotions as neutral; increased both explicit and implicit emotional empathy scores for positive emotional stimuli; increased openness, trust and closeness	Identifying and misclassifying happiness, sadness, anger and fear; inferring mental state of another, how concerned (explicit emotional empathy) they were for them, how aroused (implicit emotional empathy) they were by the scene; identifying false belief, persuasion, faux pas, metaphor and sarcasm in social contexts; maximising resources for self and others, minimising difference between the two; judging between utilitarian outcomes involving aversive effects to others
Schmid 2018 [305]	Adverts, word of mouth		Healthy		24		12 female, 12 male		22.6			125 mg (~ 1.9 mg/kg)		4 sessions, 7/+-day intervals		Mood: 75 mins.; emotion recognition: 150 mins.	Negative emotional states, facial emotion recognition, fearful-face processing	Trend for impaired facial emotion recognition	Emotion recognition: happiness, sadness, anger, fear
Tancer 2007 [306]	NA		MDMA-experienced, healthy, Caucasian, polydrug users		8		2 female, 6 male		23.9			1.5 mg/kg		6 sessions, 2/+-day intervals		60 mins.	Mood states	Increased friendly, talkative scores	Friendliness scale; friendly, self-conscious, social, talkative scales
Tancer 2003 [113]	NA		MDMA-experienced, mostly smokers, mostly marijuana users		12		6 female, 6 male		22.3			1, 2 mg/kg		7/+-day intervals		60 mins.	Mood states	2 mg/kg: increased friendly, social, talkative scores	Friendliness scale; friendly, self-conscious, social, talkative scales
Tancer 2001 [307]	NA		MDMA-experienced, mostly Caucasian		22		14 female, 8 male		23.6			1.1-2.1 mg/kg		2 sessions, 7/+ days apart		60 mins.	Mood states	Increased friendly score	Friendly scale
van Wel 2012 [127]	Newpaper adverts, word of mouth		Healthy, MDMA-experienced		17		8 female, 9 male		22.76			75 mg (~ 1.1 mg/kg)		7/+ days apart		1.5 hours	Mood states	Increased friendliness	Friendliness score
Vizeli 2018 [156]	University campus		Healthy, Caucasian, mostly non-drug users		124		64 female, 60 male		24.8			125 mg (~ 1.9 mg/kg)		7/+ days apart		Mood: 0; emotion recognition, empathy: 90 mins.	Mood states, facial emotion recognition, cognitive and emotional empathy	Increased closeness to others, talkativeness, trust, wanting to be hugged and to hug; impaired recognition of fearful, sad, angry faces; decreased cognitive empathy for all emotions, increased explicit emotional empathy for positive emotions	Mood scales: “closeness to others,” trust,” “want to be hugged,” “want to hug,”“want to be alone,” “want to be with others”, “talkative”. Facial expressions: happiness, sadness, anger, and fear. Empathy: cognitive (inferring mental state of another), implicit emotional (arousal by another's emotional state), explicit emotional (concern for another)
Study		Recruitment		Population Sampled		No. Participants		Sexes		Mean Ages (years)	Oral MDMA Dose		Dosage Timing		Time till First Test (after baseline)		Relevant Tests	Relevant Results	Relevant Parameters Tested
Vollenweider 2005 [123]		University hospital staff, medical school		Healthy, mostly MDMA-naïve		42		10 female, 32 male		25.4-27	1.5 mg/kg		NA		45 mins.		Mood states	Increased self-confidence and extroversion	Self-confidence, extroversion, introversion
Wardle 2014 [116]		Flyers, online adverts		Healthy, MDMA-experienced, mostly Caucasian		36		18 female, 18 male		24.6	0.75, 1.5 mg/kg		3 sessions, 7/+ days apart		Mood: 30 mins. Emotion recognition: 70 mins. Conversation: 2 hrs., 20 mins.		Mood states, facial emotion recognition; interpersonal perception	Mood: increased loving, insignificantly increased playful. Emotion recognition: 1.5 mg/kg: impaired recognition of angry expression; decreased frown response to happy expressions, only in females; increased smile response to happy expressions. Increased positive emotion words (both doses). 1.5 mg/kg: insignificantly increased perceived regard, increased empathy, insignificantly increased percepton of empathy from others	Mood: playful, loving, lonely. Emotion recognition: angry, fearful, sad, happy faces. Interpersonal perception: picked 3 personally important people; percentage of positive and negative emotion words to describe each person; participant's perceptions of investigator and scales of regard (“S/he was truly interested in me”), empathy (e.g., “S/he understood me”), and congruence (“I felt that s/he was real and genuine with me”)
Wardle 2014 [308]		Flyers, online adverts		MDMA-experienced, healthy, mostly Caucasian		101		43 female, 58 male		24.1	0.75, 1.5 mg/kg		3 or 4, separated by 5/+ days		Mood: 30 mins. Picture ratings: 1 hr., 10 mins.		Mood states, responses to emotional pictures, identifying emotional facial expressions	Dose-dependently increased playfulness, lovingness. 1.5 mg/kg: increased positivity of positive social pictures; 0.75 mg/kg: decreased the positivity of positive non-social pictures (1.5 mg/kg insignificantly had this effect)	Mood: playful’ and ‘loving’. Pictures: social vs. non-social, yielding participant-rated scores on scales of positive/negative and arousal

Between 0.2 and 0.75 mg/kg, recognition of emotions from others’ facial expressions is unaffected. A dose of 1.5 mg/kg and above shows impaired recognition of negative emotions and a tendency to misclassify emotional expressions as neutral or happy (although the studies using 2 and 2.1 mg/kg did not test for facial emotion recognition). 1.9 mg/kg shows increased accuracy in identifying positive emotions (chronic or singular dosing). Likewise, relevant tests show social anxiety increased at 0.5-1 mg/kg, but decreased at 1.1-1.9 mg/kg (including higher self-confidence) (chronic or singular dosing). At a chronic 2 mg/kg, however, one study showed self-consciousness not being affected [113], but this was a self-rating component as part of a wider questionnaire as opposed to a behavioural activity testing for social anxiety. This suggests that perhaps 0.75-1.5 mg/kg MDMA taken orally and chronically may be best to alleviate social impairments in humans, without impairing emotion recognition in others.

There was insufficient variation in age between the studies, to assess age effects. All studies tested young adults in their 20s-30s, with the exception of Corey et al. (2016) whose mean participant age was 40.5 years old. Here, they also chronically used the unusual dose of 62.5 mg/kg [114]. Therefore, the effects of MDMA found in these participants are difficult to attribute a certain cause to, although the common theme of increased empathic utterances continues at this dose.

Sex effects were apparent in some studies. In Liechti et al. (2001), chronic 0.2 and 0.75 mg/kg only increased thoughtfulness and sensitivity in women, but not men [115]. At a chronic 0.5 mg/kg, Kirkpatrick et al. (2015) found that only in females was generosity towards stranger persons increased [46]. At a chronic 1.5 mg/kg, Wardle et al. (2014) found decreased frowns in response to others’ happy faces only in women [116]. At a chronic 1.9 mg/kg, Hysek et al. (2014) found that openness, closeness to others and empathy were increased for positive emotions in men specifically, but only to levels comparable to those of placebo women [45]. Also at this dose, the resource-allocation task showed increased prosocial behaviour only in men. Impaired accuracy of facial emotion recognition occurred more in women than men, with impairments specific to negative expressions only seen in women [45]. Hence, lower MDMA doses seemed to have prosocial effects in females exclusively, whereas higher doses were required for men to attain the same empathy levels as women. Interestingly, however, the higher chronic dose of 1.9 mg/kg impaired recognition of facial expressions more in women than in men [45]. Altogether, these results may indicate a lower tolerance for, or a higher sensitivity to, MDMA in women than in men. This suggests that men may require a higher clinical dose, such as chronic oral 1.9 mg/kg MDMA, to improve social impairments.

3.2 Core ASD Behaviour 2: Repetitive Behaviours

3.2.1 Rodents

3.2.1.1 Open-field Tests

The open-field test places a rodent in the centre of an open apparatus and observes its solitary behaviours. With respect to ASD-specific behaviours, investigators are able to observe motor stereotypies (repetitive purposeless movements), including compulsive self-grooming or repetitive routes travelled. A hole-board can be included to provide holes into which the rodent is allowed to poke its nose or dip its head, as an exploratory behaviour. Whether it tends to poke in the same or different holes, can be an indicator of repetitive stereotypy.

We found 72 studies giving MDMA singularly or chronically postnatally to rodents, and observing them for solitary behaviours. The studies show that stereotypies (such as self-grooming, head dipping or hole-pokings, headshakes, digging and repetitive locomotive patterns) are actually increased at 5-20 mg/kg, and there were fewer varied holepokes at chronic 10 mg/kg (Table 6). At a singular 0.63 and 0.94 mg/kg, there were perseverative locomotor patterns (Table 6). However, stereotypies are decreased at 1-20 mg/kg in other studies (singular or chronic dosing) (Table 6). At 1.25-10 mg/kg, there were dose-dependently decreased repeated, as opposed to varied, holepokes (Table 6).

Table 6.

Solitary behaviours in postnatally MDMA-treated rodents, compared with control rodents.

Study	Species		Sexes		Sexes Combined or Separated	Sample Size		Singular/Chronic Dosing		Chronic Timing		Dose (mg/kg)		Route		Treatment Age (PD)		Testing Age (PD)		Temperature (°C)		Males		Females	Both
Abad 2014 [191]	Rat		Males		NA	16-24		Chronic		Twice daily for 4 days		20		s.c.		35		46		26		More time spent in centre.		NA	NA
Ball 2011 [238]	Rat		Males		NA	64		Chronic		2 daily for 5 consecutive days (6 hours apart)		5		i.p.		“Adult” (300-400g)		Same days		NA		Paired MDMA: increased locomotor activity (15 days post-injection). 100 days post-injection: only homecage-treated had hyperlocomotion.		NA	NA
Ball 2011 [238]	Rat		Males		NA	63		Singular		15 or 100 days later		2.5		i.p.		“Adult” (300-400g)		Same day		NA		Hyperlocomotion in unpaired, 100 days post-injection = hyperlocomotion in paired, 15 days post-injection. 100 days post-injection: paired had higher stereotypy (at 15 days post-injection, higher than MDMA-treated unpaired).		NA	NA
Biezonski 2009 [239]	Rat		Males		NA	16		Chronic		Twice daily, 4 hours apart, every 5th day		10		s.c.		35-60		67		23-24		No effect.		NA	NA
Braida 2002 [240]	Rat		Males		NA	40		Chronic		Daily		1, 2, 3, 4		i.p.		90		104		22		4 mg/kg: increased line crossings and stereotypy		NA	NA
Bull 2003 [133]	Rat		Males		NA	24		Chronic		Twice daily		15		i.p.		28-30		28		21		15 mg/kg: higher sustained locomotion (controls: low decreasing locomotion), over 30 mins. Fewer rears, decreased to low plateau (controls: rearing progressively declines). Serotonin syndrome: reciprocal forepaw treading, lateral head weaving, flat body posture.		NA	NA
Bull 2004 [132]	Rat		Males		NA	32		Chronic		Each of 4 hours		5		i.p.		28		84		21		Sustained hyperlocomotion, fewer rearings.		NA	NA
Callaway 1990 [241]	Rat		Males		NA	55		Singular		NA		0.3, 1, 3, 10		s.c.		“Adult” (300-400g)		Same day		NA		1, 3, 10 mg/kg: decreased exploration (holepokes, rearings). 10 mg/kg: increase of routes taken repetitively. 3, 10 mg/kg: increased sustained hyperlocomotion. Centre avoidance, direction alternation.		NA	NA
Callaway 1992 [242]	Rat		Males		NA	32		Chronic		1-week intervals		10, 30 microg.		i.c.		300-400g		Same day		NA		Hyperactivity (30 microg. slightly later than 10 microg.). 30 microg.: hypolocomotion in 1st 10 mins., increased rearings. 10 microg.: decreased holepokes. Results dependent on cerebral area injected.		NA	NA
Clemens 2004 [217]	Rat		Males		NA	63		Chronic		4 injections in 1 day, one every 2 hours		2.5		i.p.		376g		Same time		28		Higher total locomotion.		NA	NA
Study	Species		Sexes		Sexes Combined or Separated	Sample Size		Singular/Chronic Dosing		Chronic Timing		Dose (mg/kg)		Route		Treatment Age (PD)		Testing Age (PD)		Temperature (°C)		Males		Females	Both
Clemens 2005 [218]	Rat		Females		NA	56		Chronic		Every 2 hours in one day (4 times)		4		i.p.		281g		Same day		28		NA		Hyperactivity.	NA
Clemens 2007 [136]	Rat		Females		NA	16		Chronic		1 injection per week for 16 weeks		8		i.p.		238g		7, 56, 112 days later		28		NA		Constant hyperactivity between weeks.	NA
Cohen 2005 [196]	Rat		Males		NA	30		Chronic		Twice a day (8 hours apart)		20		s.c.		11-20		5 weeks, 1 day later		21		Neonatal: less total and central distance travelled, increased thigmotaxis.		NA	NA
Cohen 2005 [196]	Rat		Males		NA	30		Chronic		4 times, at 2 intervals		15		s.c.		82-100		5 weeks, 1 day later		21		Neonatal and adult: increased thigmotaxis.		NA	NA
Colussi-Mas 2008 [243]	Rat		Males		NA	21		Chronic		Once daily for 5 consecutive days		10		i.p.		“Adult” (250–300g)		Same ages		21		No effect.		NA	NA
Colussi-Mas 2008 [243]	Rat		Males		NA	21		Singular		NA		5		i.p.		“Adult” (250–300g)		Same ages		21		5, 10 mg/kg: increased locomotion graph. 5 mg/kg: increased central time.		NA	NA
Colussi-Mas 2008 [243]	Rat		Males		NA	21		Chronic		Once daily for 5 consecutive days + this one after 2 days		5		i.p.		“Adult” (250–300g)		Same ages		21		Hyperlocomotion in, and time spent in, central zone		NA	NA
Cox 2014 [244]	Rat		Males		NA	26		Chronic		Daily for 4 days		5, 10		i.p.		38-41		46		NA		10 mg/kg: decreased centre time.		NA	NA
Daza-Losada 2009 [221]	Mouse		Males		NA	315		Singular		NA		5, 10, 20		i.p.		28		28		21		10, 20 mg/kg: increased hyperlocomotion.		NA	NA
Ebrahimian 2017 [245]	Mouse		Males		NA	40		Both		Single dose, twice		20, 60		p.o.		NA		0 and 9 days later		22-24		20 and 60 mg/kg: total distance travelled dose-dependently increased, rearing decreased.		NA	NA
Fantegrossi 2004 [246]	Mouse		Males		NA	12		Singular		NA		32		i.p.		20-30g		Same age		22		Long-lasting hyperlocomotion (3 hours post-injection).		NA	NA
Ferraz-de-Paula 2011 [247]	Mouse		Males		NA	14-16		Singular		NA		0.2-20		i.p.		60		67		22-26		5, 8, 10, 20 mg/kg: increased total distance travelled, peripheral time, locomotor activity; decreased frequency of rearing and grooming. 8, 10, 20 mg/kg: increased peripheral distance travelled. 1, 5, 20 mg/kg: increased central distance travelled. 1, 5, 10, 20 mg/kg: increased central time. 5, 10 mg/kg: increased distance travelled, mean locomotor activity; decreased number and time of head-dipping (hole-board).		NA	NA
Study	Species		Sexes		Sexes Combined or Separated	Sample Size		Singular/Chronic Dosing		Chronic Timing		Dose (mg/kg)		Route		Treatment Age (PD)		Testing Age (PD)		Temperature (°C)		Males		Females	Both
Fone 2002 [131]	Rat		Males		NA	36		Chronic		Twice daily		7.5		i.p.		39-41		39		21		7.5 mg/kg: sustained hyperlocomotion. Serotonin syndrome: extensive reciprocal forepaw treading, lateral head weaving, flat body posture.		NA	NA
García-Pardo 2017 [94]	Mouse		Males		NA	14-16		Singular		NA		5, 10		i.p.		42		70, 71		35-37		More centre entries.		NA	NA
Gold 1988 [248]	Rat		Males		NA	60		Singular		NA		1.25, 2.5, 5, 10		s.c.		“Adult” (250-400g)		Same day		NA		Hyperlocomotion in first 10 mins. 2.5-10 mg/kg: hypolocomotion for rest of 2 hours. Initially decreased, then increased, holepokes. Dose-dependent sustained decreased repeated:varied holepokes. Decreased, then increased, rearings. Dose-dependent centre avoidance, hyperlocomotion. 10 mg/kg: increased rearings in 2nd hour. 10 mg/kg: hyperlocomotion.		NA	NA
Gold 1988 [249]	Rat		Males		NA	60		Singular		NA		0.63, 0.94		s.c.		“Adult” (250-400g)		Same day		NA		More perseverative locomotion patterns. 0.63 mg/kg: decreased repeated:varied nosepokes. 0.94 mg/kg: decreased rearing.		NA	NA
Gold 1988 [249]	Rat		Males		NA	60		Singular		NA		10		s.c.		“Adult” (250-400g)		Same day		NA		Hyperlocomotion.		NA	NA
Gurtman 2002 [134]	Rat		Males		NA	26		Chronic		Each of 4 hours		5		i.p.		90		118		22		Hyperlocomotion.		NA	NA
Hegadoren 1995 [250]	Rat		Males		NA	24		Singular		NA		NA		i.p.		250-400g		Same day		NA		1st 30 mins.: rapid locomotor increase. Decreased yawning, mouth movements, lying down, stretching. Increased body adjustments, clockwise rotations. Complete insomnia.		NA	NA
Hiramatsu 1989 [251]	Rat		Males		NA	21-27		Singular		NA		5, 10, 20		s.c.		200-300g		Same ages		23		Stereotyped sniffing, head-weaving, backpedalling, turning. NB: S(+) MDMA: more head-weaving, backpedalling, turning than R(-).		NA	NA
Ho 2004 [252]	Rat		Males		NA	65		Singular		NA		7.5, 15		i.p.		271g		9-10 days later		22-24		No effect.		NA	NA
Kalivas 1998 [253]	Rat		Males		NA	24		Chronic		5 days: 5 mg/kg once daily, or 20 mg/kg twice daily		5, 20		s.c.		NA		NA		NA		Acute, 5 mg/kg, Day 2: increased horizontal activity, distance travelled, stereotypy. Chronic 5, 20 mg/kg for 4 days: increased activity. Day 19: more increase in horizontal activity, distance travelled, response greater. 2 days washout, daily 5 mg/kg: more horizontal activity, distance travelled. More activity at 30-90 mins. post-injection.		NA	NA
Study	Species		Sexes		Sexes Combined or Separated	Sample Size		Singular/Chronic Dosing		Chronic Timing		Dose (mg/kg)		Route		Treatment Age (PD)		Testing Age (PD)		Temperature (°C)		Males		Females	Both
Kindlundh-Högberg 2009 [254]	Mouse		Males		NA	12		Chronic		3 injections every 7th day for 4 days		5		i.p.		120-180		Same ages		20		Increased horizontal activity. 1st week: increased locomotor:horizontal ratio.		NA	NA
Kurling 2008 [225]	Rat		Males		NA	12		Singular		NA		5		i.p.		“Adult” (300-380g)		Same day		21		Increased locomotion, burst of rearings, slight agitation, stereotypies (intensive sniffing, head/body weaving, head-bobbing).		NA	NA
Lebsanft 2005 [255]	Rat		Males		NA	20		Singular		NA		5		s.c.		47-56		Same day		22.5		No effect.		NA	NA
Llorente-Berzal 2013 [199]	Rat		Both		Separated	60		Chronic		Every 5 days, twice daily (4 hours apart)		10		s.c.		30-45		71		22		Increased thigmotaxis.		Increased thigmotaxis.	NA
Lorens 1990 [256]	Rat		Males		NA	36-48		Chronic		Every 12 hours for 4 consecutive days		10, 20, 40		s.c.		225-250g		2-3 weeks later		NA		No effect.		NA	NA
Ludwig 2008 [200]	Rat		Males		NA	39+		Singular		Single injection		5		s.c.		“Adult” (242-275g)		“Adult” (242-275g)		26		5-10 mins.: increased total locomotion. 10-20 mins.: decreased rearing		NA	NA
Ludwig 2008 [200]	Rat		Males		NA	39+		Chronic		5 daily injections		5		s.c.		“Adult” (242-275g)		“Adult” (242-275g)		26		After 10 mins.: increased locomotion. Multiple MDMA injections: increased central and total locomotion, decreased peripheral locomotion.		NA	NA
Ludwig 2008 [200]	Rat		Males		NA	39+		Both		Chronic, then single		5		s.c.		“Adult” (242-275g)		“Adult” (242-275g)		26		1st 10 mins., multiple MDMA injections: decreased rearing. Multiple MDMA injections: increased central and total locomotion, decreased peripheral locomotion. Multiple doses: increased rearing. Check - Low doses had higher locomotor activity than high doses. - check.		NA	NA
McNamara 1995 [257]	Rat		Males		NA	24		Chronic		Twice daily for 4 days		5, 10, 20		i.p.		200-250g		7 days later		22-24		10 and 20 mg/kg increased locomotor activity (experimental day 1, 2, 4). 20 mg/kg: increased locomotion (experimental day 3).		NA	NA
Mechan 2002 [258]	Rat		Males		NA	16		Singular		NA		12.5		i.p.		“Adult” (160-200g)		9–11, 30–32, 71–73 days post-injection		21		White light: no effect. Red light: hyperlocomotion till significant on PD 73.		NA	NA
Miczek 1994 [227]	Mouse		Males		NA	29		Singular		NA		0.3-10		NA		“Adult”		Same day		21		10 mg/kg: decreased rearing.		NA	NA
Morley 2000 [228]	Rat		Males		NA	48		Singular		NA		1.25, 2.5, 5		i.p.		85-95		99-109		22		Decreased centre time, increased defaecation. 5 mg/kg: hyperlocomotion.		NA	NA
Study	Species		Sexes		Sexes Combined or Separated	Sample Size		Singular/Chronic Dosing		Chronic Timing		Dose (mg/kg)		Route		Treatment Age (PD)		Testing Age (PD)		Temperature (°C)		Males		Females	Both
Morley 2001 [91]	Rat		Males		NA	48		Chronic		Each of 4 hours over 2 days		5		i.p.		75-95		160-180		28		Hyperlocomotion.		NA	NA
O'Loinsigh 2001 [259]	Rat		Males		NA	12-14		Chronic		Twice daily for 4 consecutive days		20		i.p.		200-300g		200-300g		20-22		Increased locomotion and stereotypy, decreased rearing		NA	NA
Olsen 2016 [260]	Rat		Males		NA	NA		Chronic		Twice		0.75, 1.5, 3		i.p.		105		105		NA		1.5, 3 mg/kg: dose-dependently decreased response rate for repeated, as opposed to varied, tasks		NA	NA
Palenicek 2005 [261]	Rat		Both		Separated	79		Singular		NA		2.5, 5, 10		s.c.		50		50		22-24		Dose-dependently increased locomotion		Dose-dependently increased locomotion. 2.5 and 5 mg/kg: longer trajectory than males, increased thigmotaxis.	NA
Paleníček 2007 [262]	Rat		Both		NA	88		Singular		NA		2.5, 5, 10		s.c.		150-220g		150-220g		22-24		Females, 5 mg/kg: sniffing stereotypy. 10 mg/kg: sniffing stereotypy (only males in 2nd session). 2.5, 10 mg/kg in 1st session; 10 mg/kg in 2nd session: males > females. Females, 10 mg/kg: decreased stereotypy
Paulus 1992 [263]	Rat		Males		NA	47		Singular		NA		MDMA: 1.25, 2.5, 5, 10		s.c.		250-300g		250-300g		NA		Increased ratio of short:long microevent durations. 5, 10 mg/kg: straighter paths travelled		NA	NA
Paulus 1992 [263]	Rat		Males		NA	61		Singular		NA		(+)MDMA: 0.3, 1, 3, 10		s.c.		250-300g		250-300g		NA		3, 10 mg/kg: increased thigmotaxis. 10 mg/kg: straighter paths travelled. Hyperlocomotion		NA	NA
Paulus 1992 [263]	Rat		Males		NA	62		Singular		NA		(-)MDMA: 1, 3, 10, 30		s.c.		250-300g		250-300g		NA		Long straight paths with thigmotaxis. 10, 30 mg/kg: straighter paths. 30 mg/kg: hyperactivity		NA	NA
Piper 2004 [203]	Rat		Males		NA	16		Chronic		On every 5th day on PD 35-60, twice daily, 4 hours apart each		10		s.c.		35-60		65		22		No effect.		NA	NA
Piper 2005 [204]	Rat		Males		NA	20		Chronic		Hourly intervals over 4 hours, once every 5 days		5		s.c.		35-70		35, 45, 60, 65		22		PD 35: increased headweaving. Hyperlocomotion at 2-4 mins., hypolocomotion at 7-10 mins.		NA	NA
Piper 2008 [205]	Rat		Males		NA	20-24		Chronic		4 doses, 1 each hour		10		s.c.		“Young adult” (307.7g)		Young adult		23		Hole-board: fewer hole entries after MDMA on 2nd injection.		NA	NA
Study	Species		Sexes		Sexes Combined or Separated	Sample Size		Singular/Chronic Dosing		Chronic Timing		Dose (mg/kg)		Route		Treatment Age (PD)		Testing Age (PD)		Temperature (°C)		Males		Females	Both
Powell 2004 [264]	Mouse		Both		Combined	15-33		Singular		NA		10, 20, 30		i.p.		150		150		NA		NA		NA	Hyperlocomotion, straight perseverative movements
Procopio-Souza 2011 [85]	Mouse		Males		NA	19		Singular		NA		10		i.p.		90		Same day		22-23		Increased bouts of locomotion.		NA	NA
Procopio-Souza 2011 [85]	Mouse		Males		NA	19		Singular		NA		+10		i.p.		97		Same day		22-23		More bouts of hyperlocomotion with MDMA pretreatment.		NA	NA
Quinteros-Munoz 2010 [265]	Rat		Males		NA	56-77		Singular		NA		0.25, 0.5, 1, 3, 5, 10		i.p.		200-230g		200-230g		NA		1 mg/kg: increased grooming; declined after higher doses. 10 mg/kg: decreased head shakes		NA	NA
Shen 2011 [233]	Rat		Males		NA	60		Chronic		2, every 5th day		10		s.c.		50-60		70		26		No effect.		NA	NA
Shen 2011 [233]	Rat		Males		NA	19		Chronic		2, every 5th day		10		s.c.		50-60		74		26		Holeboard: decreased varied entries.		NA	NA
Shen 2011 [233]	Rat		Males		NA	19		Chronic		2, every 5th day		10		s.c.		48-58		68		26		No effect.		NA	NA
Slamberova 2018 [266]	Mouse		Males		NA	16		Singular		NA		5		s.c.		77-97		77-97		22-24		Increased locomotion, distance travelled, average speed. No effect on grooming duration.		NA	NA
Stanley 2007 [267]	Rat		Males		NA	8		Singular		NA		10		i.p.		250-300g		250-300g		22		Stereotypic-included behaviour increased.		NA	NA
Thompson 2009 [82]	Rat		Males		NA	36		Singular		NA		5		i.p.		“Adult” (345g)		Same day		28		Hyperlocomotion, more central time.		NA	NA
Walker 2010 [268]	Rat		Males		NA	30-34		Singular		NA		5		i.p.		28, 42, 65		28, 42, 65		NA		Early hyperlocomotion (PD 28 < 42, 65). No stereotypy effects.		NA	NA
Yamamotová 2012 [269]	Rat		Males		NA	20		Singular		NA		5		s.c.		85-90		85-90		NA		Increased stereotypy.		NA	NA
Yang 2011 [270]	Rat		Females		NA	26		Singular		NA		5		i.p.		“Adult” (180-190g)		“Adult” (180-190g)		21		NA		Increased horizontal activity and total distance travelled (experimental day 2 > day 1).	NA
Study	Species		Sexes		Sexes Combined or Separated	Sample Size		Singular/Chronic Dosing		Chronic Timing		Dose (mg/kg)		Route		Treatment Age (PD)		Testing Age (PD)		Temperature (°C)		Males		Females	Both
-	-		-		-	-		-		-		-		-		-		-		-		-		Decreased vertical activity (experimental day 2 > day 1). 5 mg/kg: increased total distance travelled, horizontal activity, stereotypies; decreased vertical acitivity (10-110 mins. post injection).
Yang 2011 [270]	Rat		Females		NA	26		Singular		6 consecutive days		5		i.p.		85-87		85-87		21		NA		Increased stereotypies, decreased vertical activity (0-20 mins. post-injection). Increased horizontal activity (30 mins.-2 hours post-injection). 5 mg/kg: increased total distance travelled, horizontal activity, stereotypies; decreased vertical acitivity (10-110 mins. post injection). Chronic dosing had no effect. 5 mg/kg: increased/decreased locomotion (experimental day 11 > day 2).	NA
Study	Species	Sexes		Sexes Combined or Separated			Sample Size		Singular/ Chronic Dosing		Chronic Timing		Dose (mg/kg)		Route		Treatment Age (PD)		Testing Age (PD)		Temperature (°C)		Males	Females	Both
Young 2008 [271]	Mouse	Males		NA			48		Singular		NA		(±)-MDMA: 0.3, 1, 3, 10, 30		i.p.		27-34g		27-34g		22		Hyperlocomotion. 30 mg/kg: decreased thiogmotaxis.	NA	NA
Young 2008 [271]	Mouse	Males		NA			48		Singular		NA		S(+)-MDMA: 0.3, 1, 3, 10, 30		27-34g		27-34g		22		Hyperlocomotion.		NA	NA
Young 2008 [271]	Mouse	Males		NA			64		Singular		NA		R(-)-MDMA: 0.3, 1, 3, 10, 17, 30, 50		27-34g		27-34g		22		Hyperlocomotion.		NA	NA

Each study is shown alongside the species, sex and sample size of the rodents tested (control and relevant treatment groups), whether the study used singular or chronic (multiple) dosing, and if they used chronic dosing, the timing of the doses; the dose and route (mode of injection) used, the ages the rodents were treated and tested at, the temperature of the testing environment, and the results obtained from the experiment in the treated rodents (where it is different from the results in the corresponding control rodents). i.p. = intraperitoneal mode of injection; s.c. = subcutaneous mode of injection; p.o. = oral route of ingestion; i.c. = intracerebral mode of injection.

Therefore, singular or chronic 1-5 mg/kg MDMA may be able to reduce stereotypies in these rodents.

Thigmotaxis (the anxious tendency to remain close to the walls) decreased when singularly given 1-20 mg/kg, and even chronically 5-10 mg/kg. When rodents were chronically given 10 mg/kg, however, it gave mixed results. When the chronic dose increased even more to 15-20 mg/kg, it increased thigmotaxis with higher sustained hyperlocomotion (possibly indicating anxiety). It also increased at 1.25-5 mg/kg, with accompanying increased defaecation (also taken as an indicator of anxiety), further lending support to the increased-anxiety theory. Interestingly, there seemed to be a sex effect in one study, where females showed a greater thigmotaxic increase than males, at 2.5 and 5 mg/kg MDMA [117]. Hyper-locomotion tended to occur at 5 and 10 mg/kg, as well as decreased exploration (holepokes and rearings). This hyper-locomotion was increased at 3-20 mg/kg, and also when chronically given. At lower doses, hyper-locomotion appeared early on (1.25-10 mg/kg). At 2.5-10 mg/kg, hypo-locomotion followed. Holepokes decreased first, then increased. At 10 mg/kg, rearings also increased, later. There were fewer rearings, but dose-dependently increased locomotion at 20 and 60 mg/kg. When 5 or 20 mg/kg is chronically given, there is also hyper-locomotion. Interestingly, under red light (which triggers less anxiety in rodents), there was increasing hyper-locomotion at 12.5 mg/kg, whereas the same dose under white light had no effect. When chronically given 10 or 15 mg/kg, there was less exploration (rears and holepokes). When 7.5-30 mg/kg was chronically given, symptoms of serotonin syndrome also appeared in the rodents, namely, reciprocal forepaw treading, lateral head weaving, flat body posture.

3.2.1.2. Marble-burying Tests

Testing for repetitive/compulsive behaviours can be undertaken via the marble-burying test in rodents [118, 119]. Here, 16 or 20 marbles are embedded in a 4x4 or 4x5 pattern in a corncob bedding inside a cage [118, 119]. The test rodent is free to roam in the cage for 15 mins., and the number of marbles buried by the rodent is then counted [118, 119].

We found 3 studies which delivered MDMA to rodents chronically postnatally, and tested for marble-burying behaviour (Table 7). The results show that fewer marbles are buried when 2.5 mg/kg MDMA is given intraperitoneally thrice, 3 hours apart, possibly providing a means of decreasing compulsion/stereotypy. At higher doses, however, compulsivity/stereotypy was either increased (10 mg/kg x 4, for 10 days) or was not affected (15 mg/kg x 4 weekly over 5 weeks).

Table 7.

Marble-burying propensity in postnatally MDMA-treated rodents, compared with control rodents.

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Males	Females	Both
Saadat 2006 [272]	Mouse	Males	NA	16	Chronic	3 times at 3-hour intervals	2.5	i.p.	25-30g	Same day	20	Reduced	NA	NA
Skelton 2008 [211]	Rat	Males	NA	21	Chronic	4 times on 1 day a week, over 5 weeks	15	s.c.	225-250g	5 days later	22	No effect	NA	NA
Skelton 2009 [273]	Rat	Both	Combined	80	Chronic	4 times daily	10	s.c.	11-20	29	21	NA	NA	Increased

In terms of repetitive behaviour or stereotypies in rodents, the open-field and marble-burying tests show that when 10-5 mg/kg MDMA is given acutely, it may be possible for repetitive and compulsive behaviours to subside.

3.2.2. Other Animals

Three studies assessed stereotypies in non-rodent animals, specifically monkeys, after chronic MDMA administration. All of them found no effect on stereotypy levels [120-122]. All studies tested only males, however, with singular and chronic doses of 0.32-7.8 mg/kg by intramuscular, oral or intragastric administration [120-122].

3.2.3. Humans

There were no studies specifically investigating the effect of MDMA on repetitive behaviours in human subjects. This indicates a gap in the literature, which may be an opportunity to test potential treatment drugs (such as MDMA) on this core impairment of ASD. However, MDMA may increase stereotyped behaviour in humans, as seen in one study where when given singularly at a dose of 1.5 mg/kg orally [123].

3.3. Core ASD Behaviour 3: Cognitive Rigidity

3.3.1. Rodents

3.3.1.1. T/Y-maze Tests

The Y/T-maze can be used to test cognitive rigidity, or insistence on sameness in routine, in rodents via the spontaneous-alternation task or the food-reward task [124]. Spontaneous alternation monitors whether rodents will

Each study is shown alongside the species, sex and sample size of the rodents tested (control and relevant treatment groups), whether the study used singular or chronic (multiple) dosing, and if they used chronic dosing, the timing of the doses; the dose and route (mode of injection) used, the ages the rodents were treated and tested at, the temperature of the testing environment, and the results obtained from the experiment in the treated rodents (where it is different from the results in the corresponding control rodents). i.p. = intraperitoneal mode of injection; s.c. = subcutaneous mode of injection.

spontaneously alternate arms in successive trials [124], capitalising on natural exploratory behaviour. Animals that persist in selecting the same arm to travel down suggests cognitive rigidity or memory impairment [124]. Thus, this test is also confounded by working spatial memory, which is independent of ASD [124]. One study stated that statistically, it was more likely due to a perseverative tendency than cognitive impairment [125]. Similarly, in a trained version (which either uses a food reward, or blocks off one arm to train, before testing), rodents are explicitly reinforced to travel down one arm [124]. Once trained (training/acquisition phase), the rewarded arm is switched, and the rodent is monitored to see how quickly it explores the previously non-rewarded/non-forced arm (testing/reversal phase) [124]. Frustrated responses (such as bouts of stereotypy) to going towards the previous/wrong arm could also be monitored.

We found 21 studies where MDMA was given mostly chronically postnatally, and the rodents were assessed for their inclinations to spontaneously alternate arms, or to change habit for food (Table 8). The results show that 10-20 mg/kg MDMA seems to have less influence on cognitive rigidity than do doses either side of this range (less than 10 or more than 20 mg/kg), but this may have to do with technique inconsistencies between studies. It did not seem to make a difference whether investigators used Y- or T-shaped mazes. In those using radial mazes, we wonder if their results were confounded by spatial learning and memory as factors influencing spontaneous alternation.

Table 8.

Resistance to change travel direction in postnatally MDMA-treated rodents, compared with control rodents.

Study	Y-/T-maze	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Food Restriction	Food	Males	Females	Both
Adeniyi 2016 [194]	Y	Mouse	Males	NA	10	Chronic	5 times over 10 days, at 2-day intervals	2	s.c.	21	31, 32	NA	Nil	NA	No effect	NA	NA
Cassel 2005 [274]	T	Rat	Males	NA	20	Chronic	4 times, 24 hours apart	10	i.p.	“Adult”	4 days after last injection	23	Nil	NA	No effect	NA	NA
Edut 2011 [197]	Y	Mouse	Males	NA	37	Singular	NA	10	i.p.	25-30g	“Juvenile”	23	Nil	NA	No effect	NA	NA
Edut 2014 [198]	Y	Mouse	Males	NA	NA	Singular	NA	10	i.p.	25-30g	Same age	23	Nil	Visual cues (shapes)	No effect	NA	NA
Kolyaduke 2013 [157]	Y	Rat	Both	Separated	80	Chronic	10 days, once a day	10	i.p.	35, 45	90	22	Nil	NA	No effect	No effect	NA
Odland 2019 [275]	Y	Mouse	Males	NA	32	Singular	NA	1,3,10	i.p.	8-16 weeks	8-16 weeks	20-24	Nil	NA	No effect	NA	NA
Ricaurte 1993 [276]	T	Rat	Males	NA	21	Chronic	Twice daily for 4 days	20	s.c.	300g	84 days later	NA	Not specified	Chocolate milk	No effect.	NA	NA
Schwarting 2005 [277]	T	Rat	Males	NA	40	Singular	NA	7.5, 15	i.p.	409.7g	Same day	22-24	3 days before, for 23 hours each		NA	NA	NA
Schulz 2013 [208]	T	Rat	Males	NA	24	Chronic	Once daily for 10 days, twice daily (4 hours apart) for 5 days	7.5	s.c.	“Adult” (230-300g)	40-65, 80-105	22	Not specified	Food pellets	Increased	NA	NA
Young 2005 [278]	Y	Rat	Males	NA	40+	Singular	NA	1.5, 2.5, 5	i.p.	“Adult”	“Adult”	21	4 days	Froot loops (cereal)	5 mg/kg: increased	NA	NA
Reversal learning through odour-span task:
Hawkey 2014 [279]	NA	Rat	Males	NA	6	Singular	NA	0.3, 1.0, 1.8, 3.0	i.p.	90-150	90-150	NA	Not specified	Sugar pellets	No effect	NA	NA
Study	Y/T maze	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Food Restriction	Food	Males	Females	Both
Hawkey 2014 [279]	NA	Rat	Males	NA	6	Chronic	Twice daily over 4 days	10	i.p.	90-150	90-150	NA	Not specified	Sugar pellets	Increased	NA	NA
Acquired operant-alternation task:
Vinals 2013 [280]	NA	Mouse	Males	NA	19-21	Chronic	Twice daily over 4 days	3, 30	i.p.	24g	24g	21	Not specified	Chocolate pellets	30 mg/kg: increased	NA	NA
Radial-arm maze:
Braida 2002 [240]	8-arm	Rat	Males	NA	32	Chronic	Daily	1, 2, 3	i.p.	90	104	22	Restricted (by 15% free-feeding weight) for 2 weeks before	Food pellets	Increased	NA	NA
Canales 2014 [159]	8-arm	Rat	Females	NA	12	Chronic	Twice daily, 8 hours apart	10	NA	13-15	97	21	Nil	Noyes pellets	NA	No effect	NA
Compton 2016 [158]	Plus	Rat	Both	Separated	26	Chronic	2 injections 5 times, with 5-day intervals.	5	NA	35-57	131	24	Nil	NA	Increased	Increased	NA
Harper 2013 [281]	8-arm	Rat	Males	NA	13	Chronic	4 times, 2-hour intervals + once a week	10 + 4	i.p.	90-120	90-120	NA	Nil	Chocolate chips	No effect	NA	NA
Harper 2013 [281]	8-arm	Rat	Males	NA	13	Chronic	Once a week	4	i.p.	90-120	90-120	NA	Nil	Chocolate chips	No effect	NA	NA
Kay 2011 [282]	NA	Rat	Males	NA	19	Chronic	4 times, 1 every 2 hours, for 1 day	10	i.p.	90-120	90-120	NA	Not specified	Chocolate chips	No effect	NA	NA
Kay 2011 [282]	NA	Rat	Males	NA	19	Chronic	Twice, 24 hours apart	4	i.p.	90-120	90-120	NA	Not specified	Chocolate chips	Increased	NA	NA
Ros-Simo 2013 [207]	8-arm	Mouse	Males	NA	24-30	Chronic	Twice, 6 hours apart	20	i.p.	25	28	22	Not specified	Chocolate pellets	Increased	NA	NA

3.3.2. Other Animals

Studies using other animals are explored in Table 9. One study assessed cognitive rigidity in non-rodent animals given MDMA, specifically cynomolgus monkeys. Using only males and chronic dosing of 1.5 mg/kg p.o., they found that when given orally, MDMA animals tended to have more reversal-learning errors, predominated by persevering at the same option [101], indicating that MDMA at this dose and route may even exacerbate cognitive inflexibility. When the dose was given intramuscularly, the same study found no such effect [101], indicating perhaps that the intramuscular route may not be as bioavailable (as the oral route) to create this exacerbating effect.

Table 9.

Non-rodent animal studies testing core ASD behaviours.

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age	Testing Age	Temperature (°C)	Males	Females	Both
Ali 1993 [102]	Rhesus monkey	Females	NA	14	Chronic	Twice daily for 4 consecutive days	1.25, 2.5, 20	i.g.	Varying weights	Same day	NA	NA	20 mg/kg: serotonin behaviour	NA
Ballesta 2016 [98]	Long-tailed macaque	Males	NA	3	Chronic	3 doses at 1-week intervals	1, 1.5, 2	s.c.	3 years	3 years	NA	1 mg/kg: nil. 1.5 mg/kg: increased time spent being groomed	NA	NA
Capurro 1997 [103]	Electric fish	Both	Combined	100	Singular	NA	1, 5	i.m.	5-50g	3 days later	18	NA	NA	Decreased aggression, increased adjacent swimming, longer latency to first (aggressive) bite
Crean 2006 [120]	Rhesus macaque	Males	NA	6	Singular	NA	0.56, 1, 1.78, 2.4	i.m.	6-10 years	6-10 years	23, 27	No effect on stereotypy	NA	NA
Crean 2007 [121]	Rhesus macaque	Males	NA	10	Chronic	Intervals of at least 1 week	1.78, 5	i.m., p.o.	6-10 years	6-11 years	23-27	No effect on stereotypy	NA	NA
Edsinger 2018 [111]	Octopus	Both	Separated	NA	Singular	NA	0.5-0.005	Immersion	9 months	9 months	22	Increased social preference	Increased social preference	NA
Edsinger 2018 [112]	Octopus	Both	Separated	7	Singular	NA	0.5-0.005	Immersion	9 months	9 months	20-23	Increased social preference and exploratory body contact	Increased social preference and exploratory body contact	NA
Goodwin 2013 [122]	Baboon	Males	NA	4	Chronic	5-day intervals	0.32–7.8	i.g.	“Adult” (18.4-33.2kg)	“Adult” (18.4-33.2kg)	NA	No effect on stereotypy	NA	NA
Green 2012 [104]	Zebrafish	Both	Combined	16-24	Singular	NA	80 mg/L	Immersion	5-8 months	5-8 months	25-27	NA	NA	Increased inter-fish distance, decreased duration of proximity
Insel 1989 [283]	Rhesus monkey	Males	NA	9-12	Chronic	Twice daily for 4 consecutive days	2.5, 10	i.m.	“Adult” (7-12kg)	1 hour later	24	Decreased exploration, increased passivity. 10 mg/kg may induce serotonin-syndrome-like features, upright posture and later continuous activity	NA	NA
Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age	Testing Age	Temperature (°C)	Males	Females	Both
Iravani 2003 [99]	Common marmoset	Both	Combined	26	Chronic	4-7-day intervals	12	p.o.	280-330g	280-330g	25	NA	NA	Decreased motor activity and exploration, vocalisation. Serotonin behaviour
Pitts 2017 [100]	Squirrel monkey	Males	NA	4	Singular	NA	0.03–3.0	i.m.	11-16 years	11-16 years	23	Both enantiomers: increased huddling, decreased activity, increased number of affiliative vocalisations. Lower doses: S(+) MDMA increased huddling, decreased activity, more than did R(−) MDMA. R(−) enantiomer: more affiliative calls at higher doses, than S(+) enantiomer	NA	NA
Ponzoni 2016 [105]	Zebrafish	Both	Combined	20	Singular	NA	0.1–20	i.m.	6-12 months	6-12 months	28	NA	NA	0.5-7.5 mg/kg: increased social preference
Ponzoni 2017 [106]	Zebrafish	Both	Combined	40	Singular	NA	10, 0.5, 0.1	i.m.	6-12 months	6-12 months	28	NA	NA	Increased social preference
Stewart 2011 [107]	Zebrafish	Both	Combined	142	Singular	NA	0.25, 40, 80, 120 mg/L	Immersion	5-7 months	5-7 months	25-27	NA	NA	Dose-dependently decreased time taken to travel to the top of the tank, increased time spent up there, as well as the total number of fish swimming at the top. 40, 80, 120 mg/L: impaired zebrafish habituation
Verrico 2008 [101]	Cynomolgus monkey	Males	NA	4	Chronic	Twice, 1 week apart, over 3 weeks	1.5	p.o.	4-6 years	4-6 years	NA	Increased cognitive rigidity and social vigilance	NA	NA
Verrico 2008 [101]	Cynomolgus monkey	Males	NA	5	Chronic	Once a week for 5 weeks	1.5	i.m.	4-6 years	4-6 years	NA	No effect on cognitive rigidity, increased social vigilance.	NA	NA

3.3.3. Humans

As shown in Table 11, there were only two placebo-controlled studies showing how MDMA affects cognitive rigidity in humans, and only one showed some effect. Given that 1.5 mg/kg MDMA is comparable to 100 mg [126], 75 mg should be comparable to approximately 1.1 mg/kg, based on the assumption of a linear positive correlation of drug mass to human body weight. Therefore, the studies compare the effects of chronic 1.1 mg/kg [127] and singular 1.5 mg/kg [123] MDMA in humans. The first study shows no effect of chronically given 1.1 mg/kg [127], but this was tested in MDMA-experienced users who may have higher tolerance levels to MDMA. Alternatively, it may be an effect of the different tests used, despite both testings for the attribute of cognitive rigidity. Van Wel et al. (2012) tested a response to changing rules in a visual game, where latency to detection of the change in rule could be a confounder, and found no effect [127]. However, in a second study which showed an inflexibility-exacerbating effect at 1.5 mg/kg, Vollenweider et al. (2005) tested the pattern of unchanging responses which may also be more subtle and subconscious, thereby less consciously manipulatable [123]. In the former study, a “cue-dependent reversal-learning task” has subjects respond to target stimuli and not respond to non-target stimuli. The target and non-target stimuli are reversed several times throughout the task, and the acquisition and reversal learning rates are measured in each subject [127]. In the latter study, a “two-choice prediction task” has subjects predict where a stimulus will be presented on a computer screen, based on previous outcomes or responses. Here, stay/switch responses are measured, as well as how much the response is influenced by the previous outcome or response, and the predictability of subject responses [123]. Notably, Vollenweider et al. (2005) had a male-biased sample, which may explain the increased vulnerability to stereotypy, as shown in males in the rodent studies [123]. There were too few studies to determine age or sex effects.

Table 11.

Resistance to change in behaviour, in postnatally MDMA-treated subjects, compared with placebo-treated subjects.

Study	Recruitment	Population Sampled	No. Participants	Sexes	Ages	Oral MDMA Dose	Dosage Timing	Time till First Test	Relevant Tests	Relevant Results	Relevant Parameters Tested
van Wel 2012 [127]	Newspaper adverts, word of mouth	Healthy, MDMA-experienced	17	8 female, 9 male	22.76	75 mg (~1.1 mg/kg)	7/+ days apart	105 mins.	Reversal learning	No effect	Response to changing rules of shapes presented
Vollenweider 2005 [123]	University hospital staff, medical school	Healthy, mostly MDMA-naïve	42	10 female, 32 male	25.4-27	1.5 mg/kg	Singular	120 mins.	Decision-making rigidity	Increased likelihood of previous response predicting current response; fewer different strategies used, but frequency of switching unchanged	Degree to which previous response determines next response; changing of choice strategies, and the frequency of this change

4. DISCUSSION

We will focus our discussion on rodent and human studies only, as the other animal studies are too sparse and inconsistent for deriving substantial conclusions from.

Each study is shown alongside whether a T- or Y-shaped maze was used, species, sex and sample size of the rodents tested (control and relevant treatment groups), whether the study used singular or chronic (multiple) dosing, and if they used chronic dosing, the timing of the doses; the dose and route (mode of injection) used, the ages the rodents were treated and tested at, the temperature of the testing environment, the duration of food-restriction (deprivation) enforced before testing, the food used, and the results obtained from the experiment in the treated rodents (where it is different from the results in the corresponding control rodents). i.p. = intraperitoneal mode of injection; s.c. = subcutaneous mode of injection.

Each study is shown alongside the species, sex and sample size (control and relevant MDMA groups) of the animals tested, whether the study used singular or chronic (multiple) dosing, and if they used chronic dosing, the timing of the doses; the dose and route (mode of administration) used, the ages the animals were treated and tested at, the temperature of the testing environment, and the results obtained from the experiment in the treated animals (where it is different from the results in the corresponding control rodents). i.p. = intraperitoneal mode of administration; s.c. = subcutaneous mode of administration; p.o.: oral mode of administration; i.m.: intramuscular mode of administration; i.g.: intragastric mode of injection.

Each study is shown alongside how the participants were recruited, the type of population sampled, the number of participants, the sex ratio of the participants, the ages of the participants, the amount of MDMA consumed orally, the dosing schedule, the time from MDMA administration to the first relevant test, the relevant tests used in the study, the relevant results from those tests, and the relevant parameters measured in the relevant tests.

4.1. Social Impairment

4.1.1. Rodent Studies

The outstanding result of this positional systematic review lies in the social-impairment experiments relating to autism. The set of studies show that social behavioural results are mixed between the studies, when 5-10 mg/kg MDMA is given singularly or chronically postnatally (Table 5), with these effects persisting over hours and days (Table 5). Adding value to this treatment, since MDMA has prosocial effects chronically in rodents, humans treated with MDMA would hopefully also benefit chronically beyond the timeframe of therapy sessions.

Given the widely used face and construct validity of any rodent model to represent autistic-like features in humans [128], further investigation needs to be undertaken in rodent ASD models to explore and fine-tune possible effects of MDMA as a treatment for autism-related social impairments in future clinical research. In 2016, there was a paper considering MDMA as a potential social anxiolytic in people with ASD [129]. In fact, a recent study published by Danforth et al. (2018) showed decreased social anxiety in autistic humans who were treated with MDMA [48], further adding substance to our argument. In addition, in this study, sustained long-term effects were also present [48]. However, limitations of this study include small sample size, a wide range of results, some insignificant differences between groups, potentially confounding comorbidities unaccounted for, uncertain autism diagnostic methods, observable MDMA effects interfering with blinding (especially with inability to verify prior drug abstinence), and recruitment challenges [48]. The author follows up with a subsequent observational study of autistic MDMA users, finding both short- and long-term alleviating effects of even non-clinical use of the drug on trauma and social anxiety, with no adverse effects [130].

However, chronic/high dosing or late testing had the opposite effects in rodents. Studies that tested 12-70 days after administering the MDMA, for example evidenced decreased prosocial [91-93] and increased asocial [93] behaviour. Two authors attributed this behaviour to increased anxiety [91, 92], and one author attributed it to either a change in serotonin-receptor function (namely, especially increased 5-HT1A receptor function in the dorsal hippocampus, basolateral amygdala or lateral septum), or a reorganisation of developing serotonergic innervation (in particular the dorsal raphe neurons, notably critical to social interaction) [131]. Morley et al. (2001) support their claim by quoting increased anxiety shown by the same rodents in other experiments in their study [91]. They also provide the alternative explanation that

Each study is shown alongside how the participants were recruited, the type of population sampled, the number of participants, the sex ratio of the participants, the ages of the participants, the amount of MDMA consumed orally, the dosing schedule, the time from MDMA administration to the first relevant test, the relevant tests used in the study, the relevant results from those tests, and the relevant parameters measured in the relevant tests.

it might be due to decreased environmental exploration (which would include the conspecific, in a social test), as a high dose caused decreased locomotion in this test [91]. So although both low and high doses of MDMA were anxiogenic, low or acute doses increased social interaction (with only low doses showing upregulation of the SERT receptor), and high or chronic dosing caused decreased social interaction [91]. The lattermost, they attribute to MDMA-induced neurotoxicity or adaptation to a neurotoxic effect, creating long-lasting effects in their rats, although they concede that from other studies, this probably improved over the last 3 months [91]. Fone et al. (2002), however, who tested only 12 days after treatment, claimed there was no serotonergic neurotoxicity involved in their decreased social interaction [131].

In addition, with the same doses, chronic dosing has the opposite effect: decreasing prosocial behaviour and increasing asocial behaviour. One study was an exception to this [78], where 5-20 mg/kg MDMA given twice daily over 3 days actually increased the duration of social investigation. This may have been anomalous; the only differences the authors highlight are that their studies used a control (instead of treatment-matched) conspecific, and that asocial and exploratory behaviours are not included in that statistic [78].

We found one study where 5 or 10 mg/kg MDMA, given chronically intra-peritoneally on PD 28-52, increased mouse social novelty preference when tested later on PD 120 (Table 3). This study also shows a long-lasting prosocial effect of the MDMA, which would increase the value of MDMA as a treatment. Interestingly, one study showed subsequent MDMA administration to cause a decrease in sensitivity to MDMA-increased social behaviour in rats [83]. Here, MDMA caused decreased social interaction in the long run in rats, and also inhibited prosocial effects of subsequent MDMA doses [83].

Other studies gave surprising results, too: social and prosocial behaviours were decreased when MDMA was given at a singular 5-10 mg/kg [95-97]. Navarro et al. (2004) attributed this to an anxiogenic effect, with c-fos as a molecular marker to support this (heightened in central amygdala, associated with increased anxiety) [96]. Bull et al. (2004) found anxiogenic effects with their chronic dosing, accompanied by a modest serotonin depletion [132]. They attributed their fewer rears to an acute efflux of vesicular serotonin, and decreased 5-HT2A receptor function in the cortex/brainstem [132]. Bull et al. (2003) also found an anxiogenic effect, with reduced social interaction 20 days later, accompanied by 20-40% decreased serotonin in the hippocampus and frontal cortex [133]. They suggest that high-dose MDMA could more directly cause compensatory re-innervation via long-term amygdalar/hippocampal hyperinnervation, causing these effects, which is significant because these are the areas which modulate serotonin receptor function and/or response to negative stimuli [133]. Gurtman et al. (2002) also noticed increased anxiety, with only minimal tissue serotonin concentrations and axon density being recovered, but a large amygdalar serotonin depletion, weeks to years after MDMA administration in rats [134]. The amygdala, of course, is strongly associated with anxiety. Having said that, Welsh et al. (2005) assert that autism may be due to a disruption of the inferior olive which hinders the processing of stimuli, because its neurons are not electrically synchronised, which jeopardises rhythmic output [135]. One study used a moderate chronic dose, but found no significant serotonin depletion (unlike in high acute doses) [136]. According to Clemens et al. (2007), with an acute high MDMA dose, endogenous free-radical-scavenging is exhausted, leading to free-radical-induced damage. With chronic moderate doses, more prefrontal-cortical serotonin depletion still occurs than single-dose, as the recovery time between doses does not seem fast enough [136]. It is unclear whether this is due to neurotoxicity or natural neuroplasticity over time [136].

Some authors attribute these results to specific parts of social interaction. Thompson et al. (2008) specified that this decrease was more applicable to the general-investigation part of social behavior [83]. In fact, prior MDMA exposure’s acute effects of decreased adjacent lying (involving 5-HT1A receptor) can be rescued by increasing the MDMA dosing even more [83]. Adjacent lying is the social passive behaviour of lying side-by-side with another rodent. Thus, the authors propose that the decrease in social behaviour involves non-5-HT1A receptors, such as persistent changes in receptor densities or the serotonin transporter in the amygdala and hypothalamus, mediating social behavior [83]. Thompson et al. (2008) assert that previous MDMA dosing reduces the organism’s sensitivity to future acute MDMA doses, partly by eliciting serotonin release less, and there may also be persistent desensitisation of the oxytocinergic network [83]. Homberg et al. (2007) found these effects specific to playful social behaviours and not confounded by effects on locomotion, with accompanying increased extraneuronal serotonin [95]. They state that it could also be due to increased anxiety, as reflected by their other, anxiety-specific tests. Their increased non-social exploration could just be an indication of the rodent attempting to escape from the arena. This does not explain why only playful social behaviours are affected, however [95]. Whether social interaction can be used as a measure of social anxiety is debated [137]. They used defaecation and freezing behaviours, as well as an unfamiliar and lit environment, as indicators of how anxious the test rats must be. They found that as these variables increased, rat social interaction decreased [137]. Hence, it is possible that social interaction can indicate decreased anxiety in a rodent. Another point of note is that Kang et al. (2015) found that a novel bright environment induced stereotypies, whereas a dark familiar environment did not, in a valproic-acid-induced rodent model of autism [138].

A glaring question may be why oxytocin should not be used directly, as MDMA acts to ultimately induce oxytocin release contributing to its prosocial effects. One such study used a “cyberball” task, whereby each subject was tested on their prosocial helping behaviour towards a known socially excluded person. This study found that with intranasally administered oxytocin, subjects compensated for other players’ ostracism by throwing the ball more often towards the excluded player [139]. Another study gave intranasal oxytocin to autistic adults, and found that there was improved social cognition after 6 weeks’ treatment [140]. But in fact, one paper says that, when intra-nasally administered, very little oxytocin enters the central nervous system, and most accumulates in the peripheral bloodstream causing a myriad of adverse effects such as cardiac arrhythmias [141]. Another study directly administered intranasal oxytocin to autistic pre-teens and teenagers, and found no effects on social behavior [142]. Yet another found no increase in empathic concern after subjects were given intranasal oxytocin [143].

We also examined sex and age effects. There were some sex differences, in studies where both female and male test subjects were employed. In terms of male behaviour, a study found that gonadotrophin-releasing hormone and testosterone were suppressed in MDMA-treated rats [144], which might explain the result found above that MDMA decreased copulating behaviour in rats [84]. Females were more affected with investigation (sniffing) behaviours, whereas males were more affected with other social-interaction behaviours.

4.1.2. Human Studies

The studies suggest that a chronic oral dose of 0.75-1.5 mg/kg might be optimal to address social impairments, depending on the sex and metabolism of the individual. Caution is warranted to ensure the dose minimally affects the ability to recognise emotions in others.

For social interaction, a few specialist tests are worth mentioning to inform future clinical studies, namely a virtual ball-throwing task [145], an ultimatum game [146], the Prisoner’s Dilemma [147], and a welfare trade-off task [46]. The ball-throwing task simulates social rejection and tests for the participant’s self-ratings of self-esteem and mood, whereby a virtual “cyberball” is tossed between the participant and two experimenter-controlled virtual “players”. For the first part, social inclusion was simulated by having the ball tossed to each player equally; the second part simulated social exclusion, by tossing the ball mostly between the two virtual players only. After each part, participants self-rate positive mood and self-esteem. With MDMA, chronic 0.75 and 1.5 mg/kg reduced the decreasing effect of rejection on mood and self-esteem, and increased perceived percentage of inclusive throws under rejection condition [145]. The ultimatum game assesses social decision-making (trust and cooperation), whereby participants are paid based on their acceptance of first-person, third-person and computer-generated monetary offers [146]. The study found that chronic 1.5 mg/kg MDMA decreased the probability of rejecting unfair offers in the first-person condition, increased average percentage offer from the participants themselves, and increased prosocial interaction [146]. The Prisoner’s Dilemma also assesses choice to trust and cooperate, and the study found that chronic 1.5 mg/kg MDMA increased cooperation, especially with a trustworthy opponent, that is maintained over time [147]. Interestingly, chronic 1.5 mg/kg did not increase probability of cooperating with an untrustworthy opponent or game server (neutral), possibly indicating retained interpersonal insight [147]. Indeed, self-ratings of insightfulness have been shown to increase with singular dosings of 0.75 and 1.5 mg/kg [148], although the reliability of self-ratings needs to be checked The same dose given chronically also decreased vigilance [149].

Likewise, in another study, self-reported trust increased at a chronic 1.1 mg/kg [150]. The welfare trade-off task assesses generosity, whereby the participant chooses when to switch between monetarily benefiting themselves to benefitting others [86]. The study found that chronic 1 mg/kg increased generosity to a friend, and that chronic 1.5 mg/kg increased average percentage offer [46]. Hence, it seems that chronic 0.75-1.5 mg/kg MDMA given orally to humans is effective in increasing prosocial feelings towards another person.

A prominent advantage of using human subjects is that they are articulate, so their vocabulary under different treatments can be analysed. 0.75 and 1.5 mg/kg MDMA increased positive emotion words, whereas 1.5 g/kg increased words describing another that were social and reflective of theory of mind (insight into another person’s mental state, which is often considered lacking in ASD [151]); increased time verbally interacting, words closer to “intimacy”, “friend”, “rapport” and “support”. One study administered an oral dose of 62.5 mg/kg with subsequent top-ups of 1.9 mg/kg, and held talking sessions, where they found that those participants exhibited increased ensuic (describing a change in their sense of self), empathic (regarding others' emotions) and entactic (inclination towards physical touch) utterances [114].

Empathy has been suspected to be lacking in ASD individuals [152]. In terms of empathy, these studies showed that at a chronic 1.1 mg/kg MDMA, participants were equally aroused by positive and negative sounds, whereas in control participants, negative sounds produced more arousal than did positive [153]. Chronic 1.1 mg/kg MDMA also increased both explicit and implicit emotional empathy scores for positive emotional stimuli [150]. There was also more concern (especially for positive stimuli) and arousal (for both positive and negative (as opposed to just positive, as in controls) stimuli) for people depicting emotions, at chronic 1.1 mg/kg [154] but also at chronic 1.9 mg/kg [155]. In the middle, chronic 1.5 mg/kg MDMA decreased frown response, and increased smile response, to happy expressions [116]. And at chronic 1.9 mg/kg, there was decreased cognitive empathy for all emotions, but increased explicit emotional empathy for positive emotions [156]. Chronic 1.9 mg/kg MDMA promoted shift from joint gain maximization to inequality aversion, as well as increased prosociality via a resource-allocation task [45]. As a cautionary note, increased trust due to MDMA may even make autistic individuals more prone to be swindled, especially given that they are already thus susceptible if they lack theory of mind.

4.2. Repetitive Behaviours (Rodent and Human Studies)

The studies indicate that singular 10-5 mg/kg MDMA may reduce repetitive and compulsive behaviours in rodents, but more studies need to be conducted specifically on this, as the open-field test is unspecific for stereotypy and the marble-burying test may include compulsive behaviour unaccounted for. Only one study found significant results of stereotypy in humans [123], and this is not enough to draw conclusions from. Further controlled studies in humans are warranted, to look at the effects of MDMA on stereotypy as a core impairment in ASD.

4.3. Cognitive Rigidity (Rodent and Human Studies)

Rodent studies indicate that mostly chronic 10-20 mg/kg MDMA seems to have the least aggravating effect on cognitive rigidity. Older rodents tended to have more cognitive rigidity, but were more likely to investigate their surroundings and perform self-grooming. Younger rodents made fewer pinnings, but more head-weavings as a serotonin behaviour. For cognitive rigidity, the radial-arm maze suggested that older rodents were not as impaired compared to their placebo counterparts, as were younger rodents which showed more working-memory impairments (Table 8). This may be because older rodents naturally have impaired memory, so the difference between MDMA-treated and placebo rats may not be so significant. Only three studies tested females [157-159], so there is insufficient data to properly assess sex effects here. There were too few human studies measuring cognitive rigidity as a parameter to conclude the optimal dose at which this core impairment is most alleviated, but there is a possibility that a singular 1.5 mg/kg p.o. may actually exacerbate an inflexibility, as shown by the indirect measure of the likelihood of changing choices [123].

4.4. Accessory ASD Behaviours (Rodent and Human Studies)

Accessory ASD traits (impairments not included in the 3 core symptoms we have explored in this review) are also worth investigating. For example, because an empathy deficiency is considered to be characteristic of some ASD individuals, we will also introduce an empathy test which could, in future, be used to test behavioural impairment in autistic-like rodents. Conveniently, Bartal et al. (2011) have developed a means to assess empathic tendency in a rodent, by setting up an apparatus with a restrained conspecific inside. The test subject is placed inside the chamber, and is timed to see how quickly they open the restrainer door deliberately in order to free the conspecific [160]. Other infrequent impairments in autism include motor coordination [8]. Since some studies already use variants of motor function tests like the vermicelli-handling task or the sunflower-seed-eating task [161], we wonder whether the MDMA tests could also be extended to accessory behaviours of ASD. Anxiety is also a common comorbidity in ASD individuals [162], and an elevated plus-maze could be used to test MDMA effects in rodents for generalized anxiety [163]. For treatment purposes, however, it would be essential to look at the core impairments first, since these are by definition shared by all autistic individuals. Future directions could also include tracking for sleep or gastrointestinal disturbances, as well as seizures, as other ASD comorbidities [164].

4.5. Recent and Ongoing Clinical Trials

The first randomised double–blind placebo-controlled clinical trial being performed to test MDMA’s prosocial effects on humans with autism has yielded encouraging results [48]: improvement on the Leibowitz Social Anxiety Scale (LSAS) was substantial and statistically significant in the MDMA–treated group, and positive scores were maintained in the follow-up at 6 months. The small sample size of the study (n = 12) limits firm conclusions in regard to the potential impact of the MDMA treatment [48]. Nevertheless, the solid basis of this randomised double-blind placebo-controlled study justifies larger randomised trials to assess MDMA effects on social anxiety in subjects with autism. Of note, another trial is ongoing (ClinicalTrials.gov #NCT04053036) in which the relevant parameter being tested is change in responses to affective touch in autistic adults. No final results are reported at the time of our writing.

Based on MDMA’s well-established prosocial effects, the studies reviewed and the results obtained from the completed trial mentioned above, for this new ongoing trial, we are expecting decreased social anxiety, increased empathy/perception/response to positive emotions/touch, and decreased empathy/perception/response to negative emotions/touch. These trials are based on MDMA’s known effects of increasing oxytocin release, via serotonergic pathways, such that oxytocin deactivates amygdalar alarm signals to the brainstem which then no longer induces behavioural and sympathetic fear responses.

4.6. MDMA Neurotoxicity

The potential toxicology for new pharmacotherapeutic agents is a necessary consideration for adoption. Helpfully, MDMA has been in clinical and recreational use for several decades, and there is now considerable data describing its adverse effects [165]. The most prominent toxicological finding in the literature arises from the excessive administration of MDMA which can cause structural and physiological changes in serotonin neurons. For example, administration of 20 mg/kg MDMA (s.c.) twice daily for 4 days causes decreased brain serotonin in the forebrain in rats by selectively degenerating serotonergic axons [166, 167]. The serotonin axon terminals are selectively ablated, resulting in fragmented projections and causing the serotonin to stay in the pre-terminal fibres, in rats [166]. Serotonin and dopamine are deaminated via monoamine-oxidase (MAO) enzymes (subtypes A and B), forming hydrogen peroxide in serotonergic nerve terminals in vitro, which seems to contribute to serotonergic neurotoxicity [168]. This is substantiated by a 26% decreased 5-HTIAA concentration in the cerebrospinal fluid of individuals with previous MDMA use, suggesting impaired central serotonergic activity [169]. It has therefore, been argued that these adaptive changes in serotonin neurons cause long-term neuropsychological and affective impairments, as has been shown in recreational MDMA users [170, 171].

However, recreational MDMA users also tend to use other drugs in addition to ecstasy [172-174], which could also be the reason for these adverse effects. In fact, one study showed that people who attempt to restrict their drug intake exclusively to ecstasy seem to have negligible adverse neuropsychological sequelae from repeated use [175]. Other potential adverse effects arising from acute ingestion of MDMA in uncontrolled settings include mild serotonin-syndrome symptoms including hyperthermia and resultant hyponatremia, particularly in warm crowded active environments [172, 176]. Further consideration of putative MDMA toxicity also requires consideration of toxic synthetic by-products produced during the illicit synthesis of MDMA. In particular, paramethoxyamphetamine (PMA) is a highly toxic potential by-product of illicit MDMA synthesis that targets serotonin neurons [177]. However, irrespective of potential toxicity from recreational doses and uncontrolled ecstasy manufacture, the clinical use of MDMA has not been shown to produce serious persistent adverse effects in any of the clinical trials reported to date [178-180]. Functional adverse effects are reflective of these serotonin-neuron structural changes, and there are both short- and long-term effects of MDMA use in humans [44, 51, 127, 181, 182].

Hyperthermia has also been associated with worsening neurotoxicity, thus less hyperthermia may mean less serotonin depletion, with their weekly doses [136]. The hyperthermic effects of MDMA have been seen in both humans and rodents. In fact, rodent prosocial behaviour is amplified when the temperature is increased from the normal ~21-22⁰C to ~28⁰C during testing [69]. According to Clemens et al. (2007), repeated MDMA doses decrease the hyperthermic effect (measuring at 8-16 weeks of dosing). This may produce tolerance against serotonin depletion [136]. In terms of its neurotoxicity, MDMA is known to deplete serotonergic terminals in rodents, as well as cortical, hippocampal and striatal neurodegeneration [183]. In addition, MDMA-induced hyperthermia, monoamine oxidation (of dopamine and serotonin), dopamine oxidation, serotonin transports nitric oxide, neurotoxin and peroxinitrite formation, glutamate excitotoxicity, 5-HT2A agonism all contribute to this neurotoxicity [183]. Therefore, its dose should be monitored.

4.7. Dosing Regimen

We are aware that there is a narrow range of doses testable for MDMA. One of the studies found that when MDMA was given on PD 1-5 to rodents, there was increased offspring mortality [184]. Therefore, it would be inadvisable on ethical grounds to test MDMA given within this timeframe.

Previous studies have used interspecies scaling to convert animal drug doses to human drug doses (Fig. 3), but Green et al. (2009) emphasise that bioavailability, active metabolites, plasma-protein binding and systemic exposure patterns all differ between species, and would all affect onset, intensity and duration of the dug’s effect on the subject [185]. Green et al. (2009) compare MDMA dose-plasma concentration response curves between humans and rats, and highlight that an autoinhibition of MDMA metabolism occurs in humans that is unseen in rats, implicating that rats would require 4 times the dose given to humans, to produce similar peak concentrations in the bloodstream [185]. This would need to be taken into account when transitioning from pre-clinical to clinical studies.

Fig. (3).

Proposed equation to calculate equivalent dosages between animals and humans, where D = dose (mg) and W = weight (kg) [185].

There are limitations to administering MDMA in healthy animals and humans, also. MDMA may have unseen spurious effects around the body, which may modulate the parameters measured. Further, the ethics of giving an active drug to already healthy organisms are questionable.

4.8. Summary

In rodents, administering the investigated 5-10 mg/kg MDMA, singularly/chronically intra-peritoneally/ subcutaneously, seems to ameliorate only social impairments significantly in some studies, whereas its effects on cognitive inflexibility and motor stereotypies are either exacerbated or have no effect. Therefore, MDMA’s dose-response curve needs to be assessed, through varying dose, route, temperature and timing, in an effort to find a way to reduce these core impairments in ASD. In any particular autism patient, clinicians may then prescribe a dosing regimen according to their unique proportions of social impairment, cognitive rigidity and motor stereotypies. This is only if more definitive clinical trials support its use, and if the agent can then be legally prescribed.

In humans, the optimal dose at which maximal social impairment alleviation and minimal emotion recognition impairment seems to be 0.75 mg/kg in women and 1.9 mg/kg in men, taken orally and chronically. At 1.5 mg/kg, taken orally chronically, repetitive stereotypy may increase, so doses will have to be adjusted to the individual accordingly. Again, this is only if more definitive clinical trials support its use, and if the agent can then be legally prescribed.

In non-rodent animals, different species have different doses and routes having the same effect of social impairment alleviation, no effect on stereotypy, and similarly to humans, a chronic 1.5 mg/kg p.o. dose causing cognitive rigidity in monkeys. However, sample sizes for non-rodent animal studies are low, especially as the size of the animals is bigger (costs of upkeep), so further pre-clinical studies are likely warranted before making further conclusions.

4.9. Limitations

Our review has several limitations. Firstly, only English-language papers were reviewed. Thus, there is a significant gap in the literature for the MDMA treatment of ASD behaviours. Furthermore, whilst the authors have attempted to provide the most up-to-date evidence possible, there is still this temporal limitation that precludes papers published after the time of study identification from being included in this review. In addition, only six databases (Google Scholar, NCBI, ProQuest, Scopus, Web of Science and Wiley Online Library) have been screened, given the time constraints of this project. Behaviours not screened in this review may have affected the core ASD behaviours studied. In addition, molecular analogues of MDMA may be worth investigating, to optimise treatment drugs to maximise alleviations to the addressed impairments, and minimise the adverse effects and exacerbations of impairments. The use of molecular MDMA analogues to affect human behavioural effects has been explored in a recent review [186]. Methylenedioxymethamphetamine (MDA), 3,4-methylenedio-xyethylamphetamine (MDE) and N-methyl-1,3-benzodioxolbutanamine (MBDB), in particular, have also been proposed as potential psychotherapeutic agents due to their entactogenic properties [186]. Since there are also a wide variety of rodent strains, each of which may have different responses, this would need to be monitored in the future, and may help explain some of the mixed results in the literature. There are also limitations of construct and face validity in mapping healthy rodents to ASD humans: healthy rodents would not have the neurological wiring aberrations present in ASD humans, on top of their differences in species. Hence, their respective MDMA effects may not be so comparable.

CONCLUSION

Whilst social impairment may possibly be addressed in this way, we have surveyed the effects of MDMA on the other core autism-related impairments in rodent studies too, and we find we cannot apply MDMA to reverse these other core behavioural impairments. There are gaps in the current knowledge in this area, as well. Stereotypy and cognitive rigidity in humans and animals have not been studied adequately. Overall, the clinical implications of these studies are that MDMA may be a cost-effective therapeutic for the social disability experienced by ASD individuals, but more pre-clinical trials are needed to establish MDMA as an effective medication for this impairment; and that MDMA may have persistent effects on the prosocial behaviour induced, aiding long-term therapy for autistic individuals. The latest results from recent clinical trials corroborate the hypothesis of MDMA as a valuable tool in addressing social impairment in subjects with autism, but this is far from proven yet.

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Social Restriction	Males	Females	Both
Cornish 2003 [69]	Rat	Males	NA	24	Singular	NA	5	i.p.	NA	NA	21, 30	Nil	Increased social interaction, more so at 30°C than 20°C.	NA	NA
Curry 2018 [219]	Mouse	Males	NA	20	Singular	NA	3-30	i.p.	49-70	47-70	22	30 mins.	7.8 mg/kg SR-MDMA increased social interaction. 17 mg/kg R-MDMA increased social interaction. S-MDMA had trend towards significance at 7.8 mg/kg	NA	NA
Curry 2019 [220]	Mouse	Males	NA	20	Chronic	Every alternate day, 4 sessions	7.8	i.p.	49-70	49-70	22	25 mins.	Dose-dependently increased duration of social interaction (non-anogenital sniffing and adjacent lying)	NA	NA
Daza-Losada 2008 [78]	Mouse	Males	NA	43-64	Chronic	Twice daily	5, 10, 20	NA	28-30	51	21	Nil	Increased social-investigation time.	NA	NA
Daza-Losada 2009 [221]	Mouse	Males	NA	315	Singular	NA	5, 10, 20	i.p.	28	28	21	Nil	5 mg/kg: increased social investigation and contact time. 20 mg/kg: decreased social interaction and contact time time. 10 mg/kg: increased distant exploring. 5, 10 mg/kg: decreased non-social exploration.	NA	NA
Dornan 1991 [222]	Rat	Males	NA	19	Chronic	Every 12 hours, for 4 consecutive days	40	s.c.	350-475g	7, 14 days later	21	NA	After 7 days: smaller percentage of MDMA rats ejaculated; post-ejaculatory interval increased after repeated injections. After 14 days: no effect.	NA	NA
Fone 2002 [131]	Rat	Males	NA	36	Chronic	Twice daily	7.5	i.p.	39-41	51-53	21	Nil	Decreased social exploration (sniffing, following conspecific).	NA	NA
Garcia-Pardo 2015 [93]	Mouse	Males	NA	30	Singular	NA	1.25, 10	i.p.	60	81	NA	1 min.	Less time in attack, social contact. More time in avoidance/flee, defence/submission, distant exploration of conspecific.	NA	NA
Gurtman 2002 [134]	Rat	Males	NA	26	Chronic	Each of 4 hours	5	i.p.	90	132	22	Nil	Decreased time and frequency of social interaction.	NA	NA
Homberg 2007 [95]	Rat	Males	NA	NA	Singular	NA	0.5, 2, 5	s.c.	28-35	Same day	21	3.5 hours	Dose-dependently decreased pinning, pouncing, boxing. Decreased following/chasing. 2, 5 mg/kg: decreased social exploration, social grooming.	NA	NA
Kirilly 2006 [223]	Rat	Males	NA	16	Singular	NA	15	i.p.	49	70	21	14 days	Increased kicking, decreased grooming and social behaviour. Acute decreased biting, boxing, kicking, wrestling time, increased time to start wrestling. Decreased social behaviour and grooming. When young intruders introduced, increased locomotion and general exploration. 2nd MDMA dose increased locomotion and general exploration more than first dose.	NA	NA

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Social Restriction	Males	Females	Both
Kirilly 2010 [224]	Rat	Males	NA	NA	Chronic	Twice	15	i.p.	42-49	21 days later, and once on testing day	21	14 days	Acute: decreased aggressive-type behaviour. 1st dose: decreased biting, boxing.	NA	NA
Kurling 2008 [225]	Rat	Males	NA	12	Singular	NA	1, 5	i.p.	“Adult” (300–380g)	Same day	21	6 days	Increased locomotor activity, rearing bouts, agitation, stereotyped sniffing, head/body weaving, head bobbing.	NA	NA
Machalova 2012 [79]	Mouse	Males	NA	98	Singular	NA	2.5, 10, 30	i.p.	“Juvenile” (18-20g)	Same day	22	Not specified	Aggressive mice: decreased aggression, increased timidity (defensive posture; 30 mg/kg: escape; 10, 30 mg/kg: alert posture); 2.5, 10 mg/kg: increased social behaviour (2.5 mg/kg: social sniffing; 2.5, 10 mg/kg: conspecific-following); 30 mg/kg: walking. Timid mice: 10, 30 mg/kg: increased timidity (increased alter- posture frequency); 30 mg/kg: increased escape frequency. Decreased social behaviour frequency (social sniffing; 10, 30 mg/kg: conspecific-following). 30 mg/kg: walking.	NA	NA
Maldonado 2001 [226]	Mouse	Males	NA	NA	Singular	NA	0.5, 1.25, 2.5	i.p.	NA	Same day	NA	NA	0.5, 1.25 mg/kg: decreased shorter inter-attack intervals	NA	NA
Maldonado 2001 [88]	Mouse	Males	NA	60	Singular	NA	1, 8, 15	i.p.	“Juvenile” (25-30g)	Same day	21	30 days (for aggression)	8, 15 mg/kg: decreased grooming, digging, social exploration, threat/attack time. 8, 15 mg/kg: increased non-social exploration, defence/submission, distance exploration, avoidance. 1 mg/kg: increased distant exploration, avoidance.	NA	NA
McGregor 2003 [92]	Rat	Males	NA	24	Chronic	Each of 4 hours over 2 consecutive days	5	i.p.	90	160	28	Nil	Decreased social interaction time and frequency.	NA	NA
McGregor 2003 [92]	Rat	Males	NA	24	Singular	NA	5	i.p.	90	160	28	Nil	Decreased social interaction time.	NA	NA
McGregor 2003 [77]	Rat	Males	NA	32	Chronic	Each of 4 hours over 2 consecutive days	5	i.p.	60-75	56-70 days later	28, 16	Nil	Less social interaction, fewer social-interaction bouts.	NA	NA
Miczek 1994 [227]	Mouse	Males	NA	29	Singular	NA	0.3, 3, 6, 10	NA	“Adult”	“Adult”	21	Nil	3-10 mg/kg: decreased frequency of attacks > decreased dose-dependent sideways threats.	NA	NA

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Social Restriction	Males	Females	Both
Morley 2000 [228]	Rat	Males	NA	56	Singular	NA	1.25, 2.5, 5	i.p.	85-95	99-109	22	Nil	Decreased aggression time. 5 mg/kg: increased social interaction time, locomotion. 1.25 and 2.5 mg/kg had no effect on locomotion or social interaction duration	NA	NA
Morley 2001 [91]	Rat	Males	NA	32	Chronic	Each of 4 hours on 2 consecutive days	5	i.p.	75-95	160-180	22	Nil	Decreased total interaction time and frequency, rearing frequency, locomotion.	NA	NA
Morley 2001 [91]	Rat	Males	NA	32	Singular	NA	5	i.p.	75-95	160-180	22	Nil	Decreased total interaction time and frequency, rearing frequency, locomotion, social interaction.	NA	NA
Morley 2005 [81]	Rat	Males	NA	112	Singular	NA	5	i.p.	50-90	50-90	28	5 mins.	Increased total social interaction time (adjacent lying, approach), decreased anogenital sniffing and rearing. Serotonin syndrome: greater turning, low body posture, piloerection.	NA	NA
Morley-Fletcher 2002 [190]	Mouse	Males	NA	30	Chronic	Once on each of 3 days, 2 days apart	5, 10	i.p.	28, 38, 52	80	21	Nil	Altered environmental exploration time (dosed at early and late adolescence: dose-dependent increase; dosed at middle-adolescence: decrease). Decreased self-grooming. Decreased freezing time (dosed at early adolescence).	NA	NA
Navarro 1998 [89]	Mouse	Males	NA	96	Singular	NA	0.5, 1.25, 2.5, 5, 10, 15, 20	i.p.	42	42	20	30 days (for aggression)	Decreased aggression (threat, attack) and social exploration; increased distant exploration, avoidance and defence. 5-20 mg/kg: decreased threat/attack time. 2.5-20 mg/kg: increased distant exploration. 5-20 mg/kg: increased non-social exploration, avoidance/defence; decreased social exploration. 1.25-20 mg/kg: decreased digging. 2.5, 20 mg/kg: decreased grooming.	NA	NA
Navarro 2004 [96]	Mouse	Males	NA	40	Singular	NA	1, 8, 15	i.p.	25-30g	30 mins. later	20	Nil	1, 8, 15 mg/kg: decreased social-investigation time. 8, 15 mg/kg: decreased digging, increased avoidance/flee.	NA	NA
Navarro 2004 [90]	Mouse	Males	NA	120	Singular	Experimental day 7	1.25, 2.5, 5	i.p.	25-30g	30 mins. after last injection	21	30 days	Increased non-social exploration. 2.5, 5 mg/kg: decreased digging time, threat, attack; increased distant exploration. 5 mg/kg: increased avoidance/flee, defence/submission.	NA	NA
Navarro 2004 [90]	Mouse	Males	NA	120	Chronic	Experimental days 1-7, daily	1.25, 2.5, 5	i.p.	25-30g	31 mins. after last injection	21	30 days	1.25 mg/kg: increased digging. 2.5 mg/kg: increased exploration from distance, decreased non-social exploration behaviors.	NA	NA

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Social Restriction	Males	Females	Both
Piper 2008 [205]	Rat	Males	NA	20-24	Chronic	4 doses, 1 each hour	10	s.c.	“Young adult” (307.7g)	“Young adult”	23	Nil	No effect.	NA	NA
Procopio-Souza 2011 [85]	Mouse	Males	NA	24	Chronic	Acute dose, then repeat 10 days later	10	i.p.	90	90, 100	22-23	NA	Increased locomotor frequency (housing: group > individual). Group-housed: increased social-interaction time. 2nd dose: higher locomotion frequency; group-housed: repeated dosing did not modify social behavior; but increased body contact, peaceful following; decreased anogenital sniffing.	NA	NA
Ramos 2013 [229]	Rat	Males	NA	32	Singular	NA	2.5, 5	i.p.	“Adult” (250-300g)	Same day	23	1 hour	5 mg/kg: increased adjacent-lying time, decreased anogenital sniffing and rearing.	NA	NA
Ramos 2015 [230]	Rat	Males	NA	10	Singular	NA	5	i.p.	“Adult” (250-300g)	“Adult” (250-300g)	21	Nil	More time spent in physical contact with a conspecific (but this also occurred with an inanimate object).	NA	NA
Rodriguez-Arias 2011 [231]	Mouse	Males	NA	40	Chronic	Twice, 4 hours apart, on each of 2 days, 6 days apart	10, 20	i.p.	41, 42	64	21	Nil	20 mg/kg: less social-investigation time, less threat and attack times, more non-social exploration time.	NA	NA
Rodriguez-Arias 2015 [232]	Mouse	Males	NA	179	Chronic	Twice daily (4-hour intervals), over 2 consecutive days a week	10, 20	i.p.	33, 34, 40, 41	75	21	Nil	More aggression (10, 20 mg/kg: more attacks; 10 mg/kg: more threats). 10 mg/kg: more time in social investigation, less time in non-social investigation.	NA	NA
Shen 2011 [233]	Rat	Males	NA	60	Chronic	Twice every 5th day	10	s.c.	52-62	72	26	7 days	Reduced crawling frequency and time.	NA	NA
Slais 2005 [234]	Mouse	Both	NA	NA	Chronic	2.5 mg/kg: once daily for 5 consecutive days; 30 mg/kg: 8 days later	2.5, 30	p.o.	NA	NA	NA	Nil	Decreased aggression in aggressive mice, decreased prosocial behaviour in timid mice. Increased defence-escape behaviour. MDMA pre-treatment (2.5 mg/kg) had no effect.	-	-
Slais 2005 [235]	Mouse	Males	NA	NA	Singular	NA	2.5, 10, 30	p.o.	NA	NA	NA	Always	Decreased aggression, increased sociability and defence-escape behaviours in aggressive mice. Increased defence-escape behaviour and decreased sociability in timid mice.	NA	NA

Study	Species	Sexes	Sexes Combined or Separated	Sample Size	Singular/ Chronic Dosing	Chronic Timing	Dose (mg/kg)	Route	Treatment Age (PD)	Testing Age (PD)	Temperature (°C)	Social Restriction	Males	Females	Both
Slais 2009 [236]	Mouse	NA	NA	49	Chronic	Twice, 1-week interval	2.5, 10, 30	p.o.	NA	NA	NA	Nil	Aggressive mice: decreased aggression and sociability, increased defence-escape behaviour; 30 mg/kg: increased walking. Timid mice: increased defence-escape behaviour, decreased prosociality; 30 mg/kg: increased walking	-	-
Slamberova 2015 [97]	Rat	Males	NA	246	Singular	NA	2.5, 5.0, 10	s.c.	“Adult”	Same day	22-24	Nil	Decreased time and frequency of mutual sniffing. 10 mg/kg: decreased frequency of mutual sniffing and following. Decreased time and frequency of climbing over conspecific. 5, 10 mg/kg: decreased rearing frequency, time and frequency of allogrooming.	NA	NA
Thompson 2004 [237]	Rat	Males	NA	25	Chronic	Each of 4 hours on 2 consecutive days	5	i.p.	332g	84-105 days later	28	Nil	Less time in social interaction.	NA	NA
Thompson 2007 [52]	Rat	Males	NA	16	Singular	NA	5	i.p.	403g	403g	28	Nil	Increased adjacent lying; decreased general investigation, anogenital investigation and rearing.	NA	NA
Thompson 2008 [83]	Rat	Males	NA	48	Chronic	Each of 4 hours over 2 consecutive days	5	i.p.	6-16	90-100	28	20 mins.	Chronically decreased total social interaction.	NA	NA
Thompson 2008 [83]	Rat	Males	NA	48	Chronic	Each of 4 hours over 2 consecutive days	2.5	i.p.	9-19	90-100	28	20 mins.	Decreased investigation.	NA	NA
Thompson 2008 [83]	Rat	Males	NA	48	Singular	NA	5	i.p.	8-18	90-100	28	20 mins.	2.5 mg/kg: decreased total social interaction, adjacent lying.	NA	NA
Thompson 2009 [82]	Rat	Males	NA	36	Singular	NA	5	i.p.	“Adult” (345g)	Same day	28	20 mins.	Increased total social interaction and general exploration; decreased anogenital sniffing and rearing.	NA	NA
van Nieuwenhuijzen 2010 [212]	Rat	Males	NA	24	Chronic	Daily over 10 days	5	i.p.	220-300g	220-300g	21	Nil	Decreased general-investigation time.	NA	NA
Wallinga 2009 [80]	Rat	Males	NA	21	Chronic	Thrice, 3-hour intervals	6	i.p.	425g	133	21	1 day	Low-aggressive: 4-month-old intruder caused increased aggressiveness.	NA	NA

Table 5.

Definitions of specific physical interactions.

Behaviour	Definition	Type
Anogenital sniffing	The rodent sniffing the anogenital areas of the conspecific	Social investigative (neither friendly nor hostile)
Crawling/mounting	The rodent crawling over the conspecific	Prosocial
Digging	The rodent digging a hole into the bedding of the apparatus	Compulsive/repetitive/anxious
Fighting	Fighting in adults (approximately PD 90-120)	Hostile
Non-anogenital sniffing	The rodent sniffing the non-anogenital areas of the conspecific	Prosocial investigative (friendly)
Pinning	The rodent standing over the conspecific which lies flat on its back	Prosocial
Play fighting	Fighting in juveniles (approximately PD 30-50)	Prosocial
Play responsiveness	The probability of being pinned in response to being pounced on	Prosocial
Rearing	The rodent standing up on its hind legs	Environmentally investigative

LIST OF ABBREVIATIONS

5-HT

serotonin

ASD

autism spectrum disorder

DSM

Diagnostic and statistical manual (of mental disorders)

FDA

Food and drug administration

i.g.

Intragastric (mode of administration)

i.m.

Intramuscular (mode of administration)

i.p.

intraperitoneal (mode of administration)

ICD

International classification of diseases

MAO

Monoamine oxidase

MBDB

N-methyl-1,3-benzodioxolbutanamine

MDA

Methylenedioxymethamphetamine

MDE

3,4-methylenedio-xyethylamphetamine

MDMA

3,4-methylene-dioxy-methamphetamine

OXTR

Oxytocin receptor

p.o.

Per os (mode of administration)

PD

Postnatal day

PMA

Paramethoxyamphetamine

PRISMA

Preferred Reporting Items for Systematic Reviews and Meta-Analyses

s.c.

Subcutaneous (mode of administration)

SERT

Serotonin

USV

Ultrasonic vocalisation

CONSENT FOR PUBLICATION

Not applicable.

STANDARD OF REPORTING

PRISMA guidelines and methodology were followed.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.